The Influence of Mechanical Compression On The Induction of Osteoarthritis-Related Biomarkers I N A R T I C U L A R Cartilage Explants
The Influence of Mechanical Compression On The Induction of Osteoarthritis-Related Biomarkers I N A R T I C U L A R Cartilage Explants
The Influence of Mechanical Compression On The Induction of Osteoarthritis-Related Biomarkers I N A R T I C U L A R Cartilage Explants
ª 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.joca.2005.07.003
International
Cartilage
Repair
Society
Summary
Objective: Macromolecules of the articular cartilage extracellular matrix released into synovial fluid, blood, or urine can serve as potentially
useful biomarkers of the severity of osteoarthritis (OA). Biomechanical factors play an important role in OA pathogenesis, yet their influence on
biomarker production is not well understood. The goal of this study was to examine the hypothesis that dynamic mechanical stress influences
the release of these biomarkers from articular cartilage.
Methods: Explants of porcine cartilage were subjected to dynamic compression at 0.5 Hz for 24 h at stresses ranging from 0.006 to 0.1 MPa.
The concentrations of cartilage oligomeric matrix protein (COMP), keratan sulfate (KS measured as the 5D4 epitope), total sulfated
glycosaminoglycan (S-GAG), and the KS (keratanase-digestible) and chondroitin sulfate (CS) (chondroitinase-digestible) fractions of S-GAG
were measured. Radiolabel incorporation was used to determine the rates of proteoglycan and protein synthesis.
Results: The magnitudes of mechanical stress applied in this study induced nominal tissue strains of 4e23%, consistent with a range of
physiological to hyperphysiologic strains measured in situ. COMP release increased in proportion to the magnitude of dynamic mechanical
stress, while KS, CS and total S-GAG release increased in a bimodal pattern with increasing stress. Protein and proteoglycan synthesis were
significantly decreased at the highest level of stress.
Conclusion: Mechanical stress differentially regulates the turnover of distinct pools of cartilage macromolecules. These findings indicate that
mechanical factors, independent of exogenous cytokines or other stimulatory factors, can influence the production and release of OA-related
biomarkers from articular cartilage.
ª 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Key words: Proteoglycan, Cartilage oligomeric matrix protein, COMP, Keratan sulfate, Chondroitin sulfate, Biomechanics, Mechanical
compression, Arthritis.
1092
Osteoarthritis and Cartilage Vol. 13, No. 12 1093
measure of proteoglycan, total S-GAG, also increases in the applied stress magnitude. A range of compressive loads
synovial fluid in clinical7,27 and animal models28 of OA. was applied to articular cartilage explants to induce
Despite the extensive consequences of OA, the etiopa- physiologic and hyperphysiologic magnitudes of tissue
thogenesis of this disease is not fully understood and strain (deformation). The release of COMP, the proteogly-
appears to involve a complex interaction of biomechanical can epitope 5D4 (KS), and total S-GAG, as well as the KS
stress as a cofactor together with the local biochemical (keratanase-digestible) and CS (chondroitinase-digestible)
environment1,29e31. Both clinical and animal studies have fractions of S-GAG, into the media was measured.
provided strong evidence that mechanical factors can Radiolabel incorporation of 35S-sulfate and 3H-proline was
initiate and contribute to the imbalance of cartilage used to determine the rates of proteoglycan and protein
metabolism in OA. Alterations in the normal pattern of joint synthesis, respectively, as a function of stress magnitude.
loading may predispose to OA and may be caused by
a variety of factors such as immobilization, joint instability,
overuse, trauma, injury, or obesity32e34. Studies of OA Materials and methods
progression following meniscal or ligamentous instability
ARTICULAR CARTILAGE EXPLANTS
link alterations in joint loading and kinematics to specific
biomechanical changes at the tissue level that may Full-thickness explants of articular cartilage were har-
predispose the joint to OA, such as decreased cartilage vested from the femoral condyles of skeletally mature female
stiffness (tensile and compressive moduli) and increased pigs (1.5e3 years old) using a 5 mm dermal biopsy punch
hydraulic permeability35e37. (Miltex Inc, Bethpage, NY) and separated from the underlying
To better understand the role of mechanical stress on bone. The explants were harvested in adjacent pairs to allow
articular cartilage, several in vitro models have been de- site-matching of control (uncompressed) and compressed
veloped that allow the application of mechanical stress to specimens to reduce site-to-site variability among explants.
articular cartilage in a controlled biochemical environment. In Before testing, the explants were allowed to stabilize in
such models, continuous or intermittent compression can culture for 72 h at 37(C, 5% CO2, in a 48-well plate containing
alter the biosynthetic rates of proteoglycan and colla- 1 ml/well of standard culture medium consisting of Dulbec-
gen35,38e47. The consensus of these studies is that static co’s Modified Eagle Medium (Gibco, Gaithersburg, MD) with
compression suppresses matrix biosynthesis, while cyclic 10% fetal bovine serum (heat inactivated at 56(C for 30 min)
and intermittent loading can either stimulate or suppress (Sigma Chemicals, St. Louis, MO), 0.1 mM modified eagle
matrix synthesis, depending on the frequency or magnitude medium non-essential amino acids (Gibco), 100 U/ml peni-
of loading. High rates or magnitudes of stress can induce an cillin/streptomycin (Gibco), 10 mM N-(2-Hydroxyethyl)piper-
‘‘injurious’’ response that has been associated with increased azine-1-ethanesulfonic acid buffer (Gibco), and 37.5 mg/ml
degradation, cell death, and the production of matrix metal- ascorbate-2-phosphate64. Synthetic rates and levels of
loproteinases40,48e52. These responses have been reported biomarkers released into the culture media were normalized
over a wide range of loading magnitudes and frequencies, to the wet weight of cartilage measured before compression.
and exhibit a stressedose dependency53.
Evidence exists for an interaction between mechanical
stress and OA-related biomarker release. In vivo studies MECHANICAL COMPRESSION EXPERIMENTS
show that physical exercise (running on a treadmill for 60 min
or playing soccer for 90 min) can increase the levels of the To determine the effects of mechanical compression on
5D4 epitope54, as can running in horses55. However, other biomarker release, individual explants were dynamically
studies have reported no statistically significant difference in loaded in unconfined compression at stresses of
serum KS levels in marathon runners (before and after a 42- 0.006e0.1 MPa using a modified version of the Biopress
km marathon)56,57 or in patients with arthritis in response to system, a computer-controlled instrument for compressing
a more chronic (3 months) conditioning activity58. A recent tissue explants (Flexcell International, Hillsborough,
study has shown that serum COMP levels are increased NC)64e66. Briefly, individual cartilage explants were placed
immediately following 30 min of walking in healthy subjects59. in Biopress culture plates (Flexcell International) consist-
Several in vitro studies also provide evidence for a link ing of a Delrin chamber attached to the bottom of a flexible
between dynamic loading of cartilage and the release of silicone rubber membrane. A range of calibrated air
various biomarkers of OA. Dynamic compression of cartilage pressures was applied to the membrane, and the corre-
in culture increased immunolabeling for the 3B3() epitope of sponding compressive stress (s) applied to the explant was
CS, which is believed to be an early indicator of alterations in determined from the applied force (F ) and the initial cross-
cartilage metabolism60 and a marker of cartilage develop- sectional area (A) of the explant, where s Z F/A. The loads
ment41. Other studies have shown increased 3B3() labeling were applied at a frequency of 0.5 Hz for 24 h at 37(C, 5%
in adult bovine cartilage under a relatively severe compres- CO2. Unloaded (control) explants were incubated for 24 h
sion regimen (5 MPa, 24 h at 0.5 Hz) that was also under the same culture conditions.
associated with cell death and tissue damage40. Cyclic
mechanical compression or tension can upregulate the
MEASUREMENT OF TISSUE STRAIN UNDER
expression of the COMP gene at different frequencies and DYNAMIC LOADING
magnitudes of stress in vitro61e63. These few studies suggest
an interaction between mechanical stress and OA-related To determine the deformation of the explants under the
biomarkers; however, the doseeresponse relationship be- range of applied stresses, a set of explants (n Z 5) was
tween the magnitude of dynamic compression and the loaded under the same conditions in an electromechanical
synthesis and release of these biomarkers is not fully materials testing system (EnduraTec ELF 3200, Minnetonka,
understood. MN) using a closed-loop, load-controlled test regimen. Each
The goal of this study was to test the hypothesis that explant was first placed under a tare load of 10 gf and
cyclic mechanical compression of cartilage alters the allowed to equilibrate for 1 h. Cyclic compression was
release of biomarkers of OA in a manner that depends on applied at 0.006, 0.0125, 0.025, 0.05, or 0.1 MPa at 0.5 Hz
1094 J. L. Piscoya et al.: Influence of mechanical compression on induction of OA-related biomarkers
and allowed to reach a steady-state response for 3 h. At this individually, with significance reported at the 95% confidence
point, the root-mean-square (RMS) and the range of strain level (P ! 0.05). Correlation analysis was performed using
amplitudes over a loading cycle were determined at steady the Pearson ProducteMoment Correlation test.
state as a function of the applied stress.
Results
MEASUREMENT OF BIOMARKER RELEASE
TISSUE STRAIN UNDER DYNAMIC LOADING
After 24 h of compression, levels of COMP and KS in the
media surrounding the explants were measured by en- Explants showed increasing peak-to-peak strain magni-
zyme-linked immunosorbent assay. KS was measured with tudes with increasing mechanical stress magnitudes from
mAb 5D4 as previously described10. COMP was measured 0.006 to 0.1 MPa, resulting in nominal tissue strains of
with mAb 12C421, which cross-reacts to porcine COMP, as 4e23% (Fig. 1).
previously described67.
RELEASE OF COMP AND 5D4 EPITOPE WITH LOAD
MEASUREMENT OF TOTAL S-GAG AND KERATANASE- AND COMP release into the media increased with increasing
CHONDROITINASE-DIGESTIBLE FRACTIONS OF S-GAGS magnitude of mechanical loading, with significant increases
Total S-GAG was measured with the 1,9-dimethylmethy- at 0.05 MPa (P ! 0.05) and at 0.1 MPa (P ! 0.01) com-
lene blue (DMB) assay68,69. For this assay, eight standards pared to uncompressed explants (Fig. 2). KS release (5D4
were used, prepared with control media and CS C (Sigma epitope) significantly increased at 0.05 MPa, with a peak
Chemicals) at concentrations 0e100 mg/ml. Twenty micro- release observed at 0.0125 MPa (Fig. 3).
liters per well of standards and sample media were pipetted
into a 96-well plate, and 125 ml/well of DMB (pH 3.00) was RELEASE OF TOTAL S-GAG AND KERATANASE- AND
added. Optical absorbance was read at 540 nm within 5 min CHONDROITINASE-DIGESTIBLE FRACTIONS
of DMB addition. OF S-GAG WITH LOAD
To determine the relative proportions of KS and CS, the
predominant S-GAGs in cartilage, the media were digested Mechanical compression significantly increased total
using purified keratanase I (KI, Pseudomonas sp) and II S-GAG release into the media at all magnitudes of com-
(KII, Bacillus sp) or chondroitinase ABC (ABC, P. vulgaris) pression above 0.006 MPa (P ! 0.01), with the highest
(Seikagaku, Tokyo, Japan and MP Biomedicals, Costa release at 0.0125 MPa [Fig. 4(a)].
Mesa, CA), respectively. KI hydrolyzes monosulfated di- The proportion of total S-GAG released due to total KS
saccharide repeats in KS, while KII hydrolyzes mono- and (KI C KII-digestible fraction) and CS (ABC-digestible frac-
disulfated disaccharide repeats. ABC hydrolyzes CSs A, B, tion) paralleled the increases in total S-GAG (undigested)
and C. Briefly, 25 ml aliquots of experimental media were release into the media [Fig. 4(a and b)] as compared to site-
incubated with no enzyme (undigested), 37.5 mU of KI, matched, uncompressed controls. Significant increases in
6.5 mU of KII, 37.5 mU of KI and 6.5 mU of KII, or 37.5 mU total KS (KI C KII digestion) were observed at
of ABC. All digestions were performed in 0.1 M Tris acetate 0.025e0.1 MPa (P ! 0.05), and in CS at 0.0125e0.1 MPa
and 0.05 M sodium acetate digestion buffer (pH 6.5) for 4 h (P ! 0.01), except at 0.025 MPa [Fig. 4(b)]. The chondroi-
at 37(C. Total S-GAG concentration was measured in the tinase-digestible fraction of S-GAG contributed to the
undigested and digested samples using the DMB assay as majority of the S-GAG levels in both control (68e89%)
described above. The difference in total S-GAG levels and loaded groups (72e83%). The KI-digestible fraction of
between the undigested and digested samples was
attributed to the amount of KS or CS originally present in
25
the samples.
Surface-to-Surface Strain (%)
300 Discussion
Control **
Compressed
|____|
The results of this study show that dynamic mechanical
compression can alter the release of the OA-related
biomarkers COMP, KS, and S-GAG, and alter the rates of
extracellular matrix synthesis in a manner that varied with
COMP (% Control)
200
the magnitude of applied stress. These findings also
*
|____| indicate that the magnitude of mechanical stress can
differentially regulate the turnover of distinct pools of
cartilage macromolecules. These data suggest that the
production and release of cartilage biomarkers in vivo may
100 be influenced by the local mechanical environment of the
joint, and support the hypothesis that biomechanical factors
may contribute to metabolic changes in cartilage that are
characteristic of OA10,30,31,37,59.
The magnitudes of mechanical stress applied in this
0 study were selected to induce nominal tissue strains of
0.006 0.0125 0.025 0.05 0.1
4e23%, which represent a range of physiologic to hyper-
Stress (MPa) physiologic strains observed in human cartilage in vitro or in
vivo70,71. For example, previous radiographic studies have
Fig. 2. COMP release into the culture media following mechanical reported strains of 2e20% in femoral head cartilage under
loading of 0.006e0.1 MPa at 0.5 Hz. Results are normalized to the
wet weight of the articular cartilage explants and expressed as the
a static load of five times body weight70, while other
percent change of compressed over control values. Data are magnetic resonance imaging studies on healthy individuals
presented as mean G S.E.M., n Z 18, *P ! 0.05, **P ! 0.01. showed mean changes in cartilage thickness of 2.8% and
4.9% after several minutes of recovery following dynamic
loading (knee bends) or static loading (squatting), re-
S-GAG release increased significantly at 0.05 MPa spectively71. While it is difficult to correlate directly the
(P ! 0.005) [Fig. 4(c)], while the KII-digestible fraction stressestrain parameters between our explant loading
increased significantly at 0.025e0.1 MPa (P ! 0.05) system and the in vivo state, we presume that the tissue
[Fig. 4(d)]. strains induced in vitro by the applied stresses in our model
represent a physiologic range from 0.006 to 0.05 MPa,
while the higher stress magnitude (0.1 MPa) may corre-
PROTEOGLYCAN AND PROTEIN SYNTHESIS spond to a hyperphysiologic magnitude of tissue strain.
No significant changes were observed in the rates of The release of COMP into the media increased with
proteoglycan and protein synthesis at low magnitudes of increasing magnitude of mechanical stress at magnitudes
mechanical stress (0.006e0.0125 MPa) [Fig. 5(a and b)]. greater than 0.025 MPa. This increased COMP may have
Higher levels of magnitude of mechanical stress resulted from facilitated release due to dynamic loading or
(0.025e0.01 MPa) decreased proteoglycan and protein degradation of COMP, rather than increased biosynthesis
synthesis relative to control, non-compressed samples as the overall matrix biosynthetic rates did not increase with
(P ! 0.05 for 0.1 MPa). increasing stress. Our data, in conjunction with previous
studies showing increased COMP gene expression follow-
ing cyclic compression of bovine articular cartilage ex-
plants61,63, support the hypothesis that the synthesis and
250
release of COMP in cartilage can be altered by mechanical
Control
compression. Furthermore, a recent study has shown
Compressed
increased serum COMP levels in healthy subjects following
30 min of walking59, providing in vivo supporting evidence
200
*
|____| that local mechanical factors influence COMP production
and release from the articular cartilage.
5D4 (% Control)
(a) (b)
KS or CS Proportion (% Control)
**CS Control CS
**
|____| Control |____|
400 Compressed 400 Compressed CS
Control KS
Compressed KS
GAG (% Control)
300 **
|____| 300 **CS, KS
|____|
**CS, KS
***
|____|
|____|
*CS
200 **
|____| 200 |____| *KS
|____|
100 100
0 0
0.006 0.0125 0.025 0.05 0.1 0.006 0.0125 0.025 0.05 0.1
Stress (MPa) Stress (MPa)
(c) (d)
100 100
Control Control
Compressed Compressed
80 80
60 60 **
*
|____|
|____|
*
40 40
|____|
**
|____|
20 20
0 0
0.006 0.0125 0.025 0.05 0.1 0.006 0.0125 0.025 0.05 0.1
Stress (MPa) Stress (MPa)
Fig. 4. The effects of mechanical compression at 0.006e0.1 MPa on (a) S-GAG release; (b) proportion of total S-GAG release in the culture
media due to CS and KS via enzymatic digestion with 37.5 mU chondroitinase ABC or 37.5 mU of KI and 6.5 mU of KII, respectively; (c)
proportion of total S-GAG release following enzymatic digestion with 37.5 mU of KI alone; and (d) proportion of total S-GAG release following
enzymatic digestion with 6.5 mU of KII alone. Results are normalized to wet weight of the articular cartilage explants and expressed as the
percent change of compressed over control values. Data are presented as mean G S.E.M., n Z 18, *P ! 0.05, **P ! 0.005, ***P ! 0.01.
facilitated release due to dynamic loading or aggrecan behaved differently from that digestible by KII with in-
catabolism, rather than increased proteoglycan synthesis creasing mechanical loading, indicating differences in the
was responsible for the observed effect. In addition, our amounts of mono- and disulfated disaccharides present.
findings showed that the fraction of KS digestible by KI Previous studies have characterized the disaccharide
(a) (b)
Incorporation (% Control)
150 150
Incorporation (% Control)
Control Control
Compressed Compressed
*
|____|
*
120 120 |____|
90 90
60 60
3H-proline
30 30
4
35SO
0 0
0.006 0.0125 0.025 0.05 0.1 0.006 0.0125 0.025 0.05 0.1
Stress (MPa) Stress (MPa)
Fig. 5. (a) Proteoglycan synthesis and (b) protein synthesis measured by the uptake of 35SO4 and 3H-proline, respectively, by cartilage
explants following mechanical loading of 0.006e0.1 MPa at 0.5 Hz. Results are normalized to wet weight of the articular cartilage explants and
expressed as the percent change of compressed over control values. Data are presented as mean G S.E.M., n Z 18, *P ! 0.05.
Osteoarthritis and Cartilage Vol. 13, No. 12 1097
composition73 and level of sulfation of KS and determined synthesis and reorganization. Agents Actions Suppl
that the ratio of unsulfated:monosulfated:disulfated disac- 1993;39:3e13.
charides in the KS-rich regions of aggrecan is approxi- 4. Poole AR, Nelson F, Hollander A, Reiner A, Pidoux I,
mately 1:3:574. The digestible fractions tested in this study Ionescu M. Collagen II turnover in joint diseases. Acta
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release is differentially regulated by different levels of 5. Eyre DR. The collagens of articular cartilage. Semin
mechanical load. Due to the differences in composition and Arthritis Rheum 1991;21:2e11.
mechanical properties of the extracellular matrix, compres- 6. Hardingham TE, Fosang AJ, Dudhia J. The structure,
sive loading will likely result in a non-uniform stressestrain function and turnover of aggrecan, the large aggre-
state in the tissue75e77, and the observed differences in gating proteoglycan from cartilage. Eur J Clin Chem
biomarker release may reflect the influence of mechanical Clin Biochem 1994;32:249e57.
stress on chondrocytes from different zones within the 7. Ratcliffe A, Flatow EL, Roth N, Saed-Nejad F, Bigliani
articular cartilage77. LU. Biochemical markers in synovial fluid identify early
In summary, we observed decreased biosynthesis rates osteoarthritis of the glenohumeral joint. Clin Orthop
and increased matrix degradation at the highest stress 1996:45e53.
applied (0.1 MPa). These findings are consistent with 8. Heinegård D, Oldberg A. Structure and biology of
previous observations that cyclic tensile strains of low cartilage and bone matrix noncollagenous macro-
magnitude (3e8% equibiaxial strain) and physiological molecules. FASEB J 1989;3:2042e51.
levels of cyclic compressive forces (15% compression) 9. Hazell PK, Dent C, Fairclough JA, Bayliss MT,
elicit an anabolic response78e83, while strains of high Hardingham TE. Changes in glycosaminoglycan
magnitude (10e15% equibiaxial strain) initiate cartilage epitope levels in knee joint fluid following injury.
damage78,79. While the mechanisms involved in these Arthritis Rheum 1995;38:953e9.
processes are not fully understood, our findings indicate 10. Lindhorst E, Vail TP, Guilak F, Wang H, Setton LA,
that mechanical factors can increase the production and/or Vilim V, et al. Longitudinal characterization of synovial
release of several OA-related biomarkers. In this study, fluid biomarkers in the canine meniscectomy model of
a doseeresponse relationship between mechanical stress osteoarthritis. J Orthop Res 2000;18:269e80.
and OA-related biomarker release was strongest for COMP. 11. Saxne T, Glennas A, Kvien TK, Melby K, Heinegard D.
Moreover, the differences in the patterns of biomarker Release of cartilage macromolecules into the synovial
release with stress magnitude, namely between COMP and fluid in patients with acute and prolonged phases of
total S-GAG, and the KI- and KII-digestible fractions of KS, reactive arthritis. Arthritis Rheum 1993;36:20e5.
suggest that the magnitude of stress applied to the articular 12. Saxne T. Differential release of molecular markers in
cartilage may differentially regulate chondrocytes from joint disease. Acta Orthop Scand Suppl 1995;266:
different zones and thus the turnover of distinct pools of 80e3.
molecules in the cartilage extracellular matrix. 13. Caterson B, Mahmoodian F, Sorrell JM, Hardingham
TE, Bayliss MT, Carney SL, et al. Modulation of native
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and in disease. J Cell Sci 1990;97:411e7.
Acknowledgments 14. Oldberg A, Antonsson P, Lindblom K, Heinegard D.
COMP (cartilage oligomeric matrix protein) is structur-
This study was supported by NIH grants AR50245, ally related to the thrombospondins. J Biol Chem
AR48182, AR50898, AR49790, AG15768, and the Claude 1992;267:22346e50.
D. Pepper Older Americans Independence Center 15. Lohmander LS, Saxne T, Heinegard DK. Release of
AG11268, NASA grant NNJ04HC72G, and by the Pratt cartilage oligomeric matrix protein (COMP) into joint
Fellows Program at Duke University. We would like to thank fluid after knee injury and in osteoarthritis. Ann Rheum
Julie Cernanec, Bradley Estes, Steve Johnson, and Robert Dis 1994;53:8e13.
Nielsen for their excellent technical assistance, Dr Vladimir 16. Sharif M, Saxne T, Shepstone L, Kirwan JR, Elson CJ,
Vilim (Prague, Czech Republic) for his kind gift of anti- Heinegard D, et al. Relationship between serum
COMP 12C4 mAb, and Dr Bruce Caterson (Cardiff, Wales, cartilage oligomeric matrix protein levels and disease
UK) for his kind gift of 5D4 mAb. progression in osteoarthritis of the knee joint. Br J
Rheumatol 1995;34:306e10.
17. Jordan JM, Luta G, Stabler T, Renner JB, Dragomir AD,
Vilim V, et al. Ethnic and sex differences in serum
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