The Effect of Mechanical Compression on
Chondrocyte Gene Expression
by
Paula M. Ragan
B.S., Mechanical Engineering,
Tufts University, 1992
M.S., Biomedical Engineering,
Boston University, 1994
Submitted to the M.I.T.-Harvard
Division of Health Sciences and Technology
in Partial Fulfillment of the
Requirements for the Degree of
SCHERING PLO"GH
MASSACHUSETTS INSTITUTE
Doctor of Philosophy
OF TECHNOLOGY
at the
Massachusetts Institute of Technology
May 1999
@
LIBRARIES
Massachusetts Institute of Technology
Signature of Author
M. I.T.-Harv rd Division of Health Sciences and Technology
May 1999
Certified by
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Thesis Supervisor
Accepted by
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-2-
The Effect of Mechanical Compression on
Chondrocyte Gene Expression
by
Paula M. Ragan
Submitted to the M.I.T.-Harvard Division of Health Sciences and Technology
in partial fulfillment of the requirements for the Degree of Doctor of Philosophy,
May 1999.
Abstract
Chondrocytes are the cells responsible for the growth, maintenance, and
repair of articular cartilage. Recent research has shown that chondrocytes are
very sensitive to mechanical forces in their environment, and they can alter
their synthesis of extracellular matrix (ECM) molecules to meet the functional
demands of loading in vivo. Presently, the mechanisms by which mechanical
forces cause changes in gene expression or other intracellular pathways that
regulate chondrocyte synthesis of matrix molecules are not known. The goal
of this thesis is to investigate the influences of mechanical compression chondrocyte gene expression (through messenger RNA analyses) and the resulting
down-stream synthesis of matrix macromolecules.
In previous studies, static mechanical compression has been shown to
decrease chondrocyte synthesis of aggrecan and type II collagen. However,
there have only been limited studies examining the effects of static loading
on chondrocyte gene expression and aggrecan and type II collagen. We have
shown that static compression can cause a down-regulation in expression of
both aggrecan and type II collagen that is dependent on the duration and
magnitude of the compression. In addition, our studies suggest that static
compression does not act to accelerate the decay of existing mRNA molecules,
rather it acts to inhibit new transcription of these mRNA molecules. Finally,
our studies have shown that inhibition of macromolecular synthesis occurs
earlier after the application of static compression then corresponding changes
in expression. This suggests that the total decrease in chondrocyte production
of aggrecan and type II collagen is only in part due to down-regulation in the
expression levels of these molecules.
Motivated by examining mechanical loads more closely resembling forces
experienced in vivo, we also examined the affects of dynamic compression on
-3chondrocyte gene expression of aggrecan and type II collagen. Previous reports
have shown that dynamic stimulation can cause significant increases in production of aggrecan and type II collagen molecules. Our series of experiments
have shown that dynamic loading can result in increased expression of aggrecan and type II collagen. Interestingly in some cases, we have seen an increase
in macromolecular synthesis of some ECM molecules without a corresponding
upregulation of mRNA expression. This phenomena appears to be related to
the bulk levels of the surrounding extracellular matrix.
Together our data show that chondrocytes are influenced by mechanical
compression at the gene-expression level. In trends similar to those previously
reported for macromolecular protein synthesis, static compression appears to
down-regulate matrix molecule mRNA expression and dynamic loading can
upregulate ECM-specific messages. However, it still remains to be determined
what intracellular mechanisms are responsible for these changes and what
initiates the cascade that ultimately leads to alterations in extracellular matrix
production.
-4-
Acknowledgments
This is the best part of my thesis: saying 'thanks' to all of the people who
helped me so much over my Ph.D. years. To everyone who reads this, pull up a
chair and grab a cold beverage, as this will take some time.
Firstly, I must thank my advisor, Alan Grodzinsky for giving me the opportunity to work with him in his lab. I've learned a great deal about research,
and more importantly, about life getting to work with Al. I appreciated his
ever-present optimism and insights, and know I'll carry many lab memories
with me in the years to come.
I'd also like to thank my thesis committee Drs. Spector, Lauffenburger,
and Housman for their help and guidance. It was a pleasure and honor to
work with such exceptional scientists. The SmithKline group, Dr. Mike Lark,
Dr. Alison Badger, and Mike Cook got the 'RNA' ball rolling and helped launch
my thesis forward. Not to mention, they're great people to work with. The
Variagenics folks, Cassie Gibson and Drs. Jim Basilion, Ling Shen, Andrea
Schievella, and Christine DeMaria were generous teachers, especially for an
engineer working with molecular biology. I really enjoyed working with Dr.
John Sandy, aggrecan king. He was an excellent mentor in my understanding
of aggrecan Western blot analysis and it's applications in cartilage research.
The folks of HST cannot go unnoticed. Dr. Gray, Dr. Gehrke, and Dr.
Mark helped guide me in my career path. I really appreciate their honesty and
their willingness to listen. I hope I can return the favor to any future students
I might encounter.
My years in the lab would have not been as great without the wonderful
people that made the lab "the lab". Firstly, I'd like to thank Vicki Chin. Her
dedication and hard work were essential to my thesis work and I am proud
to have gotten to work with her. I'd like to thank Han-hwa Hung and Linda
Bragman. They are the corner stones of the lab, keeping it moving on all levels.
Not to mention, they added a lot of fun and sunshine. I'd also like to thank Andy
Loening, my scuba partner and friend. His scientific nature always pushed me
to think harder, and not to mention, he was just plain fun to be around. Bodo,
Emerson, Fen, Cyndi, Moonsoo, Parth, and Marc were always helpful and added
color. People make the place, and everyone made the lab special.
My roommates, Michelle, Danielle, Catie and Sooz made my home away
from lab an even more wonderful place to be. I appreciate their support,
warmth, and laughter. I especially thank Brandon (the spring in my step)
for his kindness, love, and for simply listening. Outside of lab, I couldn't have
-5been more blessed being surrounded by such good people.
In life, there are always a set of people who make the difference, no matter
what the situation. It's these people I'd like to thank now. To Sol Eisenberg,
"ditto" to my master's thesis acknowledgements. I now know I was right 5 years
ago when I realized what a tremendous advisor he is. His friendship has been
invaluable. To Ernie Cravalho, I give my thanks for being so supportive and
encouraging. He took the time with me when I really needed it and always
made me feel like I could do anything if I set my mind to it. To Steve Treppo
(super fantastic super duper Steve!), I owe the best thing that came out of my
MIT experience: my friendship with him. Steve, Sandra and Markus have been
a second family to me (especially when it comes to having me over for dinner!).
To put it simply Steve, "You are my friend!".
Saving the best for last: I owe everything I have to my family. My mom
and dad have been unyielding in their support, encouragement, and love. Their
lives have set examples for me that are truly inspiring. My sister, Terry, has
given me great advice, encouragement, and a positive attitude, always lifting
my spirit. I'm lucky to have such a friend in her. My brother, Chris, has always
been a strong example of what it means to be steady. His humor has made me
laugh many times when I needed it. I look forward to more time fly fishing with
him. Mom, Dad, Terry, and Chris, I love you all very much.
This research was funded by NIH grants AR45779, AR33236, SmithKline Beecham, and Genzyme Tissue Repair.
-6-
Contents
4
Acknowledgments
I
17
Introduction
1.1 Explant Model Systems and Chondrocyte Cellular
Pathw ays . . ....
.. .. .. . .. . . .. . . . .. .. . . . . . . . 18
1.2 Mechanical Forces on Isolated Chondrocytes ................
20
II
Down-Regulation of Chondrocyte Aggrecan and Type II Collagen Gene Expression Correlates With Increases in Static Compression Magnitude and Duration
2.1 Sum m ary ................................
.
2.2 Introduction ..............................
.
2.3 Methods ........
.................................
2.4 Results .........
..................................
2.5 D iscussion . .. ....
.. .. . ...
.. . . . . .. .. . . . . . . .
III
Chondrocyte Extracellular Matrix Synthesis and Turnover in
Alginate Culture Is Influenced by Mechanical Compression
3.1 Introduction ..............................
.
3.2 Methods ........
.................................
3.2.1 Chondrocyte isolation and alginate gel culture .........
3.2.2 Evolution of cell and matrix constituents in the alginate
disk system .......
...........................
3.2.3 Compression and radiolabeling protocols ..........
3.2.4 Biochemical analysis of rates of incorporation, GAG content, total collagen, and DNA content: ............
3.2.5 Static Compression and Aggrecan Turnover ............
3.3 Results .........
..................................
3.3.1 Evolution of cell and matrix constituents in the alginate
disk system ..................................
3.3.2 Effects of Static and Dynamic Compression on 3 5S-sulfate
Incorporation ..........................
3.3.3 Effects of Compression on Aggrecan Turnover . . . . . . .
3.4 Discussion . ..........
........
.. ......
....
.
23
23
24
26
29
31
39
39
40
40
42
43
44
44
47
47
49
50
52
IV
Dynamic Loading Upregulation of mRNA Expression is a
Function of the Surrounding Extracellular Matrix
59
4.1 Introduction ..............................
. 59
4.2 Methods ........
.................................
60
4.2.1 Experiments with Explanted Tissue: ................
60
CONTENTS
-74.2.2
Experiments with Isolated Chondrocyte Culture in Alginate: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 R esults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1 Effect of Dynamic Loading Chondrocyte mRNA Levels in
Tissue Explants . . . . . . . . . . . . . . . . . . . . . ...
*.
4.3.2 Effect of Dynamic Loading on Isolated Chondrocytes Suspended in Alginate Disks . . . . . . . . . . . . . . . . . . . .
4.4 D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
mRNA Stability is Unaltered during Static Mechanical ComV
pression
.
5.1 Introduction ..............................
5.2 Methods ........
.................................
5.2.1 Effects of Blocked Transcription on Down-Stream Protein
Synthesis: . . . .. .. . . . . . . . . . . . . . . . . . . . . .
5.2.2 mRNA Stability in Isolated Chondrocytes Cultured in Alginate Disks: ..................
.........
5.3 Results .........
..................................
5.4 D iscussion . .. ........
. . . . . .. . . . .. . . . . .. . . .
A
62
64
64
68
70
72
72
73
73
74
75
78
Alginate Material Properties are Stable in Long-Term Culture 83
A. 1 Introduction ..............................
. 83
A.2 Methods ........
.................................
83
A.3 Results and Discussion ...............................
85
B
Human Chondrocytes In Native Tissue, and as Primaries and
90
Dedifferentiated Cells
B. 1 Introduction ..............................
. 90
B.2 Methods ........
.................................
90
B.2.1 Human Chondrocytes in Native Tissue ..............
90
B.2.2 Primary Human Chondrocytes in Alginate Disks ......
91
B.2.3 Dedifferentiated Human Chondrocytes in Alginate Slabs
or Cylinders ..........................
. 92
B.3 Results and Discussion ...............................
92
B.3.1 Chondrocytes Maintained in Native Tissue ...........
92
B.3.2 Human Primary Cells Cultured in Alginate Disks: A Time
Course Study ..........................
93
B.3.3 Dedifferentiated Human Chondrocytes Cultured in Alginate 99
B.4 Conclusions .......
...............................
104
C
Response of a Cartilagenous Tissue Construct to Dynamic
Compression
107
C. 1 Introduction .......
...............................
107
CONTENTS
-8........
.. . 108
........
C.2 M ethods . .............
C.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . 108
C.4 Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
CONTENTS
-9-
List of Figures
1.1
Chpndrocytes synthesize and maintain the macromolecules
which form the bulk extracellular matrix (ECM) of articular cartilage. Aggrecan and type II collagen, as well as other macromolecules, cytokines, and degradative enzymes can influence cell
metabolism. Mechanical compression can influence the synthesis
of these components, and can mediate chondrocyte metabolism of
the E C M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
2.1
Schematic of the cartilage disk explant protocol. Larger 12-mm
plugs are cored from the medial and lateral femoropatellar grove.
Four 3-mm disks are punched from the 1-mm by 12-mm diameter
cores. Groups are matched for disk depth and location. . . . . . .
2.2 Expression of aggrecan and type II collagen mRNA in response
to static compression as assessed by Northern blot. Expression of
A) aggrecan G1 domain; B) collagen type Ila; and C) GAPDH for
normalization was evaluated at the compressed thickness shown.
Lanes 1 through 4 show increasing amplitudes of static unconfined compression (1.15-mm (FS) to 0.5-mm); compression duration was 24 hours. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 Aggrecan and type II collagen gene expression and biosynthesis in response to graded static compression of cartilage disks.
A) mRNA levels for aggrecan E and collagen type Ila El were
quantified and normalized to GAPDH as a function of increasing
amounts of static unconfined compression. Values are reported as
mean +/- SD (n=3) normalized to the 1.15-mm (free-swell) value.
* p < 0.05 vs 1.00-mm. B) 3 5S-sulfate E and 3 H-proline 1I incorporation into macromolecules as a function of increasing amounts
of static unconfined compression. Values are reported as mean
+/- SD (n=10) normalized to the 1.15-mm (free swell) value. * p
< 0.05 vs 1.00-mm; * p < 0.0001 vs all other thicknesses. The
correlation coefficients, r, relating mRNA expression to radiolabel incorporation rates were calculated for aggrecan and type IIA
collagen. The values are: raggrecan = 0.90; rcoIlagenIIA = 0.89. . . . .
2.4 Northern blot analysis for one of three similar experiments to
examine kinetics of mRNA change due to 50% static compression
using bovine probes for: A) aggrecan G1 domain; B) collagen
type Ia; C) iNOS; and D) GAPDH for normalization. Lanes 1
through 4 show increasing duration of compression for disks held
at 0.5-mm thickness (0 hrs (control) to 24 hrs). . . . . . . . . . . .
26
32
33
34
LIST OF FIGURES
-102.5
Aggrecan and type II collagen gene expression and biosynthesis
in response to increased duration of static compression to 0.5-mm
thickness. A) mRNA levels for aggrecan G1 El and collagen type
Ha E were quantified and normalized to GAPDH as a function
of increased duration of compression. Values are reported as
mean +/- SD (n=3) normalized to the 0 hour (control) value. *
p < 0.005 vs 0.5 hours; ** p < 0.005 vs 0.5 and 4 hrs; * p <
0.05 and 4 hrs. B) 3 5S-sulfate EL and 3 H-proline l incorporation
into macromolecules as a function of increasing compression time.
Values are reported as mean +/- SD (n=10) normalized to the 24
hour free-swell (control) value. * p < 0.001 vs 0 hrs, * p < 0.01 vs
0 and 4 hrs, * * p < 0.01 vs 0 and 4 hrs. . . . . . . . . . . . . . . .
35
3.1
Alginate casting frame. A) Exploded view showing the steel casting frame (thickness = 1.5 mm) sandwiched between Whatman 2
filter paper supported by an 80 pm polyester mesh. B) The mesh,
filter paper and frame are clamped together and the alginate with
cells is injected into the 1.5 mm space between the two pieces of
filter paper. The casting frame with alginate is then submerged
into a bath of 102 mM CaCl 2 , 0.15 M NaCl for 10 minutes to
polymerize the alginate. . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Schematic diagram showing the epitope sites used for analysis
of the aggrecan core protein. ATEGQV and CDAGWL (HAL)
are used to examine the presence of the G1 region. VDIPES
and NITGE are sites of aggrecanase activity in the interglobular
domain. TFKEEE is used to assess aggrecanase activity in the
CS-region of the core protein. LEC-7 detects the presences of the
G 3 dom ain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3 Cell number and matrix constituents as a function of time in culture (n=4/time point, avg +/-SD). A) Cell number was calculated
from DNA content using 7.9 X 10-12 g/cell [42] on days 0, 3, 6,
13, and 20. B) Total collagen content in one 6-mm alginate disk
(n=4/time point) was measured from total hydroxyproline content
[14] on days 0, 3, 6, 13, and 17. C) Total GAG was measured using DMMB reactivity methods on days 0, 3, 6, 13, 20, 35 and
49. GAG content (pg GAG) was normalized to DNA content (pg
DNA). Corrections for the alginate background were made [131.
D) Macromolecular 3 5S-sulfate incorporated into the disk and released into the media was measured on days 0, 3, 6, 13, 20, 35,
and 49. Incorporation rates were normalized to DNA content. . .
41
47
48
LIST OF FIGURES
-113.4
3.5
3.6
3.7
The effect of static compression on chondrocyte proteoglycan synthesis. 31S-sulfate incorporation into alginate disks decreased
with increasing static compression. Maximal inhibition of macromolecular synthesis occured at compression levels between 60%
and 80% of the disks original thickness (* p < 0.05 vs FS; * p <
0.01 vs FS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The effect of dynamic mechanical compression on chondrocytes
grown in alginate disks. A sinusoidal dynamic compression (0.5
Hz, 4% dynamic amplitude) superimposed on a 80%static compression increased 3 5S-sulfate incorporation in alginate disks by
1.9 times than in control disks subjected to 80% static compression alone (* p < 0.05 vs DYN and FS). . . . . . . . . . . . . . . .
Western analysis of media samples from alginate disks cultured
for 40 hours at 75% of original disk thickness, 100% of original
thickness, and under free swelling conditions. Media was pooled
from 12 wells of the compression chamber or culture dish for
each condition. Samples were analyzed with anti-CDAGWL (H)
and anti-KEEE (E). Release of full length (400 kDa) aggrecan
core protein and core protein fragments (300 kDA and 220 kDA)
consistent with CS-domain aggrecanase activity are evident in
all three conditions. . . . . . . . . . . . . . . . . . . . . . . . . . .
Western analysis of the cell-associated matrix of chondrocytes cultured in alginate disks for 40 hours at 75% of original disk thickness, 100% of original thickness, and under free swelling conditions. Disks (n=12) were pooled, dissolved in a calcium chelating
agent, and cells were pelleted via centrifugation. Samples were
analyzed with anti-CDAGWL (H), anti-ATEGQV (A), anti-KEEE
(E), an 8-A-4 monoclonal antibody for link protein (L), and LEC7 (3) for the G3 domain. Full length aggrecan core protein (400
kDa), a link protein doublet band (60 kDA) and core protein fragments consistent with normal chondrocyte catabolism (300 kDa)
were evident for all conditions. There was no reactivity with the
anti-NITEGE or anti-VDIPEN epitopes in all conditions. . . . . .
49
50
51
53
LIST OF FIGURES
-123.8 Western analysis of the further-removed matrix of chondrocytes
cultured in alginate disks for 40 hours at 75% of original disk
thickness, 100% of original thickness, and under free swelling
conditions. Disks (n=12) were pooled, dissolved in a calcium
chelating agent, and the further-removed matrix was separated
from cells via centrifugation. The supernatants were subject
to analysis with anti-CDAGWL, anti-NITEGE and anti-KEEE.
The FRM contained only small G1 fragments (detected with antiCDAGWL) that result from proteolysis of the interglobular dom ain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1
4.2
4.3
Expression of aggrecan and type II collagen mRNA in response to
static (S) and dynamic (D) compression as assessed by Northern
blot. Expression of mRNA for A) aggrecan G1 domain; B) collagen
type Ila; C) GAPDH and D) EF-1a for normalization was evaluated for each condition. The blots shown are characteristic of the
three repeats of the experimental protocol. Dynamic compression of 0.01 Hz, 4% dynamic strain amplitude was superimposed
on a static compression to 1.0-mm (cut thickness); compression
duration was for 12 hours. . . . . . . . . . . . . . . . . . . . . . . .
Aggrecan and type II collagen gene expression and biosynthesis
in response to 12 hours of dynamic loading of cartilage disks. A)
mRNA levels for aggrecan LI and collagen type IIA LI were quantified and normalized to GAPDH. Values are reported as mean +/SD (n=3) normalized to the static (STAT) control value. B) 35Ssulfate L and 3 H-proline LI incorporation into macromolecules.
Values are reported as mean +/- SD (n=20-24) normalized to the
static control. * p < 0.05 vs static control for 3 H-proline incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Expression of aggrecan and type II collagen mRNA in response
to static (S) and dynamic (D) compression as assessed by Northern blot. Expression of A) aggrecan G1 domain; B) collagen type
Ila; and C) EF-la for normalization was evaluated for each condition. Dynamic loading of 0.1 Hz, 4% dynamic amplitude was
superimposed on a static compression to 1.0-mm (cut thickness);
compression duration was for 48 hours. Lanes 1 through 4 represent 2 repeats of the loading protocol, with static (S) and dynamic
(D) conditions alternating lanes. . . . . . . . . . . . . . . . . . . .
54
65
66
67
LIST OF FIGURES
-134.4
Aggrecan and type II collagen gene expression and biosynthesis
in response to 48 hours of dynamic loading of cartilage disks.
A) mRNA levels for aggrecan D and collagen type Ia E were
quantified and normalized to EF-1a. Values are reported as mean
+/- SD (n=3) normalized to static (STAT) control value. B) 35S_
sulfate l and 3H-proline l incorporation into macromolecules.
Values are reported as mean +/- SD (n=18) normalized to the
static control. * p < 0.001 vs static control. . . . . . . . . . . . . .
4.5 Expression of aggrecan and type II collagen mRNA in response to
static (S) and dynamic (D) compression in chondrocytes cultured
in alginate disks as assessed by Northern blot. Expression of
A) aggrecan G1 domain; B) collagen type Ila; and C) GAPDH
for normalization was evaluated for each condition. Dynamic
loading of 0.1 Hz, 4% dynamic amplitude was superimposed on a
90% static compression; compression duration was for 20 hours.
Lanes 1 through 6 represent 3 repeats of the loading protocol,
with dynamic (D) and static (S) conditions alternating lanes. . .
4.6 Aggrecan and type II collagen gene expression of chondrocytes
in alginate in response to 20 hours of dynamic loading. mRNA
levels for aggrecan El and collagen type Ia l were quantified and
normalized to GAPDH. Values are reported as mean +/- SD (n=3)
normalized to static (STAT) control value. In all cases, aggrecan
mRNA was greater in the dynamic condition compared to static
controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1
The biosynthetic response of chondrocytes in native explants to
two dosage levels of actinomycin D. 1-mm by 3-mm diameter
cartilage disks were cultured for 1, 4, 9, 12 and 24 hours in 0,
10pg/ml and 50pg/ml. Disks were radiolabeld for 1 hour prior to
being removed from culture with 3 5S-sulfate (10[Ci/ml) and 3 Hproline (20piCi/ml). Disks (n=4/condition) were then removed and
washed 4 by 15 minutes in PBS to remove unincorporated label.
Disks matched for joint depth and location were used for each
time point group. Values are normalized to the control (0 [g/ml)
for each time point. A) kinetics of 3 5 S-sulfate incorporation; B)
kinetics of 3 H-proline incorporation. . . . . . . . . . . . . . . . . .
67
68
69
76
LIST OF FIGURES
-14The biosynthetic response of chondrocytes in native explants subjected to mechanical compression and dosed with actinomycin
D. 1-mm by 3-mm diameter cartilage disks were cultured for 1,
2, 5, 11 and 24 hours in either 0 pg or 10 p/g of actinomycin
D with or with a superimposed 75% static compression. Disks
were radiolabeld for 1 hour prior to being removed from culture with 3 5S-sulfate (l0pCi/ml) and 3 H-proline (20Ci/ml). Disks
(n=4/condition) were then removed and washed in PBS to remove
unincorporated label. Disks matched for joint depth and location
were used for each time point group. Values are normalized to the
control (0 pg/ml Free Swelling) for each time point. A) kinetics of
35
S-sulfate incorporation; B) kinetics of 3 H-proline incorporation.
5.3 Northern blot analysis of chondrocytes cultured in alginate disks
and dosed with 10 pg of actinomycin D for 1, 2, 4, 8, 12 14 and
18 hours. After time in free swelling culture, total RNA was
extracted using the Qiagen RNeasy protocol for animal cells. Approximately 2 pg of RNA was run in each lane. Expression of A)
both aggrecan (9.0 kb) and GAPDH (1.4kb); B) collagen type IIa;
and C) EF-1 was evaluated at each time point. D) 28S (ethidium bromide) was used for normalization. Each lane represents
increasing times in drug-treated culture. . . . . . . . . . . . . . .
5.4 Kinetics of changes in mRNA levels after transcription-inhibition
via actinomycin D. Values are normalized to 28S. Increasing stability is seen with A) aggrecan and B) collagen type Ha. C) Elongation factor-la remained unchanged. D) GAPDH decreased to
less than half of its starting levels. . . . . . . . . . . . . . . . . . .
5.2
A. 1 Equilibrium stress-strain behavior for alginate disks from day 0
of culture. The dynamic stiffness and streaming potential were
measured in the linear region of the data (30% static strain). . .
A.2 Equilibrium confined compression modulus HA of alginate disks
versus time in culture. These were calculated from the slope of
the stress-strain curve in the linear region of the data for each
sam ple. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A.3 Amplitude of the streaming potential for frequencies ranging
from 0.02 to 1.0 Hz, at four time point during a 7 week culture of
alginate. Displacement amplitude was 45pm. . . . . . . . . . . . .
A.4 Amplitude of the dynamic stiffness of alginate at frequencies between 0.02 and 1.0 Hz at days 0, 15, 29, 56 in culture. Displacement amplitude was 45pm. . . . . . . . . . . . . . . . . . . . . . .
77
78
79
86
87
87
88
LIST OF FIGURES
-15A.5 Dedifferentiated human chondrocytes (2 nd passage) were cast in
alginate slabs and cultured for up to 56 days. At days 0, 15, 29,
and 56, the mechanical integrity of newly synthesized matrix was
assessed. No significant differences in equilibrium modulus, HA,
and streaming potential were evident between alginate samples
with and without cells (data not shown). Significant differences
in dynamic stiffness were evident at all frequencies (p < 0.005).
B. 1 Total GAG content of human cartilage explants maintained in
culture for up to eleven days. Glycosaminoglycan content was
assess using the DMMB dye method. . . . . . . . . . . . . . . . .
B.2 Aggrecan synthesis (as measured by macromolecular sulfate incorporation) as a function of time in culture. . . . . . . . . . . . .
B.3 Total protein synthesis (as measure by macromolecular proline
incorporation) as a function of time in culture. . . . . . . . . . . .
B.4 Cartilage disks (1-mm by 3-mm diameter) were cored from human
cartilage and statically compressed to either 1.0-mm (control) or
to 0.5-mm (50% compression). Compression lasted either 12 or
24 hours, with 3 5S-sulfate incorporation assessed during the last
12 hours of culture. . . . . . . . . . . . . . . . . . . . . . . . . . . .
B.5 Cartilage disks (1-mm by 3-mm diameter) were cored from human
cartilage and statically compressed to either 1.0-mm (control) or
to 0.5-mm (50% compression). Compression lasted either 12 or
24 hours, with 3 H-proline incorporation assessed during the last
12 hours of culture. . . . . . . . . . . . . . . . . . . . . . . . . . . .
B.6 Cell density of primary human chondrocytes in alginate disks
versus time in culture. Cell number was calculated from DNA
content using 7.9 X 1012 g/cell [42]. . . . . . . . . . . . . . . . . .
B.7 Total GAG content accumulated by primary human chondrocytes
in alginate versus time. GAG content was assessed using a modified DMMB method that accounted for the background charge of
the alginate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
B.8 Macromolecular 31S-sulfate incorporation into the alginate disk
and released into the media versus time in culture. The multiscreen 3 5S-sulfate incorporation assay [52] was used in analysis.
B.9 Cell density of dedifferentiated human chondrocytes in alginate
disks versus time in culture. Chondrocytes were initially seeded
at 1 X 105 or 1 X 106 cells/ml. Cell number was calculated from
. . . . . . . . . . . . .
DNA content using 7.9 X 10-12 g/cell [42].
35
B. 10 Macromolecular S-sulfate incorporation into the alginate disk
and released into the media versus time in culture. The multiscreen 3 5S-sulfate incorporation assay [52] was used in analysis.
89
94
95
96
97
98
99
100
101
102
103
LIST OF FIGURES
-16B. 11 Cell density of dedifferentiated human chondrocytes in alginate
disks versus time in culture. Chondrocytes were initially seeded
at 6 X 10' cells/ml. Cell number was calculated from DNA content
using 7.9 X 10-1 g/cell [42]. . . . . . . . . . . . . . . . . . . . . . . 104
B. 12 3 5S-sulfate incorporation by dedifferentiated human chondrocytes
into the alginate disk and released into the media versus time in
culture. The multiscreen 31S-sulfate incorporation assay [52] was
used in analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
C.1
35
S-sulfate incorporation of chondrocytes grown in ARC method
subjected to dynamic, static and free swelling conditions. The top
figure shown radiolabel incorporation into the cartilage-like disk.
The bottom figure shows macromolecular radiolabel released into
...........
the media during culture . . . . . . . . . . ..
3
C.2 Top: Total 1S-sulfate incorporation of chondrocytes grown in
ARC method subjected to dynamic, static and free swelling conditions. Bottom: Total GAG normalized to DNA content of the
cartilage-like disks for each compression condition. . . . . . . . .
C.3 3 5S-sulfate incorporation of chondrocytes grown in ARC method
subjected to dynamic, static and free swelling conditions. The top
figure shown radiolabel incorporation into the cartilage-like disk.
The bottom figure shows macromolecular radiolabel released into
the media during culture. This is a repeat of the first series of
experim ents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C.4 Top: Total 3 5S-sulfate incorporation of chondrocytes grown in
ARC method subjected to dynamic, static and free swelling conditions. Bottom: Total GAG normalized to DNA content of the
cartilage-like disks for each compression condition. . . . . . . . .
110
111
112
113
LIST OF FIGURES
-17-
Chapter I
Introduction
Articular cartilage covers the bony surfaces in synovial joints and functions to withstand mechanical loads and reduce friction and wear. It is avascular, aneural, alymphatic and highly hydrated. This connective tissue is primarily comprised of extracellular matrix (ECM) molecules, which include proteoglycans (PGs), glycoproteins,
and collagens. Aggrecan, the proteoglycan found most abundantly in articular cartilage, has a large core protein (400 kDa) to which glycosaminoglycan (GAG) chains
are attached. The negatively charged GAG chains provide cartilage's resistance to
compressive forces normally occurring during joint loading. Type II collagen is the
dominant collagen type in articular cartilage. Type II collagen molecules are homotrimers of a single type of alpha chain. Molecules of types II, IX, and XI together
form the heteropolymeric fibrils, which account for the most of the tensile strength
of the tissue. Aggrecan and the type II collagen network, along with other matrix
molecules, form the functional matrix of cartilage that withstands static and dynamic
loads associated with physiologic conditions.
Articular cartilage is synthesized and maintained by a specialized cell population known as chondrocytes 1.1. Recent research has shown that chondrocytes are
very sensitive to mechanical forces in their environment, and they can alter their synthesis of extracellular matrix (ECM) molecules to meet the functional demands of in
vivo loading [68, 43]. Presently it is not known how mechanical forces cause changes
in the gene expression or other intracellular pathways that regulate chondrocyte synthesis of ECM molecules. One difficulty impeding such investigations has been the
technical challenges associated with isolating critical intracellular molecules, such as
message RNA (mRNA) for analysis of gene expression [1] due to the sparse cell population of chondrocytes in cartilage. Yet another challenge has been the inability
to isolate living chondrocytes immediately following exposure to physiologic loading
Chapter I
-18conditions for analysis of other signal transduction marker molecules. Some progress
Collagen
GAG chain
PG Iy
/
*(
11%) '4
cytet
Chondro
Figure 1.1: Chpndrocytes synthesize and maintain the macromolecules which form
the bulk extracellular matrix (ECM) of articular cartilage. Aggrecan and type II
collagen, as well as other macromolecules, cytokines, and degradative enzymes can
influence cell metabolism. Mechanical compression can influence the synthesis of
these components, and can mediate chondrocyte metabolism of the ECM.
has been made to elucidate the influence of mechanical forces on chondrocyte gene
expression and intracellular pathways using various explant or isolated cell culture
systems. The following sections describe previous research that has focused on such
investigations.
1.1
Explant Model Systems and Chondrocyte Cellular
Pathways
Tissue explant systems have been used extensively to elucidate intracellular pathways for chondrocyte synthesis of extracellular matrix molecules [30, 53] and how
these pathways may be regulated. Research has shown that rates of synthesis are
Section 1.1
-19sensitive to media, pH, potassium and sulfate concentrations and to the concentrations of serum and various growth factors [53, 24, 8]. Mechanical forces also affect
chondrocyte metabolism. Increased levels of static compression has been shown to
decrease synthesis of aggrecan and total protein either as measured by
3 5S-sulfate
and 3 H-proline radiolabel incorporations [24, 68, 411, whereas application of dynamic
compression at frequencies 0.01 to 1 Hz and
1-5% dynamic amplitude have been
shown to increase synthesis of aggrecan and total protein [68, 43].
Others investigations have focused more specifically on the interplay between
mechanical compression and aggrecan synthesis.
The aggrecan core protein con-
tains three globular domains, G1, G2 and G3, and intervening regions to which glycosaminoglycan (GAG) chains and oligosaccharides are attached [82]. Synthesis of
aggrecan involves RNA-dependent synthesis of the core protein on ribosomes of the
rough endoplasmic reticulum (ER) followed by post-translational events in which
GAG chains and oligosaccharides are enzymatically added to the polypeptide core.
Previous experiments have shown that static compression inhibits the synthesis of new
core protein, but does not seem to alter the processing rate of existing core protein
and addition of GAG chains [41] These results suggest that mechanical compression
can influence aggrecan synthesis at the transcription level or prior to translation of
the core protein.
Additional studies in which aggrecan RNA transcription was inhibited using
actinomycin D (which blocks total RNA transcription) in bovine cartilage explants
showed an exponential decay in aggrecan synthesis measured by
ration [54]. The presence of benzyl
#-xyloside
3 5 S-sulfate
incorpo-
(when added to actinomycin D dosed
media) delayed the inhibition of synthesis by approximately 10 hours, but ultimately
aggrecan synthesis decreased at the same rate as non-#-zyloside treated controls [54].
Therefore, the decrease in aggrecan synthesis could be a result of inhibition of mRNA
encoding for the aggrecan core protein or mRNA encoding for other enzymes necessary for GAG synthesis, such xylosyl transferase or glycosyl transferases [41].
Section 1.1
-20Ultimately, mechanisms by which mechanical compression affects chondrocyte
aggrecan and type II collagen gene expression in cartilage explants still remains to be
understood. However recent study of gene expression in explant cultures of bovine
tendons implicated the importance of mechanical compression and expression of aggrecan and type II collagen messages [65]. Investigators harvested bovine fetal and
adult deep flexor tendons (DFT) which have regions which are exposed to predominantly tensile and compressive forces. The goal of the study was to determine if
any differences in gene expression existed between adult and fetal tissue and in the
compressed versus stretched regions of the tendon [65]. Using isolated total RNA and
Northern blot techniques with probes for aggrecan, type II collagen and GAPDH,
Robbins and Vogel showed that aggrecan and type II collagen mRNA levels were at
least 50 times larger in the compressed regions of the adult DFT compared to regions loaded in tension in the same adult tendon, and also compared to both tension
and compression regions in fetal tissue. This strongly supports the hypothesis that
mechanical forces can alter cell phenotypic expression.
Most recently, there have been a reports of the influence of mechanical compression on aggrecan mRNA levels in bovine cartilage explants [78, 81, 63]. Static
compression applied for times greater than 12 hours caused a 50% decrease in normalized values of aggrecan mRNA [81, 63]and in type II collagen mRNA as compared
to uncompressed controls [63].
In addition, normalized
3
1S-sulfate
and 3 H-proline
incorporation as markers for aggrecan and type II collagen synthesis, respectively,
decreased in compressed tissue versus controls with trends similar to those found
in mRNA levels [63]. However, more rigorous studies are necessary to elucidate how
mechanical forces affect stability and synthesis of the mRNA inside the chondrocytes.
1.2
Mechanical Forces on Isolated Chondrocytes
Because of the technical difficulty and the hours required for isolating viable chondrocytes from explanted tissue, it is often difficult to measure intracellular marker
Section 1.2
-21molecules that rapidly degrade. Therefore, researchers have turned to initially isolating chondrocytes from native tissue, plating them in high density ( 2 X 10' cells/cm 2 )
monolayer cultures, and then subjecting the cells to various mechanical forces, such
as fluid shear [75] hydrostatic [80, 76] or tensile forces [38]. While these systems are
useful models, there have been reports of altered gene expression or protein synthesis
for chondrocytes cultured in such conditions as compared to chondrocytes suspended
in agarose gels or in native tissue [45, 36]. Specifically, Hering et al reported drastic
decreases in type II collagen, link protein, and aggrecan gene expression during the
first 5 days of monolayer culture, and also show an increase in mRNA for type I
collagen and decorin which more characteristic of fibroblastic cell lines [36] .
Isolated chondrocytes seeded in three-dimensional matrices have been reported
to maintain chondrocyte morphology and phenotype that closely resembles that of
chondrocytes in native tissue [29, 56, 45, 11, 32, 48, 64]. When seeded in gel matrices such as agarose and alginate, cells retain their rounded shape but undergo some
mitoses, as demonstrated by a 50-100% increase in cell number over the initial three
to four weeks of culture [45, 11, 64]. Typically by week 4 of culture, chondrocyte
synthesis rates of proteoglycans and collagens approach those of chondrocytes in native cartilage, and total GAG and collagen content increase over the course of the
culture confirming matrix synthesis [11, 64]. More detailed studies of chondrocytes
in alginate have shown production of aggrecan as part of cell-associated matrix production [56]. These studies demonstrate the effectiveness of chondrocyte suspension
cultures in retaining metabolic and morphologic states that more closely resemble
that of chondrocytes in articular cartilage.
Chondrocyte/agarose cultures have been used to investigate the influence of
mechanical compression on isolated cells during culture [20, 11, 48]. Using the agarose
system, Buschmann and colleagues have shown that chondrocyte 3"S-sulfate and 3Hproline incorporation rates decrease with increasing amounts of static compression.
Compressive effects were more pronounced and similar to native cartilage at day 41
Section 1.2
-22compared to day 2 in culture. They also demonstrated that dynamic compression at
frequencies and amplitudes similar to previous cartilage explant experiments stimulated chondroctye production of proteoglycans and collagen; again more pronounced
effects were evident at later times (day 25 versus day 4) in culture [11].
Lee and
Bader demonstrated stimulatory effects of dynamic loading on 3 H-thymidine uptake,
suggesting that chondrocyte mitosis is upregulated by dynamic compression frequencies ranging between 0.3 Hz to 3 Hz [48]. Together, these studies have shown that
both static and dynamic loads can affect isolated chondrocyte metabolism and extracellular matrix synthesis similar to effects demonstrated with cartilage explants. The
advantages of the agarose system relate to maintaining chondrocyte morphology;
however cells maintained in agarose, like those within cartilage, cannot be rapidly
released from the gel matrix for intracellular analyses.
Other models systems for
isolated chondrocyte culture are necessary for further investigation of the affects of
mechanical forces on intracellular pathways of ECM molecule synthesis.
Section 1.2
-23-
Chapter II
Down-Regulation of Chondrocyte Aggrecan and
Type II Collagen Gene Expression Correlates
With Increases in Static Compression Magnitude
and Duration
2.1
Summary
The goal of this study was to examine the simultaneous effects of mechanical compression of chondrocytes on messenger RNA (mRNA) expression and macromolecular
synthesis of aggrecan and type II collagen. Bovine cartilage explants were exposed
to different magnitudes and durations of applied mechanical compression and levels of aggrecan and type Ila collagen mRNA normalized to GAPDH were measured
and quantified by Northern blot. Synthesis of aggrecan and type II collagen protein was measured using radiolabel incorporation of "S-sulfate and 3 H-proline into
macromolecules.
Results showed a dose-dependent decrease in mRNA levels for aggrecan and
type II collagen with increasing compression relative to physiological cut thickness
applied for 24 h.
Radiolabel incorporation into glycosaminoglycans (GAGs) and
collagen also decreased with increasing compression in a dose-related manner similar
to the changes seen in mRNA expression. The modulation of both aggrecan and
type II collagen mRNA and protein synthesis were dependent on the duration of
the compression. Aggrecan and type II collagen mRNA expression increased during
the initial 0.5 hours of static compression, but by 4 to 24 hours after application
of compression, total mRNA levels had significantly decreased.
The synthesis of
aggrecan and collagen protein decreased more rapidly than the rate of decrease in
ChapterII
-24mRNA levels after the application of a step compression.
Together, these results
suggest that mechanical compression rapidly alters chondrocyte aggrecan and type
II collagen gene expression rapidly upon application of load. However, our results
indicate that the observed decreases in biosynthesis may not be related solely to
changes in mRNA expression. The mechanisms by which mechanical forces affect
different segments of the biosynthetic pathways remain to be determined.
2.2
Introduction
Articular cartilage functions to withstand compressive load and allow fluid joint articulation. Like other biological tissues, cartilage can adapt its structure to meet the
functional demands of its surrounding environment. For example, moderate cyclic
loading has been observed to stimulate proteoglycan synthesis and accumulation in
vivo [34]. In contrast, reduced joint loading or immobilization results in more rapid
degradation and loss of extracellular matrix (ECM) and decreased synthesis of ECM
macromolecules [5, 67]. These changes in cartilage matrix turnover may ultimately
result in loss of articular cartilage, causing joint pain, decreased mobilization, and
significant morbidity.
The functional mechanical properties of cartilage depend on the composition
and quality of the ECM. Cartilage contains aggregating and non-aggregating proteoglycans, collagen fibrils and non-collagenous proteins. These molecules are distributed
in defined patterns throughout the cartilage matrix and interact to efficiently maintain water balance and prevent collapse of the tissue. These ECM components are
synthesized, deposited, and maintained by articular chondrocytes.
Recent studies
have shown that chondrocytes are very sensitive to mechanical forces and that they
respond by altering the synthesis of ECM molecules, such as aggrecan, type II collagen, link protein, and hyaluronan [24, 68, 41, 28]. Additionally, studies have shown
that mechanical compression can lead to the restructuring of intracellular organelles
such as the rough endoplasmic reticulum and golgi apparatus which are involved in
Section 2.2
-25the synthesis of the ECM [26].
Mechanical forces can also lead to changes in chondrocyte gene expression. In
recent studies [80, 76, 38], the effect of various mechanical forces on gene expression
were investigated using chondrocytes in monolayer culture. For example, static and
intermittent hydrostatic pressure increase the expression of transforming growth factor 3-1 (TGF-31) as well as aggrecan and type II collagen mRNA in high-density
monolayer cultures [80, 76]. Additionally, constant fluid shear forces stimulate expression of mRNA for tissue inhibitor of metalloproteinase-1 (TIMP-1) in isolated
human chondrocytes grown in monolayer [75]. Dynamic mechanical forces have also
been shown to influence matrix gene expression. When isolated bovine and human
chondrocytes are cyclically stretched on flexible membranes, aggrecan and type II collagen mRNA expression are increased [38]. Isolated chondrocyte systems are useful
models for investigating chondrocyte response to mechanical load. However, extrapolating information obtained from isolated cells in monolayer culture to chondroctyes
maintained in their native tissue is difficult due to the complex physicochemical interactions that exist between the chondrocyte and the ECM in vivo.
Presently, there is limited information regarding the effects of compression on
chondrocyte gene expression within native articular cartilage. One study reported
that static loads of 0.1 MPa applied for 1 hour can transiently increase levels of
chondrocyte aggrecan mRNA [81]. In addition, aggrecan mRNA levels are found to be
independent of the magnitudes of applied stress during 24 hours of static compression
[81]. In contrast, many previous studies have reported a dose-dependent reduction of
aggrecan synthesis (as measured by
3
1S-sulfate
incorporation) with increased amounts
static compression [73, 24, 68, 43, 28]. The goal of the present study was to examine
the influence of static mechanical compression on chondrocyte synthesis of aggrecan
and type II collagen both at the protein and mRNA levels. Our data suggest that
mechanical compression can modulate both chondrocyte gene expression, as well as
post transcriptional biosynthesis.
Section 2.2
-26-
2.3
Methods
Explant and Culture: Cylindrical disks of cartilage were prepared as previously
described by Sah et al (Fig.
2.1)[68]. Briefly, the saddle sections of 1-2 week old
calves were obtained from a local abattoir within 4 hours after slaughter (Research '87,
Boston, MA). Four to five cylindrical cores of cartilage and underlying bone (9.5-mm
in diameter and 20-mm deep) were drilled from each facet (medial and lateral) of the
femoropatellar groove. During the process, the cartilage was kept moist using sterile
phosphate buffered saline (PBS) supplemented with antibiotics (100 U/ml penicillin,
100 pg/ml streptomycin, and 0.025 gm/ml amphotericin B).
'xCyHinders
Cartilag e-one
1mm Thick
3mm Diameter
2
CARTILAGE DISKS
G D
~eeee
ee e 5
CALF ARTICULAR
CARTILAGE
femoropatellar groove
Figure 2.1: Schematic of the cartilage disk explant protocol. Larger 12-mm plugs are
cored from the medial and lateral femoropatellar grove. Four 3-mm disks are punched
from the 1-mm by 12-mm diameter cores. Groups are matched for disk depth and
location.
Each cartilage-bone core was then inserted into the sample holder of a sledge
microtome (Model 860, American Optical, Buffalo, NY). After removing the superficial layer of cartilage to obtain a level surface, two 1-mm thick plane-parallel cartilage
slices were dissected and placed in 1-ml PBS with antibiotics. From these slices (9.5
mm diameter, 1-mm thick), four 3-mm disks were obtained using a sterile dermal
punch (Miltex Instruments, Lake Success, NY) giving a total of 76 disks from each
joint surface. The disks were incubated in medium (Dulbecco Modified Eagle Medium
(DMEM) with 10 mM HEPES, 10% fetal bovine serum (FBS), 0.1 mM nonessential
Section 2.3
-27amino acids, antibiotics as described previously, 1 mM sodium pyruvate, an additional 0.4 mM proline and 20 pg/ml ascorbate) at 370 C in 5% CO 2 . The total time
from slaughter to incubation was < 8 hours. Disks were cultured for four days and
the medium was changed daily.
Static Compression: Dose Dependence:
Using specially designed polysul-
phone chambers described previously [68], cartilage disks were uniaxially compressed
in a radially unconfined manner.
Cartilage disks were maintained between fluid-impermeable platens at their
free swelling thickness of 1.15-mm or compressed to 1.0 mm (cut thickness), 0.7-mm,
and 0.5-mm. Each disk was bathed in > 100 volumes of medium and maintained in an
incubator at 370 C and 5% CO 2 for 24 hours. Cartilage disks in each condition were
matched for joint position and tissue depth, as previous studies have reported joint
location dependent variations in chondrocyte response to mechanical compression
[68].
After compression for 24 hours, disks ( 300 mg: 32 disks/condition) were flashfrozen in liquid nitrogen within 1 minute of removal from the compression chambers,
and stored at -800 C prior to RNA isolation for Northern blot analysis. Total RNA
was isolated from the cartilage plugs by a modified method of Adams et al. [1].
All reagents were nuclease-free. Frozen cartilage was powdered in a Spex Freezer
Mill (SPEX, Metuchen, NJ) and immediately homogenized in 4M guanidinium isothiocyanate solution, 0.3% 0-mercaptoethanol (Gibco BRL, Gaithersburg, MD). The
extracts were clarified by centrifugation at 1,500 X g for 10 minutes at 40 C and the
supernatant was removed. The extraction of the pellet was repeated and the supernatants were pooled and brought to 1% Triton X-100 and 1.5 M sodium acetate, pH
6.0. The supernatants were then extracted with an equal volume of chlorform/phenol
(1:4) on ice for 10 minutes. The extraction and separation of the aqueous phase was
repeated twice in equal volumes of chloroform/isoamyl alcohol (1:1). The RNA in
the aqueous phase was precipitated with equal volumes of isopropanol on ice for 5
Section 2.3
-28minutes followed by centrifugation at 15,000 X g for 30 minutes. The RNA pellet was
subsequently purified using the Qiagen RNeasy kit according to the manufacturer's
instructions. RNA was quantified spectrophotometrically and structural integrity was
confirmed by electrophoresis. Equivalent amounts of RNA (5 jg) from each condition were loaded onto agarose gels and mRNA expression was analyzed on Northern
blots using
32
P-labeled probes for bovine collagen Ila, collagen Ilb, the GI region
of aggrecan [70], and GAPDH. Northern blots were scanned and quantified using
phosphorimaging. Aggrecan GI and collagen Ila levels were normalized to that of
GAPDH. [65, 9]. Other normalization standards including total DNA, total RNA
and elongation factor-la (EF-la) were measured. mRNA levels at each compression
were normalized to the those at 1.15 mm (FS) and are reported as the mean from
three separate experiments (n=3) +/- SD.
To measure matrix biosynthesis, 4-6 disks per condition were dual-radiolabeled
with 10 pCi/ml
3 1S-sulfate
and 20 pCi/ml 3 H-proline during the 24-hour compression
period. Previous control studies using this same newborn calf explant culture system
showed that more than 98% of
3
1S-sulfate
incorporation was into macromolecules,
predominantly aggrecan, in both compressed and free swelling (FS) control disks
[68, 69]. Furthermore, in free swelling controls, over a 24 hour label period, 90% of
the 3 H-proline incorporation was into macromolecules, 83% of which was 3H-collagen
[69]. After the 24 hours of compression, disks were washed four times for 15 minutes
each in PBS with 0.8-mM sodium sulfate and 1 mM proline to remove free label,
lyophilized, and then digested overnight with papain. Digests were analyzed for DNA
content using the Hoechst 33258-dye assay [42], total sulfated glycosaminoglycan
(GAG) content by the DMMB dye [16], and 3 H proline and
by scintillation counting [68]. Incorporation levels of
3
3
1S-sulfate
1S-sulfate
incorporation
and 3 H-proline were
normalized to that in free swelling control disks. Values are reported as the mean of
4-6 disks from 2 experiments (n=10) +/- SD.
Section 2.3
-29Static Compression: Kinetics of Response Using the same chambers
described above, cartilage disks were statically compressed to 0.5-mm thickness and
maintained in culture for 0 (uncompressed controls), 0.5, 4 and 24 hours. Matched
cartilage disks were used in each condition as described above. After culture, disks
( 300 mg: 32 disk/condition) were flash-frozen in liquid nitrogen and RNA was isolated
for Northern blot analysis. Northern blots were examined for mRNA expression of
aggrecan, types Ila anb IIb collagen, and inducible nitric oxide synthase (iNOS) [21].
Signals were standardized using GAPDH, and data were normalized to uncompressed
controls. Values are reported as the mean of three separate experiments (n=3) +/SD. In addition, 4-6 disks per condition were radiolabeled with 10 pjCi/ml
35
S-sulfate
and 20 puCi/ml 3 H-proline during 4 and 24 hours of static compression to 0.5-mm. An
additional 4-6 plugs were maintained under free swelling conditions for 24 hours. This
condition was used as the "0-hour" control for evaluation of the kinetics of radiolabel
incorporation after application of static compression. After culture, these disks were
washed to remove free label, lyophilized, and then digested with papain. Digests
were analyzed for DNA content, GAG content, and radiolabel as above. Radiolabel
incorporation values were normalized to those of 0-hour control disks. Values are
reported as the mean of 4-6 plugs from 2 experiments (n=10) +/- SD.
2.4
Results
Dose Dependence of Aggrecan and Type II Collagen Expression and
Synthesis: Cartilage disks were compressed up to 50% of the cut thickness and
compared to a free-swelling control. Aggrecan GI (9.0 kb), collagen Ila (4.4 kb),
and GAPDH (1.4 kb) signals were evident in all lanes on Northern blot analysis for
the four compression conditions (Fig. 2.2). Normalized aggrecan mRNA expression
decreased significantly (p < 0.05 )with increasing amounts of static compression from
cut thickness (1.0 mm) to 0.5-mm (Fig. 2.3A). Collagen Ila mRNA expression showed
similar trends, although compressed values were not statistically different from the
Section 2.4
-301.15-mm control (Fig. 2.3A). Collagen lIb mRNA levels showed identical results to
collagen Ila levels (data not shown). Maximum normalized aggrecan (1.34 +/- 0.18)
and collagen Ila (1.01 +/- 0.13) expression occurred at cut thickness (1.0 mm) (Fig.
2.3A). These same dose-related trends were also observed when aggrecan and collagen
Ila mRNA levels were normalized to total DNA, total RNA, or EF-1a mRNA levels
on a specimen-by-specimen basis (data not shown). To determine if these changes
in gene expression were reflected in biosynthesis of aggrecan and type II collagen,
parallel cultures were compressed and dual-labeled with 35 S-sulfate and 3H-proline to
quantify proteoglycan or protein synthesis, respectively. Normalized values for
35 S_
sulfate incorporation and 3 H-proline incorporation decreased significantly (p < 0.05)
with increasing compression from cut thickness (1.0 mm) to 0.5-mm thickness with
maximum levels occurring at cut thickness (Fig. 2B). Total GAG and DNA values
were not statistically different between groups.
Kinetics of Changes in Aggrecan and Type II Collagen: To determine the kinetics of the reduction in gene expression observed at 0.5 mm-thickness,
disks were compressed for times ranging between 30 minutes and 24 hours. Aggrecan G1 (Fig.
GAPDH (Fig.
2.4A), collagen Ila (Fig.
2.4B), collagen Ilb (data not shown) and
2.4D) mRNA signals were evident in all lanes for the different du-
rations of compression; collagen Ila and Ilb showed identical trends. In contrast, no
expression of chondrocyte inducible nitric oxide synthase (iNOS) mRNA was seen
under any condition (Fig. 2.4C). Within the first 30 minutes of compression, there
was an increase in aggrecan (1.24 +/-0.07) and collagen Ila (1.54 +/-0.22) mRNA
compared to 0 hour controls. However, after the initial 30 minutes, total aggrecan
and collagen Ila normalized to GAPDH showed statistically significant decreases (p
< 0.05) with increasing amounts of time under static compression (Fig.
2.5A). By
24 hours of compression, total aggrecan GI and collagen Ila mRNA levels decreased
to approximately 35% of 0-hour values. In contrast, we have previously shown that
IL-1 stimulates chondrocyte iNOS mRNA expression and iNOS production [4] in ex-
Section 2.4
-31planted bovine tissue, whereas in our similar culture system with applied mechanical
load, no increased expression of iNOS mRNA was observed under conditions where
aggrecan and type II collagen expression was reduced.
Normalized values for
31S-sulfate
and 3 H-proline incorporation also decreased
significantly (p < 0.01) with increasing compression time (Fig. 2.5B). Due to the limitations of equilibration of the radiolabel precursors, a 0.5 hour time point could not
be measured for incorporation studies. However at later times, there was a monotonic
decrease in the incorporation of 3 5S-sulfate and 3H-proline into ECM macromolecules.
This time-related decrease was also observed for aggrecan and collagen Ila mRNA
(Fig.
2.5A). The data presented in these graphs represent averages of data from
3 separate experiments. However, the trends evident in the averages were present
within individual experiments and are not artifacts of data analysis (data not shown).
Finally, total GAG and DNA values were not statistically different between groups.
2.5
Discussion
The major findings during application of a 24 hour static compression were that aggrecan and collagen Ila mRNA levels decreased with increasing compression relative to
the levels at physiological cut thickness (Fig. 2.3A). Concomitantly,
3 H-proline
35
S-sulfate and
incorporation also decreased in a dose-dependent manner with increasing
compression up to 50% of cut thickness (Fig.
2.3B). Previous control studies us-
ing this same newborn calf explant culture system showed that more than 98% of
35 S-sulfate
incorporation was into macromolecules, predominantly aggrecan, in both
compressed and free-swelling control disks [68, 69]. Furthermore, in free swelling controls, over a 24 hour label period, 90% of 3 H-proline incorporation was into macromolecules, 83% of which was 3 H-collagen [69]. Thus, a 24-hour static compression
caused decreases in both aggrecan and collagen gene expression and protein synthesis. Previous studies [41, 43] showed that such compression to 50% did not cause
cellular damage, and that the decreases seen in proteoglycan and protein synthesis
Section 2.5
-32-
A
B
C
1.15
1.0
0.75
0.5
Thickness (mm)
Figure 2.2: Expression of aggrecan and type II collagen mRNA in response to static
compression as assessed by Northern blot. Expression of A) aggrecan GI domain; B)
collagen type Ha; and C) GAPDH for normalization was evaluated at the compressed
thickness shown. Lanes 1 through 4 show increasing amplitudes of static unconfined
compression (1.15-mm (FS) to 0.5-mm); compression duration was 24 hours.
Section 2.5
-33-
U)
2
mRNA Levels
* Aggrecan G1
o Collagen Ila
1.5-
I
0.51-
E
1.15
1.0
0.70
0.50
Thic kness (mm)
2
"-
Label Incorporation
* 35S-sulfat
o 3H-proline
n = 10
1.5-
C
0
0.5-
Z
T*
1.15
1.00
0.70
0.50
Thickness (mm)
Figure 2.3: Aggrecan and type II collagen gene expression and biosynthesis in response
to graded static compression of cartilage disks. A) mRNA levels for aggrecan l and
collagen type Ila l were quantified and normalized to GAPDH as a function of
increasing amounts of static unconfined compression. Values are reported as mean
+/- SD (n=3) normalized to the 1.15-mm (free-swell) value. * p < 0.05 vs 1.00-mm.
B) 1 5 S-sulfate l and 3 H-proline L incorporation into macromolecules as a function
of increasing amounts of static unconfined compression. Values are reported as mean
+/- SD (n=10) normalized to the 1.15-mm (free swell) value. * p < 0.05 vs 1.00-mm;
* p < 0.0001 vs all other thicknesses. The correlation coefficients, r, relating mRNA
expression to radiolabel incorporation rates were calculated for aggrecan and type
IIA collagen. The values are: raggrecan = 0.90; rcojuagenIIA= 0.89.
Section 2.5
-34-
A
C
0
0.5
4
Time (hrs)
24
Figure 2.4: Northern blot analysis for one of three similar experiments to examine
kinetics of mRNA change due to 50% static compression using bovine probes for: A)
aggrecan GI domain; B) collagen type Ila; C) iNOS; and D) GAPDH for normalization. Lanes 1 through 4 show increasing duration of compression for disks held at
0.5-mm thickness (0 hrs (control) to 24 hrs).
Section 2.5
-35-
2
A.
1.5kCo
Mi
1
-1
jL
*
0.51
E
0
0
0.5
4
24
Compression Time (hrs)
2
B.
Co
1.511
0
T
0.5 1-
z
0L
I
0
4
24
Compression Time (hrs)
Figure 2.5: Aggrecan and type II collagen gene expression and biosynthesis in response
to increased duration of static compression to 0.5-mm thickness. A) mRNA levels for
aggrecan GI D and collagen type Ila LI were quantified and normalized to GAPDH
as a function of increased duration of compression. Values are reported as mean
SD (n=3) normalized to the 0 hour (control) value. * p < 0.005 vs 0.5 hours; **
p < 0.005 vs 0.5 and 4 hrs; * p < 0.05 and 4 hrs. B) 3 5 S-sulfate LI and 3 H-proline
l incorporation into macromolecules as a function of increasing compression time.
Values are reported as mean +/- SD (n=10) normalized to the 24 hour free-swell
(control) value. * p < 0.001 vs 0 hrs, * p < 0.01 vs 0 and 4 hrs, * * p < 0.01 vs 0
and 4 hrs.
Section 2.5
-36were reversible upon release of compression.
Recently, Valhmu et al [81] reported that after 24 hours of static load applied to
4-6 month calf cartilage explants, aggrecan mRNA levels did not vary with increased
levels of static stress and were similar to unloaded controls.
Our results do not
support these findings. The reason for this difference is unclear, but may be related to
differences in loading techniques (load- versus displacement-control) and/or methods
for mRNA analysis. In addition, the younger bovine calves (1-2 weeks old) used as
the tissue source in the present experiment may also introduce variation, as others
have shown an age-dependent response in chondrocyte synthesis levels to mechanical
loading [50]. Since proteoglycan synthesis was not directly measured by Valhmu et al.
[81], the effects of static load on changes in biosynthesis at the protein level was not
observed and therefore could not be used for comparison of the chondrocyte metabolic
response most often seen upon static compression.
We next examined the kinetics of the change in mRNA levels and radiolabel
incorporation following a step compression to 0.5-mm thickness. Interestingly, the
applied compression caused an initial increase in mRNA levels for both aggrecan and
type Ila and IIb collagens by 25%-50% after 0.5 hours of compression (Fig. 2.5A).
This initial increase in mRNA levels was followed by significant monotonic decreases
in mRNA with increasing duration of compression up to 24 hours (Fig. 2.5A). Application of a step compression causes an initial radially directed fluid flow, pressure
gradients, and deformation of cells and matrix in the radially unconfined geometry
imposed by the compression chamber platens [40]. Mechanical stress relaxation to
a new static equilibrium state (with no fluid flow or pressure gradients) has been
demonstrated experimentally to be 90% completed by 15-20 minutes after application of compression, using similar 3-mm diameter cartilage disks in these chambers
[68]. Therefore, the initial upregulation in aggrecan and collagen Ia gene expression
observed within the first 30 minutes may be associated with non-equilibrium flows
and deformation still present at the earliest times after application of compression.
Section 2.5
-37This observation is consistent with previously reported transient increases in aggrecan mRNA levels by one hour after application of static load in the load-controlled
application of compression [81]. Previous studies have demonstrated that dynamic
(oscillatory) compression can stimulate increased synthesis of aggrecan and proteins
[43, 68]. We are now examining changes in the corresponding mRNA levels for aggrecan and collagen Ila in response to dynamic compression to further confirm our
hypotheses regarding the nature of the mechanical stimuli.
From the data reported in this study, it is unclear what signals may be involved in altering gene expression. Cytokines such as IL-1 have been reported to
down-regulate chondrocyte matrix biosynthesis [57, 22]. Therefore, it is possible that
loading could result in an immediate increase in local cytokine levels which could
secondarily down-regulate matrix biosynthesis. It is unlikely, however, that these
signaling pathways in the loaded cartilage system involve IL-1. IL-1 has been shown
to regulate chondrocyte iNOS expression
[4],
as well as reduced aggrecan and type
II collagen expression [57, 22]. In contrast, in the loaded cartilage explant system,
aggrecan and type II collagen expression was reduced while no increase in iNOS
gene expression was observed. There are several stress activated signaling pathways
within cells which control gene expression. Further studies will be required to determine which signaling pathways are involved in modulating chondrocyte aggrecan and
type II collagen gene expression, independent of iNOS expression.
The kinetics of changes in mRNA levels following a step compression to 0.5mm (Fig. 2.5A) were in marked contrast to the more rapid decreases in radiolabel
incorporation (biosynthesis) measured in parallel specimens from the same joints
(Fig.
2.5B). Significant inhibition of proteoglycan and protein synthesis following
static compression has been shown to occur as rapidly as one hour after application of
compression [25, 68]. The data in Fig. 4B are consistent with these previous reports
and show that biosynthesis is significantly and rapidly inhibited. In contrast, the
decreases in aggrecan and collagen Ila mRNA levels from 0.5 to 24 hours occur with
Section 2.5
-38best-fit exponential decay times of 5.0 hours and 16.4 hours, respectively (Fig. 2.5A).
These differences in the kinetics of changes in gene expression versus biosynthesis may
suggest that the observed decreases in synthesis are not related solely to changes in
total endogenous mRNA levels for these genes. To further test this hypothesis, it is
necessary to quantify the effects of compression on the stability of pre-formed mRNA,
as well as changes in levels of transcription and other post-transcriptional events such
as mRNA stability.
In summary, recent studies suggest that there are multiple pathways by which
chondrocytes sense and respond to mechanical stimuli, including upstream signalling
pathways and mechanisms which alter transcription, translation, post-translational
modifications, and cell-mediated assembly and degradation of matrix (see [27] for
review). Correspondingly, there may be multiple pathways by which physical stimuli
can alter not only the rate of matrix production, but the quality and functionality
of newly synthesized proteoglycans, collagens, and other molecules. In this manner,
specific mechanical loading regimes may either enhance or compromise the ultimate
biomechanical function of cartilage. In the present study, new evidence demonstrates
that static compression can significantly alter gene expression of two mechanically
functional matrix molecules. These results indicate that chondrocytes within intact
tissue are very sensitive to mechanical stresses and can modulate matrix gene expression and biosynthesis quickly (< 0.5 hours). Ongoing studies are focused on chondrocyte responses to dynamic compression and on elucidating the effects of mechanical
compression on transcription and stability of mRNA molecules.
Section 2.5
-39-
Chapter III
Chondrocyte Extracellular Matrix Synthesis and
Turnover in Alginate Culture Is Influenced by
Mechanical Compression
3.1
Introduction
Articular cartilage is a connective tissue responsible for load bearing in synovial joints.
Adult cartilage is avascular; aneural and alymphatic. Diffusion of nutrients from the
synovium through the tissue maintains the cell population of chondrocytes responsible
for synthesis, maintenance and gradual turnover of the cartilage matrix. The extracellular matrix (ECM) is composed of densely packed proteoglycans and collagens
among other proteins enabling it to withstand the complex physiologic loads.
The compressive resistance of articular cartilage is associated with aggrecan,
a densely sulfated proteoglycan. The high net charge density associated with the
carboxylate and sulfate groups of the glycosaminoglycan (GAG) chains attached to
the aggrecan core protein exerts significant electrostatic swelling forces that helps
to maintain the high water content characteristic of cartilage that helps to resist
mechanical compression and reduce joint wear. A collagen network provides a mesh
to retain aggrecan molecules and the associated swelling pressures, and enables the
tissue to resist tensile and shear forces.
The functional demands placed on articular cartilage can result in altering the
cellular response of chondrocytes and their production of ECM components. Previous
studies have shown that static compression decreases chondrocyte proteoglycan and
protein synthesis in tissue explants [73, 24, 68], while dynamic compression at certain
frequencies and amplitudes has a stimulatory effect on chondrocyte ECM production
[46, 60, 68]. However, the low ratio of cell volume to extracellular matrix volume
Chapter III
-40makes it difficult to extract some cell-specific molecules (e.g. mRNA) that could
provide additional insight into the pathways by which mechanical forces can regulate
chondrocyte behavior [58].
Isolated chondrocytes cultured in three-dimensional matrices continue to synthesize key ECM molecules, such as aggrecan and types II, IX, and XI collagen
[3, 6, 79] and produce a mechanically functional matrix similar to that of native cartilage [12]. These model systems have provided insights into cellular-level functions
that are difficult to measure in native tissue, particularly in understanding the effects
of mechanical forces on new matrix synthesis [11, 48]. In the present study, we present
a new alginate culture system in which chondrocytes can continue to synthesize ECM
molecules characteristic of articular cartilage and can be subjected to mechanical
loading in culture. In addition, we take advantage of the unique material properties
of alginate, which can be depolyzmerized rapidly by calcium chelation, to examine
how mechanical loads affect newly synthesized aggrecan and link protein associated
with the cell in addition to the matrix components further removed from the cell.
3.2
3.2.1
Methods
Chondrocyte isolation and alginate gel culture
Saddle sections of 1-2 week old calves were obtained from a local abattoir within 4
hours after slaughter (Research '87, Boston, MA). The femoropatellar groove was
isolated as previously described by Sah et. al. [68] and full-thickness cartilage pieces
were shaved off using a sterile surgical blade. Care was taken not to include the
underlying subchondral bone. Chondrocytes were isolated from whole cartilage using
sequential digestions of 0.4% pronase and 0.08% collagenase [56].
Total cell number was determined using a Coulter counter and viability was
assessed using a live/dead fluorescence assay (fluorescein diacetate and ethidium bromide). Isolated cells were mixed with 7- 10 ml of 2% alginate and injected into a
Section 3.2
-41-
A)
Steel Frame
Whatman 2
Filter Paper
80 Micrometer
Polyester Mesh
B)
Syringe with
Cell/Alginate
Mixture
0
0
0
0
0
0
0
0
0
0
0
0
000
Clamps
Figure 3.1: Alginate casting frame. A) Exploded view showing the steel casting frame
(thickness = 1.5 mm) sandwiched between Whatman 2 filter paper supported by an
80 prm polyester mesh. B) The mesh, filter paper and frame are clamped together
and the alginate with cells is injected into the 1.5 mm space between the two pieces
of filter paper. The casting frame with alginate is then submerged into a bath of 102
mM CaCl 2 , 0.15 M NaCl for 10 minutes to polymerize the alginate.
Section 3.2
-42diffusion-permeable casting frame using 16 G needle and syringe. The casting frame
consisted of a stainless steel, rectangular frame 14 cm X 10 cm X 0.15 cm thick,
sandwiched between Whatman 2 filter paper supported by an 80 pm polyester mesh
(Fig.
3.1). The mesh, filter paper, and frame were clamped together and wetted
with 102 mM CaCl 2 0.15 M NaCl prior to the addition of alginate. The frame with
alginate was then submerged in a 102 mM CaCl 2 0.15 M NaCl bath. After 10 minutes, the frame was removed from the bath, disassembled, and the alginate slab was
sequentially washed twice for 5-10 minutes in 0.15 M NaCl and once for 5-10 minutes in DMEM to remove residual calcium chloride. The gel slabs were then cut
into 16 mm diameter cores using a stainless steel punch and maintained in medium
(Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 10
mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,100 U/ml
penicillin, 100 [g/ml streptomycin, and 0.025 pm/ml amphotericin B, and 20 pg/ml
ascorbate) at 370 C, 5% CO 2 for up to 56 days. Three milliliters of media were added
to each disk for the first 3 weeks, and then increased to four after day 21. Media
was changed daily. The data presented below represent a compilation of five cultures
ranging from 14 to 56 days. Cell densities of 15-20 X 106 cells/ml of alginate gel were
used in cultures to be analyzed with Western blot techniques. For all other cultures,
cells were seeded at an initial density of 7.5 X 106 cells/ml.
3.2.2
Evolution of cell and matrix constituents in the alginate disk
system
At various times during the course of a sever week culture, 6-mm diameter disks were
punched from the larger 16-mm cores. Four groups of 6-mm diameter disks (n=4
disks/group) were labeled with 20 pCi/ml "S-sulfate for 12-14 hours. After labeling,
disks were removed from culture and radiolabel incorporation rates, cell viability,
GAG content, total collagen content, and DNA content were measured (see below).
Macromolecular radiolabel released into the culture media wa also quantified.
Section 3.2
-433.2.3
Compression and radiolabeling protocols
Disks (6-mm diameter) to be used for compression were placed in culture dishes with
unlabeled medium (defined above) and maintained in culture for 24 hours prior to
mechanical compression. The chambers used for static and dynamic compression have
been previously described [68].
Static Mechanical Compression: On day 21, 6-mm disks were compressed
uniaxially in a radially unconfined manner. The disks were compressed to 60%-100%
of original thickness (n=4/condition), and labeled during the 22 hour compression
period with 20 pCi/ml
31S-sulfate.
Alginate disks were removed from media and
washed in PBS with 0.8 mM sodium sulfate. Free swelling (FS) control disks in culture
dishes were also radiolabeled during this time. Macromolecular sulfate incorporation
into the alginate disks and macromolecular radiolabel released into the media was
analyzed and normalized to total DNA content of the disks (see below). Chondrocyte
viability after static compression for all conditions was also assessed.
3 5S-sulfate
incorporation levels were normalized to that in free swelling control disks. Values are
reported as the mean of 6 disks +/- SD.
Dynamic
Mechanical
Compression:
On day 56,
6-mm disks
(n=6
disks/condition) were placed in three different loading conditions: 1) disks were statically compressed (STAT) to 80% of original thickness; 2) disks were statically compressed to 80% of original thickness and a dynamic (DYN) sinusoidal compression
(0.5 Hz, 5% dynamic strain amplitude) was superimposed on the static offset compression; and 3) disks were remained uncompressed in free swelling (FS) conditions.
In all conditions, plugs were radiolabled with 20 pCi/ml
3
5
S-sulfate and maintained in
culture. After 12 hours, disks were removed from culture and macromolecular sulfate
incorporation into the alginate disks and any released into the media was analyzed
and normalized to total DNA content (see below).
35
S-sulfate incorporation levels
were normalized to that in free swelling control disks. Values are reported as the
mean of 6 disks +/- SD.
Section 3.2
-443.2.4
Biochemical analysis of rates of incorporation, GAG content,
total collagen, and DNA content:
After each radiolabel experiment, the disks were washed 4 times over one hour in
cold PBS (with
1 mM unlabeled sulfate and proline but without CaCl 2 or MgCl 2 ).
Wet weights were taken and disks were then lyophilized and digested in 1 ml of
papain (Sigma P3125, 125 pg/ml in 0.1 M sodium phosphate, 5 mM Na 2-EDTA,
and 5 mM cysteine-HCl, pH 6.5) for 16 hours at 60 0 C. Portions (25 pl) of the digest
and media were analyzed for
35
S-sulfate incorporation using the multiscreen system
[52]. The content for sulfated GAG was measured by a modified DMMB dye method
designed to account for the alginate background
[13].
Total DNA content within
the disks was measured by reaction of 100 pl of the digest with Hoechst 33258 dye
solution and fluorometry [42]. Total collagen content using one 6-mm disk digested in
1 ml of papain was assessed by hydroxyproline methods [14]. Viability was assessed
by releasing cells from one 6-mm diameter alginate disk (n=4) using 55 mM sodium
citrate, 0.15 M NaCl, pH 6.8 using a live/dead fluorescence assay (fluorescein diacetate
and ethidium bromide).
3.2.5
Static Compression and Aggrecan Turnover
Static Compression and Culture: On day 21, thirty-six 6-mm disks were cut
from the larger alginate cores and divided evenly between three compression conditions. One group of disks was statically compressed to 75% of original thickness in
the static chambers similar to the ones described above. A second group of disks was
placed in chambers and held at original (100%) thickness. A third group was maintained in free-swelling (FS) conditions in a 24-well culture dish. In each condition,
the 12 disks were maintained in medium ( 2.0 ml/disk) at 370, 5% CO 2 . After 40
hours, the disks were removed from culture, pooled together, and chondrocytes were
released from alginate by adding 3 mls of 0.15 M sodium chloride containing of 55
mM sodium citrate and 30 mM EDTA. Chondrocytes and their cell-associate ma-
Section 3.2
-45trix (CM) separated from the dissolved alginate by centrifugation. The supernatant,
which contained the dissolved alginate and the further-removed matrix (FRM) [56],
was removed and stored at -80"C. The cell pellet, containing the chondrocytes and the
CM, was washed twice with PBS and stored at -80 0 C. Finally, the media from each
condition was pooled and stored at -80 0 C. The CM and FRM fractions and media
were examined for proteoglycan core protein cleavage fragments by Western analysis
(see below).
Analysis of Culture Media and CM Proteoglycans: The aggrecan which
accumulated in the medium during the 40 hours was isolated by CPC precipitation
of quadruplicate portions of 1 ml of medium from each condition. This was followed
by an ethanol wash and chondroitinase ABC digestion to remove the sulfated GAG
chains. The GAG content of the medium was determine by DMMB reactivity of the
isolated GAGs. Yields from the CPC precipitation averaged 35% of total GAG based
on DMMB estimates. Sample data was adjusted accordingly to determine total yield.
The cell-associated (CM) fraction underwent chondroitinase ABC digestion followed
by centrigation of the cells. The yield of the CM was considered to be 100% since
no fractionation was required. The aggrecan content of the CM was determined by
semi-quantitative Western analysis (see below).
Analysis of FRM Proteoglycans: The dissolved alginate contained in the supernatant fraction makes Western analysis of proteoglycans in the further removed
matrix difficult.
To eliminate the alginate background, the supernatant was dia-
lyzed against 2-4 L of deionized water for 36 hours at 4' C. The retentate was then
lyophilized, and the remaining alginate-FRM powder was mixed with pure alginate
and 0.15 M NaCl to again form a viscous mixture ( 1.5-2% alginate). Beads of the
alginate+FRM solution were made by dropping the solution through a 16-G needle
and syringe into 102 mM barium chloride, 0.15 M NaCl solution. The barium chloride
irreversibly re-polymerizes the alginate. Beads were polymerized for 8 minutes under
constant stirring. The beads of alginate+FRM were then extracted in 4 M GuHCl
Section 3.2
-46and protease inhibitors (10 mM N-ethylmaleimide, 5 mM PMFS) for 4 days at 40 C.
Under these conditions, proteoglycans can be selectively extracted from the beads
with minimal contamination from the background alginate. Aggrecan was isolated
by ethanol precipitation of the extracts. Recovery through this protocol was assessed
by experiments with standard aggrecan taken through the barium chloride repolymerization and the guanidium extraction steps. We estimate that about 50% of the
starting material was recovered. The total recovered was determined by DMMB reactivity, taking into account 50% losses and some alginate contribution to the DMMB
reaction. The proportion attributed to GAG was that which could be eliminated by
chondroitinase ABC digestion.
Western Analyses Deglycosylated core protein samples were fractionated on a
4-20% gradient SDS-PAGE and transferred to PVDF for Western analysis with different anti-peptide antisera (Fig.
3.2) (ask Dr. Sandy if correct). Anti-CDAGWL
(HAL) was used to examine if a link-stabilized interglobular domain was present. To
examine the structure of Gi-bearing species, blots were probed with anti-ATEGQV
antisera [71] and LEC-7 was used to establish the presence of the G3 domain. The
anti-VDIPEN antisera has been used to establish evidence of MMP-generate GI domain cleavage products []. Anti-NITEGE and anti-KEEE were used to examine if
aggrecanase products were detectable. Anti-NITEGE is a antiserum which specifically detects the aggrecanase-generated cleavage of the Glu 373-Ala 374 bond in the
GI domain [47]. Anti-KEEE detects aggrecanase cleavage sites with the chondrotinsulfate (CS) attachment regions of the core protein. Finally, an 8-A-4 monoclonal
antibody was used to detect the presence of link protein.
Section 3.2
[CAGWL
LEC-7
ATEGQV
Ti
!H
K'
~
.
I
*.I11H
VDIPES
NITEGE
.TFKEEE
Figure 3.2: Schematic diagram showing the epitope sites used for analysis of the
aggrecan core protein. ATEGQV and CDAGWL (HAL) are used to examine the
presence of the G1 region. VDIPES and NITGE are sites of aggrecanase activity
in the interglobular domain. TFKEEE is used to assess aggrecanase activity in the
CS-region of the core protein. LEC-7 detects the presences of the G3 domain.
3.3
3.3.1
Results
Evolution of cell and matrix constituents in the alginate disk
system
Viability of chondrocytes prior to casting, immediately after casting and during the
culture was stable, averaging 90% +/-4% over the 6 weeks of culture. Spherical morphology was maintained throughout the culture. Total cell density increased monotonically from the original 7.5 X 106 cells/ml, and plateaued to 12 X 106 cells/ml by
day 13 (Fig. 3.3A). Total collagen increased monotonically with time in culture, with
the largest increase occurring between days 13 and 17 (day 17: 1.8 Mg collagen/disk)
(Fig. 3.3B). Total GAG content increased throughout 49 days of culture (Fig. 3.3C),
with larger increases occurring at later days in culture. 31S-sulfate incorporation was
initially low during the first 14-21 days of culture (Fig. 3.3D); however, by day 35,
35S-sulfate
incorporation reached a plateau level( 0.15 nmol/ugDNA/hr) similar to
that of chondrocytes in native articular cartilage [68].
On all days of culture, less
than 10% of the total incorporated sulfate was released into the medium
Section 3.3
-48-
15
0)
A.
B.
2. 5
10-
1.
C.)
(0
0
0
5tM 0.
C.)
0
0
5
0
10
C.
15
20
5
0
Day
Z10 0
10
15
20
D.
0. 2
0Total
8
0 60
-
40
-
z0.15 0.
A Media
0. 1 -
UO
Mt
0.0
0)
02
N Disk
E~o
0-
5
i
U
a
.E
WA1
0
10
20
30
Day
40
50
0
10
20
30
40
50
Day
Figure 3.3: Cell number and matrix constituents as a function of time in culture
(n=4/time point, avg +/- SD). A) Cell number was calculated from DNA content
using 7.9 X 10-12 g/cell [42] on days 0, 3, 6, 13, and 20. B) Total collagen content
in one 6-mm alginate disk (n=4/time point) was measured from total hydroxyproline
content [14] on days 0, 3, 6, 13, and 17. C) Total GAG was measured using DMMB
reactivity methods on days 0, 3, 6, 13, 20, 35 and 49. GAG content (pg GAG) was
normalized to DNA content (pg DNA). Corrections for the alginate background were
made [13]. D) Macromolecular 31S-sulfate incorporated into the disk and released
into the media was measured on days 0, 3, 6, 13, 20, 35, and 49. Incorporation rates
were normalized to DNA content.
Section 3.3
-493.3.2
Effects of Static and Dynamic Compression on
35
S-sulfate Incor-
poration
On day 21, the effect of increasing static compression on resulting chondrocyte incorporation of 35S-sulfate was examined. Our results show that chondrocyte proteogylcan (PG) synthesis rates decreased with increasing amounts of static compression
(Fig. 3.4). The synthesis rate in compressed disks plateaued between 60% and 80%
of original thickness and was approximately 75% of that in the free swelling (FS)
controls on day 21 of the culture (p < 0.001).
Cell viability was not affected by
the applied compression and was approximately 90% in all conditions. On day 56,
1.5
0
1 -
N
0.5
0
z
0FS
100%
80%
60%
Compression Condition
Figure 3.4: The effect of static compression on chondrocyte proteoglycan synthesis.
35
S-sulfate incorporation into alginate disks decreased with increasing static compression. Maximal inhibition of macromolecular synthesis occured at compression levels
between 60% and 80% of the disks original thickness (* p < 0.05 vs FS; * p < 0.01
vs FS).
the influence of dynamic compression on chondrocyte synthesis was examined. Our
results show that chondrocytes in alginate disks subjected to a dynamic compression
had a significantly increased PG synthesis rate (2 times greater) compared to disks
Section 3.3
-50subjected only to static compression (Fig. 3.5). In addition, the dynamic synthesis
rate was i.25 times higher than the FS control, although the difference was not significant. These trends in PG synthesis are similar to those previously reported for
chondrocytes in native cartilage subjected to similar loading protocols [43].
2
.
N
0
Z
1.51
0.5-
0
DYN
STAT
FS
Condition
Figure 3.5: The effect of dynamic mechanical compression on chondrocytes grownin
alginate disks. A sinusoidal dynamic compression (0.5 Hz, 4% dynamic amplitude)
superimposed on a 80%static compression increased 3 1S-sulfate incorporation in alginate disks by 1.9 times than in control disks subjected to 80% static compression
alone (* p < 0.05 vs DYN and FS).
3.3.3
Effects of Compression on Aggrecan Turnover
Aggrecan Contained in the Medium:
The aggrecan released the culture
medium during the 40 hour experiment was isolated by CPC precipitation of quadruplicate portions of 1 ml of medium from the FS, 100% and 75% compressed disks.
The mean concentration in the medium (ug GAG/ml) was 14.7, 12.0 and 21.3 Pg;
therefore the total in the medium from the 12 disks was therefore 221 pg, 180 Pg,
and 320 pg for the FS, 100% and 75% conditions respectively.
Section 3.3
-51Western analysis of the medium aggrecan (Fig. 3.6) showed essentially identical patterns for each condition. Probing with the anti-CDAGWL (HAL) antiserum
gave a single rather broad band at the molecular weight of full-length aggrecan core
protein present in calf cartilage aggrecan preparations. Probing with the KEEE antiserum revealed two distinct bands of reactivity at about 300 kDa and 220 kDa
respectively. These are presently believed to be the Gi-KEEE species (300 kDa) and
the Ala 374-KEEE species (220 kDa) [31].
75%
400
FS
100%
-
-
H
E
300
220
H
E
H
E
Figure 3.6: Western analysis of media samples from alginate disks cultured for 40
hours at 75% of original disk thickness, 100% of original thickness, and under free
swelling conditions. Media was pooled from 12 wells of the compression chamber
or culture dish for each condition. Samples were analyzed with anti-CDAGWL (H)
and anti-KEEE (E). Release of full length (400 kDa) aggrecan core protein and core
protein fragments (300 kDA and 220 kDA) consistent with CS-domain aggrecanase
activity are evident in all three conditions.
Section 3.3
-52Cell-Associated Aggrecan:
Since this fraction was analyzed following chon-
droitinase ABC digestion, it was not possible to determine the GAG content by
DMMB reactivity. However the Western analyses (with antisera HAL and KEEE)
were standardized with known loading of bovine AlDi. This semi-quantification indicated that there was between 1.5 - 2.0 mg (HAL reactivity) or 250 - 350 Mg aggrecan
(KEEE reactivity) in the 12 disks for each of the three conditions. The discrepancy
between the HAL and KEEE reactivities can not be explained at present.
Western analysis of these samples (Fig.
3.7) again showed a very similar
pattern of products for each condition. The major HAL-reactive and ATEGQVreactive product ran as a single band at about 400 kDa and this represents full-length
aggrecan core protein. In addition, the HAL antiserum detected a doublet at about
40 kDa which was confirmed as link protein by reactivity with antibody 8-A-4. These
samples also contained a high reactivity for the KEEE antiserum at the 300 kDa
position.
Further-Removed Matrix Aggrecan: Aggrecan was isolated by ethanol precipitation of guanidinium extracts of repolymerized (BaCl 2 ) alginate. Based on our
extraction methods, the total GAG for each condition was estimated to be 33 pg
(FS), 54 pg (100%), and 16 pg (75%).
Western analysis of the FRM (Fig. 3.8) with the ATEGQV showed a similar
pattern for each sample and interestingly, no evidence for full-length core protein.
The major G-reactive species were in the 50-60 kDa size range consistent with free
GI generated by interglobular domain cleavage.
3.4
Discussion
Our study has shown that articular chondrocytes grown in a three-dimensional alginate gel synthesize extracellular matrix components and respond to mechanical
loading in ways similar to that for chondrocytes in explant tissue culture and in vivo.
The accumulation of total GAG and total collagen throughout the culture period
Section 3.4
-53-
75% Compressed
300
1e
-
0
Free Svelling
100% Compressed
it W I
HAEs
60-
H A E L 3
3
HA E L
3
Figure 3.7: Western analysis of the cell-associated matrix of chondrocytes cultured
in alginate disks for 40 hours at 75% of original disk thickness, 100% of original
thickness, and under free swelling conditions. Disks (n=12) were pooled, dissolved in
a calcium chelating agent, and cells were pelleted via centrifugation. Samples were
analyzed with anti-CDAGWL (H), anti-ATEGQV (A), anti-KEEE (E), an 8-A-4
monoclonal antibody for link protein (L), and LEC-7 (3) for the G3 domain. Full
length aggrecan core protein (400 kDa), a link protein doublet band (60 kDA) and
core protein fragments consistent with normal chondrocyte catabolism (300 kDa)
were evident for all conditions. There was no reactivity with the anti-NITEGE or
anti-VDIPEN epitopes in all conditions.
Section 3.4
-54-
Calf
PSW
100%
75%
220kDa
220kDa
60kDa
60kDa
Figure 3.8: Western analysis of the further-removed matrix of chondrocytes cultured
in alginate disks for 40 hours at 75% of original disk thickness, 100% of original thickness, and under free swelling conditions. Disks (n=12) were pooled, dissolved in a
calcium chelating agent, and the further-removed matrix was separated from cells
via centrifugation. The supernatants were subject to analysis with anti-CDAGWL,
anti-NITEGE and anti-KEEE. The FRM contained only small GI fragments (detected with anti-CDAGWL) that result from proteolysis of the interglobular domain.
Section 3.4
-55suggests that the chondrcytes are able to assemble molecules into an intact extracellular matrix. In addition, the response of the chondrocytes to mechanical stimuli
further supports the notion that the newly synthesized ECM is capable of transducing
macroscopic loads to the cell surface. On closer examination of specific aggrecan components, we have found that chondrocytes in alginate culture can produce full-length
aggrecan and the spectrum of aggrecan cleavage products normally found in healthy
articular cartilage [72].
These data taken together suggest that chondrocytes in a
new alginate disk culture system behave similarly to their in vivo counterparts, and
that the advantage of quick cell isolation from the alginate system can provide new
methods for gaining insight into chondrocyte synthesis, maintenance and turnover of
extracellular matrix molecules.
Time Evolution of ECM Constituents and Chondrocyte Biosynthetic
Rates Over the course of 56 days in culture, the chondrocyte/alginate disks were
assessed for viability, total GAG, collagen, and DNA content, and "S-sulfate incorporation. Over 90% of the chondrocytes were viable immediately after tissue digestion,
after casting in the alginate system and throughout the several weeks of culture,
showing that our methods enable maintenance of viable chondrocytes in a slab/disk
geometry in long term culture. Although chondrocytes were transiently exposed to
high calcium concentrations (102 mM CaCl 2 ), their overall viability was not affected,
consistent with previous reports of viable cells polymerized in alginate using calcium
chloride [29, 23, 33].
The initial increase in cell number over the first 14 days of culture suggests
that some chondrocytes are undergoing proliferation. However, the net effect is small
as the total cell number at day 21 is approximately 1.5 times the original seeding
density and from that time onward cell density remained constant (Fig.
3.3A).
Chondrocytes in both agarose and alginate cultures have shown proliferative trends
within the first 14 days; however cell number had been shown to stabilize for later
times in culture [12, 23, 32], suggesting that cells do not develop into hyperplastic
Section 3.4
-56chondrocytes characteristic of those found in osteoarthritic cartilage [61].
Total collagen content in the alginate disks increased throughout the course
of the culture. Although the hydroxyproline method used to measure total collagen
does not differentiate between type I and type II collagen, previous reports for human
chondrocytes in cultured in alginate for three months showed that type II collagen
was the major (> 90%) form of collagen present [33]. In addition, the calf chondrocytes used in the present study are known to actively produce type II collagen. Our
data placed in the context of previous reports suggest that chondrocytes cultured in
alginate disks maintain a chondrocytic phenotype and produce type II collagen.
Proteoglycan synthesis by bovine chondrocytes in alginate (as measured by
31S-sulfate
incorporation) occurred initially at one-third to one-half the rate found in
native tissue explants. However, by day 35 in culture,
35
S-sulfate incorporation rates
very closely resembled those found in native calf cartilage [68]. The total GAG accumulated over 49 days of culture was less than that reported for chondrocytes cultured
in agarose (75 pg GAG/pg DNA vs. 100 pg GAG/pg DNA [12]) and is consistent
with the lower biosynthesis rates seen in the first 2 weeks of culture. Longer culture
times for the alginate system would most likely show continued GAG accumulation
and build up of the extracellular matrix.
Biosynthetic Response to Static and Dynamic Compression PG synthesis
decreased with increasing amounts of static compression showing trends similar to
those observed in cartilage explant systems [68]. Static compression to 80% of original
thickness resulted in approximately a 25% decrease in biosynthesis levels compared to
uncompressed controls. This demonstrates that the important ability of chondrocytes
to respond to mechanical compression is maintained in alginate disk culture. However
unlike that in explants, sulfate incorporation did not decrease with larger compression
levels (60%). By this time in culture (day 21), the accumulation of matrix molecules
such as aggrecan and other proteoglycans, which are responsible for the compressive
stiffness of cartilage, is still well below that found in articular cartilage. Longer culture
Section 3.4
-57times are needed to further investigate the relationship between PG accumulation and
effects of static compression on cell metabolism [11].
Dynamic compression increased PG synthesis compared to both static and free
swell conditions with similar trends as those seen in bovine explant systems. A 2-fold
increase was seen with the superposition of a 5% dynamic strain compared to the
static controls. This increase was larger than the 25% increase seen in chondrocytes
seeded in agarose disks stimulated with a 3% dynamic amplitude at the same 0.1
Hz frequency [11]. However in the present study, chondrocytes were cultured for 30
days longer than those cultured in the agarose system. Chondrocyte response to
dynamic load has been shown to increase with longer times in culture and therefore
with greater ECM accumulation [11].
Together, this suggests that by 8 weeks in
culture, matrix deposition around chondrocytes in the alginate disks is sufficient to
elicit responses to mechanical load and resembles responses seen by chondrocytes in
intact articular cartilage.
Effects of Mechanical Compression on Chondrocyte-Mediated Processing and Turnover of Aggrecan The amount of aggrecan present in the 12 disks
from the FS, 100% and 75% conditions was estimated (using KEEE Western data
only for the CAM) at 554 Mg, 534 pg, and 636 pug respectively. In each condition,
less than 10% was in the further-removed matrix (FRM), and 40-60% was in the
medium or cell-associated matrix (CM). This culture system appears to assemble a
tight CAM (link-stabilized) and a loose FRM which releases rapidly into the medium
under compression conditions applied for 40 hours during culture. The application of
compression to 75% appeared to enhance the release of aggrecan from the CM/FRM
to the medium compartment, relative to the 100% or FS controls.
Western analysis showed no major qualitative differences between the three
groups but significant differences in the composition of the medium, FRM, and CM
compartments. The CM appears to be composed of link-stabilized aggregates and
high content of aggrecan GI which results from proteolysis of the interglobular domain
Section 3.4
-58(aggrecanase or MMP). The medium compartment from all cultures was similar and
contains both full-length aggrecan and two KEEE-positive species which we believe to
be the petides Gi-KEEE (300 kDa) and the Ala374-KEEE (220 kDa). The presence
of the putative Ala374-KEEE peptide in the medium fraction only suggests that
proteolysis occurs during the 40 h culture at the aggrecanase site and possibly the
MMP site in the interglobular domain as well.
A very interesting finding unique to this culture system is that it appears to
model the processing which occurs in the normal bovine articular cartilage, in which
a proportion of the newly synthesized full length aggrecan is rapidly processed to the
G1-KEEE form. Further processing of this species to the Ala374-KEEE catabolic
product is generally only seen under catabolic conditions (serum removal or IL-I
addition) with cartilage explants, although in vivo, there is a steady state generation
of this product which appears in the synovial fluid.
Together, these data show that chondrocyte function is maintained in an alginate disk culture system. Specifically, the alginate disk culture system is a useful
model to simulate chondrocyte extracellular matrix metabolism and catabolism normally found in vivo. Our data also show that the response of chondrocytes within
alginate disks subjected to mechanical compression is similar to that of chondrocytes
maintained in native tissue. This is very important, as the rapid release of cells from
the alginate culture system will allow analysis of cellular-level biological markers that
would otherwise be difficult to measure from explanted tissue. We are presently
examining the effects of mechanical compression on signal transduction and resulting changes in mRNA levels of aggrecan, type II collagen and other related ECM
molecules.
Together with the data presented here, we hope to better understand
how mechanical compression alters chondrocyte behavior. This information will be
useful in understanding normal cartilage physiology, and the degradative processes
associated with osteoarthritis.
Section 3.4
-59-
Chapter IV
Dynamic Loading Upregulation of mRNA
Expression is a Function of the Surrounding
Extracellular Matrix
4.1
Introduction
Chondrocytes are the cells responsible for synthesis and turnover of the extracellular
matrix (ECM) molecules that comprise the bulk of articular cartilage. Specifically,
they synthesize proteoglycans, predominantly aggrecan, and various collagens, including types II and IX. Together, these molecules form a highly charged scaffold that
serves to maintain the high hydration levels characteristic of cartilage, and helps to
resist the shear, compressive and tensile forces experienced in vivo.
Chondrocytes housed in this ECM scaffold are subjected to the same mechanical forces that the matrix experiences. Many previous studies have shown that
chondrocytes respond to mechanical load in native tissue by altering their synthesis
of ECM macromolecules, either decreasing synthesis in cases of static compression
[73, 10, 24, 43] or increasing production in cases of dynamic loading [68, 43]. Isolated chondrocytes synthesizing new matrix in three-dimensional scaffolds have been
shown to respond similarly [11, 48]. Together these data demonstrate an important
physiologic phenomena and also present the deeper question of which intra-cellular
mechanisms are changing in response to mechanical compression.
Towards answering this question, research has focused on examining how mechanical forces influence messenger RNA levels, as protein synthesis is initiated by
mRNA translation. Fluid shear has been shown to upregulate mRNA expression for
tissue inhibitor of metalloproteinase-1 (TIMP-1) [37] and interleukin-6 (IL-6) [75].
Heat shock protein 70 (HSP70) mRNA expression increases with constantly applied
Chapter IV
-60hydrostatic pressure [80, 39]. Intermittent hydrostatic pressure [76] and cyclic mechanical stretching [38] upregulated aggrecan and type II collagen mRNA levels. Together these studies show that various mechanical forces can alter mRNA expression
of isolated chondrocytes grown in monolayer.
Due to the two dimensional geometry of monolayer culture, it is difficult to
impose compressive forces similar to those found in vivo on chondrocytes and examine how mRNA levels change. In addition, chondrocytes grown in high-density
monolayer can drastically change mRNA expression of ECM molecules [36]. Previously, it has been shown that static compression can decrease aggrecan [81, 62] and
type II collagen mRNA expression in chondrocytes maintained in native tissue [62].
This down-regulation in gene expression can be in part responsible for the decrease
in macromolecular synthesis of aggrecan and type II collagen induced by static compression [73, 25, 68]. In the present study, we have examined the effects of dynamic
mechanical compression on mRNA expression of aggrecan and type II collagen motivated by past reports of increased macromolecular ECM synthesis in response to
dynamic compression [68, 43]. Our results show that dynamic loading caused an increase in type II collagen mRNA but no changed in aggrecan levels in chondrocytes
maintained in native tissue. Chondrocytes cultured in alginate responded differently
as aggrecan mRNA was upregulated, and type II collagen was unaffected by dynamic
compression. From these data, we hypothesize that upregulation of ECM-specific
mRNA is influenced by the surrounding extracellular matrix.
4.2
4.2.1
Methods
Experiments with Explanted Tissue:
Cylindrical disks of cartilage were prepared as previously described by Sah et al [68].
Briefly, the saddle sections of 1-2 week old calves were obtained from a local abattoir within 4 hours after slaughter (Arena, Hopkinton, MA). Four to five cylindrical
Section 4.2
-61cores of cartilage and underlying bone (9.5-mm in diameter and 20-mm deep) were
drilled from each facet (medial and lateral) of the femoropatellar groove. During the
process, the cartilage was kept moist using sterile phosphate buffered saline (PBS)
supplemented with antibiotics (100 U/mil penicillin, 100 pg/ml streptomycin, and
0.025 pm/ml amphotericin B).
Each cartilage-bone core was then inserted into the sample holder of a sledge
microtome (Model 860, American Optical, Buffalo, NY). After removing the superficial layer of cartilage to obtain a level surface, two 1-mm thick plane-parallel cartilage
slices were dissected and placed in 1-ml PBS with antibiotics. From these slices (9.5
mm diameter, 1-mm thick), four 3-mm disks were obtained using a sterile dermal
punch (Miltex Instruments, Lake Success, NY) giving a total of 76 disks from each
joint surface. The disks were incubated in medium (Dulbecco Modified Eagle Medium
(DMEM) with 10 mM HEPES, 0.1 mM nonessential amino acids, antibiotics as described previously, 1 mM sodium pyruvate, an additional 0.4 mM proline and 20
pg/ml ascorbate) at 370 C in 5% CO 2 . Medium was supplemented with 10% FBS
throughout culture for the 12 hour loading protocol (see below). Disks were cultured
for four days and the medium was changed daily.
Dynamic Compression Using specially designed polysulphone chambers described previously [68], cartilage disks were uni-axially compressed in a radially unconfined manner. Cartilage disks were static compressed in the chambers to their cut
thickness of 1.0 mm (Static) or were placed into a incubator-housed compression device where the disks were statically compressed to 1.0 mm with a sinusoidal dynamic
compression superimposed on the static offset. Disks were loaded for either 12 or 48
hours. During the 12 hour loading protocol, disks were dynamically loaded at 0.01
Hz with a dynamic amplitude of 4% (40 pm). During the 48 hour protocol, disks
were dynamically loaded at a frequency of 0.1 Hz and 4% dynamic amplitude. Each
disk was bathed in > 100 volumes of medium and maintained in an incubator at 37'
C and 5% CO 2 . Cartilage disks in each condition were matched for joint position and
Section 4.2
-62tissue depth. Each 12 hour and 48 hour experiment was repeated three times.
After compression, disks (200 mg: 24 disks/condition) were flash-frozen in liquid nitrogen within 1 minute of removal from the compression chambers, and stored
at -80' C prior to RNA isolation for Northern blot analysis. Total RNA was isolated
from the cartilage plugs by a modified method of Adams et al. [1, 62]. Equivalent
amounts of RNA (5 pg) from each condition were loaded onto agarose gels and mRNA
expression was analyzed on Northern blots using
32 P-labeled
probes for bovine col-
lagen Ila, collagen IIb, the GI region of aggrecan [70], and GAPDH. Northern blots
were scanned and quantified using phosphorimaging. Aggrecan G1, and collagen Ila
levels were normalized to that of GAPDH or EF-oz [65, 9]. mRNA level of each
dynamic loading condition was normalized to that of static compression condition
and are reported as the mean from two or three separate experiments (n=2,3) ±/SD.
To measure matrix biosynthesis, 4-6 disks per condition were dual-radiolabeled
with 10 pCi/ml
3
1S-sulfate
and 20 [Ci/ml 3 H-proline during compression periods. Af-
ter the 12 or 48 hours of loading, disks were washed four times for 15 minutes each in
PBS with 0.8-mM sodium sulfate and 1 mM proline to remove free label, lyophilized,
and then digested overnight with papain. Digests were analyzed for DNA content using the Hoechst 33258-dye assay [42], total sulfated glycosaminoglycan (GAG) content
by the DMMB dye [16], and 3 H proline and
counting [68]. Incorporation levels of
3 5S-sulfate
35S-sulfate
incorporation by scintillation
and 3 H-proline were normalized to
that in free swelling control disks. Values are reported as the mean of 6 disks from 3
experiments (n=18) +/- SM.
4.2.2
Experiments with Isolated Chondrocyte Culture in Alginate:
Saddle sections of 1-2 week old calves were obtained as before, and the femoropatellar
groove was isolated as previously described by Sah et. al. [68]. Full-thickness cartilage
Section 4.2
-63pieces were shaved off using a sterile surgical blade. Care was taken not to include
the underlying subchondral bone. Chondrocytes were isolated from whole cartilage
using sequential digestions of 0.4% pronase and 0.08% collagenase [56].
Viability was assessed using a live/dead fluorescence assay (fluorescein diacetate and ethidium bromide). Isolated cells were mixed with 8 mls of 2% alginate
(20 X 106 cells/ml and injected into a diffusion-permeable casting frame described in
Chapter 3. The frame with alginate was then submerged in a 102 mM CaCl 2 0.9%
NaCl bath. After 10 minutes, the frame was removed from the bath, disassembled,
and the alginate slab was sequentially washed twice for 5-10 minutes in 0.9% NaCl
and once for 5-10 minutes in DMEM to remove residual calcium chloride. The gel
slabs were then cut into 16 mm diameter cores using a stainless steel punch and maintained in medium (Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine
serum (FBS), 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,100 U/ml penicillin, 100 pg/ml streptomycin, and 0.025 pm/ml amphotericin B,
and 20 pg/ml ascorbate) at 370 C, 5% CO 2 for up to 56 days. Three milliliters of
media were added to each disk for the first 3 weeks, and then increased to four after
day 21. Media was changed daily.
Dynamic Compression and mRNA Expression: On days prior to loading,
20 1.5 mm X 5-mm diameter disks were cut using a sterile biopsy punch from the
larger 16 mm diameter alginate cores and cultured for 24 hours. On days 26, 27 and
28, 10 disks were loaded into a compression chamber, and placed into the dynamic
loading apparatus [19] housed in an incubator. Disks were statically compressed to
90% of original thickness and a 4% sinusoidal dynamic strain (60 pm) was superimposed at 0.1 Hz. A chamber containing disks statically compressed to 90% of original
thickness (no dynamic loading) served as controls. The disks were maintained under
compression conditions for 20 hours, removed from culture, and pooled together. The
chondrocytes were then released from the alginate gel and total RNA was extracted
from these cells using the Qiagen RNEasy protocol (see below). mRNA levels for
Section 4.2
-64aggrecan, type IIA collagen and GAPDH were evaluated using Northern blot techniques. mRNA-levels were quantified using phosphoimaging techniques. Values are
reported as the average of three loading times +/- SD.
Isolation of Total RNA: Chondrocytes/alginate samples were dissolved using 4
volumes of ice cold 55 mM sodium citrate, 30 mM EDTA, 0.9% for 5- 7 minutes.
Cells were pelleted by centrifugation (300 g). The supernatant containing the alginate was aspirated off, and the pellet was washed with > 10 volumes of sterile 1X PBS
by gentle mixing for 2 minutes. Cells were re-pelleted and the supernatant was aspirated off. Total RNA was isolated from the pelleted chondrocytes using the Qiagen
RNeasy protocol for animal cells. Briefly, cells were lysed and homogenized simultaneously using the guanidinium isothyocianate (GITC)-containing solution, RLT
(Qiagen).
The mixture was centrifuged and the RNA-containing supernatant was
then processed and passed through a column to which the RNA adheres. After a
series of washes, the RNA was then eluted out using PCR-grade water. RNA was
quantified by spectrophotometric analysis and structural integrity was confirmed by
electrophoresis. The time lapse between cell release from the alginate and cell lysis
was approximately 20 minutes.
4.3
4.3.1
Results
Effect of Dynamic Loading Chondrocyte mRNA Levels in Tissue
Explants
Cartilage disks subjected to dynamic loading were assessed for mRNA expression and
compared to a static control. Aggrecan GI (9.0 kb), collagen Ila (4.4 kb), GAPDH
(1.4 kb) and EF-la signals were evident in all lanes on Northern blot analysis for
the two loading conditions (Fig. 4.1). Normalized aggrecan mRNA expression was
unchanged with with dynamic loading (Fig. 2.3A). Collagen Ila mRNA expression
increased under dynamic compression compared to the static controls (Fig.
4.1B).
Section 4.3
-65Normalized values for aggrecan and collagen Ha mRNA, presented in Figure 4.2A,
show the 17% increase in type IIA collagen expression compared to controls. These
same trends were also observed when aggrecan and collagen Ha mRNA levels were
normalized to total RNA or EF-la mRNA levels on a specimen-by-specimen basis.
To determine if these changes in gene expression were reflected in biosynthesis of
aggrecan and type II collagen, parallel cultures were compressed and dual-labeled
with
3 5 S-sulfate
and 3 H-proline to quantify proteoglycan or protein synthesis, respec-
tively. Normalized values for
35S-sulfate
incorporation and 3 H-proline incorporation
increased in for both proteoglycan and protein synthesis, although only the change
in 3 H-proline incorporation was significant (p < 0.05) (Fig. 4.2B). Total GAG and
DNA values were not statistically different between groups.
A.C.
D.
SD
DS
D
S D
Figure 4. 1: Expression of aggrecan and type II collagen mRNA in response to static
(S) and dynamic (D) compression as assessed by Northern blot. Expression of mRNA
for A) aggrecan G1 domain; B) collagen type Ha; C) GAPDH and D) EF-1a for
normalization was evaluated for each condition. The blots shown are characteristic
of the three repeats of the experimental protocol. Dynamic compression of 0.01 Hz,
4% dynamic strain amplitude was superimposed on a static compression to 1.0-mm
(cut thickness); compression duration was for 12 hours.
A second series of experiments were initiated to determine if a longer duration
of dynamic compression would subsequently increase changes seen in mRNA expression. Cartilage disks were cultured serum-free for 48 hours under dynamic loading
Section 4.3
-66B.
A.
2
U Aggrecan G1
mRNA Levels
0
*15n=3±SM
>
2
Collagen Ila
20-24±SM
1-
0.5-
0.5-
12hr-DYN
12hr-STAT
Loading Condition
U 35S-sulfate
0 3H-proline
1-
0
Incorp.
_
>n
0
12hr-DYN
12hr-STAT
Loading Condition
Figure 4.2: Aggrecan and type II collagen gene expression and biosynthesis in response
to 12 hours of dynamic loading of cartilage disks. A) mRNA levels for aggrecan E and
collagen type IIA l were quantified and normalized to GAPDH. Values are reported
as mean +/- SD (n=3) normalized to the static (STAT) control value. B) 3 5S-sulfate
L and 3 H-proline l incorporation into macromolecules. Values are reported as mean
+/- SD (n=20-24) normalized to the static control. * p < 0.05 vs static control for
3
H-proline incorporation.
(0.1 Hz, 4% dynamic amplitude) superimposed on a 1.0-mm static compression (cut
thickness). Control disks were statically held 1.0-mm. Aggrecan G1, collagen Ila, and
EF-1a signals were evident in all lanes on Northern blot analysis for the two loading
conditions (Fig.
4.3).
After 48 hours, normalized aggrecan mRNA expression was
unchanged with with dynamic loading (Fig. 2.3A). Collagen Ila mRNA increased by
47% compared to static controls.
3
1S-sulfate and 3 H-proline were measured to quan-
tify proteoglycan or protein synthesis and to determine if the changes in seen in gene
expression were reflected in biosynthesis. Parallel cultures were compressed and dual-
labeled with 31S-sulfate (10 pCi/ml) and 3 H-proline (20 pCi/ml). Normalized values
for 3 1S-sulfate incorporation and 3H-proline incorporation significantly increased in
for aggrecan and collagen synthesis, (p < 0.001) (Fig. 4.4B).
Section 4.3
-67-
B.
A.
S D
S
D
C.
S
D
S
S D
D
S
D
Figure 4.3: Expression of aggrecan and type II collagen mRNA in response to static
(S) and dynamic (D) compression as assessed by Northern blot. Expression of A)
aggrecan G1 domain; B) collagen type Ila; and C) EF-1a for normalization was
evaluated for each condition. Dynamic loading of 0.1 Hz, 4% dynamic amplitude
was superimposed on a static compression to 1.0-mm (cut thickness); compression
duration was for 48 hours. Lanes 1 through 4 represent 2 repeats of the loading
protocol, with static (S) and dynamic (D) conditions alternating lanes.
2
A.
B.
mRNA Levels
1. 5
U Aggrecan G1
Incorp.
3 Collagen Ila
n=2
-
1.51
-j
U)
cc
E
0.5DYN
II
AL
STAT
Loading Condition
.
E
1
0 0.51
Z
0
Li
L
* 35S-sulfate
0
3H-proline
n =18± SM
STAT
DY N
Loading Condition
Figure 4.4: Aggrecan and type II collagen gene expression and biosynthesis in response
to 48 hours of dynamic loading of cartilage disks. A) mRNA levels for aggrecan El and
collagen type Ha E were quantified and normalized to EF-1a. Values are reported as
mean +/- SD (n=3) normalized to static (STAT) control value. B) 35S-sulfate 0 and
3 H-proline E] incorporation into macromolecules. Values are reported as mean
SD (n=18) normalized to the static control. * p < 0.001 vs static control.
Section 4.3
--.
=~
~
-68-
A.
B.
C.
Figure 4.5: Expression of aggrecan and type II collagen mRNA in response to static
(S) and dynamic (D) compression in chondrocytes cultured in alginate disks as assessed by Northern blot. Expression of A) aggrecan GI domain; B) collagen type IIa;
and C) GAPDH for normalization was evaluated for each condition. Dynamic loading of 0.1 Hz, 4% dynamic amplitude was superimposed on a 90% static compression;
compression duration was for 20 hours. Lanes 1 through 6 represent 3 repeats of the
loading protocol, with dynamic (D) and static (S) conditions alternating lanes.
4.3.2
Effect of Dynamic Loading on Isolated Chondrocytes Suspended
in Alginate Disks
After 21 days in culture, isolated chondrocytes maintained in alginate disks were subjected to 20 hours of dynamic loading (0.1 Hz, 4% dynamic amplitude) superimposed
on a 90% static compression. Disks statically compressed to 90% of original thickness served as controls. Messenger RNA bands for aggrecan, type IIA collagen and
GAPDH were evident in all lanes for each set of dynamic (D) and static (S) conditions (Fig. 4.5). Band intensities were quantified using a phosphorimager, and values
were normalized to GAPDH. In all three repeats, normalized aggrecan was greater
than static controls, showing an average increase of 45% (Fig. 4.6). Differences in
mRNA levels for collagen IIA in response to dynamic and static compression collagen
were not consistent between the three sets of experiments, although the normalized
average of type IIA collagen for the dynamic condition was larger than the control
(Fig. 4.6).
Section 4.3
-69-
2
1.51-
mRNA Levels
T
Aggrecan G1
Collagen Ila
3 SM
-I-
<i
1
z
0.5
E
0
E-
24hr-Dyn
24hr-Stat
Loading Condition
Figure 4.6: Aggrecan and type II collagen gene expression of chondrocytes in alginate
in response to 20 hours of dynamic loading. mRNA levels for aggrecan EL and collagen
type Ha ELwere quantified and normalized to GAPDH. Values are reported as mean
+/- SD (n=3) normalized to static (STAT) control value. In all cases, aggrecan
mRNA was greater in the dynamic condition compared to static controls.
Section 4.3
-70-
4.4
Discussion
From this series of experiments, it is evident that dynamic mechanical compression
can stimulate expression of mRNA molecules encoding for aggrecan and type IIA collagen. In addition, dynamic loading caused increases in macromolecular biosynthesis
levels of aggrecan and type II collagen, as measured via radiolabel techniques. Interestingly in the case of aggrecan, increases in biosynthesis still occurred although the
aggrecan mRNA levels remain unchanged (Figs. 4.2 and 4.4). Increases in type IIA
collagen mRNA follow a similar trend as that seen with macromolecular 3 H-proline
incorporation, suggesting that the increase in biosynthesis is at least in part due to
increases in gene transcription.
Isolated chondrocytes cultured in alginate also responded to dynamic compression, and increased expression of matrix molecule mRNA. Aggrecan mRNA was
increased by 45% of static controls, and collagen IIA mRNA levels were slightly increased by 10% (Fig. 4.6). Although no measurements of macromolecular aggrecan
synthesis were made in the present study, previous experiments have shown that
similar dynamic stimulation of chondrocytes in alginate disks increased
3 5 S-sulfate
incorporation by almost two-fold compared to static controls (see Results section,
Chapter 3).
These data taken together suggest that dynamic loading can selectively upregulate gene expression of extracellular matrix molecules characteristic of articular
cartilage. Perhaps more interesting are the questions that have arisen as to what
causes the differences in upregulation between aggrecan mRNA levels and resulting
macromolecular synthesis, and the difference seen between chondrocyte response in
native tissue versus those cultured in a three-dimensional gel matrix.
On closer examination of aggrecan synthesis, other enzymes (and therefore
other mRNA's) are necessary for addition of the sGAG chains to the core protein.
Glycosyltransferases are a set of such enzymes responsible for chondroitin sulfate
synthesis. Dynamic loading could upregulate the mRNA expression and resulting
Section 4.4
-71production of these products, and result in an increased macromolecular 3"S-sulfate
incorporation.
The effects of dynamic loading could be related to the bulk of ECM surrounding the cell, and would explain the difference response of the chondrocytes in the
alginate compared to those in native tissue, as total GAG content is an order of magnitude less in the alginate system than that in explanted cartilage. This most likely
has an effect on microscopic fluid flow and induced streaming potential. Both of these
effects can stimulate cellular response and could be the mechanisms through which
dynamic stimulation is influencing chondrocyte gene expression.
In summary, dynamic compression can upregulate chondrocyte gene expression of aggrecan and type II collagen. In addition, down-stream synthesis of these
macromolecules is also upregulated by dynamic compression, and in some cases, is independent of increases in mRNA levels. Together these data show that chondrocytes
can increase synthesis of extracellular matrix molecules under physiologic loading. It
suggests the importance of considering therapies, such as continuous passive motion
for patients undergoing cartilage transplantation or other cartilage repair procedures.
Section 4.4
-72-
Chapter V
mRNA Stability is Unaltered during Static
Mechanical Compression
5.1
Introduction
Mechanical forces have been shown to affect chondrocyte production of new extracellular matrix aggregates and proteins. Chondrocytes subjected to static compression
decrease matrix synthesis [43, 68, 24, 73], and dynamic compression has been shown
to stimulate these cells to produce more ECM molecules [68, 40, 11]. However, many
questions still remain as to which segments of ECM biosynthesis pathways are sensitive to the application of mechanical forces.
Studies have begun to explore in more detail the biosynthetic pathways of
aggrecan, the predominant proteoglycan found in articular cartilage that provides for
the tissue's compressive resistance. Kimura et al. demonstrated that chondrosarcoma
cells are still able to synthesize chondroitin sulfate at levels close to base-line even
with inhibition of aggrecan core protein synthesis via cyclohexamide treatment
[44].
Their study suggested that the down-stream cellular mechanisms responsible for the
final stages of aggrecan synthesis and secretion were still functioning even in the
absence of the aggrecan core protein. A related study by Kim et al. showed that
static compression can markedly decrease the available pool of aggrecan core protein,
although the processing time during which chondroitin sulfate chains were added
did not change [41]. Together, these studies show that the various stages involved
in aggrecan synthesis are regulated differentially and can be individually altered by
applied static compression.
Messenger RNA levels for the aggrecan core protein have been shown to be
decreased by applied static compression [81, 62].
In part, this could provide the
reason for the previously shown decrease seen in aggrecan core protein pool size with
Chapter V
-73application of static mechanical compression. However, it is yet unclear as to whether
static mechanical compression causes a decrease in transcription of new aggrecan
mRNA or causes a more rapid degradation of existing message molecules, as both
can cause a decrease in total measured aggrecan message. The present study is an
attempt to elucidate how mechanical compression alters mRNA stability and resulting
ECM macromolecule synthesis. Specifically, we have examined how an applied static
compression alters aggrecan synthesis in the presence and absence of the transcription
inhibitor actinomycin D, and have examined the stability of existing mRNA after
transcription has been blocked. Our results show that separate decreases seen due
to both static mechanical compression and mRNA inhibition can be superimposed to
result in even more drastic decreases in total ECM molecule synthesis. In addition,
our results demonstrate the stability of ECM mRNA molecules after transcription
inhibition. Together, these data suggest that mechanical compression does not alter
the stability of existing mRNA for ECM molecules.
5.2
5.2.1
Methods
Effects of Blocked Transcription on Down-Stream Protein Synthesis:
Cartilage Explant Culture Cartilage Explant Culture: 1-mm by 3-mm diameter
cartilage disks were harvested from the femoropatellar groove of a 1-2 week old calf
[68]. The cartilage disks were cultured in medium (Dulbecco Modified Eagle Medium
(DMEM) with 10 mM HEPES, 10% fetal bovine serum (FBS), 0.1 mM nonessential
amino acids, 100 U/ml penicillin, 100 pg/ml streptomycin, 0.025 pm/ml amphotericin
B, 1 mM sodium pyruvate, and 20 pg/ml ascorbate) at 370 C in 5% CO 2.
Effect of Actinomycin D Dosage Levels The effect of blocking mRNA transcription on downstream production of aggrecan and type II collagen was examined.
After eight days in culture, cartilage disks (n=4/condition) were placed in one of three
Section 5.2
-74conditions: a) 0 ug/ml actinomycin D; b) 10 ug/ml actinomycin D; and c) 50 ug/ml
actinomycin D. Plugs were matched for location and depth between groups. Plugs
were radiolabeled with
3
S-sulfate and 3 H-proline for 1 hour at times 0, 3, 8, 11 and 23
hours, removed from culture, and washed 4 X 15 minutes in cold IX PBS, with cold
proline and sulfate to remove residual radiolabel. The cartilage disks were digested
overnight with papain, and assayed for total DNA using the Hoechst method [42],
and for
3
1S-sulfate
and 3 H-proline incorporation using scintillation methods. Values
reported are the average of n=4/time point/condition +/- SD.
Effect of Mechanical Compression and Actinomycin D on Protein Synthesis: The effect of mechanical compression on mRNA stability was examined by
blocking new transcription with actinomycin D, subjecting explanted samples to 75%
static compression, and measuring down-stream protein synthesis. After four days
in culture, cartilage disks (n=4/condition) were placed in one of four conditions: a)
O-FS: 0 ug/ml actinomycin D with no compression (free swelling); b) 10-FS: 10
ug/ml actinomycin D with no compression; c) 0-75C:10 ug/ml actinomycin D with
75% static compression; and d) 10-75C: 10 ug/ml of actinomycin D with 75% actinomycin D. Plugs were matched for location across the four groups for each time
point. Plugs were radiolabeled with
3
1S-sulfate
and 3 H-proline for 1 hour at times 0,
1.5, 4, 10 and 23 hours, removed from culture, and washed 4 X 15 minutes in cold
1X PBS, with cold proline and sulfate to remove residual radiolabel. Cartilage disks
were digested overnight with papain, and total DNA [42] and
35
S-sulfate and 3 -H
proline incorporation were measured. Values reported are the average of n=4/time
point/condition +/- SD.
5.2.2
mRNA Stability in Isolated Chondrocytes Cultured in Alginate
Disks:
Chondrocyte Culture: Chondrocytes were isolated from whole fresh cartilage
from the femoropatellar groove of 1-2 week old calves using sequential digestions of
Section 5.2
-750.4% pronase and 0.08% collagenase [56]. Isolated cells were mixed with 8 mls of 2%
alginate and injected into a diffusion-permeable casting frame (see Methods Section,
Chapter 3). The frame with alginate was then submerged in a 102 mM CaCl 2 0.9%
NaCl bath for 10 minutes. The frame was removed from the bath, disassembled, and
the alginate slab was sequentially washed twice in 0.9% NaCl and once in DMEM to
remove residual calcium chloride. The gel slabs were cut into 16 mm diameter cores
using a stainless steel punch and maintained in 3-4 mls of medium (as define above)
for up to 25 days. Media was changed daily.
mRNA Stability after Blocked Transcription: The stability of various ECM
messenger RNA levels was examined by blocking new transcription with actinomycin
D. After 20 days in culture, 10 X 106 chondrocytes suspended in alginate disks ( 0.5
ml) were placed in 4 mls of feed medium (as defined above) containing 10 Pg/ml
of actinomycin D at time zero. At times 1, 2 , 4, 8, 12, 14 and 18 hrs, chondrocyte/alginate samples were removed from culture, released from the alginate gel, and
total RNA was extracted using the Qiagen RNEasy protocol for animal cells. Total
RNA (2 ug/well) was electrophoresed on a formaldehyde/agarose gel and examined
for structural integrity. Northern blot techniques were used to examine the levels
of aggrecan, type IIA collagen, EF1-oa and GAPDH. Autoradiography signals were
quantified by using a phosphoimager and the values obtained were normalized against
28S rRNA. Values reported are normalized to the first one-hour time point.
5.3
Results
Actinomycin D Dose Response in Cartilage Explants: The addition of
10 pg/ml and 50 pg/ml of actinomycin D decreased the rate of "S-sulfate and 3 Hproline incorporation in a similar fashion (Fig. 5.1). By 24 hours, both dosage levels
showed a 50% and 90% decrease in radiolabled sulfate and proline incorporation
rates, respectively. The incorporation of radiolabeled proline dropped exponentially
after an an initial increase during the first one hour of exposure to actinomycin D.
Section 5.3
-7635
S-sulfate incorporation was essentially constant for the first eight hours and then
decayed slowly over the remaining time course.
Mechanical Compression of Transcription-Inhibited Chondrocytes in
Cartilage Explants: The addition of actinomycin D (10-FS) caused a 50% decrease
in
35
S-sulfate incorporation after 24 hours in free-swelling (no compression) culture,
compared to no-dose (0-FS) controls (Fig. 5.2A). Similarly, the actinomycin-treated
cartilage disks subjected to 75% static mechanical compression (10-75C) had a 50%
decrease in sulfate incorporation compared to the untreated compressed samples (075C). Comparing the individual effects of compression (0-75C) to of that of actinomycin D (10-FS) reveals that both conditions result in a 50% decrease in aggrecan
synthesis, as measured by sulfate incorporation.
B.
A.
2.5
2.5
0
4-2
1,4,12.
S
-
ad5
S0 Ug/m1
10 Ug/ml
2
50 ug/ml an
0 Ug/ml
0
5[
4
10 Ug/ml
A 50 ug/ml
1.6
1
.
0
4
8
12
16
20
24
Time (hrs)
1
0
4
8w
2
1
5620
24
Time (hrs)
Figure 5. 1: The biosynthetic response of chondrocytes in native explants to two dosage
levels of actinomycin D. 1-mm by 3-mm diameter cartilage disks were cultured for
1, 4, 9, 12 and 24 hours in 0, 10pg/ml and 50pg/ml. Disks were radiolabeld for 1
hour prior to being removed from culture with 31 S-sulfate (10PCi/ml) and 3 H-proline
(20pCi/ml). Disks (n=4/condition) were then removed and washed 4 by 15 minutes
in PBS to remove unincorporated label. Disks matched for joint depth and location
were used for each time point group. Values are normalized to the control (0 pg/ml)
for each time point. A) kinetics of
3
1S-sulfate
incorporation; B) kinetics of 3H-proline
incorporation.
The effects static compression with and without actinomycin D treatment
on 3 H-proline incorporation were also examined. After 24 hours of culture, protein
synthesis (as measure by proline incorporation) in drug-treated samples (10-FS) deSection 5.3
-77creased to 75% of untreated controls (Fig.
5.2B). Similarly, statically compressed
samples with no drug treatment (0-75C) had a 25% decrease in protein synthesis
compared to uncompressed untreated (0-FS). Finally, the superposition of static compression onto samples treated with 10 pg of actinomycin D showed the largest drop
in synthesis rates, to approximately 55% of the untreated uncompressed controls.
A.
B.
2
0 0-FS
0
0O)-FS
1 O-FS
0
O1-FS
-1
0-75C
010-75C
1.0
E0
2
Z-
0-75C
0
10-75C
1
0
0
4
___
____
___
____
0
8
12
Time (hrs)
16
___
20
24
0
I
I
I
I
I
4
8
12
16
20
24
Time (hrs)
Figure 5.2: The biosynthetic response of chondrocytes in native explants subjected
to mechanical compression and dosed with actinomycin D. 1-mm by 3-mm diameter
cartilage disks were cultured for 1, 2, 5, 11 and 24 hours in either 0 pg or 10 P/g
of actinomycin D with or with a superimposed 75% static compression. Disks were
radiolabeld for 1 hour prior to being removed from culture with 3 5S-sulfate (10PCi/ml)
and 3 H-proline (20pCi/ml). Disks (n=4/condition) were then removed and washed in
PBS to remove unincorporated label. Disks matched for joint depth and location were
used for each time point group. Values are normalized to the control (0 pg/ml Free
Swelling) for each time point. A) kinetics of 3 5S-sulfate incorporation; B) kinetics of
3 H-proline incorporation.
In order to directly examine changes in mRNA stability, new transcription in
isolated chondrocytes cultured alginate disks was inhibited using 10 Pg/ml of actinomycin D and mRNA levels were measured using Northern blot techniques. Northern
blots of aggrecan (9kb) and GAPDH (1.4kb), type IIA collagen (4.4 kb), and EF1-a
(size 3.0?kb) are shown (Fig. 5.3A-C), and the ethidium bromide staining is shown
in Fig. 5.3D. mRNA signals are evident in all lanes for each time point. Figure 5.4
shows the quantified values for each of these bands normalized to the 1 hour time
point. Both aggrecan and type IIA collagen show trends suggesting an increasing
Section 5.3
-78stability throughout the culture (Fig. 5.4A and B). Elongation factor-la stability
was essentially constant throughout culture (Fig. 5.4C), and GAPDH decreased to
50% of it original value after 18 hours in culture (Fig. 5.4D).
A.
B.
C.
D.
Figure 5.3: Northern blot analysis of chondrocytes cultured in alginate disks and
dosed with 10 pg of actinomycin D for 1, 2, 4, 8, 12 14 and 18 hours. After time in
free swelling culture, total RNA was extracted using the Qiagen RNeasy protocol for
animal cells. Approximately 2 pg of RNA was run in each lane. Expression of A)
both aggrecan (9.0 kb) and GAPDH (1.4kb); B) collagen type Ha; and C) EF-la was
evaluated at each time point. D) 28S (ethidium bromide) was used for normalization.
Each lane represents increasing times in drug-treated culture.
5.4
Discussion
Together our results show that inhibition of transcription and mechanical compression
can separately cause a decrease in macromolecular ECM synthesis and that together
the superposition of their effects result in an even more drastic reduction in 3"S-sulfate
and 3 H-proline incorporation. Previous studies examining the effects of actinomycin D
alone have shown a more rapid decrease in
31S-sulfate
incorporation with the addition
Section 5.4
-79-
A.
B.
2.5
2.5
2
Ch
Go
-
C,)
Go~ 1.5
-
1
21.5-
0
0
1- -
CD)
0.5
0
0.5 -
0
I
5
I
15
0
20
U-
LU
I
10
5
D.
I
15
20
I
15
20
Time (hrs)
2.5
2
C,,
0
Time (hrs)
C.
2.5
I
10
2-
1.51 -A
C,)
CO,
A
1
1.5-
CL
0.5
1
**
*
0.5 -
01
C
I
5
I
10
Time (hrs)
I
15
20
0[
0
5
10
Time (hrs)
Figure 5.4: Kinetics of changes in mRNA levels after transcription-inhibition via
actinomycin D. Values are normalized to 28S. Increasing stability is seen with A)
aggrecan and B) collagen type Ila. C) Elongation factor-la remained unchanged. D)
GAPDH decreased to less than half of its starting levels.
Section 5.4
-80of actinomycin D [53, 55].
Estimates of half-lives based on their studies suggest
synthesis decay rates that are 3-4 times shorter than those reported in the present
study. These differences could be due to animal age differences, as young calf cartilage
was used in the present study compared to the adult-derived tissue used in other
studies [53, 55]. There have been previous reports of decreased mRNA stability of
link protein with increases in age, however aggrecan mRNA stability was shown to
be indepdent of age [7].
From our data, it appears that the addition of static compression (10-75C)
does not change the kinetics of the decreases in "S-sulfate seen with addition of
actinomycin D alone (10-FS) (Fig. 5.2A). This would suggests that mechanical compression does not alter the stability of existing aggrecan mRNA. It also suggests that
mechanical compression does not solely act to decrease mRNA at the transcription
level resulting in downstream decrease in protein synthesis. If mRNA transcription
was only pathway through which mechanical compression acted, the inhibition of
transcription would effectively block this pathway and therefore inhibit the effects
seen with mechanical compression. This is not the case, as is evident by the more
pronounced decrease in radiolabel incorporation with the addition of mechanical compression to samples dosed with actinomycin D.
Total protein synthesis as measured by 3 H-proline incorporation decreased
with the addition of actinomycin D and static compression (Fig.
5.2B). However,
the decreases seen with the addition of actinomycin D varied between the dosage and
compression studies. The results from a repeat study of the mechanical compression protocol were similar to those reported within. This suggests that total protein
synthesis can vary within the time course studied here and makes extrapolating information about changes in mRNA stability difficult. To our knowledge, no other studies
have shown changes in total protein synthesis due to the addition of actinomycin D
to place our data in perspective.
Finally, our direct examination of mRNA stability has shown that message
Section 5.4
-81levels for extracellular matrix molecules such as aggrecan and type IIA collagen are
constant or increase for 18 hours after inhibition of transcription. The increase in
mRNA stability is more reflective of a decrease seen in ribosomal RNA (as values reported here are normalized to 28S rRNA), which can also be influenced by inhibition of
transcription. Previous reports have estimated a 4 hour half-life for aggrecan mRNA
and a 10 hour half-life for EF-1a [35]. In these studies, chondrocytes obtained from
bovine cartilage of adult animals were plated in high-density monolayer and cultured
in the presence of 5 pg/ml of actinomycin D. Differences between animal ages could
account for these inconsistencies. In addition, previous reports have shown markedly
altered mRNA expression in chondrocytes maintained in high-density monolayer during the first seven days of culture [36]. Chondrocytes cultured in three-dimensional
matrices, such as alginate used in this study, have been shown to maintain phenotypic
expression of extracellular matrix molecules for long term culture [56, 33]. Perhaps the
three-dimensional environment more typical of that found in native tissue influences
chondrocyte message stability.
The changes in aggrecan mRNA levels found the alginate-cultured chondrocytes dosed with actinomycin D would not account for the decrease seen in 35 S-sulfate
incorporation evident in the explant system. This would suggest that alternative
pathways are acting to decrease the level of GAG-sulfation.
Changes in individ-
ual protein synthesis would be reflective of the stability differences between various
messenger RNA's. As all mRNA's are inhibited by the drug, it is difficult to postulate which protein(s) involved in macromolecular aggrecan synthesis is/are the rate
limiting factor that causes the decrease in
1 5 S-sulfate
uptake. Perhaps the mRNAs
encoding the glycosyltransferases involved in chondroitin sulfate synthesis could have
a more rapid decay and result in the decrease seen in GAG sulfation. One study
showed that the addition of actinomycin D resulted in a 16% to 26% decrease in four
different glycosyltransferase enzyme activities, although this would not account for
the 82% decrease in sulfate incorporation seen in their system [53]. However, decrease
Section 5.4
-82in enzyme activity is perhaps due to decrease in enzyme production resulting from loss
of mRNA expression and can account for some inhibition of sulfate incorporation. It
would be interesting to examine mRNA expression of the various glycosyltransferases
during both mechanical loading and transcription inhibition to further elucidate the
initiation of changes in previously seen in these enzymes.
Our study has given evidence that mechanical compression and transcription
inhibition result in decreases in macromolecular ECM protein synthesis, and that the
two effects are additive when simultaneously applied. In addition, our results show
that messenger RNA for aggrecan and type IIA collagen are relatively stable after
transcription inhibition and suggest that the addition of mechanical compression does
not change aggrecan mRNA stability. Further studies that directly examine mRNA
levels during applied loading are being developed for chondrocytes maintained in alginate disk culture. Together, we hope to elucidate the mechanisms through which
mechanical forces influence chondrocyte metabolism, and hope to gain an understanding normal cartilage physiology and disease processes that are function of mechanical
wear, such as osteoarthritis.
Section 5.4
-83-
Appendix A
Alginate Material Properties are Stable in
Long-Term Culture
A.1
Introduction
Alginates are polysaccharides derived from brown algae and are composed of
#-D-
manuronic acid and a-L guluronic acid monomers. Divalent cations, such as Ca2+,
can form ionic bridges between adjacent sequential repeats of guluronic acid and cause
gelling of the liquid form of alginate [66]. These calcium bridges can be eliminated
using a calcium chelator which allow for a rapid release of cells or macromolecules
suspended in the alginate. This quality makes alginate an appealing material to use
for tissue engineering purposes, as it allows for study of newly synthesized matrix
molecules and while simultaneously allowing for measurements of intracellular markers that define the metabolic state of the cell.
Previous studies have examined the physico-chemical properties of alginate
[59], as well as their long-term stability [74]. The present study evaluated the longterm mechanical and electromechanical properties of alginate maintained as if it were
used for cell culture purposes. The goals were to establish it's potential as a stable
scaffold to deliver mechanical loads to cells seeded in the alginate.
A.2
Methods
Liquid 2% alginate was injected into diffusion permeable dialyzing cassettes and submerged into 102 mM CaCl2 for 20-30 minutes. The gelled alginate slab was cut free
from the cassette and washed sequentially 3 times in 1 X PBS and then placed in
culture medium (DMEM, 10% FBS, 100 U/ml penicillin, 100 pg/ml streptomycin,
and 0.025 pm/ml amphotericin B). Alginate disks were maintained in a humid envi-
Appendix A
-84ronment at 37'C 5% CO2 for up to 7 weeks.
On days 1, 15, 29, and 56, three to four 3-mm cylinders were punched from
the larger alginate slab. Because of the gelling methods, the ends of the cylinder were
uneven. Therefore, cylinders were placed through a cylindrical tube (1.5 mm by 3mm diameter), and the ends were shaved off to obtain the parallel surfaces necessary
for accurate mechanical testing.
The mechanical and electromechanical properties of the of the 1.5 mm by
3-mm diameter alginate disks were measured in uni-axial confined compression an
electrically insulating testing chamber [17]. The chamber was mounted in a Dynastat
mechanical spectrometer which was interfaced with a computer to control displacement. At eacht time point, individual disks were placed in the chamber between
two Ag/AgCl electrodes and bathed in PBS. The bottom of the alginate disk was in
contact with one electrode, whereas the second was electrode was suspended in the
surrounding bath. The top of the disk was in contact with a porous platen, which
separated it from the load cell used to record the strain-induced stress of the disk.
The equilibrium stress-strain behavior of the specimen was assessed in the
0-30% compression range. Sequential step compressions of 3% were slowly applied,
and the resulting load was recored after stress- relaxation of the disk was reached (5
minutes). The equilibrium stress was defined as the measured load divided by the
circular contact area of the disk. When the 30% static offset limit was reached, a
sinusoidal displacement of 45pm (3%) was superimposed at frequencies ranging from
0.02 Hz to 1 Hz. The imposed displacement and the resulting oscillating load and
streaming potential were measured and stored on the computer for further analysis.
The amplitude of the dynamic stiffness was defined as the measured load
normalized to the cylindrical disk area and to the dynamic strain (the dynamic displacement normalized to the original specimen thickness). The streaming potential
amplitude was computed as the measured voltage difference between the two Ag/AgCl
electrodes normalized to the dynamic strain. For review of the physical mechanisms
Section A.2
-85giving rise to the measured streaming potential see Frank et al. [17].
Finally, dedifferentiated chondrocytes were seeded in the alginate slabs described above. They were cultured in parallel with the unseeded alginate in the same
medium, lacking ascorbate. Ascorbic acid is necessary for production of collagen by
chondrocytes maintained in culture [15]. Therefore, the dedifferentiated chondrocytes
used in this experiment were most likely unable to produce the extracellular matrix
molecules necessary for a functional matrix . This became evident during material
properties evaluation, as the mechanical properties of alginate seeded with the chondrocytes had virtually no differences in equilibrium modulus, or streaming potential
compared to unseeded controls throughout the eight weeks of culture. There was
significant increase in dynamic stiffness of alginate slabs seeded with dedifferentiate
chondrocytes after eight weeks of culture. Those values are reported in the following
sections.
A.3
Results and Discussion
Figure A.1 shows a typical stress-strain relationship for the alginate disks tested in
uniaxial confined compression. A toe region is evident between 3% and 9% strain,
and is consistent with other poroelastic materials [18]. The slope of the linear region,
between 10% and 30% was used to calculate the equlibrium modulus for each sample.
Figure A.2B reports the average equlibrium modulus of 3-4 disks at various time
points in culture. Equilibrium moduli ranged from 9-18 kPa for all times points in
culture, and shows that there is no loss in mechanical integrity of the gel throughout
the seven weeks of culture. Streaming potential (Fig. A.3)amplitude did not vary
significantly with time in culture of with changes in frequency. Dynamic stiffness (Fig.
A.4) did not very with time in culture, but stiffness increased with corresponding increases in the applied frequency for all time points. This is characteristic behavior of
poroelastic material, such as cartilage where the frequency-dependent increase in dynamic stiffness is due to the increase in fluid velocity produced at higher compression
Section A.3
-86rates [18].
The data presented in Figure A.5 shows that dedifferentiated chondrocytes
maintained for 8 weeks in culture produced ECM molecules that resulted in an increased dynamic stiffness compared to the no-cell alginate disk controls at all frequencies. Although culture conditions were sub-optimal for ECM production (no ascorbic
acid was used), these results suggest that alginate is a suitable scaffold for chondrocyte culture and evaluation of the material properties of developing extracellular
matrix.
Alginate has been used to support cells in culture for other tissue engineering
purposes [49, 2] and appears to be equally beneficial for maintenance and culture
of chondrocytes derived from articular cartilage. Our data shows that alginate mechanical and electromechanical properties are stable in long term culture. Therefore,
alginate appears to be a suitable material to evaluate the physical properties of developing engineering tissues. In addition, it has added advantages over other gel
suspension systems due to the ability to rapid cell release for molecular analysis of
cell metabolic states.
3
2
0.T
*
II
0
10
20
30
Static Strain, %
Figure A.1: Equilibrium stress-strain behavior for alginate disks from day 0 of culture.
The dynamic stiffness and streaming potential were measured in the linear region of
the data (30% static strain).
Section A.3
-8750
40
.
30
0
20
w
W- 10
0
0
I
I
20
40
60
Days
Figure A.2: Equilibrium confined compression modulus HA of alginate disks versus
time in culture. These were calculated from the slope of the stress-strain curve in the
linear region of the data for each sample.
0.5
0 Day
*Day
A Day
EDay
0
15
29
56
0.4-
0-
0.3|
0)
C
0.21
-N
I
0.11-
01
0.0 1
0.1
1
Frequency (Hz)
Figure A.3: Amplitude of the streaming potential for frequencies ranging from 0.02
to 1.0 Hz, at four time point during a 7 week culture of alginate. Displacement
amplitude was 45pm.
Section A.3
-88-
1
*Day 0
*Day 15
A Day 29
* Day 56
0.8
cc
0.
0.61
C
E
0.4F
cc
C
0.2
-I
A
I
F
0'
O.0 1
L
0.1
1
Frequency (Hz)
Figure A.4: Amplitude of the dynamic stiffness of alginate at frequencies between
0.02 and 1.0 Hz at days 0, 15, 29, 56 in culture. Displacement amplitude was 45pm.
Section A.3
-89-
1
*No Cells: 8 wks
0 3rd HC Cells: 8 wks
0.8-
(.
0.6-
E
0.4 -
0.2 --
0
0. 1
0.1
-
Frequency (Hz)
Figure A.5: Dedifferentiated human chondrocytes ( 2 nd passage) were cast in alginate
slabs and cultured for up to 56 days. At days 0, 15, 29, and 56, the mechanical
integrity of newly synthesized matrix was assessed. No significant differences in equi-
librium modulus, HA, and streaming potential were evident between alginate samples
with and without cells (data not shown). Significant differences in dynamic stiffness
were evident at all frequencies (p < 0.005).
Section A.3
-90-
Appendix B
Human Chondrocytes In Native Tissue, and as
Primaries and Dedifferentiated Cells
B.1
Introduction
It is more difficult to examine chondrocyte behavior in human cartilage due to the
limited availability of human tissue, the lower cell density, and the wide age-related
differences in tissue samples. At the initiation of my Ph.D., we collaborated with
others who were examining the influence of mechanical forces on dedifferentiated
human chondrocytes in alginate. Difficulties arose in that we were unable to place
our results in context with the behavior of differentiated chondrocytes in intact human
cartilage. This series of studies that follow were my attempts to understand base-line
metabolism of human chondrocytes in intact tissue, as isolated primary cells, and as
dedifferentiated cells maintained in long term culture.
B.2
B.2.1
Methods
Human Chondrocytes in Native Tissue
Stability of Biosynthesis Cartilage shavings from the human femoral condyle
of a 54 year old were obtained within 36 hours of death.
Cartilage pieces (5-10
mg/well) were cultured for up to 11 days in medium (Dulbecco Modified Eagle
Medium (DMEM) with 10 mM HEPES, 10% fetal bovine serum (FBS), 0.1 mM
nonessential amino acids, antibiotics as described previously, 1 mM sodium pyruvate,
an additional 0.4 mM proline and 20 pg/ml ascorbate) at 370 C in 5% CO 2 . Medium
was changed daily. On days 3, 4, 5, 6, 8, and 10, cartilage shavings were cultured
in medium with l0pCi/ml
3
S-sulfate and 20pCi/ml of 3 H-proline. After 24 hours,
the cartilage pieces were removed from culture and washed in 1 X PBS with cold
Appendix B
-91proline and sulfate to remove residual label. Each sample was weighed, lyphilized,
and papain digested overnight. Total GAG, DNA, and radiolabel incorporation were
measured. Values presented are the average of n=6/time point +/-SM.
Response to Mechanical Compression 1-mm by 3-mm cartilage disks
were obtained from the femoral condyle of a 45 year old male within 36 hours of death.
Disks were cultured in medium (defined above) for 2 days. On day 3, cartilage disks
(n=6/chamber) were compressed to either 0.5-mm (50% compression) or 1.0-mm (cut
thickness). The first series of disks were compressed at time 0, and dual-labled with
35
S-sulfate and 3 H-proline. After twelve hours, these disks were removed from cul-
ture, washed, weighed, and lyophilized. The second series of disks were compressed
at time 0 and maintained in culture with unlabeled medium for twelve hours. After
twelve hours of compression, radiolabel was added to the culture medium, and the
compressed disks were culture for another twelve hours. These disks were then removed from culture, washed to removed free label, weighed and lyophilized. Cartilage
disks were analyzed for total DNA and macroscopic radiolabel incorporation. Values
presented are the mean of n=6/condition +/- SD.
B.2.2
Primary Human Chondrocytes in Alginate Disks
Human chondrocytes were obtained from whole tissue using sequential pronasecollagenase digestion [56].
Cells were seeded in 2% alginate sheets (see Methods
section, Chapter 3) at an initial density of 5 X 106 cells/ml. On days 0, 5, 10, 15
and 20 16 6-mm disks were punched from the larger cores. Disks in diameter were
grouped into 4 samples (n=4, 4 disks/sample) and labeled with 20 PuCi/ml
35
S-sulfate
for 24 hours. After labeling, disks were removed from culture and were assayed for
incorporation rates, viability, total GAG content and DNA content (see below). Culture medium was also assessed for macromolecular radiolabel incorporation that may
have been released into the media.
Section B.2
-92B.2.3
Dedifferentiated Human Chondrocytes in Alginate Slabs or
Cylinders
Human chondrocytes were plated in monolayer and cultured for 7 days or until 90%
confluency was reached. Cells were trypsinized to release them from the culture plates,
divided and subcultured for another 7 days or until cells reached 90% confluency.
Cells were trypsinized a second time, washed in PBS and total cell number was
counted. Dedifferentiated chondrocytes were seeded into 2% alginate in using either
alginate slabs created using a dialyzing cassette (Slide-a-Lyzer, 2-3 mm thick) or
dialysis tube (SpectraPore, 10 mm Diameter) [77]. Our collaborators were responsible
for cell casting and culture. The data are derived from using samples that were
made available to us at the designated times in culture. Total GAG content, DNA
content and
3 5S-sulfate
incorporation were measured for each sample and values are
presented as the mean (n=4) +/- SD. In one series of experiments, dedifferentiated
chondrocytes obtained from a 24 year old patient were expanded to third passage in
10% FBS/DMEM in monolayer culture. Cells were seeded at two densities (1 X 105
and 1 X 106) in order to assess the influence of cell density on chondrocyte biosynthesis
and redifferentiation. In a second experiment, dedifferentiated chondrocytes obtained
from a 24 year old patient (different than above) and seeded at 6 X 105 cells/ml
cultured for up to three weeks. On days 1, 7, 14, 21 and 28, samples of the culture
were assessed for
3 5S-sulfate
incorporation rates, total GAG and total DNA content.
Values are reported as the mean (n=3-4) +/- SD.
B.3
B.3.1
Results and Discussion
Chondrocytes Maintained in Native Tissue
Total GAG (Fig.
B.1), and biosynthesis levels (Figs.
B.2 and
B.3) remained
relatively constant over the last 7 days of an 11 day culture, suggesting that human
explant system is a reasonable model to examine changes in chondrocyte behavior
Section B.3
-93during culture.
Biosynthesis levels increased slightly from days 4 to 7, but were
not significantly different than day 4 levels. In addition, cartilage shavings were not
matched for depth or location which could account for variations from time point to
time point. In addition, biosynthesis levels of human chondrocytes are approximately
1/ 5 1h/10h of that of chondrocytes from young bovine tissue.
Biosynthesis levels in human cartilage compressed to 50% of its original thickness decreased compared to controls, particularly in the last 12 hours of a 24 hour
compression (Figs. B.4 and B.5). This suggests that human chondrocytes are sensitive to mechanical compression in similar ways to that reported in bovine tissue.
However, response appears to be delayed in comparison. It is important to note that
synthesis levels are dropping two orders of magnitude in comparison to those typically found in bovine tissue, bordering on the limitations of the incorporation assays
typically used.
B.3.2
Human Primary Cells Cultured in Alginate Disks:
A Time
Course Study
Human primary cells in culture tended to increase in cell number by day 20 in culture
to approximately 3 times the original seeding density (Fig. B.6). Total GAG accumulation was minimal, and bordered the resolution of the assay (Fig. B.7). Finally
macromolecular incorporation of
3
1S-sulfate
was relatively constant, but significantly
reduced compared to the synthesis levels of human explants from which the cells were
isolated (Fig. B.8). At day 20, chondrocytes did not appear to recover to the explant
baseline value, unlike isolated bovine chondrocytes cultured in the alginate system
(see Results section, Chapter 3). Human chondrocytes were viable throughout the
culture, as assessed by a live/dead fluroscence assay. This set of data suggests that
the isolation of human chondrocytes from tissue could irreversibly alter the chondrocytes capabilities of synthesizing ECM molecules. Other methods of isolation were
not tested to optimize the protocol for human tissue and might be considered in the
Section B.3
-94-
-3UUI
200-
z
M
100 -
0
0
I
2.5
I
5
I
7.5
10
12.5
Culture Time (Days)
Figure B.1: Total GAG content of human cartilage explants maintained in culture
for up to eleven days. Glycosaminoglycan content was assess using the DMMB dye
method.
Section B.3
-95-
3
C
2
0
L.
0
N
0
z
1
ni
0
I
2.5
I
5
I
7.5
I
10
12.5
Culture Time (days)
Figure B.2: Aggrecan synthesis (as measured by macromolecular sulfate incorporation) as a function of time in culture.
Section B.3
-96-
3
20.
0
G0±
N
E
z0
1 -J
CutreTm
01I
0
2.5
5
(as
7.5
10
12.5
Culture Time (days)
Figure B.3: Total protein synthesis (as measure by macromolecular proline incorporation) as a function of time in culture.
Section B.3
-97-
0.05
0
Control
o
50% Compression
0.04-
o
0.03-
0
E
0.02-
0.01
0
0-12 hrs
12-24 hrs
Labeling Time During Compression
t
Figure B.4: Cartilage disks (1-mm by 3-mm diameter) were cored from human cartilage and statically compressed to either 1.0-mm (control) or to 0.5-mm (50% compression). Compression lasted either 12 or 24 hours, with 35S-sulfate incorporation
assessed during the last 12 hours of culture.
Section B.3
-98-
0.05
0
O
Control
50% Compression
0.04-
0.030
E
0.02-
0.01
0
12-24 hrs
0-12 hrs
Labeling Time during Compression
Figure B.5: Cartilage disks (1-mm by 3-mm diameter) were cored from human cartilage and statically compressed to either 1.0-mm (control) or to 0.5-mm (50% compression). Compression lasted either 12 or 24 hours, with 3 H-proline incorporation
assessed during the last 12 hours of culture.
Section B.3
-99future.
20
e.
15-
E
10-
E
z
L)
0
0
5
10
15
20
Day in Culture
Figure B.6: Cell density of primary human chondrocytes in alginate disks versus time
in culture. Cell number was calculated from DNA content using 7.9 X 10-1 g/cell
[42].
B.3.3
Dedifferentiated Human Chondrocytes Cultured in Alginate
Effect of Cell Density on Biosynthesis and GAG Accumulation The effect
of cell seeding density on chondrocyte biosynthesis levels was examined. Dedifferentiated chondrocytes proliferated during the initial 2-3 weeks of culture (Fig.
B.9.
Chondrocytes initially seeded at 1 X 105 proliferated at a higher rate, reaching a
seeding densitiy similar to alginate seeded initially at 1 X 106 by day 21. Bioynthesis
rates of dedifferentiated chondrocytes at both seeding densities were similar throughout the three weeks of culture (Fig.
B.10). Interestingly, synthesis levels were very
similar to that of native cartilage, although it is difficult to compare dedifferentiated chondrocytes derived from the cartilage of a 24 year old patient to chondrocytes
Section B.3
-100-
2
z
0
1.5-
IMI
T
1
.
0
0
S0.5-
0
5
10
15
20
Day in Culture
Figure B.7:
Total GAG content accumulated by primary human chondrocytes in
alginate versus time. GAG content was assessed using a modified DMMB method
that accounted for the background charge of the alginate.
Section B.3
-101-
0.02
OTotal
ODisk
AMedia
Explants Day O
0.015
z
a
C,)
0.01
LO
CO
E
0.005--
0
5
10
15
20
Day
Figure B.8: Macromolecular 3 5S-sulfate incorporation into the alginate disk and released into the media versus time in culture. The multiscreen 3 5S-sulfate incorporation
assay [52] was used in analysis.
Section B.3
-102maintained in the native tissue from that of a 45 year old patient. The specific type
of proteoglycan synthesized remains to be examined.
10
*
1 X 105 Cells/mi
*
1 X 106 Cells/mi
-I
8
U
6
ID
0
4
2
II
h
0
I
I
I
I
7
14
21
28
35
Day in Culture
Figure B.9: Cell density of dedifferentiated human chondrocytes in alginate disks
versus time in culture. Chondrocytes were initially seeded at 1 X 105 or 1 X 106
cells/ml. Cell number was calculated from DNA content using 7.9 X 10-12 g/cell
[42].
A Second Culture with Dedifferentiated Human Chondrocytes
Chondrocytes isolated from tissue of a second 24 year old patient were expanded in
monolayer and cast into 1-cm diameter alginate cylinders. Cells were cultured for up
to 28 days. On days 1, 7, 21 and 28, samples were examined for total DNA content and
35
S-sulfate incorporation. Total cell density remained constant throughout culture
(Fig.
B.11).
Incorporation levels decreased during the course of the culture to
one-half of that on day 1 (Fig.
B.12). Biosynthesis levels were simlar to that of
chondrocytes maintained in native cartilage, although again comparisons due to cell
state and patient age differences make direct comparisons difficult.
Section B.3
-103-
0.04
OTotal-10 5 cells/mi
ODisk-10 5 cells/mi
AMedia-10 5 cells/mi
*Total-106 cells/mI
UDisk-106 cells/ml
AMedia-10 6 cell/ml
0.03
0
z
I=
U,)
0.02
E
0.01
0
7
14
21
Day in Culture
28
35
Figure B.10: Macromolecular 3 1S-sulfate incorporation into the alginate disk and released into the media versus time in culture. The multiscreen 31S-sulfate incorporation
assay [52] was used in analysis.
Section B.3
-104Long Term Culture in Alginate 'Sausages'
1.5
E
1
0
0.5 l -
0L
0
I
I
10
20
30
Day in Culture
Figure B.11: Cell density of dedifferentiated human chondrocytes in alginate disks
versus time in culture. Chondrocytes were initially seeded at 6 X 10' cells/ml. Cell
number was calculated from DNA content using 7.9 X 10-12 g/cell [42].
B.4
Conclusions
Experimentation with human tissue is difficult on many levels. Firstly, biosynthesis
rates are at least 20% lower than that typically seen in bovine explants, so care must be
taken that raw incorporation counts are well above background limits of scintillation
assays.
The explant system was well above these limits, however dedifferentiated
chondrocyte biosynthesis was on the border of detectable levels for the protocols used
in this series of experiments.
GAG accumulation in alginate was difficult to assess as accumulation often
was lost in the background values inherent using the alginate system. Alginate is
Section B.4
-105-
0.05
0.04-
Z
0
0.03Total
C
0.02-
Media
E*
0.01
-A'
.Alinate
01
0
1W
1
10
20
30
Day in Culture
Figure B.12: 35S-sulfate incorporation by dedifferentiated human chondrocytes into
the alginate disk and released into the media versus time in culture. The multiscreen
35S-sulfate incorporation
assay [52] was used in analysis.
Section B.4
-106highly charged, therefore DMMB assays (which are designed to measure charge) are
modified to deal with the alginate background.
In summary, these experiments help place in context the relative differences in
biosynthesis levels for chondrocytes in native tissue, as primary cells in alginate, and
as dedifferentiated cells in alginate. There is an order of magnitude range in these levels, which would be larger if chondrocytes were subjected to mechanical compression,
as static compression has been shown to significantly decrease chondrocyte aggrecan
synthesis. Refined measurement techniques would have to be considered to ensure
accurate assessment of chondrocyte behavior.
Section B.4
-107-
Appendix C
Response of a Cartilagenous Tissue Construct to
Dynamic Compression
Vicki I. Chin, Koichi Masuda, Paula M. Ragan, E. J-M. A. Thonar and Alan J.
Grodzinsky
C.1
Introduction
Osteogenic protein-1 (OP-1), also known as bone morphogenic protein-7(BMP-7) has
been shown to increase production of extracellular matrix macromolecules. A novel
method of producing cartilaginous tissue in vitro using primary chondrocytes treated
with OP-1 has been developed by collaborators at Rush Medical College in Chicago,
IL[51]. In this system, chondrocytes are grown in alginate beads to maintain their
phenotype and produce enough cell-associated matrix to form a cohesive matrix when
the alginate is dissolved away. After the alginate has been dissolved, this procedure
does not require the further use of a scaffold, eliminating any biocompatiblity or
degradation issues. The cells and the associated matrix are then placed on a cell culture insert with a porous membrane. Masuda et al. have demonstrated that the cells
remain phenotypically stable after 10 days on the insert and that the cartilaginous
mass stains strongly for Safarnin-O [51]. Previous studies have shown that dynamic
compression increases the level of synthesis of matrix macromolecules in cartilage explants. Our objective was to determine whether dynamic compression would have a
similar effect on the in vitro-engineered cartilaginous tissue.
Appendix C
-108-
C.2
Methods
The cartilaginous matrix was grown at Rush Medical College in Chicago, IL using
the alginate recovered chondrocyte (ARC) method [51]. Briefly, chondrocytes from
the articular cartilage of 14-18 month-old bovine steers were isolated and seeded in
alginate beads. These beads were cultured for 10 days in complete culture medium
(DMEM/F-12+20% FBS) or complete culture medium plus 200 ng/mL recombinant
OP-1(Creative BioMolecules, Boston, MA). After 10 days, the beads were dissolved
and the chondrocytes and cell-associated matrix were placed on a cell culture insert
and cultured for 12 more days. The tissue was then shipped overnight to MIT on wet
ice. The tissue on the membrane was then removed and cylindrical disks (0.5 mm x
5mm diameter) were cored using a sterile biopsy punch (Premier Medical Products,
King of Prussia, PA). Disks (n=6) were compressed to 100% thickness between impermeable platens and a sinusoidal strain of 4% at a frequency of 0.1 Hz was imposed
on the tissue. In a second chamber, disks (n=6) were compressed statically to 100%
thickness to serve as a control. A third group of disks were cultured in free swelling
conditions. Disks in all groups were cultured in the presence of 20 Ci/ml
1 5 S-sulfate
for 24 hours under the different loading conditions. The disks and associated media
were analyzed for
3 5S-sulfate
incorporation into macromolecules,total GAG content,
and total DNA content using methods previously described in this thesis. Values are
reported as the mean of 6 disks +/- SD.
C.3
Results and Discussion
Two experiments were performed using the above protocol. In the first experiment
performed, dynamic mechanical compression of the tissue constructs cultured in 20%
FBS resulted in a more than eight-fold increase in 35S-sulfate incorporation over the
statically compressed controls after 24 hours in culture (Fig.
C.1). For the disks
cultured in OP-1, the dynamically compressed group had double the incorporation
Section C.2
-109rate as the statically compressed group. There was no statistical difference between
the free swelling group and the dynamically compressed group in either the 20% FBS
case or the OP-1 treated case.
There were no statistical differences in the total DNA or total GAG content between the two groups in either case (Fig. C.2). These results suggest that dynamic
compression can significantly stimulate glycosaminoglycan production by chondrocytes in this new matrix. In addition, this study suggests that mechanical loading
(e.g., CPM) after transplantation of such a cartilaginous tissue construct may help
stimulate in vivo remodeling of the tissue.
In the second repeat of the experiment, similar trends were observed. The level
of 35S-incorporation of the dynamically compressed disks was on the same order of
magnitude as the level found in the free swelling disks. The incorporation level of the
statically compressed group was significantly lower when compared to the levels of the
other groups, but in this experiment, the incorporation of the statically compressed
group was exceedingly low (Fig. C.3). This may have indicated that viability was
compromised in this group and therefore the disks were not synthesizing any new
proteoglycan, but a viability assay was not performed on this experiment. Therefore,
it is uncertain whether the low incorporation level resulted from low viability or a
lowered synthesis rate. Total GAG and DNA were not significantly different in any
group (Fig. C.4)In future experiments, this problem will be addressed by performing
a viability assay on the samples at the end of mechanical compression.
Together, these experiments show that dynamic compression increases 35Ssulfate incorporation in tissue formed by the ARC method when compared to statically compressed controls. This suggests that dynamic compression and the OP-1
growth factor can act synergistically to increase 35S-sulfate incorporation.
Section C.3
-110-
0.3
Sample
n:6
IOP-1 treated
020% FBS only
0.20.1 -
011
Dynamic
Static
Free Swell
Dyn Media
Stat Media
Free Swell Media
0.3
Media
n:6
0.2-
0.1-
Figure C.1: "S-sulfate incorporation of chondrocytes grown in ARC method subjected to dynamic, static and free swelling conditions. The top figure shown radiolabel
incorporation into the cartilage-like disk. The bottom figure shows macromolecular
radiolabel released into the media during culture.
Section C.3
-111-
0.35
6 0.28
0
Sample +Media
n:6
0.14
C
020% FBS only
-I
0.21
0)
E
I ON treated
0.07
n%
U~
-
Dynamic
Static
Free Swell
Static
Free Swell
12
.5
4
z
0
Total GAG
I0 -n=6
7.50)
52.51Al-
I
.
Dynamic
Figure C.2: Top: Total 31S-sulfate incorporation of chondrocytes grown in ARC
method subjected to dynamic, static and free swelling conditions. Bottom: Total
GAG normalized to DNA content of the cartilage-like disks for each compression
condition.
Section C.3
-112-
0.2
Sample
0.1 5 -n=6
z
IOP-1 treated
020% FBS only
0.1
U)
5-
E
C
01
Dynamic
Static
Free Swell
Dyn Media
Stat Media
FS Media
0.2
Media
z 0.1 n=6
0
0 .
0. 5 -
E
C
A
Figure C.3: 31S-sulfate incorporation of chondrocytes grown in ARC method subjected to dynamic, static and free swelling conditions. The top figure shown radiolabel
incorporation into the cartilage-like disk. The bottom figure shows macromolecular
radiolabel released into the media during culture. This is a repeat of the first series
of experiments.
Section C.3
-113-
0. 2
Sample +Media
z 0.1 5 -n=6
IOP-1 treated
020% FBS only
0
(0
0.
ILn
0.0 5 Dynamic
Static
Free Swell
Static
Free Swell
E
5
Total GAG
4 -n=6
0
0)
32-
Dynamic
Figure C.4: Top: Total 3 1S-sulfate incorporation of chondrocytes grown in ARC
method subjected to dynamic, static and free swelling conditions. Bottom: Total
GAG normalized to DNA content of the cartilage-like disks for each compression
condition.
Section C.3
-114-
C.4
Future Studies
The possible benefits of dynamic mechanical compression on tissue engineered cartilage have only begun to be considered.
Our results show that dynamic loading
increases proteoglycan synthesis and suggests that the use of CPM would be beneficial for cartilage transplant patients. The next step in this series of experiments would
be to test the OP-1 treated tissue with media containing OP-1. In our studies, chondrocytes were cultured in OP-1 only during the first 14-18 days in culture. It would
be important to use OP-1 during the mechanical compression studies to determine
whether dynamic compression was capable of further stimulating 35S-incorporation
or if OP-1 had maximally stimulated the proteoglycan production in the sample.
This series of experiments would give further insight into the benefit of administering
growth factors to patients once tissue transplantation has occurred.
Section CA
-115-
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