BEHAVIOR OF PASSAGED CHONDROCYTES IN COLLAGENGLYCOSAMINOGLYCAN SCAFFOLDS: EFFECTS OF CROSSLINKING, MECHANICAL LOADING, AND GENETIC
MODIFICATION OF THE SCAFFOLD
by
Cynthia R. Lee
B.S. Bioengineering
University of California, Berkeley, 1997
S.M. Mechanical Engineering
Massachusetts Institute of Technology, 1999
Submitted to the Department of Mechanical Engineering in Partial
Fulfillment of the Requirements for the degree of Doctor of Science
at the
Massachusetts Institute of Technology
February 2002
©Massachusetts Institute of Technology, 2002
Signature of Author
4/
Department of Mechanical Engineering
September 2001
Certified by
w
Mvyon Spector, Thesis Supervisor
/ Senior Lecturer Mechanical Engineering
Professor of Orthoydic Surgery (B materials), Harvard Medical School
Certified by
Pr
MASSAC HUSETTS N
OF TECHNOLOGY
. Grod nsky, Thesis Supervisor
ical, and Bioengineering
ssor of Electrical, Me
Ain A. Sonin
Chairman, Departmental Committee on Graduate Students
MAR 2 9 2002
LIBRARIES
BARKER
BEHAVIOR OF PASSAGED CHONDROCYTES IN
COLLAGEN-GLYCOSAMINOGLYCAN SCAFFOLDS:
EFFECTS OF CROSS-LINKING, MECHANICAL
LOADING, AND GENETIC MODIFICATION OF THE
SCAFFOLD
by
Cynthia R. Lee
Submitted to the Department of Mechanical Engineering in partial fulfillment of the
requirements for the Degree of Doctor of Science, February 2002.
ABSTRACT
Tissue engineering is a promising solution to the problematic healing of cartilage
defects. The purpose of this thesis was to establish a foundation for the development of a
collagen-glycosaminoglycan (CG) scaffold for articular cartilage tissue engineering by
exploring the behavior of passaged chondrocytes in the CG scaffolds under the influence
of a variety of environmental factors.
Using in vitro studies, the first two parts of the thesis evaluated the effects of the
physical environment on the behavior of adult, passaged chondrocytes in the CG scaffold.
Scaffold cross-linking procedures increased cross-link density and scaffold stiffness and
increased resistance to cell-mediated degradation and contraction as follows:
dehydrothermal treatment (DHT) < ultraviolet irradiation (UV) < gluteraldehyde (GTA)
< carbodiimide (EDAC). EDAC scaffolds also provided for the highest levels of cell
proliferation and protein and GAG synthesis throughout a 4-week culture. Static
mechanical compression (0-50% strain) applied to cell-seeded EDAC cross-linked
scaffolds decreased rates of protein and GAG synthesis while dynamic compression (3%
sine amplitude, 0.1 Hz) increased rates of biosynthesis over a 24-hour period. These
results were similar to those of prior studies of loading of intact cartilage explants.
Unlike the explant studies, however, dynamic compression failed to increase the
accumulation of matrix molecules within the construct compared to unloaded ("freeswelling") controls because of a large increase in the release of newly synthesized
macromolecules into the media.
To evaluate the in vivo performance of the chondrocyte-seeded EDAC crosslinked CG scaffold, repair tissue formed 15 weeks after implantation of a 4-week in vitro
cultured construct was evaluated. The majority of the repair tissue was hyaline and
fibrocartilaginous. However, it displayed decreased levels of type II collagen and GAG
staining compared to normal articular cartilage, and had a compressive stiffness that was
20-fold lower than normal.
Finally, in anticipation of future work utilizing gene therapy to improve cartilage
repair, the CG scaffolds were modified for direct delivery of genetic material to cells in
situ. Scaffold cross-linking and plasmid pH altered the ability of the CG scaffolds to
carry plasmid DNA to local cells. EDAC cross-linked scaffolds and scaffolds prepared
with plasmid at a neutral pH bound the lowest amount of DNA but, over two to eight
week in vitro culture periods, these scaffolds led to higher levels of gene expression
compared to non- or DHT cross-linked scaffolds and plasmid preparations at an acidic
pH (pH 2.5).
Although current knowledge is not sufficient to successfully repair articular
cartilage wounds, the understanding of the responsiveness of passaged chondrocytes in
CG scaffolds gained in this thesis can be used to further the development of this system.
In brief, the construct that is recommended for future investigation as an implant for
articular cartilage repair is a chondrocyte-seeded, EDAC cross-linked CG scaffold
cultured in vitro under dynamic compression prior to implantation.
Thesis Supervisors:
Senior Lecturer Mechanical Engineering
Massachusetts Institute of Technology
and Professor of Orthopaedic Surgery (Biomaterials)
Harvard Medical School
Myron Spector......................................................
Alan J. Grodzinsky.......................Professor of Electrical, Mechanical, and Bioengineering
Massachusetts Institute of Technology
Thesis Committee:
Loannis Yannas ....................... Professor of Mechanical Engineering and Materials Science
Massachusetts Institute of Technology
Principal Research Scientist
Gordana Vunjak-Novakovic ....................................................
Massachusetts Institute of Technology
and Adjunct Professsor of Chemical Engineering
Tufts University
ACKNOWLEDGEMENTS
I am deeply grateful to my advisors, Professors Myron Spector and Alan J.
Grodzinsky, and my thesis committee members, Drs. Vunjak-Novakovic and Yannas for
all of their valuable advice and guidance. Professors Spector and Grodzinsky have been
great mentors during the past four years. They allowed me the freedom to explore my
own ideas, while giving me structure and guidance. Through Professor Spector's
Orthopaedic Research Laboratory at Brigham's and Women's Hospital, I gained valuable
exposure to the world of orthopaedics, both research and clinical. Through Professor
Grodzinsky's Continuum Electromechanics Laboratory at MIT, I remained rooted in
engineering (mechanical, biomedical, electrical, aeronautical, chemical, etc...).
To all of the people in the ORL and Al's gang, thank you many times over for
everything you taught me - especially Eliot Frank's lessons on the Dynastat; Han-Hwa
Hung's rigorous training in lab safety, maintenance, organization, supply ordering and
everything in between; Steve Treppo's teachings of biochemistry; Andy Loening's
tutorials in enzymes and cell isolation; Moonsoo's words of wisdom with the Incustat;
Dr. Hsu's crash courses in knee anatomy, cartilage harvesting and suturing; Sandra
Zapatka-Taylor's wealth of knowledge in all aspects of histology; Xiuying Zhang's
lessons in Western blotting; John Kisiday's various cell culture and collagen tricks - and
did for me - all those animal surgeries (Dr. Hsu); ALL those samples processed
embedded, sectioned, and stained (Sandra); ALL those gels for Western blots and
autoradiography (Han-Hwa); the troublesome SMA Western blots (Robyn Marty-Roix);
and caring for my cultures whilst I traveled (John). Thank you also to Professor Yannas
and members of the Fiber and Polymers Laboratory (Lila Chamberlain, Mark Spilker,
Toby Freyman, and, in the end, Brendan Harley) for use of the collagen-GAG technology
and cell culture space, and for giving me my first "home" when I came to MIT. Thank
you Ray Samuel, MD/PhD, for introducing me to gene therapy and bringing me into the
project with the "gene-seeded scaffolds" (Chapter 5!). Thank you also to all the students
who worked with me as UROPs and helped with various experiments - Wendy Liu,
Katherine Oates, Juwell Wu, Julie Watts, Aileen Wu, and Karen Riesenburger.
Thank you also to all of my friends here in Cambridge - Helen and Camille, you
made adjusting to life on the east coast so much easier - I guess us California girls won't
melt in the eastern summers or turn to popsicles during the winters after all (tho' it sure
felt like it at times). My roommates, Chris(tina), Alan, and Tim suffered through grad
school at MIT with me and provided drama as a reminder that there was life beyond the
sterile confines of the lab. Of course, I could not have kept my sanity through four plus
years at MIT without some fun, whether it be playing field hockey with the
Minutewomen (and Minutemen), soccer with the Kickbacks, mountain biking with Helen
and Chris, kayaking with Andy, triatholoning with Robin Evans (when she wasn't
crashing into one thing or another!), or random adventures with Linda Bragman, Bodo
Kurz, and Parth Patwari (yes Al, when you were gone, we did play...).
And, last, but not least, thank you to my family who have always given me the
love and support to help me succeed (even when they thought I was majoring in
recreation)!
CONTENTS
ABSTRACT
..........................................................................................................
ACKNOWLEDGEMENTS..........................................................................................
CONTENTS
..........................................................................................................
2
4
5
LIST OF FIGURES .......................................................................................................
8
LIST OF TABLES .......................................................................................................
9
LIST OF EQUATIONS.................................................................................................9
CHAPTER .
GENERAL INTRODUCTION......................................................
10
1.1.
STRUCTURE AND FUNCTION OF ARTICULAR CARTILAGE ...............................
10
1.2.
CARTILAGE DEGENERATION ..........................................................................
1.3.
SURGICAL TREATMENT OF CARTILAGE WOUNDS ..........................................
11
12
14
1.4. TISSUE ENGINEERING OF ARTICULAR CARTILAGE ..........................................
1.4.1.
GeneralApproach.................................................................................
14
1.4.2.
Review of Previous Work .....................................................................
15
1.4.2.1.
Scaffolds........................................................................................
15
1.4.2.2.
Growth Factors...............................................................................
16
1.4 .2.3.
C ells..............................................................................................
. 16
1.4.2.4.
Regulation by physical forces ........................................................
17
1.4.2.5.
The Collagen-Glycosaminoglycan System....................................
18
1.5 . SPECIFIC AIM S..................................................................................................
18
CHAPTER 2: EVALUATION OF CROSS-LINKING METHODS FOR
COLLAGEN-GLYCOSAMINOGLYCAN SCAFFOLDS ......................................
21
2.1.
INTRODUCTION................................................................................................
21
2.2. MATERIALS AND METHODS.............................................................................
22
2.2.1.
Collagen-GlycosaminoglycanScaffold Fabrication............................. 22
2.2.1.1.
Freeze-Drying...............................................................................
22
2.2.1.2.
Cross-Linking Methods.................................................................
22
2.2.2.
Physical Characterizationof Scaffolds .................................................
23
2.2.2.1.
Swelling Ratio Determination ........................................................
23
2.2.2.2.
Compression Testing of Scaffolds .................................................
24
2.2.2.3.
Glycosaminoglycan Content of Scaffolds.....................................
24
2.2.3.
Cell-Seeded Assays.................................................................................
25
2.2.3.1.
Chondrocyte Isolation and Culture ..............................................
25
2.2.3.2.
Cell Seeding and Culture of CG Scaffolds....................................
25
2.2.3.3.
Measurement of Cell-Mediated Contraction..................................26
2.2.3.4.
Radiolabel Incorporation..............................................................
26
2.2.3.5.
Dry Weight Determination............................................................
26
2.2.3.6.
2.2.3.7.
2.2.3.8.
DN A A nalysis ...............................................................................
Glycosaminoglycan Content of Cell-Seeded Scaffolds ................
Immunohistochemistry..................................................................
26
27
27
StatisticalA nalysis ...............................................................................
27
RESU LTS..........................................................................................................
28
Physical Characterizationof Unseeded Scaffolds ................................
2.3.1.
Sw elling R atio ................................................................................
2.3.1.1.
Compressive Stiffness ....................................................................
2.3.1.2.
GAG Content of Scaffolds .............................................................
2.3.1.3.
Cell-Seeded Assays................................................................................
2.3.2.
Cell-mediated Contraction .............................................................
2.3.2.1.
28
28
30
30
31
31
2.2.4.
2 .3 .
2.3.2.2.
Dry W eights .................................................................................
Radiolabel Incorporation................................................................
2.3.2.3.
DN A A nalysis ...............................................................................
2.3.2.4.
2.3.3.
GAG Content of Seeded Scaffolds.........................................................
2.3.4.
2 .4 .
Immunohistochemistry ...........................................................................
D ISCU SSION ....................................................................................................
33
34
36
37
39
39
CHAPTER 3: EFFECTS OF MECHANICAL COMPRESSION ON
BIOSYNTHETIC ACTIVITY OF PASSAGED CHONDROCYTES IN TYPE II
48
COLLAGEN-GLYCOSAMINOGLYCAN SCAFFOLDS ......................................
3 .1.
3.2.
INTRODUCTION ................................................................................................
MATERIALS AND METHODS............................................................................
48
49
49
49
3.2.1.
3.2.2.
Collagen-GlycosaminoglycanScaffolds ...............................................
Cell Culture and Cell-Seeding of Scaffolds ..........................................
3.2.3.
Mechanical Compression of Cell-Seeded Scaffolds..............................49
49
50
Static Compression - Dose Response ...........................................
3.2.3.1.
Static Compression - Kinetics ......................................................
3.2.3.2.
Dynamic Compression..................................................................51
3.2.3.3.
Proteinand GAG Biosynthesis..............................................................
3.2.4.
51
3.2.5.
Analysis of Macromolecules Released to the Medium..........................
52
3.2.6.
Newly Synthesized ProteoglycanSize and Affinity for HyaluronicAcid .. 52
3.2.7.
StatisticalA nalysis ...............................................................................
3 .3 .
RESU LTS..........................................................................................................
3.3.1.
Static Compression - Dose Response ....................................................
Static Compression - Kinetics ...............................................................
3.3.2.
53
53
53
55
56
3.3.3.
Dynamic Compression ...........................................................................
3.3.4.
3.3.5.
Newly Synthesized Macromolecules Released to the Medium..............57
59
ProteoglycanAnalysis...........................................................................
3 .4 .
D ISCU SSIO N ...............................................................................................
CHAPTER 4: REPAIR OF CANINE CHONDRAL DEFECTS IMPLANTED
WITH AUTOLOGOUS CHONDROCYTE-SEEDED TYPE II COLLAGEN
SCAFFOLDS ...........................................................................................................
4 .1.
INTRODUCTION ................................................................................................
60
65
65
4.2.
4.2.1.
4.2.2.
4.2.3.
4.2.4.
4.2.5.
4 .3 .
66
68
68
69
70
A nimal M odel ........................................................................................
Type II Collagen Scaffolds ...................................................................
Cell Culture and Preparationof Cell-Seeded Implants.........................
Histomorphometry..................................................................................
Mechanical Testing ...............................................................................
RESU LTS.............................................................................................................72
4.3.1.
4.3.2.
4.3.3.
4.3.4.
4 .4.
66
M ATERIALS AND M ETHODS ...............................................................................
72
72
74
76
77
Histology and Immunohistochemistry of Cell-Seeded Scaffolds............
GeneralGross and Histological Observations......................................
HistomorphometricEvaluation of Reparative Tissue ...........................
MechanicalPropertiesof Repair Tissue...............................................
D ISCU SSION ....................................................................................................
CHAPTER 5: FABRICATION OF GENE-SEEDED COLLAGENGLYCOSAMINOGLYCAN SCAFFOLDS.............................................................
81
81
82
5.2. MATERIALS AND METHODS ............................................................................
82
Preparationof Collagen-GAG Scaffolds ...............................................
5.2.1.
83
CG
scaffolds..........................
Addition of Plasmid DNA to Preformed
5.2.2.
83
5.2.3.
Electron Microscopy of the CG Scaffolds .............................................
83
PlasmidDNA Content of GSCG Scaffolds.............................................
5.2.4.
84
5.2.5.
PlasmidDNA Leaching Studies ............................................................
5.2.6.
Transfection of Canine Articular Chondrocytes in Gene-Supplemented CG
84
Scaffolds ...................................................................................................................
84
5.2.7.
Stability of Chondrocyte Transfection .................................................
85
5 .3 . RESULTS.........................................................................................................
5.3.1.
Morphology and Ultrastructureof the GSCG Scaffolds........................ 85
87
5.3.2.
Loading of PlasmidDNA ......................................................................
88
Release Kinetics ...................................................................................
5.3.3.
5.3.4.
In Situ Transfection of Chondrocytes Seeded into GSCG Scaffolds.........91
93
5.3.5.
Stability of Chondrocyte Transfection .................................................
93
5 .4. D ISCU SSION ......................................................................................................
5.1.
INTRODUCTION ................................................................................................
96
CHAPTER 6:
CONCLUSIONS .................................................................................
CHAPTER 7:
LIMITATIONS AND FUTURE WORK........................................100
REFERENCES .............................................................................................................
104
APPENDICES................................................................................
115
LIST OF FIGURES
Figure 1.1.
Figure 1.2.
Figure 1.3.
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 2.5.
Figure 2.6.
Figure 2.7.
Figure 2.8.
Figure 2.9.
Figure 2.10
Figure 2.11.
Figure 2.12.
Figure 2.13.
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6
Figure 4.1.
Figure 4.2.
Figure 4.3.
Figure 4.4.
Figure 4.5.
Figure 4.6.
Figure
Figure
Figure
Figure
Figure
5.1.
5.2.
5.3.
5.4.
5.5.
Anatomy of articular cartilage..................................................................
10
Approved clinical procedures for treatment of articular cartilage defects ... 13
A porous collagen-GAG scaffold.............................................................
15
Inverse swelling ratio of cross-linked scaffolds........................................
28
Compressive stiffness of cross-linked scaffolds ......................................
29
Compressive stiffness vs. inverse swelling ratio...................................... 29
31
GAG content of unseeded scaffolds........................................................
32
Cell-mediated contraction ........................................................................
33
Correlations for normalized cell contraction.............................................
34
Dry weight of seeded scaffolds ...............................................................
35
Protein and GAG biosynthesis .................................................................
DNA content of scaffolds.........................................................................
36
GAG content of seeded CG scaffolds ......................................................
38
Type II collagen immunostaining of cell-seeded scaffold ....................... 39
44
Proposed cross-linking mechanisms ........................................................
46
Type II collagen western blot....................................................................
Chambers for compressive loading ...........................................................
50
Biosynthetic dose response to static compression....................................
54
55
Kinetics of radiolabel incorporation.........................................................
Effects of compression on macromolecular accumulation ....................... 56
Total rates of biosynthesis.........................................................................
58
Size distribution of newly synthesized proteoglycans .............................
59
Surgical creation of chondral defects ......................................................
67
Light micrographs of cell-seeded type II collagen scaffolds cultured for four
weeks.......................................................................................................
. 72
Gross appearance of joint surfaces at necropsy ........................................
73
Histological sections from the center of repair tissue filling the canine
75
chondral defects 15 weeks after implantation ..........................................
Indentation stiffness of repair tissue and articular cartilage.....................76
Histomorphometric comparison of repair tissue filling defects subjected to
different treatm ents ...................................................................................
78
Electron micrographs of gene-seeded collagen-GAG (GSCG) scaffolds.... 86
Total DNA loaded into scaffolds ............................................................
87
D N A leaching profiles ............................................................................
89
D N A release rate ......................................................................................
90
In situ transfection of chondrocytes in GSCG scaffolds ........................... 92
LIST OF TABLES
Table 2-1. Summary of cross-linking treatments .......................................................
Table 5-1. Plasm id DNA release rates .......................................................................
23
91
LIST OF EQUATIONS
Equation 2-1. Calculation of swelling ratio from wet and dry weights ......................
Equation 2-2. Calculation of cell-mediated contraction...............................................
23
26
CHAPTER 1: GENERAL INTRODUCTION
Normal joint function depends on the low joint friction and absorption and
transmission of loads afforded by healthy articular cartilage. For over two and a half
centuries it has been known that articular cartilage has limited natural healing [Hunter,
1743] and that injury to articular cartilage can lead to acute and chronic pain. Despite
this knowledge, relatively little progress has been made towards improving cartilage
healing. Recently, the development and advances of tissue engineering - the ex vivo
growth of tissue - have given renewed hope for articular cartilage repair. To take full
advantage of any tissue engineering scheme, a thorough understanding of the role of
various environmental factors in the natural and engineered development of articular
cartilage is necessary. The focus of this thesis is the development of a tissue engineering
system
employing
a collagen-glycosaminoglycan
scaffold
with passaged
adult
chondrocytes.
1.1.
STRUCTURE AND FUNCTION OF ARTICULAR CARTILAGE
Articular cartilage is a highly specialized connective tissue. It is the avascular,
aneural, glassy-like (hyaline) connective tissue lining the ends of long bones (Figure 1.1).
proteoglycan
cartilage
y
n
Type 11 collagen chondrocyte
Figure 1.1. Anatomy of articular cartilage. Articular cartilage lines the ends of long bones in
diarthroidal joints (i.e. hip, knee, elbow, knuckles). The solid components of the extracellular
matrix of articular cartilage are primarily rope-like type II collagen fibers and negatively-charged
proteoglycans. The chondrocyte is the cell responsible for the synthesis and organization of the
matrix.
10
Articular cartilage serves as a natural bearing material that absorbs and transmits loads
across diarthroidal joints (i.e. knee, hip, knuckles, etc.).
Healthy articular cartilage
supports compressive, tensile, and shear loads while providing low friction between
articulating joint surfaces.
The mechanical properties of articular cartilage arise from the seemingly simple,
though unique structure and composition of the tissue. Articular cartilage is primarily
water (65-75% wet weight), with an arcuate collagen network (50-90% dry weight as
type II collagen with smaller amounts of type VI, IX, X and XI collagens) and highly
negatively charged proteoglycans (5-10% dry weight) [Muir, 1995] making up the solid
components of the matrix. The collagen fibers provide tensile and shear strength while
the proteoglycans and interstitial fluid impart compressive strength. Even small changes
in the composition or organization of the extracellular matrix can profoundly alter the
mechanical properties of articular cartilage. Thus, a crucial aspect in the natural and
engineered regeneration of articular cartilage is the realization of the precise cartilage
matrix substance and architecture.
1.2.
CARTILAGE DEGENERATION
The specialized articular cartilage matrix is maintained by chondrocytes. Mature
chondrocytes are sparsely distributed throughout the tissue (approximately 10,000
cells/mm 3 in adult human cartilage [Muir, 1995]) and have relatively low mitotic and
biosynthetic activity. As a result, once damaged, articular cartilage has limited capacity
for repair. In particular, even relatively small changes to the integrity of the cartilage
matrix will alter its mechanical properties, making damaged cartilage prone to more
severe degradation. As the extracellular matrix of cartilage degenerates, bone wears on
bone, resulting in pain, deformity and loss of joint motion. This condition, known as
arthritis, is the leading cause of disability in the United States with some 43 million
Americans suffering from arthritis in 1998, at a cost to the US economy of $65 million in
medical care and lost wages [AAOS, 2000]. By 2020, it is estimated that as many as 60
million people will suffer from arthritis [AAOS, 2000].
Osteoarthritis, or "wear and tear" degeneration, is the most common form of
arthritis, affecting 30 million Americans in 1998 [AAOS, 2000]. Although the etiology
11
of osteoarthritis is largely unknown, it is generally accepted that unrepaired focal lesions
that may initially be due to trauma can extend themselves and predispose the joint to
more wide-spread degeneration due to excess friction and uneven joint loading [Grande
et al., 1989; Minas and Nehrer, 1997]. The avascular and aneural nature of the cartilage
matrix, coupled with the limited mobility, proliferative, and biosynthetic activity of the
mature chondrocyte severely impairs the healing of defects limited to the cartilage
("partial thickness" or "chondral").
While these partial thickness lesions do not heal
[Ghandially et al., 1977; Mankin, 1982], wounds penetrating the cartilage and underlying
bone ("full thickness" or "osteochondral") are more likely to fill with repair tissue. This
repair tissue, however, lacks the mechanical integrity of normal articular cartilage and
tends to deteriorate over time [Shapiro et al., 1993].
1.3.
SURGICAL TREATMENT OF CARTILAGE WOUNDS
Left untreated, isolated cartilage wounds may eventually progress to severe
cartilage degeneration and joint pain. The only relief for severely arthritic joints is total
joint replacement (TJR). While TJR is successful for most patients, problems exist,
particularly for young, active patients. Early treatment of cartilage lesions to restore the
integrity of the joint surface may prevent osteoarthritic degeneration of the joint and
future TJR. In an effort to relieve pain and preempt TJR, it has been estimated that each
year 1 million Americans have surgery to treat articular cartilage injuries [AAOS, 2000].
Several surgical techniques aim to improve cartilage healing [Minas and Nehrer,
1997; Newman, 1998] by promoting filling of the cartilage wound.
Since cartilage
lesions which penetrate the subchondral bone can fill with repair tissue, techniques such
as microfracture (Figure 1.2a) and abrasion arthroplasty have been developed to draw
blood and multi-potent stem cells from the underlying subchondral bone into the wound
to promote filling of the defect. These techniques increase the amount of repair tissue,
providing at least temporary pain relief. The inferior mechanical properties of the repair
tissue, however, predispose the repair tissue and surrounding cartilage to degeneration
under the demanding joint loads [Minas and Nehrer, 1997; Temenoff and Mikos, 2000].
Transplantation
procedures
such as
autologous
cell implantation
(ACI),
osteochondral transplantation (OCT) and mosaicplasty aim to fill the defect with articular
12
cartilage synthesized in situ by transplanted chondrocytes (ACI) or with mature cartilage
translocated from "non-weight-bearing" locations (OCT and mosaicplasty).
(Figure 1.2b),
chondrocytes are harvested from "healthy,"
In ACI
"non-weight-bearing"
cartilage, expanded in monolayer culture, and reinjected into the cartilage wound under a
periosteal flap. This procedure has been shown to alleviate pain [Brittberg et al., 1994;
Minas and Nehrer, 1997], but the retention of the cells within the defect [Breinan et al.,
1998], the degeneration induced by suturing of the periosteal flap [Breinan et al., 2000;
Breinan et al., 1997], the quality and durability of the repair tissue synthesized by the
cultured cells [Breinan et al., 2001], and the integration of the repair tissue with the host
tissue [Breinan et al., 1998; Brittberg et al., 1994] are problematic.
With OCT
[Outerbridge, 1995 #481] and mosaicplasty [Matsusue et al., 1993] (Figure 1.2c), an
OKA Stone M.D.
a
b
Figure 1.2. Approved clinical procedures for treatment of articular cartilage defects. (a) In
microfracture, the surgeon creates tiny holes in the underlying subchondral bone to stimulate bleeding
into the defect (image downloaded from www.stoneclinic.com). (b) In autologous chondrocyte
implantation (ACI), chondrocytes are harvested from a cartilage biopsy, expanded in monolayer
culture, resuspended, and injected under a periosteal flap (image downloaded from
www.drmendbone.com). (c) In mosaicplasty, multiple cartilage-bone plugs are harvested from one
(less load-bearing) site and transplanted to fill the larger defect (image downloaded from
www.maitrise-orthop.com).
13
intact cartilage matrix and underlying subchondral bone are transplanted into the defect.
Major disadvantages to these procedures include donor site morbidity [Temenoff and
Mikos, 2000], limited availability and suitability (surface contour and load-bearing
capacity) of graft tissue [Hunziker, 1999; Laurencin et al., 1999; Temenoff and Mikos,
2000], and instability and degeneration of the graft [Laurencin et al., 1999].
Although the above procedures improve pain and joint function for many patients
[Minas and Nehrer, 1997; Temenoff and Mikos, 2000], thus far no surgical solution has
been able to fully regenerate articular cartilage and proven to be a long-term solution.
1.4.
TISSUE ENGINEERING OF ARTICULAR CARTILAGE
An alternative, or possible augmentation, to the above-mentioned surgical
techniques, is tissue engineering of articular cartilage. Tissue engineering, as coined by
Langer and Vacanti involves the use of cells, scaffolds, and/or regulators to grow
functional tissue ex vivo [Langer and Vacanti, 1993].
More recently, "regenerative
medicine" has been adopted to refer to procedures in which the majority of the
regeneration process occurs in vivo. This thesis is motivated by the latter approach.
However, for the purpose of this write-up, the term "tissue engineering" will be used.
1.4.1.
General Approach
The relatively simple structure of articular cartilage (avascular, aneural, single cell
population), limited self- and surgically-induced repair, and the clinical consequences for
cartilage repair, have made tissue engineering of articular cartilage an area of intense
research over the past decade [Temenoff and Mikos, 2000].
In addition to the three
traditional pillars of tissue engineering: 1) the scaffold to serve as an analogue of the
natural extracellular matrix, 2) the cells, and 3) regulators and/or growth factors [Langer
and Vacanti, 1993], it is important to also consider the role of physical forces in the
development of musculoskeletal tissues, such as articular cartilage. Due to the complex
physical loads to which orthopaedic tissues are subjected to in vivo, the optimal tissue
engineering schemes for articular cartilage may involve a combination of in vitro and in
vivo stages.
14
1.4.2. Review of Previous Work
1.4.2.1.Scaffolds
A wide array of materials has been used in various in vitro and in vivo studies for
articular cartilage engineering.
Candidate scaffolds must be biocompatible and
accommodate cell proliferation and matrix synthesis. The scaffold serves as an analog of
the natural three-dimensional extracellular matrix, and can also be used as a carrier of
cells, growth factors, and/or genetic material.
Scaffold composition and porosity is known to affect cell viability, attachment,
morphology, and macromolecular biosynthesis [Coutts et al., 1994; Frenkel et al., 1997;
Grande et al., 1997; Nehrer et al., 1997b].
Materials that are most often studied in
cartilage tissue engineering include hydrogels made up of collagen [Kawamura et al.,
1998; Rich et al., 1994; Wakitani et al., 1994], agarose [Buschmann et al., 1992; Cook et
al., 1997; Rahfoth et al., 1998], and synthetic peptides [Kisiday et al., 2001]; sponge-like
scaffolds manufactured from collagen [Frenkel et al., 1997; Grande et al., 1997; Nehrer et
al., 1998b; Nehrer et al., 1997a; Nixon et al., 1993; Sams et al., 1995; Toolan et al., 1996;
Toolan et al., 1998], polyglycolic acid [Freed et al., 1994; Freed et al., 1993; Grande et
al., 1997], and/or polylactic acid [Chu et al., 1997; Coutts et al., 1994; Freed et al., 1993];
and materials with a naturally-occurring porous structure, such as coral [Shahgaldi, 1998]
and devitalized articular cartilage [Toolan et al., 1998].
100 AM
Figure 1.3. A porous collagen-GAG scaffold. This scanning electron microscope image shows the
porous structure of a typical scaffold resulting from freeze-drying of a type I collagen-GAG coprecipitate used in the studies described in Chapters 2 and 5 of this thesis.
15
While each of these scaffolds has particular advantages and disadvantages, there
are general characteristics of the different types of scaffolds. For example, hydrogels are
easy to fabricate and allow for uniform and predictable cell-seeding of the gels. The gels,
however, have poor mechanical properties and are difficult to handle during implantation
surgery. In contrast, the porous scaffolds have higher mechanical integrity and can be
sutured or press-fit into cartilage defects. The porous nature of such scaffolds, however,
result in a lower retention of newly synthesized macromolecules.
1.4.2.2.Growth Factors
Tissue engineering scaffolds can also be used as carriers for therapeutic proteins
or genes for the proteins.
Various growth factors (i.e. fibroblast growth factor-2,
transforming growth factor-$, insulin-like growth factor-1, and osteoprogenitor factor-1)
have been used to modulate chondrocyte phenotype, proliferation, and biosynthesis rates.
Such growth factors may be tethered to insoluble scaffolds to permit localized dosing in
vivo. Alternatively, advancing research in gene therapy may utilize tissue engineering
scaffolds for direct, localized delivery of specific genes to cells for prolonged in situ
production of growth factors in therapeutic quantities. Recently, it has been reported that
polymer scaffolds loaded with genetic material can be used to transfect cells over
prolonged periods
[Bonadio
et al.,
1999; Lauffenburger and Schaffer,
1999].
Furthermore, biodegradable scaffolds can be designed to deliver the desired proteins or
genes gradually over a period of time dependent on the degradation rate of the scaffold.
1.4.2.3. Cells
Due to the low metabolic rate of native, mature chondrocytes, most approaches to
tissue engineering of articular cartilage involves transplantation of cells along with the
scaffold. Traditionally, autologous articular chondrocytes are used [Breinan et al., 2000;
Breinan et al., 1998; Breinan et al., 1997; Brittberg et al., 1994; Frenkel et al., 1997;
Kawamura et al., 1998], but allogenic chondrocytes [Rahfoth et al., 1998; Toolan et al.,
1998], chondrocytes from other cartilaginous tissues [Bouwmeester et al., 1997; Chu et
al., 1997; Klein-Nulend et al., 1998], and chondroprogenitor cells [Johnstone and Yoo,
1999; Nixon et al., 1999; Wakitani et al., 1997] have also been used.
The low cell density of cartilage requires the expansion of the harvested cell
population if autologous articular chondrocytes are used.
16
Chondrocytes expanded in
monolayer cultures (passaged cells) demonstrate increased rates of proliferation [Green,
1971], but de-differentiate and lose their characteristic phenotype - namely they lose
their spherical morphology and synthesize type I rather than type II collagen [Benya and
Shaffer, 1982]. Fortunately, the chondrocyte phenotype can be re-induced (from these
de-differentiated passaged cells) or induced (from progenitor cells) by the appropriate
matrix environment [Benya and Shaffer, 1982], growth factors [Benya and Padilla, 1990;
Benya et al., 1988; Benya and Padilla, 1993; Borge et al., 1997; Jakob et al., 2001;
Johnson et al., 2001; Kulyk and Reichert, 1992; Martin et al., 2001a; Martin et al.,
2001b], or other culture variables [Domm et al., 2000].
1.4.2.4.Regulation by physicalforces
Although the precise mechanisms by which physical loads effect biological
responses have yet to be resolved, it has been well-established that mechanical loads
regulate chondrocyte behavior both in vivo [Grumbles et al., 1995; Kiviranta et al., 1988;
Paukkonen et al., 1986] and in vitro [Buschmann et al., 1995; Buschmann et al., 1999;
Gray et al., 1989; Grodzinsky et al., 1998; Jin et al., 1999; Kim et al., 1995; Kim et al.,
1994; Lee et al., 1981; Martin et al., 2000; Quinn et al., 1998a; Quinn et al., 1998b;
Quinn et al., 1999; Ragan et al., 1999b; Sah et al., 1989]. Specifically, static compression
[Buschmann et al., 1995; Kim et al., 1996; Ragan et al., 1999a] and high-impact
compressive loading [Loening et al., 1999; Quinn et al., 1998b] impair chondrocyte
metabolism, while dynamic compression [Kim et al., 1994; Quinn et al., 1998a; Sah et
al., 1989] and shear [Jin et al., 1999] at moderate amplitudes can increase biosynthetic
activity.
Comparatively little research has been done on chondrocytes in tissue engineering
systems.
It has been established that chondrocytes in non-native scaffolds such as
agarose [Buschmann et al., 1995] and alginate gels [Ragan et al., 2000] respond to
mechanical compression in a similar manner to chondrocytes in cartilage explants, but
only once the cells become encapsulated in extracellular matrix. In the development of
cartilage constructs, dynamic compression of chondrocyte-seeded agarose gels [Mauck et
al., 2000] and laminar fluid flow on chondrocyte-seeded PGA fibrous meshes [Martin et
al., 2000] have been used over extended periods of time to increase cartilage-like matrix
production.
17
1.4.2.5.The Collagen-GlycosaminoglycanSystem
Porous collagen-glycosaminoglycan (CG) scaffolds, developed for the purpose of
dermal tissue engineering [Yannas and Burke, 1980; Yannas et al., 1980; Yannas et al.,
1989], have also shown promise in promoting the healing of peripheral nerve
[Chamberlain et al., 1998; Chamberlain et al., 2000] and conjuctiva [Hsu et al., 2000].
Previous studies in our laboratory have shown that the CG scaffold may also be used to
promote articular cartilage repair. In vivo studies using a canine model for articular
cartilage repair, indicate that implantation of a CG scaffold, either alone or seeded with
cells [Breinan et al., 2000; Nehrer et al., 1998b] improved healing of surgically created
cartilage defects compared to untreated or ACI-treated defects. The repair tissue formed
after implantation of the scaffolds, however, was principally fibrocartilage rather than the
desired hyaline cartilage.
The cell-seeded scaffolds utilized autologous chondrocytes that were expanded in
monolayer culture prior to seeding into the CG scaffolds. Although the extent of dedifferentiation and re-differentiation were not quantified, in vitro studies have shown that
passaged articular chondrocytes will proliferate and synthesize glycosaminoglycans, and
may also synthesize type II collagen when seeded into the CG scaffolds [Nehrer et al.,
1998a; Nehrer et al., 1997a; Nehrer et al., 1997b].
In order to further improve cartilage repair stemming from implantation of cellseeded CG scaffolds, a more thorough understanding of the chondrocyte behavior in the
scaffolds is necessary.
In particular, the physical properties of the scaffold and the
response of the seeded chondrocytes to mechanical forces are important to the
progression of cartilage-specific tissue engineering systems. Additionally, it may be of
great interest to use the CG scaffolds to transfect chondrocytes and/or infiltrating stem
cells in situ.
1.5.
SPECIFIC AIMS
To lay the groundwork for future development of the CG scaffolds for articular
cartilage tissue engineering, the purpose of this thesis was to explore the interactions of
passaged chondrocytes with the porous CG scaffolds. To better understand how the
physical properties of the CG scaffold affect chondrocyte behavior, the effects of scaffold
18
cross-linking and mechanical loading of cell-seeded scaffolds were investigated in vitro
and used to design an implant for in vivo study. Additionally, to determine how the
scaffold could be used in future work involving therapeutic growth factors, experiments
were conducted to determine the feasibility of transfected chondrocytes in situ with
plasmid DNA attached to the CG scaffold. The working hypotheses were:
1.
Increasing cross-link density would decrease
chondrocyte-mediated
contraction and increase chondrocyte proliferation and biosynthesis;
2.
Mechanical compression of cell-seeded CG scaffolds could be used to
stimulate chondrocyte biosynthetic activity;
3.
In vitro culture of cell-seeded CG constructs prior to implantation into
chondral defects would improve repair of the cartilage lesions; and
4.
CG scaffolds loaded with genetic material could be used to transfect
chondrocytes over an extended period of time.
To test these four hypotheses, in vitro and in vivo experiments were conducted
with the following specific aims:
L.a.
To determine the effects of dehydrothermal (DHT), ultraviolet (UV),
glutaraldehyde (GTA), and carbodiimide (EDAC) cross-linking treatments
on compressive stiffness and glycosaminoglycan (GAG) content of
unseeded CG scaffolds;
1.b.
To quantify the extent of in vitro cell-mediated contraction of scaffolds
with
different
compressive
stiffnesses
by passaged,
adult canine
chondrocytes;
1.c.
To evaluate in vitro chondrocyte proliferation and protein and GAG
accumulation rates in the cross-linked scaffolds;
2.a.
To evaluate the effects of 24 hours of static and dynamic compression on
protein and GAG synthesis by passaged adult chondrocytes seeded in
EDAC cross-linked scaffolds;
2.b.
To quantify protein and GAG accumulation rates in cell-seeded CG
scaffolds subjected to up to 24 hours of static or dynamic compression in
vitro;
19
3.a.
To evaluate the histological make-up of repair tissue formed in vivo 15
weeks after the implantation of a 4 week in vitro cultured chondrocyteseeded CG construct for comparison to previous results in the same animal
model; and
3.b.
To compare the mechanical indentation properties of the repair tissue to
normal articular cartilage;
4.a.
To assess the effects of the pH of the solution of plasmid DNA added to
the CG scaffold on the leaching characteristics of DNA from genesupplemented CG (GSCG) scaffolds;
4.b.
To evaluate the effects of GSCG scaffold cross-linking on plasmid
leaching characteristics; and
4.c.
To measure transfection levels of chondrocytes cultured in GSCG
scaffolds for 2, 4, or 8 weeks in vitro.
20
CHAPTER 2: EVALUATION OF CROSS-LINKING
METHODS FOR COLLAGEN-GLYCOSAMINOGLYCAN
SCAFFOLDS
2.1.
INTRODUCTION
Previous studies investigating the behavior of passaged adult canine chondrocytes
in collagen-glycosaminoglycan
(CG) scaffolds have noted significant dimensional
changes in the cell-seeded scaffolds over time [Lee et al., 2000a; Nehrer et al., 1997a].
That articular chondrocytes have the potential for contraction, was recently supported by
immunohistochemical findings of a contractile muscle actin, a-smooth muscle actin
(SMA), in human [Kim and Spector, 2000] and canine [Wang et al., 2000] articular
chondrocytes in situ and in the reparative cartilaginous tissue in healing defects in a
canine model [Wang et al., 2000].
This finding represents a potential problem in the
application of such scaffolds for tissue engineering. As the scaffold contracts, there is a
reduction in the pore volume that could restrict cell proliferation. Additionally, in vivo
deformation of the construct could result in a loss of contact between the implanted
device and the host tissue, thereby decreasing the chances for successful integration of
the repair tissue.
Recognizing the potential importance of chondrocyte-mediated contraction of
scaffolds employed for tissue engineering, one objective of this experiment was to
evaluate the ability of various cross-linking methods to increase the cross-linking density
and stiffness of the CG scaffold. The hypothesis was that increased levels of crosslinking could be used to sufficiently increase scaffold stiffness to thwart chondrocytemediated contraction. The cross-linking treatments evaluated here were: dehydrothermal
treatment (DHT) [Weadock et al., 1983; Weadock et al., 1995; Weadock et al., 1996;
Yannas et al., 1989]; ultraviolet irradiation (UV) [Weadock et al., 1983; Weadock et al.,
1995; Weadock et al., 1996]; glutaraldehyde (GTA) [Petite et al., 1994; Weadock et al.,
1983; Yannas et al., 1989], and carbodiimides (EDAC) [Olde Damink et al., 1996;
Osborne et al., 1998; Osborne et al., 1999; Weadock et al., 1983]. In addition to affecting
stiffness, different cross-linking protocols can affect scaffold degradation rate [Petite et
21
al., 1994; Weadock et al., 1983; Weadock et al., 1996], cell proliferation [Osborne et al.,
1998; Petite et al., 1994; Weadock et al., 1983] and biosynthesis [Chevallay et al., 2000].
Therefore, another objective of this experiment was to evaluate the effects of the selected
cross-linking treatments on the proliferative and biosynthetic activity of adult canine
articular chondrocytes seeded in the scaffolds. The hypothesis was that the changes in
physical and chemical properties afforded to the CG scaffolds by the different crosslinking methods would affect adult canine chondrocyte proliferation and protein and
GAG biosynthesis within the scaffolds.
2.2.
MATERIALS AND METHODS
2.2.1. Collagen-Glycosaminoglycan Scaffold Fabrication
2.2.1. 1.Freeze-Drying
The porous CG scaffold was produced by freeze-drying a co-precipitate of type I
bovine tendon collagen (Integra Life Sciences, Plainsboro, NJ) and shark chondroitin-6sulfate (Sigma Chemical, St. Louis, MO) as previously described [Yannas et al., 1989]
and outlined in detail in Appendix A. The final concentration of the slurry was 5.0 mg
collagen/ml and 0.44 mg chondroitin sulfate/ml.
The scaffolds have been previously
reported to have a porosity of approximately 87% and an average pore size of 84 pm
[Nehrer et al., 1997a]. Nine-millimeter diameter disks (approximately 3.5 mm thickness)
were used for subsequent mechanical tests and cell culture experiments.
2.2.1.2. Cross-LinkingMethods
All scaffolds were sterilized and minimally cross-linked for 24 hours by
dehydrothermal treatment (DHT) [Yannas et al., 1989].
Certain scaffolds were further
cross-linked as follows: a) by exposure to ultraviolet radiation (UV) for one hour (5 cm
from a 258nm source rated at 4510 pW/cm 2 at 5 cm), with the scaffolds being turned
over after thirty minutes, b) immersion in a 0.25% glutaraldehyde solution in 0.05 M
acetic acid for 24 hours at room temperature (GTA), or c) immersion in a carbodiimide
solution (14 mM 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5.5
mM N-hydroxysuccinimide; Sigma) for two hours at room temperature (EDAC). Excess
glutaraldehyde was removed from the scaffold through a series of four washes in sterile
22
distilled water. Excess EDAC was rinsed from the scaffold using phosphate-buffered
saline (PBS) followed by two washes in sterile distilled water.
Table 2-1. Summary of cross-linking treatments.
Group
DHT
GTA
EDAC
Cross-linking Treatment
24 hours dehydrothermal treatment at 105'C, 30 mtorr
24 hours DHT plus 30 minutes ultraviolet irradiation (4510 gW/cm 2
X=258 nm) to each side of scaffold
24 hours DHT plus 24 hours immersion in 0.25% gluteraldehyde in
0.05 M acetic acid, followed by 4x15 minute rinse in sterile distilled,
deionized water
24 hours DHT plus 2 hours immersion in 14 mM EDAC/5.5 mM NHS
solution in distilled water, followed by 2 hour rinse in sterile phosphate
buffered saline
2.2.2. Physical Characterization of Scaffolds
2.2.2. 1.Swelling Ratio Determination
Cross-link density for randomly coiled polymer networks is inversely related to
the swelling ratio [Weadock et al., 1983].
Thus, as an approximate measure of the
density of cross-links that were formed by the different cross-linking methods, the
swelling ratios of the scaffolds were determined as described by Weadock et al.
[Weadock et al., 1983]. Cross-linked CG samples placed in a water bath at 90'C for two
minutes to swell and denature the collagen.
Water within the pores was expelled by
pressing the swollen scaffolds between sheets of filter paper with a 1.0 kg weight placed
on top for 20 seconds. The sample was weighed and the weight recorded as wet weight
(WW). Samples were then dried in an oven (1 10 0 C) overnight and the dry weights (DW)
of the collagen scaffolds determined. The swelling ratio, defined as the inverse of the
volume fraction of dry collagen (Vf), was calculated from the wet and dry weights and
the densities of water (Pwater=.00 g/cm 3 ) and collagen (p,=1.32 g/cm 3) as follows:
r
r
=-
Vf
)7J]
(WW -D W
+DW
DDP
23
Eq. 2-1
2.2.2.2. Compression Testing of Scaffolds
Disks (9 mm in diameter) from each cross-linking group were hydrated in
phosphate buffered saline (PBS) and stored at 40 C prior to mechanical testing. On the
day of testing, the thickness of the fully hydrated specimen was measured using a
micrometer. Disks were then placed in a PBS-filled polymethylmethacrylate (PMMA)
chamber mounted in the lower jaw of a Dynastat Mechanical Spectrometer (IMASS,
Hingham, MA). A 50-gram load cell (Sensotec, Cleveland, OH) fitted with a 9.5-mm
diameter PMMA cylindrical plunger was fixed in the upper jaw of the Dynastat and the
distance between the plunger and the lower chamber set to the thickness of the hydrated
scaffold.
Using displacement-feedback control, successive ramp-and-hold displacements
were applied in radially-unconfined compression, giving sequential strain increments of
1-5% up to a maximum of 40% strain (see Appendix C).
Radially-unconfined
compression was used because it was desired to design a single chamber that would
accommodate future testing of cell-seeded scaffolds which are known to contract in
diameter over time. At each strain level stress relaxation was achieved in 30-75 seconds,
depending on the magnitude of the displacement, and the equilibrium loads were
recorded. Stress was computed as the load normalized to the initial unstrained disk area.
Compression testing of samples occurred within 48 hours of hydration (DHT and
UV) or the completion of cross-linking (GTA and EDAC). Additionally, DHT and GTA
samples stored in sterile PBS at 4C for six weeks were tested to determine if the
stiffness changed over time.
2.2.2.3.Glycosaminoglycan Content of Scaffolds
To determine the amount of GAG tightly bound to the collagen network after
cross-linking, the sulfated GAG content of unseeded scaffolds was determined by the
dimethylmethylene blue (DMMB) dye assay (Appendix E.3). Unseeded, cross-linked
scaffold disks were hydrated in PBS to rinse away unbound GAG. The disks were then
lyophilized and solubilized overnight at 60'C with 1 ml of papain buffer (6 pg/ml papain
and 10 mM cysteine-HCl in 0.1 M sodium phosphate and 5 mM Na 2EDTA, PBE). An
aliquot of the digest (20 pl) was mixed with 200 p1 of the DMMB dye in a 96-well
24
microplate and the absorbance at 520 nm was measured. Shark chondroitin-6-sulfate was
used as the standard.
2.2.3. Cell-Seeded Assays
2.2.3.1. Chondrocyte Isolation and Culture
Chondrocytes were isolated from articular cartilage from the patellar, femoral and
tibial surfaces of knee (stifle) joints of three adult mongrel dogs using sequential pronase
(one hour at 37'C; 20 U/ml; Sigma Chemical, St. Louis, MO) and collagenase (overnight
at 37*C; 200 U/ml; Worthington Biochemical) digestion as described by Kuettner, et. al
[Kuettner et al., 1982] and detailed in Appendix D.
Isolated chondrocytes were
resuspended in culture medium (DMEM/F12, Gibco Life Sciences, Grand Island, NY)
supplemented with 10% fetal bovine serum (FBS, Hyclone Technologies, Logan, UT), 25
gg/ml
ascorbic
acid
(Wako
Chemical,
Osaka,
Japan),
and
a
Penicillin/Streptomycin/Fungizone cocktail (Gibco) and plated in 75-cm 2 flasks at a
density of 2 million cells/flask. The culture flasks were incubated at 37C with 5% CO 2.
Cells were cultured to confluence, trypsinized, resuspended and replated into 75-cm 2
flasks.
Chondrocytes from the three different animals were isolated and cultured
separately throughout.
2.2.3.2.Cell Seeding and Culture of CG Scaffolds
Third passage cells were collected by trypsinization and resuspended in complete
medium at a concentration of 4x10 6 cells/ml. CG disks were incubated with 0.5 ml of
cell suspension per disk for 1.5-2 hours on a rocking table (n=16 for each of the 3
animals). This targeted seeding density (2x 106 cells/disk) aimed to seed the cells at a
near physiological density for adult cartilage (10,000 cells/mm 3 [Muir, 1995]).
Approximately 50% of the chondrocytes (lx106 cells/scaffold, 5,000 cells/mm 3 ) attach to
the scaffolds by this seeding method (Appendix J).
Disks were then transferred to
agarose-coated wells (12-well plates) with 1.0 ml of complete media per well and
returned to the incubator overnight. The following day, an additional 0.5 ml of medium
was added to each well. Media (1.5 ml) were changed every other day. Cultures were
terminated after 2, 7, 15, or 29 days. Unseeded disks were cultured as controls.
25
2.2.3.3.Measurement of Cell-Mediated Contraction
The diameters of the seeded and unseeded scaffolds were measured 1, 3, 5, 7, 15,
21, and 29 days post-seeding.
Contraction was calculated as the relative change in
scaffold diameter from the day 1. Cell-mediated contraction (CMC) was determined by
subtracting the contraction of the unseeded scaffolds from the contraction of the seeded
scaffolds.
CMC =
OriginalDiameter- Diameter
OriginalDiameter- Diameter
OriginalDiameter
OriginalDiameter
nveage
Eq.
2-2
2.2.3.4.RadiolabelIncorporation
On days 2, 7, 15, and 29, nine seeded (three per animal) and one to three unseeded
scaffolds from each cross-linking group were terminated for biochemical analyses of
synthesis rates and proliferation. During the last eight hours of culture, constructs were
cultured in complete media supplemented with 10 gCi/ml of 3 H-proline and 10 RCi/ml of
35
S-sulfate,
to assess rates of total protein and GAG synthesis, respectively. At the end of
the labeling period, disks were washed (4x15 minutes at 40C) in PBS supplemented with
unlabeled proline (1 mM) and sulfate (0.8 mM), lyophilized and digested with 1 ml
papain buffer as described above for the digestion of the unseeded scaffolds for GAG
determination. Radiolabel incorporation was determined by mixing 100 gl of the papain
digest with 2 ml scintillation cocktail (EcoLume, Costa Mesa, CA) and measuring 3 H and
35S
counts per minute (cpm) in a liquid scintillation counter (Rack-Beta 1211 LKB,
Turku, Finland), with corrections for spillover (Appendix E.5). Counts were normalized
to DNA content (see below).
2.2.3.5.Dry Weight Determination
In order to determine the net rates of matrix accumulation and degradation for the
different scaffolds, the weights of the scaffolds were measured after lyophilization (DW).
Both the total DW and the net change in DW were analyzed.
2.2.3.6.DNA Analysis
The DNA content of the constructs was measured using Hoechst 33258 dye
(Appendix E.4). A 20 gl aliquot of the papain digest was mixed with 80 pl of phosphate
buffered EDTA and 2 ml of Hoechst dye solution (10% Hoechst dye in 10 mM Tris, 1
26
mM Na 2EDTA, and 0.1 M NaCl, pH 7.4) and assayed fluorometrically. Calf thymus
DNA was used as the standard.
The background fluorescence of the scaffold was
accounted for by subtracting the values obtained for the unseeded scaffolds.
2.2.3.7.Glycosaminoglycan Content of Cell-Seeded Scaffolds
The GAG content of cell-seeded scaffolds was determined from the digests of the
constructs using the DMMB assay described above for the unseeded scaffolds.
2.2.3.8.Immunohistochemistry
At each sacrifice time point (days 2, 7, 15, and 29), three seeded samples (one
from each animal) and one unseeded sample were fixed in 10% neutral buffered formalin,
dehydrated, and embedded in paraffin. Specimens were sectioned (7 gm thick) in crosssection and stained for type II collagen.
Deparaffinized slides were prepared for
immunostaining by digestion in 0.1% protease XIV (diluted in Tris-buffered saline, pH
7.4, TBS; Sigma) for 60 minutes and non-specific staining was blocked with application
of 30% goat serum (Sigma) for 20 minutes. Sections were incubated with the primary
antibody to either a-smooth muscle actin (Sigma) or type II collagen (II-116B3, prepared
by T. Linsenmayer and obtained from the Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City, IA; diluted 1:20 in TBS) or TBS alone (negative control)
for 2 hours, followed by incubation with biotinylated goat anti-mouse IgG antibody
(Sigma; 1:200 in TBS) for 45 minutes. Endogenous peroxidase was quenched with 3%
hydrogen peroxide (10 minutes) and sections were then incubated with ExtrAvidinConjugated Peroxidase (Sigma; 1:50 in TBS) for 20 minutes and developed with AEC
(Zymed Laboratories, Inc., S. San Francisco, CA) and counter-stained with Mayer's
hematoxylin for 20 minutes.
2.2.4. Statistical Analysis
There was no significant effect of animal on any of the measured parameters by
one-way analysis of variance (ANOVA). Therefore, the data from all three animals were
pooled and are reported as the mean ± standard error of the mean (SEM). ANOVA and
Fisher PLSD post-hoc testing were performed using StatView (SAS Institute, Inc, Cary,
NC).
27
2.3.
RESULTS
2.3.1. Physical Characterization of Unseeded Scaffolds
2.3.1. 1.Swelling Ratio
The swelling ratio of the CG scaffolds, calculated as the inverse of the volume
fraction of collagen, ranged from 5.3 ± 0.2 (mean ± SEM) for the DHT scaffolds to 3.2 ±
0.1 for the EDAC scaffolds (Figure 2.1). Taking the cross-link density to be proportional
to the inverse of the swelling ratio, the density of cross-links increased with the different
cross-linking methods as follows: DHT<UV<GTA<EDAC (p<0.05 for each Fisher
PLSD post-hoc test).
0.35
-
0.3 *4
0.25 -
0.2
0.15 0.1 0.05 0DHT
I
I
I
UV
GTA
EDAC
Figure 2.1. Inverse swelling ratio of cross-linked scaffolds. Cross-link density is proportional to
the inverse of the swelling ratio and increased with UV, GTA, and EDAC cross-linking treatments of
DHT cross-linked scaffolds. Mean ± SEM; n=4. All groups were significantly different from each
other.
28
1400
14.17
12001000800-
NS
600-
*
'0
369
346
400-
145
2000-
~~1~~
EDAC
GTA
UV
DHT
____r_
Figure 2.2. Compressive stiffness of cross-linked scaffolds. Unconfined compressive stiffness of
hydrated DHT cross-linked scaffolds was the lowest. UV and GTA cross-linking more than doubled
scaffold stiffness, while EDAC cross-linking increased stiffness 9-fold over DHT cross-linking alone.
Mean ± SEM; n=5-10. All groups significantly different except the pair marked NS.
1400
1200 1000~
~p.
*-
800
-
600
-
F-f-I
y
=
7952x - 1364
R 2 = 0.98
(6wk storage)
x
-
1266
2
R = 0.80
4000
U
200
-
0-
0.15
0.25
0.2
0.3
0.35
1/Swelling Ratio
Figure 2.3. Compressive stiffness vs. Inverse swelling ratio. Unconfined compressive stiffness
increase linearly with increasing cross-link density (R2 =0.80). The correlation improved to R2 =0.98
when the compressive stiffness of the GTA scaffold after 6 weeks storage was utilized (open
diamond).
29
2.3.1.2. Compressive Stiffness
The equilibrium, uniaxial unconfined compressive stress-strain relation was
remarkably linear (see Appendix C) with coefficients of determination above 95%. The
slope of the linear fit to the stress-strain data was used to define the "apparent
compressive modulus" of the scaffolds. This method yielded moduli ranging from 145 +
23 Pa (mean ± SEM) for the DHT samples to 1117 ± 109 Pa for the EDAC samples
(Figure 2.2). Compared to the minimally cross-linked DHT scaffolds, the compressive
modulus was doubled by UV and GTA cross-linking protocols and increased another
three-fold by the EDAC cross-linking protocol. ANOVA revealed the highly significant
effect of cross-link treatment on modulus (p<0.0001). Post-hoc testing showed that each
group was different from the others except for the UV and GTA groups.
The stiffness of DHT scaffolds evaluated after six weeks of storage in sterile PBS
did not change significantly. The stiffness of the GTA scaffolds, however, increased
from 369 ±56 to 663 ±64 Pa.
Increasing cross-linking density, as indicated by the inverse of the swelling ratio,
correlated with an increase in compressive stiffness (R2 =0.80, Figure 2.3).
The
correlation increased to R2 =0.98 when the compressive stiffness of the GTA scaffold
after 6 weeks in storage was used.
2.3.1.3.GAG Content of Scaffolds
All scaffolds were fabricated from similar batches of collagen-GAG slurry
containing 8.8% GAG/DW. The GAG content of the rinsed, unseeded scaffolds was less
than that of the original slurry, indicating that not all of the added chondroitin sulfate
became tightly bound to the collagen fibrils during the mixing and freeze-drying process.
Samples cross-linked by UV irradiation or EDAC had significantly more GAG than the
samples cross-linked by DHT or GTA (Figure 2.4; p<0.01). The EDAC scaffolds had
110 ± I ptg GAG/disk (3.4 ± 0.06% GAG/DW), compared to only 33.7 ± 3.6 [tg
GAG/disk (1.2 ± 0.09% GAG/DW) in the DHT cross-linked scaffolds. These values
span the range measured for adult canine articular cartilage (1.5-3% GAG/DW [Lee,
1999]). The GAG content of the unseeded scaffolds decreased slightly from the day 1 to
30
4.0%-
3.5%3.0%
2.5%
2.0%
1.5%
0
1.0%0.5%
0.0%
DHT
UV
GTA
EDAC
Figure 2.4. GAG content of unseeded scaffolds. GAG content normalized to dry weight of crosslinked scaffolds was higher for UV and EDAC cross-linked scaffolds. Mean ± SEM; n=4-8; GAG
content was significantly different for all comparisons except the pairing marked by t.
day 7 readings, indicating that not all of the unbound GAG was rinsed from the scaffolds
in the initial PBS rinse (see also GAG analysis of seeded scaffolds, section 2.3.3). There
was no noticeable change in GAG content of the unseeded scaffolds beyond the first
week.
2.3.2. Cell-Seeded Assays
2.3.2.1. Cell-mediated Contraction
The diameters of the unseeded scaffolds changed less than 5% between the first
and
2 8 th
day in culture. In contrast, all cell-seeded constructs underwent a minimum of
30% reduction in diameter by the end of the four-week culture period (Figure 2.5). The
patterns and the magnitudes of contraction were different for the different cross-linking
protocols. The average diameter of the seeded DHT and UV scaffolds decreased to 60%
of their original value during the first week, and by the end of four weeks these scaffolds
had contracted to 40% and 45% of their original dimensions, respectively. In contrast,
the seeded GTA and EDAC scaffolds did not contract during the first week in culture.
Thereafter, the seeded GTA scaffolds contracted at an approximately constant rate to a
31
100 -
90 -
1
80 -70
-
-60
-
-50
-}NS
o
#
4030
20-
-+-DHT
0
+UV
-*-GTA --
20
10
EDAC
30
Days
Figure 2.5. Cell-mediated contraction. Contraction of cell-seeded scaffolds, minus shrinkage of
unseeded scaffolds, for DHT (+), UV (m), GTA (A), and EDAC (o) scaffolds. DHT and UV
constructs contracted rapidly during the first week and then more slowly during the next three weeks
whereas GTA and EDAC constructs contracted very little during the first week and gradually
thereafter. Mean ± SEM; n=9-12; NS=groups not significantly different.
final diameter that was 60% of the original value. The seeded EDAC constructs had the
most dimensional stability, maintaining a diameter 70% of the original value throughout
the four-week period.
As expected, there was a decrease in CMC with increasing cross-link density and
scaffold stiffness. Using a simple linear regression model, when CMC was normalized to
the number of cells in the scaffold (total CMC/DNA content) 98% of the variation in cellmediated contraction could be attributed to differences in cross-link density, as measured
by the inverse of the swelling ratio (R2=0.98, Figure 2.6a).
With regards to the
compressive stiffness, nearly 70% of the variation in contraction could be explained by
differences in the compressive modulus of the scaffold (R2=0.69, Figure 2.6b). The main
discrepancy was found with the UV and GTA scaffolds - while the compressive
stiffnesses of these two scaffolds were nearly the same, there was greater CMC in the UV
32
3
3
b
y = -0.0015x+ 2.11
R2 =0.69
2.5
2 -
2.5 -
q2
-
1.5
1.5 -
0.5
6wk storage stiffness
00 0.5
0
0
500
1000
=
y -13.8x+4.77
0
0.15
1500
Stiffness (Pa)
R=0.98
0.2
0.25
0.3
1/Swe Bing Ratio
Figure 2.6. Correlations for normalized cell contraction. 4 week contraction normalized to total
DNA content at 28 days decreased with increasing scaffold (a) compressive stiffness (R2 =0.69) and
(b) cross-link density (R 2=0.98). Correlation of contraction with stiffness increased to R2 =0.94 when
the GTA stiffness after 6 weeks of storage was used.
scaffolds. If the stiffness of the GTA scaffolds after 6 weeks in storage was considered,
however, the correlation increased to R 2=0.94.
2.3.2.2.Dry Weights
The dry weight of the unseeded scaffolds was approximately constant throughout
the four-week culture period, with an initial dry weight of 3.3 ± 0.16 mg. The weight of
the GTA scaffolds tended to be slightly higher (3.7 ± 0.17 mg).
The dry weights of the cell-seeded constructs depended on cross-linking and time
in culture (Figure 2.7; 2-way ANOVA, interaction p<0.001).
Seeded UV constructs
maintained an approximately constant mass throughout the four-week culture period,
indicating a balance between deposition of newly synthesized matrix and scaffold
degradation. In contrast, there was a net loss of approximately 50% of the original mass
in the DHT constructs (3.5 ± 0.2 mg on day 2 to 1.7 ± 0.1 mg on day 28), while the
EDAC constructs demonstrated a 50% net gain in dry weight, increasing from 3.6 ± 0.2
mg to 5.4 ± 0.1 mg during the four-week culture period.
33
6
':
5
*
2 1 -- DHT -U-
5
GTA -@-
EDAC
1
1
1
1
1
10
15
20
25
30
0
0
UV -A-
Day
Figure 2.7. Dry weight of seeded scaffolds. Dry weight of seeded DHT constructs decreased over
the 4 week culture period, indicating greater matrix degradation than deposition, while UV construct
mass remained constant (equal degradation and deposition), and GTA and EDAC construct mass
increased (greater deposition than degradation). Mean ± SEM; n=9; *p<0.002 compared to day I
mass.
2.3.2.3.Radiolabel Incorporation
There were significant effects of cross-linking treatment and time in culture on
proline incorporation (Figure 2.8a; 2-factor ANOVA, p<0.0001). On day 2, the lowest
rate of proline incorporation was seen in the GTA constructs (post hoc p<0.003) and the
highest rate of incorporation was seen for the UV constructs (post hoc p<0.03). At the
final time-point, proline incorporation rates on day 29 in DHT scaffolds were
significantly lower than in the other three groups (p<0.01). The general trends in the
DHT and UV groups indicate decreasing rates of protein synthesis with increasing culture
time while the EDAC constructs maintained relatively high rates of synthesis throughout
the first week before decreasing by day 15. The GTA scaffolds, after supporting only
low rates of incorporation on day 2, had the highest rates on days 7 and 15 (p<0.005)
before decreasing by day 29.
34
a
0.15T
T
C
EDHT
ElIUV
i GTA
N EDAC
*
I-
0.1-
NS
eCz
0 PO
t
0.05*
02
0.03
b
-
t
Cu
*0-
0.02
-
0.01
-
NS
T
T~
29
15
7
tt
TT
rL
NS
t t
0
0
0
0
NS
NS
'5,
0-f-
2
15
7
29
Day
Figure 2.8. Protein and GAG biosynthesis. Accumulation of newly synthesized a) total protein, as
measured by 3H-proline and b) GAG, as measured by 35S-sulfate incorporation was influenced by
cross-linking and time in culture. Total amount of radiolabel incorporated into the constructs was
normalized to the radiolabel incubation time and DNA content. Mean ± SEM; n=9; *p<0.01
compared to all other groups at same time point; NS=groups not significantly different from each
other, but all other comparisons significantly different; comparing within a cross-linking group the
rates on day 2 were significantly different than at later time points, except where noted (t).
35
In contrast with the significantly lower level of proline incorporation observed in
the GTA scaffolds on day 2, there were no significant differences in the rates of sulfate
incorporation on day 2 among the cross-linking groups (Figure 2.8b). As with proline
incorporation, the rates of sulfate incorporation decreased from day 2 to day 7 for the
DHT and UV groups. The rate of sulfate incorporation for the DHT and UV scaffolds
remained at this lower level throughout the remainder of the culture period. At all time
points assayed, the rates of sulfate incorporation for the EDAC and GTA scaffolds were
approximately constant (0.018 nmol/hr/gg DNA).
2.3.2.4.DNA Analysis
During the first week in culture, there was an increase in DNA content for all
seeded scaffolds except those cross-linked by GTA (Figure 2.9a). In the following week,
DNA levels in the DHT and UV constructs remained constant while the amount of DNA
in the EDAC constructs continued to increase. During this time period, the cells in the
60
+DHT
-E-UV
50
40 -
-kGTA
eEDAC
30
a
20 10 0
0
10
20
30
Days
Figure 2.9. DNA content of cell-seeded scaffolds. Total DNA content of proteinase K digests of
the cell-seeded constructs. All scaffolds were initially seeded with 2x10 6 cells/disk. The total amount
of DNA increased during only the first week in culture in the DHT (*) and UV (i) cross-linked
scaffolds. There was no increase in DNA content during the first week in the GTA (A) cross-linked
scaffolds, but did increase over the next three weeks. The largest increase in DNA was seen during
the first two weeks in the EDAC (e) cross-linked scaffolds.
36
GTA constructs began to proliferate. Over the next two weeks, the DNA content in the
GTA constructs increased, whereas there was no significant change in DNA content in
the other three groups.
On day 29, the EDAC constructs still had the highest DNA
content, but the difference between the EDAC and GTA groups was no longer significant
(post-hoc p=0.07).
At this time point, the DNA content in the EDAC and GTA
constructs was 20-40% higher than in the DHT and UV constructs.
As mentioned above, the diameter of the scaffolds decreased over the four week
culture period. Normalizing the DNA content to the area of the constructs, the DNA
density (pg DNA/mm 2) was seen to increase with time in culture for all cross-linking
methods (Figure 2.9b). Due to the high degree of contraction, the DNA density on day
28 was highest for the DHT and UV cross-linked constructs.
2.3.3. GAG Content of Seeded Scaffolds
The amount of GAG in the cell-seeded constructs depended on cross-linking and
time in culture (Figure 2.10a; two-way ANOVA p<0.0001 for both variables). The total
GAG content in the DHT cross-linked scaffolds did not change over the four-week
culture period, indicating a balance between loss of GAG in the scaffold due to
degradation of the scaffold and deposition of newly synthesized GAG. In contrast, the
total GAG content of the seeded UV, GTA, and EDAC scaffolds increased over the fourweek period. It should be noted that the total GAG content reflected in these values
includes small GAGs, such as the chondroitin-6-sulfate originally added to the scaffolds.
As a better indicator of the behavior of the passaged chondrocytes in these CG
scaffolds, net GAG content (conservatively estimated as GAG content of seeded
contstructs minus GAG content of unseeded scaffolds) was normalized to DNA content
of the constructs (Figure 2.10b). The negative values seen on day 1 for the EDAC and
UV seeded scaffolds is likely reflective of more thorough rinsing of the unbound GAG
from the scaffold during the seeding process than the one-time rinse of the unseeded
scaffolds prior to GAG analysis.
With the exception of the seeded GTA scaffolds,
GAG/DNA increased significantly throughout the four-week culture period.
37
a
140
-+-DHT
120
-
100
-
80
-
60
-
40
-
20
-
-UUV
-A-
GTA
0-
5
0
15
10
--
EDAC
I
2
20
25
30
Day
b
3 ----
-E-UV
DHT
-A-GTA
---
EDAC
1 -
0S0-
I
I
'
15
20
25
30
-1
-2
-
-3
-
Day
Figure 2.10. GAG content of seeded CG scaffolds. (a) Total GAG content of proteinase K digests
of seeded CG scaffolds including GAG immobilized to collagen network (Figure 2.5). (b) Net GAG
accumulation (total GAG minus GAG originally in unseeded CG constructs) normalized to dry
weight.
38
2.3.4. Immunohistochemistry
Immunostaining for type II collagen demonstrated that the cultured chondrocytes
were able to synthesize and deposit type II collagen in the CG scaffolds by day 15 in the
DHT and UV cultures (Figure 2.11). The presence of type II collagen was seen only in
the interior of the constructs where the cells displayed a more rounded morphology.
None of the GTA or EDAC constructs stained positive for type II collagen at any time
point assayed.
Figure 2.11. Type II collagen immunostaining of cell-seeded scaffold. Immunohistochemical
staining in a DHT scaffold fixed after 15 days in culture. Red staining indicates the presence of type
II collagen deposited within the pores of the scaffold. a) Low magnification with negative control as
inset. b) Higher magnification of interior of same sample. Arrows point to examples of positive type
II collagen staining.
2.4.
DISCUSSION
As anticipated, the cross-link density and compressive stiffness of CG scaffolds
were altered by the different cross-linking protocols. The inverse of the swelling ratio,
which may be taken to be a measure of cross-link density, increased nearly 60% when
DHT cross-linked scaffolds were further cross-linked by immersion in an EDAC/NHS
39
solution. Compared to DHT cross-linking alone, EDAC cross-linking also provided a
nine-fold increase (to 1.1 kPa) in the apparent compressive modulus of the scaffold. The
degree of cross-linking afforded by the GTA and EDAC protocols used in this study
imparted sufficient stiffness to the scaffolds to reduce in vitro chondrocyte-mediated
contraction by 50% over a four-week in vitro culture period.
The stiffness of the EDAC CG scaffolds was still far lower than the equilibrium
modulus of articular cartilage (0.5-3 MPa [Athanasiou et al., 1991; Hale et al., 1993;
Jurvelin et al., 1987; Lee et al., 2000b; Setton et al., 1994]). The deformation of the cellseeded constructs was not measured, although it was observed that the constructs were
much less compliant after the first week in culture. It would be constructive to quantify
the increase in stiffness of the constructs, but consideration of the cell-mediated
deformation must be considered in designing the appropriate mechanical test.
The mechanical properties of polymer scaffolds, such as the CG scaffold in this
study, are usually evaluated in tension. One advantage to using tensile testing is that the
cross-link density may be related to tensile deformation behavior. Shulz Torres, et al.
[Schulz Torres et al., 2000] previously reported that different cross-linking protocols
could alter the tensile stiffness of the CG scaffolds and that tenocyte-mediated
contraction was moderately correlated to the tensile stiffness of the scaffold (R2 =0.65).
In this study, we sought a mechanical test that might better represent the changes that
occur during cell-mediated contraction of the scaffolds. Lee, et al. recently investigated
the ability of chondrocytes to bend collagen fibrils [Lee and Loeser, 1999].
Such
behavior may be akin to buckling of the collagen struts of the porous collagen scaffold
used in our laboratory. Thus, it was anticipated that cell-mediated contraction would be
correlated to compressive stiffness. The stiffest scaffolds (EDAC cross-linked) were, in
fact, able to slow contraction and limit the amount of contraction to approximately 30%
(of the original diameter) over the four-week period. The moderate correlation between
contraction and compressive stiffness (R2 =0.69) was similar to that observed by Shulz
Torres, et al. It should be noted, however, that the UV and GTA scaffolds had similar
compressive stiffnesses (346 and 369 Pa, respectively) but the CMC of the GTA
scaffolds was approximately half that of the UV scaffolds. The discrepancy in CMC may
be due to differences in cell-matrix adhesion and/or toxic by-products due to the different
40
cross-linking treatments. The noted increase in GTA scaffold stiffness with storage time
may also be attributed to the decreased contraction of GTA scaffolds. When the GTA
stiffness (unseeded) measured after six weeks of storage (663 ± 64 Pa) is used, the
correlation coefficient increases to R2 = 0.89.
The increase in stiffness with time in
storage may be due to residual gluteraldehyde molecules forming additional cross-links.
Other investigators have also reported that chondrocytes are capable of
contracting collagen gels [Hunter et al., 2000; Ohsawa et al., 1982]. It is possible that
degradation of the scaffolds or gels plays a role in the cell-mediated contraction. The
DHT scaffolds, which decreased in diameter by 60%, also demonstrated a net loss in dry
weight of approximately 50%.
There are several findings, however, indicating that
degradation is not the major contributor to the dimensional changes observed here. The
UV scaffolds also decreased in diameter by more than 50%, but did not exhibit a net loss
in mass, while the EDAC and GTA scaffolds, which had decreases in diameter of more
than 30% had a net gain in mass. Macroscopically, the borders of the scaffolds remained
well-defined and the samples displayed a decrease in transparency with shrinkage.
Additionally, under histological observation the pores of the scaffold were found to
collapse while the walls comprising the CG material remained essentially intact. Also of
importance in this regard is the previous finding that chondrocytes (human and canine),
both in situ and in vitro synthesize a contractile cytoskeletal protein, ct-smooth muscle
actin [Kim and Spector, 2000; Wang et al., 2000].
Furthermore, in studies involving
fibroblast-mediated contraction, it has been reported that collagen synthesis and
degradation does not affect the rate of contraction [Allen and Schor, 1983; Ehrlich et al.,
1989; Nishiyama et al., 1988].
While it does not appear that degradation plays a primary role in the decrease in
construct diameter, it is possible that the degradation rate of the scaffolds affects the
kinetics of contraction by reducing the stiffness of the scaffold with time in culture. The
delayed contraction seen in the EDAC scaffolds may be due to the need for a certain
amount of degradation to occur before the cells can contract the scaffold as well as a need
for a greater number of cells to begin to contract the stiffer scaffold. Additional studies
are needed to further quantify the rates of degradation of the different scaffolds as well as
the effects of degradation on scaffold stiffness and geometry.
41
In addition to the effects of cross-linking on stiffness and cell-mediated
contraction, we found significant differences in the proliferative and biosynthetic
behavior of the chondrocytes in the different scaffolds. All scaffolds supported some
degree of proliferation and biosynthetic activity. Although direct comparisons with other
tissue engineering systems are not possible due to inconsistencies of cell source (species
and age of donor and primary or passaged cells), cell density, culture medium, etc., a
rough assessment of this CG system can be made.
Consistent with trends seen for
newborn bovine chondrocytes in agarose [Buschmann et al., 1995] and peptide gels
[Kisiday et al., 2001] and adolescent bovine chondrocyte in porous collagen-GAG
scaffolds [van Susante et al., 2001], respectively, radiolabel incorporation decreased with
increasing time in culture.
The radiolabeled proline incorporation rates for the
chondrocytes in this system (0.023-0.09 nmol/gg DNA/hour) were one to five times
lower than that measured for calf chondrocytes in explant cultures (0.10 nmol/tg
DNA/hour measured 5 days after explant, Patwari, unpublished data) and primary calf
chondrocytes in agarose hydrogels (0.10-0.15 nmol/pg DNA/hour measured at various
time points up to four weeks after seeding, Kisiday, unpublished data). The difference in
sulfate incorporation between this system and the calf chondrocytes was more marked.
Sulfate incorporation for chondrocytes in explant or agarose gels was higher than proline
incorporation and ranged from 0.1-0.5 nmol/ptg DNA/hour. In the CG system, sulfate
incorporation was lower than proline incorporation and approximately 10-100 times
lower than that measured for the calf chondrocytes (0.005-0.02 nmol/pg DNA/hour).
The relatively higher rates of proline incorporation compared to sulfate incorporation
may be a consequence of the monolayer expansion of the chondrocytes, with the
passaged cells preferentially synthesizing proteins (collagenous and/or non-collagenous)
over proteoglycans. Finally, it should be noted that the measured incorporation rates
reflect the rate of accumulation of newly synthesized macromolecules within the
construct. In the experiments discussed in Chapter 3, it was found that a large proportion
(up to 70%) of the newly synthesized macromolecules were released to the media.
Assuming similar trends for the constructs in this experiment, protein biosynthesis of the
passaged chondrocytes in these type I CG scaffolds is on par with that of the primary calf
42
chondrocytes, while proteoglycan biosynthesis remains at least an order of magnitude
lower.
A comparison using the net GAG/DNA values (Figure 2.10b) is a conservative
comparison since it assumes that none of the chondroitin sulfate in the unseeded scaffold
is lost due to cell-mediated degradation of the scaffold. After two weeks of culture,
GAG/DNA in the CG scaffolds (1-2.5 pg GAG/gg DNA) was markedly lower than in
alginate (50 pg/pg [Ragan et al., 2000]) and peptide (22 pg/gg; Kisiday, unpublished
data) hydrogels and approaching that measured in porous PLA [Freed et al., 1993], PGA
[Freed et al., 1993], and collagen [Mizuno et al., 2001] scaffolds.
The lower GAG
content in the porous scaffolds compared to the hydrogels is likely due to lower rates of
retention of newly synthesized macromolecules. Finally, although the GAG/DW ratios
of the four-week constructs (1.9-3.6%, calculated from data represented in Figures 2.7
and 2.10a) were in the range of normal articular cartilage (1.5-3% [Lee, 1999]), the
GAG/DNA ratio for all of these constructs is still far below typical values (-200 pg/pg)
for normal adult articular cartilage [Lee, 1999].
The delayed proliferation, lower initial rates of synthesis, and initially stable dry
weight of GTA cultures indicate that the GTA samples may have been slightly cytotoxic
(see below).
In the other groups there appeared to be some association between cell
proliferation and biosynthesis with cell contraction.
In the DHT and UV scaffolds,
biosynthetic activity was highest near the beginning of the culture period, before the
scaffolds underwent significant contraction; this also applied to cell proliferation in the
UV group. During the active phase of contraction (days 2 to 5), mitosis and biosynthesis
decreased in these groups.
Similarly, in the EDAC group proliferation and synthesis
were reduced at 15 days and after as contraction occurred. These patterns of proliferation
and synthesis as they relate to cell contraction are consistent with previous studies that
have found that fibroblast proliferation and collagen synthesis are down-regulated after
contraction of collagen gels [Nakagawa et al., 1989; Paye et al., 1987].
Whether our
findings are due to the effects of the change in the microenvironment of the cells in the
collapsed pores or to an association of cell signaling pathways among contraction,
mitosis, and biosynthesis warrants further investigation in the chondrocyte-seeded CG
scaffold model.
43
NH 2
N
HC
CH
HC
0
0
+
HC
N
NH 2
R1
0
0
b
NH
R
+
R
=N-R
2
R
OH
carboxylic group
R=chondroitin-sulfate
or collagen
O-C
N
AC
R2
R1
Ri
0
O
0
NH
NH
O-C
R
+
+
R--C-O-N
HO-N
0-=LC
NH
N
O
0
NHS
substituted urea
0
R -C-O-N
||0
00
0
+
H2 N-COII
-P
R-
C-N-
Col
+
HO-N
0
free amine groun
on collagen
amide crosslink
Figure 2.12. Proposed cross-linking mechanisms. Formation of cross-links by a) glutaraldehyde
reaction with collagen amino groups and b) EDAC reaction of collagen or chondroitin sulfate
carboxyl groups with amino groups of collagen. The proposed reaction with chondroitin sulfate
carboxyl groups would explain the higher GAG content of the unseeded EDAC scaffolds (Fig 2.4).
44
Glutaraldehyde cross-linking is often cited as being undesirable as it introduces
cytotoxic aldehyde molecules. The aldehyde may remain non-specifically bound to the
scaffold even after exhaustive rinsing and the aldehydes that have been incorporated into
the cross-links (Figure 2.12a) may also be released from the scaffold as the collagen
network degrades [Huang-Lee et al., 1990]. The decreased proliferation and synthesis
observed at early time points in this study might be the result of the cytotoxic
glutaraldehyde.
In EDAC cross-linking, on the other hand, the cross-linking agent is not
incorporated into the amide cross-links that form, thus allowing the cytotoxic
carbodiimide to be completely rinsed from the scaffold. For the CG system used in this
study, EDAC cross-linking also has the advantage that it forms collagen-collagen and
collagen-GAG cross-links [Osborne et al., 1998; Pieper et al., 1999] (Figure 2.12b),
thereby immobilizing greater amounts of chondroitin sulfate in the network compared to
DHT and GTA treatments.
The chemical reactions involved in EDAC cross-linking (see above) and the
random formation of radicals and subsequent cross-links by UV irradiation provided for
increased amounts of GAG tightly bound to the collagen compared to the DHT and GTA
cross-linking methods that are more specific to collagen-collagen cross-link formation.
Previously, it has been reported that the addition of GAGs to collagen scaffolds increases
the synthesis of GAGs by cells seeded into the scaffolds [Sechriest et al., 2000]; van
Susante et al., 2001]. In this study, however, in which the EDAC and UV cross-linked
scaffolds had higher amounts of GAG immobilized in the scaffold than the DHT and
GTA scaffolds, there was no increase in the rates of GAG synthesis (35S-sulfate
incorporation) with increasing amounts of immobilized GAG.
It is possible that the
amount of GAG in the DHT and GTA scaffolds is enough to stimulate GAG synthesis by
the seeded chondrocytes.
It is also possible that the presence of increased amounts of
chondroitin sulfate may regulate which types of GAGs are being synthesized. Since only
general rates of GAG synthesis were measured in this study, it is not possible to make the
distinction between cartilage-specific GAGs (i.e. aggrecan) and other sulfated GAG
molecules.
45
I
IE-12
II
"-lkDa
.7;11
Figure 2.2. Western blot for type II collagen. Western blot reveals presence of type II
collagen in the chondrocyte-seeded EDAC cross-linked scaffold after 12 weeks of in
vitro culture. I=purified type I collagen standard; II=purified type II collagen standard;
IE-12=extract from 12-week culture of passaged canine chondrocyte-seeded type I CG
scaffold with EDAC cross-linking
Although we initially sought to develop a scaffold for cartilage tissue engineering
that would not be subjected to cell-mediated contraction and severe deformation,
immunohistochemical
staining indicated that cartilage-specific
type II collagen
accumulation occurred only in the contracted constructs (DHT, days 15 and 29 and UV
day 29. It should be noted that Western blot analysis of chondrocyte-seeded EDAC
constructs cultured for twelve weeks (performed for a separate study) has shown that
there is indeed deposition of type II collagen in these constructs over the longer term
(Figure 2.13). It is unknown whether the early identification of type II collagen in the
DHT and UV constructs is due to simply to increased matrix molecule retention or due to
differences in cell phenotype. The contraction of the DHT and UV scaffolds during the
first week in culture could likely have led to a higher retention of matrix molecules and,
therefore, accumulation of the type II collagen.
Alternatively, the contraction of the
scaffold may promote the chondrocyte redifferentiation and type II collagen synthesis.
Previous studies have reported on the use of high-density cultures to promote
chondrogenesis [Fedewa et al., 1998]. It is possible that the synthesis of type II collagen
46
in our system relies on the high density of the extracellular matrix resulting from
contraction of the DHT and UV scaffolds (Figure 2.9b). Additionally, contraction of the
scaffolds could lead to increased type II collagen synthesis by slowing diffusion of
nutrients and gases through the construct [Clark et al., 1991; Domm et al., 2000;
Obradovic et al., 1999; Rajpurohit et al., 1996]. Future studies are needed to specifically
delineate the effects of cross-linking, cell density, and extracellular matrix density on
chondrocyte differentation.
In choosing appropriate cross-linking methods for scaffolds used in the tissue
engineering of articular cartilage, the effects of the treatment on cell proliferation and cell
synthesis of matrix specific molecules must be considered in addition to the more
obvious effects on degradation rate and mechanical properties. EDAC and GTA crosslinking slowed chondrocyte-mediated contraction of CG constructs and limited the
amount of contraction to roughly 30% over a four-week period. EDAC cross-linking
method also appeared to favor cell proliferation and biosynthetic activity in contrast to
GTA cross-linking which inhibited proliferation and biosynthesis at early time points.
Neither of these cross-linking methods, however, permitted detectable levels (by
immunohistochemistry) of cartilage-specific type II collagen accumulation over the first
four weeks in culture.
47
CHAPTER 3: EFFECTS OF MECHANICAL
COMPRESSION ON BIOSYNTHETIC ACTIVITY OF
PASSAGED CHONDROCYTES IN TYPE 11 COLLAGENGLYCOSAMINOGLYCAN SCAFFOLDS
3.1.
INTRODUCTION
An important parameter to consider in evaluating potential scaffolds for
orthopaedic tissue engineering is the cell-scaffold interaction and, more specifically, the
ability of the scaffold to transmit mechanical stimuli to the cells. It is well-known that
mechanical loading plays an important role as a regulator in the metabolic processes of
chondrocytes in situ [Gray et al., 1989; Grodzinsky et al., 2000; Grodzinsky et al., 1998;
Grumbles et al., 1995; Guilak et al., 1995; Kiviranta et al., 1988; O'Connor et al., 1988;
Paukkonen et al., 1986; Sah et al., 1991; Sah et al., 1989; Urban, 1994; Wilkins et al.,
2000]. Chondrocytes isolated from cartilage and cultured in non-native environments are
also capable of responding to mechanical loading [Lee and Bader, 1997; Lee et al.,
2000c; Ragan et al., 2000].
Consequently, in recent years, the response of tissue
engineering-motivated cell-scaffold systems to mechanical loading has become a part of
the in vitro development of engineered cartilage [Buschmann et al., 1995; Davisson et al.,
2001; Mauck et al., 2000].
Buschmann, et al. [Buschmann et al., 1995] found that
chondrocytes encapsulated in agarose gels could respond to mechanical loading in a
manner similar to chondrocytes in cartilage explants. The agarose-seeded chondrocytes,
however, only responded to mechanical stimuli after three weeks in culture, presumably
because the response required the deposition of an extracellular matrix through which
mechanical stimuli could be transmitted to the cells.
Long-term culture of freshly
isolated (primary) chondrocytes seeded into scaffolds and subjected to compressive
loading [Mauck et al., 2000] or hydrodynamic shear stress [Freed et al., 1998; VunjakNovakovic et al., 1999] has been shown to enhance in vitro culture of cartilage-like
tissue. Recently, short-term compressive loading of primary chondrocytes seeded into
polyglycolic
acid (PGA)
scaffolds has been shown to increase the rates of
glycosaminoglycan synthesis (GAG) [Davisson et al., 2001].
48
The purpose of this experiment was to test the hypothesis that mechanical
compression of cell-seeded CG scaffolds would regulate passaged chondrocyte
biosynthesis.
The specific aim of this experiment was to evaluate the biosynthetic
response of passaged chondrocytes seeded in the porous CG scaffolds to short-term (up
to
24 hours)
compressive
glycosaminoglycan
loading by
biosynthesis
comparing
the rates
of protein
and
of the seeded chondrocytes under free-swelling,
statically compressed, and dynamically compressed conditions at various times in culture.
3.2.
MATERIALS AND METHODS
3.2.1. Collagen-Glycosaminoglycan Scaffolds
Type II CG scaffolds were fabricated from a slurry of type II collagen and
glycosaminoglycans (Geistlich Biomaterials, Wolhusen, Switzerland) using the same
freeze-drying method used for the fabrication of type I CG scaffolds described in Chapter
2 (Appendix A). Disks (4 mm diameter) were punched from the 1.5-2 mm thick sheets
and EDAC cross-linked as described in Chapter 2.
3.2.2. Cell Culture and Cell-Seeding of Scaffolds
Adult canine chondrocytes harvested and cultured as described in Chapter 2.
Third passage chondrocytes (8-10 cell doublings over approximately 20 days) were
resuspended in culture medium at a density of 7.5x 106 cells/ml. Prepared scaffolds were
incubated with the cell suspension (15 disks/ml suspension) for 1.5-2 hours with
continuous rocking. Assuming a 50% seeding efficiency (Appendix J), this corresponded
to an initial cell density (10,000 cells/mm 3 ) similar to adult human articular cartilage
[Muir, 1995]. Seeded scaffolds were transferred to agarose-coated 24-well plates with
0.5 ml media/well. An additional 0.5 ml media was added the following day. Media was
exchanged every 2-3 days.
3.2.3. Mechanical Compression of Cell-Seeded Scaffolds
3.2.3.1.Static Compression - Dose Response
After 7 or 14 days of free-swelling culture in the 24-well plates, cell-seeded
scaffolds were transferred to polysulfone compression chambers described previously
[Sah et al., 1989] (Figure 3.1a).
Each chamber could accommodate up to 12 disks.
49
Using Teflon spacers to adjust the height between the base and top halves of the chamber,
cell-seeded scaffolds (n=6 per condition) were held at 1.2, 1.05, 0.9, or 0.6 mm
(corresponding to approximately 0%, 10%, 25%, or 50% strain, respectively, relative to
an average construct thickness of 1.2 mm). After fixing the chamber strain level and
adding radiolabeled media (0.6 ml) to each chamber well (see below), cultures were
returned to the incubator. Control cultures were transferred to identical chambers and
Teflon spacers were used to ensure that the top half was not in contact with the scaffolds
(free-swelling).
3.2.3.2.Static Compression - Kinetics
In a separate study, the kinetics of the biosynthetic response to static compression
was evaluated. Cell-seeded scaffolds were cultured under free-swelling conditions for 7
days and then subjected to 50% static compression for 1, 2, 4, 8 or 24 hours, during
a
b
Figure 3.1. Polysulfone compression chambers. (a) Static chambers accommodated up to 12
samples, each held to the same thickness. The height of the chamber was adjusted using Teflon
spacers. (b) Dynamic chambers accommodated up to 12 samples with the height for each sample
individually adjusted. The top half of the dynamic chamber was fixed into the upper jaw of the
loading device and its axial position controlled by computer feedback.
50
which time the cultures were incubated in media containing radiolabeled sulfate and
proline.
Compressed cultures were compared to free-swelling cultures that were
radiolabeled for the same period of time as the compressed cultures.
3.2.3.3.Dynamic Compression
After 2, 7, 14 or 30 days of culture in the 24-well plates, cell-seeded scaffolds
were transferred to a similar polysulfone chamber that had a top half which attached to
the top jaw of an incubator-housed mechanical spectrometer [Frank et al., 2000]. The top
half of the chamber had platens that could each be raised and lowered independently
(Figure 3.1b). The EDAC cross-linked scaffolds had sufficient compressive stiffness to
support the weight of the individual polysulfone platens. After adjusting the height of the
platens and adding media with radiolabeled sulfate and proline (see below), scaffolds
were first subjected to 10% or 50% strain by applying a ramp-and-hold displacement
(10% strain/15 seconds) using computer-controlled displacement.
A 3% sinusoidal
displacement was then superimposed on the static offset strain and cycled at 0.1 Hz for
the next 24 hours (n=8). The static and dynamic compression amplitudes were chosen to
be similar to those used in loading experiments of cartilage explants. The single loading
frequency (0.1 Hz) represents the low end of the physiological loading frequency for
articular cartilage. Separate sets of both free-swelling cultures and cultures held at 10%
or 50% static compression were used as controls.
3.2.4. Protein and GAG Biosynthesis
To determine rates of protein and GAG biosynthesis in all static and dynamic
compression studies, media (600 pl) containing 20 gCi/ml
35S-sulfate
and 10 gCi/ml 3H-
proline was added to each well and cultures were returned to the incubator (37 0 C and 5%
C0
2
). Following compression, unincorporated radiolabel was rinsed from the scaffolds
through 4x15 minute washes in cold phosphate buffered saline supplemented with 100
mM unlabeled proline and 500 mM unlabeled sulfate. Scaffolds were then lyophilized
dry and digested with proteinase K (100 [tg in 1 ml 50 mM Tris-HCl buffer with 1 mM
CaCl 2 ; overnight at 60'C). Aliquots of the digest (100 jil) were mixed with 2 ml of
scintillation buffer (EcoLume) and assayed for radioactivity content by scintillation
counting (Rack-Beta 1211, LKB, Turku, Finland). Scintillation counts were normalized
51
to the DNA content of each scaffold, as determined using the Hoechst 33258 dye assay
[Kim et al., 1988].
3.2.5. Analysis of Macromolecules Released to the Medium
Portions of media collected from selected cultures were analyzed for newly
synthesized
35S
and 3 H macromolecules released into the medium during the 24 hour
compression and radiolabeling period. Medium was fractionated on a PD-10 column
(Sephadex G-25, Biorad) equilibrated and eluted (1 ml/minute) with 2M guanidine-HCl,
0.5M sodium acetate, 1mM sodium sulfate, 1mM L-proline, and 0.02% sodium azide, pH
6.8. A 100 g1 aliquot of the void volume (3.0 ml) from each sample (n=3 per loading
group) was mixed with 2 ml of scintillation buffer and assayed for radioactivity content
by scintillation counting.
3.2.6. Newly Synthesized Proteoglycan Size and Affinity for Hyaluronic Acid
Separate samples, cultured for 7 days, were incubated in medium containing 10
gCi/ml
3
S-sulfate for 24 hours under free-swelling conditions.
5
Following the
radiolabeling period, samples were washed in PBS containing cold (unlabeled) sulfate
(4x15 minutes), and the proteoglycans extracted from the samples overnight at 4'C with
4M GnHCl in 0.05 M sodium acetate buffer, pH 6.8, in the presence of 2% (v/v) Triton
X-100 and the proteinase inhibitors: phenylmethanesulphonyl fluoride (1 mM), Nethylmaleimide (1 mM), dithiothreithol (1 mM), leupeptin (0.1 gg/ml), and pepstatin (0.1
pl/ml) [Rong et al., in submission].
Extracts containing the newly synthesized
proteoglycans were desalted on a column of Sephadex G-25 (Pharmacia PD-10 columns,
#17-0851-01) with 0.5 M sodium acetate/0.1% Triton X-100/0.02% sodium azide as the
eluent. The proteoglycans were collected in the void volume indicated by the elution of
Dextran blue. The collected volume was lyophilized and dissolved in 400 pl 0.5 M
sodium acetate with 0.1% Triton X-100. A portion of this solution (200 pl) was analyzed
on a Sephacryl S-1000 column (1.0 x 50 cm, BioRad), run at 9 ml/hour, with 0.5 M
sodium acetate/0.1% Triton X-100/0.02% sodium azide as the eluent [Sah et al., 1990].
Each 0.75 ml fraction was mixed with 2 ml of scintillation fluid and assayed for
radioactivity content. The remaining portion (200 [d) was incubated with hyaluronic acid
(4 mg/ml of rooster comb hyaluronic acid, Sigma Chemical Co.) for 24 hours at 4'C
52
[Rong et al., in submission] prior to S-1000 analysis. For comparison, aggrecan extracted
from calf cartilage was also eluted through the S-1000 column and fractions analyzed for
GAG content by DMMB analysis (20 il of each fraction mixed with 200 gl of 1,9dimethylmethylene blue dye and absorbance read at 520 nm).
3.2.7. Statistical Analysis
Radiolabel incorporation rates (nmol/gg DNA/hr) for compressed samples were
normalized to the average rates measured in the respective free-swelling controls. Values
are reported as mean ± standard error of the mean (SEM). One-way analysis of variance
(ANOVA) and Fisher protected least squares difference (PLSD) post-hoc testing were
performed at each time point using StatView (SAS Institute, Inc, Cary, NC), as described
in Chapter 2, to compare the effects of various levels of static compression (compared to
0% compression) or the effects of dynamic compression versus static compression and
free-swelling. Statistical significance was taken to be p<0.05.
3.3.
RESULTS
3.3.1. Static Compression - Dose Response
When transport through the top of the culture was limited by bringing the platens
into contact with the samples (0% compression), there was a notable decrease in the
accumulation of newly synthesized macromolecules compared to free-swelling cultures
(Figure 3.2).
The accumulation of newly synthesized proteins in 0% compressed
cultures, as measured by 3 H-proline incorporated into the scaffolds, was 35-55% lower
than in free-swelling controls after one and two weeks of initial free-swelling culture,
respectively.
Similarly, the accumulation of newly synthesized GAG in the 0%
compressed scaffolds, as measured by
35S-sulfate
incorporation, was 20-40% lower than
free-swelling after one and two weeks of initial free-swelling culture, respectively.
Static compression applied to cultures after one or two weeks in free-swelling
culture resulted in a further dose-dependent decrease in newly synthesized protein
accumulation in the construct (Figure 3.2a; p<0.01). Except for 10% compression on the
day 7 cultures, all cultures subjected to all levels of static compression contained
significantly lower amounts of radiolabeled protein compared to the 0% compressed
cultures (p<0.01).
53
I
10 0% Compression
0.8
3-
0.6
M10%
-
25%
050%
-
T
0
0.4
-
0.2
-
0
7
Z
1.2
I
14
*
-
El 0% Compression
*10%
10.8
025%
-
-I-
050%
0.6
0
_r -]
0.4
-
0.2
-
*
07
I -
14
Days in Free-Swelling Culture
Figure 3.2. Biosynthetic dose response to static compression. Radially-unconfined static
compression (0%, 10%, 25%, or 50% strain) applied to constructs after 7 or 14 days of free-swelling
3
culture led to decreased rates of (a) protein accumulation, as measured by H-proline incorporation
35
into the scaffold, and (b) glycosaminoglycan (GAG) accumulation, as measured by S-sulfate
incorporation over a 24-hour period. Radioactive counts were normalized to DNA content of
individual samples. Data (mean ± SEM; n=6) are presented normalized to the average values for the
free-swelling control cultures. *p<0.01 compared to 0% strain.
Rates of glycosaminoglycan (GAG) deposition, as measured by
3 5S-sulfate
accumulation in the scaffold cultures, were similarly lowered when 14-day cultures were
subjected to 24 hours of static compression (Figure 3.2b). The cell-seeded scaffolds that
were compressed after only 7 days of culture, however, did not display lower levels of
GAG accumulation, compared to 0% controls. In fact, 10% compression of the 7-day
cultures increased the amount newly synthesized GAG within the scaffold relative to the
0% compressed cultures.
Static Compression - Kinetics
3.3.2.
The amount of 3 H-proline and
35S-sulfate
incorporated into the constructs
normalized to cell (DNA) content, increased linearly with time for both the free-swelling
3
a
0.08
*Free-swelling
9
- 2.5-
150% Static
b
0.07
R
2
=
0.9303
±
0.06-
R2= 0.989
2-
1 0.050.04
1.5
0.032
1
R
20
R
0.5
=
0.8686
0.02
= 0.7564
0.01 -
8
00
0
5
10
15
20
0
25
5
10
15
Time (hrs)
Time (hrs)
35
3
Figure 3.3. Kinetics of radiolabel incorporation. Radioactive (a) H-proline and (b) S-sulfate
incorporation into the scaffolds increased linearly with free-swelling (+) radiolabeling time. Static
3
compression at 50% strain (m) decreased the rate of H-proline incorporation at all time points
35
(p<0.05), while decreasing the rate of S-sulfate incorporation only over the 24-hour period.
Cultures were labeled after 7 days in free-swelling culture; mean ± SEM, n=4.
55
20
25
and statically compressed samples (Figure 3.3). Protein accumulation, as measured by
3
H-proline incorporation, was significantly lower in 50% statically compressed samples
at all time points evaluated (Figure 3.3a; p<0.02). In contrast, while GAG accumulation,
as measured by
35
S-sulfate
incorporation, also tended to be lower in statically compressed
samples (p>0.05), it was only significantly lower for 24 hours of compression (Figure
3.3b; p<0.02).
3.3.3. Dynamic Compression
Dynamic compression, applied as a continuous 3% amplitude sinusoidal
superimposed on a 10% or 50% static offset compression for 24 hours, upregulated the
rates of protein biosynthesis compared to static controls (Figure 3.4a, p<0.01) when
applied to cultures at all time points except for day 7. The rate of proline incorporation
into the scaffold in the dynamically compressed samples, however, was still lower than
i
3-
1
a
Z
0 Static 0 Dynamic
0.8-
0.8
0.6-
0.6
0.4
T
. 0.4
0
0
0.2
0.20
I
I
2
7
14
z 0
30
Days in Free-Swelling Culture
2
7
14
30
Days in Free-Swelling Culture
Figure 3.4. Effects of compression on macromolecular accumulation. 24-hours of continuous
dynamic compression (3% sine, 0.1 Hz) superimposed on a 10% static offset strain (a) increased rates
of protein accumulation, relative to 10% static compression, for all cultures except those compressed
after 7 days in free-swelling culture, but (b) did not affect GAG accumulation at any time point
evaluated. Radiolabeled protein and GAG accumulation of all compressed samples were lower than
for free-swelling samples (normalized values <1); n=6. *p<0.01 compared to static control
56
those measured in the free-swelling controls (normalized incorporation rates less than 1).
The protein biosynthesis rates in the dynamically compressed cultures were significantly
lower than the free-swelling rates at all time points except day 2.
In contrast, the rate of
35
S-labeled-GAG accumulation in the scaffolds was not
affected by dynamic compression (Figure 3.4b, p> 0 .15). As with proline accumulation,
however, the rate of sulfate accumulation into the scaffold for both the statically and
dynamically compressed cultures were lower than that of the free-swelling cultures at
each time point (p<0.01).
3.3.4. Newly Synthesized Macromolecules Released to the Medium
PD-10 fractionation of radiolabel in the medium revealed that a significant
portion of the newly synthesized macromolecules had been released into the medium. Of
the total amount (construct radiolabel + macromolecular label in the medium) of newly
synthesized proteins and GAGs in the free-swelling cultures, 14-60% of the newly
synthesized proteins
(3H-labeled
macromolecules) and 7-70% of the newly synthesized
GAGs (3 5S-labeled macromolecules) were in the medium (Figure 3.5). The percent of
radiolabeled macromolecules in the media decreased by the second week in culture
(p<0.01).
The effects of mechanical compression on macromolecular release were evaluated
for day 2 and day 7 cultures (Figure 3.5 c,d). Mechanical loading affected the release of
3H-
and
35S-labeled
macromolecules to the media in a similar manner. Although the total
average radiolabel incorporation for both 10% and 50% static compression on day 2
tended to be lower than for free-swelling cultures, neither level of static compression
significantly affected the amount of macromolecular radiolabel released to the medium.
Statically
compressed
day 7 cultures
tended to have
higher proportions
of
macromolecular radiolabel in the media than free-swelling controls, with the increase at
50% static strain being significant (p<0.05).
The effects of dynamic loading on the release of newly synthesized proteins and
glycosaminoglycans were dependent on the offset strain.
On days 2 and 7, dynamic
compression superimposed on a 10% strain offset had the highest total rates of both 3Hproline
35
S-sulfate
incorporation (p<0.05). Compared to both the free-swelling and 10%
static controls, dynamic loading superimposed on a 10% offset strain significantly
57
-0.16
0.014
a
0.14
mrdia
(
construct
0.12
b
0.012
0.01
0.1
0.008
0.08
0.006
0.06
& 0.004
0.04
n
0.02
0.002
2
7
2
14
0.45
0.4
-
-
0
0
7
14
0.035
C
*
0.350.3
0.25-
0.03
*
d
*
0.025± 0.02-
0.2-
iiI~ I
0.015-
0.15
0.01.
0.1
0.05
0
0~
kn
ui
0.005
11
2
W
eJ+W
+
n>
-
7
Days in Free-S
2
welling Culture
7
Days in Free-Swelling Culture
Figure 3.5. Total rates of biosynthesis. The total rates of (a, c) protein and (b, d) GAG was
measured as the sum of the radiolabel in the construct (o) and the macromolecular radiolabel in the
media (m) for (a, b) free-swelling cultures and for (c, d) compressed cultures. (a, b) For free-swelling
cultures, the rate of release of newly synthesized macromolecules decreased as the cultures matured
(day 14 media values < day 2, 7 values; p<0.01). (c, d) Dynamic compression, superimposed on a
10% static offset strain significantly increased the rate of release and the total rate of biosynthesis of
both (c) protein and (d) GAG; n=3.
increased the amount of 3H- and
(p<0.005).
35S-labeled
macromolecules released to the medium
In contrast, dynamic loading superimposed on a 50% static strain had no
significant effect on macromolecular release on either day, relative to the 50% static
control.
Compared
to
the
free-swelling
cultures,
however,
the
increase
in
macromolecular release with dynamic compression superimposed on the 50% static
offset on day 7 was significant (p<0.05).
58
The effects of mechanical compression on the total rates of biosynthesis (sum of
radiolabeled macromolecules in the media and radiolabel retained in the scaffold) were
proportionally the same as those reported for the release of macromolecules to the media.
on a 10% static offset
Most importantly, dynamic compression superimposed
significantly increased total synthesis of protein and GAG compared to free-swelling
controls on days 2 and 7.
3.3.5. Proteoglycan Analysis
To begin to determine the nature of the newly synthesized proteoglycans, the
radiolabled proteoglycans were extracted from the construct and analyzed on S-1000
columns (Figure 3.6). Nearly 90% of the aggrecan extracted from calf articular cartilage
1
1
1.2
S
1--
1.0
0.8
0.6
0.4
0.2
0.0
5S -HA
3--- 35S +HA
- - Calf PG
e0.8
~0.6
0.40.2 -I
AL-
A.
015
20
30
25
35
40
45
Elution Volume (ml)
Figure 3.6. Size distribution of newly synthesized proteoglycans. Proteoglycans synthesized during
a 24-hour radiolabeling period after the 7t day in free-swelling culture were extracted, desalted on a
Sephadex G-25 (PD-10) column, and fractionated on a Sephacryl S-1000 column both in the absence
and presence of 4 mg/ml hyaluronic acid. Proteoglycans extracted from calf articular cartilage were
similarly fractionated and identified through the DMMB colorimetric assay.
59
ran in the free monomer position (Kay -0.38) as seen previously, confirming their
isolation from endogenous hyaluronate [Sah et al., 1990].
Compared to the elution
profile of this calf aggrecan the monomer peak from the chondrocyte-CG samples was
smaller, constituting -35% of the total
35S
activity. The largest peak from the culture
extracts peaked at Kav=0.7, suggesting that the majority of the newly synthesized
proteoglycans in the CG cultures were smaller proteoglycans and/or smaller aggrecan
fragments. The addition of the hyaluronic acid did not significantly affect the elution
profile of
35
S-labeled species eluting near the Vo, suggesting no increase in their
aggregatability.
3.4.
DISCUSSION
Similar to previously established patterns for compressive loading of primary
chondrocytes in intact tissue and hydrogel systems [Buschmann et al., 1995; Gray et al.,
1989; Ragan et al., 1999a; Ragan et al., 2000; Sah et al., 1989; Steinmeyer et al., 1999],
static loading decreased biosynthesis of the passaged chondrocytes, while dynamic
compression (3% sine, 0.1 Hz) superimposed on a 10% strain offset increased the rates of
biosynthesis.
Due to increased release of macromolecules, however, the rates of
accumulation of newly synthesized macromolecules did not always follow the same
pattern.
As with other chondrocyte systems, static compression led to a decrease in the
amount of newly synthesized protein deposited within the scaffold in a dose-dependent
manner. Relative to statically compressed cultures, most of the dynamically compressed
cultures accumulated more newly synthesized protein during a 24-hour continuous
loading period. Compared to free-swelling cultures, however, there was no change or a
decrease in protein accumulation in the dynamically compressed cultures.
After two
weeks in free-swelling culture, glycosaminoglycan (GAG) deposition also tended to
decrease when cultures were subjected to static compression but, in contrast to protein
deposition, did not increase when dynamic compression was applied.
The cultures that did not show an increase in the amount of newly synthesized
protein in the scaffold with dynamic compression, relative to static compression, were
those that had been in free-swelling culture for one week prior to loading. It should be
60
noted, however, that the total amount of newly synthesized macromolecules (construct
plus medium) did increase with dynamic loading (superimposed on 10% offset strain)
even at this time point. As reported previously (Chapter 2), the DNA content of these
cultures (data not shown) indicated active cell proliferation the first week in culture.
Thus, one possible explanation for the inability of dynamic compression to increase
macromolecular accumulation at this time point is the altered response of the actively
proliferating cells mechanical stimuli.
The chondrocytes used in these experiments had been expanded in monolayer
culture for up to three weeks (approximately 8-10 doublings) prior to seeding into the CG
scaffolds. It has been well-established that such expansion of chondrocytes can lead to
the loss of the chondrocyte phenotype, namely decreased expression of type II collagen
and increased synthesis of type I collagen [Benya and Shaffer, 1982]. Previous studies
with a similar type I CG scaffold have shown that there is type II collagen deposition in
the scaffolds within the first two weeks (Figure 2.11 and [Nehrer et al., 1997a]). It is
unknown, however, the extent of dedifferentiation prior to seeding or the rate of
redifferentiation once the cells are seeded into the scaffolds.
This study focused on
tracking the overall rates of synthesis of protein (collagenous and non-collagenous) and
glycosaminoglycan (including cartilage-specific aggrecan and smaller non-cartilaginous
GAGs), without regard for whether or not the newly synthesized macromolecules were
cartilage-specific.
Analysis of selected free-swelling day 7 chondrocyte-seeded CG
cultures using S-1000 size-exclusion chromatography for newly synthesized
35S_
proteoglycans indicated that 8-10% of the proteoglycans being synthesized by the
passaged cells eluted in the aggregate form. There was also, however, a substantial
portion of
35S-labeled
species that were smaller than cartilage aggrecan monomer. At
present, it is unknown whether these represent aggrecan fragments (e.g., partially
degraded) or small molecular weight proteoglycans such as decorin.
Clearly, future
studies are needed to more specifically determine the nature of the proteins and
proteoglycans that are being synthesized in this system.
Since the composition and structure of cartilage is essential to the mechanical
function of cartilage in situ, it is possible that mechanical loading may influence which
genes/proteins are expressed/synthesized. Previous studies have shown that mechanical
61
regulation can influence cell differentiation and maturation [Elder et al., 2000; Wu and
Chen, 2000] and the response of chondrocytes to growth factors [Bonassar et al., 2001].
Thus, it may be possible that although dynamic compression failed to increase deposition
of newly synthesized macromolecules within constructs in day 7 cultures, dynamic
loading may have influenced cell differentiation and the nature of the molecules that were
synthesized. Future studies will focus on the influence of mechanical loading on the
cell's phenotype and the structure of the newly synthesized matrix molecules.
Third passage chondrocytes seeded into the porous CG scaffolds and cultured
under free-swelling conditions released up to 70% of their newly synthesized
macromolecules into the medium. Although this portion decreased with extended time in
culture, it remained higher than the reported release into the medium (<2%) of newly
synthesized glycosaminoglycans produced by chondrocytes in intact cartilage explants
[Sah et al., 1991] or in agarose gels [Buschmann et al., 1995]. The noted decrease with
extended culture periods is likely a result of decreased transport as the density of the
extracellular matrix increased, due to both the gradual accumulation of matrix molecules
synthesized by the seeded cells and the cell-mediated contraction of the scaffold's pores
(Chapter 2). In contrast to cartilage explants or agarose gels, the original structure of the
CG scaffolds was highly porous, allowing for easy diffusion of the newly synthesized
molecules out of the construct.
In previous explant studies [Sah et al., 1991], it was reported that the percentage
of newly synthesized glycosaminoglycans released to the medium increased with
dynamic compression. Similar increases were seen for the dynamically compressed CG
scaffolds evaluated in this study, using a similar dynamic compression amplitude
superimposed on a 10% offset strain on days 2 and 7 and for the CG scaffolds with a 50%
offset strain on day 7 only. These changes may be attributed to a number of factors,
including fluid flow-induced increases in the rate of transport of macromolecules out of
the scaffold, physical disruption of the matrix, and/or increased synthesis of matrix
proteinases leading to increased matrix turnover.
While it is important to note the
overall changes in total biosynthetic activity of the chondrocytes in the CG scaffolds, the
long-term objective of this work is to develop a method for tissue engineering of articular
cartilage. Thus, it is the accumulation and assembly of a functional macromolecular
62
extracellular matrix within the scaffold that is ultimately important. In order to improve
the quality of the implant developed under both free-swelling and mechanically
stimulated cultures, future studies will need to investigate how to improve the retention of
the newly synthesized matrix molecules within the scaffold while stimulating increased
synthesis.
The results reported in this study are limited to cultures subjected to an initial 24
hours of continuous loading. Different loading patterns may have different effects. In
preliminary studies, similar results for dynamic loading relative to free-swelling cultures
were obtained using first passage chondrocytes. Preliminary studies also indicated that
dynamic compression over a range of frequencies
(0.01 to 1.0 Hz) affected
macromolecular accumulation within the scaffolds in the same manner. It is apparent,
however, that there were differences in cultures with 10% and 50% offset strains.
Dynamic compression superimposed on a 10% offset strain yielded the highest rates of
biosynthesis
(total
3
H-proline
and
macromolecular form, Figure 3.5).
35S-sulfate
radiolabel
incorporation
into
Additionally, whereas dynamic compression
superimposed on a 10% offset strain led to increased biosynthetic activity compared to
the static control, dynamic compression superimposed on a 50% offset strain did not.
Furthermore, it may be possible that 24 hours of continuous loading may be "over
exercising" the cells and the deleterious effects of static compression may offset the
stimulation of biosynthetic activity by dynamic loading. Since it was seen that static
compression up to 8 hours did not lead to a decrease in GAG accumulation, it may be
possible that shorter compression periods may lead to a net increase in matrix molecule
accumulation. Long-term studies that have shown a beneficial effect of physical forces
on tissue engineered cartilage [Freed et al., 1997; Mauck et al., 2000; Vunjak-Novakovic
et al., 1999] have used intermittent loading protocols to achieve their results. Further
studies are needed to determine how different loading protocols (i.e. intermittent loading
and total duration of loading) will affect biosynthetic activity and overall construct
properties.
Compressive loading elicited changes in biosynthetic activity as early as two days
after cells were seeded into the CG scaffolds. This is in contrast to previously established
systems using chondrocytes in gel systems, in which two to three weeks of culture were
63
required before the cells were able to respond to mechanical stimuli. In the gel cultures,
the two to three week culture period was necessary in order for the cells to deposit an
extracellular matrix around the individual cells. In the CG scaffolds, the cells were able
to attach directly to the pre-fabricated scaffold, but at early time points, at least, the cells
were not completely surrounded by an extracellular matrix. The ability of the day 2
cultures to respond to compressive loading, therefore, suggests that establishing cellmatrix attachment may be sufficient and the cell need not be completely surrounded by
newly synthesized extracellular matrix. Therefore, this system may also be useful for
studying the mechanisms by which mechanical stimuli are transmitted to the cell (i.e.,
cell deformation, fluid flow, nutrient and metabolite transport, etc).
Finally, the effects of mechanical loading on the cultured chondrocytes in the CG
scaffolds are likely not limited to in vitro conditions. The effects of mechanical loading
after implantation in vivo should also be considered, and appropriate post-operative care
(i.e. continuous passive motion) will likely be important.
64
CHAPTER 4: REPAIR OF CANINE CHONDRAL
DEFECTS IMPLANTED WITH AUTOLOGOUS
CHONDROCYTE-SEEDED TYPE II COLLAGEN
SCAFFOLDS
4.1.
INTRODUCTION
There are two basic approaches to cartilage tissue engineering: (1) implantation of
cells [Breinan et al., 1997; Brittberg et al., 1994; Brittberg et al., 1996; Chu et al., 1997]
and/or scaffolds [Breinan et al., 2000; Coutts et al., 1994; Frenkel et al., 1997; Nehrer et
al., 1998b; Rich et al., 1994] to promote regeneration in vivo; or (2) in vitro formation of
cartilaginous tissue for subsequent implantation [Fortier et al., 1999; Freed et al., 1998;
Freed et al., 1993; Toolan et al., 1996]. The direction of this research aims to combine
the two approaches through the development of an appropriate CG scaffold and optimal
in vitro culture conditions to prepare a cell-seeded CG scaffold prior to implantation to
promote in vivo cartilage regeneration.
A canine model has been developed in our laboratory by Breinan, et al., to
evaluate in vivo repair of full thickness chondral (i.e., not penetrating the underlying
calcified cartilage or subchondral bone) [Breinan et al., 1998]. This animal model has
been used to evaluate various surgical treatments including implantation of autologous
cultured chondrocytes (the ACI treatment described by Brittberg, et al., [Brittberg et al.,
1994]) [Breinan et al., 1997], implantation of non-cell-seeded type II CG scaffolds
[Breinan et al., 2000], and implantation of chondrocyte-seeded type II CG scaffolds
[Breinan et al., 2000].
The cell-seeded CG scaffolds were seeded into the scaffolds
within a 12 hour-period prior to implantation. Other investigations using chondrocyteseeded collagen gels have suggested that culturing the cells in the scaffold prior to
implantation may better prepare the implant for in vivo mechanical loading [Kawamura et
al., 1998]. Indeed, the compressive stiffness of the unseeded scaffolds (up to 1100 Pa
[Chapter 2]) is much lower than that of normal adult canine articular cartilage (-1 MPa
[Athanasiou et al., 1991; Hale et al., 1993; Jurvelin et al., 1987; Lee et al., 2000b; Setton
et al., 1994]).
65
The purpose of this experiment was to test the hypothesis that in vitro culture of a
cell-seeded scaffold prior to implantation would improve the healing of canine chondral
defects. This experiment employed a previously established canine model for articular
cartilage repair in which defects were surgically created down to, but not through, the
calcified cartilage [Breinan et al., 2000; Breinan et al., 1998; Nehrer et al., 1998b]. Prior
work with this model has indicated that the majority of the reparative tissue forms by 15
weeks post-surgery [Breinan et al., 2001].
Therefore, this single time point was
evaluated in the present study, as it was in previous evaluations of several treatment
modalities [Breinan et al., 2001; Breinan et al., 2000; Nehrer et al., 1998b].
A
histomorphometric method was employed to evaluate the percentages of specific tissue
types in the defects, and indentation testing was performed to determine selected
mechanical properties of the reparative tissue. The cell-seeded construct evaluated was a
type II CG, EDAC cross-linked scaffold, similar to the type I CG scaffold described in
Chapter 2.
4.2.
MATERIALS AND METHODS
4.2.1. Animal Model
Six male skeletally mature outbred hound dogs (ages 1.5-3 years) weighing
approximately 25 kg were used. The animal experiment was approved by the Veterans
Prior to surgery, the knee joints were
Administration Animal Care Committee.
roentgenographically examined to exclude animals with degenerative joint disease or
other orthopaedic problems.
All operations were performed on the canine stifle (knee) joint with the animal
under general anesthesia and sterile conditions. The joint was opened by an anteromedial
approach and the patella was displaced laterally to expose the trochlea.
To obtain
autologous chondrocytes for the cell-seeded implants, articular cartilage shavings were
harvested from the trochlear ridges and trochlear notch of the left knee joint of each
animal seven weeks prior to the treatment of the right knee. Chondrocytes were isolated
from the shavings and cultured as described below.
Articular cartilage defects were created and treated in the right knee joint of each
animal. Two 4-mm-diameter defects were produced in the trochlear groove as previously
66
Figure 4.1. Surgical creation of chondral defects. (a) Two 4-mm diameter trochlear defects are
outlined with a dermal punch. Blood from the joint space is then wiped over the surface to make the
defect borders more pronounced. (b) Using a customized curette under loupe visualization, all of the
articular cartilage within the borders of the defect is removed, down to, but not penetrating the
underlying calcified cartilage. (c) Two trochlear defects ready for implantation of the cell-seeded CG
implants.
described [Breinan et al., 1997]. A 4-mm-diameter dermal punch was used to outline the
two defects (Figure 4.1a), placed approximately 1.25 cm and 2.25 cm proximal to the
intercondylar notch and slightly lateral or medial to the mid-line.
Using loupe
visualization, a customized curette was used to remove all non-calcified cartilage from
the defect (Figure 4.1b). An attempt was made to remove as much articular cartilage as
possible, so as not to leave residual articular cartilage in the defect, while preserving the
integrity of the calcified cartilage and subchondral plate (Figure 4.1c). In only one defect
was there evidence of small amounts of punctate bleeding from the base of the lesion
intraoperatively. The cell-seeded scaffold was then placed into the defect and sutured
into place using three to four 8-0 coated Vicryl sutures.
Before the joint was closed, bleeding vessels were cauterized and the patella
relocated.
The joint was sutured closed, and the knee was immobilized by external
fixation (IMEX Veterinary, Longview, TX, USA) for 10 days as described previously
[Breinan et al., 1997].
The animals were then allowed to ambulate normally until
67
sacrifice at 15 weeks. Upon sacrifice, the joint was opened and the defect sites were
photographed and grossly evaluated. The proximal defects (n=6) were prepared for
histological evaluation and the distal defects (n=6) were allocated for mechanical testing.
Based on an expected standard deviation of a=20% [Breinan et al., 2000; Breinan et al.,
1997] and a desire to detect differences of 33% with a power of 80% (P=0.20) and
c-0.05, a sample size of 5 was required.
Untreated defects were not included as controls because they have already been
extensively evaluated and reported in previous studies performed by our laboratory
[Breinan et al., 1997; Wang et al., 2000]. The prior and present studies employed the
same surgeon (H.-P. Hsu) and animal care facilities, and the same histological processing
and evaluation methods.
4.2.2. Type II Collagen Scaffolds
Type II CG scaffolds, prepared from porcine cartilage, were obtained as prefabricated sponges (Chondrocell; Geistlich Biomaterials, Wolhusen,
Switzerland).
Investigation of implants prepared from this type II collagen material and implanted into
canine defects has previously been reported [Breinan et al., 2000]. In the current work,
scaffolds were sterilized by gamma-irradiation by the manufacturer. Cylindrical disks, 9mm-diameter and 1.5-2 mm thick, were punched from the sheets of scaffold using a
corneal trephine (Katena Products, NJ, USA).
In order to increase the stiffness and
decrease degradation rate, disks were cross-linked for two hours in an aqueous solution of
14 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma) hydrochloride and 5.5
mM N-hydroxysuccinimide (Sigma), as described in Chapter 2.
4.2.3. Cell Culture and Preparation of Cell-Seeded Implants
Chondrocytes were isolated from harvested articular cartilage shavings and
cultured in monolayer as described in Chapter 2.
Third passage chondrocytes were resuspended in culture medium at a density of
2x10 6 cells/ml. The type II CG scaffolds were incubated with the cell suspension (0.5 ml
suspension/scaffold disk) for two hours with continuous rocking. Seeded scaffolds were
transferred to agarose-coated 12-well plates with one scaffold per well and 1 ml
medium/well. An additional 1 ml of medium was added the following day. Medium was
68
exchanged every other day. After 28 days of in vitro culture, two disks per animal were
trimmed to 4-mm-diameter using a dermal punch and transported to the operating room
in a sterile container for implantation.
Previous experiments with porous type I CG scaffolds determined that
chondrocyte-seeded carbodiimide-cross-linked constructs underwent approximately 30%
contraction over a 28-day culture period (Chapter 2). This contraction was attributed to
chondrocytes containing the contractile muscle actin isoform, ox-smooth muscle actin. To
accommodate this contraction, an initial 9-mm-diameter scaffold disk was seeded, and
then trimmed to the appropriate size (4-mm-diameter) prior to implantation. It was also
determined that in the type I CG constructs there was an approximate 5-fold increase in
cell number during the 28 day period for the EDAC cross-linked 9-mm-diameter disks
(Chapter 2).
4.2.4. Histomorphometry
Proximal defects were removed from the joint 15 weeks after implantation, fixed
in 10% neutral buffered formalin for at least 1 week and decalcified in a solution of
ethylenediamine tetraacetic acid (EDTA) for 4 weeks.
The decalcified samples were
embedded in paraffin and microtomed to sections 7 gm in thickness. The sections were
stained with hematoxylin and eosin, Safranin O/Fast Green, a monoclonal antibody for
type II collagen mouse IgG1 (CIICI; Developmental Studies Hybridoma Bank, Iowa), or
a monoclonal antibody for c-smooth muscle actin (Product No. A-2547, Clone 1A4,
Monoclonal Anti-cL Smooth Muscle Actin, Sigma) using previously reported protocols
[Breinan et al., 1997; Kinner and Spector, In press; Wang et al., 2000].
A quantitative histomorphometric analysis [Breinan et al., 1997; Wang et al.,
2000] of specific tissue types was used to evaluate the tissue filling the defects. The
inter-observer error associated with this quantitative histological method was evaluated in
a recent study [Breinan et al., 2001]. One histological section from the center portion of
each defect, stained with hematoxylin and eosin, was evaluated. Digital micrographs of
the defect and surrounding tissue were recorded. The total area of the tissue and the
percentages of the specific tissue types (hyaline cartilage, fibrocartilage, and fibrous
tissue) filling the original defect region were measured using ImageJ (NIH). Classical
criteria relating to the morphology of the cells and the presence or absence of lacunae and
69
the composition and structure of the extracellular matrix were used to distinguish these
tissue types, as has previously been reported [Breinan et al., 1997; Wang et al., 2000]. In
brief, tissue was classified as hyaline cartilage if the cells were in well-defined lacunae
and there was no fibrous appearance to the extracellular matrix.
Normally, hyaline
cartilage exhibits positive Safranin-O staining, however, in this and other studies to
which these results are compared [Breinan et al., 2000; Breinan et al., 1997], tissue
lacking Safranin-O staining but meeting the other criteria for hyaline cartilage was
classified as hyaline. Fibrocartilage was identified as having rounded cells in lacunae but
with a distinct fibrous appearance to the matrix. Cells in fibrous tissue were elongated,
with no lacunae, and were surrounded by an extracellular matrix with a distinct fibrous
appearance.
The linear percentage of the calcified cartilage layer that was intact and the linear
percentage of the reparative tissue continuous with the calcified cartilage and adjacent
articular cartilage at the edges of the defect were also measured [Breinan et al., 1997].
4.2.5. Mechanical Testing
Indentation testing of the mechanical properties of the reparative tissue was
performed on the distal implantation sites. This method was selected because of the
anticipated limited thickness of the reparative tissue (less than 1 mm) and the difficulty of
removing the tissue for confined or unconfined compression testing, without disturbing
its structure and previous experience with indentation testing of cartilage from the canine
model [Lee et al., 2000b].
After the proximal defect was removed for histomorphometry at the time of
sacrifice, the remainder of the joint was wrapped in saline soaked gauze and packed on
ice. A 9.5-mm-internal-diameter coring bit, fixed to a standard drill press, was used to
remove an osteochondral core containing the repair tissue filling the distal defect, the
surrounding articular cartilage, and approximately 10 mm of subchondral bone. The
bony
portion
of the
osteochondral
core
was
then
mounted
in
self-curing
polymethylmethacrylate (Quickmount; Fulton Metallurgical Products, Saxonburg, PA,
USA).
The cartilage surface was kept moist with phosphate-buffered saline (PBS)
throughout the coring and mounting process.
70
The
test
specimen
was
then
mounted
in
a
chamber
with
five
rotational/translational degrees of freedom. The multi-axial chamber was clamped into
the lower jaw of a Dynastat mechanical spectrometer (IMASS, Hingham, MA, USA) and
used to position the testing site (center of original defect) perpendicular to the axis of the
indenter.
A 1-mm-diameter cylindrical, plane-ended polymethylmethacrylate indenter
was mounted into the upper jaw of the Dynastat.
Once the specimen was in place, it was immersed in PBS with EDTA and allowed
to regain any fluid lost during the mounting process. The indenter was lowered to contact
the specimen and three successive computer-controlled ramp-and-hold displacements
were applied to achieve approximately 10%, 15% and 20% strain. Tissue thickness was
estimated based on the thickness measured in the distal portion of the trochlear groove in
unoperated canine joints (animals were the same breed and approximately the same age)
[Lee et al., 2000b].
After each ramp-and-hold displacement, stress relaxation was
achieved within 5 minutes and the resulting equilibrium load was recorded and
normalized to indenter area to calculate effective stress.
In addition, sinusoidal
displacements corresponding to approximately 1% strain amplitude at frequencies of
0.01, 0.1, and 1.0 Hz were superimposed on the 15% static strain, and the resulting
dynamic loads were recorded. All mechanical testing was completed within six hours of
sacrifice.
Due to the lack of a well-defined tidemark and resorption of the subchondral
bone, it was not possible to calculate the applied strain (applied displacements
normalized to tissue thickness).
The displacements were therefore normalized to the
average thickness (0.5 ± 0.05 mm) of the original defect. The slope of the linear portion
of the resulting curve of effective equilibrium stress versus effective strain was taken to
be the equilibrium stiffness of the tissue. The dynamic stiffness was normalized to the
stiffness at the static offset (approximately 15% strain).
The same anatomical site in each animal's contralateral (left) knee joint was
similarly tested and the resulting equilibrium and dynamic stiffnesses were used as
control values corresponding to normal articular cartilage.
71
4.3.
RESULTS
4.3.1. Histology and Immunohistochemistry of Cell-Seeded Scaffolds
Histological examination of cell-seeded scaffolds similar to those implanted in the
cartilage defects showed that, while cells could be found distributed throughout the
construct, the majority of the cells were in a cell-continuous layer (8-10 cells thick)
around the periphery of the construct after 28 days in culture (Figure 4.2a).
Immunohistochemical staining of these cell-seeded constructs indicated the presence of
ct-smooth muscle actin in the cytoplasm of many of the cells (estimated to be up to 20%
of the cells in the center of the disk; Figure 4.2b).
Figure 4.2. Light micrographs of cell-seeded type 11 collagen matrices cultured for four weeks.
These matrices, similar to the constructs implanted into canine chondral defects, were stained for asmooth muscle actin (SMA; positive staining indicated by reddish-brown cytoplasm). (a) There was a
cell-continuous layer (8-10 cells thick) around the periphery of the construct. (b) A lower density of
cells was found in the center of the matrix. Approximately 20% of these cells stained positive for
SMA (indicated by arrows).
4.3.2. General Gross and Histological Observations
At the time of sacrifice all animals were ambulating normally although one
animal seemed to be favoring its left (harvest) leg. Upon opening of the joint, there were
no visible joint abnormalities in this animal. Another animal had slight inflammation
around the patella in both the right and left knee joints. A third animal appeared to have
softened articular cartilage and subchondral bone in the right knee joint. All other joints
appeared normal.
The harvest sites in the left joints were visible with the naked eye and had
undergone various degrees of repair (Figure 4.3a). The borders of all trochlear defects in
the right knee joints were also visible with the naked eye (Figure 4.3b). Additionally,
72
Figure 4.3. Gross appearance of joint surfaces at necropsy. (a) Harvest sites along trochlear ridges
were still visible 22 weeks post-harvest surgery. (b) Defect borders and suture tracts were visible 15
weeks post-implantation.
there were visible suture tracts around most of the defect sites. There were no gross signs
of residual type II collagen implant. Gross observations at the time of sacrifice revealed
that all 12 defects were at least half-filled with reparative tissue and 10 of the 12 defects
were judged to be at least 90% filled, including one of which had filled to a level above
the surface of the adjacent cartilage. Upon gentle probing, it was apparent that the repair
tissue was much softer than the surrounding articular cartilage.
One defect was essentially empty upon histological evaluation (Figure 4.4a).
Gross examination at the time of sacrifice, however, revealed that this defect was
completely filled with reparative tissue. This suggested that the tissue must have been so
poorly integrated with the host site that just the slight mechanical perturbation of the
tissue during post-mortem handling and histological processing resulted in its
dislodgment.
Histologically, only one defect showed signs of residual implanted collagen
scaffold (Figure 4.4b). As in previous studies [Breinan et al., 2001; Breinan et al., 1997],
the suture tracts created when suturing the implants into the defects were still visible in
histological sections. Moreover, a routine finding of decreased Safranin-O staining of the
articular cartilage near the edges of the defect indicated a depletion of matrix
proteoglycans.
73
The majority of the tissue filling the proximal defects was fibrocartilage and
hyaline cartilage (Figure 4.4c). Hyaline cartilage generally appeared at the edges of the
defects and fibrocartilage was found in the center of the defects. In one defect there was
a significant amount of fibrous tissue that appeared in the center of the defect and
transitioned to fibrocartilage and hyaline cartilage moving towards the host articular
cartilage (Figure 4.4d).
The majority of the tissue classified as hyaline cartilage on the hematoxylin and
eosin-stained
sections
contained
type
II
collagen
as
demonstrated
by
immunohistochemical staining of adjacent sections (Figure 4.4e). There was substantial
Safranin-O staining of the reparative tissue filling one of the defects (Figure 4.4f).
4.3.3. Histomorphometric Evaluation of Reparative Tissue
Excluding the one defect that was empty at the time of histological observation,
the total amount of tissue filling the defect was 88 ± 6% (mean ± SEM; n=5; range, 70100%) of the original defect.
Hyaline cartilage constituted 42 ± 10% of the original
cross-sectional area of the lesion (range, 7-67%), and fibrocartilage most of the balance,
52 ± 11% (range, 33-93%). Only a small amount of fibrous tissue was recorded (5 ± 5%;
range, 0-26%).
In the one defect that was empty, the calcified cartilage was intact along virtually
the entire length of the base of the defect (Figure 4.4a). In contrast, 78 ± 10% (range, 4090%) of the calcified cartilage and subchondral bone in the other defects was disrupted
(Figures 4.3 b and f). In those defects, 64 ± 12% (range, 30-90%) of the reparative tissue
was continuous with the underlying remodeling subchondral bone. With the exception of
the empty defect and one edge of another defect, the repair tissue was continuous with the
adjacent articular cartilage over at least part of the defect border (Figures 4.3 c and f).
74
Figure 4.4. Histological sections from the center of repair tissue filling the canine chondral
defects 15 weeks after implantation (original magnification I0x). (a)-(d) Hematoxylin and eosin
(H&E) staining. (a) The one defect that was empty upon histological examination had an intact
subchondral plate. Hyaline appearance of normal articular cartilage is seen in the tissue to the left of
the defect. (b) One defect showed signs of residual implanted matrix near the center of the defect
(indicated by arrows). (c) Most defects were filled predominately with hyaline (HC) and
fibrocartilage (FC). Arrowhead indicates suture tract that did not heal. Host articular cartilage (dark
purple tissue on the left side of the micrograph) in this defect was continuous with the repair tissue.
(d) Fibrous tissue (FT), when present, was found near the center of the defect with fibrocartilage (FC)
and hyaline cartilage towards the edge of the defect. (e) Type II collagen immunohistochemical
staining was positive in most of the tissue classified as hyaline cartilage from the H&E staining, as
shown in this sample taken from a region near the edge of a defect (edge just to right of this
micrograph). (f) Safranin-O/Fast-Green staining revealed that repair tissue in only one defect.
4.3.4. Mechanical Properties of Repair Tissue
The equilibrium stiffness of the repair tissue could not be related to an
equilibrium Young's modulus due to the disruption of the underlying subchondral bone.
Therefore, the stress-strain stiffnesses for the control and repair tissues are reported here.
The equilibrium stiffness of the control articular cartilage in the left knee was 4.8 ± 0.8
MPa. This value is consistent with previous values for the same site in the same animal
model with a reported Young's modulus of 3.7 ± 0.7 MPa [Lee et al., 2000b].
As expected, based on qualitative observations made at the time of sacrifice, the
repair tissue filling the distal defects had significantly lower equilibrium and dynamic
stiffnesses than the articular cartilage at the same anatomical site in the contralateral (left)
joint (Figure 4.5, Student's t-test, p<0.0001). The equilibrium stiffness of the articular
cartilage in the left joint was more than six-fold higher than that of the repair tissue.
Moreover, the dynamic stiffness of the cartilage was more than 20-fold higher than that
measured in the repair tissue.
80 u
6-
E]
5
4 -60
Repair
70 2
-
AC70e
50
S-40
y
a))
-0W
30c
22
-
1
10
Cl)
-
0
0
0.1 Hz Dynamic
Stiffness
Equilibrium
Figure 4.5. Indentation stiffness of repair tissue and articular cartilage. Repair tissue from
chondral defects had equilibrium and dynamic stiffness that was only a fraction of that measured for
articular cartilage at the same anatomical position in the contralateral joint (p<0.0001).
76
4.4.
DISCUSSION
In order to minimize the number of animals that had to be sacrificed for this
study, the histomorphometric results of this study were compared to results from previous
studies using the same animal model and performed by the same surgeon. The amount of
reparative tissue filling the defects implanted with the cell-seeded scaffolds in the current
study (88 ± 6%) was considerably greater than the amount of tissue (34 ± 7%) found in
empty (untreated) defects evaluated previously using the same model after a comparable
implantation period (Figure 4.6) [Breinan et al., 1997; Nehrer et al., 1998b].
This
indicates that the cell-seeded scaffolds remained in the defects for a sufficient amount of
time to meaningfully affect the reparative process. Prior studies also demonstrated that
implantation of cultured autologous chondrocytes alone increased the amount of
reparative tissue filling the defect and significantly increased the percentage of hyaline
cartilage when compared to the untreated defects [Breinan et al., 2001] (Figure 4.6).
Subsequent work demonstrated that the percentage of reparative tissue filling the defect
could be meaningfully increased (by 75%) by employing chondrocyte-seeded type II
collagen scaffolds [Breinan et al., 2000] (Figure 4.6). The predominant tissue, however,
was fibrocartilage rather than hyaline cartilage as seen in this study (Figure 4.6).
In the previous investigation [Breinan et al., 2000], the cell-seeded collagen
scaffolds were implanted within twelve hours of being seeded with cells [Breinan et al.,
2000; Nehrer et al., 1998b].
It was subsequently hypothesized that allowing the
constructs to enhance their ultrastructure and mechanical properties through the
deposition of a more extensive extracellular matrix during in vitro culture could further
improve in vivo repair. In the present study, cell-seeded constructs were implanted after
four weeks of in vitro culture in order to allow for the accumulation of cell-associated
matrix within the porous scaffolds. Compared to the histomorphometric results of the
previous study [Breinan et al., 2000] (Figure 4.6), which used the same animal model,
surgeon, and animal care facilities, implantation of the four-week cultured constructs led
to a significant increase the amount of hyaline-like tissue (51 ± 12% here versus 1 ± 0.2%
in the previous study; p<0.001) while also tending to increase the total amount of fill (88
± 6% vs. 71 ± 16%; p=0.28) and decreasing the amount of fibrous tissue filling the
defects (5 ± 5% vs. 15 ± 7%; p=0.13).
77
100%cU
90%. 1 hyaline
El fibrocartilage
*
80%-
8
prior work in
same model
fibrous
70%60%-
o
50%-
40%.
30%-
o
20%-
10%-
empty
CAC
CAC+Il
cultured
CAC+II
Figure 4.6. Histomorphometric comparison of repair tissues filling defects subjected to various
treatments. The four week in vitro culture of the construct prior to implantation increased the total
amount of fill and the percentage of the repair tissue that was hyaline-like, while decreasing the
amount of fibrous tissue. Empty, CAC (cultured autologous chondrocytes implanted without a
matrix), and CAC + II (CAC seeded into a type II collagen matrix just before implantation) treatments
were performed on chondral defects created in the same manner and by the same animal surgeon (H.P. Hsu) responsible for this study (cultured CAC + II).
With the exception of one defect (Figure 4.4b), there was no gross or histological
evidence of the implanted collagen scaffold.
It is known that degradation rate is an
important variable in the design of scaffolds for regeneration of other tissues [Yannas,
1992], so it is possible that different cross-linking treatments may yield better results in
this model relative to the amount and type of tissue in defects in the articular surface.
Care was taken to not disrupt the calcified cartilage and underlying subchondral
bone during the creation and treatment of the defects. Bleeding was only observed in one
defect and was stopped prior to the implantation of the cell-seeded construct. At the time
of sacrifice, however, the calcified cartilage and subchondral bone had been disrupted in
the majority of the defects and the repair tissue was continuous with the remodeling bone.
These findings are consistent with our previous reports using a similar collagen scaffold
implanted into the same animal model [Breinan et al., 2000; Nehrer et al., 1998b].
It is
therefore unlikely that the different cross-linking treatment (carbodiimide used here
versus UV used in the previous study) or the in vitro culture prior to implantation led to
the bony resorption.
Implantation of cells alone [Breinan et al., 2001; Breinan et al.,
78
2000; Nehrer et al., 1998b] does not lead to as significant a disruption of the tidemark,
indicating that the scaffold contributes to bony resorption in some way.
Possible
pathways include a direct bone cell reaction to the scaffold or to degradation products of
the scaffold or bone cell regulation by factors released by other cells (i.e. synovial cells or
chondrocytes) in response to the scaffold. Additional studies are necessary to determine
what triggers remodeling and the extent to which the tidemark will reform. While the
continuity of the reparative tissue with the underlying bone is useful in enhancing
attachment and retention of the repair tissue, the extent to which and rate at which the
tidemark reforms will likely be an important parameter in terms of the ability of the
repair tissue to remodel to a suitable cartilaginous material capable of bearing the applied
mechanical loads.
It is unknown how the metabolic activity of the cells changed after implantation
of the constructs into the cartilage defects. In a separate experiment, it was found that the
biosynthetic and proliferative activity of chondrocytes cultured in a similar scaffold had
slowed down by four weeks of in vitro culture (Chapter 2). While implantation at an
earlier time period would have meant that the constructs would have had a less dense
extracellular matrix, the cells would have had a higher biosynthetic rate that may have led
to better repair. Additionally, the in vivo chemical and mechanical environment can
influence the type of matrix molecules that are synthesized as well as the architecture of
the extracellular matrix.
Further studies are needed to address the optimal in vitro
culture period prior to implantation of the scaffolds.
Constructs were cultured in vitro under free-swelling conditions (i.e. no
mechanical shear, compressive or other physical forces were applied) with media
supplemented only with 10% fetal bovine serum. Other researchers have reported on the
beneficial effects of shear [Freed et al., 1998; Martin et al., 2000] and compressive forces
[Davisson et al., 2001; Mauck et al., 2000] on the in vitro culture of constructs for
articular cartilage tissue engineering. It has been demonstrated that compressive forces
influence the biosynthetic activity of passaged chondrocytes in scaffolds similar to those
used in this study (Chapter 3), but additional studies are necessary to determine the
optimal loading protocols.
79
Furthermore, it is known that various soluble regulators can affect the
chondrocyte phenotype and metabolic activities. Future studies utilizing this model may
therefore also include treatment of the cell-seeded constructs with growth factors in vitro,
injection or attachment to the scaffold of the growth factor for in vivo dosage, or use
incorporation of genes into the collagen scaffold [Bonadio et al., 1999] for in situ
transfection and production of the desired proteins.
Due to bone resorption under the cartilage defects, the mechanical properties of
the repair tissue reported are dependent on the test geometry. It was not possible to
accurately normalize to the tissue thickness, nor was it possible to apply traditional
models of indentation testing of articular cartilage to determine material properties.
Nonetheless, it was obvious that the mechanical properties of the repair tissue were far
inferior to that of normal articular cartilage.
Another problem with the indentation test used in this study was the damage that
it caused to the repair tissue. An attempt was made to preserve the distal defect repair
tissue for histological analysis after indentation testing. Two of the six defects, however,
were severely damaged during testing.
Histological sections of another two defects
revealed severe disruption of the repair tissue where the indenter contacted the surface of
the repair tissue. Future studies seeking to evaluate the mechanical properties of cartilage
repair tissue should take into consideration these limitations.
80
CHAPTER 5: FABRICATION OF GENE-SEEDED COLLAGENGLYCOSAMINOGLYCAN SCAFFOLDS
5.1.
INTRODUCTION
The repair of defects in adult articular cartilage may be improved by the administration of
certain therapeutic factors. Selected growth factors, given as a single bolus dose at the beginning
of the cartilage repair process, have been shown to accelerate the production of a hyaline-like
reparative cartilage matrix [O'Connor et al., 2000]. None of these cytokines, however, have been
able to maintain their effectiveness during the remodeling phase that ensues a few weeks to
months after the initial repair procedure. The limited efficacy of the bolus dosing of growth
factors may be due to its inherent inability to maintain therapeutic levels of the cytokine for
prolonged periods. The transitory effects of bolus dosing of polypeptide growth factors are a
consequence of the relatively short in vivo half-life of protein molecules (minutes to hours), the
temporal nature of growth factor signaling on cellular differentiation and metabolic function, and
the fact that the exogenous cytokine does not likely stimulate endogenous production.
Transfer of the gene for a selected cytokine to the cells involved in the reparative process
is one means of maintaining therapeutic levels of the protein through the later phases of the
cartilage repair process [Smith et al., 2000]. Non-viral vector systems offer the advantages of
low level of immune response, simplicity of vector design, and relative ease of large-scale
production [Cohen et al., 2000]. The disadvantage of this approach is normally related to the
lower efficiency of transfection. However, in the case of the reparative process even relatively
small amounts of the cytokine produced by a few transfected cells may be of significant value.
This approach has provided promising results in recent studies directed toward enhancing bone
regeneration using polymeric scaffolds as a carrier for selected genes [Bonadio et al., 1999; Fang
et al., 1996; Sato et al., 2001].
There are two principal advantages in the use of such a scaffold as a carrier for selected
genes. First, the attachment of the DNA to the insoluble scaffold to which cells can also attach
allows for the sustained, local presence of the genetic material in close proximity to the high
concentration of DNA. Second, the use of a degradable material, such as collagen [Bonadio et
al., 1999; Fang et al., 1996] or chitosan [Sato et al., 2001], could allow for prolonged release of
the genetic material as the scaffold degrades. This would allow for continued transfection of
81
local cells over a longer period of time, rather than a single transfection time as in the case of in
vitro transfection prior to implantation. The rate of release could be controlled by the extent of
cross-linking employed in fabricating the collagen scaffold. Prolonged release (over several
weeks or months) of DNA from an implant is necessary in cases where there is a benefit in
transfecting selected cells that do not appear at the implant site until days or weeks
postoperatively, and in which there is a premature loss of expression in transfected cells or in
which transfected cells migrate from the defect site.
The objective of this experiment was to test the hypothesis that CG scaffolds could be
modified to deliver genetic material to chondrocytes in situ.
With the goal of providing
prolonged therapeutic levels of growth factors, we sought to fabricate a novel collagenglycosaminoglycan (CG) scaffold for impregnation with a plasmid DNA vector. The design
objectives were to manufacture a dry gene-supplemented collagen-GAG (GSCG) complex to
have a long shelf-life, for molding into any shape, and for delivery of plasmid DNA to transfect
repair cells over a period of several weeks, when implanted into an articular cartilage defect.
The specific aims of this in vitro study were to (1) determine the release kinetic profiles of
plasmid DNA from the GSCG scaffolds, and (2) determine the ability of the released plasmid
DNA to transfect target cells.
To achieve these aims, we fabricated GSCG scaffolds using
different cross-linking methods, in order to obtain scaffolds with different degradation profiles,
and using buffers at different pH in order to vary the affinity of the CG scaffold for the plasmid
DNA. In this report of the initial in vitro characterization of several formulations of our GSCG
scaffolds, we show that the GSCG scaffolds are able to provide sustained release of the plasmid
DNA and continually transfect canine articular chondrocytes, providing sustained production of
the gene product for up to eight weeks.
5.2.
MATERIALS AND METHODS
5.2.1. Preparation of Collagen-GAG Scaffolds
Collagen-GAG scaffolds were fabricated using a modification of a previously described
protocol [Yannas et al., 1989]. One-milliliter aliquots of a suspension of microfibrillar type I
collagen (5.0 mg/ml bovine tendon collagen; Integra LifeSciences, Plainsboro, NJ) and
chondroitin-6-sulfate (2.6 mg/ml; Sigma Chemical, St. Louis, MO) in 0.05 M acetic acid, pH 2.5,
were freeze-dried in 24-well tissue culture plates. The scaffolds disks were then cross-linked by
82
one of the following methods: 1) 24 hours of dehydrothermal (DHT) treatment; 2) exposure to
ultraviolet (UV) light for 24 hours; and 3) 2 hour immersion in l-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide (EDAC; Fluka Chemie, Milwaukee, WI).
These cross-linking
methods were similar to those described in Chapter 2, although these scaffolds were not
subjected to DHT cross-linking prior to UV and EDAC treatment. Non-cross-linked scaffolds
were also employed in the study.
5.2.2. Addition of Plasmid DNA to Preformed CG scaffolds
The circular double-stranded plasmid DNA used in this study carried the firefly luciferase
reporter gene driven by the CMV promoter.
The plasmid DNA stock solution in Tris-
HCl/EDTA (TE) buffer, pH 7.5 was diluted either in TE buffer, pH 7.5, or in 0.05 M acetic acid,
pH 2.5, to a concentration of 125 or 250 pg/ml. Individual scaffolds, prepared as described
above, were placed in 24-well tissue culture plates and incubated at room temperature in a 1 ml
aliquot of the diluted plasmid DNA solution for 24 hours. Control scaffolds were produced by
addition of buffer solution without plasmid DNA.
The gene supplemented collagen-GAG
(GSCG) complex was then freeze-dried using the same protocol employed to produce the
original CG scaffolds. The dry GSCG scaffolds were stored in a desiccator until use.
5.2.3. Electron Microscopy of the CG Scaffolds
The morphology of control and GSCG scaffolds was examined by both scanning electron
microscopy (SEM) and transmission electron microscopy (TEM). SEM images were obtained
on a JEOL JSM-6320FV microscope at 1.0 KV after sputter coating a 5-nm layer of gold
palladium on the surface of the scaffolds.
Samples allocated for TEM were post-fixed in
osmium tetroxide and embedded in Epon. TEM images of ultra-thin sections (approximately
100nm) of scaffolds were obtained on a JEOL 1200EX microscope.
5.2.4. Plasmid DNA Content of GSCG Scaffolds
To determine the initial amount of plasmid DNA loaded into the scaffolds under each
cross-linking and pH condition, six GSCG scaffolds from each formulation (250 gg/disk) were
digested by papain (50 gg/ml in 0.1 M sodium phosphate, 5 mM sodium EDTA, and 5 mM
cysteine-HCl, pH 6.2) in a 65'C water bath.
The concentration of DNA in the digest was
determined by a fluorometric assay using the Hoescht 33258 dye (Polysciences, Warrington, PA)
83
with a calf thymus DNA standard curve [Kim et al., 1988]. Control scaffolds prepared without
added plasmid were similarly digested and assayed to account for any background fluorescence.
5.2.5. Plasmid DNA Leaching Studies
The kinetics of release of the plasmid DNA from the GSCG scaffolds loaded with 250 pg
DNA were investigated by immersion of the specimens in 1 ml of TE buffer, pH 7.5 at room
temperature. The full 1 ml volume of TE buffer was removed and replaced at 30 minutes, 1
hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, and 28 days.
After leaching of plasmid DNA into TE buffer for 28 days, the scaffolds were digested by
papain. The concentration of plasmid DNA in the release buffer collected at the serial time points
and in the digested scaffolds were determined by the Hoescht 33258 dye assay. Scaffolds
prepared without plasmid were leached and digested as controls. The slopes of the log-log plots
of the cumulative DNA released versus time of leaching served as measures of the "early" and
"late" release rates (jig/hour). The release rates in Table 1 are given as pg/day.
5.2.6. Transfection of Canine Articular Chondrocytes in Gene-Supplemented CG Scaffolds
Canine articular chondrocytes were seeded into scaffolds loaded with either 250 or 125
jg DNA/scaffold disk or into control scaffolds (no DNA) as described in Chapter 2.
For
scaffolds with 125 jg DNA/disk, only DHT and EDAC cross-linked scaffolds were used. In
brief, the scaffolds were rinsed in complete culture medium, immersed in a cell suspension (1 ml
culture medium containing 4 million chondrocytes), and then continuously mixed on a rocking
surface for 2 hours. Chondrocyte-seeded scaffolds were maintained in culture for 15 and 28 days
(250 pg DNA/disk) or 56 days (125 pg DNA/disk due to a limited supply of the plasmid) with
medium changed every 2-3 days. At the end of the culture periods, chondrocytes in both the
control and GSCG scaffolds were lysed by a freeze-thaw protocol previously described
[Manthorpe et al., 1993]. Cell extracts were stored at -20 *C until the luciferase activities were
assayed using the Promega Luciferase Assay Kit protocol (Promega, Madison, WI).
5.2.7. Stability of Chondrocyte Transfection
To determine the stability of chondrocyte transfection with the luciferase plasmid, the
luciferase activity of transfected chondrocytes was measured 2, 16, and 29 days posttransfection.
Third passage canine chondrocytes, cultured in monolayer until reaching 80%
confluence, were transfected with the stock luciferase plasmid vector using the Gene Porter
84
Transfection Kit (Gene Therapy Systems, San Diego, CA). Two days post-transfection, the cells
were collected by trypsinization and either lysed and luciferase activity measured as described
above, or seeded into CG scaffolds (not containing any plasmid). The seeded CG scaffolds were
cultured for an additional 14 or 27 days, at which time points the cells were lysed by the freezethaw protocol (see above) and luciferase activity determined.
5.3.
RESULTS
5.3.1. Morphology and Ultrastructure of the GSCG Scaffolds
The GSCG scaffolds treated by various cross-linking methods and prepared under the
different pH conditions displayed morphological features similar to CG scaffolds previously
described [Yannas et al., 1989]. The interconnecting pores were formed by thin strands of the
CG material and films of the substance (Figure 5.1a).
The average pore diameter of
approximately 100 pm was consistent with that previously reported for these types of CG
scaffolds [Nehrer et al., 1997b]. Except for the collapse of the pores in the 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide (EDAC)-cross-linked scaffolds, consistent with prior reports in the
literature [Pieper et al., 1999], there were no remarkable differences in the morphology of the
scaffolds cross-linked with the various treatment protocols.
The fibrous appearance (Figures 5.1 b-f) and the occasional banded collagen fibrils
(Figure 5.1f) of the CG scaffolds was also similar to that previously reported [Yannas et al.,
1989].
There were no noticeable differences in the ultrastructure of the GSCG scaffolds
prepared under the various conditions. Of particular note was the generalized deposition of a
fine filigree-like network on the surface of the GSCG scaffolds (Figures 5.1 b, d, and f). These
features were not seen in the CG scaffolds that were not treated with the plasmid DNA solution
(Figure 5.1 c and e). This presumed plasmid DNA layer extended 50 to 800 nm from the CG
surface (Figure 5.1d).
Infrequent globular deposits ranging in diameter of 2 to 5 gm were
observed, particularly in the pH 2.5 formulations (Figure 5.1b). The pores of the EDAC crosslinked formulations were often collapsed, with plasmid DNA entrapped within the narrowed
spaces.
85
Figure 5.1. Electron micrographs of gene-seeded collagen-GAG (GSCG) scaffolds. (a) Scanning
electron micrograph demonstrating the interconnecting pore structure. (b-f) Transmission electron
micrographs of CG scaffolds prepared at pH 2.5 (b,d: with plasmid; c,e: no plasmid control) and pH
7.5 (e: no plasmid control; f: with plasmid). Pore walls are comprised of a fine fibrillar form of
collagen with occasional portions of banded fibrils, displaying the quaternary structure of collagen (f).
Plasmid DNA appears as a fine filigree-like network (d and f) that was not seen on any of the control
scaffolds (c and e). Infrequent globular deposits were also seen (b).
86
5.3.2. Loading of Plasmid DNA
In the initial experiment, each dry GSCG scaffold was loaded with approximately 250 Rg
of plasmid DNA in 1.0 ml of either TE buffer or 0.05M acetic acid. Assay of papain digests of
the GSCG scaffolds digested without prior leaching revealed that different amounts of plasmid
DNA were bound to the surfaces (Figure 5.2). The measurement of more than 250 gg of plasmid
DNA in the digest of selected scaffolds reflected the variations in the amount of plasmid solution
added to the samples during their preparation. Near maximal amounts of plasmid DNA were
absorbed by the CG scaffolds that were non-cross-linked, dehydrothermal (DHT)-cross-linked,
In contrast, the CG scaffolds cross-linked by
and ultraviolet (UV)-cross-linked (Figure 5.2).
EDAC treatment were found to absorb only about 50% of the approximately 250 pg of plasmid
DNA that was added. Two-factor analysis of variance (ANOVA) revealed a significant effect of
the cross-linking treatment on the amount of plasmid DNA absorbed (p<0.001) but not of the pH
The processing pH was only found to have a
at which the samples were prepared (p>0.2).
meaningful effect on plasmid DNA absorption for the samples cross-linked by the DHT
treatment (Figure 5.2). In that case there was 30% less plasmid in the DHT-cross-linked sample
350*
300 -pH3
* pH7
250 S200150 10050 0
no xlink
DHT
UV
EDAC
Figure 5.2. Total DNA loaded into scaffolds. DNA content of papain digests of the non-leached
scaffolds initially loaded with 250 pg DNA/scaffold disk. DNA content varied by cross-linking and
pH of plasmid addition. EDAC cross-linked scaffolds absorbed only 50% of the loaded plasmid.
Plasmid load was significantly different for pH only in the DHT cross-linking. Mean ± standard error
of the mean (SEM); n=6. *p<0.05 compared to pH 2.5 preparation with same cross-linking. tp<0. 0 5
compared to other cross-linking methods.
87
processed at pH 7.5 when compared to the specimen treated at pH 2.5. Analyzing this treatment
group separately, this difference was statistically significant by Student's t test (p=0.05).
5.3.3. Release Kinetics
There were marked differences in the cumulative amounts of plasmid DNA released by
the various CSCG scaffolds through 28 days of leaching ("passive release").
For GSCG
scaffolds prepared at pH 2.5, the total amount of plasmid DNA recovered in the TE buffer
ranged from 78 ptg (EDAC-cross-linked) to 184 jig (non-cross-linked). At pH 7.5 the amount of
plasmid DNA released ranged from 126 pg for the EDAC-cross-linked to 331 itg for the UVcross-linked scaffolds. Three-factor ANOVA indicated significant effects of time, cross-linking
method, and pH on the amount of released DNA (p<0.001 for each).
To account for variations in the initial plasmid loading of the scaffolds, the cumulative
amount of DNA leached (Figure 5.3 a-b) and the amount of the DNA detected after digesting the
leached scaffolds were used to calculate the residual fraction of plasmid DNA bound to the
scaffold at each time point (Figure 5.3 c-d). The residual fraction of the total DNA varied among
the formulations.
Three-factor ANOVA indicated significant effects of time, cross-linking
method, and pH on the fraction of residual DNA (p<0.001 for each). In general, there was a
marked increase in the amount of DNA retained in the samples prepared at pH 2.5 compared to
those prepared at pH 7.5. After four weeks in TE buffer, the pH 7.5 preparations retained 1%
(UV), 2% (EDAC), 4% (DHT), and 27% (non-) cross-linked plasmid DNA (Figure 5.4b). With
the exception of the non-cross-linked groups, significantly higher amounts of DNA were retained
by the samples prepared at pH 2.5: 28% (UV-), 13% (EDAC-), 32% (DHT-), and 19% (non-)
cross-linked (Figure 5.3 c-d).
Only 28 ± 11% (mean ± SEM) of the total amount of plasmid DNA released during the
28 day incubation period occurred during the late release phase. The majority (72 ± 11 %) of the
plasmid DNA load was released during the initial 4 hours of incubation in the buffer. The
biphasic pattern of plasmid DNA release was reflected in the kinetic curves (Figures 5.4; Table
5-1). All preparations exhibited an initial ("early") rapid release over the first 4 hours followed
by a "late" slower release phase extending from 4 hours to 28 days of incubation in TE buffer,
pH 7.5 at room temperature.
The plasmid DNA release rate of the pH 2.5 GSCG scaffolds
showed a 2-fold difference between the early (4.0 ± 1.0 Itg/day; mean ± SEM) and late (2.0 ± 0.5
pg/day) phases. In contrast, there was a 7-fold difference between the early (7.0 ± 1.4 ptg/day)
88
and late (0.9± 0.2 jyg/day) phases in the plasmid DNA release rate of the pH 7.5 GSCG scaffolds.
Three-factor ANOVA again indicated significant effects of time (early or late phase), crosslinking method, and pH on the release rates (p<0.001 for each).
0.80.61
A
0.4
0
~rh
-
0.20
0
I
I
5
10
I
15
I
20
I
25
3
30
a
0.84
II
4
0
0.6
0
0.41
.....---
0.2
0
-A-
0
b
I
5
I
10
15
20
25
30
Time (days)
Figure 5.3. DNA leaching profiles. Non-cell-seeded scaffolds were incubated in Tris-EDTA (TE,
pH 7.5) buffer at room temperature for a four week period (30 minutes to 28 days). Buffer was
exchanged periodically and assayed for DNA content to determine the amount of plasmid released
from non- (0), DHT (o), UV (A), and EDAC (o) cross-linked scaffolds modified by the addition of
250 pg plasmid at either (a) acidic (0.05M acetic acid, pH 2.5) or (b) neutral (TE buffer, pH 7.5). The
residual fraction of the original amount of plasmid loaded into the scaffolds was affected by crosslinking (p<0.0001) and pH (p<0.0001). Mean ± SEM; n=6.
89
2.4
-
a: pH 2.5
* No X-linkO DHT A UV O EDAC
2.3 2.2
-
2.1
-
2 -
z
1.9
-
1.8 1.7 1.6
-
1.5 -0.5
2.7
0
0.5
1
1.5
2
2.5
3
-
b: pH 7.5
+No X-link E DHT A UV e EDAC
2.5-
A-k
~--A
A
-A-A
2.3-
z
2.1-
1.9
1.7 -
1.5
-0.5
0
0.5
1
1.5
2
2.5
3
log Time (hr)
Figure 5.4. DNA release rate. Logarithmic plots were used to determine the rate of DNA release
from the scaffolds. All scaffolds prepared at (a) pH 2.5 and (b) pH 7.5 had initial (early) rates of
release that were higher than subsequent (late) rates. Early and late release rates (slopes from the loglog plot) are tabulated in Table 3.1.
90
Table 5-1. Plasmid DNA release rates. Mean values for the "early" and "late" plasmid DNA
release rates. Release rates (tg/day) are the slopes of the linear fits to log-log plots of cumulative
release vs. time (Figure 5.5; all R2 > 0.9).
GROUP
Non-x-link
DHT
UV
EDAC
Mean±SEM
pH 2.5
"Early"
"Late"
Rate,
Rate,
pg/day
pg/day
6.6
1.3
2.7
1.7
4.9
3.5
2.1
1.4
4.0±1.0
2.0±0.5
pH 7.5
"Early"
"Late"
Rate,
Rate,
pg/day
gday
7.2
1.3
9.6
0.8
8.0
0.5
3.3
1.1
7.0±1.4
0.9±0.2
5.3.4. In Situ Transfection of Chondrocytes Seeded into GSCG Scaffolds
Three-factor ANOVA revealed significant effects of cross-link method, pH, and time on
the luciferase expression by the seeded chondrocytes (all p<0.02). The chondrocytes seeded in
all of the GSCG scaffolds prepared at pH 2.5 and the non- and DHT-cross-linked GSCG material
prepared at pH 7.5 demonstrated measurable but relatively low levels of luciferase gene
expression after 14 days in culture (Figure 5.4a). There were 5- to 7-fold higher levels of
luciferase gene expression in chondrocytes seeded in the UV-and EDAC-cross-linked scaffolds
prepared at pH 7.5. In the ensuing 14 days in culture there was a decrease in the expression of
luciferase in the pH 2.5 non-cross-linked, and the pH 7.5 UV- and EDAC-cross-linked scaffolds
(Figure 5.4b). In contrast, the cross-linked pH 2.5 scaffolds and the pH 7.5 non- and DHT-crosslinked scaffolds displayed at most only a minor change in their generally low luciferase gene
expression level over this latter 14-day period. After 28 days of culture in the scaffolds loaded
with 250 ptg DNA/disk, the highest level of luciferase gene expression was produced by
chondrocytes seeded into the pH 7.5 UV-cross-linked CG scaffolds (Figure 5.4b).
Only DHT- and EDAC-cross-linked scaffolds loaded with 125 gg DNA/disk were seeded
and cultured for 8 weeks (Figure 5.4c). All of these cultures exhibited low luciferase activity at
8 weeks. In the pH 2.5 preparations, there was higher activity in the DHT-cross-linked scaffolds,
whereas in the pH 7.5 preparations, there was a much higher activity in the EDAC-cross-linked
scaffolds. The luciferase activity in the DHT-cross-linked scaffolds was approximately the same
for the pH 2.5 and pH 7.5 groups (p=0.86), but in the EDAC pH 7.5 preparation the activity was
nearly 20-fold higher than in the pH 2.5 preparation.
91
2500-
..
a. Day 15, 250 gg/disk
2000-
O No
Xlink
E DHT
OUV
1500~
NEDAC
1000-
50001600
b. Day 28, 250 pg/disk
1200-
800 -
400-
T~
0
400
300-
I
I
T
c. Day 56, 125 pg/disk
*DHT
*EDAC
200-
100-
0pH 2.5
pH 7.5
Figure 5.5. In situ transfection of chondrocytes in GSCG scaffolds. Luciferase activity
(above background) of lysates from cell-seeded GSCG scaffolds (original plasmid load 125250 pg/scaffold disk) decreased over time, but showed sustained transfection for (a) 15, (b)
28, or (c) 56 days (note that scaffold cultured for 56 days were initially loaded with only 125
ptg of plasmid/disk). Mean ± SEM; n=4.
5.3.5. Stability of Chondrocyte Transfection
The luciferase activity of chondrocytes transfected prior to seeding into the CG scaffolds
was significantly reduced by 16 days post-transfection, indicating the transient nature of the
transfection. After two weeks in the CG scaffolds, the previously transfected chondrocytes had
less than 0.5% of the original luciferase activity. By four weeks post-transfection, there was less
than 0.001% of the original luciferase activity.
5.4.
DISCUSSION
A notable finding of this study was that selected CG scaffolds could be formulated to
provide for the prolonged (greater than 1 month) release of plasmid DNA and that the plasmid
DNA loaded into the scaffolds could transfect chondrocytes cultured in those scaffolds for up to
eight weeks in vitro. A significant percentage (20-40%) of the plasmid DNA added to the CG
scaffolds was tightly enough bound to the scaffold to resist passive (non-enzymatic) release into
the leaching buffer [Langer, 1990].
For comparison, in a prior study [Shea et al., 1999]
investigating release of plasmid DNA from copolymers of D,L-lactide and glycolide, less than
10% of the DNA remained in the synthetic polymer construct after 28 days in leaching studies
performed using Tris-EDTA buffer. An additional advantage in the use of a CG scaffold for
gene delivery is that this class of materials has proven successful as an implant to facilitate
regeneration of dermis [Yannas et al., 1989] and has enhanced peripheral nerve [Chamberlain et
al., 2000] and articular cartilage repair [Breinan et al., 2000] in certain procedures.
With the exception of the EDAC cross-linking protocol employed here, cross-linking did
not affect the total amount of plasmid that was initially loaded into the CG scaffolds. The lower
plasmid loads of the EDAC scaffolds can be attributed to the collapsed pore structure resulting
from the cross-linking methods used here. Future preparations using EDAC cross-linking should
seek to retain the open pore structure, either by stabilization through DHT cross-linking prior to
EDAC treatment or by EDAC cross-linking in the presence of ethanol [Pieper et al., 1999].
Cross-linking had a more marked effect on the plasmid leaching profiles. It should be
noted that the leaching profiles illustrated in Figure 5.3 represent only the passive (nonenzymatic) release of plasmid that was not tightly bound to the CG network. In practice, the
GSCG scaffolds will also be subjected to enzymatic degradation. The degradation rates of the
93
scaffolds would be predicted to be non>DHT>UV>EDAC, such that the longer residence time of
the EDAC cross-linked scaffolds may provide for a longer DNA delivery compared to the more
slowly releasing, but more quickly degrading non-cross-linked scaffolds. The data from the
cultures sacrificed after 8-weeks does, in fact, support the notion that the degradation rate may
play an important role in the long-term transfection as the more highly cross-linked EDAC
scaffolds provided a much greater luciferase activity than the DHT scaffolds (pH 7.5).
The higher residual plasmid DNA and longer predicted passive release duration of the pH
2.5 cross-linked GSCG scaffolds, when compared to the CSGC scaffolds prepared at pH 7.5, are
consequences of the biophysical properties of collagen in an acidic environment [Dick and
Nordwig, 1966; Hayashi and Nagai, 1973].
Under acidic conditions the fibrillar collagen
bundles tend to unwind or dissociate, thus increasing the available surface area for non-specific,
electrostatic interactions between the loaded plasmid DNA molecules and the reactive side
chains of the amino acids in the collagen fibrils. The subsequent freeze-drying cycle in the
fabrication process leads to physical compaction of the dissociated collagen fibrils and the
entrapment of a fraction of the plasmid DNA into regions of the GSCG scaffolds that are
resistant to solvent mediated, non-enzymatic release.
On day 14, the levels of luciferase activity revealed the highest levels of transfection in
the pH 7.5 EDAC- and UV-cross-linked GSCG scaffolds, indicating that these scaffolds
transferred the largest amount of transcriptionally functional plasmid DNA to the target cells
("high response" group). The intermediate response group consisted of the pH 2.5 non-crosslinked and the pH 7.5 DHT cross-linked GSCG scaffolds. The low response group consisted of
all of the pH 2.5 cross-linked scaffolds (DHT, UV, and EDAC) and the pH 7.5 non-cross-linked
GSCG scaffolds.
The sustained vector delivery and continued reporter gene expression in
chondrocytes seeded into the various formulations were reflected in the 28- and 56-day results.
There was, however, a notable decrease in luciferase gene expression for the high response group
of GSCG scaffolds from the early time (14 days) to the later time (28 days). In contrast, the low
response group produced approximately the same level of the luciferase gene activity at the late
time (28 days) as at the earlier time (14 days). Although the transfection rates seen here are
much lower than are typically attained with other transfection methods (i.e. lipid or viral
transfection systems such), this union of tissue engineering and gene therapy may still hold
therapeutic value. Growth factors are typically effective in nano- to microgram doses so it may
94
be possible that only a few transfected cells producing the therapeutic protein in situ will be
sufficient to elicit the desired response.
Although all scaffolds were able to provide for long-term delivery of plasmid DNA to the
resident chondrocytes for up to 28 days, lower transfection levels were seen in the pH 2.5
scaffolds compared to the pH 7.5 preparations, despite the higher levels of DNA retained in the
pH 2.5 scaffolds. While the acidic pH allows for collagen swelling and therefore higher and
tighter DNA loading, the acidic pH may be damaging to the plasmid DNA (see Appendix L), as
DNA is susceptible to proton-induced depurination at low pH. Such damage could explain the
lower transfection levels measured in cells seeded into the pH 2.5 scaffolds compared to cells
were seeded into pH 7 scaffolds.
In order to determine the actual duration of transfection, future in vivo studies are
necessary. Further studies are also needed to determine how best to preserve the integrity of the
plasmid DNA incorporated in the GSCG scaffolds to further increase the level of gene
expression attainable by these gene delivery devices. Nonetheless, this study has provided a
rational basis for the future development of collagen-GAG scaffolds for the controlled and
prolonged release of therapeutic levels of plasmid DNA for articular cartilage repair and other
procedures.
95
CHAPTER 6: CONCLUSIONS
The purpose of this thesis was to form a basis for the development of an articular
cartilage tissue engineering system based on porous collagen-glycosaminoglycan
scaffolds by systematically investigating variables related to chondrocyte-matrix
interactions. In chapter 2, the effects of the physical properties of the scaffold, varied by
selected cross-linking treatments, on the proliferative, contractile, and biosynthetic
behavior of passaged chondrocytes in these scaffolds were investigated.
Chapter 3
investigated the effects of mechanical compression of the scaffolds on chondrocyte
biosynthesis. In chapter 4, the effects of implantation of one of the cell-seeded scaffolds
studied in the in vitro studies on in vivo cartilage repair were examined. Finally, to
establish a foundation for future investigations on the effects of genetic modification of
chondrocytes, chapter 5 explored potential modifications of the scaffolds to allow for
direct delivery of genetic material from the scaffold. This chapter briefly summarizes the
development and conclusions of each study.
The primary objective of chapter 2 was to find a cross-linking method that would
minimize cell-mediated contraction. Chondrocyte proliferation and biosynthesis patterns
were also evaluated, since cross-linking methods can also affect cell proliferation and
biosynthesis, by virtue of changes to chemical composition and/or scaffold stiffness.
Prior work with passaged canine chondrocytes in porous collagen-glycosaminoglycan
scaffolds revealed a tendency for the cell-seeded scaffolds to shrink ("cell-mediated
contraction") during in vitro culture. It was hypothesized that such deformation of the
scaffold would be detrimental to in vivo repair as it would impair implant-host contact
and, therefore, integration. Studies with other cell types indicated that increasing scaffold
stiffness by varying the cross-linking treatment could alter contraction patterns. The
main conclusions from this study are:
.
The following methods, applied under the specific conditions in this study,
increase cross-linking density and resistance to cell-mediated contraction in the
following order - dehydrothermal (DHT) < ultraviolet (UV) < gluteraldehyde
(GTA) < and carbodiimide (EDAC).
96
.
Radially unconfined compressive stiffness (140-1100 Pa) increases with crosslink density, except for UV and GTA scaffold stiffnesses, which are not
significantly different.
*
UV and EDAC methods cross-link GAGs to the collagen network.
.
All cross-linking methods permit chondrocyte proliferation and protein and GAG
biosynthesis, with EDAC scaffolds permitting maximum proliferation and
biosynthesis.
The focus of chapter 3 was the effects of mechanical compression on the adult,
passaged canine chondrocytes seeded into the porous collagen (type I and type II)glycosaminoglycan scaffolds at different time points in culture. In addition to its role as a
carrier of cells and genetic material, the collagen scaffold is an important link between
external mechanical forces and the cell. It is well-known that mechanical compression of
cartilage regulates biosynthesis of chondrocytes in their native environment. However,
relatively little work has been done to evaluate the effects of mechanical compression in
articular cartilage tissue engineering systems. Due to the stabilization of the scaffold
geometry and the stiffness imparted by EDAC cross-linking, EDAC cross-linked
scaffolds were used exclusively these studies. The following conclusions are based on
these studies of up to 24 hours of continuous compression:
.
Third passage chondrocytes in the porous collagen-glycosaminoglycan scaffolds
respond to static and dynamic compression as early as the third day post-seeding.
" Consistent with results for cartilage explants, unconfined static compression (1050% strain) leads to a dose-dependent decrease in protein and GAG biosynthesis
and matrix molecule accumulation within the constructs.
.
Consistent with results for cartilage explants, unconfined dynamic compression
(3% sinusoidal amplitude, 0.1 Hz superimposed on a 10% strain offset) leads to
increases in protein and GAG biosynthesis, relative to both free-swelling and
static controls.
97
.
Dynamic compression decreases the rate of matrix molecule accumulation within
the scaffold, relative to free-swelling controls, due to increased rates of release of
the molecules to the media.
.
Thus, passaged adult chondrocytes in this system were found to be capable of
responding to static and dynamic compression with trends similar to those for
primary chondrocytes in native tissue and in three-dimensional gel (agarose)
culture.
Ultimately, it is the in vivo results that dictate the success of any articular cartilage
regeneration system. Hence, the next chapter presented the results of in vivo cartilage
repair of canine chondral defects that were treated by implantation of an autologous
chondrocyte-seeded type II collagen (EDAC cross-linked)-glycosaminoglycan scaffold.
In contrast to prior studies with the same animal model, the cell-seeded scaffolds in this
study were cultured for four weeks prior to implantation into the surgically-created
defects. This in vitro culture period was included in order to allow for matrix deposition
and stabilization of the scaffold geometry. From this study, the main conclusions are:
.
Four-week in vitro free-swelling culture of autologous passaged chondrocyteseeded CG scaffolds improves canine cartilage repair compared to previous
treatments with cells alone or scaffolds seeded just prior to implantation
.
Repair tissue formed at 15 weeks post-implantation is still far inferior to normal
articular cartilage from both histological and mechanical perspectives.
In looking forward to the further development of the collagen-glycosaminoglycan
system for articular cartilage tissue engineering, it is likely that growth factors will be
necessary to increase biosynthesis of cartilage-specific matrix molecules.
One of the
promising methods for delivery of the growth factors is to manipulate the cells to produce
the proteins through gene therapy. The collagen-based scaffold is an insoluble, slowly
degrading material that can serve as a localized delivery vehicle for the genetic material.
With these promising prospects in mind, chapter 5 was a first look at potential
modifications that can be made to the collagen-glycosaminoglycan
scaffold to
incorporate genetic material and transfect chondrocytes in situ. Conclusions from this
work are:
98
*
Up to 90% of the loaded plasmid is released to an aqueous buffer within the first
few hours of hydration.
.
Acidic pH increases the retention of plasmid within the scaffold, likely due to
swelling of the collagen fibrils and entrapment of the plasmid within the swollen
fibers.
.
Despite the higher plasmid levels, acidic pH leads to lower levels of chondrocyte
transfection. This is likely due to acidic denaturation of the plasmid, but may
also be due to unavailability of the plasmid trapped within the collagen fibers.
.
EDAC and UV cross-linked, gene-seeded scaffolds produce the highest levels of
chondrocyte transfection.
Based on these results, the system commended for further investigation as a
potential implant for articular cartilage repair is an EDAC cross-linked scaffold,
impregnated with genes coding for selected growth factors added at a neutral pH, seeded
with passaged chondrocytes, and subjected to cyclic compressive loading.
99
CHAPTER 7: LIMITATIONS AND FUTURE WORK
This final chapter highlights some of the limitations of this work - including: the
choice of cross-linking methods, measurement of biosynthetic activity and the
relationship to cartilage matrix synthesis, mechanical loading conditions, animal model
for in vivo repair, and methods of genetic modification of the scaffolds - and highlights
some of the areas for future research to address these limitations and to further develop
the appropriate CG scaffold.
In the first set of experiments, four different cross-linking methods were
evaluated, each under very specific conditions. The results stated should be considered
valid only for the specified conditions as altering the time of cross-link treatment or the
conditions under which cross-linking is performed (i.e., pH and temperature) can alter the
nature and rate of formation of cross-links. Future studies may focus on a single crosslinking method carried out under a variety of conditions in order to isolate the influence
of specific scaffold properties (i.e., stiffness, GAG content, etc.). Future studies may also
investigate the degradation rate of the cross-linked scaffolds (in vitro and in vivo) and
evaluate the relationship of scaffold degradation and cell-mediated contraction.
In the studies evaluating rates of biosynthesis, the primary parameters were the
rates of accumulation of 3H-proline and
35
S-sulfate into the scaffolds.
These are
measures of general protein and sulfated-glycosaminoglycan accumulation, respectively.
For chondrocytes in their native environment these parameters measure cartilage matrix
synthesis, with 3 H-proline incorporation correlating to the synthesis of type II collagen
and other proteins and
35
S-sulfate
primarily aggrecan synthesis.
incorporation correlating to proteoglycan synthesis,
As chondrocytes are expanded in monolayer culture
(passaged), they gradually de-differentiate such that they are no longer necessarily
synthesizing predominately
(see Appendix
type II collagen
N)
and aggrecan.
Consequently, 3H-proline incorporation may represent type I collagen or non-collagenous
protein synthesis in significant quantities and
35
small (non-aggrecan) proteoglycan biosynthesis.
S-sulfate
incorporation may represent
Although a detailed accounting of
molecule-specific biosynthesis was not performed in any of the studies in this thesis,
100
qualitative analysis of some specimens was conducted as a general measure of how likely
it was that at least some of the measured biosynthesis was cartilage-specific.
Immunohistochemical staining for type II collagen revealed accumulation of type
H collagen only in the DHT and UV cross-linked scaffolds.
There was no
immunohistochemical evidence that passaged canine chondrocytes in EDAC and GTA
scaffolds deposited significant amounts of type H collagen in the scaffold during the first
four weeks of in vitro, free-swelling culture.
Western blot analysis did reveal the
presence of type H collagen in twelve-week EDAC cross-linked scaffolds. Additionally,
in an effort to ascertain the nature of the
35S-labeled
species, size-exclusion column
chromatography (S-1000) of selected samples (EDAC cross-linked, 7 day free-swelling
culture) was conducted. There were elution peaks corresponding to the molecular weight
of aggrecan.
However, there was also a significant peak corresponding to smaller
molecular weight molecules, which could represent aggrecan fragments as well as small
proteoglycans that are produced in larger quantities by non-chondrocytic cell types.
There was some difficulty in obtaining good histological sections of the EDAC
cross-linked scaffolds and this may explain the lack of positive type Id collagen
immunostaining. More likely, however, is that 1) redifferentiation may be slower in the
EDAC scaffolds, as compared to the DHT scaffolds, and/or 2) retention of newly
synthesized macromolecules is lower in the non-contracting EDAC scaffolds. To the
first point, once the cells cease to contract the scaffolds, their metabolic patterns may
shift towards remodeling the surrounding extracellular matrix. Additionally, once the
scaffolds have contracted, the cells are in a high cell-density environment that may
simulate the "micromass" cultures, which have been used to induce chondrocyte
differentiation. Treatment of the cell-seeded scaffold cultures with selected agents may
be used in the future to allow the cells to more rapidly recover a chondrocyte phenotype.
One such study has already been conducted using staurosporine (Appendix N).
Regarding the retention of macromolecules,
analysis of the medium in
compression experiments (EDAC cross-linked scaffolds) did reveal that a significant
percentage of the newly synthesized macromolecules were not retained within the
scaffold, especially during the first week of culture. As discussed in Chapter 2, the rate
of protein accumulation within the constructs was of the same order of magnitude as
101
measured for calf articular chondrocytes in intact cartilage and in agarose hydrogels
while GAG accumulation was 10-100 times lower. The large proportion of the newly
synthesized macromolecules that were released from these CG scaffolds is much greater
than that normally seen in cartilage explant or chondrocyte-seeded agarose cultures
(typically <2%) and this release may explain some of the discrepancy. It is clear that
more work should be done with regards to characterizing the molecules synthesized by
the chondrocytes once they have been passaged and cultured in the scaffolds.
Additionally, future work should seek to increase the retention of matrix molecules
within the scaffold.
The experiments with mechanical compression described in chapter 3 only begin
to explore the utility of physical loading in the development of articular cartilage
implants. A great deal of work is needed to further characterize the effects of loading and
to maximize the benefits. As mentioned previously, only overall rates of biosynthesis
were measured with 3 H-proline and
35
S-sulfate incorporation rates. Therefore, the effects
of mechanical loading on chondrocyte differentiation and the relative rates of cartilagespecific molecule synthesis were not analyzed.
The loading conditions used for the
studies in chapter 3 were likely not optimal. All of the studies were with continuous
loading,
whereas
physiological
conditions
would
dictate
intermittent
loading.
Furthermore, loading was only applied for up to 24 hours, but the effects on implant
properties would only be realized with long-term loading.
Also, as mentioned
previously, the retention of newly synthesized macromolecules was relatively low. In
order to take advantage of the increased biosynthetic activity, modifications should be
made to maximize matrix molecule retention.
The major limitation of the in vivo study was the single time point (15 weeks)
evaluated. Prior studies with untreated and chondrocyte-only implantations indicated that
the maximum repair was seen at this time. The introduction of the scaffold material (and
the specific cross-linking of the scaffold and the in vitro pre-culture of the implant),
however, may alter the time course of repair.
The work with the gene-seeded scaffolds described in chapter 5 should be
regarded as just the first step towards the development of collagen-GAG gene-delivery
scaffolds.
There are many subtleties to the scaffold fabrication that could improve
102
plasmid retention and plasmid integrity (i.e. cross-linking before or after plasmid
addition, addition of plasmid under weakly acidic or basic conditions, the temperature
and length of scaffold incubation with the plasmid prior to freeze-drying, etc.).
Additionally, transfection patterns during both the shorter and longer term in vitro, as
well as transfection in vivo should be evaluated. It is likely that such studies will reveal
greater differences among the cross-linking treatments, as degradation rates will become
important.
103
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APPENDICES
I have decided to include all (or at least almost all) of the protocols that I have
used over the past 3 years in this appendix. Hopefully, they will be of use to someone at
some point. The first 9 appendices are filled with protocols. The last 5 summarize pilot
experiments and studies that did not make the final cut into the body of the thesis...
APPENDIX A:
FABRICATION OF COLLAGEN-GLYCOSAMINOGLYCAN
121
MATRICES
..................................................
A.1.
I COLLAGEN-GLYCOSAMINOGLYCAN...................................................
Slurry.......................................................................................................
Freeze-drying..........................................................................................
TYPE II COLLAGEN-GLYCOSAMINOGLYCAN ................................................
Old protocol (from H.A. Breinan thesis, 1998).......................................
New p rotocol...........................................................................................
TYPE
A .1.1.
A .1.2.
A.2.
A.2.1.
A .2.2.
121
121
121
122
122
122
122
A .3.
C ROSS-LINKING ...........................................................................................
A.3.1.
Dehydrothermal (DHT)(fromskin regenerationtemplate protocols,F&P
Lab )
.................................................................................................................
12 2
A.3.2.
Ultravioletirradiation(UV) (modification of J. Hsiao thesis, 1999).....122
A.3.3.
Gluteraldehyde (GTA) (from skin regneration template protocols, F&P
L ab )
.................................................................................................................
12 3
A.3.4.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDAC) (modification of
Olde Damink, et al., Biomaterials, 1996 protocol).................................................
123
APPENDIX B:
FABRICATION OF GENE-SEEDED COLLAGENGLYCOSAMINOGLYCAN MATRICES..................................................................125
B . 1.
M ATRIX PREPARATION.....................................................................................
125
B .2.
ADDITION OF PLASM ID .....................................................................................
125
B.3.
ALTERNATIVE METHODS ATTEMPTED .............................................................
125
B.3.1.
B.3.2.
125
Plasmid Addition to Slurry......................................................................
PlasmidAddition in the Presence of Monovalent Salt............................125
APPENDIX C:
C. 1.
ASSESSMENT OF COMPRESSIVE STIFFNESS OF UNSEEDED SCAFFOLDS.............
127
G eneralp rotocol:....................................................................................127
Dynastat Set-up .......................................................................................
Calibration..............................................................................................
Placing Matrix in Chamber....................................................................
Comp uter Set-up ......................................................................................
Data analysis...........................................................................................128
127
12 7
128
128
C .1.1.
C .1.2.
C .1.3.
C.1.4.
C .1.5.
C.1.6.
C.2.
UNCONFINED COMPRESSION OF COLLAGEN MATRICES
127
.
.................................................
MECHANICAL COMPRESSION OF CELL-SEEDED CONSTRUCTS .........................
C.2.1.
Static Compression..................................................................................131
115
131
C.2.2.
Dynam ic Compression ............................................................................
APPENDIX D:
CELL CULTURE METHODS ....................................................
D. 1.
CHONDROCYTE HARVEST PROTOCOL.............................................
D.1.1.
OR Supplies.............................................................................................133
Lab Supplies ............................................................................................
D.1.2.
D .1.3.
Solutions..................................................................................................133
M ethods..................................................................................................134
D.1.4.
M ON OLA YER CELL CULTURE .............................................................
D .2.
D .3.
CELL SEED IN G OF M A TRICES .............................................................
D .3.1.
M aterialsneeded.....................................................................................
M atrixpreparation..................................................................................136
D .3.2.
D .3.3.
Cell preparation......................................................................................
D.3.4.
Seed cells.................................................................................................137
RADIOLABELING PROTOCOL FOR SEEDED COLLAGEN DISKS..
D.4.
General Outline.......................................................................................138
D .4.1.
D .4.2. M aterialsneeded.....................................................................................
Preparationof radioactivem edia ...........................................................
D.4.3.
D.4.4. Labeling disks..........................................................................................139
D.4.5. W ashing disks..........................................................................................139
APPENDIX E:
BIOCHEMICAL ASSAYS ...........................................................
E . 1.
LYOPHILIZATION ..............................................................................................
E .2.
SAMPLE D IGESTION..........................................................................................
E.2.1.
E.2.2.
E.3.
133
133
133
135
136
136
136
138
138
138
140
140
140
PapainDigestion.....................................................................................140
140
ProteinaseK Digestion ...........................................................................
GAG ASSAY USING DIMETHYLMETHYLENE BLUE (DMMB) DYE .................... 141
Sum m ary..................................................................................................141
E.3.1.
Equipment...............................................................................................141
E.3.2.
E.3.3.
Protocol...................................................................................................141
GAG standards........................................................................................141
E.3.4.
E.3.5.
DMMB dye solution ................................................................................
E.4. DNA ASSAY USING HOECHST 33258 FLUORESCENT DYE ................................
E.4.1.
Sum m ary..................................................................................................143
Equipment................................................................................................143
E.4.2.
Protocol...................................................................................................143
E.4.3.
DNA standards........................................................................................
E.4.4.
DNA dye solution ....................................................................................
E.4.5.
E.5.
131
SCINTILLATION COUNTING OF RADIOLABELED SAMPLES ................................
142
143
143
144
145
Sum m ary..................................................................................................145
Protocol...................................................................................................145
145
Calculations............................................................................................
E.6. DowEx-50W SEPARATION OFPROLINE AND HYDROXYPROLINE .................... 147
147
E.6.1.
Sum m ary..................................................................................................
Column Preparation................................................................................147
E.6.2.
E.5.1.
E.5.2.
E.5.3.
116
E.6.3.
Running a Sample....................................................................................149
E.6.4.
Calculations............................................................................................
150
E.7. PD-10 SEPARATION OF MACROMOLECULAR AND FREE LABEL ....................... 152
E.7.1.
Sum m ary..................................................................................................152
E.7.2.
Equipment................................................................................................
152
E.7.3.
Column preparation................................................................................152
E.7.4.
Sample loading and collection................................................................153
E.8.
S-1000 MOLECULAR WEIGHT SEPARATION OF GAG MOLECULES..................155
E.8.1.
E.8.2.
E.8.3.
E.8.4.
E.8.5.
E.8.6.
Sum m ary..................................................................................................
Equipment...............................................................................................155
Column preparation................................................................................
Column calibration.................................................................................
Sample loading and collection ................................................................
Variations................................................................................................156
155
E .9. COLLAGEN EXTRACTION ..................................................................................
E.9.1.
Sum m ary..................................................................................................157
E.9.2.
Materials.................................................................................................
E.9.3.
Procedure................................................................................................
E. 10.
CYANOGEN BROMIDE (CNBR) CLEAVAGE OF COLLAGEN ...........................
E.10.1. Sum m ary..................................................................................................
E.10.2. Reagents ..................................................................................................
E.10.3. Procedure................................................................................................
157
APPENDIX F:
155
155
155
157
157
158
158
158
158
GEL ELECTROPHORESIS METHODS................160
F. 1.
SD S-PA GE ......................................................................................................
160
F.1.1.
Sum m ary..................................................................................................160
F.1.2.
Reagents:.................................................................................................160
F.1.3.
Procedures..............................................................................................
161
F.2. TYPE I AND II COLLAGEN WESTERN BLOT PROTOCOL....................................... 163
F.2.1.
Sum m ary..................................................................................................163
F.2.2.
SDS-PAGE - see previous section, do not stain gel! .............................. 163
F.2.3.
Weste blotting ......................................................................................
163
F.3. C-SMOOTH MUSCLE ACTIN WESTERN BLOT PROTOCOL.................................. 165
F.3.1.
Sum m ary..................................................................................................165
F.3.2.
Cell LS .................................................................................................
165
F.3.3.
ProteinAssay (need to determine how much of each sample to load). . 166
F.3.4.
Gel electrophoresis(start making gels before protein assay)- See also
section on SD S-PA GE .............................................................................................
166
F.3.5.
W estern blotting ......................................................................................
167
F.3.6.
Solutions neededfor Western Blot ..........................................................
167
F.4. FLUOROGRAPHY OF RADIOLABELED PROTEINS................................................. 169
F.4.1.
Summ ary..................................................................................................169
169
F.4.2.
Sample preparation.................................................................................
F.4.3.
Protein/peptideseparation......................................................................169
F.4.4.
Fluorography..........................................................................................
169
117
APPENDIX G:
HISTOLOGY METHODS...........................................................170
170
TISSUE FIXATION AND EMBEDDING PROTOCOL ...........................................
G. 1.
(All protocolsfrom S. Zapatka-Taylor,BWH OrthopaedicsResearch Laboratory,
170
unless specified otherwise)......................................................................................
170
Fixation ...................................................................................................
G .1.1.
170
Decalcification........................................................................................
G.1.2.
170
Glycol Methacrylate (JB-4) Embedding .................................................
G.1.3.
170
................................................................................
Embedding
Paraffin
G.1.4.
HISTOLOGY AND IMMUNOHISTOCHEMISTRY PROTOCOL .............................. 171
G.2.
Safranin-O Staining.................................................................................171
G.2.1.
171
Hematoxylin and Eosin (H&E) Staining.................................................
G.2.2.
Immunohistochemical Stainingfor type II collagen (paraffinsections). 172
G.2.3.
ImmunohistochemicalStainingfor a-Smooth Muscle Actin (paraffin
G.2.4.
1 73
sections) .................................................................................................................
slides)
(chamber
Immunofluorescent Stainingfor Type II procollagen
G.2.5.
(modification of Borge, et al., In Vitro Cell Dev Biol Anim, 1997 protocol)..........174
APPENDIX H:
INDENTATION TESTING OF CARTILAGE-BONE PLUGS
............................................
... 175
H.1.
H.2.
H.3.
SAMPLE PREPARATION.................................................................................
175
DYNASTAT SET-UP .......................................................................................
H.4.
TESTING OF SAMPLES...................................................................................
H.5.
THICKNESS MEASUREMENTS .......................................................................
175
176
176
177
COMPUTER SET-UP.......................................................................................
STATISTICS .................................................................................
178
1.1.
POWER CALCULATION .....................................................................................
1.2.
ANOVA ANALYSIS AND POST-HOC TESTING .....................................................
178
178
APPENDIX I:
APPENDIX J:
EVALUATION OF SEEDING TECHNIQUES ........................ 179
INTRODUCTION .................................................................................................
J. 1.
J.2. MATERIALS AND METHODS .............................................................................
Cell density ..............................................................................................
J.2.1.
Incubation tim e........................................................................................180
J.2.2.
Suspension volume ..................................................................................
J.2.3.
J.3 . RESU LTS...........................................................................................................
Cell density ..............................................................................................
J.3.1.
Incubation tim e........................................................................................
J.3.2.
Suspension volume ..................................................................................
J.3.3.
179
179
180
J.4.
C ON CLU SION S ..................................................................................................
180
18 1
181
183
183
184
J.5.
ACKNOWLEDGEMENTS .....................................................................................
184
EFFECTS OF PASSAGE ON CELL-MEDIATED
APPENDIX K:
CONTRACTION AND RESPONSE TO MECHANICAL LOADING .................. 185
118
K.1.
INTRODUCTION .............................................................................................
185
186
MATERIALS AND METHODS .........................................................................
K.2.
Contraction Studies.................................................................................186
K.2.1.
K.2.2.
Dynamic Compression Studies................................................................186
186
K.3 .
R ESULTS.......................................................................................................
Cell-mediated Contraction......................................................................186
K.3.1.
K.3.2.
Compressive Loading..............................................................................187
K.4 .
D ISCU SSION ..................................................................................................
189
K.5 .
REFERENCES ................................................................................................
189
INTEGRITY OF PLASMID IN GENE-SEEDED COLLAGENAPPENDIX L:
GLYCOSAMINOGLYCAN MATRICES..................................................................191
L .1.
INTRODUCTION .................................................................................................
191
191
L.2. MATERIALS AND METHODS ....................................................................
L.2.1.
ElectrophoreticAnalysis of Released Plasmid DNA...............................191
Transfection of Canine Articular Chondrocytes in Monolayer Culture. 191
L.2.2.
192
L .3 . RE SU LT S ........................................................................................................
ElectrophoreticAnalysis of Released PlasmidDNA...............................192
L.3.1.
193
L.3.2.
In Vitro Transfection Assays ...................................................................
194
L.4. DISCUSSION .................................................................................................
195
L .5 . REFERENCES ....................................................................................................
APPENDIX M: ALTERNATIVE METHODS FOR THE FABRICATION OF
196
THE GSCG MATRICES ..............................................................................................
M . 1.
INTRODUCTION.............................................................................................
196
196
MATERIALS AND METHODS .........................................................................
M .2.
M.2.1.
Addition of Plasmid to Collagen-GlycosaminoglycanSlurry.................196
197
M.2.2.
Plasmidleaching.....................................................................................
197
M.2.3. In situ transfection of chondrocytes ........................................................
197
M .3 .
RESU LTS.......................................................................................................
197
M.3.1.
Plasmidleaching.....................................................................................
197
transfection..............................................................
M.3.2. In situ chondrocyte
197
D ISCU SSION ..................................................................................................
M .4 .
STAUROSPORINE MODULATION OF THE PHENOTYPE
APPENDIX N:
OF MONOLAYER-PASSAGED CANINE CHONDROCYTES SEEDED IN
COLLAGEN-GAG MATRICES.................................................................................199
N .1.
A B STRA CT ....................................................................................................
199
N .2 .
N .3 .
INTRODUCTION .............................................................................................
199
N.3.1.
N.3.2.
N.3.3.
N.3.4.
20 1
201
Chondrocyte Isolation and Culture.........................................................
ImmunohistochemicalStaining of Type II Procollagenin Monolayer ... 201
202
Proliferationof cells in monolayer .........................................................
Western Blot Analysis of a-Smooth Muscle Actin................................... 203
M ETH OD S .....................................................................................................
119
N.3.5.
N.3.6.
N.3.7.
N.3.8.
N.3.9.
N.3.10.
N .4 .
Collagen-GlycosaminoglycanScaffold Synthesis ...................................
Culture of Chondrocytes in Collagen-GAG Scaffolds ............................
Measurement of Cell-Mediated Contraction...........................................
Radiolabel Incorporationinto Chondrocyte-CG Constructs..................
DNA Analysis ..................................
StatisticalAnalysis ..................................................................................
RESU LTS.......................................................................................................
204
204
205
205
205
206
207
N.4.1.
Light Microscopy and Type II Collagen Immunohistochemistry of Cells in
207
Monolayer ...............................................................................................................
207
N.4.2.
Monolayer Proliferation.........................................................................
Western Blot Analysis for a-Smooth Muscle Actin................................. 208
N.4.3.
209
N.4.4.
Cell-Mediated Contraction.....................................................................
DNA Content in the Cell-Seeded CG Matrices....................................... 210
N.4.5.
Radiolabel Incorporationto Determine Protein and GAG Synthesis..... 211
N.4.6.
N .5 .
N.6.
N .7 .
ACKNOWLEDGEMENTS .................................................................................
2 13
215
REFERENCES ................................................................................................
215
D ISCU SSION ..................................................................................................
120
APPENDIX A: FABRICATION OF COLLAGENGLYCOSAMINOGLYCAN MATRICES
A.1.
TYPE I COLLAGEN-GLYCOSAMINOGLYCAN
(from Fiber & Polymers Lab, protocolfor skin regenerationtemplate)
A.1.1. Slurry
1)
2)
Cool blender for 30 minutes to 4C according (directions for cooling blenders on
cooling bath next to blenders)
Fill blender with 600 ml of 0.05M acetic acid (2.9 ml glacial acetic acid + distilled
H20 to 1 L)
3)
4)
5)
6)
7)
8)
9)
Add 3.6 g of dry bovine tendon collagen (Integra Life Sciences)
Blend on high for 90 minutes
Dissolve 0.32 g chondroitin-6-sulfate in 120 ml 0.05M acetic acid (do this while
blending the collagen as it will take some time for the c-6-s to dissolve)
Add c-6-s solution to blender over 15 minutes using peristaltic pump (continue
blending)
Blend for an additional 90 minutes
If using slurry immediately, transfer to a vacuum flask and de-gas for 10 minutes
(be careful not to suck slurry into vacuum!!)
If using later, pour out slurry and refrigerate. When ready to use, blend slurry for
15 minutes at 4C and then de-gas as above.
A.1.2. Freeze-drying
1)
2)
3)
4)
5)
Turn on VirTis freeze-drier
Make sure drain plug is in drain and turn on condenser
After condenser has cooled, turn on freeze
Wait for shelf to cool down to -45'C (may only reach -40'C), at least 1 hour
Pipette slurry into freeze-drying trays - usual tray is divided into 3 sections - -220
ml/section yields foam thickness of -3.5-4 mm; -180 ml/section yields foam
thickness of -2.5 mm
Make sure there are no air bubbles in slurry
6)
Place tray of slurry into freeze drier and freeze (-1.5 hours)
7)
When slurry has frozen, turn on vacuum (condenser must be at -50'C before
8)
vacuum is turned on) and press door shut to make sure it seals (also, make sure
chamber release is off; do not leave until you are sure the door is sealed and the
pump is pulling a vacuum!)
Pull vacuum at -45'C until it is below 200 mtorr (-1-2 hours)
9)
10) Once vacuum is below 200 mtorr, set temperature to 00 C and turn on heat
11) Sublimate overnight (at least 12 hours)
12) In the morning, set temperature to 20'C
13) When temperature reaches 20'C (- 30 minutes), turn off heat, freeze, vacuum,
condenser. Turn on chamber release
121
14)
A.2.
After vacuum is released, turn off VirTis, remove tray, and open drain plug on
condenser
TYPE II COLLAGEN-GLYCOSAMINOGLYCAN
Using Chondrocell slurry (unspecified GAG type and amount) from Geistlich
Biomaterials. Slurry should be stored at 4'C.
A.2.1. Old protocol (from H.A. Breinan thesis, 1998)
1)
2)
3)
4)
Warm slurry to 42'C to melt slurry
De-gas slurry
Pipette slurry into molds (Aluminum weighing cups work well)
Freeze-dry as for type I CG slurry (section A.2 above)
A.2.2. New protocol
1)
2)
3)
4)
Transfer slurry to 50ml centrifuge tube
Centrifuge 5 minutes at highest speed (3500 rpm) to de-gas slurry
Pipette slurry into molds - 6-well plates work well with -3.5 ml slurry/well (pipette
reading, not actual volume due to slurry sticking to inside of pipette) yielding
matrices -2-2.5 mm thick; pipette slurry into center of well and tap/bang plate on
countertop to evenly distribute slurry, remove bubbles with 1cc syringe and needle
Freeze-dry (freeze with lids on, remove lids before pulling vacuum) as for type I
CG slurry (section A.2 above)
Note: Other molds/freeze-drying temperatures could be used, but I found that freezedrying in the 6-well plates (with the lids on) using the standard freeze-drying protocol
seemed to yield similar pore sizes to the standard type I CG matrices (visual inspection
only)
A.3.
CROSS-LINKING
A.3.1. Dehydrothermal (DHT) (from skin regeneration template protocols, F&P Lab)
1)
2)
3)
4)
Place matrix in aluminum foil bags (leave one end open)
Place in vacuum oven set at 105'C
Pull vacuum (to 50 mtorr)
Dehydrate for 24 hours
Note: Longer treatment times and higher temperatures can increase cross-linking density
A.3.2. Ultraviolet irradiation (UV) (modification of J.Hsiao thesis, 1999)
1)
2)
3)
Place matrix (or pre-cut disks) 2" under UV lamp (UVP #XX15S; 254 nm, rated
1680 pW/cm 2 at 12"; 2" corresponds to highest shelf on exposure stand)
Irradiate for 30 minutes
Flip matrix and irradiate for another 30 minutes
Note: If you move the UV source closer/further, decrease/increase the exposure time.
Irradiation intensity is proportional to the square of the distance. Longer treatment times
may increase cross-linking, but will also increase damage to collagen. For my samples,
122
the above time/distance produced matrices with the greatest resistance to collagenase
digestion.
A.3.3. Gluteraldehyde (GTA) (from skin regneration template protocols, F&P Lab)
1)
2)
3)
4)
5)
6)
7)
8)
Rehydrate matrix in 0.05M acetic acid
Remove acetic acid via suction
Add 0.25% sterile gluteraldehyde (in 0.05M acetic acid - use fresh dilution)
Treat 24 hours at room temperature
Remove gluteraldehyde (dispose of as hazardous waste)
Rinse 3 times in sterile, distilled, deionized water
Store in fresh water (sterile) 24 hours
Store at 4*C for up to one week before use (extended storage may increase/alter
cross-linking)
Note: The acidic pH of the cross-linking slows the rate of cross-link formation, possibly
allowing for more uniform cross-linking throughout the matrix. In cross-linking the 9mm
diameter disks, I did not notice a difference in samples cross-linked in the presence of
0.05M acetic acid versus in water.
A.3.4. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) (modification of Olde
Damink, et al., Biomaterials, 1996 protocol)
EDAC (Sigma #E-7750; store in freezer)
Take out and let warm up (so that moisture doesn't condense inside bottle when
opened - EDAC is moisture sensitive)
Weigh on balance (MW=197 g/mol)
ie: for 100 ml, use 0.276 g
NHS (Sigma #H-7377) store in dessicator at room temperature)
Weigh on balance (MW= 115 g/mol)
ie: for 100 ml, use 0.064 g
1) Hydrate matrices in half the final volume (ie: for 100 ml final volume, hydrate in 50
ml) of sterile, dI H 2 0
2) Dissolve EDAC and NHS in half the volume of dI H2 0 water (ie: for 100 ml final
volume, dissolve in 50 ml; make up fresh for each use)
3) Sterile filter (0.2 gm) into sterile container (or directly into container with hydrated
matrices)
4) Cross-link at room temperature for 2 hours
5) Discard solution as hazardous waste
6) Rinse in sterile PBS, change to fresh, sterile PBS and incubate 2 hours
7) Rinse 2x10 minutes in sterile dI H2 0
8) Store at 4C for up to one week before use (effects of longer storage unknown)
Notes: Use -6 mmol EDAC/g collagen
5:2 ratio EDAC:NHS
9mm diam CG disk (3-4 mm thick) -0.005 mg
For 48 disks, use 100 ml 14.4 mM EDAC/5.6 mM NHS
123
If you have not DHT cross-linked your matrices, you may want to EDAC crosslink in the presence of ethanol to prevent pore collapse (Pieper, et al, Biomaterials: 20,
847-858, 1999)
124
APPENDIX B: FABRICATION OF GENE-SEEDED
COLLAGEN-GLYCOSAMINOGLYCAN MATRICES
B.1.
1)
2)
3)
4)
B.2.
MATRIX PREPARATION
Matrix slurry was prepared as described in Appendix A for type I collagen-GAG
matrices
1 ml of slurry was placed in each well of 24 well plates
Slurry was freeze-dried (with the lid on the well plates) as described in Appendix A
Matrices were either not cross-linked at all, or subjected to:
a. 24 hours DHT,
b. 24 hours UV (2" from source, one side only; no pre-treatment with DHT)
c. 2 hours EDAC treatment (no pre-treatment with DHT); matrices were refreeze-dried after EDAC treatment to again provide a dry matrix
ADDITION OF PLASMID
1)
Plasmid DNA with the gene for firefly luciferase (3.43 mg/ml) was diluted in either
0.05M acetic acid (pH 2.5) or Tris-EDTA (TE) buffer (pH 7.5) to 0.5 mg/ml
2) Diluted plasmid was then added to dry matrix disks (500 gl/disk; total of 250 g
DNA/disk)
3) Matrix-plasmid formulations were left covered at room temperature for 24 hours
4) Matrix-plasmid formulations were freeze-dried as described for matrices (Appendix
A)
B.3.
ALTERNATIVE METHODS ATTEMPTED
(see Appendix M for details of results)
B.3.1. Plasmid Addition to Slurry
Plasmid was added to slurry prior to freeze-drying at a final concentration of 250 gg/ml
slurry using a peristaltic pump with the slurry being continuously mixed by a small stir
bar at the bottom of the 50ml conical tube
B.3.2. Plasmid Addition in the Presence of Monovalent Salt
Plasmid was added to pre-formed matrices (DHT cross-linked) as described in section
B.2 with the following changes
1) Matrices were pre-soaked for 1 hour prior to plasmid addition in 250 gl of one of the
following:
a. 0.05M acetic acid
b. 0.05M acetic acid + 0.1M NaCl
c. TE buffer
d. TE buffer + 0. 1M NaCl
e. Sodium phophate buffer (pH 11)
f. Sodium phosphate buffer + 0.1M NaCl
125
2)
3)
4)
Plasmid was mixed with each of the above buffer solutions at a concentration of 0.5
mg/mi and 250 pl of the mixture was added to the soaked matrix disk (125 g
plasmid/matrix disk)
Matrix-plasmid formulations were left covered at room temperature for 1 hour
Matrix-plasmid formulations were freeze-dried as described for CG matrix
fabrication (Appendix A)
126
APPENDIX C: UNCONFINED COMPRESSION OF
COLLAGEN MATRICES
C.1. ASSESSMENT OF COMPRESSIVE STIFFNESS OF UNSEEDED
SCAFFOLDS
C.1.1. General protocol:
1) Specimens: 9-mm diameter disks; hydrated in PBS
2) Sequential ramp and hold displacements, corresponding to 1% (1%-5% strain), 2.5%
(10-20% strain), or 5% (20-40% strain) strain
3) Record load and convert to stress
4) Plot stress versus strain and use slope of best-fit line as modulus
C.1.2. Dynastat Set-up
1) Wiring:
Load cell 4 filter
Filter - ADC 2 (box below chart recorder)
Hi-R Displacement (back of Dynastat) - ADC 3
2) Amplifier Settings:
Scale 100%
Zero suppression -.01 volts
3) Dynastat Servo Settings:
0
1.0
5.75 5.0
8.3
7.0
C.1.3. Calibration
a) Calibrate load cell
i) Insert aluminum end of load cell into lower jaw and plug in load cell to filter
ii) Got to Set-up menu, 50g-Load, hit 'F9' and follow directions to calibrate
iii) Calibrate with Og, -1g, -2g, -5g and -10g weights
b) Move load cell to upper jaw and insert chamber in lower jaw, tighten small
collets, make sure wire on load cell doesn't get caught on anything
c) Push TRIP/RESET button
d) Push CLOSED loop
e) Push PROGRAM OFF
f) Calibrate Displacement
i) Make sure B is active
ii) Set B to COMPRESSION mode
iii) Put displacement control in Lo-R and read in Hi-R
iv) Push and hold ZERO button, use screwdriver to set to 0.000
v) Push and hold CAL button, use screwdriver to set to 4.945
vi) Put displacement control in Hi-R and read in Lo-R
vii) Push and hold ZERO button, set to 0.000
viii) Push and hold CAL button, set to 6.692
ix) Put read in Hi-R (leave control in Hi-R too)
127
C.1.4. Placing Matrix in Chamber
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
1)
m)
n)
o)
p)
Set offset under dynssp control menu to 0 (this will be changed later)
Push PROGRAM ON on the Dynastat
Switch toggle to "Compression" position
Set B odometer to 800 and dial scale knob to 10.0 (800 means 800 mV which
corresponds to 4mm)
Screw down load cell using silver knob at top of testing apparatus until plunger on
load cell touches chamber (load cell reading around -29g).
Tighten large collet. Computer reading should be around -1-0g.
Dial knob from 10.0 to 0.0. (The plunger should now be 4mm above the surface
of the chamber.)
Set odometer to 999
Switch toggle to "Transient" position
Change dynproc control offset to 5mm
Measure sample thickness of "almost" completely immersed specimen using
micrometer (make sure you subtract the thickness of the plate/dish)
Set dynproc set-up Hi-R offset to 2x(sample thickness - 4.0)
(ex: Sample thickness = 5.75 mm; 4mm of thickness is taken from 800 setting in
step (e). 5.75-4.0=1.75mm is remaining distance; 2x1.75=3.5 -> 3.5V should be
entered as the offset voltage in the Set-up: Hi-R menu)
Read offset voltage of load cell: Set-up: 50g Load: Read offset
Dial scale knob on Dynastat to 10.0
Insert matrix, center it as best as possible under plunger
Make sure DYN/EXT is on
C.1.5. Computer Set-up
a) Start program dynssp from c:\cyndi directory
(Notes: Hit 'Esc' to get out of pull-down menus. Hit 'Tab' to toggle between
command list and the screen that shows current channel readings)
b) File menu:
Open output file
Enter sample data (name, description, thickness, area =63.62 mmA2)
Protocol file should be cc.pro
c) Control menu (set-up ramp, sine, goto commands):
Waveform: Ramp/Sine/Goto
Acquisition List: Hi-R, 50g Load/Hi-R only for Goto
Iterations: (whatever is desired)/1/1
Iteration hold time: 0
Ext Scale Setting: 1/0.032/1
Offset: Change to 5 mm after matrix in chamber (step 3k)
Amplitude: (whatever is desired)IOIGoto 0
Control units: strn/m
Run protocol
C.1.6. Data analysis
a) Convert equilibrium loads to stresses
128
b) Plot stress versus strain
c) Apply line fit through data
d) Slope of line is compressive modulus
If R2 <0.9, re-run test
-100
R= 0.9971
-80
-60
-40
-20
0
0
-0.1
-0.2
-0.3
-0.4
-0.5
Strain
Figure C.1: Typical stress-strain data for unconfined compression of type
I collagen-glycosaminoglycan matrices. The slope of the fitted line was
used as the compressive modulus of the matrix. For accepted moduli, the
data was linear over the range 0-40% strain compression (R 2>0.9).
129
Table C-1: Matrix compressive stiffness. Stiffness (modulus), as measured in
unconfined compression, of type I collagen-glycosaminoglycan matrices cross-linked by
various methods and by various people. Bold entries correspond to the cross-linking
methods that were used for the cell-seeded studies reported in Chapter 2.
Prepared by
Cross-linking
Time
Stiffness (Pa)
Joyi (n=6)
DHT
24 hrs
145 ± 12
Cyndi (n=6)
DHT
24 hrs
145 ±23
Cyndi (n=7)
DHT
72 hrs
280 ± 14
Cyndi (n=6)
DHT+70% EtOH
10 min
146 ± 19
Cyndi (n=5)
DHT+Cyanamide
24 hrs
167 ± 15
Cyndi (n=6)
DHT+Cyanamide
72 hrs
199 ± 17
Cyndi (n=12)
DHT+UV
8 hrs @ 30 cm
217 ± 18
Joyi (n=6)
DHT+UV
8 hrs @ 15 cm
256 ± 11
Dawn (n=10)
DHT+UV
16 hrs @ 30 cm
318 ±24
Cyndi (n=8)
DHT+UV (1 hr)
Joyi (n=6)
DHT+GTA
1 hr
373 ±39
Joyi (n=6)
DHT+GTA
24 hrs
371 ± 16
Cyndi (n=5)
DHT+GTA
24 hrs
369
Joyi (n=6)
DHT+GTA
24 hrs*
663 ± 64
Cyndi (n=8)
DHT+EDAC
0.5 hrs
767 ± 37
Cyndi (n=10)
DHT+EDAC
2 hrs
1140
Cyndi (n=4)
DHT+EDAC
5 hrs
956 ± 169
1 hr (2")
346
40
54
85
stored for 6 weeks in sterile PBS at 4C between cross-linking and
testing
*
130
C.2.
MECHANICAL COMPRESSION OF CELL-SEEDED CONSTRUCTS
C.2.1. Static Compression
C.2.1.1.
Equipment
Loading chambers - 12-well polysulfone chambers, top and bottom halves (A
chambers have a well depth of 250 pm with no Teflon spacers)
2) Teflon spacers (there are spacers of varying thicknesses, chose the appropriate
spacers to hold the chamber at the desired height, remember that the chambers have a
well depth, usually 250 pm, even with no spacers)
3) Stand (metal base with bolt to go through center of chamber), washer, and nut to
tightly fix chamber height
4) Spatula/forceps to transfer samples
5) Media containing radiolabeled species, if desired
6) P-1000 pipetman and sterile 1000 gl tips
1)
C.2.1.2.
1)
2)
3)
4)
5)
6)
7)
8)
Procedure
Autoclave chambers and spatula/forceps
Transfer samples to center of wells in base of loading chamber (one sample per well)
Place spacers, if needed, on top of bottom half and position top half of chamber
Slowly tighten bolt (you don't want to do this too fast otherwise you could be doing
"injurious compression"
Add 600 pl of media to each sample (remember to use appropriate precautions if you
are working with radiolabel-containing media
Check to make sure bolt is tight
Place chamber in incubator along side free-swelling control (I usually use an extra
12-well chamber with extra spacers to make sure top is not contacting samples)
When loading protocol is finished, remove samples from wells (radiolabel wash if
needed) and clean chambers thoroughly
C.2.2. Dynamic Compression
C.2.2.1.
1)
2)
3)
4)
5)
6)
Incustat - incubator-housed loading apparatus (sign up for use a week or so
in advance to make sure it is free when you need it)
Loading chamber - polysulfone base consists of 12 wells arranged around the
circumference; top attaches to the upper jaw of the Incustat and has 12 little
pegs whose positions can be fixed individually with the set screws
Spatula/forceps to transfer samples
Loading protocol on computer controlling the Incustat you're using (controls
amplitude of compression, cycling frequency, length of loading, etc)
Media containing radiolabeled species, if desired
P-1000 pipetman and sterile 1000 pil tips
C.2.2.2.
1)
Equipment
Procedure
Adjust height of load cell so that you will be "in range" over the displacement range
you need
131
a. Fix top into upper half of Incustat
b. Position bottom half on the base
c. Lower upper chamber until the pegs are about as far above the bottom of
the base of the wells as the height of your samples (computer control)
d. Adjust position of load cell so that displacement reads -0 pm (the range is
2 mm, so if you're going to need to compress more than 2 mm, you will
want to set the distance higher)
2) Autoclave chamber and spatula/forceps
3) Fix top half of chamber into upper jaw of Incustat (make sure all of the pegs are
pulled up as far as possible) and move upper jaw to near maximum height
4) In sterile hood transfer samples to center of wells in base of loading chamber (one
sample per well)
5) Cover samples with sterile lid/half a petri dish (i.e. use from the well-plate you just
took your samples from if you're not going to culture anything else in that plate) and
transfer to base of Incustat
6) Lower top half of chamber (computer control) until bottom of pegs near the top of
the samples
7) Loosen the set screws and let the pegs (gently) fall until they touch the top of the
samples and then retighten screws
8) Add 600 g1 media to each well
9) Begin loading protocol - watch to make sure the machine is doing what you wanted
it to do
10) When loading protocol is finished, raise top half of chamber (computer control),
remove samples and thoroughly wash chambers
132
APPENDIX D: CELL CULTURE METHODS
D.1.
CHONDROCYTE HARVEST PROTOCOL
(modifiedfrom H. A. Breinan, BWH OrthopaedicsResearch Lab and Kuettner et al.,
1982 [1])
D.1.1. OR Supplies
Scalpel handle (#3)
Scalpel blades (2 #10)
Sterile gloves
If removing whole joint:
Surgical saw
Sterile specimen bags (plastic bags in histo room)
Sterile towels/wrap
Sterile PBS to moisten towels
If removing shavings (ie: for subsequent autologous seeding)
Extra scalpel blades
50 ml tubes with complete PBS
40 ml D-PBS (Gibco #14190-144)
0.4 ml Pen/Strep/Fungizone cocktail (100X; Gibco #15240-062)
(OPTIONAL: label and weigh tubes w/fluid to later determine amount of
tissue harvested)
D.1.2. Lab Supplies
Sterile petri dish (100 mm diameter)
Sterile spatula
Sterile forceps (2)
Sterile razor blades (flat edges are easiest to use)
Sterile centrifuge tube/bottle with sterile stir bar
Clean stir plate in incubator (one that doesn't heat up too much during prolonged
operation)
10-20 ml/joint complete PBS (see above)
D.1.3. Solutions
Pronase solution (20 U/ml) - approx 40ml/2 joints
Dissolve appropriate amount of pronase (Sigma protease type XIV #P 5147) in
10ml DMEM/F12 (Gibco #11320-033, although I don't see any reason not to use the
DMIEM/F12 with HEPES buffer)
133
pronase(mg) = Vx20Ulml
#Units / mg
where V=volume of solution
Sterile filter (0.2 jtm) solution (I use syringe filter; it will take a while for pronase
to dissolve and it will be difficult to pass solution through filter)
Add remaining volume of DMEMIF12 (ex: 30 ml if making 40 ml/2 joints)
Add appropriate amount of 100X pen/strep/fungizone cocktail (ex: 0.4 ml)
Collagenase solution (200 U/ml) - approx 40ml/2 joints
Dissolve appropriate amount of collagenase (Worthington Biochemical CLS2) in
10 ml DMIEM/F12 (this dissolves quickly)
200U / ml
collagenase(mg) = V x
#Units / mg
where V=volume of solution
Sterile filter (0.2 pim) solution (again, I use syringe filter and it should be much
easier to filter than the pronase solution)
Add remaining volume of DMEM/F12
Add appropriate amount of 100X pen/strep/fungizone cocktail
Complete media
1 bottle DMEM/F12 (-530 ml; Gibco 11320-033)
60 ml Fetal Bovine Serum (Hyclone SV 3002.01)
6 ml 10OX Penicillin/Streptomycin/Fungizone cocktail (Gibco #15420-062)
6 ml 100X ascorbic acid solution (final concentration 25 pg/ml)
10OX solution is 0.25 g ascorbic acid (Wako Chemical #D13-12061) in
100 ml DMEM/F12; sterile filtered, store frozen
note: B. Kinner says it should be 25 gg/ml of ascorbic acid and Wako
ascorbic acid is not all ascorbic acid - he uses 50 gg/ml of the Wako
powder
T-75 and/or T-25 tissue culture flasks
D.1.4. Methods:
1. Using scalpel blades, remove slices of articular cartilage from joint surfaces and
place in complete PBS (or in tubes and store on ice if harvesting in OR)
- Don't dig into the hard calcified cartilage or subchondral bone, cartilage
should offer little resistance to the blade; full thickness slices should be 0.51.5 mm thick depending on joint location
- If harvesting whole joint, open joint in sterile hood, cutting ligaments and
removing menisci and you can take cartilage from tibial, femoral and patellar
surfaces
- If slices are large, use razor/scalpel blade to cut into pieces no larger than
3x3x1mm
OPTIONAL: Weigh tubes with tissue to determine amount of AC harvested
134
2. Using forceps and spatula, transfer cartilage pieces into tube/bottle with stir bar
and pronase solution, cap loosely
3. Incubate 1hr (37 0 C, 5% CO 2 ) on spinner plate (or shake every 15 minutes or so if
can't use spinner plate in incubator); cartilage will become sticky in this step and
may stick to the sides of the bottle
4. Remove pronase, taking care not to remove cartilage (if desired, you can
centrifuge the solution you remove and resuspend any pellet in collagenase
solution and return to bottle) and add collagenase solution
5. Incubate overnight (maybe as short as 4-6 hours if you want to do it all in one day
- all tissue should be digested) on spinner plate; make sure cap is loose!!
6. Strain through 70 pm pore strainer into new 50m] centrifuge tube
7. Centrifuge (10 min @ whatever speed you normally spin down cells, -300g)
8. Resuspend in -30 ml complete media; spin and resuspend
9. Count cells and assess viability (should have -7-8x 106 cells/joint with >90%
viable if you remembered to loosen the cap during digestion)
10. Spin (again) and resuspend at -2x10 6 cells/17 ml for monolayer culture OR 4x10 6
cells/ml for seeding into 9mm diameter matrices OR 7.5x 106 cells/ml for seeding
into 4mm diameter matrices
For monolayer cultures, wait 3 days (or until cells are attached) before changing media
D.2.
MONOLAYER CELL CULTURE
(modifiedfrom H. A. Breinan,BWH OrthopaedicsResearch Lab)
Plate chondrocytes at a density of 1-2x10 6 cells/T-75 flask (2x10 6 primary
cells/flask)
2. Add 15-17 ml cell culture media per flask
3. Change media every 2-3 days
4. Passage cells when confluent (-5-7 days):
a. Aspirate media
b. Rinse with -5 ml PBS
c. Add 2-3 ml trypsin (0.05%)-EDTA (0.53 mM) (Gibco #25300-062)
d. Incubate -5 minutes until cells detach from surface
e. Add 3-5 ml media with FBS to inactivate trypsin and collect cell
suspension
f. Rinse flask with additional 3-5 ml media
g. Spin, count, and resuspend as needed
1.
135
D.3.
CELL SEEDING OF MATRICES
D.3.1. Materials needed
Sterile pickups/forceps
Sterile spatula
Sterile pipettes
Sterile pipette tips
Pipetteman (10 pl, 200 pl)
Centrifuge tubes (50 and/or 15 ml)
6ml round bottom tube (for 4mm diameter disks)
Well plates coated with sterile 2% agarose (24-well plate for 4mm diam disks; 12well plate for 9mm diam disks)
Complete DMEMI/F12
IX antibiotics/antimycotics
25 gg/ml ascorbic acid
10% FBS or 1X ITS
Trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; Gibco #25300-062)
D.3.2. Matrixpreparation
Cut to desired dimensions (4mm diameter or 9mm diameter)
Cross-link as desired
Rinse 2x in complete DMEM/F12
Aspirate media and transfer disks to tube
6ml round bottom tube for 4mm diameter disks
15ml or 50ml centrifuge tube for 9mm diameter disks
D.3.3. Cell preparation
Aspirate and rinse media from cells with PBS (-5ml PBS/T-75)
Trypsinize cells from flasks (-3 ml trypsin/T-75; -5 minutes)
Stop trypsinization with FBS containing media (-5ml/T-75)
Spin cell suspension; aspirate media; resuspend cells and take 10-100pl for cell
count
Dilute cell count sample with trypan blue or erythrosin B vital dye - usually 1
part cell suspension: 1 part vital dye (dilution factor=2)-1:4 (dilution factor=5)
- and count cells (viable cells clear, dead cells blue with trypan blue, red with
erythrosin B)
# cells
=
# CellsCounted
x DilutionFactorx SuspensionVolume x 104
# SquaresCounted
Spin cells and resuspend at desired concentration
7.5x10 6 cells/ml for 4mm diameter disks
4x10 6 cells/ml for 9mm diameter disks
136
D.3.4. Seed cells
Add cell suspension to matrices in tube and mix gently
15 disks/ml for 4mm diameter disks
2 disks/ml for 9mm diameter disks
Place on nutator in incubator for 1.5-2hrs
Transfer disks to agarose coated wells
Add media
0.3-0.5 ml for 4mm diameter disks
Iml for 9mm diameter disks
Next day add additional media
To 1ml total volume for 4mm diameter disks
To 2ml total volume for 9mm diameter disks
Change media every 2-3 days
137
D.4.
RADIOLABELING PROTOCOL FOR SEEDED COLLAGEN DISKS
(slightly modifiedfrom MIT Continuum ElectromechanicsLab protocols)
D.4.1. General Outline
1)
2)
3)
4)
Label disks during final 24 hrs of culture with 35-S and 3-H to measure GAG and
collagen synthesis, respectively
Wash unincorporated radiolabel
Lyophilize tissue and measure dry weights
Papain or Proteinase K digestion - use digest for GAG, DNA, and scintillation
counts
D.4.2. Materials needed
1) Latex gloves (double glove)
2) Tape for labcoat sleeves
3) Aluminum foil to line hood
4) Complete DMIEMI/F12 (10%FBS, 1% Antibiotic/Antimicotic, 25gg/ml Ascorbic
Acid)
5) Sterile 15ml or 50ml centrifuge tube
6)
Pipetman - 20ul and/or 200ul
7)
8)
9)
10)
11)
12)
13)
14)
Sterile pipette tips - 200ul capacity
Vacuum flask and sterile pasteur pipettes
Radionuclide (35-S and 3-H)
PBS
Na 2SO 4 and proline
Clean spatula/forceps
48 well culture plate (doesn't have to be sterile, can reuse)
Sterile pipets
D.4.3. Preparation of radioactive media (1.5 ml media/disk; V=1.5n+2ml):
1)
Calculate volume of isotope needed:
For 35-S half-life is 87.4 days:
C(pCi I ml) = 10000MCi / ml x 2
volume 31S = V(ml)x10Ci / ml
C(pCi / ml)
where (x = #days past calibration date)
For 3-H half-life is 8-9 years:
Always at 1000gCi/ml
3
volume H = V(ml)x20yCilml
1000MCi I ml
2)
Wipe hood with 70% EtOH
138
Y?^
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
Line with aluminum foil
Double glove and tape lab coat sleeves
Aliquot calculated volume of media into centrifuge tube
Place radionuclide in hood on aluminum foil and loosen cap
Use sterile technique to aliquot calculated volume of 35-S
Save lml media for calibration
Add calculated volume of 3-H
Save 1ml media for calibration
Remove pipette tips to original wrapper and dispose of in radioactive waste container
Return radionuclides to container and note amount used; return to refrigerator
Warm media in incubator
Rinse pipetman in cold water
Check pipetman and hood with geiger counter
D.4.4. Labeling disks:
1)
2)
3)
4)
Aspirate spent media
Feed disks with radiolabeled media
Return pipette to paper wrapper and dispose of in radioactive waste container
After 8-24 hours, radiolabel is complete
D.4.5. Washing disks:
1)
a.
b.
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
Complete PBS with 0.8 mM Na 2SO 4 and 1.0 mM proline
For 500ml PBS: 0.057 g Na2 SO 4 and 0.057 g proline
Recommended to make fresh wash solution each time; can make frozen stock
solutions of proline and sulfate and dilute in PBS
Aliquot lml PBS/well in 24 well culture plate
Line hood with aluminum foil
Use clean spatula to transfer disk to top row of 48 well plate
Place in refrigerator for 15 minutes
Transfer disks to 2 "d row and refrigerate for 15 minutes
Repeat for a total of four washes
Dispose of radiolabeled media in sink and note amount of radioactivity disposed
Place each sample in a labeled vial
Optional: Save PBS from last row (1 from each group) to make sure thoroughly
washed (radioactive counts - background)
Lyophilize, measure dry weight, digest... (for DNA, GAG, scintillation counting)
REFERENCES
Kuettner, KE, Pauli, BU, Gall, G, Memoli, VA, and Schenk, RK. Synthesis of
1.
cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture
characteristics, and morphology. J Cell Biol 1982; 93:743-750.
139
APPENDIX E: BIOCHEMICAL ASSAYS
(from MIT Continuum ElectromechanicsLab protocols)
E.1.
LYOPHILIZATION
Lyophilize samples for 16-24 hours (Labconco Freeze Dry System. Temperature set at 60*C; vacuum pulled to at least 100 pmHg)
E.2.
SAMPLE DIGESTION
E.2.1. Papain Digestion
Digest samples overnight (12-24 hours) at 60 0 C in a 125 pg/ml papain solution:
For 100 ml solution: 90 ml phosphate buffered EDTA (PBE, see below)
0.01M L-cysteine (0.176 g dissolved in 10 ml PBE and sterile
filtered (0.2 ptm) into 90ml PBE)
0.5 ml papain (Sigma suspension 25 mg/ml)
PBE (1L):
0.04M Na 2HPO 4 (5.68 g/L)
0.06M NaH2PO 4*H 20 (8.28 g/L)
0.01M Na 2EDTA*2H 20 (3.72 g/L)
Dissolve above salts in 900 ml distilled water
Adjust pH to 6.5 with IN HCl or IN NaOH
Bring to volume with distilled water
E.2.2. Proteinase K Digestion
Digest samples overnight (12-24 hours) at 60'C in a 100 pg/ml proteinase K solution:
For 100 ml solution: 99 ml Tris-HCl buffer (see below)
1 ml proteinase K stock solution (50 mg proteinase K powder;
Roche #745-723, resuspended in 5 ml Tris-HCl buffer)
Tris-HCl buffer (IL): 0.05M Tris HCl (7.88 g/L)
1mM CaCl 2 (147 mg/L)
Dissolve above salts in 900 ml distilled water
Adjust pH to 8.0 with iN NaOH
Bring to volume with distilled water
140
E.3.
GAG ASSAY USING DIMETHYLMETHYLENE BLUE (DMMB) DYE
E.3.1. Summary
DMMB dye binds to sulfated glycosaminoglycans and changes color from blue to
purple based on concentration of GAG in solution (adapted from JM protocol 3/1/94)
E.3.2. Equipment
Perkin Elmer, Lambda 3B spectrophotometer
Warm up for 30 minutes
Set wavelength to 525 nm
Blank the spec with cuvette (4.5 ml polystyrene cuvette) of air
OR
Maxy Plate Reader (Molecular Devices, Vmax)
Set to MOD2, OPD
Set wavelength to 520 nm
E.3.3. Protocol
Run Standards (0, 31.25, 62.5, 125, 250, 500, 1000 pg/ml for Elmer OR 0, 3.125, 6.25,
12.5, 25, 50, 100 gg/ml for Maxy) in triplicate
20 pl standard
2 ml (for Elmer) OR 200 pl (for Maxy) DMMB dye
Blank with 0
Create standard curve (should be quadratic)
Run Samples in duplicate
20 pl sample
2 ml (for Elmer) OR 200 p1 (for Maxy) DMMB dye
Record absorbance readings
Determine GAG concentration from standard curve
E.3.4. GAG standards
0.01 g shark chondroitin 6-sulfate
10 ml PBE
Sterile filter (0.45 gm)
Stock concentration 1000 pg/ml
Serial dilutions in PBE (for papain-digested samples) OR Tris-HCl (for proK-digested
samples)
141
E.3.5. DMMB dye solution
0.016 g 1,9 dimethylmethylene blue dye
5 ml 100% ethanol
Mix in 15 ml centrifuge tube to dissolve (2-3 hours)
2.37 g NaCl
3.04 g glycine
950 ml distilled water
Add dissolved DMB to solution
Rinse tube 2x w/100% EtOH and add wash to solution
8.7 ml 1N HCl
0.2 g sodium azide (toxic!!)
pH to 3.0 with 1N HCl
Bring to final 1L volume with water
Filter solution through large filter paper
OD reading at 525 nm (4.5 ml cuvette) should be in range 0.20-0.28
Store in brown/amber bottle
Solution stable 3 months
Anything with DMMB dye is ORGANIC WASTE!
GAG standards 093099
120
100
E
80
0D 60
y = 309.55x 2 + 122.84x - 26.997
R2 = 0.9996
40
20
0
0
0.1
0.2
0.3
Optical Density (520 nm)
0.4
0.5
Figure E.1: Typical standard curve for GAG (shark chondroitin-6-sulfate) standards on
Maxy
142
E.4.
DNA ASSAY USING HOECHST 33258 FLUORESCENT DYE
(Method developed by Kim, et al., 1988; protocol updated/detailedby A. M. Loening,
1997)
E.4.1. Summary
Bisbenzimidazole fluorescent dye Hoechst 33528 interacts with DNA dissociated
from proteins of the nucleoprotein complex during standard papain digestion
E.4.2. Equipment
SPF-550c, SLM Instruments Spectrafluorometer
Excitation wavelength 365 nm
Emission wavelength 458 nm
Excitation bandpass 10 nm
Emission bandpass 5 nm
Reference voltage 445
Reference gain 1
Fluorescence voltage 850
Fluorescence gain 1
Filter 3 seconds
48 mm acrylic cuvettes (#67.755 Sarstedt, Newton NC)
(for details on use of the spectrometer, ie: how not to burn out very expensive equipment,
see detailed protocol outlined by AML 11/18/97)
E.4.3. Protocol
Run standards (0, 0.3125, 0.625, 1.25, 2.5, 5, 10 jig/ml) in triplicate
100 pl standard
2 ml dye
Create standard curve (should be linear)
Run samples in duplicate
100 p1 sample
2 ml dye
Determine DNA content from standard curve
Sample/dye complex should reach equilibrium in about 15 seconds and should be stable
for at least 2 hours (less than 5% decrease in fluorescence)
Record emission readings automatically on computer by hitting 'space bar'
E.4.4. DNA standards
Stock concentration 10 gg/ml
1 mg calf thymus DNA
100 ml sterile PBE
143
Serial dilutions in PBE (for papain-digested samples) OR TrisHCl (for proteinase Kdigested samples) to 5, 2.5, 1.25, 0.625, 0.3125, 0 pg/ml
E.4.5. DNA dye solution
450 ml distilled water
50 ml 10x TEN buffer
50 pl Hoechst 33258 dye (10000x) (light sensitive; carcinogenic)
Cover beaker in tin foil or use amber bottle to minimize exposure to light
TEN buffer (10x)
100 mM Tris
10 mM Na 2EDTA
1.0 M NaCl
pH 7.5
Anything with Hoechst dye is ORGANIC WASTE!
DNA Standards 093099
.
E
12
10-
y =13.98x - 0.6106
R= 0.9996
8
c,)
86-
0
20
0
0.2
0.4
0.6
Absorbance
0.8
Figure E.2: Typical standard curve for DNA (calf thymus DNA) standards
144
1
SCINTILLATION COUNTING OF RADIOLABELED SAMPLES
E.5.
E.5.1. Summary
Radioactive counts of sample digests and calibrated media are counted with LKB
E.5.2. Protocol
Combine 100 [d of sample digest or calibrated media with 2 ml of scintillation fluid
(EcoLume, ICN #88247002). Assay samples in duplicate.
Count 3 minutes/sample, with 3 H counts in channel 1 and 3 5S counts in channel 2 (scint
H,S+C+R 3:00 filename).
After samples are counted, the vials with scintillation fluid and radioactive digest should
be dumped into the barrel for liquid scintillation vials.
E.5.3. Calculations
The amount of radioactivity, [3 H] or [3 5S], is calculated based on the counts per minute
(cpm) from channels 1 (C1 ) and 2 (C 2 ) as follows:
([
H f]
[35j]
k il
k12
k 21
k22
C ,
C2
The coefficients k11 , k12, k21, and k22 are determined from the counts of the media
samples taken when adding the isotopes to the media:
From the first media sample (35S only, no 3H) the matrix equation becomes:
([ 35 S] reflects concentration of 3 5S added to media; superscript "S" reflect that counts
are from first media sample with 3 5S only, subscripts denote the channel counted)
0= k Cs +
[ 35S] =
CS
Cs +
CS
From the second media sample (35 S and 3 H) the appropriate equations are:
([ 3 5S] and [3 H] reflect concentrations of 35 S and 3 H, in gCi/ml, added to media,
respectively; superscript "S,H" reflect that counts are from second media sample
with both 3 5 S and 3H, subscripts denote the channel counted)
[ 3H] = k CS'H + k2C
[ 35S] = k CSH +
2
,H
145
Solving these 4 equations for the 4 unknown k's:
k1 = -k
[3 H ]
C_
CS C sH
_
2
[-H
C~S
k1 k= =-k CS
CH
C
CS,H
S3 I c~SH
k2 =
CSH
k
C
~
-1
C
CI
2
C
2
s
2
S
-
S
Use the calculated values of [3H] and [35S] to determine the percent of the total available
radiolabeled proline and sulfate that was converted into macromolecular form (%
incorporated, aproeine and asunfate, respectively). Assuming that the same percent of
radiolabeled proline/sulfate and unlabeled proline/sulfate was converted. Furthermore,
assume that the amount of proline/sulfate added in radiolabeled form is insignificant
compared to the concentrations of (unlabeled) proline/sulfate in the media. There is 150
nmol/ml proline and 406 nmol/ml sulfate in DMEM/IF12 media. The amount of
proline/sulfate incorporated into macromolecular form during the radiolabel period is
then determined as follows:
pro =
pn(1
5nmol / ml)V
suif = cxsulfate (4O6nmol / ml )V
where V is the volume of media fed to the cultures, in milliliters.
Incorporation data can be normalized to the time of radiolabel and the amount of DNA in
the construct to yield the rate of incorporation normalized to cell content (nmol/pg
DNA/hr).
146
DOWEX-50W SEPARATION OF PROLINE AND HYDROXYPROLINE
E.6.
(taken almost verbatim from Doong, Sah, and Kerin)
E.6.1. Summary
Ion-exchange chromatography separates substances by reversible absorption to a
matrix (resin) with exchangeable cations and anions. The resins (stationary phase) of a
cation exchanger are negatively charged and are able to bind cations in the mobile phase.
Dowex 50W-X8 (50W means it is a strong acidic cation exchanger, X8 means 8% of
cross-linker [2]) is a cation exchanger.
In ion-exchange chromatography, the separation of several substances in a
solution is based primarily on charge, but also secondarily, on charge density (solvated
equivalent volume), charge distribution, polarizability and molecular size [3,4]. The
separation of proline and hydroxyproline (both carrying a charge of +1 when protonated
in 0.9N HCl) is due to the following properties: (1) hydroxyproline is more polarizable
(hydrophilic) than proline. The hydroxyl group on hydroxyproline can form a hydrogen
bond with water molecules. The aromatic hydrocarbon backbone of Dowex resins is
strongly hydrophobic. (2) hydroxyproline has a lower charge density (a bigger solvated
volume). Therefore, hydroxyproline elutes out of the Dowex column earlier than proline.
This is true for all hydroxylated amino acids and their non-hydroxylated analogues, such
as serine (-CH2-OH) and alanine (-CH3).
E.6.2. Column Preparation
E.6.2.1.
Equipment needed:
0.7cm x 10cm column (BioRad #737-0711)
IL 0.9N HCl
250ml erlenmeyer flask
DOWEX 50W-X8 beads - 400 mesh size (Sigma, Dowex - 50W #50X8-400)
Stock solution: 20mg/ml Proline in 0.9N HCl
Stock solution: 20mg/ml Hydroxyproline in 0.9N HCl
Stock solution: 0.2pCi/ml labelled proline in 0.9N HCl
1.5ml Methacrylate Cuvettes (not standard polystryrene ones - Fisher #14-385938)
E.6.2.2.
Packing the ion-exchange column
1.
Wash 50g Dowex 50W-X8 four times with 80ml 0.9N HCl each time in a 250ml
erlenmeyer flask and resuspend it with another 80ml 0.9N HCl (It takes about 3.35gm
Dowex per 0.7cm x 10cm column).
2. Degas the elution buffer 0.9N HCl with a stirring bar for 5 minutes and degas Dowex
suspension (Dowex 50W-X8 + 0.9N HCl) without a stirring bar for 15 minutes.
(Degas using vacuum in sterile hood) 3. Mount a 0.7cm x 10cm column. The volume
of this column is
-
3.85ml.
147
3. Set up the peristaltic pump speed at 0.68ml per minute (for the TRIS pump, this is 25
(lOX)). That is, equivalent to a linear flow rate of 1.77cm / minute (0.68ml/3.1416 x
(0.7/2) x (0.7/2) ).
4. Fill half of the column with 0.9N HCl (use siphon method from bottle above), turn
the pump on and add Dowex suspension gradually. When the gel beads are added up
to about 1/2 inch from the top of the column, stop packing the column.
E.6.2.3.
1)
2)
3)
4)
5)
6)
7)
8)
Conditoningthe column
To make up the conditioning solution, mix the following:
a) 12.5pl of proline stock (0.25mg)
b) 12.5gl of hydroxyproline stock (0.25mg)
c) 475gl of 0.9N HCl.
Set up the pump speed at 1 ml per 3 minutes (14.5 (10X) on the TRIS pump. That is,
the linear flow rate is about 0.87cm/minute).
Remove the siphon feed of HCl, turn on the pump, and allow the level in the column
to drop to the top of the beads. Stop the pump.
Carefully add the 500pl of conditioning solution to the column using a pipette.
Allow beads to settle.
Turn pump on, and lower level to top of beads again.
Reattach the HCI siphon feed.
Elute 35 mls of 0.9N HCl. (-1:45 hrs)
E.6.2.4.
Calibratingthe column
1) Proline
a) To calibrate proline peak make up solution of:
100ul of stock labelled proline (= 0.02 Ci, which is about 20,000 cpm, and
12.5ul standard proline stock (carrier)
387.5pl 0.9M HCl.
b) Set up the pump speed at 1 ml per 3 minutes (14.5(1OX) on the TRIS pump, add
the solution as before, set the automated collector to collect a fraction every
3minutes, and collect next 35 fractions.
c) Add 2ml scintillation fluid (Fisher, ScintiVerse Bio-HP) to each fraction and
count for 5 minutes.
2) Hydroxyproline
a) To calibrate hydroxyproline peak make up:
100pl hydroxyproline stock solution (2mg)
4 0 0 gI 0.9N HCl
b) Set up the pump speed at 1 ml per 3 minutes (14.5(1OX) on the TRIS pump), load
the solution and collect next 35 fractions.
c) Transfer each fraction into methacyrylate 1.5ml cuvettes (standard polystryene
cuvettes are no good at the wavelength used) and read the absorption in the
spectrophotometer at 210nm (against dH2O).
d) Alternatively, one can use 3H-Hydroxyproline with carrier hydroxyproline (e.g.
0.25mg) to calibrate hydroxyproline peak. However, it is very expensive to do
148
that ($576.00 for 1 mCi of 3H-Hydroxyproline vs $241.00 for 1 mCi of 3Hproline).
E.6.3. Running a Sample
E.6.3.1.
Sample preparation
Transfer 500pl of the sample to a 2ml borosilicate vial
Freeze and lyophilize the sample.
Using disposable 1 ml pipet, add 2 50p 6N HCl to the pyrex tube after
lyophilization. If using pipetman, it must be disassembled and cleaned afterwards
(since the vapor of 6N HCl may erode the piston inside the pipetman and it costs
$83.00 to replace that piston).
4) Place the pyrex tubes containing samples in the oven at 1 10 0 C overnight [5].
5) After hydrolysis, cool down the samples in air for about an hour.
6) Dilute the sample solution from 6N HCl to 0.9N HCl by adding 1416g1 dH20 into
the pyrex tube.
7) Add carrier proline or hydroxyproline, if the amount of proline or hydroxyproline in
the sample is less than 200gg/ml. For human I added 10pl of Proline and 10pl of
hydroxyproline to give 200gg of each
8) Filter the diluted sample solution through a sterile Millex-GS 0.22 m filter unit.
1)
2)
3)
E.6.3.2.
Sample loading andfraction collecting
Set up the pump speed at 1 ml per 3 minutes (14.5(1OX) on the TRIS pump), and the
fraction collector at 3 minutes per fraction.
2) Using pipetman with blue pipet tip, load 500ul sample to the column.
3) Discard the first 0.5ml fraction and collect the next 40 fractions.
4) Before turning the peristaltic pump off, disconnect the column from the pump.
Remove the tubes from the pump (otherwise the tubes get crimped). If you're not
going to use the columns for a long time, pump out the 0.9N HCl in the tubing with
DI water, since HCl can corrode the tube.
5) After the separation by ion-exchange column, add 2ml scintillation fluid (Fisher,
ScintiVerse Bio-HP) to each fraction. The vials need to be vortexed on their side.
The contents will go cloudy. You have the option of shaking for longer until it goes
clear, or waiting for a few hours for them to clear on their own. Count for 5 minutes.
6) The total cpm of the 500ul sample loaded to the column should also be determined
by taking another 50ul of the sample mixed with 950ul 0.9N HCl and 2ml
scintillation fluid. After scintillation counting, the cpm obtained times 10 is the total
cpm loaded to the column.
1)
E.6.3.3.
1)
2)
3)
Counting
For dual labelled (3H and 35S) samples, "scint D+C+R 5 filename".
For single labeled (3H) samples, "scint H+C 5 filename".
If the S35 read has peaks in the 3H fractions, the spillover should be corrected.
149
E.6.4. Calculations
1)
2)
To compute the average background:
Take the sum of cpm of the last 5 fractions (31+32+33+34+35)/5
To calculate the total cpm of hydroxyproline peak (TH):
a) Make a table of accumulated 3H-cpm for each fraction
b) Let: EH = the accumulated cpm of the ending fraction of hydroxyproline peak
BH = the accumulated cpm of the beginning fraction of hydroxyproline
peak
SAB = Sum of the average backgrounds
then:
3)
TH = (EH - BH) - SAB
To calculate the total cpm of proline peak (TP):
a) Use the table of accumulated 3H-cpm of each fraction
b) Let EP = the accumulated cpm of the ending fraction of proline peak
BP = the accumulated cpm of the beginning fraction of proline peak
SAB = Sum of the average backgrounds
then:
TP = (EP - BP) - SAB
4)
To compute the % of recovery:
The total cpm eluted from the column is divided by the total cpm loaded to the
column. That is, (TH + TP)/(the cpm of 50ul sample + 950ml 0.9M HCl) x 10 Usually,
above 90-110% recovery is acceptable.
5) The % of hydroxyproline peak is calculated by TH / (TH + TP) and the % of proline
peak is calculated by TP / (TH + TP).
Sample Calculation
1. The average cpm background is 13.2 (= (10.2+12.4+18.7+12.7+12.2)/5)
2. EH is the 13th fraction and the accumulated cpm is 888.9
BH is the 7th fraction and the accumulated cpm is 172.5
SAB = 13.2 x (13 - 7) = 79.2
TH = (888.9 - 172.5) - 79.2 = 637.2
3. EP is the 25th fraction and the accumulated cpm is 4268.3
BP is the 14th fraction and the accumulated cpm is 925.2
SAB = 13.2 x (25 - 14)= 145.2
TP = (4268.3 - 925.2) - 145.2 = 3197.9
4. The cpm of 50ul sample + 950ul 0.9M HCl is 456.62
The % of recovery = (3197.9 + 637.2) / (456.62 x 10) = 84%
5. The % of hydroxyproline = 637.2 / (3197.9 + 637.2) = 16.6%
The % of proline = 3197.9 / (3197.9 + 637.2) = 83.4%
NOTES
1. With a short labelling time (less than 8 hours), there is a significant unknown peak
eluting between the hydroxyproline and proline peaks (fraction numbers 12-16,
usually). This unknown peak becomes even more significant when the protein
synthesis and 3H-hydroxyproline formation is blocked by cycloheximide. However,
with bovine/human cartilage explants, this unknown compound disappears almost
completely from radiolabelled disks during a one-day chase.
150
Hydroxyproline and Proline separation
25002000-
35S
- -
3H
--
1500 -
1000 -
500-
01
5
9
13
21
17
25
29
33
37
Fraction Number
Figure E.3: Typical radiolabel counts from Dowex 50W-X8 separation
of proline and hydroxyproline in canine chondrocyte-CG cultures. First
and second peaks are hydroxyproline, third peak is proline, first peak is
unknown.
151
E.7.
PD-10 SEPARATION OF MACROMOLECULAR AND FREE LABEL
(modifiedfrom Sah protocol)
E.7.1. Summary
Gel filtration chromatography separates substances based on molecular weight.
PD-10 columns are Bio-Rad's pre-packed, disposable columns containing hydrophilic
Sephadex G-25 beads (dextran cross-linked with epichlorohydrin) for rapid desalting and
buffer exchange [2,6]. They are used in this instance to separate macromolecular label
from free (unincorporated label), for example in spent media following a radiolabel.
Macromolecules elute with the void volume while the unincorporated label runs through
the column more slowly.
E.7.2. Equipment
Peristaltic pump set at 1 ml/minute (TRIS pump set at (10X) 38) - this is
approximately the flow rate if using gravity
Fraction collector and vials
Pre-packed PD-10 columns (Pharmacia #17-0851-01)
Dextran blue (10 mg/ml) - this is used to mark the void volume
Phenol red (5 mg/ml)
1% chondroitin sulfate
4% bovine serum albumin
Running buffer
2M Guanidine HCl
0.5M sodium acetate
1mM sodium sulfate (Na 2 SO 4 )
1mM L-proline
pH to 6.8 with glacial acetic acid
filter through 0.2 pm membrane and degas
Add sodium azide (NaN3 ) to 0.02% (prevents bacterial growth)
(I made stock solutions of 4M GnHCl, 1M sodium acetate, 1M Na2 SO 4 , IM
L-proline, and 2% NaN 3 and mixed in appropriate quantities)
E.7.3. Column preparation
1)
2)
3)
4)
5)
6)
7)
Let column (stored at 4'C) to equilibrate to room temperature
Cut off bottom tip and place cap on end
Pour off excess fluid from top of column
Mount the column on support (could use large test tube rack)
Equilibrate column with running buffer by running 20-25ml of running buffer
through the column
Mix 1 ml 4% BSA, 10 p1 chondroitin sulfate solution, 30 p1 Dextran blue solution,
10 p phenol red solution
Run solution through column until all phenol red has eluted (-15 ml)
152
E.7.4. Sample loading and collection
1)
2)
3)
4)
5)
6)
7)
8)
9)
Mix 200 pl of sample, 20-40 g1 of Dextran blue solution, and 20 pl of phenol red
solution
Remove line drawing buffer from reservoir and run fluid level down to top of
column
Load 200 pl of mix to top of column
Run column until sample fully enters column (discard eluent)
Replace reservoir line and add 1-2 ml buffer to top of column
Start running sample through column (should see blue band running ahead of red
band)
Collect 30 fractions (0.5ml = 0.5 minutes per fraction), if you want elution profile
OR
Discard first 2.5-3.0 ml and collect next 3.0 ml (whatever elutes with the Dextran
blue), if you only want a macromolecular count. Continue running column until
phenol red runs through (total elution volume of -15ml).
Add 2 ml scintillation fluid to 0.5ml fractions (or to 100 p of pooled Dextran blue
eluent) and count radioactivity
Wash column with -5ml buffer
Store columns at room temperature. Make sure top and bottom of columns are capped.
Do not let columns run dry.
PD-10
coLO
0.2 0.15
o CIC0.1
0= 2
0
0
20
10
30
f raction
Figure E.4: Elution profile for sample digest mixed with 0.01 pCi free
35S-sulfate. Dextran blue eluted with the first peak (fractions 6-11) which
corresponds to the void volume in which the macromolecules are found.
The second peak is the free 35 SS04
153
600500400CD)
300-
1 200100-
00
5
10
15
20
25
30
fraction
Figure E.5: Elution profile for digest from a 7 day, free-swelling type II
CG EDAC cross-linked sample. Dextran blue eluted with the first peak
(fractions 6-11) which corresponds to the void volume in which the
macromolecules are found. Counts from the macromolecular peak
accounted for 86% of the 35S-sulfate counts in this sample.
154
E.8.
S-1000 MOLECULAR WEIGHT SEPARATION OF GAG MOLECULES
E.8.1. Summary
Again, this is a gel filtration method used to separate molecules based on their
molecular weights. S-1000 is a superfine Sephacryl gel (allyl dextran covalently crosslinked with N,N'-methylene bisacrylamide) for the fractionation of large polysaccharides
with molecular weights ranging from 5x10 5 - >108 [6]. In this case, we are using the S1000 column to separate proteoglycan aggregates and monomers. Aggregate will elute
first. Chromatography with the S-1000 serves the same purpose as chromatography with
the CL-2B columns (Sepharose 2B reacted with 2,3-dibromopropanol [6]), but the higher
peak Kay on the S-1000 allows for a more clear resolution of proteoglycan aggregates
from monomers on the S-1000 [7].
E.8.2. Equipment
Peristaltic pump set at 9 ml/hr (on the TRIS pump this is IX 69)
Fraction collector and scintillation vials
S-1000 beads (Amersham Pharmacia Biotech #17-0476-01)
Glass column 50cm x 1cm (BioRad #737-105 1)
Running buffer
0.5M sodium acetate
0.1% Triton X-100
0.02% sodium azide
pH 6.8
E.8.3. Column preparation
1)
2)
3)
4)
5)
6)
Resuspend and rinse S-1000 beads in 0.5M sodium acetate
Degas solution
Fill column -1/4 full and start running pump (lml/3min)
Pour S-1000 slurry into column (use a glass rod to help get slurry into column)
Pack column until -5 cm from top
Run 80 ml through column (9 ml/hour)
E.8.4. Column calibration
1)
To calibrate V0 , I used 50 jig of calf thymus DNA (50 jl of 1 mg/ml stock in PBE
further diluted in 150 pl of S-1000 running buffer). Sample was eluted through
column and fractions collected every 8 minutes (1.5 ml/fraction). 100 pl of fractions
#11-20 were assayed for DNA content using the Hoechst dye assay. (Note: 5 pg
DNA would have been enough).
2)
To calibrate Vt, I ran 0.1
[tCi
of 3 H 2 0 (1 pA of 5mCi/ml solution dated 1994 diluted
in 1000 gl of S-1000 buffer) in 200 pl of buffer through the column and collected
fractions every 8 minutes (1.5 ml/fraction). Fractions #21-50 were assayed for
radioactivity by scintillation counting.
E.8.5. Sample loading and collection
1)
Resuspend lyophilized sample in -1 ml running buffer (adjust volume to get same
count concentration for all samples if you want to compare peaks across samples)
155
2)
3)
4)
5)
6)
Add 200 pl of sample to top of column
Run sample through column (9 ml/hour, 11.5 cm/hr)
Collect 60 fractions, 0.6 ml (4 minutes) per sample
If desired, take 20 gl of each fraction for DMMB GAG analysis
Add 2 ml scintillation fluid and count 35S radioactivity (5 minutes/fraction)
Kav
A
1.2
0
1.0
0.8
0.6
0.4
0.2
0.0
no HA
1
-- 4 mg/m I HA
- ---GAG
o 0.8-
E
0.4E
0
E 0 0.X
0
15
20
30
25
35
40
45
Elution Volume (ml)
Figure E.1. S-1000 elution profile. Elution profile for proteoglycans synthesized during a 24 hour
(day 7-8) 35S-sulfate radiolabel under free-swelling conditions compared to the total sulfated GAGs
extracted from calf articular cartilage (A). KavO= corresponds to Vo, the peak of the DNA elution and
3
Kav=1.0 corresponds to Vt, the peak of the H 2 0 elution.
E.8.6. Variations
The above protocol will separate aggrecan and monomer in the digest. Some of the
monomer may have the ability to aggregate.
1) To determine the percentage of proteoglycan monomer with a high affinity for
hyaluronate (i.e. high affinity for aggregation), incubate samples with 4mg/ml
rooster comb hyaluronic acid (Sigma #H-5388) 24 hours at 4'C prior to
chromatography
2) To determine the maximum aggregatability, mix samples with 5% (w/w) hyaluronic
acid and 5% (w/w) link protein, adjusted to 4M guanidium chloride for 2 hours at
4'C, followed by 24 hour dialysis against 0.15M sodium acetate, pH 6.8 at 4'C prior
to chromatography (I never did this, but it is described in Sah, et al., 1990)
156
E.9.
COLLAGEN EXTRACTION
E.9.1. Summary
Collagen peptides are extracted from cultures using pepsin. This is done prior to
SDS-PAGE (Western analysis) or CnBr digestion. An alternative method for extraction
is to boil the samples in sample buffer, but this is non-specific and gels won't be as clean;
same goes for doing the CnBr digestion of samples directly.
E.9.2. Materials
Pepsin (Sigma #P-6887)
95% ethanol
0.4M NaCl + 0.1M Tris-HCl pH7.
Washing solution: 3 Parts 95% Ethanol + 1 Part) 0.4M NaCl ,0. 1M Tris-HCl
Pepsin digest solution; 1-2 mg pepsin in 0.2M NaCl
E.9.3. Procedure
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
Lyophilize sample
Add 0.5ml of pepsin solution
Stir it for 48 hours at 4'C
Neutralize it with 1ON NaOH (about 25gl)
Raise [Na ] to IM NaCl with Xg NaCl
0.8=X/58 x1000/0.5
Stir overnight at 4*C
Spin at 600g for 20 minutes, Save the supernatant
Precipite collagen by adding NaCl up to 4.5M with Xg NaCl
3.5=X/58 x 1000/0.5
Stir overnight at 4*C
Spin at max speed in microcentrifuge for 1 hour
Wash pellet with washing solution (3 parts 95% ethanol+ 1 part 0.4M NaCl, 0.1M
Tris-HCl pH7) 2 x 10minutes
Lyophilize dry
Keep it at -20'C until ready to analyze
157
E.10. CYANOGEN BROMIDE (CNBR) CLEAVAGE OF COLLAGEN
*** Cyanogen bromide is a very toxic and unstable chemical uses it under chemical hood
and keep it at 40C****
E.10.1.Summary
Cyanaogen Bromide cleaves collagen into well-characterized peptides. The SDSPAGE (see Appendix F.1, except use a 15% acrylamide gel to separate the peptides)
pattern of collagen peptides has been established and can be used to determine the type of
collagen that was cleaved.
E.10.2.Reagents
Cyanogen Bromide (CnBr): JT Baker F-946-03 or Sigma #C-6388
Chondrocyte Culture
70% formic acid.
550mg CnBr/ ml in 70% Formic Acid.
E.10.3. Procedure
1)
2)
3)
4)
5)
6)
7)
8)
Constructs may be wet or dry but cannot be digested using other methods.
Approximate concentrations of collagen/weight of constructs are as follows:
In order to have 3 mg collagen:
Articular cartilage: 6mg dry or 35mg wet
Tissue Engineered: 15mg dry or 150 mg wet
Place Tissue containing about 3 mg of collagen in 1 ml 70% formic acid in a 1.5m1
eppendorf tube. (For more or less tissue use more or less formic acid respectively to
keep the concentration approximately the same.)
Place formic acid/tissue in 60'C water bath for 1 hour.
Bubble N2 or other inert gas into mixture.
Add CnBr to 70% formic acid to make a stock containing 550mg CnBr/1ml 70%
formic acid. Add 100 ul of the CNBr/formic acid stock per 1 ml of tissue formic
acid. (Note: carefully use CnBr in the hood only)
Stir or mix overnight at room temperature.
Add water to stop reaction (5X volume).
Freeze dry to remove solvent. Can store in freezer now if desired.
158
REFERENCES
1. Kim, Y.J., Sah, R.L., Doong, J.Y., and Grodzinsky, A.J. Fluorometric assay of DNA
in cartilage explants using Hoechst 33258. Anal Biochem 1988; 174:168-176
2. BIO-RAD, Ion Exchange Manual
3. Freifelder, D. Physical Biochemistry. W.H. Freeman and Company. New York.
1982; 248-255
4. Peters, D.G., J.M. Hayes and G.M. Hieftje. A Brief Introduction to Modem Chemical
Analysis. W.B. Saunders Company. 1976; 374-382
5. Stem, B.D., G.L. Mechanic, M.J. Glimcher and P. Goldhaber. The resorption of bone
collagen in tissue culture. Biochem. Biophy. Res. Commu. 1963; 13: 137-143
6. APBiosystems Gel Filtration theory and practice
7. Sah, R.L., Grodzinsky, A.J., Plaas, A.H.K., and Sandy, J.D. Effects of tissue
compression on the hyaluronate-binding properties of newly synthesized
proteoglycans in cartilage explants. Biochem J 1990; 267: 803-808
159
APPENDIX F: GEL ELECTROPHORESIS METHODS
This is the collection of protocols for various assays utilizing SDS-PAGE. Note
that I did NOT actually use most of these protocols myself as Han-Hwa Hung and Robyn
Marty-Roix graciously helped me in these areas.. .Thank you, thank you, thank you!!
These are their protocols.
F.1.
SDS-PAGE
(fromH.-H.Hung, MIT Continuum ElectromechanicsLab)
F.1.1. Summary
Vertical gel electrophoresis used to separate proteins by molecular weight. After
running an SDS-PAGE, the gel can also be used for Western blot analysis
(immunostaining) or fluorography (of radiolabeled proteins).
F.1.2. Reagents:
(a) 30% Acrylamide/bis
29.2g
Acrylamide
N-N- Bis
0.8g
Dissolve in 100ml dd H2 0
Keep in brown bottle or use aluminum foil wrap the bottle.
(b) 1.5 M Tris-Cl+ 0.4% SDS pH=8.8 (Running Buffer for Running Gel)
Tris
18.15gm
SDS
0.4g
pH=8.8
Dissolve 18.15 gm Tris in 90 ml ddH 20 and use HCl to PH=8.8.
Add 0.4g SDS bring up to 100ml
(SDS is a very sticky solution. It will sticky on the electrode. The SDS will not
change the pH. Always pH the solution before you add SDS.)
(c) 0.5M TrisCi +0.4% SDS pH=6.8
Tris
6.05g
SDS
0.4g
Use HCl to pH to 6.8 in 100ml.
(d) 10% Ammonium Persulfate (Make fresh every time)
0.lg in 1.0ml ddH 20
(e) TEMED (N,N,N',N'-Tetramethylethylamediamine)
Sigma #T-8133
(f) 1oX Chamber Buffer (25mM Tris, 1.92M Glycine, 1% SDS PH-8.3)
Tris
30.275g
Glycine
144.192g
in 1 liter
SDS
log
160
(g) 2X Sample buffer; 0.125 M Tri-Cl pH6.8, 4% SDS, 20% glycerol, 5% 2mercaDtoethnol, 0.002% Bromophenol Blue.
mlI/00ml
mlI10ml
Final Conc.
Stock solution
25.Oml
2.50ml
0.125 M pH 6.8
0.5 M Tris-Cl, PH6.5
40ml(4.0g)
4.Oml(0.4g)
4%
10%SDS
20.Oml
2.00ml
20%
Glycerol
5.Oml
0.5ml
5%
2-mercaptoethanol (bME)
2.0ml
0.20ml
0.002%
0.1% Bromophenol Blue
(in ethanol,=100x)
Water
0.8ml
T_
8.Oml
(h) 0.1% Coomassie blue in 50% methanol
Weigh 1 g of Coomassie blue dissolve in 500ml methanol.
Stir it about 30 min then add 500ml ddH20.
(i) Destain solution (7% Acetic acid)
70 ml of Glacial acetic acid + 930 ml of ddH 20.
(j) 7% Acrylamide gel:
5.Oml
ddH20
2.33ml
30% Acrylamide
2.5ml
1.5% Tris-Cl runing buffer(pH=8.8)
100 ul
persulfate
10% Ammonium
10 ul
TEMED
the acrylamide will start to
TEMED
add
you
Once
last.
TEMED
the
add
Always
polymerize.
(k) Stacking Gel
3.1ml
ddH 20
1.25ml
Stacking buffer (0.5M Tris-Cl, pH=6.6)
650ul
30% Acrylamide
10Oul
10 % Ammonium Persulfate
loul
TEMED
(1) Running gel:
dd H 2 0
5.Oml
30 % Acrylamide
1.5M Tris running buffer (pH=8.8)
10% NIl 4 persulfate
TEMED
2.33ml
2.50ml
10Oul
loul
F.1.3. Procedures
F.1.3.1.
Making the Running Gel
Warm up the 30% Acrylamide and running buffer to room temperature.
Make the 7% running gel according to recipe.
Assemble the casting stand: place on gray strip, screw the 2 pieces of CLEAN glass into
place with 0.75mm spaces in between (wipe glass with Kimwipe). The smaller glass is in
front, close to you, while the larger glass is behind the smaller one. Place the combs on
top (for reference only; we won't use the combs until later)
161
Use 5" pasture pipet to deliver the gel solution in between the glass plates up to the level
right below the comb. Don't exceed the level of the comb or else the wells made later will
not hold as much sample.
Remove the comb.
Immediately squirt ddH 20 on top of the gel solution and fill to the very top. Don't disturb
it for 40 min until gel polymerizes and separates from water such that water will be on
top and can be poured off.
F.1.3.2.
Making the stacking gel
Make stacking gel solution according to recipe.
Put the comb on top of the running gel.
Use 5" pasture pipet to fill space up to the top with stacking solution.
Wait for 30 min until gel polymerize.
Gently remove comb.
Gel is ready to load sample.
F.1.3.3.
Loading the ProteinSamples:
Each properly formed well (use 0.75 mm spacers) can hold a maximum of 30pl of
sample.
Protein samples need to be mixed with sample buffer (contains 2-mercaptoethanol) and
boiled for 4 min before being loaded.
For rainbow (molecular weight) marker mix 10pl of 2x sample buffer +
10pl of rainbow marker (NEL312) in eppendorf tube.
For protein sample mix 20pl of sample buffer + 2 0pl sample in eppendorf tube.
Boil all the samples for 4 min.
Spin it in microcentrifuge.
Use loading tips to load all the samples.
F.1.3.4.
Running the gel
Fill up the Gel Running Apparatus with Ix Running buffer.
Place tank cover on top of electrodes. Red goes with red (positive) and black goes with
black. Turn on the power supply. Run at 100 V constant until all the samples go to
running gel then increase to 200 V.
Stop the power supply when you see that the blue line reaches the end of gel. This usually
takes 1 to 1-1/2 hours but watch it. You don't want your protein to run off the gel.
Turn off the power and take the gel out of apparatus and carefully take it out.
F.1.3.5.
Staining the gel
Make 45ml Coomassie blue + 5ml Acetic acid, mix well and put it into a container.
Put the gel into the dye solution. Stain for 20-30min
Pull off the dye solution. Rinsing with water few times. Then destain it with 7% Acetic
acid.
For Western blot do NOT stain it.
162
F.2.
TYPE I AND II COLLAGEN WESTERN BLOT PROTOCOL
(fromH.-H. Hung, MIT Continuum ElectromechanicsLab)
F.2.1. Summary
Proteins are separated by SDS-PAGE as described above. After the gel is run, do
NOT stain with Coomassie blue. The proteins from the gel must be transferred to a
membrane which is then incubated with the appropriate antibodies.
F.2.2. SDS-PAGE - see previous section, do not stain gel!
F.2.3. Western blotting
Incubate gel 30-45 min in a defixation buffer
F.2.3.1.
6 mM urea
192mM glycerin
25mM Tris
0.2% SDS
5mM DTT
dl H 2 0
0.036 g
1.41 g
0.303 g
0.20 g
0.077 g (stored in freezer)
to 100 ml final volume
Incubate gel in blotting (transfer)bufferfor 2-3 min.
F.2.3.2.
192 mM glycerin
25mM Tris
15% methanol
dl H 2 0
F.2.3.3.
14.1g
3.03g
100ml
to final 1000ml volume
Preparenitrocellulosemembrane
Cut nitrocellulose membrane (8x5.5cm) to just fit the gel. Do this with gloves on and
use tweezer to pick up the edge. The membrane must not be larger than the gel or else it
will result in a short-circuit during the transfer, and the protein will not transfer to the
membrane.
Soak nitrocellulose membrane in transfer buffer for 15min.
F.2.3.4.
Transfer proteinsfrom gel to nitrocellulose membrane
Wet sponge and blotting paper (Quick draw) with transfer buffer and assemble
"sandwich" as follows:
Black side of holder down
Sponge
Use pipette or test tube to make it contact
Blot paper
make sure there is no bubble in between
Gel
cellulose membrane
Blot paper
Sponge
Use pipette or test tube to make sure there is good contact (with no bubbles) between blot
paper and gel.
163
Close holder carefully.
Place holder in transfer chamber with small stirring bar and transfer at 100V for 1 hr.
Remove membrane.
Soak the membrane in TBS for 5 min.; be sure the gel is removed.
TBS:
9g
NaCl
20ml 1M tris HCl pH=7.6
dI H 2 0 to final volume of 1L
F.2.3.5.
Incubate in blocking buffer
5% Blocker blotter (Bio-Rad #170-6404) in TBS
Shake overnight at 4'C
Rinse in TBS 5min.
F.2.3.6.
Incubate with 1 antibody
The dilution and time depend on quality of first antibody:
For collagen type II antibody (Iowa C1 1C1 1:5 dilution in 1% BSA/TTBS).
For collagen type I antibody.(Sigma #C-2456 1:2000 dilution in 1% BSA/TTBS)
Be sure to use enough solution to keep membrane covered.
Incubate overnight at 4C
Wash the membrane with TTBS several times (5'x3).
TTBS:
150 [d Tween 20
300 ml TBS
F.2.3.7.
Incubate with 2' antibody
Promega #S3721 1:6000 dilution in 1%BSA/TTBS
Incubate (shaking) with 2 nd antibody for 1.5 hours.
Wash with TTBS 3 times 5 min/time.
Wash once with TBS to get rid of the Tween in TTBS.
F.2.3.8.
Detection
Use Promega's BCIP/TMB stable substrate solution (#S3771) to detect color.
It takes 5-15 min to show the color.
Wash with water to stop the reaction.
Dry membrane overnight and attach to a solid backing (membrane is fragile)
164
F.3.
a-SMOOTH MUSCLE ACTIN WESTERN BLOT PROTOCOL
(from R. Marty-Roix, BWH OrthopaedicsResearch Lab, except where noted)
F.3.1. Summary
Proteins are separated by molecular weight using SDS-PAGE and then the
proteins from the gel are transferred to a membrane. The membrane is then exposed to
the mouse anti-ccSMA antibody, the goat-anti-mouse secondary antibody, and a detection
substrate to visualize the SMA bands.
Although the principles are the same, this protocol is slightly different than that
for the type II collagen western blot, as this protocol was developed in the BWH
Orthopaedics Research Laboratory and the type II collagen protocol was developed in the
MIT Continuum Electromechanics Laboratory. The main differences are that the
membrane used here is a PVDF membrane (instead of a nitrocellulose membrane which
is supposedly better for transfer of the high molecular weight collagen bands) and the
detection method described in this protocol is a chemiluminescent method, rather than a
colorimetric method (matter of convenience and personal choice).
F.3.2. Cell Lysis
F.3.2.1.
1)
2)
3)
4)
5)
6)
Collect cells by trypsinization
Centrifuge cells and rinse with cold PBS and spin again
Add 0.5 ml lysis buffer/million cells
Shake at 4C for 20 minutes
Spin at 14,000 rpm (max speed) in microcentrifuge at 4C for 20 minutes
Remove and save supernatant (store frozen)
F.3.2.2.
1)
2)
3)
4)
5)
6)
7)
8)
Monolayer cultures
CG cultures (modifiedfrom Manthorpe, et al., 1993 protocol [1])
Rinse cultures with cold PBS
Add 0.5 ml lysis buffer/9mm disk
Break up matrices as much as possible with forceps
Freeze in liquid nitrogen
Thaw in 37'C water bath
Repeat freeze-thaw cycle 2 more times
Spin at 14,000 rpm (max speed) in microcentrifuge at 4C for at least 20 minutes
Remove and save supernatant (save frozen)
165
F.3.3. Protein Assay (need to determine how much of each sample to load)
1)
2)
3)
4)
5)
Turn on spectrometer, set wavelength at 595 nm
BSA standard curve
Dilute BSA stock 1.5 gg/gl 1:10 t ) make 0.15 gg/gl
(i)
(ii)
Make standards:
BSA (0.15 pg/pl)
dH 2 0
Dye
0 pl
1600 1
400 p1
10
1590
400
20
1580
400
40
1560
400
60
1560
400
Prepare samples (i) 40 pl sample, 1560 pl dH 2 0, 400 pl dye
(ii) Mix samples and incubate at room temp 5-10 minutes
Read absorbance at 595 nm
Calculate protein concentration (gg/pl)
F.3.4. Gel electrophoresis (start making gels before protein assay) - See also section
on SDS-PAGE
1)
2)
Make resolving gel and use -7 ml/gel (use a little water on top to get surface even)
Acryl
6.6 ml
Buffer
2.5 ml
10% SDS
200 p1
Persulfate
1000 pl
H20
9.6 ml
Temed
10 pl
Make stacking gel and use -3ml/gel (till full) and insert white comb
Acryl
1.25 ml
Buffer
2.5 ml
10% SDS
100 pl
Persulfate
500 pl
H2 0
3)
4)
5.6 ml
Temed
5 g1
Prepare samples and protein standard
Mix 1:1 with sample buffer (determine volume based on loading an
(i)
equal amount of protein/lane)
(ii)
Boil 5 minutes and load into wells
Run gel in IX Tris/Glycine/SDS (running buffer) in ice bath at 100 V until all
sample gets to resolving gel, then increase to 200 V and run 30-45 minutes
166
F.3.5. Western blotting
1)
Blot transfer
(i) Soak gel, membrane (pre-wet in 100% methanol), filter paper and sponge
in transfer buffer (dilute 160 ml 5X stock in 640 ml dH 2O and add 200 ml
100% methanol) for 10 minutes
(ii) Stack sponge - filter paper - gel -membrane
-
filter paper - sponge
(iii)Load into holder and bucket and run at 100 V for 1 hr in transfer buffer
2) Wash blot
(i) Cut membrane to size of gel
(ii) Wash membranes in IX TBS for 5 minutes
3) Block in 5% dry milk overnight in cold room (- 50 ml/membrane)
4) Incubate with primary antibody diluted 1:1000 in TBS for 1 hour at room temp (20
pl Ab + 20 ml/membrane)
5) Rinse in 1X TBST 3x10 minutes
6) Incubate with secondary antibody diluted 1:1000 in TBS for 1 hour at room
temperature (20 p Ab - 20 ml/membrane)
7) Rinse in IX TBST 3x10 minutes
8) Detection
(i) Mix substrate solutions A and B
(ii) Drop mixture on the membrane (5 ml/membrane)
(iii)Incubate 2 minutes at room temperature
(iv)Wrap in saran wrap
(v) In dark room, expose to film and develop
F.3.6. Solutions needed for Western Blot
Lysis buffer (store at 4 C)
0.1% SDS
1 mM sodium orthovanadate
1 mM PMSF
10% glycerol
Acryl (store at 4 C)
30 g acrylamide
0.8 g BIS
100 ml dH 20
Filter through #1 Whatman paper
Stacking buffer (store at 4 C)
0.5 M Tris (pH 6.8)
Filter through #1 Whatman paper
Resolving buffer (store at 4 C)
3.0 M Tris (pH 8.8)
Filter through #1 Whatman paper
167
Ammonium persulfate (store at 4 C)
150 mg ammonium persulfate
10 ml dH 20
Sample buffer (store at -20 C)
1 ml 0.5 M Tris (pH 7)
0.8 ml glycerol
1.6 ml 10% SDS
0.4 ml 2-mercaptoethanol
0.2 ml 1% bromophenol blue dye
Running buffer (lOX stock, pH 8.3)
30.3 g Tris
144 g Glycine
10 g SDS
1 LH 20
Transfer buffer (5X stock)
15.15 g Tris
72.25 g Glycine
800 ml H2 0
(After dilution to IX add in 20% methanol)
TBST (pH 7.5)
9.68 g Tris
116.96 g NaCl
4 L dH 2 0
4 ml Tween-20
168
F.4.
FLUOROGRAPHY OF RADIOLABELED PROTEINS
(modified from Bonner and Laskey, 1974 protocol [2])
F.4.1. Summary
Radiolabeled proteins are detected by x-ray film after separation by SDS-PAGE.
This can be used to identify newly synthesized proteins.
F.4.2. Sample preparation
Radiolabel cultures as desired i.e. 24 hour radiolabel with 3 H-proline to map collagen
synthesis.
Prepare samples as necessary (i.e. collagen extraction, Appendix E.9 and peptide
cleavage, Appendix E. 10)
F.4.3. Protein/peptide separation
Use SDS-PAGE protocol. (NOTE: For CnBr collagen peptides, use a 15% gel instead of
7%)
F.4.4. Fluorography
1)
2)
3)
4)
5)
Put in Amplify (Amershan NAMP100) for 30 minutes
Dry the gel on Whatman paper (in slab dryer)
Wrap gel in saran and place in contact with Kodak XOMAT film (convenient to use
x-ray exposure holder)
Keep in -80'C for 4-6 days
Develop film
REFERENCES
Manthorpe, M, Cornefert-Jensen, F, Hartikka, J, Felgner, J, Rundell, A,
1.
Margalith, M, and Dwarki, V. Gene therapy by intramuscular injection of plasmid
DNA: studies on firefly luciferase gene expression in mice. Hum Gene Ther
1993; 4:419-43 1.
2.
Bonner, W, and Laskey, R. A Film Detection Method for Tritium-Labelled
Proteins and Nucleic Acids in Polyacrylamide Gels. Eur J Biochem 1974; 46:8388.
169
APPENDIX G: HISTOLOGY METHODS
G.1.
TISSUE FIXATION AND EMBEDDING PROTOCOL
(All protocols from S. Zapatka-Taylor, BWH Orthopaedics Research Laboratory,
unless specified otherwise)
G.1.1. Fixation
Tissue is first fixed in 10% neutral buffered formalin for 5-6 days
G.1.2. Decalcification
Specimens containing calcified tissue (ie: bone) must be decalcified for at least 28 days.
15% EDTA decalcifying solution:
For 1800 ml: 1570 ml 0.01 M PBS (Sigma #P-3813 dissolved in 1 L distilled water)
44 g NaOH
270 g Disodium Ethylenediamine Tetraacetate (EDTA) (add alternately
with NaOH)
12N HCl to adjust to pH 7.4
G.1.3. Glycol Methacrylate (JB-4) Embedding
1)
2)
3)
4)
5)
6)
Dehydrate by hand or by using program 9 on Tissue-Tek (30-60 minutes in each
alcohol 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%)
Infilitrate specimens twice, 2 days each, in catalyzed solution A (9g catalyst/iL
solution A; JB-4 Embedding kit, Polysciences, Inc., Warrington, PA)
Embed in catalyzed solution A - solution B mixture (lmL solution B/25 mL
catalyzed A)
Polymerize overnight at 40 C
Remove specimens from molds and store at 40C
Sections (5 gm) placed in water bath (room temperature) and picked up with slide
and placed on hot plate (setting 2) to fix sections to slide
G.1.4. Paraffin Embedding
1)
Dehydrate and infiltrate by hand or by using program 4 on Tissue-Tek (30-60
minutes in each alcohol 50%, 60%, 70%, 80%, 90%, 95%, 100%, 100%, histoclear
and 2x hot paraffin)
2) Turn on freeze and heat switches on embedding station
3) Embed specimens in hot paraffin (56-60*C)
4) Allow wax to set (either on cold plate or freezer)
5) Remove from molds and store at 40 C
6) Sections (7 gim) placed in 40'C water bath and picked up on SuperFrostPlus slides
and baked onto slides overnight in 56'C oven
170
G.2.
HISTOLOGY AND IMMUNOHISTOCHEMISTRY PROTOCOL
G.2.1. Safranin-O Staining
G.2.1.1.
Paraffin sections
2 x 5 minutes
xylene
100% EtOH 10-20 dips
10-20 dips
95% EtOH
10-20 dips
90% EtOH
10-20 dips
80% EtOH
10-20 dips
70% EtOH
10-20 dips
dH20
2) Stain 10 minutes in Safranin-O (0.2 g Safranin-O, 1 ml acetic acid, 100 ml distilled
water)
3) Rinse with tap water
4) Counterstain 10-15 seconds with Fast-Green (stock solution, dilute 1:5 for working
solution: 0.2 g Fast Green, 1 ml acetic acid, 100 ml distilled water)
5) Dehydrate (70%, 80%, 90%, 95%, 100%, 100% EtOH, xylene)
6) Air dry sections
7) Coverslip with Permount
1)
Deparaffinze and rehydrate:
G.2.1.2.
1)
2)
3)
4)
5)
6)
JB-4 sections
Stain 30 minutes in Safranin-O (0.2 g Safranin-O, 1 ml acetic acid, 100 ml distilled
water)
Rinse with tap water
Counter-stain 5 minutes with Fast-Green (stock solution, dilute 1:5 for working
solution: 0.2 g Fast Green, 1 ml acetic acid, 100 ml distilled water)
Rinse with tap water
Allow to air dry
Coverslip with Permount
Glycosaminoglycans stain red/pink and collagen stains green.
G.2.2. Hematoxylin and Eosin (H&E) Staining
G.2.2.1.
1)
Paraffinsections
Deparaffinze and rehydrate:
xylene
100% EtOH
95% EtOH
90% EtOH
80% EtOH
70% EtOH
dH 20
171
2 x 5 minutes
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
2)
Stain 10 minutes in filtered hematoxylin (Sigma Harris' Hematoxylin Solution
#HHS-128)
3) Rinse with tap water until clear (-1 minute)
4) 5-10 dips in acid alcohol (200 ml 70% EtOH + 0.5 ml 1N HCl)
5) Rinse in tap water until foaming stops (-30 seconds)
6) 5-10 dips in ammonia water (5-10 drops ammonium hydroxide in 200 ml dH 2 O; pH
10)
7) Rinse in tap water (-1 minute)
8) Counterstain 45 seconds with Eosin (100 ml Sigma Eosin Y solution #HT1 10-2-128
+ 100 ml dH 20 + 1 ml glacial acetic acid)
9) Dehydrate (70%, 80%, 90%, 95%, 100%, 100% EtOH, xylene)
10) Air dry sections
11) Coverslip with Permount
G.2.2.2.
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
JB-4 sections
Stain 90 minutes in hematoxylin
Rinse with tap water
Dip a few times in acid alcohol
Rinse with tap water
Dip a few times in ammonia water
Rinse in tap water
Counter-stain 5 minutes with eosin
Rinse with tap water
Allow to air dry
Coverslip with permount
Nuclei stain blue/purple.
G.2.3. Immunohistochemical Stainingfor type II collagen (paraffin sections)
1)
2)
3)
4)
5)
6)
Deparaffinze and rehydrate:
xylene
2 x 5 minutes
100% EtOH 2 x 2 minute
95% EtOH
2 minute
90% EtOH
2 minute
80% EtOH
2 minute
70% EtOH
2 minute
TBS (pH 7.4) 2 x 2 minutes
Wipe of excess liquid and mark around samples with PAP pen (to minimize the
amount of solution needed in the following steps)
Protease XIV (aka: pronase) digestion (2-4 drops/sample of 10 mg pronase/10 ml
TBS) for 60 minutes
Wash in TBS (pH 7.4) 2 x 2 minutes; wipe slides afterwards
Block in 5% horse serum (1:20 dilution) for 30 minutes (do NOT wash slides
afterwards)
Incubate with primary antibody (1:20 dilution of mouse monoclonal anti-type II
collagen in TBS; Iowa Hybridoma Bank CIICI) or negative control (mouse IgG
5pg/ml) for 1 hour (room temp) or overnight (4'C in hydration chamber)
172
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
Wipe slides before wash in TBS 2 x 2 minutes; wipe slides afterwards
Incubate with secondary antibody (2 drops/sample of 1:200 dilution of biotintylated
horse anti-mouse immunoglobulin; Vector Laboratories #BA-2000) for 45 minutes*
Wash in TBS 2 x 2 minutes; wipe slides afterwards
Quench in peroxidase (3% H20 2 in dH 2 O) 10 minutes
Wash in TBS 2 x 2 minutes; wipe slides afterwards
Incubate with avidin-biotin conjugate (ABC kit, Vectastain Labs) reagent 30
minutes*
Wash in TBS 2 x 2 minutes; wipe slides afterwards
Dip slides in water
Stain with DAB staining kit (50 pl 1% hydrogen peroxide + 2.5 mg DAB/5 ml TrisHCl buffer) for approximately 8-10 minutes
Rinse in distilled water for 3-5 minutes
Counter-stain 10 minutes with Harris' hematoxylin
Rinse in running tap water
Dip in acid alcohol
Rinse in water
Dip in ammonia water
Rinse in water
Dehydrate (2 min each 70%, 80%, 90%, 95%, 100% EtOH, xylene)
Air dry
Coverslip with permount
*Make ABC reagent during last 15 minutes of 20 Ab incubation: Add 1 drop reagent A
in 5ml cold Tris-HCl buffer (pH 7.6) and mix. Add 1 drop reagent B and mix
thoroughly. Let stand 30 minutes before use.
Positive staining is indicated by brown color in the extracellular region
G.2.4. Immunohistochemical Staining for a-Smooth Muscle Actin (paraffin sections)
1)
2)
3)
4)
5)
6)
7)
2 x 30 minutes
xylene
100% EtOH 2 x 1 minute
1 minute
95% EtOH
1 minute
90% EtOH
1 minute
80% EtOH
1 minute
70% EtOH
3 minutes
PBS
Wipe of excess liquid and mark around samples with PAP pen (to minimize the
amount of solution needed in the following steps)
Trypsin digestion (2-4 drops/sample of 0.01 g trypsin/10 ml PBS) for 60 minutes
Wash in Phosphate-Buffered Saline (PBS) 2 x 3 minutes; wipe slides afterwards
Hydrogen peroxide (H2 0 2) for 5 minutes (2 drops/sample)
Wash in PBS 2 x 3 minutes; wipe slides afterwards
Block in 30% goat serum for 10 minutes
Deparaffinze and rehydrate:
173
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
Incubate with primary antibody (1:400 dilution of mouse monoclonal anti-ax smooth
muscle actin; Sigma #A2547) or negative control (mouse IgG) for 2 hours
Wipe slides before wash in PBS 2 x 3 minutes; wipe slides afterwards
Incubate with secondary antibody (2 drops/sample of 1:200 dilution of biotintylated
goat anti-mouse immunoglobulin; Sigma #B7151) for 20 minutes
Wash in PBS 2 x 3 minutes; wipe slides afterwards
Quench in peroxidase (2 drops/sample of 1:50 dilution of ExtraAvidin-Conjugated
Peroxidase; Sigma #E2886)
Wash in PBS 2 x 3 minutes; wipe slide afterwards
Stain with a smooth muscle actin immunohistochemical staining kit (4 ml distilled
water, 1 drop 3% H2 0 2 , 2 drops acetate buffer, 1 drop AEC chromagen; kit supplied
by Sigma #IiMMH-2) for approximately 10-15 minutes
Rinse in distilled water for 3 minutes
Counter-stain 20 minutes with Mayer's hematoxylin
Rinse in running tap water for 20 minutes
Mount with glycerol gelatin and coverslip (must be done in hood); try to avoid air
bubbles
Let dry in hood (overnight) and store flat.
Positive staining is indicated by red or red-brown color in the cytoplasm
G.2.5. Immunofluorescent Stainingfor Type lIprocollagen (chamber slides)
(modification of Borge, et al., In Vitro Cell Dev Biol Anim, 1997 protocol)
1)
2)
3)
4)
5)
6)
7)
Culture chondrocytes on chamber slides
Fix 10 minutes with 70% ethanol
Rinse 2x3 minutes with Tris-buffered saline, pH 8.0 (TBS)
Incubate 30 minutes with 30% goat serum
Incubate 2 hours with anti-type II collagen antibody (Iowa Hybridoma Bank; 116B3)
Rinse 2x3 minutes with TBS
Incubate 45 minutes with goat anti-mouse antibody conjugated to Cy3 fluorophore
(Jackson Immunochemical)
8) Rinse 2x3 minutes with TBS
9) Rinse 3 minutes with distilled, deionized water
10) Coverslip with Aquamount
11) Let dry and store flat protected from light
Positive staining is indicated by red fluorescence (Xabs= 550 nm, Xemiss= 570 nm) in
cytoplasm
174
APPENDIX H: INDENTATION TESTING OF CARTILAGEBONE PLUGS
H.1.
SAMPLE PREPARATION
Core 3/8" diameter osteochondral cores from desired locations (use PBS as cooling
fluid) using a standard drill press fitted with a custom-made stainless steel coring bit
(inner diameter 3/8").
2) Freeze cores in PBS until the day of testing
3) Thaw bone-cartilage plugs in PBE (phosphate buffered EDTA, see Appendix B)
4) Mix Quickmount, stirring until just viscous enough to pour (slightly less liquid
component will lead to quicker setting without severely affecting set acrylic)
5) Spray inside of holders with WD-40 and insert cardboard circle
6) Pour Quickmount into holder, filling it 1/2-2/3 full
7) Use tweezers to hold specimen in Quickmount, making sure that cartilage surface is
above Quickmount. Place specimen approximately in the center of holder, trying to
get articular surface parallel to bottom of holder
8) If needed, dispense Quickmount around specimen using a syringe
9) Place PBE soaked gauze over cartilage surface
10) Wait 20-30 minutes for quickmount to completely set
11) Place mounted specimens in bath of PBE and put in 4'C refrigerator until testing (to
minimize storage affects, do not store thawed for more than one test cycle prior to
testing)
1)
H.2.
1)
2)
3)
DYNASTAT SET-UP
Insert probe and chamber, making sure to tighten collets (small wrench)
Attach probe electrode to amplifier (VC 1)
Check connections to ADC/DAC box: Lo-R displacement to ADC 0
Load to ADC 1
Streaming Potential Amplifier (bottom-most connection) to ADC 2
Hi-R Displacement to ADC 3
Streaming Potential Amplifier (VC 2) to
DAC 4
Transient to DAC 5
DYN/EXT to Waveform
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
Calibrate load
Use Coarse/Fine screw to get reading of 0.000
Push in Cal button and use Gain screw to get 6.415
Place 1 kg weight on top of load cell and use Gain screw to get 1.000
Make sure zero suppression set at 018
Set B dial to 999, Dyn/Ext dial to 032. Push active button for each. Dial scale to 5.0
Calibrate displacement
Switch toggle to compression
Put displacement in Lo-R control and Hi-R read
Push in Zero button and use Hi-R Zero to get 0.000
175
14)
15)
16)
17)
18)
19)
20)
21)
22)
Push in Cal button and use Hi-R Cal to get 4.945
Put displacement in Hi-R control and Lo-R read
Push in Zero button and use Lo-R Zero to get 0.000
Push in Cal button and use Lo-R Cal to get 6.692
Make sure zero suppression set at 000
Put displacement in Lo-R control
Set toggle switch to transient
Set servo settings: 0 1.0 5.75 5.0 8.3 7.0
Set filter settings as follows:
High pass 0 0 0
Low pass 1 5 6
Multiplier off
Roll off 0
Mode bandpass
Gain 20dB
Roll off 12dB
Multiplier 10
23) Set amplifier settings as follows :5 mV
Zero suppression off
Vernier 10.0
Cutoff low DC
Cutoff high 10
H.3.
COMPUTER SET-UP
1) From c:\cyndi run dynssp
2) Load appropriate protocol (canindent.pro) or create new protocol file
3) Enter sample name, thickness and area (0.785 mmA2 for 1 mm indenter probe)
4) Open output file
5) Check to make sure that each control step is right
6) Check Acquisition list
7) First in list is what computer tries to control, so make sure it is displacement (not HiR displacement, except for thickness testing)
8) Ramp strains should have displacement and load in list
9) Dynamic strains should have displacement, load and potential in list
10) Thickness should have Hi-R displacement, load and reference in list
11) Check amplitude, control units (strain for stress-relaxations or mm for thickness),
frequency list (dynamic only), return to baseline, max load, samples/sec
12) Check set-up and make sure that load gain is lOv/v and that all ADC/DAC
connections correspond to the hook-up of the Dynastat to the computer
H.4.
1)
2)
3)
TESTING OF SAMPLES
Insert sample into chamber and adjust to get centered under probe and as close to
parallel as possible. Tighten all screws on chamber
Fill chamber with PBE and let equilibrate to swelling pressure 10 min
Bring load cell down until get reading of about -0.030V
176
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
Tighten upper collet to fix position of load cell and let equilibrate 5 min.
Banana plugs should be plugged in so that the tab on the side of the plug corresponds
to black
When switch on zero, the load and displacement pens should be at the right and the
streaming potential pen should be in the middle
Switch to cal
Set chart speed to 100 mm/hr
Set scales to green 5V, red 5V, blue 5V
Hit 'g' to start running the protocol
Ramp to 10% strain (180 seconds) and hold for 320 seconds
Ramp to 15% strain (180 seconds) and hold for 320 seconds
Dynamic compression with amplitude of 1% strain at frequencies of 1.0, 0.5, 0.1,
0.05, 0.01, 0.005 Hz (2 cycles at each frequency)
Ramp to 20% strain (180 seconds) and hold for 320 seconds
H.5.
THICKNESS MEASUREMENTS
When indentation is done, remove probe and insert needle (need to change to the
smaller collet)
2) Let specimen equilibrate, unloaded in PBE for 10 min
3) Switch displacement to Hi-R control
4) Remove some of the PBE from the chamber so that the surface is not submerged
5) Submerge Ag/AgCl electrode in PBE
Black alligator to metal strip on needle
6) Connect needle as follows:
Red alligator after resistor and ADC 4
Red plug before resistor and DAC 4
7) Chart recorder (blue, VC14) to ADC 4 (reference)
8) Reconnect chart recorder Lo-R (ADC 0) to Hi-R (ADC 3)
9) Set chart recorder scale to green 50V, red 10 V
10) Hit 'g' to run ramp protocol
11) Ramp to 5 mm at rate of 2mm/second
12) Electrical circuit is completed when needle probe contacts moist articular cartilage
(top of cartilage)
Step load (ideally) occurs when needle contacts subchondral bone (bottom of cartilage)
1)
177
APPENDIX I: STATISTICS
In the statistical analysis, the hypothesis tested was that there was no difference in
the mean values between groups. It was also assumed that the groups had equal
variances.
1.1.
POWER CALCULATION
A power calculation was performed in order to determine the sample size that
would be necessary to detect significant differences between experimental groups. The
sample size can be calculated as follows:
n =2( C)2(tay +
t 2 l)
where: n = sample size
a = standard deviation
8= difference desired to detect
a = desired significance level (probability of obtaining a false positive result)
= desired statistical power (probability of obtaining a false negative result)
tav= t statistic corresponding to a significance level a and v degrees of freedom
t 2 P,V= t statistic corresponding to a significance level 2$ and v degrees of freedom
The solutions of this equation for various a, 8, a and
(Orthopedic Basic Science).
1.2.
3 have
been tabulated
ANOVA ANALYSIS AND POST-HOC TESTING
All statistical analyses were performed using StatView software (SAS Institute,
Cary, NC). Multi-factor ANOVA tests were first used to determine the effects of
different independent variables (i.e. cross-linking treatment, time in culture). Since
StatView is not capable of performing post-hoc tests for combined effects, data was split
by one variable and one-way ANOVA and Fisher PLSD post-hoc testing were used to
determine specific factors that affected the outcome (dependent) variables. The Fisher
PLSD post-hoc test is one of the less stringent post-hoc tests available, but it was used
because StatView could calculate exact p-values for each post-hoc combination tested.
178
APPENDIX J: EVALUATION OF SEEDING TECHNIQUES
J.1.
INTRODUCTION
Previous studies by H. A. Breinan and S. Nehrer seeded chondrocytes into the CG
matrices by pipetting a concentrated cell suspension onto filter-dried matrix disks [Breinan, 1998
This involved soaking the matrices in complete culture medium, removing most of the
#107].
fluid from the matrices by blotting the matrices on sterile filter paper, and pipetting a small
volume of a highly concentrated cell suspension onto each side of the matrix disk. The problems
with this seeding method included:
.
deformation and sometimes damage to the matrix disks during the drying process,
especially for the lightly cross-linked (DHT) matrices
.
non-uniform cell-seeding since the cell suspension was added to the surface of the
matrix and relied on gravity to distribute the cells throughout the matrix volume
.
low seeding efficiency as much of the cell suspension "rolled" off the matrices
.
tedious pipetting when seeding many disks
.
working with very small volumes of highly concentrated cells (typically 40 gl per
side of matrix with a cell density of 25 million cells/ml
Other seeding methods that have been used by other investigators include incubation of
the disks with a cell suspension for a short period of time, either with the disks floating in
suspension or using spinner flasks to circulate fluid through the matrices, drawing a cell
suspension through the matrices using a low pressure vacuum (i.e. syringe vacuum) or low-speed
centrifugation, and injecting cells into the matrices with a needle and syringe. The purpose of
this pilot study was to develop a method for seeding the matrices using the simplest of these
methods - incubation of the floating matrix disks within a cell suspension. In these experiments,
the density of the cell suspension, the volume of cell suspension, and the time of incubation were
evaluated.
J.2.
MATERIALS AND METHODS
Adult canine chondrocytes were harvested and cultured as described in Chapter 2 and
detailed in Appendix D.1-2. Third passage chondrocytes were used for all seeding studies.
CG matrices (type I collagen) were fabricated and DHT cross-linked as described in
Chapter 2 and detailed in Appendix A.1.
Matrix disks 9mm in diameter were used for all
179
seeding studies. Matrices were hydrated in complete medium for at least 20 minutes (2 x 10
minute washes).
Prior to seeding, excess medium was removed by vacuum aspiration. For
control seedings, matrices were filter dried prior to pipette seeding.
J.2.1. Cell density
To determine the appropriate cell density of the cell suspension, matrices were incubated
in suspensions with cell densities of 1, 2, or 5 million cells/ml with 2 disks/ml of suspension,
yielding 0.5, 1, or 2.5 million cells/matrix. Matrix disks were transferred into 15ml centrifuge
tubes containing the appropriate cell suspensions. The tubes were tightly capped and then placed
in an incubator on a nutator (flat surface, with rubber nubs to keep tubes from rolling off, which
rotated and rocked) for 1.5 hours.
established pipette seeding technique.
For comparison, disks were seeded with the previously
Media was removed from these disks by blotting the
matrices on filter paper. Disks were then placed in well plates coated with 2% agarose and 25 p
of cell suspension containing 0.5 million cells was pipetted onto each side of the matrix disk.
Well plates were placed in the incubator for 1.5 hours.
After the 1.5-hour incubation, matrices were either rinsed in sterile PBS and sacrificed
for determination of DNA content by the Hoechst dye assay (n=3-5 per group) or placed in well
plates with 1.0 ml of complete medium and returned to the incubator for 24-48 hours. After 24
or 48 hours of incubation, these matrices were similarly terminated and assayed for DNA content
(n=3-4 per group).
J.2.2. Incubation time
Since the tubes are slightly inverted during part of the rotation cycle on the nutator, the
centrifuge caps must be tightly secured. This, however, prevents gas exchange and the proper
CO 2 concentration cannot be maintained. To determine the minimum incubation time at which
maximum cell attachment could be achieved, matrices were incubated in a cell suspension
containing 4 million cells/ml (2 million cells/matrix) for 0.25, 0.5, 1, 2, or 3 hours (n=3-4 per
group). Matrices were then rinsed in PBS and sacrificed for DNA determination.
J.2.3. Suspension volume
To determine the optimal suspension volume, matrix disks were incubated in cell
suspensions with 2 million cells/matrix but with volumes of 0.25, 0.33, 0.5, and 1.0 ml per disk.
Matrices were incubated on the nutator for 2 hours and sacrificed for DNA determination.
180
J.3.
RESULTS
J.3.1. Cell density
The DNA content of the matrices seeded by pipetting 1 million cells onto the disks (0.5
million cells on each side) was 5.7 ± 0.38 pg DNA/matrix. This was essentially the same as the
DNA content of the matrices incubated with 1 million cells/disk (2 million cells/ml), 5.7 ± 0.44
pg DNA/matrix.
This corresponds to approximately 0.74 million cells/disk (using the
conversion factor of 7.7 gg DNA/million cells), indicating a seeding efficiency of approximately
74% at this cell density for both techniques.
As expected, DNA content of matrices incubated in the cell suspension on the nutator and
sacrificed after the incubation period increased with increasing cell density of the suspension.
The increase in DNA content with increasing suspension cell density was linear (Figure AJ.1;
R2 =0.998) over the range of densities evaluated. The seeding efficiency ranged from 82% at the
lowest cell density to 74% at the two higher cell densities. At the highest cell density (5 million
cells/ml, 2.5 million cells/matrix), there was a higher variability among the cell densities of the
matrices in that group (coefficient of variation = 0.52; coefficient of variation for other two cell
densities <0.2).
DNA content of the matrices 24 and 48 hours post-seeding were approximately the same
as those measured directly after seeding (Figure AJ.2). The one exception was a slight decrease
in DNA content in the matrices incubated with 5 million cells/ml.
181
1614
-
12 - y = 4.6924x + 0.701
R2=0.9977
10 8-
z
6420-
0
0.5
1
2.5
2
1.5
3
#cells seeded per nmtrix (xOA6)
Figure J.1: Measurement of the DNA content of the matrices versus the seeding cell density
showed a linear increase in DNA content with increasing cell density. All matrices were
incubated for 1.5 hours with a volume of 0.5 ml/9mm matrix disk. Values are mean ± SEM (n=34).
2015
-
105-
K
-
K
010
0
30
20
40
50
Time (hours)
--
-*-
0.5x10^6 cells/matrix.
2.5x10^6 cells/matrix.
-+-
-X-
1xlO^6 cells/matrix
Pipette 1xOA6 cells/matrix
Figure J.2: DNA content of the matrices was approximately constant for the 48 hours after
seeding. All matrices were incubated for 1.5 hours with a volume of 0.5 ml/9mm matrix disk.
Matrices seeded by the pipette technique (-*-) had approximately the same amount of DNA as the
6
matrices incubated with a cell suspension at a concentration of 2x10 6 cells/ml (1x10 cells/matrix;
-.-
).
Values are mean
±
SEM (n=3-4).
182
J.3.2. Incubation time
The DNA content of the matrices increased with increasing incubation time, up to an
incubation time of 2 hours. The DNA content of matrices incubated for 0.5 and 1 hour were
approximately twice that of the matrices incubated for only 0.25 hours (Figure AJ.3; p<0.03).
Increasing the incubation of the matrices in the cell suspension to 2 hours increased the DNA
content another 50%, compared to one hour incubation (p<0.01).
A further increase in
incubation time to 3 hours, however, did not result in an increase in DNA content of the matrices
(p=0.42).
18
161412S10-
< 8O
642-
0
0
0.5
I
I
I
1
1.5
2
2.5
3
3.5
Incubation Time (hours)
Figure J.3: DNA content of matrices increased with increasing incubation time up to
approximately 2 hours. All matrices were incubated in a suspension with 4x10 6 cells/ml and 0.5
ml/9mm matrix disk. Values are mean ± SEM (n=3-4).
J.3.3. Suspension volume
The amount of DNA in the matrices was the highest for matrices incubated with 0.5 ml
cell suspension/matrix 14.9 ± 0.54 pg of DNA/matrix (Figure AJ.4). The DNA content of the
matrices incubated with 1 ml cell suspension/matrix was the lowest (9.6 ± 1.56
DNA/matrix). The differences, however, were not significant (p=0.38).
183
sg
of
18
161412'10-
4200
0.2
0.4
0.6
0.8
1
1.2
Suspension Volume/Matrix
Figure J.4: DNA content of matrices incubated with 0.5 ml/9mm matrix disk
was the highest, though the differences were not significant (p=0.38). All
matrices were incubated in a suspension with 2x10 6 cells/matrix disk for 2 hours.
Values are mean ± SEM (n=3-4).
J.4. CONCLUSIONS
Seeding of the matrices by the suspension method did not yield higher seeding
efficiencies than the pipetting method. Suspension seeding, however, eliminated the need for
filter-drying of the matrices and the use of very high density cell suspensions. The drawbacks of
the suspension method include possible loss of cell-seeded matrices when transferring the disks
from the seeding tube to the well plates and a higher likelihood of uneven seeding of matrices.
Based on the outcomes of these pilot studies, the optimal conditions for seeding the
chondrocytes into the matrices by co-incubation of the matrices and cells appears to be
incubation for 2 hours with a cell suspension density of 2 million cells/ml and 0.5 ml of
suspension per matrix. These conditions yielded high density of cell attachment, while providing
for relatively consistent levels of DNA for all matrices in the suspension and minimizing the
amount of time that the cells must be in a sealed system.
J.5.
ACKNOWLEDGEMENTS
Thank you to Katherine Oates who helped me with most of these pilot studies.
184
APPENDIX K: EFFECTS OF PASSAGE ON CELLMEDIATED CONTRACTION AND RESPONSE TO
MECHANICAL LOADING
K.1.
INTRODUCTION
The low cell density and low mitotic activity of adult articular cartilage makes it
impractical to use primary autologous chondrocytes in articular cartilage tissue
engineering. When isolated from the extracellular matrix and cultured in monolayer,
chondrocytes display increased proliferative activity (Green, 1971; Benya and Shaffer,
1982). Prolonged expansion in monolayer culture ("passaging"), however, also induces a
loss of the chondrocyte phenotype, namely a decrease in type II collagen synthesis, an
increase in type I collagen synthesis, and a flattened, fibroblastic-like morphology (Benya
et al., 1978; Benya and Shaffer, 1982). Upon return to appropriate environments, such as
encapsulation in agarose hydrogels, the "de-differentiated" cells can re-express the
typical chondrocyte phenotype (Benya and Shaffer, 1982).
It has been shown that
passaged chondrocytes in porous collagen-GAG matrices can adopt a spherical
morphology and are capable of synthesizing type II collagen (Nehrer et al., 1997a;
Nehrer et al., 1997b). The extent to which these cells have "de-differentiated" and "redifferentiated," however, has not been established.
In addition to triggering changes in collagen synthesis, it is to be expected that
monolayer culture of chondrocytes induces other changes in gene expression. Previous
studies with passaged chondrocytes in collagen-GAG matrices have noted cell-mediated
deformation of the scaffolds (Lee et al., 2000). There have been no reports, however, of
chondrocytes displaying contractile behavior in vivo. Thus, the first aim of these pilot
studies was to determine the extent to which monolayer culture (i.e. passage number)
influences cell-mediated contraction.
The response of chondrocytes to mechanical loading has been studied extensively
(Gray et al., 1989; Sah et al., 1989; Sah et al., 1991; Kim et al., 1994; Buschmann et al.,
1995; Kim et al., 1996; Grodzinsky et al., 1998; Quinn et al., 1998; Ragan et al., 1998).
All of these studies were conducted with cartilage explants or freshly isolated ("primary")
chondrocytes in hydrogels.
The behavior of passaged chondrocytes has not been
185
established. Thus, the second aim of these pilot studies were to compare the response of
first and third passage chondrocytes to dynamic compression.
K.2.
MATERIALS AND METHODS
Chondrocytes were isolated from adult canine cartilage as described in Chapter 2
and detailed in Appendix C.
K.2.1. Contraction Studies
Primary (freshly isolated), first, second, and third passage chondrocytes were
seeded into 9-mm diameter DHT cross-linked type I collagen-GAG matrices. Primary
chondrocytes from three different animals were used. Passaged chondrocytes from only
one animal were used. Matrix diameter was measured at various stages during culture
and cell-mediated contraction calculated as in Chapter 2.
K.2.2. Dynamic Compression Studies
Previously frozen first passage chondrocytes were thawed and seeded into 4-mm
diameter EDAC cross-linked type I collagen-GAG matrices. Previously frozen second
passage chondrocytes were thawed and expanded one more passage in monolayer culture
prior to seeding into 4-mm diameter EDAC cross-linked type I collagen-GAG matrices.
After 7 or 14 days in free-swelling culture, constructs were subjected to unconfined
dynamic compression (10% strain offset, 3% dynamic strain at 0.1 Hz), as described in
Chapter 4. Cultures were radiolabeled with 3 H-proline and 35S-sulfate during the 24 hour
compression. Free-swelling cultures were similarly labeled as controls.
K.3.
RESULTS
K.3.1. Cell-mediated Contraction
Over the first week in culture, freshly isolated (primary) canine chondrocytes did
not contract the collagen-GAG matrices (Figure K.1).
After 28 days in culture, the
primary chondrocytes contracted the matrices an average of 19.3 ± 2.8%. In contrast,
monolayer-expanded (passaged) chondrocytes began contracting the matrices during the
first week in culture. By the end of the first week, passaged chondrocyte contracted the
collagen-GAG matrices more than 40%. First and second passage chondrocyte cultures
186
were carried only through two weeks of culture at which point the cells had contracted
the matrices 64.8 ± 10.3% and 58.5
I
=.-
-----
100 -
0.8%, respectively.
~8
60-
.- -n40 -20
-
-
Primary
-.
P
P3
--
-5-
0
5
10
15
Day
20
25
30
Figure K.1. Cell-mediated contraction for primary (freshly isolated), first, second, and third
passage chondrocytes. Primary chondrocytes exhibited minimal contraction, but there were little
differences in contraction between first, second and third passage chondrocytes.
K.3.2. Compressive Loading
DNA analysis of cultures revealed significantly lower rates of initial cell
attachment and proliferation of thawed first passage chondrocytes, compared to third
passage chondrocytes (Figure K.2). By the end of the second week, however, the first
passage chondrocytes recovered and the DNA content of the first and third passage cells
were the same. To account for differences in cell number, biosynthesis rates of loaded
cultures were normalized to (control) free-swelling cultures of the same generation of
chondrocytes.
Unconfined dynamic compression (3% strain amplitude, 0.1 Hz, superimposed on
10% static strain) tended to decrease the accumulation of
3H-
and
35S-labeled
macromolecules within the collagen-GAG constructs compared to free-swelling cultures,
although the decrease was significant only for the third passage chondrocytes loaded after
one week in free-swelling culture (Figure K.3). Comparison of normalized incorporation
rates (normalized to free-swelling controls) for first and third passage cultures did not
187
reveal any differences for either
3H-
or
35S-labeled
macromolecules after one or two
weeks of free-swelling culture.
8
7
6
5
4
0 3
2
- -
P'
-u-P3
1
0
10
5
0
15
Day
Figure K.2. DNA content of collagen-GAG matrices seeded with first (P1) or third (P3) passage
chondrocytes. There was a significantly lower cell content in the P1 cultures through the first
week in cultures, but by the end of the second week in culture, there was no difference in DNA
content for P1 and P3 cultures.
4)
c
1.2-
OP13H
* P1 35S
0
1 0
.2
0.
.-LM
a
0
0.8
-
0.6
-
0.4
-
0.2
-
T
OP33H
* P3 35S
or
T
U
0
z
0-
8
14
Days in Free-Swelling Culture
Figure K.3. Normalized 3H-proline and 35S-sulfate incorporation rates for dynamically
compressed (3% dynamic strain, 0.1 Hz, superimposed on 10% static strain) chondrocyte-seeded
type I collagen-GAG cultures after one or two weeks in free-swelling culture. Values were
normalized to incorporation rates for free-swelling samples cultured in parallel. There were no
significant differences in the response to dynamic loading for first or third passage chondrocytes.
188
K.4. DISCUSSION
Passaged (monolayer expanded) canine chondrocytes exhibit enhanced contractile
behavior compared to primary (freshly isolated) canine chondrocytes. There were not,
however, differences in cell-mediated contraction nor biosynthetic response to dynamic
compression in first through third passage chondrocytes.
With the exception of the
primary chondrocytes in the contraction study, cells from only one dog were used in each
study. It should be noted, however, that the behavior of these cells were similar to that
seen in multiple animals evaluated in other studies (Chapters 2 and 3), so it is likely that
the results reported for this pilot study are applicable to passaged canine chondrocytes in
general.
A significant difference was noted in the proliferation patterns of first and third
passage chondrocytes. The difference may, however, be attributed to the fact that first
passage chondrocytes were seeded into collagen-GAG matrices directly after thawing. It
is likely that if the cells had been allowed time to recover prior to seeding, chondrocyte
attachment and proliferation may have been more similar to that of the third passage
chondrocyte.
Although the use of primary chondrocytes for articular cartilage tissue
engineering would be the preferred approach, the limited cellularity of adult cartilage
makes it more practical to use passaged chondrocytes. The results of this study indicate
that first through third passage chondrocytes exhibit similar behavior in regards to
contractility and response to unconfined dynamic compressive loading.
It should be
noted, however, that assays that would more specifically define the differentiated state of
the passaged cells were not conducted in these studies.
It may be possible that
chondrocytes from earlier passages synthesize higher ratios of type II to type I collagen.
K.5.
REFERENCES
Benya, P. D., et al. (1978). "Independent regulation of collagen types by chondrocytes
during the loss of differentiated function in culture." Cell 15(4): 1313-21.
Benya, P. D. and J. D. Shaffer (1982). "Dedifferentiated chondrocytes reexpress the
differentiated collagen phenotype when cultured in agarose gels." Cell 30(1): 215-24.
Buschmann, M. D., et al. (1995). "Mechanical compression modulates matrix
biosynthesis in chondrocyte/agarose culture." J Cell Sci 108(Pt 4): 1497-508.
189
Gray, M. L., et al. (1989). "Kinetics of the chondrocyte biosynthetic response to
compressive load and release." Biochim Biophys Acta 991(3): 415-25.
Green, W. T., Jr. (1971). "Behavior of articular chondrocytes in cell culture." Clin Orthop
75: 248-60.
Grodzinsky, A. J., et al. (1998). Response of the chondrocyte to mechanical stimuli.
Osteoarthritis. K. D. Brandt, M. Doherty and L. S. Lohmander. New York, NY, Oxford
University Press: 123-136.
Kim, Y. J., et al. (1996). "Compression of cartilage results in differential effects on
biosynthetic pathways for aggrecan, link protein, and hyaluronan." Arch Biochem
Biophys 328(2): 331-40.
Kim, Y. J., et al. (1994). "Mechanical regulation of cartilage biosynthetic behavior:
physical stimuli." Arch Biochem Biophys 311(1): 1-12.
Lee, C. R., et al. (2000). "Articular cartilage chondrocytes in type I and type II collagenGAG matrices exhibit contractile behavior in vitro [In Process Citation]." Tissue Eng
6(5): 555-65.
Nehrer, S., et al. (1997a). "Canine chondrocytes seeded in type I and type II collagen
implants investigated in vitro [published erratum appears in J Biomed Mater Res 1997
Winter;38(4):288]." J Biomed Mater Res 38(2): 95-104.
Nehrer, S., et al. (1997b). "Matrix collagen type and pore size influence behaviour of
seeded canine chondrocytes." Biomaterials 18(11): 769-76.
Quinn, T., et al. (1998). "Mechanical compression alters proteoglycan deposition and
matrix deformation around individual cells in cartilage explants [In Process Citation]." J
Cell Sci 111(Pt 5): 573-83.
Ragan, P. M., et al. (1998). Mechanical Compression Affects Chondrocyte Matrix Gene
Expression in Cartilage Explants. ORS, New Orleans, LA.
Sah, R. L., et al. (1991). "Effects of compression on the loss of newly synthesized
proteoglycans and proteins from cartilage explants." Arch Biochem Biophys 286(1): 209.
Sah, R. L.-Y., et al. (1989). "Biosynthetic Response of Cartilage Explants to Dynamic
Compression." Journal of Orthopedic Research 7: 619-636.
190
APPENDIX L: INTEGRITY OF PLASMID IN GENE-SEEDED
COLLAGEN-GLYCOSAMINOGLYCAN MATRICES
L.1.
INTRODUCTION
Luciferase
expression
of chondrocytes
seeded
into the gene-seeded
collagen-
glycosaminoglycan (GSCG) matrices did not follow the patterns predicted by the total amounts
of plasmid loaded onto the matrices or the leaching patterns of the different matrices.
In an
attempt to ascertain the reasons for the discrepancy, two simple in vitro assays were performed
with the plasmid collected in the first rinse of the leaching assay (Chapter 5). Electrophoretic
analysis of the released plasmid DNA was used to determine the extent of nicking and
degradation of the plasmid. The second assay evaluated the functionality of the released plasmid
by transfecting monolayer chondrocyte cultures with the released DNA with the aid of a
commercially available transfection kit.
L.2.
MATERIALS AND METHODS
L.2.1. Electrophoretic Analysis of Released Plasmid DNA
The integrity of the released plasmid DNA from each matrix formulation was evaluated
by agarose gel electrophoresis. A 10 Rd aliquot of the released plasmid DNA solution taken at
the 30-minute time point was run on a 1% agarose gel (Bio-rad, Hercules, CA) with 1% ethidium
bromide (Sigma Chemical, St. Louis, MO) in TAE buffer. Lambda DNA/HindII molecular
weight marker and restriction enzyme (EcoRI and XhoI) digests of the stock plasmid DNA
vector were also electrophoresed in parallel with the DNA released from the GSCG matrices.
The percentage of the total DNA in each lane that existed in the supercoiled and nicked forms
were determined using NIH Image 1.61 acquisition and analysis software.
L.2.2. Transfection of Canine Articular Chondrocytes in Monolayer Culture
Canine articular chondrocytes were isolated from the knee (stifle) joints of adult mongrel
dogs by sequential pronase and collagenase digestion (Kuettner et al., 1982).
Third passage
chondrocytes were cultured in 96-well tissue culture plates in DMEM/F12, 2 mM glutamine, and
10% fetal bovine serum at 37 'C and 5% CO 2 . Transfection was performed with the Gene
Porter@ In Vitro Transfection Kit protocol (Gene Therapy Systems, San Diego, CA) by adding a
10 RI aliquot of the released plasmid DNA solution taken at the 30 minute time point to
191
chondrocytes grown to approximately 90% confluence. The chondrocytes were lysed 48 hours
post-transfection
and luciferase activity measured on a Monolight® 2010 (Analytical
Luminescence Laboratory, San Diego, CA) using the Promega Luciferase Assay Kit (Promega,
Madison, WI).
M.W.
Stock
EcoRi
Non
Xhol
DHT
UV
EDC
A
B
Figure L.1: Agarose electrophoresis of plasmid released from the matrices during the first 30
minutes compared to plasmid from a stock solution (lane 2) and plasmid digested with EcoRi
(lane 3) and Xhol (lane 4) restriction enzymes. (A) At pH 2.5, plasmid released from DHT
matrices was severely degraded as indicated by the smear (lane 6) and plasmid released from UV
and EDAC matrices was almost entirely nicked to the linear form. (B) At pH 7.5, with the
exception of the plasmid from the non-crosslinked matrices, there was a substantial fraction of the
plasmid that was in the supercoiled (native) form.
L.3.
RESULTS
L.3.1. Electrophoretic Analysis of Released Plasmid DNA
The relative percentage of released plasmid DNA that demonstrated the native structure,
with respect to the mixture of supercoiled and nicked plasmid DNA, varied with cross-linking
treatment and pH (Figure L.1; Table L.1). Plasmid DNA released from the pH 2.5 non-crosslinked and the pH 7.5 DHT- and EDAC-cross-linked matrices consisted of a much larger
192
percentage of supercoiled than nicked component (>60% supercoiled), similar to the stock
solution.
In contrast, a much larger percentage of nicked than supercoiled component was
released from the pH 2.5 UV- and EDAC-cross-linked and the pH 7.5 UV-cross-linked matrices
(>50% nicked), comparable with the electrophoretic pattern seen after Xho 1 restriction enzyme
digest of the stock plasmid DNA. Additionally, the plasmid DNA released from the pH 7.5 noncross-linked and the pH 2.5 DHT-cross-linked matrices showed near complete fragmentation.
Table L-1: Densitometric evaluation of agarose gels (Figure L. 1) revealed the percentage of the
plasmid that was supercoiled (SC) and nicked (Nick).
pH 2.5
%Nick
%SC
40.7
59.3
64.3
35.7
0.0
47.3
30.1
66.6
21.0
32.6
51.4
31.5
66.5
22.7
Stock DNA
XhoI
EcoRI
NonDHTUVEDC-
pH 7.5
%Nick
%SC
34.9
65.1
48.5
24.5
0.0
48.2
34.7
23.5
35.7
64.3
63.0
27.0
20.0
80.0
L.3.2. In Vitro Transfection Assays
Third passage chondrocytes (after approximately 3 weeks in culture and 8 doublings)
transfected with the plasmid DNA released from the pH 2.5 DHT- and UV-cross-linked matrices
yielded a 2-3 fold lower expression of luciferase than the DNA from the pH 2.5 non-cross-linked
matrix (Figure L.2). In contrast, plasmid DNA released from the pH 2.5 EDAC-cross-linked
GSCG matrices gave an unexpected 2-fold higher luciferase activity than the plasmid DNA from
the pH 2.5 non-cross-linked matrices.
Transfection with the plasmid released from GSCG
matrices prepared at pH 7.5 and cross-linked by DHT or EDAC yielded luciferase activities that
were nearly 2-fold higher than that seen with transfection by DNA released from the pH 7.5 noncross-linked matrix (Figure L.2).
Of note was the 10-fold difference in the gene expression
obtained from the DNA released by the UV-cross-linked matrix compared to the DHT- and
EDAC-cross-linked matrices prepared at pH 7.5 (Figure L.2). Including all groups, two-factor
ANOVA demonstrated significant effects of cross-linking technique (p<0.0001) but not of pH
193
(p>0. 2 5 ) on the luciferase activity of transfected cultures. A separate analysis of the DHT results
demonstrated a significant effect of pH on the luciferase activity (Student's t test, p<0.02).
"S 6
U pH 2.5
5 - O pH 7.5
4 -
2 -
0
none
UV
DHT
EDAC
Figure L.2: Luciferase activity of monolayer cultured canine chondrocytes 48 hours after
transfection with plasmid leached from prepared gene-seeded collagen-glycosaminoglycan
matrices. Cultures transfected with buffer from the UV matrices had the lowest luciferase acivity
levels (p<0.05) while the EDAC had the highest (p<0.05). The pH of the matrix preparation only
affected cultures transfected with buffer from the DHT cross-linked matrices (p<0.02). All
cultures were transfected with 10 pl of the buffer removed from the matrices after the first 30
minutes. Values are mean ± SEM (n=6).
L.4.
DISCUSSION
It is clear from the electrophoretic analyses that there were alterations in the secondary
structure of the plasmid DNA released from specific GSCG matrix formulations when compared
to the stock plasmid DNA.
Particularly noteworthy were the nicking and/or severe
fragmentation of the plasmid DNA incorporated in the pH 2.5 cross-linked and the pH 7.5 nonand UV-cross-linked GSCG matrix formulations.
Proton-induced depurination and/or reactive
oxygen species (ROS) may have mediated damage of the plasmid DNA and thus been involved
in the structural alterations of the plasmid DNA (Lindahl and Nyberg, 1972; Evans et al., 2000).
The preservation of the supercoiled component when the plasmid DNA was added to the noncross-linked GSCG matrix at acidic pH suggested that the reactive side groups on the collagen
fibers may scavenge the hydrogen ions and/or ROS. In contrast, the reduction of the available
194
carboxyl and amide groups along the collagen fibrils that may have resulted from all the crosslinking methods used in this study may have caused a decreased buffering of the hydrogen ions
and/or ROS and an increase in DNA depurination and fragmentation. At neutral pH, the severe
fragmentation of plasmid DNA incorporated in the non-cross-linked matrix formulation differed
greatly from the increase in the percentage of nicked DNA in the UV-cross-linked GSCG matrix
and the completely preserved DNA of the DHT- and EDAC-cross-linked GSCG matrices. The
latter observation may likewise be a consequence of the relative effects of proton-induced
depurination- and/or ROS- mediated damage of the plasmid DNA. Future studies will explore
how specific factors such as the buffer system and ROS scavengers affect the integrity of the
plasmid DNA incorporated in various GSCG matrix formulations.
It was not clear, however, how specific structural changes induced by the GSCG
fabrication process affected the functionality of the plasmid DNA. At least some of the plasmid
DNA released from all of the GSCG matrices retained its ability to code for the luciferase
reporter gene. To some extent the in situ transfection of chondrocytes seeded in GSCG matrices
(Chapter 5) resulted in luciferase gene expression patterns which matched those that would be
predicted on the basis of the electrophoretic analyses. The ranking of matrices based on the
chondrocyte transfection found in the experiments using the chondrocytes in monolayer and the
cell-seeded scaffolds was different.
This may reflect the influence of certain cell-matrix
interactions on the process by which DNA gets transferred to the cell. Moreover this finding
underscores the importance of employing in vitro experimental conditions that begin to approach
the in vivo situation.
L.5.
REFERENCES
Evans, R. K., et al. (2000). "Evaluation of degradation pathways for plasmid DNA in
pharmaceutical formulations via accelerated stability studies." J Pharm Sci 89(1): 76-87.
Kuettner, K. E., et al. (1982). "Synthesis of cartilage matrix by mammalian chondrocytes in
vitro. I. Isolation, culture characteristics, and morphology." J Cell Biol 93(3): 743-50.
Lindahl, T. and B. Nyberg (1972). "Rate of depurination of native deoxyribonucleic acid."
Biochemistry 11(19): 3610-8.
195
APPENDIX M:ALTERNATIVE METHODS FOR THE
FABRICATION OF THE GSCG MATRICES
M.1. INTRODUCTION
The addition of plasmid DNA to pre-formed collagen-glycosaminoglycan
matrices results in only a small fraction (<35%, Chapter 3) of the DNA is bound tightly
to the matrix. In general, a higher percentage of the DNA is tightly bound at pH 2.5
compared to pH 7.5, presumably because of the swelling of the collagen fibers at acidic
pH. Unfortunately, however, the matrices prepared at low pH had lower expression
levels of the protein encoded for by the plasmid DNA. The lower expression levels are
likely due to damage of the DNA by the acidic buffer (see Appendix L). In two separate
experiments, alternative methods for the fabrication of the gene-seeded collagenglycosaminoglycan (GSCG) matrices were evaluated. In the first study, we attempted to
add the plasmid to the collagen-GAG slurry prior to freeze-drying in order to increase the
integration of the plasmid into the matrix. In the second study, we evaluated the effects
of adding salt to the plasmid-buffer solution to protect the DNA and to increase swelling
of the collagen fibers. Additionally, in the second study we investigated the possibility of
using a basic pH to swell the fibers to increase plasmid loading while maintaining
integrity of the plasmid.
M.2. MATERIALS AND METHODS
M.2.1. Addition of Plasmid to Collagen-Glycosaminoglycan Slurry
The type I collagen-GAG slurry was prepared in the usual manner (Appendix
A.1).
A small volume of the slurry was transferred to a 50-ml centrifuge tube with a
magnetic stir bar placed at the bottom of the tube. The tube was held above a magnetic
stir plate set at a slow mixing speed. The plasmid DNA was suspended in 0.05 M acetic
and added to the slurry at a final plasmid concentration of 10 or 20 gg/ml was added to
the stirring slurry. Once the plasmid had been added, 1-ml aliquots of the plasmid-CG
slurry were pipetted into the wells of a 24-well plate and transferred to the freeze-drier
for overnight freeze-drying following the standard protocol (Appendix A.1). When the
plasmid was added quickly or the slurry stirred or shaken vigorously, the negative charge
of the plasmid caused the collagen to come out of suspension. Thus, great care was taken
196
to add the plasmid slowly and to stir and pipette the slurry slowly. The freeze-dried
matrices were sterilized and lightly cross-linked by 24 hour DHT.
M.2.2. Plasmid leaching
Matrices were hydrated in 1ml of tris-EDTA buffer and the leaching profiles at
room temperature determined described in chapter 5 (5.3.4).
M.2.3. In situ transfection of chondrocytes
Chondrocytes were cultured in the matrices fabricated by adding plasmid to the
CG slurry for four or eight weeks as described in Chapter 3. The lysates from the
cultures were obtained and assayed for luciferase activity as described in Chapter 3.
M.3. RESULTS
There were problems with precipitation of the collagen after addition of the
plasmid.
Such precipitation could be minimized with low concentrations of plasmid
added at a slow rate and with gentle stirring.
M.3.1. Plasmid leaching
There was no detectable amount of DNA in the leaching buffer from these
matrices at any time point up to four weeks.
M.3.2. In situ chondrocyte transfection
There was no detectable luciferase activity in the lysates from the four-week in
vitro cultures of either 10 ig/ml or 20 gg/ml matrices. In the eight-week cultures, there
was no luciferase activity in the 20 pg/ml matrices, but each of the lysates from the four
chondrocyte-seeded 10 pg/ml matrices had readings above background levels.
M.4. DISCUSSION
It was initially anticipated that addition of the plasmid to the collagen-GAG slurry
prior to freeze-drying would result in a high percentage of the DNA being retained in the
matrices upon hydration and that as the matrix degraded, the plasmid would slowly be
released to the cells. Upon addition of the plasmid solution to the slurry, however, the
collagen began to precipitate out, likely due to the addition of the highly negatively
charged plasmid.
Slower addition led to less precipitation, but the shearing created
during pipette transfer of the slurry into the molds drastically increased precipitation.
197
Once the slurry was freeze-dried and the matrices hydrated, our initial hypothesis
did prove to be correct as there was no significant release of DNA to the buffer during the
four-week leaching period. Regarding the transfection efficiency of these gene-seeded
matrices, there was no luciferase activity in the four-week in vitro cultures and only very
low levels of activity in the eight-week cultures. The fact that there was luciferase
activity at eight weeks, but not four weeks also supports our initial hypothesis that
plasmid would be released only as the matrix degrades.
The extremely low levels of transfection that were seen with these matrices may
be due to slow matrix degradation, as mentioned above, but may also be attributed to
plasmid degradation while in the acidic environment of the slurry (see Chapter 5 and
Appendix L) or during dehydrothermal treatment. If future progress is to be made
towards incorporating the plasmid into the collagen-GAG network prior to freeze-drying,
precautions should be taked to prevent such plasmid degradation.
198
APPENDIX N: STAUROSPORINE MODULATION OF THE
PHENOTYPE OF MONOLAYER-PASSAGED CANINE
CHONDROCYTES SEEDED IN COLLAGEN-GAG
MATRICES
N.1.
ABSTRACT
Staurosporine, an antibiotic known to inhibit protein kinase C and disrupt
cytoskeletal structure, was used to modulate the chondrocytic phenotype of seriallypassaged adult canine chondrocytes. Cells in monolayer cultures treated with as little as
3nM staurosporine for four days contained type II procollagen, while few cells in the
untreated control cultures demonstrated type II procollagen synthesis. Treatment with
staurosporine also led to a decrease in the amount of C-smooth muscle actin synthesized
by the cells. Consistent with this decreased expression of the contractile actin isoform,
cells cultured in three-dimensional collagen-glycosaminoglycan
(CG) scaffolds and
treated with 5nM staurosporine contracted the scaffold significantly less than untreated
cells (15% diameter contraction by treated cells, compared to more than 50% contraction
by untreated cells). In the three-dimensional CG cultures, there was no increase in cell
number during the two-week culture period. The staurosporine-treated cells, however,
were biosynthetically active, displaying higher rates of protein and GAG synthesis, as
indicated by rates of incorporation of 3H-proline and
35S-sulfate,
respectively, compared
to untreated cells. The long-held notion that changes in cytoskeletal structure influence
phenotypic characteristics of cultured chondrocytes may now be extended to relate
expression of a specific muscle actin isoform to certain cell processes.
N.2.
INTRODUCTION
With increasing serial passaging in monolayer culture, a greater percentage of
chondrocytes adopt a fibroblastic phenotype (Benya and Shaffer, 1982).
The cells
become flattened and begin synthesizing type I instead of type II collagen.
Upon
suspension in agarose or alginate gels, however, the cells again become round and
synthesize type II collagen (Benya and Shaffer, 1982; Bonaventure et al., 1994; Martin et
al., 1999). It has been proposed that there is a causal relationship between chondrocyte
shape and phenotype; re-establishment of a spherical shape signals re-expression of the
199
chondrocyte phenotype by dedifferentiated cells (Benya, 1988). This supposition led to
the hypothesis that microfilament modification serves as a signal for chondrocyte reexpression. In support of this hypothesis, studies have shown that depolymerization of
microfilaments by dihydrocytochalasin B (DHCB) and cytochalasin D can stimulate type
II collagen and proteoglycan synthesis by chondrocytes in monolayer culture (Benya and
Schaffer, 1983; Benya and Brown, 1986; Newman and Watt, 1988) and induce
chondrogenesis in vitro (Zanetti and Solursh, 1984; Rosen et al., 1986). Related studies
(Brown and Benya, 1988) showed that a change in the organization and orientation of
microfilaments was sufficient to induce re-expression of the chondrocyte phenotype, and
that depolymerization of the microfilaments is not necessary. Moreover, microfilament
modification by DHCB was found to favor chondrocytic phenotypic expression even in
the absence of a change in cell shape (i.e., rounding) (Benya et al., 1988). Conversely,
the appearance of actin stress fibers caused a decrease in type II collagen synthesis in the
absence of cell shape change (Mallein-Gerin et al., 1991).
Recent attention has focused on the specific actin isoforms that comprise the
cytoskeleton of chondrocytes. The contractile cc-smooth muscle actin (SMA) isoform,
was found in chondrocytes in adult human (Kim and Spector, 2000) and canine (Wang et
al., 2000) articular cartilage in vivo, in adult human and canine chondrocytes in
monolayer culture, and in these same cells after seeding into collagen-glycosaminoglycan
(CG) analogs of extracellular matrix (Kinner and Spector, In press; Lee et al., in press).
Some cells expressing the SMA isoform in vitro continued to express the gene for type II
collagen.
Related work showed that SMA-expressing chondrocytes could contract the
CG scaffold (Kinner and Spector, In press; Lee et al., in press) and that this contraction
could be correlated with the SMA content of the cells (Kinner and Spector, In press).
Contraction of articular chondrocytes seeded in other matrices (fibrin gels) has also been
recently reported (Shieh et al., 2000).
Collectively, these findings motivated the present study to determine the effects of
an agent known to favor re-expression of the chondrocyte phenotype on SMA expression
and the contractile behavior of the cells cultured in CG matrices. The agent employed in
this study, staurosporine, is an antibiotic known to inhibit protein kinase C and disrupt
cytoskeletal structure. It has been shown to restore the chondrocyte phenotype in cells
200
serially passaged in monolayer (Borge et al., 1997), to stimulate cartilage formation
(Kulyk, 1991; Kulyk and Reichert, 1992), and to cause rapid disruption of microfilaments
(Hedberg et al., 1990; Mobley et al., 1994).
N.3. METHODS
N.3.1. Chondrocyte Isolation and Culture
Chondrocytes were isolated from articular cartilage from the patellar, femoral and
tibial surfaces of knee (stifle) joints of two adult mongrel dogs using sequential pronase
(one hour; 20 U/ml; Sigma Chemical, St. Louis, MO) and collagenase (overnight; 200
U/ml type 2 collagenase; Worthington Biochemical, Lakewood, NJ) digestion in a
manner similar to that described by Kuettner, et. al (Kuettner et al., 1982).
Isolated
chondrocytes were resuspended in culture medium (DMEM/F12, Gibco Life Sciences)
supplemented with 10% fetal bovine serum (FBS, Hyclone Technologies), 25 gg/ml
ascorbic acid (Wako Chemical, Osaka, Japan), and IX Antibiotic/Antimycotic cocktail
(Gibco) and plated in 75-cm 2 flasks at a density of 2 million cells/flask. The culture
flasks were incubated at 37 0C and 5% CO 2 .
Cells were cultured to confluence,
trypsinized, resuspended and replated into 75-cm 2 flasks.
Cells from each dog were
cultured separately throughout.
N.3.2. Immunohistochemical Staining of Type II Procollagen in Monolayer
Immunohistochemical
staining of chondrocytes in monolayer culture was
performed to verify that staurosporine would induce type II collagen synthesis in the
serially passaged chondrocytes.
Second passage chondrocytes (corresponding to 5-6
doublings over approximately 2 weeks of monolayer culture), were plated onto tissue
culture-treated plastic chamber slides and cultured in medium containing varying
concentrations of staurosporine (3, 5, 9, and 100 nM) for four days (n=4 chambers).
Cultures were fixed in 70% ethanol for ten minutes, followed by rinsing in phosphate
buffered saline (PBS). Cultures were then incubated with the type II primary antibody
(II-116B3, prepared by T. Linsenmayer and obtained from the Developmental Studies
Hybridoma Bank, University of Iowa, Iowa City, IA; diluted 1:20 in PBS) or PBS alone
(negative control) for 45 minutes, followed by incubation with Cy3 conjugated goat antimouse IgG antibody (1:50 in PBS; Jackson Immunochemicals, West Grove, PA) for 45
201
minutes. Freshly isolated canine articular chondrocytes were similarly cultured without
staurosporine and stained as a positive control.
N.3.3. Proliferation of cells in monolayer
Third passage cells were plated into 96-well plates at a density of 5,000 cells/well.
Cells were cultured for three days in 100 pl medium containing 0, 1, 3, 5, 10, or 20 nM of
staurosporine (n=5-6 per condition). On the third day, medium was aspirated from each
well and replaced with fresh medium containing 1 ml CellTiter 96® AQueous Assay
Reagent (Promega #G4000) per 5 ml culture medium (total volume added to each well
was 120 gl). The CellTiter 960 AQueous Assay is a colorimetric method for determining
the number of viable cells. Viable cells bioreduce the tetrazolium compound (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(sulfophenyl)-2H-tetrazolium,
inner salt; MTS) to soluble formazan with the aid of an electron coupling reagent
(phenazine methosulfate; PMS)
.
The cells were cultured in the medium with the
MTS/PMS mix for one hour and the reaction stopped by the addition of 20 p1 of 10%
0.7 0.60.6
-2
0.5 -
y =0.0057x + 0.0728
R = 0.913
0.4 0.3 ~0.2
0.1 00
0
20
40
60
80
100
Viable Cell Count (xlOOO)
Figure N.1: Effect of cell number on formazan absorbance at 490 nm. Chondrocytes
were plated in well plates at varying cell densities and cultured for 1-5 days. Cells were
incubated for 2 hours with the PMS/MTS mixture from the CellTiter kit and the
absorbance at 490 nm recorded. Viable cell counts (trypan blue exclusion) were
performed on matched cultures. A linear fit to the data shows a linear increase in
absorbance with increasing cell number (R2 =0.91).
202
sodium dodecyl sulfate (SDS). The plate was wrapped in paraffin and foil and stored at
4C overnight.
The absorbance of formazan was read at 450 and 540 nm (peak
absorbance is 490 nm ) on a microplate reader. The absorbance of the medium with the
MTS/PMS mixture was also recorded by adding the mix to wells containing no cells.
The quantity of formazan, as measured by the absorbance, is directly proportional to the
number of living cells (Promega Technical Bulletin 169) (and verified for chondrocytes,
Figure N.1).
N.3.4. Western Blot Analysis of a-Smooth Muscle Actin
Second passage cells were plated into T-75 flasks and treated with 5nM
staurosporine or left untreated (control) for six days.
Cells were then collected by
trypsinization, washed by centrifugation, and resuspended in cold PBS (4"C). The cells
were then resuspended in cold protein lysis buffer (0.1% sodium dodecyl sulfate, 1mM
sodium orthovanadate, 10% glycerol) and placed on a shaker for 20 minutes at 4C after
which the suspension was centrifuged and the supernatant frozen at -20'C.
After
determining
total
protein
concentration
of
the
supernatant
(spectrophotometric assay with Bio-Rad protein dye at 595 nm), aliquots containing 10
gg of cytoplasmic protein were mixed with buffer (0.125M Tris, 20% glycerol, 4% SDS,
10% 2-mercaptoethanol, and 0.05% bromophenol blue dye), boiled for five minutes, and
loaded into electrophoretic gels. Gels were run in Tris/Glycine/SDS buffer at 1 10V for
90 minutes and then transferred to nitrocellulose membranes (100V for 60 minutes).
After washing (Tris buffered saline, TBS, 5 minutes), the membranes were exposed to a
blocking solution (5% skim milk) for one hour followed by an overnight incubation with
the primary antibody (monoclonal mouse anti-ex-SMA, #A-2547, Sigma Chemical Co.,
St. Louis, MO). The membranes were then washed (TBS with 1% Tween-20) for ten
minutes and incubated with the secondary antibody (Horseradish peroxidase conjugated
goat anti-mouse IgG, Sigma Chemical Co., #A2304) for one hour. Following thorough
washing (TBS with 1%
20-Tween), the membranes were exposed to the detection
substrate solution for two minutes, exposed to Polaroid film and the film developed.
Cytoplasmic protein from human aorta smooth muscle (5 tg) cells was used as control.
203
N.3.5. Collagen-Glycosaminoglycan Scaffold Synthesis
Porous CG matrices were produced by freeze-drying a co-precipitate of type I
bovine tendon collagen (Integra Life Sciences, Plainsboro, NJ) and shark chondroitin-6sulfate (Sigma Chemical, St. Louis, MO) as previously described (Yannas et al., 1989).
Following freeze-drying, matrices were sterilized and cross-linked for 24 hours by
dehydrothermal treatment (DHT) (Yannas et al., 1989). Nine-millimeter diameter disks
(approximately 3.5 mm thickness) were used for cell culture experiments. The matrices
have been previously reported to have a porosity of approximately 90% and an average
pore size of 85 pm (Nehrer et al., 1997b).
N.3.6. Culture of Chondrocytes in Collagen-GAG Scaffolds
Chondrocytes frozen after the second passage were thawed and cultured in T-75
flasks with supplemented (10% FBS, 25 pg/ml ascorbic acid, 1X Antibiotic/Antimycotic
Cocktail) DMEM/F12 (control) media for three days. On the second day media were
changed and replaced with control media or media with 5 nM staurosporine.
Upon
reaching confluence, cells were collected by trypsinization and resuspended at a
concentration of 4x10 6 cells/ml. CG disks were incubated with 0.5 ml of cell suspension
per disk for 1.5-2 hours on a rocking table. Approximately 50% of the chondrocytes
attach to the matrices by this seeding method. Disks were then transferred to agarosecoated wells (12-well plates) with 1.0 ml of either control media or media containing
5nM staurosporine per well, (see Table N. 1). This staurosporine concentration was based
on the findings of the monolayer culture studies as reported in the RESULTS.
After
overnight incubation, an additional 0.5 ml of the appropriate medium was added to each
well.
Media (1.5 ml) were changed every other day.
Matrix cultures treated with
staurosporine were maintained in 5 nM staurosporine throughout the entire culture
period. Cultures were terminated after 2, 7, or 15 days (n=3-6). Unseeded disks were
cultured as controls (n=2).
Table N.1. Experimental groups for CG cultures
Group
A
B
C
D
Monolayer staurosporine concentration
0 nM (control)
5 nM
OnM
5nM
204
CG staurosporine concentration
0 nM (control)
0 nM
5 nM
5 nM
N.3.7. Measurement of Cell-Mediated Contraction
The diameter of the cell-seeded and unseeded matrices were measured 1, 3, 5, 7,
and 15 days post-seeding. The contraction of each sample was calculated as the change
in matrix diameter from the day 1 value divided by the day 1 diameter. Cell-mediated
contraction (CMC) was determined by subtracting the mean value of the contraction of
the unseeded matrices from the contraction of each of the seeded matrices.
CMC =
OriginalDiameter- Diameter
OriginalDiameter
),seeded
OriginalDiameter- Diameter
OriginalDiameter
aved
N.3.8. Radiolabel Incorporation into Chondrocyte-CG Constructs
On days 2, 7, and 15 three to six samples in each group were terminated for
biochemical analyses of matrix molecule synthesis rates and proliferation.
During the
last eight hours of culture, constructs were cultured in complete media supplemented with
10 gCi/ml of 3H-proline and 10 pCi/ml of
35S-sulfate,
to assess rates of total protein and
GAG synthesis, respectively. At the end of the labeling period, disks were washed (4x15
minutes at 4C) in PBS supplemented with unlabeled proline (1 mM) and sulfate (0.8
mM), lyophilized and solubilized in 1 ml papain buffer (6 pig/ml papain and 10 mM
cysteine-HCl in 0.1 M sodium phosphate and 5 mM Na2EDTA, PBE).
Radiolabel
incorporation was determined by mixing 100 pl of the papain digest with 2 ml
scintillation cocktail (EcoLume, Costa Mesa, CA) and measuring 3H and
35S
counts per
minute (cpm) in a liquid scintillation counter (Rack-Beta 1211 LKB, Turku, Finland),
with corrections for spillover. Counts were normalized to DNA content (see below).
N.3.9. DNA Analysis
The DNA content of the constructs was measured using the Hoechst 33258 dye
assay. A 20 p.l aliquot of the papain digest was mixed with 80 p.l of phosphate buffered
EDTA and 2 ml of Hoechst dye solution (10% Hoechst dye in 10 mM Tris, 1 mM
Na 2EDTA, and 0.1 M NaCl, pH 7.4) and assayed fluorometrically. Calf thymus DNA
was used as the standard. The background fluorescence of the matrix was accounted for
by subtracting the mean value obtained for the unseeded matrices.
205
N.3.10. Statistical Analysis
Data are reported as the mean ± standard error of the mean (SEM). Analysis of
variance (ANOVA) and Fisher protected least squares difference (PLSD) post-hoc testing
were performed using StatView (SAS Institute, Inc, Cary, NC). Statistical significance
was taken to be p<0.05.
Figure N.2:
Light microscopy of 2 "d passage chondrocytes (a) untreated or (b)
treated with 5 nM staurosporine for 6 days demonstrating the angular (arrow heads)
and spherical morphology (arrow) of staurosporine treated cells, compared to the
untreated, spindle-shaped cells. Immunofluorescence (red fluorescence of Cy3
conjugated 20 antibody) for intracellular type II procollagen demonstrating decreased
synthesis of type II collagen from (c) 10 passage to (d) 2 "d passage chondrocytes. (e)
Increased staining in the second passage cells treated with 5 nM staurosporine for 4
days demonstrates the stimulation of type II procollagen production.
206
N.4.
RESULTS
N.4.1. Light Microscopy and Type II Collagen Immunohistochemistry of Cells in
Monolayer
Observations under light microscopy revealed that chondrocytes cultured on the
chamber slides and treated with staurosporine proliferated more slowly and displayed a
more angular morphology with some of the cells adopting a more spherical shape
(Figures N.2a), typical of freshly isolated chondrocytes. At low concentrations (3-9 nM),
cells remained attached to the culture slide. When treated with 100 nM staurosporine,
however, the cells were very rounded and began to detach from the slides. Chondrocytes
that were not exposed to staurosporine were more spread and elongated, more typical of
fibroblasts or dedifferentiated chondrocytes.
Immunostaining of first passage chondrocytes was positive for intracellular type
II procollagen
(Figure N.2c).
Second passage chondrocytes
cultured without
staurosporine, however, displayed only minimal positive staining for type II collagen
(Figure N.2d). In contrast, most of the second passage chondrocytes cultured with 3, 5, 9
nM staurosporine stained positive for intracellular type II procollagen (Figure N.2e
demonstrates positive staining of the 5 nM culture).
Thus, in order to maximize the
"differentiated" phenotype of the cells while maintaining near normal levels of
proliferation, a concentration of 5 nM staurosporine was chosen for the cell-seeded
matrix studies.
N.4.2. Monolayer Proliferation
The absorbance of formazan at 450 nm (Figure N.3) indicate a decrease in the
number of viable cells treated with staurosporine in concentrations greater than 1 nM. A
staurosporine concentration of 5 nM yielded an absorbance that was approximately half
that of the absorbance at 0 nM. Similar trends were seen when the absorbance was read
at 540 nm. Unfortunately, due to failure of the lamp on the plate reader, the absorbance
of these cultures was not read at the optimal 490 nm, the wavelength for which
absorbance was compared to cell count (Figure N.1). Thus, it is not possible to quantify
the proliferation/death of the cells. It should be noted, however, that cells expanded in T75 flasks in the presence of 5 nM staurosporine, prior to seeding into the CG scaffolds,
207
usually took an average of one day longer to reach confluence compared to the untreated
cells.
0.16mean +1- SEM
n=5-6
0 4
0.14
0.12
0.1 0.080.065
0.04
O 0.0200
15
10
5
20
Staurosporine (nM)
Formazan
Effect of staurosporine on monolayer cell proliferation.
absorbance at 450 nm indicates decreased cell numbers after 3 days of monolayer culture
with increasing concentrations of staurosporine, with approximately twice as many cells in
the staurosporine-free cultures compared to the 5 nM staurosporine cultures.
Figure N.3:
N.4.3. Western Blot Analysis for a-Smooth Muscle Actin
Western blot analysis confirmed the presence of the contractile actin isoform in
monolayer-cultured cells (Figure N.4).
The lighter band corresponding to the cells
SMC
P3
Figure N.4: Western blot for a-smooth muscle actin (SMA) indicating presence of the
contractile actin isoform in third passage chondrocytes (lane 2). Six days of 5 nM
staurosporine treatment lead to decreased SMA synthesis (lane 1). Equal amounts of total
protein were loaded into each lane. As a control, cell lysate from human aortic smooth
muscle cells (SMC) were run in parallel (lane 3).
208
treated with 5nM staurosporine indicates that staurosporine inhibits the production of
SMA.
N.4.4. Cell-Mediated Contraction
Cell-mediated contraction was significantly reduced when CG cultures were
treated with 5nM staurosporine (Figure N.5a; p<0.001). Untreated chondrocytes (group
a
.A:0-0
-70
S60 -Q
-- C:0-5
-
50
B:5-0
D:5-5
40-
30209Z 10- 0 10~
e
.- A.
-- --- --- ----- - --"
1
0
0
b
.
5
15
10
Days in Culture
2.5- OA:0-0O3B:5-0 IC:0-50D:5-5
gZ
2-
=LI
..........
..........
T
1-
0.5
7
15
Days in Culture
Figure N.5: (a) Decreased chondrocyte-mediated contraction (CMC), expressed as a
percent change of diameter relative to day 1 and normalized to shrinkage of unseeded
matrices, in staurosporine-treated cultures (two-way ANOVA p<0.001). Untreated cultures
(? O nM-0 nM, group A); cultures treated with 5 nM staurosporine for 6 days in monolayer
culture only (? 5 nM-0 nM, group B); cultures treated with 5 nM staurosporine in matrix
culture only (? 0 nM-0 nM, group C); and cultures treated with 5 nM staurosporine
throughout monolayer and matrix culture (? 5 nM-5 nM, group D). (b) CMC normalized
to cell number as indicated by average DNA content of cultures sacrificed on days 7 and 15
revealed the most CMC in untreated cultures by day 7 (Fisher PLSD p<0.01). By day 15,
there was no difference in CMC of untreated and monolayer-only treated groups (Fisher
PLSD p=0.85), both of which had more CMC than staurosporine-treated CG cultures
(groups A,B > C,D; Fisher PLSD p<0.005). Values are mean i SEM; n=7-10.
209
A) contracted DHT cross-linked collagen scaffolds 57 ± 0.5% (from 9mm diameter to
3.5mm diameter) over the 15 day culture period.
Cells that were treated with
staurosporine during monolayer culture only (group B) exhibited less cell-mediated
contraction during the first five days in culture (12 ± 3.1% for Group B vs. 39 ± 2.6% for
Group A). Beyond the first five days, however, the rates of contraction for the groups A
and B were similar. Taking into account the slightly lower rates of cell proliferation in
group B (see below), the amount of matrix contraction per gg DNA (CMC; Figure N.5b)
was still lower on day 7 (p<0.01). By day 15, however, there was no difference in the
total amount of CMC in groups A and B (p=0.85).
Cells that were treated with
staurosporine during the two-week culture period in the collagen scaffolds, contracted the
collagen scaffolds significantly less than the control (A) or monolayer-only treated (B)
groups. The total two-week contraction of these matrices was 11 ± 1.4% (untreated in
monolayer, group C) and 3 ± 0.6% (treated in monolayer and CG culture, group D).
Normalizing for cell content, the CMC of these matrices were also significantly lower
than untreated matrices at both day 7 and 15 (Figure 3b; p<0.005).
N.4.5. DNA Content in the Cell-Seeded CG Matrices
Staurosporine significantly decreased cell proliferation in the CG cultures only
when the staurosporine was added to the CG cultures (Figure N.6). Although there were
initially fewer cells in groups B, C and D, compared to the untreated control group A
(p<0.02), the cultures that were treated only in monolayer (Group B) proliferated in a
similar manner to the untreated controls (Group A).
These two groups had an
approximate two-fold increase in DNA content during the first week of CG culture
(p<0.0001).
In contrast, CG cultures treated with 5nM staurosporine, regardless of
whether or not the cells were treated in monolayer (Groups C and D), did not show an
increase in DNA content throughout the two-week culture period (p>0.05).
210
30 ~A:0-0
2- -
.
20
B:5-0
25~ .....- C:0-5
.l.l- D :5-5~
~- ~-
15 -
10
1
--
-
-- - - - - - - - - - - - -- a!
---
0
10
5
0
15
Days in Culture
Figure N.6: DNA content of matrices from the four experimental groups, illustrating the
decrease in proliferation due to staurosporine treatment (two-way ANOVA p<0.0001).
DNA content doubled in the untreated CG groups (A and B) during the first week (Fisher
PLSD p<0.0001), but remained relatively unchanged in the staurosporine-treated CG
cultures (C and D; Fisher PLSD p>0.05). Values are mean ± SEM; n=6-9.
N.4.6. Radiolabel Incorporation to Determine Protein and GAG Synthesis
3
H-proline and
35S-sulfate
incorporation normalized to cell number (jtg of DNA)
were used to measure rates of total protein and sulfated-glycosaminoglycan synthesis,
respectively.
Staurosporine had significant effects on rates of GAG synthesis when
added to cultures in 3-D (CG) culture, regardless of treatment in monolayer (Figure N.7a;
two-factor ANOVA p<0.001). GAG synthesis was not significantly different among the
groups on day 2, but by day 7, treatment of CG cultures with 5 nM staurosporine (groups
C and D) led to significantly higher rates of
35S-sulfate
incorporation (p<0.05).
On day
15, the increase in the rate of GAG synthesis in staurosporine-treated CG cultures was
even more apparent. At this time point, treated cultures had synthesis rates that were
more than ten-fold higher than untreated groups (p<0.005). Additionally, rates of GAG
synthesis increased over time in culture for the staurosporine-treated groups (p<0.005),
whereas, it decreased in the untreated groups (p<0.05).
In contrast to the dramatic effects of staurosporine on GAG synthesis,
staurosporine treatment had relatively little effect on protein synthesis (Figure N.7b).
There were no differences in the rate of 3 H-proline incorporation among the groups on
days 2 and 7 (p>0.15). By day 15, however, cultures treated with staurosporine while in
211
a ci
0.120.14 0.08
Coo
ElA:0-0
B:5-0
C:0-5
D:5-5
0.04
0.02
0
b
7
2
15
0.14
a
S
0.12
-O< 0.1
0.08
S
a
O
0.06'
0.04
S
O
0.020
2
7
Days in Culture
15
Figure N.7: Relative rates of (a) 35S-sulfate and (b) 3H-proline incorporation at various
times in culture. Total amount of radiolabel incorporated into the matrices was normalized to
the radiolabel incubation time and DNA content. Staurosporine-treated CG cultures had
elevated rates of GAG synthesis on days 7 and 15 (Fisher PLSD p<0.05) and elevated rates of
protein synthesis on day 15 (Fisher PLSD p<0.05). Values are mean ± SEM; n=6-9.
the CG matrix (Groups C and D) had rates of proline incorporation approximately twice
that of the untreated cultures (A and B; p<0.05).
212
N.5.
DISCUSSION
Consistent with prior work with rabbit articular chondrocytes (Borge et al., 1997),
we found that staurosporine in concentrations as low as 3nM induced morphological
changes and up-regulated type II procollagen synthesis in monolayer cultures of second
passage adult canine chondrocytes.
A novel finding of the current study was that
staurosporine treatment of serially-passaged chondrocytes decreased expression of SMA
and slowed proliferation.
Moreover, in three-dimensional CG cultures, there was a
significant reduction in the amount of cell-mediated matrix contraction, normalized to
cell number, by the staurosporine-treated chondrocytes compared to the untreated
cultures.
These findings are consistent with the decrease in SMA content, as recent
studies have revealed the association of SMA expression and the contraction of CG
scaffolds by articular chondrocytes (Kinner and Spector, In press).
It was not possible to directly determine if the decrease in formazan absorbance
with increasing staurosporine concentrations in the monolayer proliferation assay was
due to simply decreased proliferation rates or cell death (due to failure of the primary
plate reader). Based on previous experimentation with this assay and these chondrocytes
(1 doubling in the first 3 days of culture after passaging, using staurosporine-free media),
coupled with the DNA readings of the CG cultures, it appears that there was no or very
little net proliferation with 5 nM staurosporine in the 96 well plates over the three day
period.
Interestingly, however, cells plated in T-75 flasks and treated with 5 nM
staurosporine did proliferate, taking only about one day longer to reach confluence (i.e.
growing from 2x10 6 to 8-10 x 106 cells) than cells cultured in staurosporine-free media.
Our observations that treatment with staurosporine decreased proliferation rates
and increased GAG biosynthesis rates of chondrocytes in CG cultures are consistent with
a change to a more chondrocytic phenotype. While there was also an increase in protein
biosynthesis on day 15 in the staurosporine-treated cultures, it was not as dramatic as the
increase in GAG biosynthesis. It should be noted that interpretation of the 3H-proline
incorporation rates is complicated by the fact that proline is incorporated into various
intracellular and extracellular proteins which were not identified in the present study.
Further studies are needed to investigate the association between expression of SMA and
chondrocyte behavior to determine if there is a causal relationship.
213
Previous studies have shown that the regulation of certain phenotypic traits of
cells, such as proliferation and biosynthesis, involves the cytoskeletal composition and
organization (Watters et al., 1996; Vinall et al., 2001). Recently it has been shown that
bistratene A (Johnson et al., 2001), an agent that activates protein kinase C-delta and
induces reorganization of the microfilaments (Watters et al., 1996), induces cell rounding
and induction of type II collagen synthesis in serially passaged dedifferentiated
chondrocytes in much the same way as staurosporine. Placed in the context of other data
this suggested to the authors (Watters et al., 1996) a link between signaling molecules
and the chondrocyte cytoskeleton. Additionally, it has been shown that type II collagen
and aggrecan synthesis is absent in chondrocytes with well-defined actin cables (MalleinGerin et al., 1991), and that disruption of the actin cytoskeleton leads to chondrogenesis
in mesenchymal cells (Chang et al., 1998). Our results, taken together with the previous
work, suggest that the induction of the chondrocyte phenotype by staurosporine may be
due to its effects on the composition as well as the organization of the cytoskeleton (Yu
and Gotlieb, 1992).
The expression of SMA is not merely associated with a dedifferentiation of
chondrocytes to fibroblasts. The majority of chondrocytes in certain zones of articular
cartilage have been found to contain SMA in vivo by immunohistochemistry (Kim and
Spector, 2000).
It has been proposed that SMA expression should be considered a
phenotypic trait of chondrocytes conferred on them by their progenitor, the mesenchymal
stem cell (viz., the bone marrow stromal cell) (Kinner and Spector, In press). Various
chemical and physical components of the microenvironment of the chondrocyte might be
expected to up- or down-regulate expression of SMA in these cells. The present findings
support the hypothesis of an inverse relationship between the pathways that result in
expression of the genes that code for cytoskeletal proteins and matrix molecules.
One of the underlying problems in articular cartilage regeneration is the low
mitotic activity of chondrocytes. Various efforts to promote cartilage repair have focused
on eliciting an enhanced reparative response by implanting autologous chondrocytes
expanded in monolayer culture (Brittberg et al., 1994). Thus, a potential protocol for
tissue engineering of articular cartilage may include expanding isolated chondrocytes in
monolayer, where they display increased rates of proliferation while undergoing
214
dedifferentiation.
Staurosporine treatment could subsequently be implemented in order
to re-induce the chondrocyte phenotype. It should be noted, however, that the effects of
staurosporine appear to be transient.
When cells were treated with staurosporine in
monolayer but not in the subsequent three-dimensional matrix cultures, the proliferation
and synthesis rates were not significantly different from those measured in control
cultures on days 7 and 14.
It is possible, however, that longer-term exposure to
staurosporine may lead to a more stable phenotype transition.
It should also be noted that the matrices used in this study were predominately
type I collagen.
Previous studies have reported that passaged chondrocytes are more
likely to adopt the spherical morphology typical of chondrocytes when seeded in type II
collagen matrices (Nehrer et al., 1997a).
Thus, it may be possible that the re-
differentiation triggered by staurosporine treatment of cells in monolayer may be better
retained by seeding the cells into type II collagen matrices.
It should also be noted,
however, that the specific matrix molecules that were synthesized by the seeded cells
were not determined. It is possible that although the radiolabel incorporation rates were
similar for groups A (never treated with staurosporine) and B (treated with staurosporine
in monolayer), there could have been more type II collagen and/or aggrecan synthesis in
the group B cultures. Future work is necessary to investigate the effects of the matrix
chemistry on maintaining the chondrocyte phenotype and to determine the specific matrix
molecule biosynthesis.
N.6.
ACKNOWLEDGEMENTS
Thank you to Robyn Marty-Roix for the Western blot analysis, Sandra Zapatka-
Taylor
for
histological
assistance
and
Katherine
Oates
for
assistance
with
immunofluorescent staining. Primary antibody for type II collagen, II-116B3, prepared by
T. Linsenmayer and obtained from the Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City, IA.
N.7.
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