Expression of Alpha-Smooth Muscle Actin and Contraction of
Collagen-Glycosaminoglycan Scaffolds by Cells
Derived from Canine Synovium
By
Scott M. Vickers
B.S., Mechanical Engineering
University of Kentucky, 2001
Submitted to the Department of Mechanical Engineering
in Partial Fulfillment of the Requirements for the Degree of
Master of Science in Mechanical Engineering
at the
Massachusetts Institute of Technology
MASSACHUSETTS INSTITUTE
OFTECHNOLOGY
February 2003
JUL 0 8 2003
LIBRARIES
@ 2003 Massachusetts Institute of Technology
All rights reserved
Signature of Author:
Department of Mechanical Engineering
January 14, 2003
Certified by:
//
SV
*
/Myron Spector
Senior Lecturer, Department Mech/ ical Engineering
Professor of Orthopaedic Surgery (Biomateri ), Harvard Medical School
Thesis Supervisor
Accepted by:
''....
Ain Al Sonin
Chairman, Department Committee on Graduate Students
1000:1
40C
Expression of Alpha-Smooth Muscle Actin and Contraction of Collagen-Glycosaminoglycan
Scaffolds by Cells Derived from Canine Synovium
By
Scott M. Vickers
Submitted to the Department of Mechanical Engineering
on January 14, 2003 in Partial Fulfillment of the
Requirements for the Degree of Master of Science
in Mechanical Engineering
ABSTRACT
Recent studies have demonstrated that several types of musculoskeletal connective tissue
cells - including chondrocytes, fibrochondrocytes, ligament fibroblasts, osteoblasts, and
mesenchymal stem cells - can express the gene for the contractile actin isoform, alpha-smooth
muscle actin (SMA), and can contract analogs of extracellular matrix. While the physiological
role of the SMA-enabled contraction of these cells remains to be elucidated, such contractility
may have detrimental effects when the cells are seeded in scaffolds employed for tissue
engineering.
These prior findings prompted investigation of SMA expression in synovial cells. These
cells have attracted recent interest as donor cells for tissue engineering of articular cartilage
because they have been implicated in certain cartilage repair processes in vivo and the
chondrogenic potential of the cells has recently been demonstrated in vitro. The objective of this
study was to evaluate SMA expression by adult canine synovial cells and their related contraction
of collagen-glycosaminoglycan (GAG) analogs of extracellular matrix, as well as cellular
proliferation and chondrogenic potential within the scaffolds.
Cells from synovial membranes of 6 adult dogs were isolated by outgrowth from the
tissue and expanded through seven passages in monolayer culture, with samples from each
passage allocated for Western blot analysis of SMA. Cells from passage 4 were seeded into
porous type I collagen-GAG matrices and cultured for 4 weeks. The diameters of the cell-seeded
scaffolds and non-seeded controls were measured every other day. Synovium-derived cells from
the fourth passage were formed into micro-pellets by centrifugation and others seeded into the
collagen-GAG matrices, and were incubated in chondrogenic medium with and without fetal
bovine serum. After 3 weeks the specimens were prepared for type II collagen
immunohistochemistry.
Immunohistochemistry revealed the presence of SMA in some cells in the intimal layer
of synovium from 4 of the 5 animals analyzed. Western blot analysis demonstrated a regular
increase in the amount of SMA in the synovial cells with passage number. The synovial cellmediated contraction of the collagen-GAG scaffolds reached a value of 43% of the original
diameter after 4 weeks. This cell-mediated contraction associated with SMA expression was
comparable to that found with other musculoskeletal cell types. Incubation of cultures of
synovial cells with chondrogenic medium revealed trace amounts of type I collagen production
by immunohistochemistry, suggesting that they may be capable of differentiating into
chondrocytes. The findings of this study indicate that control of SMA-enabled contraction may
be important when employing synovial cells for cartilage repair procedures, and warrant further
investigation into the physiological role of SMA expression in synovial cells.
Thesis Supervisor: Myron Spector
Title: Senior Lecturer, Department of Mechanical Engineering
Professor of Orthopaedic Surgery (Biomaterials), Harvard Medical School
3
ACKNOWLEDGEMENTS
The work presented here could not have been accomplished without the help and
guidance of a great number of people, to whom I am deeply indebted.
First, I must thank my supervisor, Prof. Myron Spector, for the opportunity to
work on a project from which I have learned so much. Your guidance, support,
encouragement, and concern for my development as a researcher and scholar are greatly
appreciated. Your enthusiasm is contagious, and has greatly stimulated my interest in the
field of tissue engineering.
Secondly, I would like to thank Prof. Yannas and Prof. Gibson for their advice
and the use of their laboratory space and equipment. You have both taught me a great
deal.
In addition, I must thank all of the individuals who have been a part of the
Orthopaedics Research Laboratory at Brigham and Women's Hospital and the Fibers and
Polymers Laboratory at MIT for your assistance in so many areas.
" Brendan, thank you for coming in at odd hours to teach me to produce matrices,
for help with classes, and for your encouragement in general.
* Dr. Hsu and Dr. Xiang, thank you for teaching me to harvest synovium, and for
the many trips you made with me to NEMC.
" Robyn, thanks for teaching me to do Western blots, DNA analysis, and general
cell culture procedures.
" Liqun, thank you for helping me improve my Western blot protocols, and for
running so many of the assays when I was pressed for time.
" Nikki, Ramille, Dan, Leonide, Dawn, Jamil, Tim, Changming, and Ricardo,
thanks for your everyday companionship, stimulating discussions, and for making
the lab such an enjoyable workplace.
The financial support for this study provided by the Cambridge-MIT Institute is
gratefully acknowledged.
To all of my family and friends who have supported me throughout this entire
process, I cannot thank you enough. Luke, thanks for keeping me on my toes, and
making my life so much fun. And finally, Jenny, the acknowledgment you deserve
cannot be adequately put into words. Thank you for your companionship in all of life, for
supporting me in my academic endeavors, for sharing my times of excitement and
sympathizing with my frustrations. Thanks for taking care of the details, and for always
being there.
4
TABLE OF CONTENTS
ACKNOW LEDGEM ENTS ......................................................................................................
4
TABLE OF CONTENTS ........................................................................................................
5
LIST OF FIGURES .......................................................................................................................
7
1. INTRODUCTION AND BACKGROUND.........................................................................
9
1.1 CLINICAL PROBLEM: CARTILAGE DAMAGE AND DEGRADATION ....................................
1.2 CURRENT CLINICAL TREATMENTS..................................................................................
9
10
1.2.1 Microfracture Technique .......................................................................................
1.2.2 Tissue and Cell Transplantation...........................................................................
10
11
1.3 PROSPECTS FOR TISSUE ENGINEERING............................................................................
12
1.4 REVIEW OF SYNOVIUM, COLLAGEN-GAG SCAFFOLDS, AND SMA-ENABLED
CO N TR A CTION .................................................................................................................
13
1.4.1 Synovium Structure and Function.........................................................................
1.4.2 Collagen-GAG matrices.........................................................................................
1.4.3 SMA-Enabled Contraction.....................................................................................
13
14
14
2 SPECIFIC AIM S AND W ORKING HYPOTHESES.....................................................
15
3 MATERIALS AND METHODS .......................................................................................
17
3.1 SYNOVIAL CELL ISOLATION AND CULTURE...................................................................
3.2 THREE-DIMENSIONAL CELL CULTURE USING COLLAGEN-GAG SCAFFOLDS................
17
18
3.2.1 Matrixfabrication..................................................................................................
18
3 .2 .2 Cell-Seeding ...............................................................................................................
18
3.2.3 Matrix ContractionMeasurements .........................................................................
18
3.3
3.4
3.5
3.6
3.7
CHONDROGENIC DIFFERENTIATION CULTURE .................................................................
W ESTERN BLOT ANALYSIS FOR SMA ............................................................................
D N A A N A LY SIS ..................................................................................................................
HISTOLOGY AND IMMUNOHISTOCHEMISTRY ..................................................................
STATISTICAL ANALYSIS ..................................................................................................
4 RESULTS ................................................................................................................................
19
19
20
21
22
23
4.1 SMA EXPRESSION IN VIVO .................................................................................................
4.2 CELL GROWTH AND IN VITRO EXPRESSION OF SMA .....................................................
23
25
4.2.1 Cell Growth in Monolayer Culture .......................................................................
4.2.2 Western Blot Analysis of SMA Content...................................................................
25
25
4.3 CONTRACTION OF COLLAGEN-GAG SCAFFOLDS...............................................................
27
4.3.1 DiameterMeasurements .........................................................................................
27
4 .3.2 D NA Content ..............................................................................................................
28
4.3.3 Histology of the Cell-SeededMatrices...................................................................
29
4.4 CHONDROGENIC DIFFERENTIATION OF SYNOVIAL CELLS .............................................
5 DISCUSSION..........................................................................................................................
5.1
5.2
5.3
5.4
SMA EXPRESSION IN VIVO .............................................................................................
IN VITRO EXPRESSION OF SMA .......................................................................................
CHONDROGENIC DIFFERENTIATION OF SYNOVIAL CELLS .............................................
CONTRACTION OF COLLAGEN-GAG SCAFFOLDS............................................................
29
35
35
35
36
36
5
6 C ON CLU SIO N S.....................................................................................................................39
7 LIMITATIONS AND FUTURE WORK..........................................................................
41
REFEREN CES ............................................................................................................................
43
APPENDIX A: FABRICATION OF COLLAGEN-GAG SCAFFOLDS ..............
49
A . 1 PREPARATION OF COLLAGEN-GA G SLURRY .................................................................
A .2 FREEZE-D RYING .................................................................................................................
A .3 D EHYDROTHERMAL (DH T) CROSS-LINKING ..................................................................
49
50
50
APPENDIX B: CELL ISOLATION AND CULTURE..........................................................
51
B .1
B .2
B .3
B.4
B .5
TISSUE H ARVEST ................................................................................................................
PASSAGING CELLS ..............................................................................................................
CELL COUNTING .................................................................................................................
FREEZING CELLS ................................................................................................................
THAW ING CELLS.................................................................................................................
51
52
53
54
54
B .7 CELL-PELLET CULTURES................................................................................................
55
APPENDIX C: BIOCHEMICAL ASSAYS............................................................................
57
C.1 W ESTERN BLOT FOR DETECTION OF SM A .......................................................................
57
C.1.1 Protein Extraction..................................................................................................
C.1.2 Protein Assay .............................................................................................................
C.1.4 Blot Transfer ..............................................................................................................
C. 1.6 D ensitometric Analysis............................................................................................
C.2 DN A ANALYSIS..................................................................................................................
C.2.1 PapainDigestion.....................................................................................................
C.2.2 Fluorometric Quantificationof DNA .....................................................................
57
58
62
64
65
65
65
APPENDIX D: HISTOLOGY AND IMMUNOHISTOCHEMISTRY ...............................
67
D .1
D .2
D.3
D.4
6
PARAFFIN EMBEDDING .......................................................................................................
H EM ATOXYLIN AND EOSIN (H & E) STAINING ..................................................................
IMMUNOHISTOCHEMICAL STAINING OF ct-SMOOTH MUSCLE ACTIN............................
IMMUNOHISTOCHEMICAL STAINING OF TYPE II COLLAGEN...........................................
67
68
69
71
LIST OF FIGURES
Figure 1: Immunohistochemical staining for cc-smooth muscle actin (SMA) in 2
samples of intact adult canine synovium.....................................................
23
Figure 2: Cell growth rate in monolayer culture..........................................................
25
Figure 3: SMA content of synovial cells passage number..........................................
26
Figure 4: Linear regression analysis of SMA content of synovial cells with time in
m onolayer culture..........................................................................................
26
Figure 5: Diameter reduction of synovial cell-seeded and control scaffolds................
27
Figure 6: Cell-mediated contraction of the collagen-GAG matrices ...........................
28
Figure 7: DNA content of the cell-seeded matrices.....................................................
28
Figure 8: Histology and immunohistochemistry of synovial cell-seeded matrices. ........ 31
Figure 9: Immunohistochemical staining of type II collagen in synovial cell
m icropellet and m atrix cultures.....................................................................
33
7
8
1. INTRODUCTION AND BACKGROUND
Articular cartilage is a specialized connective tissue that provides a low-friction
load-bearing surface in diarthroidal joints such as the knee, hip, and elbow. When
damaged, articular cartilage generally does not heal, but rather continues to degenerate,
often leading to osteoarthritis. Although several surgical procedures that attempt to
induce healing of the damaged cartilage tissue have been reported to relieve symptoms of
pain and dysfunction, none have yet achieved the desired regeneration of native cartilage
over the long term.
Recent studies have indicated the promise of tissue engineering approaches to the
problem of cartilage repair and regeneration. Much of the current work has been focused
on transplantation of cells from healthy cartilage into the damaged area, with or without a
biodegradable scaffold. Recent demonstration of the chondrogenic potential of cells from
the synovial membrane, however, has anticipated the use of such cells instead of
chondrocytes in cartilage repair procedures, thus eliminating some of the difficulties such as donor site morbidity - encountered when using native chondrocytes.
The purpose of this thesis was to lay the groundwork for the development of a
tissue-engineered implant employing synovial cells and collagen-glycosaminoglycan
(GAG) scaffolds to facilitate articular cartilage regeneration. In designing such implants,
it is necessary to understand various aspects of the cell-matrix interaction, such as cellmediated contraction of the construct, as has been reported for a variety of
musculoskeletal connective tissue cells. Toward this goal, synovial cell-mediated
contraction of the collagen-GAG scaffolds and their related expression of the contractile
actin isoform, cc-smooth muscle actin, was examined, as well as cellular proliferation and
chondrogenic potential within the scaffolds.
1.1
Clinical Problem: Cartilage Damage and Degradation
That spontaneous healing of damaged articular cartilage is rarely encountered has
been recognized for over two and a half centuries [25]. Several factors contribute to the
limited healing capability of this tissue, including:
1)
Articular cartilage is primarily avascular, and thus lacks the systemic supply
of cells and soluble regulators that typically initiate the repair process.
Additionally, the avascularity precludes the formation of a fibrin clot, which
9
in vascular tissues acts as a temporary scaffold in which cells can migrate and
begin synthesizing new tissue.
2) The cell number density in articular cartilage - approximately 10,000
cells/mm3 [50] - is low compared to that of other tissues, thus limiting the
number of native cells available to participate in the repair process.
3) The contribution of chondrocytes (cells native to cartilage) to repair of
damaged tissue is hindered by their limited migratory, mitotic, and metabolic
activity.
Instead of healing, lesions in articular cartilage compromise the mechanical
properties of the surrounding tissue, predisposing the joint to further degeneration [15,
16]. Such degradation of the joint surfaces can result in the painful and often debilitating
condition of osteoarthritis, one of the leading causes of disability affecting nearly 21
million Americans [1]. Severe cases of osteoarthritis often necessitate total joint
arthroplasty in order to ameliorate symptoms. While encouraging results of pain relief
and improvement of joint function have been reported for joint replacement surgeries in
elderly patients, such procedures have a high rate of failure in individuals under the age
of 65 [57].
1.2
Current Clinical Treatments
Several surgical procedures focusing on repair of damaged cartilage tissue have
been developed in efforts to relieve pain, restore functionality, and avoid or postpone the
need for joint replacement in younger individuals (see Hunziker [27] and O'Driscoll [55]
for review). Two approaches currently in clinical use are microfracture of the
subchondral plate and tissue or cell transplantation.
1.2.1
MicrofractureTechnique
While chondral defects (those limited to the cartilage layer) do not spontaneously
heal due in part to the reasons mentioned above, wounds that penetrate the sub-chondral
plate into the underlying bone space have much greater intrinsic healing capacity [18,
66]. Since bone is much more vascular than cartilage, wounds that penetrate into the
bone space result in the formation of a fibrin clot within the lesion and introduce a supply
of cells and growth factors that initiate a repair response. Although the native
chondrocytes do not contribute substantially to the repair process [60], mesenchymal
10
stem cells derived from bone marrow can differentiate into chondrocytes or osteoblasts
(bone-forming cells) as the new repair tissue is formed.
The microfracture technique attempts to stimulate this spontaneous healing
response by extending chondral lesions into the underlying bone space. This arthroscopic
procedure introduces small holes in the subchondral bone plate by means of an awl,
inducing bleeding from the bone space into the damaged cartilage [63].
Although positive results have been reported with use of the microfracture
technique [61, 63], there are inherent disadvantages to the procedure. The spontaneous
healing response to osteochondral defects generally results in repair with fibrocartilage,
which is not durable over the long-term under physiological loading conditions [5, 48, 60,
64]. Additionally, the microfracture technique compromises the mechanical integrity of
the subchondral plate, albeit to a lesser extent than procedures employing drills or pins
(e.g. Pridie drilling and abrasion chondroplasty) [27, 55].
1.2.2
Tissue and Cell Transplantation
Other clinical approaches to treating chondral defects involve transplantation of
tissue or cells from a relatively low-weight-bearing region of the cartilage in the same or
other joint. In the mosaicplasty procedure, osteochondral plugs are removed from the
edge of the patellar groove or proximal to the intercondylar notch and transplanted into
the cartilage defect [17, 55]. In the cell-based procedure, referred to as autologous
chondrocyte implantation (ACI), harvested tissue is enzymatically digested to obtain the
cartilage cells, which are then expanded in culture and injected into the defect underneath
a periosteal graft [8].
One readily apparent drawback to these transplantation procedures is donor site
morbidity. Although the harvest site may temporarily fill with fibrocartilaginous repair
tissue, it is expected to degenerate in the long-term [27]. Furthermore, the harvest
procedure has been shown to adversely affect the mechanical properties of surrounding
tissue [39].
In addition to donor site morbidity, animal investigations utilizing the
mosaicplasty technique have revealed short-term degeneration of both the transplanted
tissue and surrounding host cartilage [2, 37]. Additional difficulties encountered with
transplantation of chondrocytes include in vitro expansion of the cells [46, 56], retention
11
of the transplanted cells within the defect [6, 7], integration of newly synthesized tissue
with host tissue [6, 8], and damage to host tissue caused by the suturing procedure [7].
1.3
Prospects for Tissue Engineering
Tissue engineering employs the three major components of tissues - cells,
extracellular matrix, and soluble regulators - or substitutes thereof, either alone or in
combination, in efforts to restore the form and function of damaged tissue [36]. While
tissue-engineering approaches utilizing each of these components alone (such as ACI)
have yet to succeed in producing the desired results of regenerated articular cartilage,
methods employing combinations of these components (viz. cells and ECM analogs)
have been successful in regeneration of other tissues (see Yannas [68] for review) and
have shown promise for articular cartilage [5]. Many different types of matrices have
been investigated, such as fibrin, collagen, polyglycolic or polylactic acid, agarose,
alginate, and synthetic polymers such as Teflon and Dacron [27]. Cells that have been
investigated for use with these matrices, in addition to autologous and allogenic
chondrocytes, include chondroprogenitor cells from sources such as periosteum and
perichondrium [40], and bone marrow [9, 34].
Another source of cells that has drawn interest of late is the synovium. Synovial
cells have recently been implicated in various cartilage repair procedures in vivo [26, 28],
and their chondrogenic potential has been demonstrated in vitro [13, 54]. Synovium is
both spontaneously regenerative [31] - thus eliminating the problem of donor site
morbidity - and easily accessible during explorative or therapeutic arthroscopy. It is
thus anticipated that this tissue may be used as a source of donor cells for articular
cartilage tissue engineering.
In designing tissue-engineered implants it is necessary to understand various
aspects of the cell-matrix interaction. For example, a variety of musculoskeletal
connective tissue cells, when cultured in collagen scaffolds, have been shown to contract
by means of their expression of the contractile actin isoform, cc-smooth muscle actin
(SMA) [62]. Such cellular contraction results in architectural deformation of the
scaffold, which could contribute to failure of the engineered construct when implanted in
vivo.
12
The purpose of this study was to lay the groundwork for development of a tissueengineered implant, composed of cells from the synovial membrane seeded in a collagenglycosaminoglycan (GAG) scaffold, to facilitate regeneration of articular cartilage.
Toward this goal, the SMA expression of the synovial cells and the related cell-mediated
contraction of the constructs were examined, as well as cellular proliferation and
chondrogenesis within the scaffolds.
1.4
1.4.1
Review of Synovium, Collagen-GAG Scaffolds, and SMA-Enabled
Contraction
Synovium Structure and Function
Synovium is a thin membrane of connective tissue found on the innermost lining
of the joint capsule in diarthroidal joints (see Hung [24]for review). It is composed of an
intimal layer of epithelial cells and a sub-intimal stroma composed primarily of fibrous,
adipose, and areolar connective tissue. Unlike most connective tissues, synovium lacks a
basement membrane separating the epithelial and stromal regions.
The synovial intima is a layer of cells usually 2-3 cells deep comprising two cell
types referred to as type A and type B synoviocytes. The type A cells are phenotypically
similar to macrophages, being highly phagocytic and staining immunohistochemically for
a number of macrophage markers. The type B cells resemble fibroblasts ultrastructurally,
but differ from sub-intimal fibroblasts in that they stain with a specific monoclonal
antibody (MAB 67) and are marked by a high level of uridine diphosphoglucose
dehydrogenase (UDPGD) activity, a precursor to hyaluronan synthesis.
The synovium is responsible for two primary functions. The first is control of
transsynovial diffusion, which supplies nutrients to the avascular meniscal and
cartilaginous tissues and regulates intra-articular pressures. The second function is
synthesis of molecular components of synovial fluid, including hyaluronan and lubricin.
The chondrogenic potential of synovial cells under certain pathological conditions
has been recognized for some time. In synovial chondromatosis, cartilage-like nodules
are found in various parts of the synovial membrane [45]. Also, a pannus of granulation
tissue in which cells appear to display both synovial and chondrocyte-like characteristics
is often found in cases of rheumatoid arthritis [3]. In addition to pathologic conditions,
13
synovial cells have recently been shown to display a chondrocytic phenotype when
exposed to certain growth factors both in vivo and in vitro.
1.4.2
Collagen-GAG matrices
The porous collagen-glycosaminoglycan scaffolds employed in this study were
first developed by for dermal tissue engineering [69]. Recently, these scaffolds,
composed of type 1 collagen and chondroitin-6-sulfate, have demonstrated promise in
facilitating regeneration of peripheral nerves [10, 11], conjunctiva [23], intervertebral
disk annulus [19], and articular cartilage [5]. These matrices have been employed as an
analog of extracellular matrix in investigations of a variety of connective tissue cellmatrix interactions, including SMA-enabled contraction [62].
1.4.3
SMA-Enabled Contraction
Actin is a cytoskeletal protein associated with cellular shape, migration, and
contraction. In humans there are six isoforms of actin, each encoded by a different gene.
Four of these actin isoforms - y-smooth (enteric) muscle, a-skeletal muscle, a-cardiac
muscle, and a-smooth (vascular) muscle (SMA) - are critical components of the
contractile apparatus of their respective muscle cell types. It was initially thought that
SMA was only expressed by vascular smooth muscle cells, but later was found in
fibroblasts responsible for the contraction observed during healing of skin wounds [20,
41]. More recently, studies have demonstrated that some musculoskeletal connective
tissue cells - including chondrocytes [33, 43], fibrochondrocytes [49], ligament
fibroblasts [52], and osteoblasts [14, 47] - can express SMA and can contract scaffolds in
which they are grown. While the physiological roles of SMA-enabled contraction of
these cells have yet to be established, cell-mediated contraction of scaffolds can alter the
pore diameter of the matrix and distort its overall shape, and thus needs to be addressed
in the design of tissue-engineered implants.
14
2
SPECIFIC AIMS AND WORKING HYPOTHESES
In order to lay the groundwork for future investigations employing synovial cell-
seeded collagen-GAG matrices for regeneration of articular cartilage, the specific aims of
this thesis were as follows:
1. To investigate the expression of SMA by synovial cells in vivo.
2. To evaluate the in vitro expression of SMA by synovial cells with passage in
monolayer culture.
3. To evaluate the contraction of the collage-GAG matrices by the synovial cells.
4. To examine the chondrogenic potential of the synovial cells when cultured in the
collagen-GAG matrices.
The working hypotheses of the investigations were:
1.
SMA-expressing cells are present in normal, adult, canine synovium.
2. The SMA content of the cells increases with passage number in monolayer
culture.
3. The synovial cells will contract the collagen-GAG matrices into which they were
seeded.
4. The synovial cells will express a chondrogenic phenotype when cultured in
collagen-GAG matrices in the presence of a chondrogenic medium.
15
16
3
3.1
MATERIALS AND METHODS
Synovial Cell Isolation and Culture
Specimens of synovial membrane were obtained aseptically from the knee joints
of 6 dogs immediately postmortem and washed extensively in phosphate buffered saline
(PBS; Life Technologies, Grand Island, NY) supplemented with antibiotic/antimycotic
solution (100 U/ml penicillin, 100 tg/ml streptomycin, 0.25 tg/ml amphotericin B, Life
Technologies). Representative samples from 5 of the animals were allocated for
histological and immunohistochemical evaluation. Specimens were finely diced and
placed in 25 cm 2 culture flasks in complete medium consisting of Dulbecco's Modified
Eagle's Medium/Nutrient Mixture F12 (DMEM/F12, Life Technologies), 10% fetal
bovine serum (FBS; Hyclone Laboratories, Logan, UT), 2% ascorbic acid phosphate
(Sigma Chemicals, St. Louis, MO), and 1% antibiotics. Culture conditions were
maintained at 37*C in an atmosphere of 5% CO 2 and 95% humidity. Medium was first
changed after 4 to 5 days in order to allow the tissue to attach to the culture flask, then
was changed every 2 to 3 days. Once cells growing out of the tissue pieces reached a
confluent layer covering approximately 50% of the flask surface (i.e., passage 1), tissue
pieces were removed and cells were released by treatment with EDTA-Trypsin (Sigma)
and sub-cultured in 75 cm 2 flasks for seven subsequent passages (P2-P8). Seeding
density at each passage was 1.33x 104 cells/cm 2. The time required for the cells to reach
confluence ranged from 6 to 8 days. Growth kinetics were determined by the following
formula:
Population doublings per day = ln(N/No) * t- 1,
where t is the time period, N is the cell number at the end of the time period, t, and No is
the cell number at the beginning of the time period [42].
During the second passage, a portion of the cells from each animal was suspended
in complete medium containing 10% dimethylsulfoxide (DMSO, Sigma) and frozen at
-70'C for up to 6 months prior to use in the chondrogenic differentiation assays (see
section 3.3).
17
3.2
3.2.1
Three-Dimensional Cell Culture Using Collagen-GAG Scaffolds
Matrixfabrication
Porous type I collagen-GAG scaffolds were fabricated using a procedure
previously described [69] (See Appendix A for detailed protocols). A co-precipitate of
type I collagen from bovine tendon (Integra Life Sciences, Plainsboro, NJ) and
chondroitin-6-sulfate from shark cartilage (Sigma) was freeze-dried to produce sheets
approximately 3 mm in thickness. After dehydrothermal cross-linking for 24 hours, 9
mm diameter samples were cut from the sheets. Prior work has shown these matrices to
have a porosity of approximately 87% and a mean pore diameter of 83 pm [53].
3.2.2
Cell-Seeding
Prior to seeding, matrix samples were soaked in PBS for 1 hour, transferred to
complete medium for 10 minutes, and dried briefly on sterile filter paper. Each matrix
was seeded with P4 cells by pipetting a suspension of 2x 106 cells in 50 pl medium onto
the surfaces of the matrix (25 pl per side), resulting in a density of approximately lx 104
cells/mm 3. Cell-seeded matrices and non-seeded controls were cultured in 20 mm
diameter wells of 12-well plates coated with 1.5 ml of agarose (2% w/v) to prevent the
cells from attaching to the polystyrene surface. Complete medium (0.5 ml) was added to
each well 2 hours post-seeding, followed by another 1.0 ml 12-16 hours later. Media
were changed every other day.
3.2.3
Matrix ContractionMeasurements
The diameters of the cell-seeded matrices as well as non-seeded controls cultured
in parallel under identical conditions were measured by visual inspection every other day
using circular templates ranging from 1 mm to 10 mm diameter in 0.5 mm increments.
The change in diameter of the matrices was expressed as a percentage reduction in the
diameter of the scaffolds based on the starting diameter of the matrices prior to hydration
on the day of seeding. The contraction of the non-seeded scaffolds was subtracted from
the contraction of the cell-seeded samples to yield a measure of "cell-mediated
contraction." Cultures were terminated after 1, 7, 14 and 28 days post-seeding for
histology and analysis of DNA content.
18
3.3
Chondrogenic Differentiation culture
A micro-pellet culture system previously described [30] was employed to
investigate the chondrogenic potential of the synovial cells. Cells frozen at
-70'C were thawed, rinsed of DMSO by centrifugation, and expanded through two
passages in monolayer culture. Aliquots of 2.5 x 105 cells (P4) were gently centrifuged
in 15 ml centrifuge tubes to form free-floating pellets and incubated in either: 1) an FBSsupplemented medium made up of DMEM/F 12 with 10% FBS, 10 ng/ml TGF-p 1, 50
pg/ml ascorbic acid phosphate, and IO0nM dexamethasone; or 2) a defined medium
consisting of DMEM (high-glucose with 110 tg/ml sodium pyruvate, Sigma), "ITS+
Premix" (BD Biosciences) - insulin (6.25 ptg/ml), transferrin (6.25 ptg/ml), selenous acid
(6.25 pg/ml) linoleic acid (5.35 pg/ml), and bovine serum albumin (1.25 pg/ml) - and 10
ng/ml TGF-$P1, 50 pg/ml ascorbic acid phosphate, and lOOnM dexamethasone. Articular
chondrocytes cultured in parallel under identical conditions served as a control.
In order to investigate the chondrogenic differentiation potential of the synovial
cells in the collagen-GAG matrix culture system, P4 cells were seeded into matrices and
cultured in the defined or FBS-supplemented media (described above). After three
weeks, cultures were fixed in formalin. Paraffin-embedded sections were evaluated for
chondrogenic differentiation by immunohistochemical detection of type II collagen.
3.4
Western Blot Analysis for SMA
At each passage (P1-P8) of the synovial cells in monolayer culture, an aliquot of
1-2 x 106 cells was removed for Western blot analysis of SMA content. Cytoplasmic
proteins were extracted from the cells using a lysing buffer (1%
sodium dodecyl sulfate,
1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, and 10% glycerol).
The cell lysates were agitated at 4'C for 10 minutes, then centrifuged for 20 minutes at
14,000 rpm at 4*C. The supernatant containing the protein was removed and stored at
-20*C until evaluation.
The concentrations of extracted proteins relative to a BSA standard were
determined by a modification of the Bradford method [4]. Proteins were mixed with BioRad Protein Assay Dye (Bio-Rad Laboratories, Hercules, CA) and the optical density
measured at a wavelength of 595 nm using a LKB Biochrom Ultraspec 4050
19
spectrophotometer. Cell extracts containing 5 pg of total protein were diluted with
sample buffer and dH 20 to a total of 48 pil, resolved in a 12% polyacrylamide gel in a
mini-gel apparatus (Bio-Rad Mini-Protean II, Bio-Rad) for 90 minutes at 90 V, then
transferred to PVDF membranes for 60 minutes at 100 V. Extracts from human aorta
smooth muscle cells containing 5 tg of protein served as positive controls. After
transfer, blotted membranes were placed in 5% dry milk (Bio-Rad) overnight to block
non-specific binding of antibodies.
Immunoprobing of the blotted proteins was achieved by incubation with the
primary antibody (Clone 1A4, Monoclonal Anti-SMA, Sigma) for 2 hours. Bound
protein was detected by incubation with a peroxidase-conjugated secondary antibody
(goat anti-mouse IgG, Sigma) for one hour followed by reaction with a luminol-based
chemiluminescent reagent (LumiGlo, Cell Signaling Technology, Beverly, MA). Film
(Kodak Scientific Imaging Film) exposed by contact with the blotted membranes was
developed and digitized for densitometric analysis using Scion Image software (Scion
Corp., Frederick, MD). The densities of the bands corresponding to SMA (molecular
weight of 42 kDa) for the experimental samples were each divided by the density of the
band for the SMC positive control in the same blot. This provided a relative measure of
SMA content as a percentage of the SMC positive control and allowed for comparison
among blots.
3.5
DNA analysis
Cell-seeded scaffolds and non-seeded control matrices allocated for analysis of
DNA content were washed in PBS and stored at -20*C until assayed. After
lyophilization, the matrices were digested overnight in a papain buffer (6 pg papain in 0.1
M sodium phosphate, 5 mM Na2 EDTA, and 10 mM cysteine-HCL) at 60*C. DNA
content in the digests was determined by the Hoechst dye method [35] using calf thymus
DNA as a standard. One hundred pl of digest was combined with 2 ml of Hoechst dye
working solution (#33258, Polyscience Inc., Northampton, UK) and evaluated
fluorometrically (TKO 100 Fluorometer, Hoeffer Scientific Instruments, San Francisco,
CA). The background fluorescence of the matrix was accounted for by subtracting the
mean value obtained for non-seeded controls from the value of each cell-seeded construct
at each time point.
20
3.6
Histology and Immunohistochemistry
Tissue samples and cell-seeded matrices were fixed in 10% formalin, processed,
and embedded in paraffin for microtomy. Seven pm thick sections were deparaffinized
and stained with hematoxylin and eosin using standard histological techniques (see
Appendix D).
Sections allocated for immunohistochemical analysis were stained with antibodies
to SMA or type II collagen (detailed protocols in Appendix D). For SMA analysis,
deparaffinized and rehydrated sections were digested in 0.1% trypsin for 1 hour, followed
by quenching of endogenous peroxidase with 3% hydrogen peroxide. Non-specific
binding was blocked by incubation with 30% goat serum (Sigma). Samples were then
incubated with primary antibody (Clone IA4, Monoclonal Anti-SMA, Sigma) for 2 hours
at room temperature. For negative controls, one section on each slide was incubated with
non-immunologic mouse serum (Sigma) diluted to the same protein concentration,
instead of the primary antibody. Sections were then incubated with a biotinylated
secondary antibody (goat anti-mouse IgG, Sigma), followed by application of affinitypurified avidin (Sigma). Labeling was developed using an aminoethyl carbazole (AEC)
chromagen kit (Zymed Laboratories, San Francisco, CA). Counterstaining was
performed using Mayer's hematoxylin (Sigma).
For analysis of type II collagen, deparaffinized and rehydrated sections were
digested for 1 hour in 0.1% protease XIV, followed by blocking of non-specific binding
with 5% horse serum. Primary antibody (CIIC 1, mouse anit-chick type II collagen
monoclonal antibody, Developmental Studies Hybridoma Bank, Iowa City, IA) was
applied for 1 hour. Negative controls were incubated with mouse IgG (Zymed) diluted to
the same protein concentration, instead of the primary antibody. A biotinylated
secondary antibody (horse anti-mouse IgG, Sigma) was applied for 45 minutes, followed
by quenching of endogenous peroxidase with 3% hydrogen peroxide. Labelling was
detected with an avadin-biotin complex [21, 22] (ABC kit, Vectastain), Vector
Laboratories, Burlingame, CA), and diamenobenzadine (DAB, Vector). Counterstaining
was performed using Harris hematoxylin.
21
3.7
Statistical Analysis
Statistical significance was determined by analysis of variance (ANOVA) with a
significance criterion of p<0.0 5 using StatView (SAS Institute Inc., Cary, N.C.) Curvefitting was accomplished using Igor Pro4 (WaveMetrics Inc, Lake Oswego, OR).
22
4
4.1
RESULTS
SMA Expression In Vivo
Immunohistochemical staining revealed the presence of SMA in tissue from 4 out
of 5 animals evaluated. Approximately 2-5% of cells in the intimal layer of these
synovium samples stained positive for SMA (Fig. 1). No significant staining could be
detected in cells from the sub-intimal tissue, with the exception of perivascular smooth
muscle cells. The intensity of staining of cells in the intima that were SMA-positive was
similar to that of the smooth muscle cells, which were used as positive internal controls.
b
Figure 1: Immunohistochemical staining for a-smooth muscle actin (SMA) in 2 samples of
intact adult canine synovium (red chromogen, see arrows). Scale bars = 20 pim. Insets are
negative controls.
23
24
Cell Growth and In Vitro Expression of SMA
4.2
Cell Growth in Monolayer Culture
Cell growth rate in monolayer culture tended to decrease with passage number,
with P8 cells growing at approximately half the rate of P3 cells (Fig. 2). ANOVA
4.2.1
revealed a significant effect of passage number on the number of population doublings
per day (P < 0.02).
0.4
0.3
.2
0
0
IL 0
1
P2
P3
P4
P5
P6
P7
P8
Passage Number
Figure 2: Change in cell growth rate in monolayer culture with passage number (n=2-7).
Mean ± standard error of the mean (SEM)
4.2.2
Western Blot Analysis of SMA Content
Western blot analysis detected SMA in P1 cells from each of the 6 animals,
although the intensity of two samples was not strong enough for meaningful
densitometric quantification. Blots of extracted protein from P2 to P8 cells revealed a
regular increase in SMA content with passage number, reaching a level nearly equivalent
to that found in the smooth muscle cell controls by the fourth passage (Fig. 3). By the
seventh passage the SMA content of the synovial cells was elevated nearly 20-fold
compared to P1 cells. One factor ANOVA revealed a significant effect of passage
number (P<0.001) on SMA content of the cells. There was also a significant effect of
25
length of time in culture on SMA content of the cells (P<0.0002), but regression analysis
showed that there was not a meaningful linear correlation (Fig.4; R2 = 0.46).
250
0
* 2000
150
0100 0
50
0
2
U,
P1
P2
III
P3
P5
P4
P6
P7
P8
Passage Number
Figure 3: Increase in SMA content of synovial cells with passage number, expressed as a
percentage of the smooth muscle cell positive controls. Mean SEM; P2-P5, n=6, P1 and P6-P8,
n=3.
300
0
L-
1250
C 50
/0
&150
0
10
30
40
50
60
Time in Culture (Days)
Figure 4: Scatter plot showing the SMA content of synovial cells with time in monolayer
culture, and a linear regression analysis (R2 = 0.46).
26
4.3
Contraction of Collagen-GAG scaffolds
DiameterMeasurements
Non-seeded control scaffolds contracted to 71% of the original diameter after 28
days. In contrast, cell-seeded constructs contracted to 27% of the original diameter (4%
of the original volume) over the same time period (Fig. 5). After only 1 week, cell-
4.3.1
seeded matrices were reduced in diameter by more than 50%. Two-factor ANOVA
revealed a significant effect of whether the scaffolds were seeded with cells (P<0.0001)
and time in culture (P<0.0001) on contraction.
inn rr~
80
E
0)
60
-
40
-
%0
--s- Non-seeded controls
20
-o-
Cell-Seeded
0
0
5
10
15
20
25
30
Time (Days)
Figure 5: Decrease in diameter of non-seeded control matrices (n=5) and of scaffolds seeded
with P4 cells (n=36). Sample numbers of cell-seeded scaffolds decreased to 24 and 12 after 7 and
14 days. Mean ±SEM.
The cell-mediated contraction data were fit with a curve of form A(1 -e-th) having
an asymptote (A) of 43% of the original scaffold diameter and a time constant (r) of 2.4
days (Fig. 6).
27
50
IL
*
40
-
.5
30
-
Uo
20
-
.L
0
E
0
(U
(U (U
.,
Cell-mediated Contraction
.5) 10-
- Curve Fit
0
0
5
20
15
10
25
30
Time (Days)
Figure 6: Cell-mediated contraction of the collagen-GAG matrices and
exponential fit of the form A(l-exp(-t/t)) with asymptote, A = 43%, of the
original scaffold diameter and time constant, t = 2.4 days.
4.3.2 DNA Content
The DNA content of the constructs diminished throughout the 4-week culture
period (Fig 7). One factor ANOVA showed a significant effect of time in culture on the
DNA content of the constructs (P=0.01). The DNA value at one day reflected
approximately 1.2x 106 cells (assuming an average DNA content of 7.7 pg DNA per cell,
as has been reported for chondrocytes [32]), indicating that about 60% of the seeded cells
were attached to the scaffold after this time period.
15000
12500
10000
r
7500
z
a
I
T
5000
2500
01
7
14
28
Time in Culture (days)
Figure 7: DNA content of the cell-seeded matrices. Mean ±SEM.
28
4.3.3 Histology of the Cell-Seeded Matrices
Staining of cell-seeded matrices with hematoxylin and eosin revealed the
distribution of the synovial cells throughout the collagen-GAG scaffolds with a generally
greater cell density on the surface as compared to the interior. Constructs terminated 1
day post-seeding demonstrated an open porous network, with relatively uniform pore size
of about 100 pm throughout (Fig. 8a). After 1 week a continuous cell layer could be seen
forming on the periphery of the constructs that stained positive for SMA (Fig. 8b). Pores
near the periphery of these scaffolds appeared compressed and were markedly smaller
than those in the central region of the constructs. Compression and collapse of the pores
was seen to progress toward the interior of the constructs until, after 4 weeks, there was
virtually no porosity remaining in many of the samples (Fig. 8c).
4.4
Chondrogenic Differentiation of Synovial Cells
Some cell pellets (Figs. 9a and 9b) and cell-seeded matrices (Figs. 9c-d) cultured in
the defined differentiation medium stained positive immunohistochemically for type II
collagen, but only in trace amounts. Several sections from these specimens did not reveal
any positive staining. The extent of type II collagen staining was considerably less than
that found in the articular cartilage tissue control (Fig. 9e) and in the chondrocyte micropellet control (Fig. 9f).
No significant staining for type II collagen could be detected in any of the pellets or
matrices cultured with the FBS-supplemented differentiation medium.
29
30
__
Figure 8: a) Histological micrograph of a synovial cell-seeded scaffold after 1 day in culture.
Hematoxylin and eosin stain; scale bar = 250 gm.
b) Immunohistochemical staining of SMA (red chromogen, arrows) in a synovial
cell-seeded scaffold after 7 days in culture. Scale bar = 50 ptm.
c) Synovial cell-seeded scaffold after 28 days in culture. Hematoxylin and eosin
stain; scale bar = 250 ptm.
31
____
-
I
32
EF~~~fmWAkW
~
m
N
3E
Ib~l~m
Figure 9: Immunohistochemical staining of type 11 collagen. Brownish chromogen (see arrows)
indicates presence of type 11 collagen. (a) Synovial cell micro-pellet culture and (b) negative
control. Scale bars = 20 pm. (c and d) Synovial cell-seeded matrix cultures. Scale bars =20 p m.
(e) Canine articular cartilage and (f') chondrocyte micro-pellet culture used as positive controls.
Scale bars = 200 p..
33
34
5
DISCUSSION
5.1
SMA Expression In Vivo
The immunohistochemical detection of SMA in specimens of intact synovium
provides confirmation of the first hypothesis presented in Section 2. This is the first
report of SMA expression in cells comprising the intimal layer of normal adult synovium
in vivo. Previous studies have reported the expression of SMA in synovial cells under
various pathologic conditions. Murray et al. identified SMA positive cells within the
synovial layer formed during the retraction of ruptured anterior cruciate ligaments and
postulated that the contraction of such cells contributed to the retraction of the ends of the
ligament. [51]. SMA was also identified in vitro by immunohistochemistry in synovial
cells from osteoarthritic and rheumatoid patients, though no significant staining of SMA
was found in vivo [44].
That the number of cells expressing SMA in vivo varied among the samples
examined in this study, with sections from one animal demonstrating no SMA positive
cells, indicates a transient nature of the SMA expression, as has been suggested for
fibroblasts [12, 58, 67]. Of particular interest is the result that in all of the samples, SMA
expressing cells were localized only in the synovial intima and were not found in the
fibroblast population of the sub-intimal tissue. These results warrant further investigation
into the identity of the SMA-expressing cells as type A (macrophage-like) or type B
(fibroblast-like) synoviocytes and the physiological role of SMA expression in synovial
cells.
5.2
In Vitro Expression of SMA
The finding that SMA expression by synovial cells increases with in vitro
expansion provides confirmation of the second hypothesis of Section 2, and is consistent
with data reported for chondrocytes [33], fibrochondrocytes [49], and osteoblasts [47].
Unlike data reported for chondrocytes [33], the relationship between SMA content of the
synovial cells and the length of time in culture did not appear to be linear (Fig. 7). The
presence of SMA in samples from intact synovium indicates that the contractility and
SMA expression of the synovial cells was not solely due to the adoption of this
phenotype in culture. However, the possibility of contribution to the in vitro results by
35
sub-intimal cells that did not exhibit SMA expression in vivo requires further
consideration.
5.3
Chondrogenic Differentiation of Synovial Cells
The detection of type II collagen in cultures of the synovial cells both in micro-
pellet and collagen-GAG matrix culture systems provides confirmation of the fourth
hypothesis of Section 2. The synthesis of type II collagen indicates their capacity to
differentiate to a chondrocytic phenotype, as has been previously demonstrated [13, 54],
and thus supports the use of these cells in cartilage tissue engineering. That no type II
collagen could be detected in FBS-supplemented cultures may indicate the presence of a
factor in the serum that inhibits chondrogenic differentiation of the cells. Similar results
have been demonstrated for chondrocytes. Following dedifferentiation during monolayer
culture, chondrocytes exhibit a greater extent of re-differentiation to a chondrocytic
phenotype when cultured in chemically defined media than in the presence of serum [29].
That only trace amounts of type II collagen were detected in the cultures of synovial cells
with defined media raises questions of the conditions that favor the differentiation of
these cells to chondrocytes, and the degree to which such conditions exist in cartilage
defects in vivo.
5.4
Contraction of Collagen-GAG scaffolds
Results of the matrix contraction assay provide confirmation of the third
hypothesis presented in Section 2. The reduction in diameter of non-seeded control
matrices may have been due to the mild plasticizing effect of water on collagen, as has
been reported [65]. While this effect was partly responsible for the decrease in diameter
of cell-seeded scaffolds, contraction of over 40% of the original diameter can be
attributed to the presence of the synovial cells. Recent studies have demonstrated the
contractility of a variety of connective tissue cell types [33, 38, 47, 49, 59], including the
progenitor mesenchymal stem cell [9, 34]. It may thus have been expected that cells of
synovial origin are also capable of displaying a contractile phenotype.
36
Cell-mediated contraction leveled off after about 3 weeks (Fig. 6), likely due to
the fact that virtually all of the porosity of the scaffold had been lost (Fig. 8c).
Additionally, the DNA content of the cell-seeded matrices decreased throughout the
culture period, indicating cell loss that may have been due to compression of the pores in
the contracted scaffolds. Histology revealed compression of the pores on the surface of
the scaffolds as early as seven days post-seeding, with collapse of the pores proceeding
from the surface to the central regions of the scaffolds throughout the 4-week culture
period. Such architectural deformation of engineered constructs caused by cellular
contraction could contribute to failure when implanted in vivo by hindering the migration
of the cells within the construct and preventing integration with surrounding host tissue.
Although the molecular mechanisms underlying contraction of SMA-expressing
non-muscle cells are not yet known, two investigations have indicated a causal
relationship between SMA expression and cell contractility [33, 70]. The results of the
current investigation may thus be of value in informing future efforts to expand synovial
cells in vitro prior to implantation for cell-based therapies or tissue engineering
procedures for cartilage repair, as well as efforts to manage cell-mediated contraction of
engineered constructs and reparative tissue by use of regulators to control SMA
expression.
37
38
6
CONCLUSIONS
Following are conclusions related to the specific aims and working hypotheses of the
study described in section 2:
1. Immunohistochemistry revealed SMA-expressing cells in the intimal cell layer of
synovial membranes from 4 out of 5 of the animals investigated. No staining of
SMA was detected in sub-intimal tissue.
2. SMA content of the synovial cells increased with passage number in monolayer
culture.
3. Synovial cells contract collagen-GAG matrices into which they are seeded,
causing collapse of the porous structure and loss of volume.
4. Synovial cells exhibit chondrogenic potential indicated by synthesis of type II
collagen, albeit in trace amounts, when cultured as micro-pellets or in collagenGAG scaffold in the presence of a chemically defined chondrogenic medium.
39
40
7
LIMITATIONS AND FUTURE WORK
There are several limitations to the current study that should be addressed in
future work. First, tissues and cells used in the present study were from a single species
(canine), and should only be considered directly applicable to this species. Future studies
evaluating the SMA expression and contractility of human synovial cells will be
beneficial. It is reasonable to expect results similar to the present study to be found in
human synovial cells because findings of SMA expression and contractility in studies of
in other human musculoskeletal connective tissue cells have paralleled findings in canine
studies.
Immunohistochemical staining of synovial tissue revealed SMA expressing cells
in the intimal layer of the synovium, but not in cells from the sub-intimal tissue. It was
assumed in this study that the increase in SMA content of cells with passage number in
monolayer primarily reflected an increase in SMA expression by cells that expressed
SMA in vivo. However, it is possible that cells from the sub-intimal tissue that did not
express SMA in vivo may have grown out of the tissue and adopted an SMA-expressing
phenotype, thus influencing the in vitro results. Future studies employing intimal cells
and sub-intimal cells separately may allow for confirmation or rejection of this
possibility. Such studies may have to await the development of markers to easily
distinguish intimal cells from sub-intimal cells in vitro.
The method of cell-isolation (outgrowth from tissue explants) chosen for this
study did not allow quantitative evaluation of the SMA content of the cells prior to their
expansion in monolayer culture (i.e. a PO time point). Additional studies employing
alternative methods of cell isolation, such as enzymatic digestion, would allow
opportunity for quantitative evaluation of SMA in freshly isolated cells and provide
additional insight concerning the extent to which this phenotype is induced by in vitro
culture of the cells.
Assays of cell-mediated contraction and chondrogenic potential in this study
employed cells that had been cultured in monolayer through Passage 4. Further studies
should be conducted employing cells from a variety of passage numbers, as well as cells
freshly isolated from explanted tissue. One would expect such studies to reveal cellular
contractility increasing with passage number of the cells, consistent with the increase in
41
SMA demonstrated in the current study. Similar results have been demonstrated in
chondrocytes [33]. Since increased SMA expression and related cellular contractility
caused by prolonged in vitro expansion of the cells can be problematic in the context of a
cell-seeded implant, it may be advantageous to employ cells from the earliest time point
possible. As such, an arthroscopic procedure is anticipated in which cells are isolated
from the synovium, seeded in a matrix, and implanted in a cartilage defect during the
same surgical procedure. Methods of mechanically isolating the cells by means of an
arthroscopic shaver, and instruments for intra-operatively seeding the cells in a matrix are
currently under investigation.
The present study examined several cell-matrix interactions important to the
development of a synovial cell/collagen-GAG implant for cartilage regeneration. Future
studies in the design of such an implant may examine effects of collagen type (i.e., type I
versus type II collagen), methods of cross-linking the matrix to increase resistance to
contraction, and the use of soluble regulators to control SMA expression and contraction
and to promote chondrogenesis in the synovial cells.
42
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39.
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the physical and biochemical properties of articular cartilage in the canine knee".
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Lee, C.R. and M. Spector, "Status of articular cartilage tissue engineering".
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49.
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47
48
APPENDIX A:
A.1
FABRICATION OF COLLAGEN-GAG SCAFFOLDS
Preparation of Collagen-GAG Slurry
*Adapted from T. Freyman (Ph.D. Thesis, Massachusetts Institue of Technology, 2001)
Summary
A co-precipitate of type I collagen and chondroitin-6-sulfate is prepared by blending in the
presence of acetic acid. The slurry is cooled during blending in order to keep the collagen from
denaturing to gelatin. The triple-helical tertiary structure of the collagen is maintained.
Eguipment
Granco overhead blender
Cooler
Peristaltic Pump
Granco Co., Kansas City, MO
Brinkman cooler model CR-2T, Brinkman Co., Westbury, NY
Manostat Cassette Pump, CAT# 75-500-0.00, New York, NY
Materials
Acetic Acid (HOAc)
Type I Collagen
Glacial Acetic Acid, Mallinckrodt Chemical Co., Paris, KY
Chondroitin 6-sulfate
Sigma # C-4384, from shark cartilage
Dry microfibrillar bovine tendon collagen, Integra
Lifesciences, Plainsboro, NJ
Methods
1.
Turn on cooling system and allow to cool to 4'C.
2.
Prepare 0.05M acetic acid (HOAc) solution: 8.7 ml HOAc in 3 L dH20
This solution has a shelf ife of approximately 1 week.
3.
Blend 3.6 g dry microfibrillar bovine tendon collagen with 600 ml of 0.05 M acetic acid on
HIGH speed setting for 90 minutes at 4'C.
4.
Prepare chondoitin 6-sulfate solution:
0.32 g chondroitin 6-sulfate in 120 ml acetic acid, stir for approx 30 minutes to mix well
5.
Calibrate peristaltic pump to 40 ml/5 min.
6.
Add 120 ml chondroitin 6-sulfate solution dropwise to the blending collagen dispersion over
15 minutes using the peristaltic pump (maintain blender at 4'C).
7.
Blend 90 additional minutes on HIGH speed at 4'C.
8.
Degas in series of vacuum flasks until bubbles are no longer present.
9.
Store at 4'C.
Slurry is good for approximately 4 months. If stored for more than one week, blend on LOW
speed 15 minutes prior to using.
49
A.2
Freeze-Drying
*Adapted from H. Breinan (Ph.D. Thesis, Massachusetts Institute of Technology, 1998)
Summary
The collagen-GAG slurry is frozen, causing formation of ice crystals. Reducing the pressure then
causes the crystals to sublimate, resulting in a porous structure. The size of the crystals, and thus
the size of the pores, is determined by the freezing temperature.
Equipment
VirTis Genesis Freeze-Drier (Virtis, Gardiner, NY)
Methods
1.
Turn on freeze-drier.
2.
Turn on freeze switch.
3.
Ensure that the plug is in the drain and turn on condenser.
4. Wait for the shelf temperature to reach -43'C (approx. I hour).
5.
Pipette slurry into freeze-drier trays, being careful not to introduce air bubbles.
6. Place slurry into the freeze-drier and allow to freeze for 1.5 hours.
7.
When slurry has frozen, turn on vacuum. Make sure that the door is sealed, chamber release
is off, and that the condenser is less than -50'C.
8. Wait for the vacuum to reach less than 200 mtorr. Set the temperature to 00 C and turn on the
heat switch. Allow to sublimate overnight.
9.
Set temperature to 20'C and turn off the freeze switch.
10. When temperature reaches 20 (approx 30 minutes), turn off heat, vacuum, and condenser.
11. Turn on chamber release, remove sample, open the drain plug, and turn off power.
A.3
1.
Dehydrothermal (DHT) Cross-linking
Prepare "envelopes" from aluminum foil large enough to hold matrix sheets.
2. Remove the collagen-GAG matrix sheets from the freeze-drier trays and place them in the
aluminum foil envelopes. Be careful not to crush matrices.
3.
Place open envelopes in the DHT oven.
4. Close oven. Turn VACUUM knob to ON and CHAMBER RELEASE knob to OFF.
5.
Turn on vacuum pump. Make sure oven temperature is at 105'C and vacuum is <50 torr.
Leave for 24 hours.
6.
Turn of vacuum pump. Turn of the VACUUM knob and turn CHAMBER RELEASE to ON.
7.
Once the pressure returns to atmospheric, open door and quickly seal envelopes. Store
matrices in sealed packets in a dessicator.
50
APPENDIX B:
B.1
CELL ISOLATION AND CULTURE
Tissue Harvest
OR Supplies
Scalpel blades
Scalpel handles
Betadine
Betadine sponges
Sterile towels/wraps
Saw
Forceps
Gloves
Face masks
Cooler with ice
Method
Shave hair from limb and scrub with betadine. Remove either entire limb or only the joint. If
removing only the joint, first remove skin, then saw through bones on either side of the joint a
distance far enough away from the joint to ensure that the joint capsule remains closed. If
possible it is usually desirable to ligate large blood vessels prior to cutting. Wrap joint in sterile
towels and place on ice in cooler.
Lab Supplies
Plastic basin
Betadine
Sterile scalpel blades
Sterile scalpel handles
Sterile
Sterile
Sterile
Sterile
cutting board
Petri dishes
forceps
gloves
Complete PBS
Complete medium
Tissue culture flasks
Methods
1.
Prepare cutting board and scalpels. Fill petri dishes with PBS.
2.
Place joint in plastic basin and soak in betadine.
3.
Put on sterile gloves.
4.
Place joint on cutting board.
5.
Open joint capsule by cutting through patellar tendon and making a cut next to the tendon
into the joint space.
6. Remove synovium and place in petri dishes with PBS
7.
Set aside samples of synovium for histology.
8.
Dice remaining synovial tissue and transfer to 25 cm 2 culture flasks.
9.
Place just enough medium in flasks to just cover the tissue, but cause it to float.
10. Pace flasks in incubator for 2-3 days to allow tissue to attach.
11. Place synovium samples allocated for histology in 10% formalin.
51
B.2
Passaging Cells
Materials
Complete medium
Trypsin
PBS (Phosphate-buffered saline)
Glass pipettes
Vacuum setup
Sterile plastic pipettes
Centrifuge tubes
Tube holders
Tissue culture flasks
Methods
1.
Warm the medium, trypsin, and PBS in 37'C water bath.
2.
Remove medium from flasks with vacuum pipette (change pipettes for different animals).
3.
Rinse with PBS (enough to cover bottom of flask, ~ 10 ml for 75 cm 2 flask). Trypsin will not
detach the cells if it has come into contact with the medium.
4.
Remove PBS and add trypsin (0.5 ml per well of 6 well plate, 2 ml for 25 cm 2 flask, 5 ml for
75 cm 2 flask).
5.
Place in incubator for 5 minutes (unless otherwise instructed).
6.
Remove from incubator and tap on the sides of the flask to loosen the cells. Check under
microscope to ensure the cells are no longer attached. If they are, return them to the
incubator and check each minute until they are unattached.
7.
Once the cells are floating, return to the hood and add complete medium to inactivate the
trypsin (1.5 ml per well of 6 well plate, 3 ml for 25 cm 2 flask, 10 ml for 75 cm 2 flask).
8.
Using a sterile plastic pipette, transfer the medium/trypsin/cell suspension to a centrifuge
tube. At this point you can combine the contents of the flasks if they are from the same
sample.
9.
Balance the tubes and centrifuge at 1500 rpm for 10 minutes.
10. Once you have the cell pellet at the bottom of the tube, draw off the medium with the vacuum
pipette (be sure not to suck up the cells!!!).
11. Resuspend and count the cells (see Cell Counting protocol). While counting, centrifuge the
cell suspension a second time to ensure all trypsin has been removed.
12. Decant medium from second centrifugation and resuspend at desired seeding density.
Transfer to culture flasks and add complete medium to bring the flasks up to final volume.
52
B.3
Cell Counting
*Adapted from Current Protocols in Cell Biology
Materials
Complete medium
Trypan Blue
Cell counting slide
70% ethanol
Micropipetters Calculator
Cell counter
Pipette tips
Pipette Aid
coverslip
Methods
1. Clean surface of hemacytometer and coverslip with 70% alcohol.
2. Wet edge of coverslip slightly with tap water and press over
grooves on hemacytometer. The coverslip should rest evenly over
the silver counting area.
3. Beginning with a cell pellet, suspend the cells in a known amount
of complete medium
4. Collect a 100 pLd sample from the cell suspension and dilute with
trypan blue (1:2 dilution if few cells are expected, 1:5 or 1:10 if a
large number is expected).
grid
load cell
suspension
1mm
5.Mix well, and collect 15 pl of suspension in a micropipette tip.
6. Load the cell suspension into the hemacytometer, allowing it to be
drawn under the coverslip by capillary action. Load just enough
cell suspension to reach the edges of the silvered surface. Do not
overfill as this may change the volume and make the count
inaccurate.
I
1
7.Place hemacytometer on microscope stage, remove yellow glass
filter, and view with standard lOx objective.
8. Count cells in each of the four corner squares and the central
square (clear cells are viable, stained cells are dead). Count cells
that lie on the top and left lines but not those on the bottom or
right lines of each square in order to avoid counting the same cells
twice for adjacent squares. Repeat counts for other counting
chamber. A maximum cell count of 20 to 50 cells per lxl-mm
square is recommended. When a count of living cells is complete,
count the number of dead cells in order to report viability.
1mm
0 1
1
1
1
Picture from Current Protocols
in Cell Biology Online
9.Calculate total cell number from the following:
T=
xD x104
Ns
x
V
T = Total number of cells in suspension
Nc = Number of cells counted.
Ns = Number of 1mm squares counted.
D = Dilution factor
V = Volume of media used to suspend
cell pellet.
The number 104 is the volume correctionfactorfor the hemacytometer: each square is lx] mm
and the depth is 0.1 mm.
10. Begin preparing samples for culturing and/or protein extraction
53
B.4 Freezing Cells
Materials
Complete Medium
Dimethyl sulfoxide (DMSO)
Sterile Filter
Pipettes
Sterile cryogenic tubes
Methods
1.
Determine amount of medium needed (1 ml per 2x10' cells)
2. Prepare solution of 10% DMSO in complete medium.
Sterilize the medium +DMSOsolution by filtering through 0.22 or 0.45 pm sterilefilter, or by
autoclavingDMSO prior to adding it to the medium.
3.
Adjust cell concentration to 2x10 6 cells/ml of complete medium + DMSO solution.
4.
Store in cryogenic tubes (3ml per 5 ml tube, 1 ml per 2 ml tube) in the -20'C freezer for 2-4
hours (longer time in this range is preferable), then transfer to -70'C for storage.
B.5 Thawing Cells
Methods
1.
Place cells directly into a 37'C water bath. Agitate gingerly while cells thaw for 40-60
seconds.
2. When defrosted minimally (see liquid around outer edges) add a drop of complete medium.
3.
Wait a minute and add another drop of medium. Repeat until tube is full. This insures that
the cells thaw into the medium.
4.
Transfer the cells to a 50 ml tube and wash them clean of medium+DMSO 2x for 10 minutes
in the centrifuge.
5.
Count the cells, and resuspend at the proper concentration. Cells should be cultured at least
3-4 days before being used for experimentation (or before changing medium, depending on
when they attach).
54
B.6
Cell-Seeding of Collagen-GAG Scaffolds
*Adapted from D. Hastreiter (Ph.D. Thesis, Massachusetts Institute of Technology, 2002)
1.
Prepare and autoclave 2% (w/v) agarose (SeaPlaque Agarose, BioWhittaker #50101)
2.
Pipette 1.5 ml agarose into each well of 12-well plates and allow to set for at least 4 hours at
4'C. Warm in incubator at 37'C prior to use.
3.
Pre-wet matrices in PBS for 1 hour.
4.
During this time, passage cells from flasks. Suspend cells at desired concentration. Use 50
pl for each 9 nm disc. For example, for a desired concentration of 2 x 106 cells/disc, the
concentration should be 4 x 107 cells/ml.
5.
Transfer matrices to medium for 10 minutes.
6. Briefly dry matrices on sterile filter paper (allow enough time for liquid to be drawn out, but
do not allow matrices to dry completely)
7.
Transfer matrices to warmed 12-well plates.
8.
Pipette 25 pl onto one side of each matrix. After 10 min., flip matrices and pipette 25 pl onto
the opposite sides.
9.
Place matrices in incubator for 2 hours.
10. Add 0.5 ml medium (or enough to just cover matrices) to each well.
11. The following day, add another 1.0 ml medium to each well.
B.7
1.
Cell-Pellet Cultures
Suspend 2 x 10 5 cells in medium in a 15 ml centrifuge tube. Centrifuge at 500g for 10
minutes.
2. Place tubes in incubator at 37'C overnight. Cells should form a free-floating pellet within
approximately 16-20 hours.
3.
Culture as desired, being careful not to break pellets when changing media.
55
56
APPENDIX C:
C.1
BIOCHEMICAL ASSAYS
Western Blot for detection of SMA
C.1.1
ProteinExtraction
Lysing Buffer
1% Sodium Dodecyl Sulfate (SDS)
Sigma #L-4509
1 mM Sodium Orthovanadate (SO)
1 mM Phenylmethanesulfonyl fluoride (PMSF)
10 % Glycerol
Sigma #S6508
Sigma #P7626
Sigma #G8773
To make 50 ml:
45 ml of 1% SDS
5 ml of 10% glycerol
100 91 of 100 mM stock SO
250 1d of 200 mM stock PMSF
Methods
*Begin with cell pellet after centrifugation
1.
Rinse pellet with PBS after medium is drawn off. Spin for 10 min. and remove PBS
2.
Suspend cells in lysing buffer at a concentration of at least 2x 106 cells/ml.
3.
Transfer cell suspension to microcentrifuge tubes.
4. Shake tubes on shaker in cold room (4*C) for 10 minutes.
5.
Centrifuge at 4*C for 20 minutes at 14,000 rpm.
6. Draw off supernatant and put into a new microcentrifuge tube. Discard pellet
7. Label and store at -20*C until evaluation.
57
C. 1.2 Protein Assay
Summary
Based on the Bradford method (Analytical Biochemistry, 1976. 72: p. 248-54), this procedure
involves the additions of an acidic dye to a protein solution. The binding of the dye to the protein
results in a differential color change of the dye, which is measured at 595 nm with a
spectrophotometer.
Equipment
Spectrophotometer
LKB Biochrom Ultraspec 4050
Materials
Bovine Serum Albumin (BSA) BioRad #500-0007
Protein Assay Dye
Cuvettes
BioRad #500-0006
Fisher #14-386-21
Methods
1. Dilute the BSA stock solution (1.5 pg/pl) to a 1:10 with dH 20 for a final concentration of
0. 15 ptg/ptl.
Prepare25 pl BSA with 225 l dH20for a total of 250 ,l.
2.
Prepare gradient for standard curve. Each sample should be a total of 2 ml. The BSA
standard is linear within the range of 0.2-0.9 mg/ml.
d BSA (0.15p4g/pl)
0 (blank)
10 (1.5 pg)
20 (3.0 4g)
40 (6.0 4g)
60 (9.0 Vtg)
80 (12 pg)
pl dH 20
1600
1590
1580
1560
1540
1520
p1 Dye (BioRad)
400
400
400
400
400
400
3.
To prepare samples, add 20 pl of supernatant to 1580 pl of dH 20. Then add 400 ml of dye
and mix.
4.
Incubate at room temperature for 5-10 minutes.
5.
Turn on spectrophotometer and allow to warm up. Add the standard set with the blank first
(blue holder).
6.
Read wavelength at 595 nm. To read the next sample press the "cell number" button.
7.
Read samples. Leave the blank in and read it first, setting the reference to zero each time.
8.
Generate a standard curve in Excel to determine how much protein is in each sample.
58
C.1.3
SDS-Polyacrylamidegel electrophoresis (PAGE)
Summary
One-dimensional gel electrophoresis separates proteins as they move through the pores of
the gel, the size of which are determined by the concentration of acrylamide. Proteins are
denatured by heat and detergent (SDS) to ensure separation on basis of size, eliminating effects of
charge and shape. SDS also applies a negative charge to the proteins, which causes them to move
through the gel toward the anode when the electric field is applied, the smaller proteins moving
more rapidly through the porous matrix than larger proteins. After separation, the proteins are
electrophoretically transferred ("blotted") onto a PVDF membrane for immunoprobing. (see
Laemmli, 1970, Nature 227:680)
Materials
2 mercaptoethanol
Acrylamide
Ammonium Persulfate
BIS
Bromophenol Blue
Coomassie Blue
Dry Milk
Filter Paper (for blots)
Glycerol
Glycine
SDS
Primary Antibody
PVDF Membranes
Secondary Antibody
TEMED
TBS
Trizma Base
Tween 20
X-ray Film
20X Lumiglo
Sigma M-7154
Sigma A-3553
Fisher A-682-500
Sigma B-8026
Sigma B-8522
BioRad 170-6404
BioRad 162-0118
Sigma G-8773
Sigma G-8898
Sigma L-4509
Sigma A-2547
BioRad 162-0176
Sigma A-2304
Sigma T-9281
Sigma T-6664
Sigma T-1503
Sigma F-5513
Cell Signal Tech.
Cat# 7003
Prepared Solutions
30% Acrylamide-bisacrylamide
Acrylamide
BIS
dH 20
29.2 g
0.8 g
100 ml
1.5% Ammonium Persulfate
Ammonium Persulfate 150 mg
dH 20
Note: Filter through #1 Whatman filter paper, store at
41C. Good for 1 month
Note: Store at 4'C, good for 1 week
10 ml
Resolving Buffer
Tris base (3.0M)
dH 20
46.8 g
100 ml
Note: Filter through #1 Whatman filter paper, store at
7.9 g
100 ml
Note: Filter through #1 Whatman filter paper, store at
4'C. pH = 8.8 Good for 1 month
Stacking Buffer
Tris base (0.5M)
dH 20
Sample Buffer
Tris base (0.5M, pH 7.0) 1.0 ml
Glycerol
0.8 ml
10% SDS
1.6 ml
2ME
0.4 ml
Dye
0.2 ml
4'C. pH = 6.8 Good for 1 month
Notes:
1. Store at -20'C
2. 2ME = 2 mercaptoethanol, in hood in
biochem lab (reduces di-sulfide bonds)
3. Dye = 1% bromophenol blue
59
Running Buffer (10X stock solution)
Tris base
30.3 g
144.0 g
Glycine
SDS
dH 20
Note: pH = 8.3, Store at room temperature
10.0 g
1.0 L
Transfer Buffer (5X stock solution)*** Notes:
Tris
15.15 g
1. Store at room temperature
Glycine
72.25 g
2. ***For dilution to Ix, add 20% methanol.***
dH 20
800 ml
(160 ml 5X buff, 480 ml dH 2 0, 160 ml methanol)
TBS-T
Tween 20
1.0 ml
TBS
1.0 L
Methods - SDS PAGE
1. Assemble the gel-casting apparatus. Fill ice tray for blot transfer and place in freezer.
2.
Prepare the resolving gel. Load 7 ml of gel solution in each cassette. Overlay with a small
amount of dH 20.
Amounts will vary depending on the %Tofthe gelyou desire. The following is for a 12% gel.
Amounts are enough for 2 gels - dispose of extra in trash, not the sink.
Resolving gel
dH 20
Acryl-bis
Resolving buffer
8.2 ml
8.0 ml
2.5 ml
10% SDS
200 ptl
AP
1.0 ml
TEMED
20 jil
Add TEMED underfume hood.
3.
Begin boiling water for samples.
4.
Prepare samples, control, and marker. For each sample, combine 24 pl of sample buffer with
5 gg of protein, then bring to a total of 48 pl with dH20. Add sample buffer underfume
hood
5.
When the resolving gel is set (approx. 20-30 min.), remove layer of dH 2O using filter paper.
6.
Prepare the stacking gel. Load stacking gel solution and insert combs.
Stacking Gel:
dH 20
Acryl-bis
5.6 ml
1.25 ml
Stacking buffer
10% SDS
AP
2.5 ml
100 pl
500 pl
TEMED
10 jd
Add TEMED underfume hood
7.
Heat the samples in boiling water for 5 minutes, then centrifuge in cold room for 10 minutes
at the highest speed (14,000rmp).
8.
Once the gels are solid, remove the combs and move the cassettes to the gel-running
chamber.
60
9.
Fill the inside of the chamber with lx Running buffer. Fill the outside of the chamber with
the buffer to at least the first set of knobs.
10. Use the special loading pipette tips to load the samples in the gels. Before loading samples,
fill each well with running buffer to remove any bubbles.
11. Run the gel for approx. 2 hr. at 90V.
It is finished when the sample buffer has reached the bottom of the gel, so check periodically.
12. Remove the gel by using one of the spacers to carefully pry the glass plates apart. Cut away
the stacking portion of the gel. Mark the gel in a way that such that the orientation will be
preserved.
61
C. 1.4 Blot Transfer
Methods
1.
Cut PVDF membranes and filter paper to same dimensions as gel.
2.
Briefly wet membranes in pure methanol.
Be sure not to touch membranes with bare hands as oilfrom the hands can block transfer.
3.
Soak the gel, filter paper, and sponges in lx Transfer buffer (*** add methanol, see I' page
***) for 10 minutes to equilibrate the gel.
Be sure to keep track of which gel is #1 and #2. Gel equilibrationis requiredto prevent a
change in the size of the gel duringtransfer.
4.
Assemble the transfer sandwich according to the following diagram. Keep the cassette
submerged in transfer buffer to avoid air bubbles and prevent the membrane from drying.
Gently rub out any bubbles between the gel and the membrane using a stir rod.
Clear/White Plate
Sponge
Filter Paper
Membrane
Gel
Filter Paper
Sponge
Black Plate
5.
Load the cassette into the transfer chamber. Black faces black. Load ice tray into chamber.
6.
Add the remaining lx TBS for a total of 1 L.
7.
Run for 1 hour at 100 V. If desired, do this step in the cold room at 4' C.
8.
Remove membranes and wash in lx Transfer buffer for 5 minutes.
9.
Stain gels with Coomassie Blue to verify transfer of proteins to the membrane.
Membranes can be driedand stored in resealableplastic bags at 4 Cfor 1 year or longer at this
point. Priorto furtherprocessing,dried PVDF membranes must be placed into a small amount of
100% methanol to wet the membrane, then in distilled water to remove the methanol. ( Current
Protocols in Cell Biology)
If proceeding to immunoblotting and detection the following day, place membranes in blocker
(5% dry milk) overnight at 4'C.
To prepare blocker: 5g dry milk to 100 ml TBS. Mix well.
62
C.1.5 Immunoprobing and Visualization of Blotted Proteins
Summary
Immobilized proteins are probed with antibodies to the desired antigen under
investigation. All open protein-binding sites on the membrane are first filled by immersion in
blocking buffer containing nonreactive protein (dry milk). Membranes are then incubated with a
primary antibody against the desired antigen, followed by an enzyme-antibody conjugate against
the primary antibody (peroxidase). A chemiluminescent reagent that reacts with the conjugated
enzyme on the secondary antibody is applied, and X-ray film exposed by the luminous bands.
Methods - Probing
1.
If membranes have been stored, briefly wet membranes in pure methanol, then wash with
distilled water.
2. Place membranes in blocker (5% dry milk) for 30 minutes on rocker.
To prepare blocker: 5g dry milk to 100 ml TBS. Mix well.
3.
Incubate with primary antibody for 2 hours at room temperature on rocker.
1:400 dilution. Add 50 d of primary antibody to 20 ml Ix TBS-T.
The antibodies may stored at 4 C and reused, but do not store more than 24 hours.
4. Rinse in Ix TBS-T for 10 minutes, 3 times.
5.
Incubate with secondary antibody for 1 hour at room temperature on rocker.
1:5,000 dilution. Add 4 pl secondary antibody to 20 ml Ix TBS-T.
6. Rinse in Ix TBS-T for 10 minutes, 3 times.
Methods - Detection
1.
Prepare LumiGlo solutions A and B. You need a 1 ml of each solution for each membrane.
Do not mix A and B together until you are ready to use them. Once washing is complete,
thoroughly mix A and B together and put 2 ml on each membrane.
Solution A: Mix 100 ptl A to 2 ml dH 2 0.
Solution B: Mix 100 d B to 2 ml dH20.
2.
Incubate for 2 minutes at room temperature.
3.
Wrap membranes in saran wrap, carefully smooth out any air bubbles
4. Expose film in dark room (Thorn 1224C) and develop. Adjust exposure time as needed.
63
C.1.6 DensitometricAnalysis
*This protocol is adapted from Scion Image Manual
1. Open Scion Image software and load scanned image file
2.
Click Load Macros in the Special menu. Load "GelPlot2" from the Macros directory.
3. Use the rectangular selection tool to outline the lanes
4.
Select Mark First lane in the Special menu.
5.
Select Plot Lanes in the Special menu.
6. Use the line drawing tool to draw base lines and drop lines so that each peak defines a closed
area.
7. Measure the areas of the peaks by clicking inside each one in succession with the wand tool.
8. Use the text tool while holding "Scroll Lock" to label peaks, in reverse order, with the area
measurements. The area measurements are also recorded in tabular form that can be exported
to a spreadsheet.
64
C.2
DNA Analysis
C.2.1 PapainDigestion
*Samples should be lyophylized and their mass determined prior to digestion*
Materials
Sodium Phosphate, monobasic
Sodium Phosphate, dibasic
Fisher #S369
Fisher #S373
Fisher #S311
Sigma #C1276
Sigma #P3125
Disodium EDTA
L-Cystein HCL
Papain
Solutions
0.5 M Monobasic stock
NaH 2PO 4 *H 20
dH 20
6.9 g
0.5 M Dibasic stock
Na2HPO 4 *7H 20
dH 20
13.4 g
100 ml
100 ml
Papain buffer
Dibasic stock
Monobasic stock
L-Cysteine HCL
2.46 ml
17.54 ml
87.82 mg
Disodium EDTA
186.12 mg
Papain
0.5 ml (stock is 25 mg/ml)
dH 20
80 ml
Methods
Place lyophylized matrices in microcentrifuge tubes and add 1 ml papain buffer per tube. Place
tubes in 65'C waterbath overnight.
C.2.2 FluorometricQuantificationofDNA
Equipment
TKO 100 Fluorometer, Hoeffer Scientific Instruments, San Francisco, CA
Materials
Tris Base
Na 2EDTA
NaCl
Hoecsht 33258
Calf thymus DNA
Fisher
Fisher #S311
Fisher #S271
Sigma #B2883
Sigma #D-3664
Solutions
lOX TNE buffer
Tris Base
Na2EDTA
100 mM (12.1 g/L)
10 mM (3.7 g/L)
NaCl
1.0 M (58.4 g/L)
pH to 7.4, sterile filter, store at 4'C
Hoechst dye stock solution
Hoechst 33258
10 mg
Note: carcinogenic and light sensitive
65
dH 20 (sterile)
10 ml
Working dye solution
1OX TNE
dH 20
10 ml
90 ml
-+Filter through 0.45 pm filter
-+Filter through 0.45 pm filter
Hoechst stock solution 10 pl
Methods
1.
Turn on fluorometer and allow it to warm up for 15 minutes. Make sure the "scale" knob is
adjusted to 50% sensitivity (approx. 5 clockwise turns from fully counter-clockwise position).
2.
Prepare DNA standard in duplicate (or triplicate).
ig of DNA
pl of DNA
10
5
2.5
1 jU from 1 mg/mi stock
50 pil from previous
50 pl from previous
99
50
50
1.25
50 pd from previous
50
0.625
0.3125
50 l from previous
50 pl from previous
50
50
0
0
50
4l of PBE
3.
Add 20 p1 of each standard to a cuvette containing 2 ml of working dye solution.
4.
Once the machine is zeroed, add the 10 pg standard and adjust the scale knob to read 100.
Do not readjust the scale knob once the standard curve has been established.
5.
Read samples and make certain it is linear (with R2<0.95).
6.
Run samples in duplicate. Add 50 p1 of sample to 2 ml of working dye solution. Adjust
sample amount as necessary if readings are too high or low.
66
APPENDIX D:
D.1
HISTOLOGY AND IMMUNOHISTOCHEMISTRY
Paraffin Embedding
*Adapted from H. Breinan (Ph.D. Thesis, Massachusetts Institute of Technology, 1998)
Epuipment
Tissue Tek VIP 1000 model 4617
Tissue Tek tissue embedding center
Materials
Clearing Solutions: Xylene (Fisher #X5, or substitute, such as Histosolve or Citrisolve)
Paraffin (Fisher # 23-021-400)
Methods
1. Dehydration and infiltration
A. Tissue specimens are dehydrated and infiltrated by machine (Tissue Tek program 4), with
solutions changed automatically as follows:
50% ethanol
70% ethanol
80% ethanol
95% ethanol
100% ethanol
Clearing solution
Paraffin
Paraffin
1 hour
1 hour
1 hour
2x 1 hour
3x 1 hour
2x 1 hour
1 hour
2x 30 min.
Room
Room
Room
Room
Room
Room
temperature
temperature
temperature
temperature
temperature
temperature
59 0 C
59 0 C
B. Fragile collagen matrices and cell pellet cultures are dehydrated by hand. For pellet
culture specimens, the following solutions are pipetted in and out of 24 well plates.
Matrices specimens are placed in plastic tissue cassettes, which are then placed in the
following solutions:
dH 20
50% ethanol
70% ethanol
80% ethanol
95% ethanol
100% ethanol
Clearing solution
Paraffin
2.
3x 30 min.
30 min.
30 min.
30 min.
2x 30 min.
3x 30 min.
1 hour
2x 1 hour
Room
Room
Room
Room
Room
Room
Room
temperature
temperature
temperature
temperature
temperature
temperature
temperature
59 0 C
Embedding.
"
*
*
*
"
Remove specimens from tissue cassettes
Partially fill stainless steel or plastic mold with molten paraffin
Place specimen in mold with desired orientation and transfer to cooling plate briefly
Place tissue cassette on the mold and affix with additional paraffin
Place in freezer overnight prior to removing from mold
67
Hematoxylin and Eosin (H & E) Staining
D.2
Summary
Formalin fixed, paraffin embedded sections are stained with H&E for visualization of
structure. Hematoxylin stains acidic portions deep blue (such as cell nuclei rich in DNA and
RNA). Eosin stains basic substances pink (the collagenous ECM).
Solutions
Hematoxylin
Harris Hematoxylin Solution, Sigma Cat# HHS-128.
Filter 200ml of stock solution into staining dish.
Acid Alcohol
200ml of 70% ethanol (in dH20) + 0.5 ml HC1
Ammonia water
200 ml dH20 + 5-10 drops ammonium hydroxide, pH around 10.0
Eosin
Eosin Y Solution Aqueous, Sigma Cat# HTI 10-2-128
Other Materials
Cytoseal 60
Electron Microscopy Sciences Cat# 18006.
Methods
Paraffin Sections
1. Deparaffinize and Rehydrate
Xylene (or substitute)
100% ethanol
100% ethanol
95% ethanol
80% ethanol
70% ethanol
dH 20
2 x 5 minutes
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
2. Harris hematoxylin - 10 minutes.
3.
Rinse in tap water, approximately 1 min. running or swishing until almost clear.
4. Acid alcohol. Dip quickly 5-10 times, 20-30 sec. total.
5.
Rinse in tap water until foaming stops, approximately 30 sec.
6. Ammonia water. Quick dips (5 or so) until blue.
7.
Rinse in tap water approximately 1 min.
8. Eosin, 45-60 sec.
9.
Rinse in tap water, 1-2 min.
10. Dehydrate
70% ethanol
80% ethanol
95% ethanol
100% ethanol
100% ethanol
Xylene (or substitute)
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
2 x 5 minutes
11. Air dry. Coverslip with Cytoseal
68
D.3
Immunohistochemical Staining of a-Smooth Muscle Actin
Summary
Deparaffinized and rehydrated sections are incubated with trypsin to uncover antigenic
sites, followed by quenching of endogenous peroxidase with H 2 0 2 . After blocking of nonspecific
binding, primary antibody against the desired antigen (SMA) is applied. A biotinylated
secondary antibody binds to the primary antibody, and is labeled with peroxidase via avidin-
biotin binding. The bound peroxidase is then reacted with an AEC chromagen.
Solutions **Note: Amounts will vary depending on number of slides being stained**
Phosphate Buffered Saline (PBS) (Sigma # P-3813)
1 package to 1 L dH 20, make 2L for 14 slides
0.1% Trypsin (Sigma # T-7409)
0.01g trypsin
10 ml
PBS
Store at 4'C until use, dessicate
3% Hydrogen Peroxide (H 2 0 2 ) (Sigma # H-1009)
I ml
30% H 2 0 2
9 ml
d1120
Stored at 4'C
30% Goat Serum (Sigma # G-9023)
Stored at -20 0 C
0.3 ml Goat Serum
0.7 ml PBS
Primary Antibody - Mouse Monoclonal
Anti-c-Smooth Muscle Actin (Sigma #A2547)
5 pl
primary antibody
2 ml
PBS
Stored at -20'C
Use a 1:400 dilution
Secondary Antibody - Biotinylated
Goat Anti-Mouse Immunoglobin (Sigma # B715 1)
10 pl secondary antibody
Stored at -20'C
2 ml
PBS
Negative Control -Mouse Serum (Sigma # M-5905)
5 pl
mouse serum
4 ml
PBS
Extravadin-Conjugated Peroxidase (Sigma # E2886)
20 gl peroxidase
I ml
Substrate
1
1
1
1
Use a 1:200 dilution
Stored at -20'C
Dilute to same protein
concentration as diluted primary
antibody
Stored at 4'C
Use a 1:50 dilution
PBS
Reagent (Zymed # 00-2007)
ml
dH 20
drop substrate buffer (bottle A)
drop chromogen solution (bottle B)
drop hydrogen peroxide (bottle C)
Stored at 40 C
Keep away from light
Other Materials
Mayer's Hematoxylin Solution (Sigma # MHS-16)
Glycerol Gelatin (Sigma# 49927)
69
Methods
***DO NOT TOUCH THE SPECIMENS NOR LET THEM DRY OUT AT ANY POINT
DURING THIS PROCESS***
1.
Deparaffinize and rehydrate via the following baths
Xylene (or substitute)
100% EtOH
100% EtOH
95% EtOH
80% EtOH
70% EtOH
PBS
PBS
2.
1
2
2
2
2
2
2
2
hour or overnight, stir gently
minutes
minutes
minutes
minutes
minutes
minutes
minutes
Wipe off PBS from non-sample areas with Kim Wipe. DO NOT TOUCH SAMPLES.
Circumscribe the samples with a PAP pen.
3.
Drop trypsin solution onto samples using disposable pipette. Incubate 1 hour at room
temperature.
4.
During this time, prepare 3% H 2 0 2 , 30% goat serum, primary antibody, and mouse serum.
5.
Wash 2x in PBS, 2 minutes each.
6.
Incubate with 3% H 2 0 2 for 5-10 minutes.
7.
Wash 2x in PBS, 2 minutes each.
8.
Incubate in 30% goat serum for 10 minutes.
9.
Wipe off serum, but do not wash in PBS.
10. Apply primary antibody or negative control and incubate for 2 hours.
11. Rinse individual sections separately with PBS using a pipette to prevent contamination of the
negative control with the primary antibody.
12. Wash 2x in PBS, 2 minutes each.
13. Incubate with secondary antibody for 20 minutes.
14. Wash 2x in PBS, 2 minutes each.
15. Incubate with extravidin peroxidase for 20 minutes. During this time, prepare substrate
reagent and warm the glycerol gelatin.
16. Wash slides in PBS for 2 minutes, then in dH 20 for 3 minutes.
17. Dry 5 slides quickly, then incubate with AEC substrate reagent. Check slides under
microscope after 3 minutes. If darker staining is desired, leave in AEC for 1-2 additional
minutes.
18. Wash slides in dH20 for 3 minutes.
19. Place slides in Mayer's hematoxylin solution for 20 minutes.
20. Rinse with running water for 20 minutes.
21. Coverslip with warmed glycerol gelatin. If gelatin hardens too quickly, place slides on 40
degree surface or in 57 degree oven for a few minutes to re-melt the gelatin.
70
D.4
Immunohistochemical Staining of Type II Collagen
Solutions * *Note: Amounts will vary depending on number of slides being stained**
Tris-Buffered Saline (TBS) (Sigma # T-6664)
1 package to 1 L dH 2 0.
Protease XIV (a.k.a. Pronase E) (Sigma # P-5147)
0.01g Protease
10 ml
Store at -20*C until use, dessicate
TBS
3% Hydrogen Peroxide (H 2 0 2 ) (Sigma # H-1009)
1 ml
30% H 20 2
dH 20
9 ml
Stored at 4*C
5% Horse Serum (Sigma # H-0146)
0.5 ml Horse Serum
Stored at -20*C
10 ml
TBS
Primary Antibody - cIIcI mouse anti-chick type II
Collagen monoclonal Antibody (Developmental
Studies Hybridoma Bank, Iowa City, IA)
250 pl primary antibody
5 ml
TBS
Stored at -20'C
Use a 1:20 dilution
Secondary Antibody - Biotinylated
Horse Anti-Mouse Immunoglobin (Vector # BK2000)
10 pA
secondary antibody
Stored at -20'C
Use a 1:200 dilution
2 ml
TBS
Negative Control -Mouse IgG 2 A (Zymed #02-6200)
Dilute IgG in TBS to same protein
concentrationas in diluted primary antibody
Stored at -20'C
Avidin-Biotin Complex (ABC) staining kit
(Vector #PK 4000)
Stored at 4'C
Diaminobenzidine (DAB) kit
(Vector # SK-4100)
Stored at 4'C
Caution: Known carcinogen,
handle
carefully and dispose properly
Other Materials
Harris Hematoxylin Solution (Sigma # HHS-128)
Permount (Fisher #SP15)
71
Methods ***Do not let the slides dry out during this process***
1. Deparaffinize and rehydrate via the following baths
Xylene (or substitute)
100% EtOH
95% EtOH
80% EtOH
70% EtOH
TBS
2.
2
2
2
2
2
2
x 5 minutes
x 2 minutes
minutes
minutes
minutes
x 2 minutes
Wipe off TBS from non-sample areas with Kim Wipe. DO NOT TOUCH SAMPLES.
Circumscribe the samples with a PAP pen.
3.
Drop Protease solution onto samples using a disposable pipette. Incubate 1 hour at room
temperature. During this time, prepare 3% H 2 0 2 , 30% horse serum, primary antibody, and
negative control.
4.
Wash 2x in TBS, 2 minutes each. Wipe slides afterward
5.
Block with 5% horse serum for 30 minutes.
6.
Remove serum, but do NOT wash afterwards.
7.
Apply primary antibody or negative control and incubate for 1 hour.
8.
Rinse individual sections separately with TBS using a pipette to prevent contamination of the
negative control with the primary antibody.
9.
Wipe slides, wash 2x in TBS, 2 minutes each. Wipe slides again
10. Incubate with secondary antibody for 45 minutes. During last 15 minutes, prepare ABC
reagent and allow to stand for 30 min.
11. Wash 2x in TBS, 2 minutes each. Wipe slides afterwards.
12. Quench endogenous peroxidase by incubation in 3% H2 0
2
for 10 minutes.
13. Wash 2x in TBS, 2 minutes each. Wipe slides afterwards.
14. Incubate with avidin-biotin complex (ABC kit) reagent for 30 minutes.
15. Wash 2x in TBS, 2 minutes each. Wipe slides afterwards.
16. Dip slides in water.
17. Stain with DAB staining kit for 8-10 minutes.
18. Rinse in dH 20 for 3-5 minutes.
19. Counter-stain with Harris' hematoxylin for 10 minutes.
20. Rinse in running tap water.
21. Dip in acid alcohol
22. Rinse in running tap water.
23. Dip in ammonia water.
24. Rinse in water
25. Dehydrate (70%, 80%, 95%, 100%, 100%, xylene, 2 minutes each).
26. Air dry and coverslip with Permount.
72