Effects of Collagen Type on the Behavior of Annulus
Fibrosus Cells from the Canine Intervertebral Disc in
Collagen-Glycosaminoglycan Matrices
by
Leonide C. Saad
Ingenieur de l'Ecole Polytechnique (2001)
Ecole Polytechnique, Paris, France
MS Applied Mathematics (1999)
University of Paris Sorbonne, Paris, France
Submitted to the Department of Mechanical Engineering
In Partial Fulfillment of the Requirements for the Degree of
Master of Science in Mechanical Engineering
BAR ER
At the
MASSACHU'SETTS fNS'TITUtTEOF TECHNOLOGY
Massachusetts Institute of Technology
OCT 2 5 "JU
September 2002
LIBRARIES
©2002 Massachusetts Institute of Technology
All rights reserved
Signature of Author:
Department of
eJ
Engineering
ial
i
August 8, 2002
Certified by:
yron SpectoF
Mechani al Engineering
Senior Lecturer, Department
Professor of Orthopaedic Surgery (Biomateri ), Harvar Medical School
Thesis Supervisor
Accepted by:
Ain A. Sonin
Chairman, Department Committee on Graduate Students
Effects of Collagen Type on the Behavior of Annulus
Fibrosus Cells from the Canine Intervertebral Disc in
Collagen-Glycosaminoglycan Matrices
by
Leonide C. Saad
Submitted to the Department of Mechanical Engineering
On August 8, 2002
In Partial Fulfillment of the Requirements for the Degree of
Master of Science in Mechanical Engineering
ABSTRACT
The intervertebral disc (IVD) is the natural cartilaginous cushion found between the
osseous vertebrae of the spinal column. It is an essential element for the flexibility of the
spinal column but as it generally degenerates with age, with the resulting loss of structural
and functional integrity of the disc. Any damage to the IVD, be it natural or accidental, is
unlikely to heal and may even worsen with time. Tissue engineering has the potential to
provide solutions to the problems of IVD degeneration by facilitating its regeneration. The
purpose of this research was to investigate a collagen-based scaffold material for IVD tissue
engineering. Specifically, this thesis evaluated in vitro the effects of the collagen type from
which the scaffold was fabricated on the behavior of canine annulus fibrosus (AF) cells .
AF cells from 6 different canine animals were grown in monolayer culture through
Porous type I and type II collagen-glycosaminoglycan (GAG) scaffolds (also
passages.
two
referred to in this thesis as "matrices") were manufactured by a freeze-drying method and
then cross-linked by dehydrothermaland carbodiimide treatments. Specimens of the scaffolds,
9mm in diameter, were punched out of sheets of the matrices and seeded with 2 million cells
from each of the 6 animals by a pipetting method. Scaffolds were cultured for 1 day, and 2, 4,
6 and 8 weeks. Scaffold dimensions were measured every 2 or 3 days when medium was
5
and 3Hchanged. Prior to terminating the culture, samples were radiolabeled with 3 S-sulfate
proline to quantify biosynthetic activity. Scaffolds were then dried and weighed, and selected
variables were measured: dry weight, biosynthetic activity (protein and GAG synthesis),
DNA content (reflecting cell number), GAG content, protein content, and collagen content.
Histology, histochemistry, and immunohistochemistry were also performed to assess the cell
distribution and the presence of total collagen, type II collagen specifically, and
proteoglycans.
This thesis presents all the results of these experiments and the findings that are
associated with them. It also provides a comparison with prior work and provides suggestions
for a possible next step in intervertebral disc regeneration using cell-seeded collagen-GAG
scaffolds.
Thesis supervisor: Myron Spector
Title: Senior Lecturer, Department of Mechanical Engineering
Professor of Orthopedic Surgery (Biomaterials), Harvard Medical School
2
ACKNOWLEDGEMENTS
I would like to thank all the people who helped me complete this thesis. Their help
has been of a great value and I hope to have the opportunity to be as helpful to them as they
were to me.
I would like particularly to thank my advisor Professor Spector, who was always a
great guide, not only academically but also in life. From the Orthopaedic Research Lab at the
Brigham and Women's Hospital, thank you to Liqun Zou, Zhou Xiang and Huping Hsu for
helping me with my first lumbar spine resection. Lab mates and other students from MIT who
helped me much include Scott Vickers, Nicole Veilleux and all the other students of
Professor Spector. A special thank to Dawn Hastreiter who directed me during my first weeks
in the culture room and taught me so well the savoir faire she had acquired during long years
in the lab. David Bowman who helped me stain some slides and finish on time, and Joe who
works upstairs and never answered "I don't know" to whatever question I would ask him. I
would also like to thank Karen for being talkative during my waiting times at the Brigham,
and Cita who showed me the right way of doing immunohistochemistry and also gave me so
much advice.
From MIT, I would like to thank Brendan Harley and Professor Yannas for letting us
use his lab, Leslie Regan who has always been so helpful all the time even in her busiest
moments, Professor Lloyd who was my first advisor and is also always cheerful, and all the
department of mechanical engineering.
Victor Zaporojan from UMASS for bringing me a box full of doughnuts at night and
who also helped me a lot for my last experiments; I spent great moments in his lab where the
music never stops.
I would like to thank also the other people who are close enough to me to support me
when I'm tensed up or in a bad mood, and I think particularly of Jing, Ying, Jasmin, June,
Chen, Juergen and Hanh.
I could not finish without dedicating this work to my family and particularly to my
grand father and my mother and to Stephane, my cousin, who just got married.
Lastly, I would like to acknowledge the Veteran Administration for providing
financial support for this work.
3
ABSTR A CT..........................................................................................................................
2
LIST OF FIG URES.............................................................................................................
7
LIST OF TA BLES ...............................................................................................................
9
CHAP TER 1 : IN TR OD UCTION ...................................................................................
10
1.1 Purpose of Research.................................................................................................................
10
1.2 Background...............................................................................................................................
10
1.2.1
1.2.2
1.2.3
1.2.4
Structure and Function of the Intervertebral Disc ..............................................................................
Intervertebral Disc Degeneration and Low Back Pain ......................................................................
Treatment of Intervertebral Disc Degenerative Diseases..................................................................
Tissue Engineering of Intervertebral Discs........................................................................................
10
11
13
14
1.3 Specific Aim and Hypotheses ..................................................................................................
15
CHAPTER 2: MATERIALS AND METHODS................................................................
17
2.1 Experim ental Design................................................................................................................
17
2. 1.1 Outcome Variables ...................................................................................................................................
2.1.2 Sample size determination .......................................................................................................................
2.1.3 Experimental Plan.....................................................................................................................................18
2.2 Cell Culture .......................................................................................................................---....
2 .2 .1 C ell Isolation .............................................................................................................................................
2.2.2 Cell Proliferation.......................................................................................................................................18
2.3 M atrix M anufacturing and Preparation.............................................................................
2 .3 .1 Freeze D ry ing...................................................................................................................................
2 .3 .2 Cro ss L ink in g ............................................................................................................................................
17
17
18
18
19
.....-- 19
19
2.4 Cell Seeding and Scaffold Culture......................................................................................
19
2.5 Biosynthesis Assay....................................................................................................................
20
2.6 Other Assays.............................................................................................................................
20
2 .6 .1
2 .6 .2
2.6.3
2.6.4
2.6.5
2.6.6
2.6.7
D isc D ry ing ...............................................................................................................................................
D ry W eigh t................................................................................................................................................20
Digests for Biochemical Assays ..............................................................................................................
Scintillation Counting...............................................................................................................................21
DNA Quantification and Cell Counting.............................................................................................
GAG Determination..................................................................................................................................21
Aspect Ratio / Contraction.......................................................................................................................21
20
20
21
2.7 Histology and Im m unohistochem istry ...............................................................................
22
2.8 Total Collagen Content............................................................................................................
22
2.9 Total Protein Content ..............................................................................................................
22
2.10 Cell seeding efficiency............................................................................................................
22
2.11 Statistical Analysis .................................................................................................................
23
4
CHAPTER 3: RESULTS ...............................................................................................
24
3.1 B iosynthetic A ctivity ................................................................................................................
24
3.3 N umber of Cells........................................................................................................................
25
3.4 GA G Content............................................................................................................................
26
3.5 Aspect Ratio - M acroscopic Examination ...........................................................................
27
3.6 Scaffold Contraction ................................................................................................................
28
3.6.1 Overall Contraction ..................................................................................................................................
3.6.2 Cell-M ediated Contraction.......................................................................................................................28
28
3.7 D ry M ass ...................................................................................................................................
29
3.8 C ollagen C ontent......................................................................................................................
30
3.9 Protein Content ........................................................................................................................
30
3.10 Histology and Im m unohistochem istry .............................................................................
31
CHAPTER 4: DISCUSSION............................................................................................
36
4.1 Relevance of Results : Effect of collagen type on the behavior of cell seeded matrices... 36
4.1.1 GA G content and synthesis rate...............................................................................................................37
4.1.2 DN A content .............................................................................................................................................
37
4.1.3 Protein content and protein synthesis rate...........................................................................................
37
4.1.4 Collagen content .......................................................................................................................................
38
4.1.5 Contraction, Deterioration and Aspect ratio ......................................................................................
38
4.1.6 Dry M ass ...................................................................................................................................................
38
4.2 Comparison with Other Literature Results.......................................................................
39
4.3 Lim itations................................................................................................................................
40
4.4 Significance and Future W ork.............................................................................................
40
CHAPTER 5: CONCLUSION..........................................................................................
41
A PPENDICES...................................................................................................................
42
APPENDIX A - Seeding efficiency experiment ..........................................................................
43
O bjectiv e .............................................................................................................................................................
Experim ental Plan, M aterial & M ethods..........................................................................................................43
43
R e sults .................................................................................................................................................................
Discussion & Conclusion ..................................................................................................................................
43
45
APPENDIX B - Evaluation of the DNA mass of canine IVD annulus fibrosus cell ........
46
Objective.............................................................................................................................................................46
Experim ental Plan, M aterial & M ethods......................................................................................................
46
Results.................................................................................................................................................................46
Discussion & Conclusion ..................................................................................................................................
46
APPE N D IX C - Protocols .............................................................................................................
47
C-1 Annulus Digestion ......................................................................................................................................
C-2 Freezing Cells..............................................................................................................................................49
C-3 Thaw ing Cells .............................................................................................................................................
C-4 Passaging Cells ...........................................................................................................................................
C-5 Cell counting ...............................................................................................................................................
47
50
51
52
C-6 M edium preparation....................................................................................................................................54
C-7 M edium changing .......................................................................................................................................
C-8 M atrix M aking ............................................................................................................................................
C-9 EDA C Cross-Linking .................................................................................................................................
56
57
60
C-10 Cell seeding using pipetting method .................................................................................................
C-II M atrix culture in multi well-plates ......................................................................................................
61
62
C-12 Radiolabeling of 35S and 3H ...................................................................................................................
63
5
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
Lyophilization of M atrix Sam ples......................................................................................................
Papain digestion........................................................................................................................................66
Scintillation counting of 35S and 3H Radiolabeled Sam ples ..........................................................
GA G A ssay (D im ethylene Blue).............................................................................................................70
DN A A ssay using H oechst Dye...............................................................................................................72
BCA Protein A ssay...................................................................................................................................75
H ydroxyproline A ssay..............................................................................................................................76
Paraffin embedding of M atrices...............................................................................................................77
Preparation of coated slides .....................................................................................................................
.......
H em atoxylin and Eosin Staining ..................................................................................
Safranin-O Staining ..................................................................................................................................
M asson Trichrom e Staining .....................................................................................................................
.......... ...........
............
Type II imm unohistochem istry.....................................................
R EFER ENCES ..................................................................................................................
65
67
78
79
81
82
84
86
6
LIST OF FIGURES
Figure 1. Anatomy of the vertebral column (left), a detailed view of the lumbar spine (center),
longitudinal cut through an intervertebral disc (right). (source www.back.com) .....
11
Figure 2. Compression of the intervertebral disc (left) and herniated disc (right). (source
www .back.com )..........................................................................................................
12
Figure 3. Disrupted disc (left) and nucleus pulposus content pushing the nerve root (right)
(source: Bay Area Pain M edical Associates) ...........................................................
12
Figure 4. Example of fusion surgery for replacement of a cervical disc (source Medtronics) 13
Figure 5. Example of a metal/plastic intervertebral disc prosthesis. Left and bottom: SB
Charit6 III Dynamic Disc Spacer (Link Spine Group, Branford, CT, USA), Top right:
14
PRODISC@ (Spine Solutions, New York, NY, USA) ..............................................
Figure 6. Normalized rates of 3H incorporated per g of DNA, per hour......................... 24
Figure 7. Normalized rates of 35S incorporated per g of DNA, per hour........................ 24
Figure 8. Total rates of GAG biosynthesis over a 24 hour period, for type I and II seeded matrices
cultured for 8, 15, 29, 43 and 57 days...........................................................................
25
Figure 9. Total rates of protein biosynthesis over a 24 hour period, for type I and II seeded matrices
cultured for 8, 15, 29, 43 and 57 days...........................................................................
25
Figure 10. Number of cells in the matrices for type I and type II matrices ........................ 26
Figure 11. Percentage mass GAG / total Mass for type I and type II matrices.................. 26
Figure 12. Total mass of GAG for type I and type II matrices ..........................................
27
Figure 13. Aspect Ratio for type I and type II matrices...................................................
27
Figure 14. Overall contraction in percentage of initial seeded matrix size........................ 28
Figure 15. Overall contraction per cell in percentage of initial seeded matrix size. ........... 28
Figure 16. Cell mediated contraction in percentage of unseeded control size.................... 29
Figure 17. Cell mediated contraction per cell in percentage of unseeded control size........ 29
Figure 18. Dry weight of the samples for each type of matrix at different times of culture... 29
Figure 19. Collagen content contained in the type I and type II constructs for different time
30
periods. Unseeded scaffolds are represented on the right. .........................................
Figure 20. Protein content of the type I and type II constructs for different times of culture.
Unseeded constructs are represented on the right.....................................................
30
Figure 21. Type I collagen-GAG matrices after 1 week (left) and 4 weeks (right). Newly
synthesized tissue at 4 weeks is apparent..................................................................
32
Figure 22. Detailed view of the inside of type II collagen matrices after 4 weeks of culture
(left) and view on the side of the matrix after 6 weeks (right)................................... 32
Figure 23. Type II collagen matrices after 6 weeks. Notice the formation of new tissue and
the groups of cells on the sides .................................................................................
32
Figure 24. detail of 2 cells attached to a type II collagen GAG scaffold at 4 weeks........... 32
Figure 25. Type II matrices after 8 weeks under normal (left) and polarized (right) light
showing the organization and birefringence of the collagen fibers........................... 33
Figure 26. Wavy crimp-like structure after 8 weeks, type II. ...........................................
33
Figure 27. General view of a type I matrix at 8 weeks. Scaffold appears oval and irregular in
diam eter......................................................................................................................
33
Figure 28. General view and GAG localization for type I (left) and type II (right) matrices
after 1 w eek .................................................................................................................
34
Figure 29. Zooms on reddish structure (left) that could be a cell surrounded by proteoglycans
in type I matrix after 4 weeks, and GAG structure located on side of a type II matrix
after 6 w eeks of culture (right). ...............................................................................
34
7
Figure 30. Matrix type I (left) and type II (right) sides after respectively 4 and 6 weeks of
culture. GAG is mostly located on the sides. ............................................................
34
Figure 31. group of cells after 6weeks of culture of a type I matrix (left). Also after 6 weeks,
type II matrix (right) presents its very a quite evenly spread collagen content (right)... 35
Figure 32. Zoom on type I matrix after 6 weeks showing cells producing collagen (left) and
evenly spread collagen inside a matrix type II after 6 weeks....................................
35
Figure 33. type I collagen matrix after 4 weeks (left) and type II collagen matrix with little
collagen (right) after 1 week of culture....................................................................
35
Figure 34. Number of cells in the constructs after 1 and 3 days of culture for type I and type
II cell seeded matrices with different cell seeding densities. ....................................
44
Figure 35. Hemocytometer Counting Diagram...............................................................
53
Figure 36. Example GAG Standard Curve......................................................................
71
Figure 37. Example DNA Standard Curve......................................................................
74
8
LIST OF TABLES
Table 1. Outcom e Variables............................................................................................
Table 2. Performed Qualitative Assays ............................................................................
17
17
Table
Table
Table
Table
18
43
44
46
3.
4.
5.
6.
Number of seeded matrices that were used for the experiment...........................
Number of seeded matrices that were used for the seeding efficiency experiment..
Number of cells compared to initial seeded density versus time for 3 matrix types.
M ass of DNA per annulus fibrosus cell. ............................................................
9
CHAPTER 1 : INTRODUCTION
1.1 Purpose of Research
Damage to the intervertebral disc (IVD) can arise from accidents or natural degeneration.
Healing of such damage is unlikely to occur and damage often progresses to other problems
such as herniated discs which often lead to pain or numbness, and weakness. Several surgical
procedures have been developed to address this problem. Most of them ultimately replace the
IVD by a bone graft. However, long-term complications result from most of these treatment
modalities. Among these complex operations only 60% have been found to completely
relieve the pain associated with herniated discs [1, 2, 3] and 15% failed to relieve symptoms
[4, 5]. Furthermore, and of importance relative to this thesis, the current surgical management
of IVD problems is based on tissue replacement not on tissue reparation.
A tissue engineering approach using a cell-seeded collagen-glycosaminoglycan (GAG)
scaffold as a healing structure that could regenerate damaged parts of the IVD would be a
revolutionary alternative to existing surgical methods for treating IVD problems. This
approach has already shown promise for the treatment of lesions in other tissues like cartilage
[25, 26], nerves [30, 31] and skin [31]. This thesis evaluates the effects of the collagen type
from which the scaffold is fabricated on the behavior of canine IVD annulus fibrosus (AF)
cell-seeded collagen-GAG scaffolds cultured in vitro for up to 8 weeks.
1.2 Background
1.2.1 Structure and Function of the Intervertebral Disc
The structure, function and pathology of the IVD have been described in many
different studies [21]. IVDs separate the bony vertebral bodies of the vertebral column. In the
human body there are 33 vertebrae and 30 intervertebral discs, with about one-fourth of the
total length of the vertebral column accounted for by the discs.
IVDs are not rigid structures, but cartilaginous cushions which are flexible and allow
the vertebral column to bend and twist. Furthermore, during the course of a typical day, the
discs dehydrate a little and the total length of the vertebral column may vary. The discs serve
to allow motion between the vertebrae, absorb shock, and distribute the load over a large
surface of the vertebral body.
10
POSTERIOR
AMTREIt
-..
annulus
Discsi
Figure 1. Anatomy of the vertebral column (left), a detailed view of the lumbar spine
(center), longitudinal cut through an intervertebral disc (right). (source www.back.com)
The discs are mainly composed of 2 clearly separated parts: the AF and the nucleus
pulposus (NP). The AF forms the outer boundary of the disc and is composed of
fibrocartilaginous tissue with collagen fibrils arranged in concentric lamellae. Each lamella is
made principally of type I collagen fibers that run obliquely (with an angle of around 65 )
from one vertebra to another. The fibers slant in alternating directions from one lamella to
another, conferring to the AF a very strong structure with the capacity of sustaining
compression, torsion and flexion. The IVD is tightly connected to the vertebral bodies by the
cartilaginous end plates. The NP forms the central part of the IVD. It consists primarily of
type II collagen fibrils forming a gelatinous substance with the characteristics of hyaline
cartilage, and contains more water than the AF. NP cells often resemble mnorphologically
chondrocytes and fibroblasts. The NP surface accounts for about 40% of the total IVD
surface. The NP content of the IVD generally decreases with age, and the separation between
the AF and NP, while very clear in specimens from young individuals, becomes indistinct
with age. Disc thickness is also dependent upon the location on the vertebral column. Most
problems occur in the low back (lumbar) region where motion of the spine is the greatest and
lumbar IVDs are thickest. In most regions, disc height is not uniform; for example, lumbar
IVDs are thicker anteriorly than posteriorly [21].
IVD are completely without blood vessels after the first 3 decades of life, existing
vessels being gradually replaced by scars. For that reason, nutrition and disposal of metabolic
waste is difficult and only possible via 2 paths: the annulus fibrosus and the end plates. Most
of the nutrients diffuse from the cartilaginous end plates to the AP and AF and from the
surrounding blood vessels to the AF [5, 6, 7].
1.2.2 Intervertebral Disc Degeneration and Low Back Pain
IVDs, like other cartilage tissues such as articular cartilage, can degrade and wear
away with age or trauma, with a resulting loss of function. In the case of the IVDs, the
absence of a direct nutrient supply, the low mitotic activity of the disc cells, the decrease of
permeability of the end plates with age [7], and the decrease in proteoglycans and water
content are all natural biochemical factors predisposing to disc degeneration. Other risk
factors for degeneration include: high loading on the NVD, spinal deformation, vibration,
immobilization and accidental damage. Certain studies include smoking, vascular disease and
diabetes as direct factors that compromise the vascular supply and accelerate disc
degeneration [8].
11
Figure 2. Compression of the intervertebral disc (left) and herniated disc (right). (source
www.back.com)
ANUWS
of RINGS
ANULLS
fRINGS
Figure 3. Disrupted disc (left) and nucleus pulposus content pushing the nerve root
(right) (source: Bay Area Pain Medical Associates)
When IVDs degenerate, they partly lose their flexibility, elasticity and shock-
absorbing characteristics. AF lamellae become more brittle and easily torn and the content of
the AF decreases. As the IVD shrinks in height, the vertebral column loses its height, which
leads to a compression of nerve root due to the reduced available space. This can cause
several symptoms such as back pain, leg pain, weakness and numbness.
If the AF lamellae are torn after an accident or after natural degeneration, the contents
of the nucleus protrude from the disc, often compressing the nerve root, and provoking
inflammatory responses. This phenomenon is called a "herniated disc," and generally occurs
between the ages of 30 and 50 years. The pain is not only located where the nerve is
compressed, but spreads also to the location connected to the involved nerve.
IVD degeneration is unlikely to be reversed by healing processes [22,,23, 24]. Instead,
when reparative tissue is formed in the annulus, it comprises fibrous tissue and fibrocartilage
that lack the normal disc structure and predispose the disc to a continuing degenerative
process.
12
1.2.3 Treatment of Intervertebral Disc Degenerative Diseases
Challenges are multiple to treat patients suffering from IVD degenerative diseases.
First, accurate diagnosis of the problem (including the location and cause of the pain) is still
imprecise in spite of multiple techniques that include radiography, magnetic resonance
imaging (MRI), computed tomography (CT), and myelography. Second, even if accurate
diagnosis is performed, treatments are not always efficient in relieving the pain. Actually, the
pain associated with a herniated disc is naturally relieved after a few months for most cases.
It also appears in many studies that leg pain is more likely to be solved through surgery than
back pain. Non surgical options are preferred to surgical procedures for many cases; these
include: exercise, acupuncture, drugs (mostly anti-inflammatory), massage, psychological
support, heat, and ice. In approximately 20% of the cases, where no pain relief is observed,
surgery is performed.
There is currently no satisfactory procedure for prosthetic replacement or regeneration
of degenerated disc tissue. The most commonly performed procedure is the lumbar
discectomy. About 400,000 discectomies, accounting for more than 5% [9] of all orthopedic
procedures, are performed each year in the US [9] each of them costing more than 29,000$
per case [10]. These procedures consist of removing the bulging content of the NP that
compresses the nerve root. Discectomies have tried to be as minimally invasive as possible
throughout the years and presently involve technologies such as lasers and endoscopic
instruments, and arthroscopic visualization techniques. If the patients have been carefully
selected, the success rate of the operation can be as high as 90%. Even if discectomy removes
the damaged material surrounding the nerve root, it may not reduce the compression of the
nerve root.
Many investigations [9] have shown that there are many long-term complications of
disc surgery, including reherniation at the same or different location, scar tissue formation,
and additional lumbar spine problems. A second discectomy has only a 50-60% success rate
[9, 11].
When the pain cannot be solved by discectomy or when the disc degeneration is
advanced, arthrodesis, also called spine fusion, is often advised. This operation involves the
removal of the IVD and replacement by a bone graft or a titanium cage, in order to reduce
nerve root compression and future reherniation.
AR'OUZHY
Figure 4. Example of fusion surgery for replacement of a cervical disc (source
Medtronics)
13
More recently, several prosthetic devices have been developed to replace an entire
IVD while preserving mobility. These devices employed for "spinal arthroplasty" have as yet
been unproven by long term clinical studies, and may fail due to a lack of bone ingrowth into
the prosthesis and inadequate fixation, toxicity of substances leached from the device, and
wear, All these problems are critical as they are all located close to the spinal cord.
Figure 5. Example of a metal/plastic intervertebral disc prosthesis. Left and bottom: SB
Charit6 III Dynamic Disc Spacer (Link Spine Group, Branford, CT, USA), Top right:
PRODISC® (Spine Solutions, New York, NY, USA)
1.2.4 Tissue Engineering of Intervertebral Discs
The IVD is a complex arrangement of different connective tissues. Furthermore, it is
located in an avascular region of the body, and has to sustain high compressive loads and
deformations. For these reasons, creating an entire IVD using tissue engineering methods is a
very challenging problem. As a first step it may be more reasonable to consider regenerating
part of the IVD.
Even though tissue engineering is a field undergoing maturation, with products
approved by the United States Food and Drug Administration for skin and cartilage
regeneration, many uncertainties and questions remain: should a scaffold be used to help
endogenous cells proliferate and what kind of scaffold? Should that scaffold be seeded with
exogenous cells? What type of cells should be used? Should growth factors be employed?
What about gene therapy? Should the tissue be cultured in vitro or implanted directly in the
body? Should a "bioreactor" be used as a culture vessel?
Some work has already been conducted in vitro [33, 34]. Companies (e.g., Co.don)
have initiated clinical trials of implantation of autologous chondrocytes, which they have
expanded in number in vitro, in the damaged spinal disc. However, the efficacy of these
procedures has not yet been proven.
14
Repair of IVD defects should include regeneration of lost proteoglycans in the NP and
regeneration of collagen fibers in the AF. In order to repair AF tissues, recent work has
shown promising use of resorbable scaffolds as implants to facilitate regeneration of the
tissue in vivo. Scaffolds can be fabricated from synthetic (polylactic acid or polyglycolic acid)
or natural (collagen, fibrin) polymers and are designed to "maintain and substitute for the
fibrin clot necessary to support cell migration into and cell proliferation within the defect"1 .
Unseeded scaffolds have already been implanted in canine intervertebral disc defects in order
to prove the feasibility of the surgery; scar tissue was found to form at the location of the
implanted site.
Discovering the behavior of the cell-seeded scaffolds in vitro with respect to certain
factors is a first step toward the development of an implant to facilitate the regeneration of the
annulus.
1.3 Specific Aim and Hypotheses
The main objective of this research was to determine the effects of collagen type (type
I versus type II), from which the scaffold was fabricated, on the behavior of canine AF cellseeded scaffolds. The studied behavior was the capacity of the scaffolds to continue to
survive after a long period (8 weeks) of culture. This capacity was evaluated by measuring
the biosynthesis of proteoglycans and of proteins, the number of cells, the amount of
proteoglycan, and the collagen and protein contents over different periods of culture. Canine
cells have been preferred to other animal in the perspective of using a canine model for in
vitro studies in the future.
The hypotheses synthesized from previous findings include the following:
1. AF cells grown in type II collagen matrices produce more type I collagen and
GAG than cells grown in type I collagen scaffolds. The rationale for this
hypothesis is that prior preliminary work has indicated that IVD cells display a
greater degree of matrix synthesis in type II collagen matrices in vitro [40].
While the AF of the IVD is comprised principally of type I collagen, AF cell
interactions with type II collagen may provide a greater stimulation to matrix
synthesis.
2. The number of cells in the construct decreases in type I and type II matrices
between 2 and 4 weeks of culture. It may be expected that some of the seeded
cells undergo apoptosis (programmed cell death) by this time period, and the
degree of cell proliferation is no so great as to maintain a constant or
increasing cell number density.
3. An AF cell number density in the collagen-GAG matrix close to that which
occurs naturally (a few thousand cells per mm 3) can be achieved by seeding 2
million cells in a collagen-GAG scaffold that is 9mm in diameter and 3mm
thick. This hypothesis is based on a preliminary study (reported in the
appendix).
4. Carbodiimide cross-linked matrices do not undergo as much cell-mediated
contraction as non cross-linked matrices or scaffolds cross-linked by
dehydrothermal (DHT) treatment. From prior work of others, it is expected
that carbodiimide cross-linking increases the mechanical stiffness of the
1 Spector
M
15
scaffold to a level that the scaffold can resist the forces of contraction exerted
by the cells.
All of these hypotheses are important to verify as they characterize critical aspects of
the behavior of the constructs. Collagen and GAG synthesis are good indicators of the
viability of the cells. Furthermore, GAG is an essential matrix molecule (when bound with
protein to form a proteoglycan) as it binds the water which provides resistance to
compressive loading and prevents dehydration of the scaffold. Collagen is important as it is
the principal structural protein providing the mechanical integrity of the tissue, by directly
resisting tensile and shear loading and by containing the proteoglycan. The number of cells in
the construct affects directly the quantity of protein and GAG that will be synthesized and
seeding density is therefore important to optimize in order to harvest as few cells as possible
from the patient in a clinical perspective. Finally, prior work has demonstrated that AF cells
can express the gene for a contractile muscle actin isoform and can contract [40].
Contraction of the scaffold is of importance in that a large contraction of the scaffolds would
compress the pores and thus make difficult the migration in vivo of new cells or nutrition
factor in the scaffold and could also prevent the implant from maintaining its original size
and placement at the site of implantation.
The work in this thesis could be translated into the clinic in the following manner.
IVD tissue could be obtained from a patient during a surgical procedure for treatment of a
disc problem (e.g., discectomy). This tissue could be used as a source of AF cells, whose
number would be expanded by growth in monolayer culture. After two passages a sufficient
number of AF cells would be obtained to seed the collagen-GAG scaffold, and the cellseeded scaffold cultured for some additional time period. The cell-seeded construct would
then be implanted into site of herniation in the IVD of the same patient. This method would
thus allow for the use of autologous cells and obviate any immune problems resulting from
the use of AF cells from another individual. There are of course several questions that will
ultimately need to be addressed. Will the harvested herniated tissue yield a sufficient number
of healthy AF cells? How long will the AF cell-seeded collagen-GAG matrix need to be
cultured prior to implantation? The work embodied in this thesis lays the foundation for this
tissue engineering approach to solving this compelling clinical problem.
16
CHAPTER 2: MATERIALS AND METHODS
2.1 Experimental Design
The experimental design was based on the comparison of the proliferative and
biosynthetic behavior of second passage canine AF in type I and type II collagen-GAG
scaffolds.
2.1.1 Outcome Variables
Following parameters were measured or evaluated:
Var #
1
2
3
4
5
6
7
8
Variable
Dry weight
Construct size
Cell number
GAG content
GAG synthesis rate
Protein synthesis rate
Protein content
Collagen content
Measurement Method
Balance
Calibrated ruler
DNA assay
GAG assay
Radiolabeled Sulfate Incorporation
Radiolabeled Proline incorporation
Bicinchoninic Acid Assay (BCA)
Hydroxyproline assay
Table 1. Outcome Variables
Qualitative histological, histochemical and immunohistochemical assays were also performed
to verify the presence or absence of:
GAG and other proteoglycans
Cells and general structure
Collagen
Type II collagen
Safranin-O staining
Hematoxylin and Eosin Staining
Masson Trichrome Staining
Immunohistochemistry type II
Table 2. Performed Qualitative Assays
2.1.2 Sample size determination
Cells from 6 different animals were cultured and seeded separately on the scaffolds.
For each time point, each variable was measured with n=6, each sample coming from
constructs of 6 different dogs. The sample size of 6 was based on detecting as statistically
significant a 20% difference in the means of an outcome variable of two groups (e.g., cells in
the type II and type II scaffolds) each with a 10% coefficient of variation. Histology and
immunohistochemistry was performed with n=3 to allow for replication of the qualitative
findings, as were the hydroxyproline and BCA quantitative assays.
17
2.1.3 Experimental Plan
For each of 6 different time periods, 24 scaffolds, of which half the number were type
I and half were type II, were seeded with AF passage 2 cells. For each collagen type, there
were cells of 6 separately cultured animals seeded on 2 scaffolds for each animal.
Half of these scaffolds from different animals were radiolabeled 24 hours prior to
sacrifice. Three of the remaining 6 were used for histology and immunohistochemistry, and
the other 3 were used for hydroxyproline assay.
Thus, a total of 144 (6*24) scaffolds were prepared and seeded with 2 million cells
from cells of 6 animals that were maintained and seeded separately. Six type I and 6 type II
matrices were also cultured as control matrices (Table 3).
Dog #
Var 1-6
(n=6)
1
2
3
4
5
Type I
6
6
6
6
6
TypeII
6
6
6
6
6
6
6
6
Control
Measured Variables
Var 7-8
Histology /
(n=3)
Immunohistochemistry
(n=3)
Type I Type II
Type I
Type II
6
6
N/A
6
6
6
6
N/A
6
6
6
6
6
N/A
Controls
(n=6 ; 1 time
point)
Type I Type II
N/A
6
4 of each type
for var 1-8
*
* 2 of each type
for hist./immuno.
Table 3. Number of seeded matrices that were used for the experiment
2.2 Cell Culture
2.2.1 Cell Isolation
IVD AF canine cells were taken from an existing pool of frozen P1 cells that had been
isolated and frozen separately from 6 adult canines (2-7 years old). Protocols for IVD harvest,
cell isolation and cell freezing can be found in the appendices.
2.2.2 Cell Proliferation
AF cells from 6 different animals were thawed and put separately in culture in
75cmA2 flasks with 15mL of medium. Cell concentration in the flasks was 1 million. In
500mL of medium, there were 380mL of DMEM/F12 medium (#11320-033,
Life
Technologies) complemented with 5mL (1%) of antibiotic-antimycotic solution (Gibco-BRL
18
No. 15240-096, Life Technologies), lOmL (0.025g/L) of L-ascorbic acid phosphate
(magnesium salt n-hydrate; #D13-12061), 1% of L-glutamine (#25030-81, Life Technologies)
and 20% FBS (Australian fetal bovine serum; #SH30084.03, Hyclone Laboratories). Medium
was changed every other day until confluence which was reached after about 7-8 days of
culture.
For each animal, 2 tubes of 6 million cells were thawed. After thawing an average of
10.1 million cells was obtained, i.e. a yield of about 85%. After confluence, 95+34 million
(standard error of the mean SEM) cells per flask were obtained, largely more than the
required 48 million cells per animal necessary for the experiment.
2.3 Matrix Manufacturing and Preparation
2.3.1 Freeze Drying
Type I collagen-GAG matrices were produced by freeze-drying a precipitate of type I
collagen from bovine tendon (Integra Life Sciences) and chondroitin 6-sulfate from shark
cartilage (chondroitin sulface C-4384 from shark cartilage, Sigma Chemical, St. Louis, MO).
The slurry was freeze dried using a temperature ramping protocol at -40'C and less than
200mTorr (Virtis - Genesis SQ25LE). Further detail of the procedure can be found in the
appendix. Pore diameter was evaluated to be 120+18gm (Standard deviation, SD).
Type II collagen slurry was provided by Geistlich Biomaterials (Chondrocell slurry,
Lot 001511, Wolhusen, Switzerland). The same chondroitin 6-sulfate was added at a
concentration of 0.0105g per 20mL of slurry before the precipitate was freeze dried. Pore size
was evaluated by prior studies [12] to be 202±51gm (SD).
The freeze drying method produced sheets of the collagen scaffolds approximately 3
mm thick.
2.3.2 Cross Linking
Matrices were crosslinked with a DHT treatment at 100 0C for 24 hours followed by
an additional l-ethyl-3,3 dimethetylaminopropyl carbodiimide (EDAC) crosslinking (6mmol
EDAC per gram of collagen, 5:2 EDAC/NHS ratio) during 2 hours at room temperature one
day prior to the seeding day and were kept in distilled water overnight at 4'C.
2.4 Cell Seeding and Scaffold Culture
Before EDAC cross-linking, 9-mm diameter discs of the matrices were punched from
the sheets using a dermal punch (Barron Vacuum Trephine, 9mm, #K20-2062, Katena
Products). At confluence (following the protocol given in appendix), cells were detached
from the flasks and 1 million cells per side of the matrices were seeded using a pipetting
technique detailed in the appendix. A pilot study described in appendix A rationalizes the
seeding density of 2 million cells per disc.
Cell-seeded matrices were placed in 6-well plates coated with 2mL of 2% agarose (1 g
per 25mL of H20, Agarose Seaplaque, BMA, Rockland ME, Cat 50100), in order to prevent
migration of cells from the scaffolds onto the surface of the culture dish.
19
Every other day, medium was changed from the plates. The medium contained 10%
FBS medium and the same volume amount of L-glutamine, ascorbic acid and antibiotics as
previously noted. It was shown previously that 20% FBS medium induced a fast shrinkage of
the matrices [120].
To evaluate contraction, disc diameter was measured after 6 hours of culture and then
everyday during the first week of culture. After the first week, the diameter was measured
every other day. Discs were often oval and not perfectly circular. In that case, small and long
diameters of the discs were measured. A diameter template was placed under the 6-well
plates to measure the diameter.
2.5 BiosynthesisAssay
During the last 24 hours in culture, the rates of synthesis of proteoglycans and total
protein were assayed with the use of radiolabled sulfate and radiolabeled proline: 3 discs of
each matrix type for each canine were radiolabeled with tritiated proline 3H (proline, L[2,3,4,5-3H]; #NET 483, Perkin Elmer) and radioactive sulfate 35S (#NET041H, Perkin
Elmer). The medium contained l0gCi/mL of each isotope. Unseeded control matrices were
radiolabeled only for the last time point (8 weeks) because a previous study [12] already
showed that controls present negligible biosynthetic activity. Media containing the 3H label
and the double label of 3H-35S were kept for scintillation counting calibration. Cell-seeded
samples were incubated (37C, 5% CO 2) for 24 hours in 1.5mL of complete radiolabeled
medium, in 24-well plates. At the end of the 24-hour incubation, unincoroporated sulfate was
removed by rinsing 5xl5minutes at 4'C with a solution of phosphate buffered saline
supplemented with 0.8mM Na 2SO 4 (anhydrous sodium sulfate; Sigma) and 1.0mM L-proline
(Sigma, P8449). Discs were then frozen at -20*C, freeze dried and digested before
scintillation counting.
2.6 Other Assays
2.6.1 Disc Drying
Samples were freeze dried for 23 hours using a Freeze Dryer Labconco 4.5.
Appendix gives more detail on the freeze drying process.
2.6.2 Dry Weight
Dry weight was measured after freeze drying using a balance model Mettler AE240.
2.6.3 Digests for Biochemical Assays
Digestion of the samples was performed overnight in a 60 C water bath using
0.125mg of papain (Sigma #P3125) per 6.25 mg of sample. Each sample received at least
5 L of papain. 100mL of Papain buffered solution was composed of 2.45mL dibasic stock
solution, 17.54mL monobasic stock solution, 80mL distilled water, 87.82mg L(+) cysteine
HCl, 186.12mg Disodium Ethylenediamenetetraacetate (EDTA, Fischer S-657), with pH
adjusted to 6.2.
20
2.6.4 Scintillation Counting
Radiolabeled digests were assayed for radioactivity by combining 100 L of digest
with 2mL of scintillation fluid (Scintiverse II, cat #SX12-4, Fisher Scientific, Fair Lawn NJ),
and counted for 1 minute in a liquid scintillation counter (Packard Tri-Carb 4640, United
Technologies Packard, Downer's Grove, IL). Values of GAG and total protein synthesis rates
were extrapolated from counts per minute using known amounts of radiolabeled medium.
Mean counts per minute of control matrices was always negligible and therefore no
subtraction was made on the results. More information is available in the appendices.
2.6.5 DNA Quantification and Cell Counting
An aliquot of papain digested sample was read fluorometrically (Hoefer Scientific
Instruments DNA Fluorometer, model TKO 100, Xex=365nm, Xem=460nm) using Hoechst
dye no. 33258 (Polyscience Inc.). The amount of DNA was extrapolated from the standard
curve of calf thymus DNA.
The number of cells was determined from the DNA measurements by taking the value
3.6pg (3.6E-12g) of DNA content in each AF cell. That figure was obtained from a previous
study which involved digesting overnight known amounts of fresh cells in papain and for
measurement of the DNA mass fluorometrically. Amounts of cells that were aliquoted were:
0.25, 0.5, 1, 1.5, 2, 3, 4 million cells, with a concentration of 20 L per million cells of a mix
cells/medium. Each aliquot was completed with 5 L of papain solution, and completed to
lmL with papain buffer. The appendices detail that experiment.
2.6.6 GAG Determination
The GAG content was determined using a modified dimethylene blue method. A
100 L aliquot of the papain digst was assayed by addition of 2mL of 1,9 dimethyl
methylene blue dye solution (Polyscience, Northampton, UK). Absorbance at 535nm was
determined with a spectrophotometer (LKB Biochrom Ultraspec 4050, Pharmacia,
Piscataway NJ). To obtain the amount of GAG per sponge, the results were extrapolated from
a standard curve made using shark chondroitin sulfate in dH20.
2.6.7 Aspect Ratio / Contraction
Aspect ratio was evaluated as being the ratio between the longer diameter and the
smaller one.
When matrices were not circular, effective diameter was calculated as being the root
mean square of the smaller and longer diameters.
Contraction was measured using 2 different standards:
- overall contraction: the percentage of contraction of the effective diameter compared
to initial measured diameter of the same type of cells seeded scaffold.
- cell mediated contraction: the percentage of contraction of the effective diameter
compared to unseeded scaffold diameter measured at the same moment. Cell mediated
contraction per cell can also be evaluated by dividing the cell mediated contraction by the
average number of cells in each scaffold.
21
2.7 Histologyand Immunohistochemistry
After sacrifice, 6 discs for each time point (2 type I and 2 type II) were stored in 10%
buffered formalin (SF 100-20, Fischer Scientific Co.) for a period lasting from 2 to 10 weeks.
Specimens were then dehydrated and embedded manually in paraffin. Sections of 7pm were
cut through the horizontal plane of the scaffolds, and some were also cut through the vertical
plane (Microtome 2050 - Reichert Jung).
Sections were then stained with hematoxylin and eosin (see appendix) and Safranin-O.
Cell morphology, distribution, cell layers around the disc, collagen fiber location and shape,
and GAG distribution were observed through light microscopy (normal and polarized)
(Olympus Vanox-T). Slides were coverslipped and sealed with cytoseal 60 cell mounting
medium for hematoxylin and eosin stain and with permount for Safranin-O.
Immunohistochemistry for type II collagen was performed to confirm the absence of
type II collagen in the samples: previous studies had shown that the type I and type II
collagen matrices themselves did not stain positive if they had been cross-linked with the
EDAC. A positive control was also run to verify that the antibody was working. Staining
protocol used the ABC colorimetric staining method (protocol is detailed in appendices). To
assess the presence of collagen (non specific), Masson Trichrome staining was performed.
2.8 Total Collagen Content
Total collagen mass was evaluated by performing a hydroxyproline test on 3 samples
per time point per collagen type: One hundred L of the papain digestion was completed with
900pL of 6N HCl and dried overnight in covered glass tubes at 120 C. The acid hydrolyzed
sample was then transferred into a 12x75mm glass tube by mixing its content with distilled
water. The samples were then dried again. ImL of pH=6 buffer of dilution 1:10 was then
added, 500pL of Chloramine-T hydrate (98%, Sigma) was added while vortexing the samples
and the tubes were incubated at room temperature for 15 minutes. 500ptL of pDMAB
(Ehrlich's reagent, Sigma) solution was added and incubated at 60 C for 20 minutes. 200pL
of samples were read on a 96 well plate using a Perkin Elmer HTS 70000 plate reader.
Hydroxyproline concentrations were determined from a standard of hydroxyproline (Hy-Pro,
Sigma).
2.9 Total Protein Content
Total protein stored in the matrices was assayed using BCA assay (kit 323225 Pierce,
Rockford, IL). 25pL of each standard or sample was incubated with 200pL of BCA working
solution for 30 minutes at 37C on a 96 well plate. After incubation, samples were read using
a Perkin Elmer HTS 7000 plate reader at 570nm. Detail of the protocol can be found in the
appendix.
2.10 Cell seeding efficiency
P4 AF cells of one canine were seeded on type I and type II collagen discs. Type II
matrices synthesized by Geistlich Biomaterials were also seeded (Chondrocell Sponge;
Batch 019500; Geistlich Pharma AG; CH6 110 Wolhusen Switzerland).
For each matrix type and each density, 6 matrices were prepared using the same
methods as described above. For type I matrices, 1, 2 and 3 million cells were seeded on the
22
constructs, half of these amounts on each side of the matrices. For type II, 2 million cells
were also seeded half per side. Cultures was stopped at 1 and 3 days and DNA assays were
performed.
2.11 StatisticalAnalysis
Multi and single variable analysis of variance (ANOVA) or Student t-tests were used
to assess the effects of the total duration of the culture and of collagen type on all of the
measured variables. The Fisher least squares protected difference (LSPD) post-hoc test was
also utilized for selected analyses. StatView and Statistica were used to perform the analysis.
All tests were run with the assumption of equal standard deviations.
23
CHAPTER 3: RESULTS
Note: on all the following charts, error bars represent the standard error of the mean.
n=6 for each time point and each matrix type.
3.1 BiosyntheticActivity
Total protein and GAG biosynthesis continued through the 8-wk period of the
experiment for cells in the type I and II CG scaffolds (Figure 6 to 9). Two-factor ANOVA
revealed that there was no significant effect of the type of collagen from which the scaffold
was fabricated on the biosynthetic activity, but that there was a significant effect of time in
culture on biosynthesis (p<0.000 1; ANOVA). For both the type I and II scaffolds, the level of
protein synthesis increased from the I" to the 2 "d week and then again from the
6 th
to the
8
h
week (Figure 6, top). In contrast, the rate of GAG synthesis continuously increased to a
maximum at 4 weeks for cells for both types of scaffolds and then decreased to about 50% of
that level after 8 weeks (Figure 7). Normalized protein and GAG biosynthesis calculated per
cell followed the same tendency.
z.3
0 .25
-
0
.2-
E
=_.15 -
1 week
T
M.1-1
0
47
.05-
IY
0
---
h
2 w eeks
4 weeks
6 w eeks
8 weeks
II
Figure 6. Normalized rates of 311 incorporated per g of DNA, per hour.
.
Z .07
.06
0)
M
Zt
0
.05
E .04
1 week
2 w eeks
.03
0
U.)
C
4 weeks
.02
6 w eeks
8 w eeks
.01
0
II
Figure 7. Normalized rates of 35S incorporated per
g of DNA, per hour.
24
14
12
10
M
TI
-
1 week
8
E
0
2 weeks
6
4 w eeks
6 w eeks
4
8 weeks
C.)
LI)
-
2
0
Figure 8. Total rates of GAG biosynthesis over a 24 hour period, for type I and II seeded
matrices cultured for 8, 15, 29, 43 and 57 days.
45
40
E
35
30
E1 1 week
0
20
2 w eeks
4 w eeks
C.)
15
6 w eeks
0
25
8 w eeks
10
5
0
II
Figure 9. Total rates of protein biosynthesis over a 24 hour period, for type I and II seeded
matrices cultured for 8, 15, 29, 43 and 57 days.
3.3 Number of Cells
Type II matrices contained an average of 1.9 million cells whereas type I contained an
average of 1.6 million. This number decreased between 4 and 8 weeks from 1.9 million to 1.4
million for this matrix type. This phenomenon was not noticed for type II matrices for which
cell number remained nearly unchanged after the first week of culture, ceiling at about 2
million cells (Figure 10).
25
2500000
2250000
2000000
-
1750000
1500000
1250000
1000000
750000
0
E0
E
. 33
.E9
Ea
500000
250000
0
1 day
1 week
2 w eeks
4 w eeks
6 weeks
8 w eeks
Figure 10. Number of cells in the matrices for type I and type II matrices
3.4 GAG Content
GAG content was measured for samples in culture longer than 1 week. The
percentage of dry mass that is GAG, decreased slightly with time for both matrix types
(p=0.008; ANOVA) though the average mass of GAG contained in the matrix was relatively
steady around their average value. There was a significant effect of matrix type on total GAG
(p<0.0001; ANOVA) present in the constructs but not on percentage of mass that is GAG.
This percentage remained also steady for unseeded control matrices. Total GAG synthesized
was always higher for type II matrices than for type I. GAG content is shown on Figure 11.
Type I controls had an average of 50 g of GAG and type II 35 g after 8 weeks of culture.
3
2.5
2
E
0B
CU
1.5
1 week
0
1
2 w eeks
.5
4 w eeks
6 w eeks
8 w eeks
0
II
Figure 11. Percentage mass GAG / total Mass for type I and type II matrices
26
120
U)
E100
1week
-
E
2weeks
60
-
40
~4
weeks
O 6
weeks
8 weeks
20
.
A
0H
Figure 12. Total mass of GAG for type I and type II matrices
3.5 Aspect Ratio - Macroscopic Examination
Cell seeded matrices rapidly seemed like cartilaginous structures: white, shining and
resisting to forceps pressure. Unseeded scaffolds remained fragile and sponge looking during
the whole culture. They were also more transparent than cell seeded scaffolds.
Aspect ratios for matrix type I and type II differed significantly (ANOVA, p<0.001)
with type I matrices having a larger aspect ratio after 8 weeks than type II that remained just
slightly oval over all the experiment. Aspect ratio of type I matrices was furthermore less
controllable than the one of type II. This is confirmed by the higher standard deviation of
type I aspect ratio after 1 month of culture.
Mean Plot (matrk size data new 15v*1643c)
1.28
1.26
1.24
1.22
1.20
1.18
I1*12
114
1.10
1.08
1.06-
1.04
1.02
1.00 - - --10
0
10
20
Mai
Type:
30
Type I
40
s0
60
.10
0
10
20
30
40
50
60
Murk Type: Type
-e-Mean
I O.958SE
Figure 13. Aspect Ratio for type I and type II matrices
27
3.6 Scaffold Contraction
3.6.1 Overall Contraction
For the overall contraction of the matrix relative to the initial seeded matrix size,
contraction increased steadily during the culture reaching about 22% for type I and 15% for
type II. Average contraction per cell also increased with time. Resistance to cell mediated
contraction per cell for the constructs type II was much higher than the resistance of type I.
kn
Contracion of Marice. compared to thk
seeded mmbix tre for each naiml
26
24
122
-
I
Is
10
0
-10
14
40
20
20
00
00
-10
0
20
20
10
40
00
60
-~
o
MhbtrTyp.:Tai.M
MakdTyp.:Twpet
Figure 14. Overall contraction in percentage of initial seeded matrix size.
Cal
mactoted
Contraction
oompatud to tno~d
seeded malotr
ze
04-
2:tE
2E4
-
10
0
10
20
30
M" Tx oTV"iI
40
0
40-10
0
30
40
TpW.: T"p N
20
10
M
0
_Ma",
Figure 15. Overall contraction per cell in percentage of initial seeded matrix size.
3.6.2 Cell-Mediated Contraction
Cell-mediated contraction which compares seeded constructs and unseeded construct
size was, as expected, relatively low for these carbodiimide cross-linked matrices. Type II
scaffolds contracted from 4% to 10% after 8 weeks. Type I matrices appeared to contract less
with a maximum contraction of 6% after 8 weeks.
28
Contraction of th mabtcee compared to
conrol(t)
14
12
10
I
I
4
2
0
-2
-4
-8
0
-10
10
20
30
Mati Typo. Twin I
40
60
0
-10
0
10
20
S0
40
30
0
-ap- Mean
= ISE
MI 1Typa: Tpe It
Figure 16. Cell mediated contraction in percentage of unseeded control size.
Call mediated
contraction compared to contro eize
1.2E-6
I
I
I
1
I
1E-6
-
SE-7
--
6E-7
4E-7
-
2E-7
01
-2E-7
-4E-7-6E-7
0
-10
10
20
30
40
s0
60
--- Mean
-10
0
10
20
Matrk Type. Type
Matrix Type: Type I
40
30
00
60
It
Figure 17. Cell mediated contraction per cell in percentage of unseeded control size.
3.7 Dry Mass
Dry mass of the matrices increased with culture time for the seeded matrices
(p<0.0001; ANOVA). There was also a significant difference between the matrix types for
this parameter (p<0.0001; ANOVA). The mass of the type II matrices increased significantly
more than the mass of type I scaffolds.
7
6 1
5
E
.
-
-
4
1 day
3
I week
2 weeks
4 weeks
6w eeks
8 weeks
El
0
[]
U
E
aj 2
1
0
I
11
Figure 18. Dry weight of the samples for each type of matrix at different times of
culture.
29
3.8 Collagen Content
45
40
35
0)
0
0.
x
0
-o
I
1 day
10 1 week
30
2 w eeks
25
20
E
15
10
4 w eeks
6 weeks
8 w eeks
control
5
0
II
Figure 19. Collagen content contained in the type I and type II constructs for different
time periods. Unseeded scaffolds are represented on the right.
Matrix type did not have a significant effect on collagen content. Collagen content for
type I matrices at 1 week and 4 weeks appeared strangely high, but the deviation was also
high for type I at 4 weeks. The fact that only 3 samples were used per time point could be a
reason of these unexplained results. Type II matrices at 1 week also appeared to generate a
non sensible result with a collagen content value under the content of the control matrices.
Overall, collagen content was higher in the cell-seeded matrices than in the control matrices,
but not statistically significant (see figure above).
3.9 Protein Content
3500 3000
-
2j 2500
-
E)
1 day
1 week
En
2 w eeks
4 weeks
6 w eeks
8 w eeks
U
control
2000 .
0o 1500 -
1000 500
-
0
II
Figure 20. Protein content of the type I and type II constructs for different times
of culture. Unseeded constructs are represented on the right.
Matrix type did not have a significant effect on the protein content of the samples.
Cell-seeded samples did not have statistically significant higher protein content than
unseeded controls as can be observed in Figure 20. Protein content decreased slightly
throughout the culture period (ANOVA; p=0.006).
30
3.10 Histologyand Immunohistochemistry
Collagen type II immunohistochemistry was negative, indicating that cells were not
producing collagen type II collagen in the type I or type II scaffolds. Comparison of matrices
between 1 week and 4 weeks of culture for example, shows clearly that tissue has been
synthesized and shows cells grouped on the sides of the scaffold (Figure 21). It appeared to
be the case for both matrix types. More tissue seemed to have been synthesized for type II
than for type I (Figure 23). Figure 27 shows a longitudinal cut of a type I matrix after 8 weeks
of culture: the scaffold is clearly oval and many cells are present. Birefringence of the
collagen fibers can be observed (Figure 25) indicating that the collagen fibrils are highly
organized similar to the architecture found in vivo and wavy-crimp like structures are also
observable at high magnification (Figure 26).
Figure 22 and Figure 24 show very precisely the structure of the matrix around cells
and the elongated shape of the cells that attach to the matrix.
Masson trichrome staining was performed to assess the presence of collagen, in
parallel with hydroxyproline assay. Masson trichrome staining revealed the presence of
newly synthesized collagen in the matrices. More collagen appeared to be present in type II
matrices and collagen content seemed to be more prevalent in longer times of culture at least
in the periphery of the matrices (Figure 31). Also in Figure 31, most collagen appeared to be
on the periphery of groups of cells, showing collagen synthesis by the cells. Besides, collagen
seemed to be diffusing towards the inside of the matrix and did not only remain close to the
cells. After 1 week of culture, collagen appeared evenly spread in the matrix (Figure 32 and
Figure 33). While collagen was also always present in the interior pores of the matrices, it
was not the case for GAG: Safranin-O staining revealed that GAG was principally located
next to the sides of the matrices mostly surrounding cells (Figure 30). For short times of
culture however, GAG appeared evenly spread (Figure 28, 29 and 30).
31
Immunohistochemistry type H
0.5mm
O.5mm-
Figure 21. Type I collagen-GAG matrices after 1 week (left) and 4 weeks (right). Newly
synthesized tissue at 4 weeks is apparent.
A
O.1mm
WI1
Figre22 Deaiedvie
o te isie f tpeH ollge mtriesafer4;wek.o
I-
NW
41
Figure 22. Detailed view of the inside of type II collagen matrices after 4 weeks of
culture (left) and view on the side of the matrix after 6 weeks (right)
Ilk
Figure 23. Type U collagen matrices after 6
Figure 24. detail of 2 cells attached to a type
weeks. Notice the formation of new tissue
H collagen GAG scaffold at 4 weeks.
and the groups of cells on the sides
32
H&E
0.2 mm
Figure 25. Type H matrices after 8 weeks under normal (left) and polarized (right) light
showing the organization and birefringence of the collagen fibers.
Figure 27. General view of a type I matrix
at 8 weeks. Scaffold appears oval and
irregular in diameter.
Figure 26. Wavy crimp-like structure after
8 weeks, type H.
33
Safranin-O
441
7 r
500 microns
500 microns
Figure 28. General view and GAG localization for type I (left) and type II (right)
matrices after 1 week.
Ic
50 microns
Figure 29. Zooms on reddish structure (left) that could be a cell surrounded by
proteoglycans in type I matrix after 4 weeks, and GAG structure located on side of a
type H matrix after 6 weeks of culture (right).
0.1mm
0.1mm
Figure 30. Matrix type I (left) and type H (right) sides after respectively 4 and 6 weeks
of culture. GAG is mostly located on the sides.
34
Masson trichrome
0.2mm
Figure 31. group of cells after 6weeks of culture of a type I matrix (left). Also after 6
weeks, type I matrix (right) presents its very a quite evenly spread collagen content
(right).
50 microns
Figure 32. Zoom on type I matrix after 6 weeks showing cells producing collagen (left)
and evenly spread collagen inside a matrix type II after 6 weeks.
0.2mm,
0.1mm
muj,
-O
%
Figure 33. type I collagen matrix after 4 weeks (left) and type H collagen matrix with
little collagen (right) after 1 week of culture.
35
CHAPTER 4: DISCUSSION
4.1 Relevance of Results : Effect of collagen type on the behavior of
cell seeded matrices
Findings confirmed the viability of the AF cells in both types of scaffolds over a
culture period of 8 week. Cells in both matrix types synthesized GAG and proteins and even
though matrix type did not appear to be significant for these parameters, it appeared
important for cell number and contraction/deterioration of the scaffolds: despite
contraction/deterioration continuously increasing during the culture, cell number remained
constant for type II matrices showing a higher density of the cells inside the scaffolds. Type I
matrices saw their number of cells decrease, which could be due partly to the fact that
contraction/degradation was higher for that matrix type.
Hypotheses that were formulated at the beginning of the thesis can now be addressed:
- Hypothesis 1 is accepted in that the total GAG mass contained in type II
matrices was higher than in type I. For total collagen, the difference was not
statistically significant, though it appeared that collagen content was slightly
higher. One of the challenges in assessing the amount of newly synthesized
collagen was due to the fact that collagen contained in the matrices prior to
seeding was much higher than the amount of cell-synthesized collagen.
- Hypothesis 2 was rejected: the number of cells in the constructs did not
decrease with time at least for the first 4 weeks of culture. After 6 weeks, number
of cells contained in type I collagen matrices decreased.
- Hypothesis 3 was accepted: 2 million cells appeared to be a very efficient
seeding density. For type II matrices for example, the number of cells remained
steady around 2 million cells after the first week of culture.
- Hypothesis 4 was also accepted as cell-seeded contraction of the
cardodiimide-cross-linked scaffolds was not very high.
The reasons for the sudden increase in protein synthesis rate at 8 weeks are unclear, as
well as GAG synthesis rate decrease from 6 to 8 weeks: these variations could be explained
by the fact that the most active cells for protein synthesis may be in the center of the scaffold
whereas the most active cells for GAG synthesis may lie on the sides of the scaffold. Indeed,
as the scaffold degrades with time, particularly after 8 weeks of culture, cells which are on
the sides may detach from the scaffold as it degrades and the cells that remain would be more
active in protein synthesis than in GAG synthesis. This was partly observed through the
Safranin-O-stained slides which showed that most GAG was located on the surfaces of the
matrices where many cells were present, whereas Masson trichrome-stained slides showed
considerable collagen in the center of the matrices. Degradation of the scaffolds would appear
as a critical factor in that case.
As expected from previous works [15], little contraction with time was measured for
these EDAC cross-linked matrices, but contraction never actually stopped.
36
4.1.1 GAG content and synthesis rate
GAG content for type I remained relatively steady. For type II it increased slightly but
not significantly. However GAG synthesis rate was positive and even increased during the
first month of the experiment as radiolabeled sulfate incorporation showed. GAG content was
also higher for cell seeded matrices than for unseeded controls. The fact that GAG content
did not increase significantly shows that either newly synthesized GAG, either old GAG
present in the matrices is lost in the medium. In in situ IVD, proteoglycans are constantly
being turned over although this turnover is slow. The fact that incorporated sulfate rate was
positive shows that after a 24 hour period, proteoglycans had attached to the matrix, and most
probably that the old GAG content was ejected in the medium.
For unseeded matrices, GAG content was higher for type I matrices than for type II
(50 g against 35 g). For seeded matrices, GAG content was relatively identical in both
matrix types. This could come from the fact that physical properties of both matrix types are
nearly identical: wall thickness, pore density, dimensions before hydration. Therefore, total
synthesized GAG would reach the same limit in both matrix types.
4.1.2 DNA content
DNA content was significantly different for type I and type II matrices, and even if
porous size was of comparable sizes, there were slight differences: porous size was of 120 m
for type I and about 200 m for type II matrices [12]. Furthermore, wall thickness was also
larger for type II matrices.
Seeding efficiency which is detailed in the appendix showed that type II seeded with 2
million cells had the highest seeding efficiency, at around 90%, where type I has about 60%.
Furthermore type II matrices had a very regular number of cells all along the culture, which
was higher of 20% of the number of cells present in type I matrices which also decreased
after 1 month of culture. Decreasing DNA content after 4 weeks of culture which had been
observed by other studies [16, 17] for AF cells and chondrocytes was thus not observed. It
can be assumed that initial cell attachment efficiency was partly responsible for the higher
number of cells observed in type II matrices. This fact could let us think that cells in type II
collagen scaffolds produce more integrin to allow them to attach to the scaffold than cells in
scaffolds that have been fabricated from type I collagen. Other parameters may also influence
the number of cells in the matrices, such as the porous size which was relatively different for
both matrix types (120±18gm for type I and 202±51gm for type II).
4.1.3 Protein content and protein synthesis rate
Protein synthesis rate increased from 1 to 2 weeks and then from 6 to 8 weeks.
However; total protein contained in the samples decreased slightly with time as was
measured with BCA assay, showing that some of the protein content passed did not stay
inside the matrix. Matrices are very porous and the fact that protein may diffuse out of the
scaffold is not surprising. Collagen type did not appear to be significant in the synthesis rate
of proteins or on the protein content, showing that collagen type does not appear to influence
protein production in vitro.
The result was the same for GAG content for which collagen type was not influencing
GAG synthesis. Other cues like mechanical loading or presence of growth factors could
influence protein or GAG synthesis and should be investigated in future studies.
37
4.1.4 Collagen content
As demonstrated with type II immunohistochemistry, there was no type II collagen
produced by the cells: immunohistochemistry revealed to be negative for type II collagen.
Inherent type I or type II collagen that makes up the matrices does not respond to type I or
type II immunohistochemistry as was already noticed by previous studies [15]. Masson
trichrome stain, which stains collagen non specifically was positive on the cell seeded
samples and negative on the controls. This result shows that collagen was synthesized and as
type II immunohistochemistry was negative, it is judicious to think that type I collagen was
synthesized by the cells, behavior which is expected from annulus fibrosus cells which
mainly produce type I collagen. Type II collagen is however present in annulus fibrosus extra
cellular matrix, but it could be due to diffusion of collagen from the nucleus pulposus, which
is mainly made of type II collagen. Furthermore, hydroxyproline assay confirmed the
presence of collagen synthesized by the cells.
4.1.5 Contraction, Deterioration and Aspect ratio
If EDAC cross-linked matrices have been the types of matrices that contract the less
compared to other types of cross-linking (UV, DHT, GTA) [15], overall contraction was still
an important factor reaching 25% for type I and 15% for type II after 8 weeks of culture. It is
not obvious to see what part of the contraction is due to the cells and what part is due to
natural degeneration of the collagen sponge in the medium. For that reason, observing
contraction compared to control size is interesting to evaluate contraction due to cells. Cell
mediated contraction was evaluated to be continuously increasing to reach 6% for type I
matrices and 10% for type II matrices. Therefore, overall cell mediated contraction for type I
matrices was lower than the one of type II matrices. However, this result is not entirely
correct as cell seeded matrices appeared larger than controls during the first weeks of culture,
which could have been due to the fact that punching of the discs out of the matrices is often
uneven and during the cross-linking of the matrices, some parts of the matrices can be torn
out. Matrices that were selected for seeding were the ones that were the best looking in terms
of regularity and circularity.
As type II matrices contained more cells than type I, evaluating cell mediated
contraction per cell or per unit mass of DNA is also necessary, and results showed that in
type I matrices, contraction per cell continuously increased whereas contraction remained
relatively steady for type II matrices.
Another noticeable phenomenon was that the sponges did not contract
circumferentially. It was already noticed in other studies [18, 19], and it could be due to
microscopic irregularities which emphasize one axis, for example if pores are elliptic. An
analysis of the pores in that case could offer a better understanding.
4.1.6 Dry Mass
Dry Mass continuously increased in the constructs proving also that the cells in the
matrices were living. Other studies have seen the weight of control unseeded matrices
decrease with culture due to matrix degradation. If type I matrices had a weight increase of
less than 20%, type II discs weights increased of 60%. This large difference cannot be
explained by the biosynthesis rates which were nearly equivalent for both matrix types, as
well as GAG content. Difference could come from a difference of protein content,
38
particularly collagen content. However, BCA test and hydroxyproline assays showed that
global content in protein did not increase throughout the time.
4.2 Comparison with Other Literature Results
There have been few similar experiments evaluating the behavior of canine IVD AF
cells in collagen sponges. One work of interest [12] evaluated the behavior of P4 cells in
several matrix types: collagen type I, collagen type II and a mix of collagen type I and type II.
That experiment was run with 3 time points: 2 days, 2 weeks and 4 weeks. Methods that were
involved were the same as the ones of our experiment except for type I matrix in this study
produced using a temperature ramping protocol. The pore diameters of matrices for both
collagen types were evaluated as being comparable.
As for the results, differences with prior work [12] were observed on some variables:
- Total protein synthesis rate: prior work obtained no variation of that parameter
throughout the 4 week culture, the total incorporated proline staying around 0.06-0.08
nmol/hour/ g DNA. In our study, protein synthesis rate increased from 0.07 to 0.1
nmol/hour/ g DNA between 2 days and 4 weeks and increased again to reach around
0.2 nmol/hour/ g DNA after 8 weeks of culture for both matrix types.
- GAG synthesis rate: an increase of GAG synthesis rate was noticed by [12] in which
incorporated sulfate rate started from 0.02 to reach 0.03 nmol/hour/ g DNA. In our
work, the same increase was evaluated during the first 4 weeks of culture, though with
a higher range: incorporated sulfate rate started 0.02 to reach 0.06 after 4 weeks of
culture to finally decrease to a value between 0.03 and 0.04 nmol/hour/ g DNA.
- Decrease of the number of cells contained in the matrix was noted after 4 weeks of
culture in [12]. That was not the case in findings of this paper for which cell number
remained relatively steady from 1 to 8 weeks of culture for type II matrices and
started to decrease only after 6 weeks of culture for type I.
- Percentage of dry mass that is GAG was lower in our study than in [12]. Our work
measured a percentage comprised between 2 and 2.5% slightly decreasing with time
and prior work measured a percentage comprised between 5 and 7% also decreasing
with time. Type II matrices seemed to see this percentage decrease more with time
than type I which was also noticed in [12].
These differences could be explained by the fact that cell passage number was different in
both studies (P2 and P4).
Some variables presented identical results:
- Type II matrices had 20% of cells more than in type I in our experiment involving P2
cells. Prior work [12] obtained about the same ratio by using P4 cells.
- Sample mass was nearly identical in both studies with type II matrices mass
increasing much more than type I matrices.
- Aspect ratio and contraction were also not very high in both works.
Comparison with in situ intervertebral disc can also be interesting:
- Proteoglycan synthesis rate in the human intervertebral disc is comprised between 10
and 60 nmol/hour/ g DNA, whereas findings of this paper for canine annulus fibrosus
cells seeded in collagen sponges measured 0.06nmol/hour/ g DNA.
- Canine annulus fibrosus cell density was evaluated to be 15.000cells per mm 3. With
around 1.7 to 2 million cells in a disc of diameter around 0.7-0.8mm of thickness 2-
39
3mm, cell density in the collagen-GAG scaffold is evaluated around 5000 cells per
mm 3. Seeding of 2 million cells is equivalent to a density of about 2500 cells per mm 3 .
It can also be pointed that average cell density for the human inner annulus was
evaluated at around 4300 cells/mm 3 [13] and another study reports 9000 cells/mm 3
[14]
4.3 Limitations
Influence of cell passage number could be important and studied more systematically.
Even if this research has been done with P2 cells and a similar study with P4 cells,
comparison of results is difficult to realize as experimenters have been different. All that we
can say is that some parameters seemed to be higher in our research, such as biosynthesis rate,
DNA content, but others seemed lower like GAG content.
Tests of the phenotype, for example through collagen typing lacks a quantitative or
more reliable assay than simple immunohistochemistry or hydroxyproline test, the results of
which interfere with the collagen content that makes up the matrices. A type I and type II
western blot coupled could bring information on the behavior of the cells within the scaffold.
If culture has been realized through an 8 week period, clinically, such a long period of
time is unacceptable and an important question is how to accelerate the multiplication of the
cells and increase the viability of the scaffold.
No mechanical testing has been realized on the scaffolds in this research, but other
studies like [15, 12] have already demonstrated that it can play an important role in
stimulating the biosynthetic activity of articular chondrocyte [39] or generate a specific
response with annulus fibrosus cells [38].
4.4 Significanceand Future Work
The results of this thesis commend an AF cell seeded type II collagen-GAG matrix for
further study for IVD tissue engineering.
Future work should involve the incorporation of growth factors or genes that induce
the synthesis of growth factors. Seeding scaffolds using bone marrow stem cells would also
be useful to do as stromal cells are much easier to harvest than IVD cells, which would be
better from a clinical perspective. Comparing the static culture with the culture in a dynamic
environment (e.g., in a "bioreactor" or in culture chambers involving some mechanical
vibrations) could also further improve the behavior of the cell-seeded scaffolds. Some studies
have already found that the use of a bioreactor can provide a better viability of constructs [36,
37]. Finally, improving the constructs properties such as contraction and degradation would
be important, as well as determining the influence of other physical and biochemical factors
of the scaffold including initial GAG concentration, pore size, and matrix thickness. An in
vivo model using an AF cell-seeded type II collagen-GAG scaffold could also be advised to
compare the behavior of in vivo and in vitro matrices, although it appears that other
parameters should first be evaluated in vitro (growth factors, genes).
40
CHAPTER 5: CONCLUSION
AF cells seeded in type I and type II collagen-GAG scaffolds continue their
biosynthetic activity during a period of 8 weeks in culture although deterioration and
contraction of the scaffolds are clear. Tissue synthesis inside the scaffolds is apparent. There
is no significant effect of collagen type (from which the matrix is synthesized) on protein and
GAG synthesis by AF cells. A type II collagen-GAG scaffold is commended for further
study on the basis of its maintenance of cell number and the slightly higher accumulated
GAG content.
41
APPENDICES
42
APPENDIX A - Seeding efficiencyexperiment
Objective
The objective of this experiment was to evaluate the best seeding density on 9mm diameter,
3-4mm thickness type I and type II scaffolds for canine IVD annulus fibrosus cells. Best
seeding density was defined as the seeding density with highest number of cells still
remaining in the scaffold after 1 and 3 days of culture for both types of matrices. Therefore,
the ratio:
number of cells (t) / initial seeded number of cells,
is evaluated and gives a percentage of the number of cells that have attached to the scaffold
and thus an evaluation of the seeding efficiency.
Experimental Plan, Material & Methods
Number of cells was measured in the matrices through a DNA assay, for samples
sacrificed at t=24 hours and t=72 hours. 36 type I and 12 type II discs were prepared. 12 other
type II matrices were obtained by Gastlich (Chondrocell sponge, Batch 019500, Geistlich
Pharma AG, Switzerland). P4 Cells were used for this experiment. For type I matrices, 12
discs were seeded with totally 1 million cells, 12 others with 2 millions and the last 12 with 3
million cells. The type II Gastlich matrices were seeded with 2 million cells as well as the
own made type II matrices. Results obtained in the experiment described in the thesis for own
made type II matrices confirm a posteriori that it was not necessary to test the other seeded
densities of 1 and 3 million for that matrix type.
Seeded density
Matrix type
Number of seeded
matrices
Number of time points
1 million
Type I
12
TypeI
12
2 million
Type II
Geistlich type II
12
12
3 million
Type I
12
2
2
2
2
2
Table 4. Number of seeded matrices that were used for the seeding efficiency
experiment
Materials and Methods involved for this experiment were exactly similar to the
section Material and Methods of the thesis. Type I matrices that were used were made
without the temperature ramping protocol. See Matrix making protocol for more detail.
43
Results
2500000
I.
T
2250000 2000000 1750000 "
a,
1500000 -
0
1250000
E
1000000 -
z
750000 500000
-
250000
-
0-
1 million
2 million
5
U
0
Type 1,1 day
Type 1,3 days
U
U
Type 11, 3 days
Type I - Geistlich, I day
U
Type
Type 11, 1 day
II - Geistlich, 3 days
3 million
Figure 34. Number of cells in the constructs after 1 and 3 days of culture for type I and
type II cell seeded matrices with different cell seeding densities.
For type I matrices, after 1 day of culture, number of cells that had attached was:
- 62% of the initially seeded cells for 1 million initially seeded cells,
- 60% of the initially seeded cells for 2 million initially seeded cells,
- 44% of the initially seeded cells for 3 million initially seeded cells.
Seeding density was not really significant on seeding efficiency for type I matrices with
seeding densities of 2 million and 3 million (P=0.069; Fischer Adhoc). Time was significant
on seeding efficiency only for seeding densities of 2 million cells for which number of cells
dropped from 62% to 51% between 1 and 3 days of culture.
For type II own made matrices, after 1 day of culture, number of cells that had attached to
type II matrices was 89% of the initially seeded 2 million cells. Number of cells was already
higher after 3 days of culture than after 1 day. This was not observed with type I matrices
during the same period for which number of cells either decreased or did not change.
Geistlich type II matrices presented after 1 day of culture a very high attachment
efficiency, near 100%. After 3 days of culture, that figure was only of 61% of the initial
number of cells which were present in the scaffolds.
1 Million
Seeded
3 Million
2 Million
density
Time of
culture
1 day
3 days
1 day
3 days
I day
3 days
Type I
73%
73%
44%
43%
62%
51%
TypeII
89%
97%
Geistlich
110%
61%
Type II
Table 5. Number of cells compared to initial seeded density versus time for 3 matrix
types.
44
Discussion & Conclusion
Type II scaffolds presented a very high attachment rate at 2 million cells. Probably the
slightly higher porous size and larger wall thickness contributed to preventing the cells to
come out of the scaffolds and go into the medium. Cell pipetting technique appears to be an
efficient technique with good seeding efficiencies. Higher cell densities, like 3 million cells,
appeared to be unuseful for cell attachment as about the same amount of cells attached be the
scaffolds seeded with 2 or 3 million cells. The fact that attachment rate decreased between 1
and 3 days, except for type II matrices seeded with 2 million cells, indicates that the cells that
have passed in the medium after one day are less likely to diffuse inside the scaffolds. To
increase cell attachment ratio, rotating the matrices horizontally could help force the cells
migrate to the center of rotation where the scaffold would be. However, current attachment
ratios are satisfying.
45
APPENDIX B - Evaluation of the DNA mass of canine IVD annulus
fibrosus cell
Objective
Objective of this experiment was to have an estimate of the DNA mass of canine IVD
annulus fibrosus cells. Value that was used in the past was a reported value of 7.7pg of DNA
but the result was established for chondrocytes [35].
Experimental Plan, Material & Methods
P2 and P4 cells of 2 different canine specimens were used for the experiment. P2 cells
were obtained from freshly isolated IVD cells that had been frozen after one culture in flask.
P4 cells were obtained from freshly isolated IVD cells passaged 3 times in monolayer
cultures before being frozen. The 2 animal cells were always kept separately during the
whole experiment. Confluent cells were trypsinized out of the flasks, centrifuged 2 times and
counted using a hemacytometer (see protocol in appendices). The pellet was then
resuspended at a concentration of 20 L per 1 million cells. Aliquots of different volumes
were then taken and put into microcentrifuge tubes for papain digestion. Volumes that were
aliquoted were corresponding to number of cells of: 250.000, 500.000, 1 million, 1.5 million,
2 million, 3 million and 4 million. Volume was completed with papain buffer and 5 L of
papain, before being digested overnight at 60 C. DNA assay was performed using Hoechst
protocol and DNA mass was extrapolated from the standard curve of calf thymus DNA.
Knowing the number of cells in each aliquot, it was possible to determine the mass of DNA
per cell.
Results
Quantitative results are summed up in the table below:
P2 cells
P4 cells
n
7
4
Average DNA mass per cell
3.53pg
3.50pg
Std Error
0.14
0.32
Std Deviation
0.37
0.65
Table 6. Mass of DNA per annulus fibrosus cell.
One value of the samples of the P2 cells appeared quite different from the other ones.
In case that value would not be considered, average DNA mass per cell would be of 3.61pg
with a standard error of 0.1 lpg.
Discussion & Conclusion
An average value comprised between 3.5 and 3.6pg of DNA per cell seems to be
adequate to evaluate the number of cells when knowing the DNA mass through fluorometric
assay for example.
A small volume of medium which originally contained the cells also counted in the
DNA assay. It should be necessary to have a control made of medium only in the next
verification and subtract the DNA mass contained in the control from the DNA mass
contained in the samples.
46
APPENDIX C - Protocols
C-1 Annulus Digestion
(Modified from Hastreiter.)
Equipment for Annulus Digestion
-
70 pm nylon cell strainers (sterile)
Pasteur pipettes (autoclaved)
micropipette tips with extenders (autoclaved)
micropipetter
4% trypan blue solution
50 mL centrifuge tubes
complete medium (20% FBS)
-
Collagenase (355 U/mg, type IA; #C9891, Sigma)
D-PBS
trypsin (0.05%) + EDTA (0.53 mM) 4Na
Annulus Digestion
1. Enzymatic digestion must take place no longer than 4-6 hours following harvest. The
annulus tissue that has been harvested and taken into small chunks of about lmmA3 are
placed in 50 mL centrifuge tubes with just enough PBS to wash the explants.
2. These are spun for 1-2 min. The PBS in removed and the tissue is rinsed twice more with
complete PBS in the same manner.
3. For 5 lumbar discs, two 50 mL centrifuge tubes are filled with 50 mL of trypsin-EDTA.
They should be weighed right away, prior to use and after the tissue has been added so that
how much mass of tissue has been digested can be measured. The tissue is then transferred to
the trypsin tubes.
4. Once all the pieces are put into the tubes, the tubes are fixed with tape to a shaker in the
incubator at 37 C and 5% C02 After ihour and 30 minutes the shaker and the tubes are
taken out of the incubator.
5. During incubation in the trypsin, the collagenase solution is prepared. 100 mL of solution
re-quires 99 mL of medium, 1 mL of antibiotic solution (Gibco-BRL No. 15240-096, Life
Technologies), and 0.3 to 0.4 g of collagenase. The collagenase solution should be stored at
40 C until needed.
6. The trypsin is then sucked out of the tubes carefully with a pipette and the tissue is
washed in the tubes with PBS 3 times using the centrifuge.
7. 50 mL of collagenase solution is now added to each tube, and the filled tubes are again
fixed to the shaker and put into the incubator for another 2 and a half hours. The solution
should now have lost some of its transparency and the visible tissue volume should be
reduced to approximately 1/3.
8. The whole solution is then filtered through sterile 70 gm cell strainers in new 50 mL
centrifuge tubes. Residual tissue is kept and spread into two 75cmA2 cell flasks. The rest of
the solution is centrifuged at room temperature for 10 minutes at 1500 rpm. During that time,
5mL of complete medium is added in the flasks containing the small explants.
9. The supernatant is decanted and the pellet is resuspended in 20-25 mL of complete
medium. This procedure is repeated once more. At some point, the tubes are combined. 100
47
pL is removed from the cellular solution for cell counting. After the last centrifugation, the
pellet is resuspended at a concentration which suits the desired concentration for culture or
freezing.
2
10. For 75 cm tissue culture flasks, the digested cells should be plated at 2 million cells per
flask (instead of 1 million usually for already passaged cells). Medium should be changed
when most of the cells have adhered and only dead cells are floating in the medium. This
usually happens after 4-5 days, but cells may need up to 7 days before medium change for
adherence. Do not wait longer than this as the dead cells will release mediators which inhibit
cell proliferation. After 2 to 3 days, it's possible to add 1 OmL of medium to the explants and
even to transfer the explants into new flasks for even higher yield.
48
C-2 Freezing Cells
(From Hastreiter.)
General
For freezing 6 million cells in each 5 mL cryogenic tube.
Materials
Complete Medium
DMSO (Dimethyl sulfoxide): Autoclaved in light-proof bottle prior to use.
Sterile Filter
Pipettes
Sterile cryogenic tubes
Methods
1.
Determine amount of medium needed (1 mL per 2x106 cells) and add it to cells in 50
mL centrifuge tube.
2.
Add 10% DMSO (i.e., if there is 15 mL of cell/medium suspension in 50 mL tube,
add 1.5 mL of DMSO.)
3.
Store in cryogenic tubes (3.3 ml per 5 ml tube).
4.
Place in the -20*C freezer for 2-4 hours (longer time in this range is preferable), then
transfer to -70*C for storage.
49
C-3 Thawing Cells
(Modified from Hastreiter.)
Materials
Complete medium
Tissue culture flasks
Aspirating pipettes
Vacuum suction
Flame
Sterile pipettes
Pipette Aid
15 ml tubes
Methods
1.
Place cells directly into a 37'C water bath. Agitate gingerly while cells thaw for 4060 seconds.
2.
Slowly add warm medium to the tube until it is full. This insures that the cells thaw
into the medium. Close the cap and agitate up and down until all the content is thawed.
3.
Transfer the cells to a 50 mL tube. Add more warm medium. Wash them clean of
medium + DMSO for 10 minutes in the centrifuge.
5.
Count the cells, and resuspend at the proper concentration. For 75 cm 2 tissue culture
flasks, the thawed annulus cells should be plated at 1 million cells per flask. Cells should be
cultured at least 3-4 days before being used for experimentation (or before changing medium,
depending on when they attach).
50
C-4 Passaging Cells
(From Hastreiter.)
Materials
Complete medium
Trypsin
PBS (Phosphate-buffered saline)
Glass and sterile plastic pipettes
Vacuum setup
Centrifuge tubes
Tissue culture flasks
Methods
1. Warm the medium, trypsin, and PBS in 370 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) to removal
any residual medium. Trypsin will not detach the cells if it has come into contact with the
medium.
4. Remove PBS and add trypsin/EDTA (0.5 ml per well of 6 well plate, 2 ml for 25 cm 2 flask,
5 ml for 75 cm2 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 cm2 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. 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.
For 75 cm2 culture flasks, the passaged annulus cells should be plated at 0.75 x 106 cells per
flask.
51
C-5 Cell counting
(From Hastreiter.)
Materials
Complete medium
Trypan Blue
Cell counting slide
Pipette Aid
Micropipetters
Pipette tips
70% ethanol
Calculator
Cell counter
Methods
1.
Clean surface of hemocytometer and coverslip with 70% alcohol.
2.
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 pL sample from the cell suspension and put in microcentrifuge tube.
Dilute with 100 IAL of trypan blue.
5.
Mix well, and collect 15 gL of suspension in a micropipette tip.
6.
Load the cell suspension into the hemocytometer, 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.
7.
Place hemocytometer on microscope stage, remove yellow glass filter, and view with
standard loX objective.
8.
Count cells in each of the four corner and central squares (clear cells are viable, blue
stained cells are dead) (Figure 35). 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. When a count of living cells is
complet, count the number of dead cells in order to report viability.
9.
Calculate total cell number from the following:
T-
N
cx Dx 104 xV
T = Total number of cells in suspension
Nc = Number of cells counted
Ns = Number of squares counte d
D = Dilution factor, usually 2
V = Volume of media used to suspend cell pellet
The number 104 is the volume correction factor for the hemocytometer:
mm and the depth is 0.1 mm.
10.
Equationi1
each square is lxi
Cell viability is calculated by:
# dead*
I - # alive * 100%
Equation 2
52
coverslip
grid
load cell
suspension
mm
F r3 H er
y
Ig
1mm
g
Figure 35. Hemocytometer Counting Diagram
53
C-6 Medium preparation
(From Hastreiter.)
General
The following steps should be performed under sterile conditions. These directions are for
preparing 500 mL of complete cell culture medium (DMEM/F12, 10-20% FBS, 1%
antibiotics, 1% L-glutamine, and 25 pg/ml of ascorbic acid).
Materials
DMEM/F12 medium (#11320-033, Life Technologies)
Heat Inactivated FBS (Austrailian FBS; #SH30084.03, Hyclone Laboratories)
Antibiotic-Antimycotic solution (Gibco-BRL No. 15240-096, Life Technologies)
L-glutamine (#25030-081, Life Technologies)
L ascorbic acid phosphate (magnesium slat n-hydrate; #D13-12061, Wako Chemicals USA,
Inc.)
Sterile glass bottle
Pipettes
Pipette Aid
Flame
Methods
1. Thaw all frozen and refrigerated solutions for 30 minutes in a 370 C water bath.
2. Remove 70 or 120 mL (for 10 or 20% FBS medium, respectively) of DMEM/F12 from
the 500 mL bottle and place into another sterile container for storage and later use.
3. Add 50 or 100 mL of heat inactivated FBS for 10 or 20% FBS medium, respectively.
4. Add 5 mL of antibiotics.
5. Add 10 mL of ascorbic acid solution.
6. Add 5 mL of L-glutamine.
Complete medium is good for 2 weeks (due to 25% inactivation of ascorbic acid after this
time). Do not prepare more medium than you think you will use during that time period.
Heat Inactivation of FBS
1. Turn on the 56'C water bath to warm up.
2. Remove FBS from -20*C freezer (downstairs).
3. Place bottles in 56*C water bath. Once thawed, leave for an additional 30 minutes (60
minutes for 500 ml bottles), shaking every 10 minutes.
Ascorbic Acid Solution
54
1. Weigh out 0.0125 g of ascorbic acid for every 10 mL of ascorbic acid solution to be
prepared. Since you can prepare in advance and store frozen until needed it is easiest to make
a larger amount, such as 100 mL.
2. For 100 mL, weigh out 0.125 g ascorbic acid and add 100 mL incomplete DMEM/F12.
3. To sterilize, filter through a 0.45 pm sterile filter.
4. Aliquot into 15 mL tubes in 10 mL aliquots and store in -20'C freezer.
55
C-7 Medium changing
(From Hastreiter.)
Materials
Complete Medium
Pipettes
Flame
Vacuum Pipettes
Vacuum setup
Methods
1. Remove the medium from the culture dishes or flasks using the glass vacuum pipettes (use
long pipettes for flasks, short pipettes for well plates). Make sure to use a new pipette for
each sample from different animals.
2. Replace the media according to the type of culture dish you are using.
75 cm 2 flasks - 15 ml
25 cm 2 flasks - 5 ml
6 well plate - 2-4 ml per well
You can use the same pipette as long as you do not touch the flasks. If you think you might
have contaminated it at all, use a new one. Be sure to switch pipettes for samples from
different animals.
3. Quickly flame caps and lids before putting them back on to ensure they remain sterile.
4. Amounts can change depending on type of cells and desired culture conditions. If you are
changing media for someone and you do not know how much they added, draw up the
medium in the flask with a plastic pipette to measure it first.
56
C-8 Matrix Making
(From Hastreiter.)
C-8-1 Type I
C-8-l.a old protocol
Slurry making
1. Cool blenders to 4' C (takes at least 30 min.) using directions on the wall. Steps 2-4 and 6
can be done while waiting. Step 1 of the freeze-drying protocol should also be performed if
freeze-drying immediately.
2. Prepare 0.05 M acetic acid if unavailable:
17.4 ml glacial acetic acid + enough distilled water to make 6 L = 6 L of 0.05 M acetic acid
Glacial acid is in the cabinet across from the blenders labeled "acids."
3. Fill the blender with 600 ml of 0.05 M acetic acid. One blender gives enough slurry for 3
sections of 1 freeze-dryer tray.
4. Weigh 3.6 g of dry tendon collagen (kept in the refrigerator). Use right scale.
5. Place collagen in the blender and blend on high for 90 min.
6. Mix 0.32 g chondroitin 6-sulfate (in dessicator in the refrigerator) in 120 ml of 0.05 M
acetic acid with magnetic stirrer. Use the left scale for weighing.
7. Add 120 ml of chondroitin 6-sulfate solution over 15 min using the peristaltic pump.
Make sure the switch is on "reverse."
8. After the addition, blend the mixture for an additional 90 min. on high.
9. Pour out slurry and refrigerate if not freeze-drying immediately. Slurry can be used for up
to one month after making. If longer than a week after making, reblend for 15-30 min. Clean
the blender with 0.05 M acetic acid.
10. De-gas the slurry with a vacuum flask for 10-30 min. (latter time for the current
machine). Clean the vacuum flask afterwards.
Vitreous Freeze-drying Protocol
1. Drain condenser (tube under condenser). Turn on the freeze button and the condenser
button.
2. Wait for shelf to cool down to -45* C (at least 1 hour).
3. Clean the freeze-drying tray with 0.05 M acetic acid. Put the amount of slurry into the
long trays based on the thickness you desire below. Avoid bubbles as much as possible and
try to pop the ones that form.
57
"skin protocol:" Pour the slurry into the tray if using a pan with one section. If using a pan
with 3 sections, pipette the slurry into the sections in equal amounts.
"1/2 thickness:" Pipette the slurry into the sections of 2 whole trays (6 sections) in equal
amounts.
"double thickness:" Pour all the slurry into the half width unsectioned tray.
"cartilageprotocol:" Pipette 180 mL of slurry per section of a tri-partitioned tray.
4. Wait for approximately 1 hour until the slurry is frozen (or more if it doesn't look frozen).
(For a half tray it takes about 1.5 hours.)
5. After the slurry is frozen, turn on the vacuum. First, make sure the chamber release button
is off. Once vacuum is on, press door shut. Make sure the door is sealed before leaving.
Often the door will not seal and the vacuum will never establish itself - this is not good for
the vacuum pump!
6. Once the vacuum is below 200 mtorr (0.5-3 hours depending on the ambient conditions
and when the freeze-dryer was last serviced), turn the temperature set to 0* C. Leave both
freeze and heat buttons on. Turn on heat button if not previously on. Leave overnight or at
least 12 hours for sublimation.
7. Set temperature to 200 C and turn off freeze button. (Leave on heat button.)
8. Turn the DHT temperature setting to slightly past 1050 C if DHTing immediately.
9. When the freezer is at 20* C, turn off the heat button, vacuum button, and condenser
button. Release the chamber. Remember to drain the condenser chamber. After defrosting,
the chamber and condenser should be wiped dry with a paper towel. Don't forget to place the
plug back in the drain for the next run.
DHT Cross-linking
After freeze-drying, the thickness of the matrix should measured with a micrometer. Then,
the matrix should be placed into the DHT oven for DHT cross-linking for 24 hours. The
conditions of the vacuum oven are 1 atm and 105* C. The matrix sheets are placed in
aluminum foil with one end open. Additionally, this can be placed in a tape-sealed autoclave
bag for added sterilely when you remove the matrix but it is unclear how this affects the
cross-linking. Be careful not to crumple the edges of the matrix sheet. After 24 hours, the
matrix should be stored in a dessicator prior to use. The instructions for starting the vacuum
on the DHT and purging the oven are on it.
Storage
Unless hydrated, all matrices should be stored in a dessicator with blue desiccant.
C-8-1-b temperature ramping protocol
Basically, the protocol is the same as above. Smaller pans are used so that only 67mL of
slurry is used to fill the pan and have the same thickness. Freezing of the slurry is done using
a temperature ramping programmed on the machine: temperature decrease is done from 20 C
to -50 C in 2 hours.
58
C-8-2 Type II
Based on procedure developed by Lee. Uses Chondrocell type II collagen slurry from
Geistlich Biomaterials. Slurry should be stored at 4*C.
Cartilage Protocol Developed by Lee
1. Transfer more than desired amount of slurry to 50 ml centrifuge tube.
2. Centrifuge for 5 minutes at 3500 rpm to de-gas slurry.
3. Pipette slurry into 6-well plate wells. 3.5 mL per well is the standard volume for
"cartilage protocol" developed by Lee (134). Pipette and release some slurry before doing
this because it sticks to the inside of the pipette. Tap/bang plate on countertop to evenly
distribute slurry. Remove bubbles with a 1 cc syringe and needle.
4. Freeze-dry (freeze with lids on, remove lids before pulling vacuum) and DHT as for type I
CG slurry.
GAG and Pore-Size Matched Protocol
This protocol will yield a type II matrix with pore size and GAG content similar to the type I
"cartilage protocol" matrix.
1. Add 0.0105 g of chondroitin sulfate (CS) per 20 mL of type II slurry. CS is added by
mixing in a beaker with a stir bar on the highest stirrer setting for 20 min. This method works
better for 20-50 mL amounts. Some congealing of the slurry occurs at higher volume
amounts, slower speeds, and longer mixing times. Results could likely be improved if a
rotating attachment to the dremel could be found. It is also good to put ice packets around
the beaker to keep it cold.
2. Transfer more than desired amount of slurry to 50 ml centrifuge tube.
3. Centrifuge for 5 minutes at 3500 rpm to de-gas slurry. Some of the slurry may separate
after this step. Gently pipette the solution until gross mixing occurs.
4. Pipette 4 mL of slurry into each 6-well plate well. (Each well will yield 6-10 9 mm discs.)
Pipette and release some slurry before doing this because it sticks to the inside of the pipette.
Tap/bang plate on countertop to evenly distribute slurry. Remove bubbles with a 1 cc
syringe/needle.
5. Freeze-dry (freeze with lids on, remove lids before pulling vacuum) and DHT as for type I
CG slurry.
59
C-9 EDAC Cross-Linking
(from Hastreiter)
General:
6mmol EDAC/g collagen
5:2 ratio EDAC:NHS
Calculations here are based on 3-4 mm thick 9 mm-diameter CG discs with an estimated
mass of 0.005 g each. (Note that different diameter matrices or matrices that weigh
differently should use different amounts of EDAC and GAG.)
For 48 9 mm discs use 100mL of 1.44mM EDAC and 0.56mM NHS.
Supplies:
EDAC (Sigma #E-7750; store desiccated in the freezer): MW = 191.7 g/mol
N-hydroxysuccinimide (NHS) (Sigma #H-7377; store desiccated): MW = 116 g /mol
Procedure:
1. Take out EDAC and let warm up one hour prior to use (so that moisture doesn't condense
inside bottle when opened - EDAC is moisture sensitive).
2. Weigh out amounts of EDAC and NHS. For 100mL final solution, use 0.276 g EDAC
and 0.064 g NHS.
3. Hydrate matrices in half the final volume (i.e.: for 100 ml final volume, hydrate in 50 ml)
of sterile, dH 20 (#15295-017, Life Technologies).
4. Dissolve EDAC and NHS in half the volume of dH 20 water (i.e.: for 100 ml final volume,
dissolve in 50 ml; make up fresh for each use). Swish gently until dissolved (few seconds).
Do not stir solution.
5. Sterile filter (0.45 pLm) into sterile container (or directly into container with hydrated
matrices).
6. With combined solution, cross-link at room temperature for 2 hours in 50mL centrifuge
tubes on a rocker. (Note that Lee cross-linked in a petri dish and stirred manually every 15
min.)
7. Rinse in sterile PBS. Change to fresh, sterile PBS and place on rocker for another 2 hours
to remove residual uncross-linked EDAC.
8. Rinse 2x10 minutes in sterile dH 20.
9. Store at 4*C for up to one week before use in sterile dH 20 (effects of longer storage
unknown). In practice no one has used EDAC cross-linked matrix more than 2 days after
cross-linking.
60
C-10 Cell seeding using pipetting method
(from Hastreiter)
1. Matrices are generally pre-wet for 1 hour in PBS if they are not already wet. Do not
prewet in medium as the FBS alone can change the diameter of the matrices.
2. The cells are passaged from the flasks. The cells should be suspended at # cells wanted
per disc/40 p.L for a 9 mm disc. For example, for 2 million cells per 9 mm disc, you want 2
million cells/40 pL, or 50 million cells/lmL.
3. Under sterile conditions, the pre-wet matrices are dried to some extent on sterile filter
paper and placed in 6-well plates coated with 2mL of agarose (1 g/25mL Seaplaque agarose
autoclaved prior to placement in wells and in cold room for at least 4 hours).
4. 20 pL of the cell suspension is then pipetted onto the surface of all of the matrices. After
10 min., the matrices are flipped over an additional 20 pL of cell suspension is added to this
opposite surface.
5. The matrices are placed in an incubator for 2 hours. Then, 0.5mL of 10% FBS medium is
added to each well very slowly and along the sides of the wells.
6. After another 4 hours (6 hours after initial seeding), another 2.5mL of medium is added to
each well.
61
C-11 Matrix culture in multi well-plates
(modified from Hastreiter)
1. Agarose coating prevents cells from attaching and growing on the bottom of well plates.
This ensures that your explants or constructs will have all medium nutrients solely available
to them. Each well of a 6-well plate is to be coated with 2mL of liquid agarose. Prepare 1015mL more agarose than you will need.
2. In a 100mL glass bottle, place agarose and distilled water. Use 1g Seaplaque agarose
(#50100, BioWhittaker/FMC Bioproducts) per 25mL water. (Or you can use ig of Biorad
agarose per 50mL water.)
3. Autoclave the bottle on the liquid setting in an autoclave bag with the cap untightened.
4. Remove the bottle from the autoclave while it is still hot.
5. Coat the wells with 2mL of the liquid agarose each. This should be done rather quickly as
the agarose will solidify as it cools.
6. Place parafilm around the well plates and put them in the cold room for at least 4 hours.
Often it is easiest to do the agarose coating the day before using the plates. Do not use the
plates after more than one day in the cold room because the agarose will crack.
7. Warm the plates in the incubator 1-2 hours prior to use.
8. Change agarose-coated well plates every two weeks because the agarose breaks down.
Change the medium every other day: remove and add it slowly from the sides of the well
plates. If the construct floats a little bit after having readded the medium, gently tap the well
plate on the table or agitate left and right.
62
C-12 Radiolabeling of 35S and 3H
(from Hastreiter)
General
1) Label disks during final 24 hrs of culture with 35S and 3H to measure GAG and collagen
synthesis, respectively.
2) Wash unincorporated radiolabel.
3) Lyophilize tissue and measure dry weights.
4) Papain or Proteinase K digestion - use digest for GAG content, DNA content, and
scintillation counts
Materials needed
1) Latex gloves (double glove)
2) Tape for labcoat sleeves
3) Aluminum foil to line hood
4) Complete medium (10% FBS)
5) Sterile 15 mL or 50 mL centrifuge tube
6) Pipetman - 20 pL and/or 200 pL
7) Sterile pipette tips - 200 pL capacity
8) Vacuum flask and sterile Pasteur pipettes
9) Radionuclides: tritiated proline (3H) (proline, L[2,3,4,5- 3H]; #NET,483, Perkin Elmer
or #TRK534, Amersham Pharmacia Biotech, Inc.) and radioactive sulfate (35S)
(#NET041H, Perkin Einer)
10) PBS
11) Na 2SO 4 (anhydrous sodium sulfate; #S421-1, Fisher Scientific Co.) and L-proline (P8449, Sigma)
12) Clean spatula/forceps
13) 24 well culture plates
14) Sterile pipettes
Preparation of radioactive media
1) Calculate volume of isotope needed:
a. With 1.5 mL media/disc; V=1.5n+2 mL, where n is the number of discs.
b. 35S has a half-life is 87.4 days:
volume
5S
_CV
=
Cvs x 2
where Res is the radioactivity concentration you desire for 35S and Cvs is the concentration of
radioactivity of your sulfate vial. The former (Rcs) is usually 10 [tCi/mL for 9 mm-diameter
CG discs. The latter is often 1000 gCi/mL when you order 1 mCi or 10,000 gCi/mL when
you order 10 mCi, but you should check the bottle. x = # days past calibration date on vial.
c.
For 3H the half-life is 8-9 years.
volume 3H =
*
,
v:H
where RcH is the radioactivity concentration you desire for 3H and CH is the concentration of
radioactivity of your proline vial. The former (R cH) is usually 10 gCi/mL for 9 mm-diameter
CG discs. The latter is often 1000 gCi/mL when you order 1 mCi, but you should check the
bottle.
63
2)
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
Wipe hood with 70% EtOH.
Line with aluminum foil.
Double glove and tape lab coat sleeves.
Aliquot calculated volume of warm media into centrifuge tube.
Place radionuclide in hood on aluminum foil and loosen cap.
Use sterile technique to aliquot calculated volume of 3S.
Save 1 mL of this single -labeled media for calibration.
Add calculated volume of 3H.
Save 1 mL of this double -labeled media for calibration.
Remove pipette tips to original wrapper and dispose of in radioactive waste container.
Return radionuclides to container and note amount used; return to refrigerator.
Rinse Pipetman in cold water.
Check Pipetman and hood with Geiger counter.
Labeling of discs
1) Place 1.5 mL of radiolabeled medium in 24 well plate (#08-772-1, Fisher Scientific Co.)
wells. Generally, use the top row only so the bottom rows can be used for washings.
2) Use a forceps to transfer the discs to the radiolabeled wells. Drop them gently. Do not
let the forceps touch the radiolabeled medium.
3) Return pipette to paper wrapper and dispose of in radioactive waste container.
4) After 24 hours, radiolabel is complete.
Washing of discs :
1) Complete PBS with 0.8 mM Na2SO 4 and 1.0 mM proline.
a. For 500 mL PBS: 0.057 g Na2SO 4 and 0.057 g proline.
b. Recommended to make fresh wash solution each time; can make frozen stock
solutions of proline and sulfate and dilute in PBS.
2) Line hood with aluminum foil.
3) Aliquot 1 mL PBS/well in 24 well culture plate. Put this into all the wells below each
radiolabeled well in the plate.
4) Place in cold room for 15 minutes.
5) Transfer disks to 3 row and refrigerate for 15 minutes.
6) Repeat for a total of five washes.
7) Dispose of radiolabeled media in sink and note amount of radioactivity disposed.
Optional: Save the medium if you wish to count it.
8) Place each sample in a labeled vial.
9) Optional: Save PBS from last row (1 from each group) to make sure thoroughly washed
(radioactive counts - background).
10) Lyophilize, measure dry weight, digest... (for DNA, GAG, scintillation counting).
64
C-13 Lyophilization of Matrix Samples
(from Hastreiter)
Freeze-drying will be performed in the jar chambers by the distilled water source via the
following method.
To start:
1. Pull knob on freeze dryer to empty any water in the chamber.
2. Turn on main switch.
3. Wait 10 min. until temperature is -50*C.
4. Turn on vacuum switch.
5. Close any open valves. Put everything on "VENT," and make sure no other valves are
open.
6. Wait until pressure is in the green zone, about 5 min. Note that the display may be
broken.
7. Attach jar with samples and turn the valve corresponding to the jar to "VAC."
8. Confirm that the temperature goes up.
9. Record operation on record sheet.
To end after overnight freeze-drying:
1. Turn the valve corresponding to the jar 900 to position in between "VAC" and "VENT."
2. Open a different valve to release the pressure.
3. Turn off the vacuum pump.
4. Turn off the main switch.
5. Gradually turn the valve corresponding to the jar to "VENT."
6. Remove the jar.
7. Measure the mass of the matrices the same day.
8. The matrices may be frozen for further use prior to papain digestion, but they will absorb a
small amount of water in that environment. Thus, mass measurements will not be accurate
after freezing, but assays can still be performed.
65
C-14 Papain digestion
(from Hastreiter)
Note: Samples should be lyophilized and their mass determined prior to digestion.
1. Prepare stock solutions (unclear how long good for, 3 months maybe):
0.5 M Monobasic stock: 6.9 g NaH2PO4-H20 in 100mL distilled H20
0.5 M Dibasic stock: 13.4 g Na 2HPO 4 -7H 2 0 in 100 mL distilled H20
Make sure to select the correct vials for these solutions. Some have different amounts
of water complexed.
2. Prepare papain buffer:
2.46 ml dibasic stock solution
17.54 ml monobasic stock solution
80 ml distilled H20
3. On day of digestion, complete papain buffer with:
87.82 mg L-Cysteine HCl (anhydrous; #C1276, Sigma)
186.12 mg Disodium Ethylenediamenetetraacetate (EDTA) (#S311-3, Fisher
Scientific Co.)
pH to 6.2 with 10M NaOH (latter is made with 4 g of NaOH/10 mL distilled 1120)
(usually 3-5 drops of latter are required)
4. Digestion:
a. Determine how much papain (Sigma #P3125) is needed based on mass of samples:
= amount of papain per sample in mL
2
t
maximum sample mass in mg x 0.x125mg pn
6.25mg sample
23mg protein
b. Add this much papain to each sample (works better if added after next step).
c. Add as much papain buffer so that each sample as 1 mL of fluid added to it. i.e. a
+ c = 1 mL.
d. Vortex or mix thoroughly.
e. Float tubes in covered 65'C bath overnight.
f. Sometimes there is some esidual left in the tubes. Digestion results may be
improved if protease digestion protocols are used.
66
C-15 Scintillation counting of 35S and 3H Radiolabeled Samples
(from Hastreiter)
Counting protocol
1. Combine 100 pL of sample digest or calibrated media with 4 mL of scintillation fluid
(ScintiVerse II; F-09-0797, Fisher Scientific Co.). Assay samples in duplicate.
2.
Spray and wipe off the scintillation vials and holders with staticide (#2005, ACL
Staticide). Then, queue them in the machine. The maximum number of samples at once is
230. The first vials go in the tray with 6 at the top. On this tray, turn the dial to 1 (means it
counts the tray only once). The rest of the vials go in trays with no number. (Yellow dot on
dial means do not count; white dot means count continuously.)
3. Count 1 minute/sample, with 3H counts in channel A and 35 S counts in channel B. This is
done by pressing the following sequence on the scintillation machine (Packard Tri-Carb 4640;
Packard Instrument Co.):
End
Start
Enter 6
Enter
Make sure isotope line says 4
End
Forward and Enable simultaneously
4. After samples are counted, the vials with scintillation fluid and radioactive digest should
be dumped into the barrel for liquid scintillation vials.
Calculations
The amount of radioactivity, [3H] or [S], is calculated based on the counts per minute (cpm)
from channels A (C i) and B (C2) as follows:
[3H]
'ki
k2
C1
kI k22 C2
k
The coefficients 1, kI 2 , k21, and k22 are determined from the counts of the media samples
taken when adding the isotopes to the media:
From the first media sample (5S only, no 3H) the matrix equation becomes: ([ 35S]
reflects concentration of 35 S added to media (i.e., 10 ptCi/mL); superscript "S" reflect that
counts are from first media sample with 35S only, subscripts denote the channel counted)
0 = kCs +kC
[35S ] = k2lC~s + k22C2
From the second media sample ( 5S and 3H) the appropriate equations are: ([1 5 S] and
3
[H] reflect concentrations of 35S and 3H, in pC i/mL, added to media, respectively;
superscript "S,H" reflect that counts are from second media sample with both 35S and 3H,
subscripts denote the channel counted)
67
['H] =k CsH + k
CS
[IS ] = k2,C,S," + k22C2SH
Solving these 4 equations for the 4 unknown k's:
k
IH]
[
-
2,H
-SC
-['s]
k
S
SH
2S
02
,
2
CS
H
-
S
H
22"S',
Note that if you accidentally add the "Hi before the 'sS, these values change to:
3
CS,H~
kk =S
=[ H]
ClS
C S,H
212
(
I
H
,HC
Y
H
C~C
k2CH
H
_CH
22
[H]
[3H
k2=CSH
H
]
a-C kH + C2k
"S1
Thioaee ae12om
DMEM/F 12 media.
proline and
od
ta
a
[S]3g
ovre
sulfate
5.
mlm
intomcrmcua
od
omplate
(Numbers based on manufacture's values for DMEM/F 12, the
percentagheof DMEM/F12 in complete medium-88%, and exclusion of any of these
constituents from FBS--has not been measured.) The concentration of cold proline and
68
sulfate far outnumbers the concentration of hot. These calculations assume that the same
percent of radiolabeled proline/sulfate and unlabeled proline/sulfate was converted and that
the amount of proline/sulfate added in radiolabeled form is insignificant compared to the
concentrations of (unlabeled) proline/sulfate in the media. The amount of proline/sulfate
incorporated (in nmol) into macromolecular form during the radiolabel period is then
determined as follows:
proline a'H (132 nrol/mL )V
sulfate
=
a 3, s(358.2 nmol/mL )V'
where V is the volume of media fed to the cultures, in milliliters (i.e., 1.5 mL).
The values for the control matrices should be subtracted from the seeded matrices.
Then, the incorporation data can be normalized to the time of radiolabel (i.e., 24 hours) and
the amount of DNA in the construct to yield the rate of incorporation normalized to cell
content (nmol/pg DNA/hr).
69
C-16 GAG Assay (Dimethylene Blue)
Modified from (21). Based on (57).
1. Prepare of the color reagent.
Dissolve 3.04 g of glycine and 2.37g NaC 1 in 950 mL of distilled water. Adjust the pH to 3.0
by adding NaOH or HCl.
Add additional water to reach 1 L.
Add 16 mg of
dimethylmethylene blue. This solution is good for 3 months and should be kept in a lightprotected bottle.
2. Turn on spectrophotometer at 535 nm and stir the color solution 30 min prior to use.
(Turn Deuterium off.)
3. Prepare the chondroitin sulfate standards as following:
a. Prepare chondroitin sulfate stock solution at 1 mg/mL. Make at least 10 mL.
b. Dispense 3 mL color reagent to each cuvette (4 optical sides; #67.755, Sarstedt) (only
touch the tops of the cuvettes with your hands) with pipettor.
c. Add the following amounts of the cuvettes for the standards.
Standards (gg)
dH 20 (pLL)
CS (L)
0
100
0
2.5
5.0
10
15
97.5
95
90
85
2.5
5.0
10
15
20
30
80
70
20
30
40
60
40
Mix each cuvette well.
d. Read absorbance of blank first and "Set Ref" with this sample.
e. Run the standards. It is often good to plot the standard curve (Figure 36) immediately
before analyzing samples just to make sure you have calibrated correctly.
4. Analyze samples.
a. Prepare cuvettes by adding 3 mL of color reagent to each cuvette.
b. Vortex the sample. Add a 100 gL aliquot of the digested sample into 3 mL dye solution,
mix well, and read it when the reading stabilizes.
c. If the reading of your sample is off the standard curve, you need to reduce the volume of
sample in the cuvette. Make up the difference with dH 2O. For example, try 50 gL sample
and 50 gL dH 20. Make sure to record how much aliquot was used and how much fluid the
aliquot was taken from.
Finding the Percent Mass of GAG
1. The mass of the dry sample should have been weighed prior to digestion.
2. Perform the DMB GAG assay.
3. The chondroitin sulfate control data should be plotted as amount of chondroitin sulfate (in
gg) vs. absorbance (in nm). These data should be fitted with a linear equation.
70
4. The above equation should be used to find the amount of chondroitin sulfate (X) in the
samples by putting their absorbance into the equation.
5.
The % weight of GAG is calculated by:
T )rm(n
amount aicuot taken
X
x x100%
mass of sample amount of aliquot used in assay (in ML)
For example,
X(in Mg)
1000 pL
x.Os
sapx
xg1%
mass of sample (in g)
Equation 3
100 pL
6. If you don't want the % mass of GAG just do the above calculation without dividing by
mass to get the GAG in jg.
GAG Standard Curve
25
Y = MO + M1*X
MO
E20
M1
R
cu
-0.76695904457
73.301352737
-
0.99550705628
~15
0
CU 10
(I)
.S
5
(9h
-C
-5
0
0.05
0.1
0.15
0.2
0.25
0. 3
Absorbance (nm)
Figure 36. Example GAG Standard Curve
71
C-17 DNA Assay using Hoechst Dye
(from Hastreiter)
1. Prepare TNE lOx buffer solution: (100 mM TRIS, 10 mM EDTA, 1.0 M NaCl)
800 ml dH20
3.72 g Disodium Ethylenediamine Tetraacetate (EDTA) (Na2CioH14O8N2-2H 20)
12.1 g TRIS (Gibco #15504-012)
58.4 g NaCl
pH to 7.4 with concentrated HCl (approximately 70.5 mL 1 M HCl)
dH20 to 1000 ml (approximately 130 ml)
Store 4'C
(unclear how long good for, 3 months maybe?)
2. Prepare concentrated Hoechst dye stock solution: (1 mg/ml)
10 mg Hoechst dye #33258
10 ml dH20
Store 4 0C, shelf life: 6 months
Protect from light: fluorescent!
3. Prepare the calf thymus DNA (Sigma D-3664) stock solution.
a. For final DNA concentrations cf 10-400 ng/mL: Stock solution of 100 ptg/mL.
Each dry bottle has 1 mg of DNA. Add 9 mL dH 20 and 1 mL lOX TNE buffer
solution. Store at 4'C for up to 6 months.
b. For final DNA concentrations of 100-2000 ng/mL: Stock solution of 1 mg/mL.
Each dry bottle has 1 mg of DNA. Add 1 mL dH 20. Must be used the day it is made.
3. On day of assay, prepare working solution of dye. There are 2 forms depending on what
your final DNA concentration will be.
a. 90 mL dH20 filtered through 0.45 jim filter
b. 10 mL TNE lOx buffer filtered through 0.45 pm
cl. For final DNA concentrations of 10-400 ng/mL: At latest possible time before
working, add 10 piL concentrated Hoechst dye stock solution. Usually, this step is
used.
c2. For final DNA concentrations of 100-2000 ng/mL: At latest possible time before
working, add 100 ptL concentrated Hoechst dye stock solution.
4. Scale fluorimeter (Hoefer Scientific Instruments TKO 100 Dedicated Mini Fluorometer):
a. Warm up fluorimeter for 15 minutes.
b. Add 2 mL of the working dye solution to a cuvette (4 optical sides; #67.755,
Sarstedt). Place in fluorometer.
Start with the "SCALE" knob adjusted to 50% sensitivity. This is approximately 5
clockwise turns of the knob from the fully counter-clockwise position. Adjust the
zero knob until the display reads "000."
c. Prepare standards of calf thymus DNA X 2. Put the following amounts in 2 mL of
the working dye solution in cuvettes. Mix well.
I. Final DNA concentrations of 10-400 ng/mL:
Standards (ng)
calf thymus stock solution (L) Cuvette Concentration
50 ng
0.5 pL
25 ng/mL
72
100 ng
I [L
50 ng/mL
150 ng
1.5 pL
75 ng/mL
200 ng
2 gL
100 ng/mL
300 ng
3 pL
150 ng/mL
400 ng
4 pL
200 ng/mL
500 ng
5 pL
250 ng/mL
II. Final DNA concentrations of 100-2000 ng/mL:
Standards (ng)
calf thymus stock solution (tL) Cuvette Concentration
500 ng
0.5 pL
250 ng/mL
1000 ng
I L
500 ng/mL
1500 ng
1.5 pL
750 ng/mL
2000 ng
2 [L
1000 ng/mL
d. Scale with highest amount of calf thymus DNA standard, typically 250 ng/mL.
Put this sample cuvette in. and adjust the "SCALE" knob until the display readout
matches the concentration of the standard (i.e., 250).
e. Repeat b and d once or twice until reproducible. Then, run the DNA calf thymus
standard curve.
f. It is often good to plot the standard curve (Figure 37) immediately before analyzing
samples just to make sure you have calibrated correctly. The slope should always be
close to 2.
5. Assay:
a. Collagen samples must be digested by papain or protease.
b. Add an aliquot of the sample to the working dye solution in the cuvette for a total
volume of 2 mL. For example if you add a 100 pl aliquot of sample to the cuvette,
add 1.9 mL final working dye solution to the cuvette. Mix.
c. Read the fluorescence intensity immediately on the fluorometer
d. Zero the fluorometer with a blank between samples.
Finding the Percent Mass of DNA
1.
The mass of the dry sample should have been weighed prior to digestion.
2.
Perform the DNA assay.
3.
The calf thymus DNA control data should be plotted as amount of DNA (in ng) vs.
fluorescence intensity. These data should be fitted with a linear equation.
4.
The above equation should be used to find the amount of DNA (X) in the samples by
putting their fluorescence intensity into the equation.
5.
The % mass of DNA is calculated by:
X
amount aliquot taken from (in ML) x100%
xx0
mass of sample amount of aliquot used in assay (in ML)
For example,
X(in ng)
lO1000L
xlxlO
xlOO%
mass of sample (in g)
100 ML
6.
If you don't want the % mass of DNA just do the above calculation without dividing
by mass to get the DNA in ng.
73
DNA Standard Curve
600
Y = MO + M1*X
500
MO
----M1
R
-1.0824419958
1.9769887905
0.99764073537
'n400
o300
E
z200
100
04
0
U.
50
100
150
I.
200
.L.
250
DNA Reading
Figure 37. Example DNA Standard Curve
74
C-18 BCA Protein Assay
(modified from Pierce kit #23225)
1. Prepare lmL of undiluted BSA standard by mixing 2.0mg of BSA in lmL of diluent.
Diluent should preferably be the same diluent as your sample (papain buffer if digested
samples, etc.).
2. dilute the undiluted BSA standard named "Stock" as follow:
Sol#
1
2
3
4
5
6
7
8
Volume of the BSA to add
300 L of Stock
375 L of Stock
325 L of Stock
175 L of (2)
325 L of (3)
325 L of (5)
325 L of (6)
100 L of (7)
Volume of diluent to add
0 L
125 L
325 L
175 L
325 L
325 L
325 L
400 L
Final BSA concentration
2000 g/mL
1500 g/mL
1000 g/mL
750 g/mL
500 g/mL
250 g/mL
125 g/mL
25 g/mL
(working range: 20-2000 g /mL)
3. Prepare the BCA working reagent (WR)
To prepare WR, mix 50 parts of BCA reagent A with 1 part of BCA reagent B. Mix well.
Prepare sufficient volume of WR based on the number of tests to be done. Each test tube
sample will require 2mL of WR and each microwell sample requires only 200 L. The WR is
stable for at least 1 day when stored in a closed container at room temperature.
Test Tube Protocol
4. Add 2mL of the WR to each tube.
5. Pipet 100 L of the sample (or standard) andmix well. Use 100 L of diluent for the blanks
6. Incubate all the tubes at 37 C for 30 minutes, increasing the incubation time increases the
measurement values but decreases the minimum detection level
7. Cool the tubes at room temperature
8. Measure the absorbance at 562nm (between 540 and 590).
9. Subtract the average reading for the blanks to each standard or sample readings
10. Prepare the standard curve and determine the unknown protein concentration in g/mL
for each unknown tube.
Microwell Plate Protocol
1. Pipet 25 L of each standard or unknown sample in the appropriate microwell plate wells.
Use 25 L of the diluent for the blank wells.
2. Add 200 L of the WR to each well, mix the plate well on a plate shaker for 30 seconds.
3. Cover the plate and incubate the plate at 37 C for 30 minutes.
4. After incubation, cool the plate at RT.
5. Measure the absorbance at or near 562nm on a plate reader.
6. Subtract the average reading for the blanks to each standard or sample readings
7. Prepare the standard curve and determine the unknown protein concentration in g/mL for
each unknown tube.
75
C-19 Hydroxyproline Assay
Preparation of the samples
1. Take 100 L of each papain digested sample in screw-cap glass tubes.
2. Add 900 L of 6N HCl, mix well and heat at 120 C overnight until completely dry.
3. Add distilled water in the glass tubes and scratch the sides to ensure that all the sample has
been diluted in the water. Transfer the acid hydrolysed sample in a disposable 12 x 75 mm
glass tube using Pasteur pipettes. Use about 2mL of distilled water for each tube.
4. Dry in the oven at 120 C until completely dry.
5. Add 1 mL of 1:10 diluted pH=6 buffer.
Reading (for a microwell plate reader)
1. Prepare standard curve in the same disposable tubes by mixing the following amounts of
Hydroxyproline starting from a stock solution of 0.1mg/mL of hydroxyproline:
Amount of stock to add
Volume of water to add
Quantity of Hydroxyproline
0 L
10 L
20 L
30 L
40 L
50 L
50 L
40 L
30 L
20 L
10 L
0 L
0
I
2
3
4
5
g
g
g
g
g
g
2. In each sample and standard tube, slowly add 500 L of Chloramine-T hydrate (98%,
Sigma) while vortexing. Incubate at room temperature for 15 minutes.
3. Add 500 L of pdimethylaminobenzaldehyde (pDMAB) solution. Incubate at 60 C for 20
minutes, cool the tubes.
4. Transfer 200 L of sample in a 96 well plate. Read the plate at 560nm.
5. Plot the standard curve of Hydroxyproline and determine unknown hydroxyproline
concentrations.
ATT: samples should be discarded it hazardous waste disposal as it contains pDMAB.
76
C-20 Paraffin embedding of Matrices
After rinsing, matrix only specimens should be fixed in formalin for 48 hours. Explantmatrix constructs should be fixed in formalin for at least a week. They will be manually
dehydrated prior to embedding in paraffin and JB-4 resin (Polysciences, Inc., Warrington,
PA).
1. Samples are gently placed in labeled Tissue Tek cassettes in between 2 blue sponges.
Another set of Tissue Tek cassettes is prepared with the same labels.
2. Dehydration. The cassettes are transferred between the following solutions for the
designated time periods. All solutions are pre-made in buckets and kept in the flammables
cabinet. The longer times in this protocol are for matrix-explant constructs; the shorter times
are for matrices only.
Solution
Time
distilled H20
distilled H20
30 min-1 hour
30 min-1 hour
50% EtOH
70% EtOH
30 min-1 hour
30 min-1 hour
80% EtOH
95% EtOH
30 min-1 hour
30 min-1 hour
95% EtOH
30 min-1 hour
100% EtOH
30 min-1 hour
100% EtOH
100% EtOH
30 min-1 hour
30 min-1 hour
3. Bisection. The matrix specimens are removed from the cassettes and cut in half with a
scalpel to result in two half-circles. One half is then placed in the original cassette and sent
for paraffin embedding. The second half is placed in the other cassette with the same label
and sent for JB-4 embedding.
4. Paraffin embedding. Cassettes for paraffin (Paraplast Cat. #8889-501006, melting temp
56*C, Oxford Labware, St. Louis, MO) embedding proceed through additional solutions as
follows:
Time
Temp
Solution
Histoclear
1 min-2 hour
room temp
paraffin
1 hour
59 0 C
paraffin
1 hour
59 0 C
Take care not to touch the cassettes when they have just been in Histoclear (Americlear
histology clearing solvent, Baxter Healthcare Corp. #C4200-1, Deerfield, IL) because the
solution will eat through gloves. The paraffin baths are located in the paraffin oven.
77
C-21 Preparation of coated slides
Slides can be coated prior to tissue sectioning in order to increase the fixation of the tissue on
the slide. It is particularly useful for operations during which slides are washed many times
such as immunohistochemistry. For more classical staining it also offers better quality slides.
Once coated, never touch the slide with bare fingers: it would leave fingerprints on the
coating. More information can be found on the product information sheet (A3648) of Sigma.
Protocol (from Sigma)
1. Mix IOmL of the silane (product number A3648) with 500mL acetone. Solution is stable
for 8 hours after which the color will change.
2. Dip clean dry slides in slidate solution for 2 minutes
3. Wash the slides in 2 changes of distilled water
4. Dry completely in oven at 60'C and cool. Autoclave if desired will not harm the coating.
5. Store treated slides in boxes at room temperature.
78
C-22 Hematoxylin and Eosin Staining
(From Hastreiter)
For formalin-fixed, paraffin samples.
Sectioning and Storage
Histological sections are cut on a microtome (Reichert-Jung model 2050 Supercut, Leica
Instruments, Nussloch, Germany). The thickness is 7 pm for paraffin sections. The sections
are placed on glass slides (Fisherbrand Superfrost Plus, Cat. #12-550-15, Fisher Scientific,
Pittsburgh, PA). Slides are placed in an oven at 40-50*C to melt off excess paraffin
overnight, and then stored at room temperature.
Solutions
HEMATOXYLIN: Filter 200 ml of stock solution into staining dish. Sigma Harris
Hematoxylin Solution, Catalog HHS-128, Modified: Hematoxylin, 7.5 g/L; sodium
iodate, aluminum and ammonium sulfate, stabilizers and preservative.
ACID ALCOHOL: 200 ml of 70% ethanol (in dH 2 O) + 0.5 ml HCl
AMMONIA WATER: 200 ml dH 20 + 5-10 drops ammonium hydroxide, generally 5
pH should be roughly around 10.0 - use pH paper.
Generally this should be mixed up fresh each day.
EOSIN: 100 ml stock solution + 100 ml dH20 + 1.0 ml glacial acetic acid
Sigma Eosin Y Solution Aqueous, catalog HT1 10-2-128.
Staining
1.
DEPARAFFINIZE AND REHYDRATE
Xylene:
2 x 5 min.
100% ethanol:
100% ethanol:
95% ethanol:
80% ethanol:
70% ethanol:
dH20:
10-20
10-20
10-20
10-20
10-20
10-20
dips
dips
dips
dips
dips
dips
2.
3.
Harris hematoxylin, 10 min.
Rinse in tap water, approx. 1 min. running or swishing until almost clear
4.
Acid alcohol. Dip quickly 5-10 times. 20-30 sec total.
5.
6.
7.
Rinse in tap water about 30 seconds.
Ammonia water. Quick dips (5 or so) until blue.
Rinse in tap water. About 1 min.
8.
Eosin, 45 sec.
9.
Rinse in tap water, 2 min.
10.
DEHYDRATE
70% ethanol:
10-20 dips
79
10-20 dips
80% ethanol:
10-20 dips
95% ethanol:
10-20 dips
100% ethanol:
10-20 dips
100% ethanol:
2 x 5 min.
Xylene:
Air dry, coverslip with Cytoseal (Cytoseal 60; #18006, Electron Microscopy
11.
Sciences).
80
C-23 Safranin-O Staining
(From Lee)
GAG will stain red/pink and collagen stains green.
1) Deparaffinize and rehydrate:
xylene
2 x 5min
100% EtOH
95% EtOH
90% EtOH
80% EtOH
70% EtOH
dH 20
10-20
10-20
10-20
10-20
10-20
10-20
dips
dips
dips
dips
dips
dips
2) Stain 10 minutes in Safranin-I (0.2g Safranin-O, lmL glacial acetic acid, 100mL distilled
water)
3) Rinse with tap water
4) Counterstain 10-15 seconds with Fast-Green (stock solution, dilute 1:5 for working
solution: 0.2g Fast Green, lmL glacial acetic acid, 1 OOmL distilled water)
5) Dehydrate (70%, 80%, 90%, 95%, 100%, 100% EtOH, xylene)
6) Air dry section
7) Coverslip with Permount, let dry 2-3 days.
81
C-24 Masson Trichrome Staining
Nuclei will stain red, and collagen blue. Cytoplasm, keratin, muscle fibers and intercellular
fibers will stain pink.
Protocol (adapted from Breinan and from Manual of Histologic Staining Methods of the
Armed Forces Institute of Pathology, 3 'd edition - American Registry of Pathology)
Solutions
-
Bouin's solution:
Picric acid, saturated aqueous solution: 750mL
37-40% formalin: 250mL
Glacial acetic acid: 50mL
-
Weigert's iron hematoxylin solution
Solution A
Hematoxylin crystals: ig
Alcohol, 95%: 100mL
Solution B
Ferric Chloride, 29% aqueous: 4mL
Distilled water: 95mL
Hydrochloric acid, concentrated: lmL
Mix equal parts of solution A and solution B
-
Phosphomolybdic-Phosphotungstic acid solution
Phosphomolybdic acid: 5g
Phosphotungstic acid: 5g
Distilled water: 200mL
-
Aniline blue solution
Aniline blue: 2.5g
Glacial acetic acid: 2g
Distilled water: 1 OOmL
-
2% light green solution
Light green, SF yellowish: 2g
Distilled water: 98mL
Glacial acetic acid: lmL
-
1% glacial acetic acid solution
Glacial acetic acid: ImL
Distilled water: 1 OOmL
Procedure
1. Deparaffinize and rehydrate:
xylene
2 x 5min
100% EtOH
95% EtOH
90% EtOH
80% EtOH
70% EtOH
dH 20
10-20
10-20
10-20
10-20
10-20
10-20
dips
dips
dips
dips
dips
dips
2. Quench in Bouin's solution for 1 hour at 56 C or overnight at RT
82
Note: Bouin's solution is used as a mordant to intensify the color of the tissue. It is hazardous
and the picric acid when in less than 10% water is very explosive. Bouin's solution should be
disposed in a hazardous container.
3. Cool and wash in running water until yellow color disappears.
4. Rinse in distilled water.
5. Weigert's iron hematoxylin solution for 10 minutes. Wash in running water 10 minutes
6. Rinse in distilled water.
7. Phosphomolybdic-phosphotungstic acid solution for 10 to 15 minutes, discard solution
8. Aniline blue solution for 5 minutes.
9. Rinse in distilled water.
10. Glacial acetic solution for 3 to 5 minutes, discard solution.
11. Dehydrate in 70, 80, 90, 95, and 100% EtOH.
12. Xylene 2 x 5 minutes (leave in xylene overnight to get good clearing of Ethanol)
13. Coverslip with Permount
83
C-25 Type I immunohistochemistry
(modified from Lee)
Positive staining is indicated by brown color in the extracellular region
Products location
- Tris buffered saline (TBS) : biochemical lab downstairs in a drawer, pouch of TBS
powder
- PAP Pen: Histology room in a small box labeled "PAP pen" on the shelves
- Protease XVI (pronase): in the freezer of the histology room
- Horse serum: in the freezer of the histology room, to be diluted
- Primary antibody (mouse monoclonal anti-type II): in the fridge of the histology room,
in the drawer for all anti bodies.
- Negative control (mouse IgG): Same as the primary antibody
- Secondary antibody (biotintylated horse anti-mouse immunoglobulin): in the freezer
of the histology room (next to the horse serum)
- Peroxydase: in the fridge of the histology room, labeled H2 0 , the commercial
2
solution is 30%, to be diluted 10 times.
- ABC reagent: box in drawer of the fridge of the histology room
- Tris HCl buffer: inside Nicole's box in the cold room
-
DAB Kit: in the fridge of the histology room, black box labeled ABC
Protocol
1) Deparaffinize and rehydrate:
xylene
2 x 5min
100% EtOH
95% EtOH
90% EtOH
80% EtOH
70% EtOH
TBS (pH 7.4)
10-20 dips
10-20 dips
10-20 dips
10-20 dips
10-20 dips
2 x 2min
ATT:
- Once the samples have been hydrated, never let them dehydrate. To remove the excess of
liquid, simply put the slides vertically and agitate. You can also use a small pipette to remove
the excess of liquid. Do not use any type of cloth, and never touch the samples. Each time it
is mentioned "wash", it means that a few drops of the washing solution should be added.
- all the dilutions are made using TBS unless specified.
- slides can be coated with silane so that the samples attach better to the slides.
2) Wipe off excess of liquid and use a small cloth to remove the water around the samples.
Do not touch the samples. Mark around the samples with a permanent market under the slide
and mark the same way with a PAP pen around each sample so as to prevent future solution
to leak from one sample to another.
3) Protease digestion: 1 to 2 drops of protease solution (10mg pronase/1OmL TBS) on each
sample during 60 minutes.
4) Remove the protease, wash with TBS for 2 x 2 minutes, remove TBS excess afterwards.
84
5) Add 1 or 2 drops of blocking solution (5% horse serum, 1:20 dilution) for 30 minutes. Do
not wash slides with TBS afterwards, simply remove the excess of blocking solution
6) Add 1 or 2 drops of primary antibody (1:20 dilution of mouse monoclonal anti-type II
collagen in TBS; Iowa Hybridoma Bank CIICI) on some samples as well as some negative
control solution (mouse IgG 5 g/mL) for 1 hour. Be careful not to mix the liquids onthe
slides.
7) Remove antibody and negative control with 2 different pipettes, be careful not to mix the
liquids. Do not put the slides vertically for that as it could mix the liquids. Wash in TBS 2 x 2
minutes. Remove excess of TBS afterwards.
8) Add 1 or 2 drops of secondary antibody (1:200 dilution of biotintylated horse anti-mouse
immunoglobulin; Vector Laboratoris #BA-2000) for 45 minutes. During the last 15 minutes,
prepare the ABC reagent by mixing 1 drop of reagent A in 5mL cold Tris HCl buffer (pH
7.6), then mixing, then adding 1 drop of reagent B and mix again. Let stand 30 minutes
before use.
9) Wash in TBS 2 x 2 minutes, remove excess of TBS afterwards
10) Add 1 or 2 drops of peroxydase (3% H 20 2 in dH2O) for 10 minutes.
11) Wash in TBS 2 x 2 minutes, remove excess of TBS afterwards
12) Add 1 or 2 drops of avidin-biotin conjugate (ABC kit, Vectastain Labs) reagent and wait
30 minutes.
ATT: ABC solution is highly cancerous. It should be dumped in the appropriate hazardous
waste bottle (labeled ABC).
13) Wash in TBS 2 x 2 minutes, remove excess of TBS afterwards
14) Wash in water
15) Stain with DAB staining kit (50 L of 1%
hydrogen peroxide + 2.5mg DAB/5mL
TrisHCl buffer : read the kit to make the DAB solution) for approximately 8-10 minutes.
ATT: From this step on, the slides can be put vertically in the regular staining plastic trays.
16) Rinse in distilled water for 3-5 minutes
1.7) Counter stain 10 minutes with Harris' hematoxylin
18) Rinse in running tap water until clear
19) Dip 10-20 times in acid alcohol
20) Rinse in water
21) Dip 10-20 times in ammonia water
22) Rinse in water
23) Dehydrate (2min each 70%, 80%, 90%, 95%, 100%, 100% EtOH, xylene)
24) Air dry
25) Coverslip with permount
85
REFERENCES
1.
McCulloch JA, Inoue S, Moriya H et al, Clinical entities : surgical indications
and techniques, in Weinstein JN, Wiesel SW (eds) : The Lumbar Spine.
Philadelphia, PA, WB Saunders, 1990, p 393-421
2.
Spangfort EV : The lumbar disc herniation : A computer aided analysis of
2504 operations. Acta Orthop Scand 1972; 142 (suppl):1-95
3.
Frymoyer JW : Epidemiology: Magnitude of the problem, in Weinstein JM,
Weisel SW (eds) : The Lumbar Spine. Philadelphia, PA, WB Saunders, 1990,
p 32-38
4. La Rocca H : Failed Lumbar Surgery: Principles of management, in
Weinstein JN, Wiesel SW (eds) : The Lumbar Spine. Philadelphia, PA, WB
Saunders, 1990, p 871-881
5. Wiesel SW, et al. The aging lumbar spine. Philadelphia: Saunders Co, 1982:
18-33
6. Bermick S, et al. Vertebral end plate changes with aging of human vertebra.
Spine 1982; 7-79
7.
Ogata K. et al, Nutritional pathways of the intervertebral disc. Spine 1981;
6:211
8. Buckwalter JA : Spine Update : Aging and degeneration of the human
intervertebral disc. Spine 20:1307-1314, 1995 Rutkow IM : Orthopaedic
Operations in the United States, 1979 through 1983 JBJS 68-A 716-719, 1986
9. Hanley EN : Surgical Outcomes for Herniated Lumbar Disk. In Low Back
Pain:A Scientific and Clinical Overview, ed by JN Weinstein, SL Gordon,
Rosemont, AAORS, 1997, pp 113-123
10. Roberts MP : Complications of Lumbar Disc Surgery. In Lumbar Disc Disease,
ed by RW Hardy, Jr. New York, Raven Press, Ltd, 1993, pp 161-170
11. Frymoyer JW, Matteri RE, Hanley EN, et al : Failed Lumbar Disc Surgery
Requiring a Second Operation. Spine 3: 7-11, 1978
12. Hastreiter D, A Collagen-GAG Matrix for the Growth of Intervertebral Disc
Tissue, MIT, 2002
13. Maroudas A, Nutrition and metabolism of the intervertebral disc, White AA,
Gordon SL, editors. Symposium on Idiopathic Low Back Pain. St Louis: C.V.
Mosby Company; 1982, 370-390
14. Hastreiter D, Ozuna R, Spector M: Regional Variations in Certain Cellular
Characteristics in Human Lumbar Intervertebral Discs, Including the presence
86
of -smooth muscle actin, Journalof OrthopaedicResearch, 19, 597-604,
2001
15. Lee C, Grodzinsky A, Spector M: The effects of Cross-Linking of Collagen
Glycosaminoglycan Scaffolds on Compressive Stiffness, ChondrocyteMediated Contraction, Proliferation and Biosynthesis. Biomaterials 22 (2001)
3145-3154
16. Schneider TO, Mueller SM, Shortkroff S, Spector M: Expression of alphaSmooth Muscle Actin in Canine Invertebral Disc Cells in situ and in Collagen-
GAG Matrices in vitro. JOR 17: 192-199, 1999
17. Breinan HA: Development of a Collagen-GAG Analog of Extracellular Matrix
to Facilitate Articular Cartilage Regeneration. MIT, 1998
18. Mueller SM, Schneider TO, Shortkroff S, Breinan HA, Spector M: alpha
smooth muscle actin and contractile behavior of bovine meniscus cells in type
I and type II collagen-GAG matrices. J Biomed. Mater Res. 45: 157-166, 1999
19. Shultz-Torres D: Effects of Modulus of Elasticity of Collagen Sponges on
Their Cell Mediated Contraction In Vitro. MIT, 1998
20. Rutkow IM : Orthopaedic Operations in the United States, 1979 through 1983
JBJS 68-A 716-719, 1986
21. DePalma AF, Rothman RH, The Intervertebral Disc, WB Saunders company,
Philadelphia, 1970
22. Hampton D, Laros G, McCarron R, Franks D: Healing potential of the annulus
fibrosus. Spine 14:398-401, 1989
23. Key JA, Ford LT: Experimental intervertebral disc lesions. J.Bone Jt. Surg.
30-A:621-630, 1948
24. Smith JW, Walmsley R: Experimental incision of the intervertebral disc. J.
Bone Jt. Surg. 33B:612-625, 1951
25. Grande et al, JBiomed Mat Res 34:211, 1997
26. Lee CR, Spector M. Status of articular cartilage tissue engineering. Curr Op
Orthop 1998;9:88-93.
27. Ibarra et al., Trans Orthop Res Soc 23:148, 1998
28. Young et al, Trans Orthop Res Soc 22:249, 1997
29. Murray MM, Spector M. Fibroblast distribution in the anteromedial bundle of
the human anterior cruciate ligament: The presence of alpha-smooth muscle
actin positive cells. J Orthop Res 1999;17:18-27
87
30. Yannas IV. Regeneration of skin and nerves by use of collagen templates. In:
Nimni M, ed. Collagen Vol. IH: Biotechnology. Boca Raton, Florida: CRC
Press, 1989:87-115.
31. Yannas IV, Lee E, Orgill DP, Skrabut EM, Murphy GF. Synthesis and
characterization of a model extracellular matrix that induces partial
regeneration of adult mammalian skin. Proc Nat Acad Sci 1989;86:933-937.
32. Kusior et al, Tissue engineering of nucleus pulposus in nude mice, Tissue
engineering laboratory, university of Massachusetts Medical Center,
Worcester, MA01655
33. Harding, J; Zaporojan, V; Vacanti, C; Bonassar, L, Tissue engineering of a
composite intervertebral disk using human cells, poster #0828 at the 48th
Annual Meeting of the Orthopaedic Research Society.
34. Mizuno et al., Tissue engineering of a composite intervertebral disc, Trans.
Orthop. Res. Soc., 26:78, 2001.
35. Kim YJ, Sah RLY, Doong JY, Grodzinsky A, Fluorometric Assay of DNA in
Cartilage Explants Using Hoechst 33258, Analytical Biochemistry, 174:168176, 1988
36. Freed LE, Vunjak-Novakovic G: Tissue culture bioreactors: Chondrogenesis
as a model system. In: Principlesof Tissue Engineering,pp 151-165: Ed by R.
Lanza, R Langer and W Chick. Austin, TX, R.G. Landes CO. 1997.
37. Freed LE, Vunjak-Novakovic G, Langer R: Cultivation of cell-polymer
cartilage implants in bioreactors. J. Cell. Biochem. 51:257-264, 1993
38. Russo AJ, Minchew JT, Banes A, The effect of Vibration on Annulus fibrosus
cell signaling, AAOS meeting, 2002
39. Davisson T, Kunig S, Chen A, Sah R, Ratcliffe A, The effects of perfusion
and compression on modulation of tissue engineered cartilage, 4 8 th ORS
meeting, poster #0488, 2002-08-23
40. Schneider TO, Mueller SM, Shortkroff S, Spector M: Expression of -smooth
muscle actin in canine intervertebral disc cells in situ and in collagen-GAG
matrices in vitro. J. Orthop. Res. 17:192-199, 1999
88