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Address correspondence and reprints requests to: R. Lane Smith, Ph.D., Stanford University School of Medicine, Department of Orthopaedic Surgery, 300 Pasteur Drive, R-144, Stanford, CA 94305-5341, USA. Tel: 1-650-725-6633; Fax: 1-650-725-6631;
Orthopaedic Research Laboratory, Stanford University School of Medicine, Stanford, CA, USARehabilitation Research and Development Center, Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA
This study tested the hypothesis that intermittent hydrostatic pressure applied to human osteoarthritic chondrocytes modulates matrix metalloproteinase and pro-inflammatory mediator release in vitro.
Human osteoarthritic articular chondrocytes were isolated and cultured as primary high-density monolayers. For testing, chondrocyte cultures were transferred to serum-free medium and maintained without loading or with exposure to intermittent hydrostatic pressure (IHP) at 10 MPa at a frequency of 1 Hz for periods of 6, 12 and 24 h. Levels of matrix metalloproteinase-2, -9 (MMP-2, -9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the pro-inflammatory mediators, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), released into the culture medium were assessed by ELISA. Matrix metalloproteinase activity was confirmed by zymographic analysis.
In the absence of IHP, levels of MMP-2, TIMP-1, IL-6, and MCP-1 in the chondrocyte culture medium increased in a time-dependent manner. Application of IHP decreased MMP-2 levels at all time periods tested, relative to unloaded control cultures maintained for the same time periods. Although 84/82 kDa bands were faintly detectable by zymography, MMP-9 levels were not quantifiable in medium from loaded or unloaded cultures by ELISA. TIMP-1 levels were not altered in response to IHP at any time period tested. IL-6 and MCP-1 levels decreased in cultures exposed to IHP at 12 and 24 h, relative to unloaded control cultures maintained for the same time periods.
IHP decreased release of MMP-2, IL-6 and MCP-1 by osteoarthritic chondrocytes in vitro suggesting that pressure influences cartilage stability by modulating chondrocyte expression of these degradative and pro-inflammatory proteins in vivo.
. In vivo, chondrocyte metabolism appears to match a pattern in which deviatoric stress is associated with matrix degeneration and compressive intermittent hydrostatic pressure is associated with matrix thickness
. The effects of loading on cartilage deformation are complex and due in part to osmotic pressure effects on the total swelling behavior of cartilage and electromechanical properties of cartilage that depend on flow of ions through extracellular constituents
Cartilage homeostasis and matrix stability also depend on biochemical factors present within the joint environment. In arthritis, degradation of the cartilage matrix macromolecules, aggrecan and collagen, is associated with increased expression of degradative enzymes, such as matrix metalloproteinases
. IL-6 is associated with bone remodeling activity, depression of chondrocyte proteoglycan synthesis, and stimulation of interleukin-1 (IL-1) dependent proteoglycan degradation. Another inflammatory factor, the C-C chemokine, monocyte chemoattractant-1 (MCP-1), is increased in cartilage organ culture and chondrocyte monolayer cultures in response to exogenous IL-1
In osteoarthritis, alterations of chondrocyte metabolism by inappropriate mechanical loading and/or with increased levels of soluble mediators may culminate in matrix degeneration and bone remodeling. This study tested the hypothesis that a physiological stress state, intermittent hydrostatic pressure (IHP), reduces secretion of matrix metalloproteinases and the pro-inflammatory mediators, IL-6 and MCP-1, from human osteoarthritic chondrocytes in vitro.
Isolation of articular chondrocytes
Articular chondrocytes were isolated from resected cartilage specimens obtained from six individuals undergoing primary total knee arthroplasty using enzymatic digestion. Osteoarthritic cartilage specimens were collected from joint surfaces without visible softening and fraying that would indicate moderate to severe fibrillation. Briefly, cartilage was removed from the joint surfaces, diced and placed in 0.25% trypsin (Gibco BRL, Gaithersburg, MD) for 30 min. The cartilage was then washed with Dulbecco's phosphate buffered saline without Ca++ and Mg++ (PBS) and placed in 20 ml of a 1:1 mixture of Dulbecco's Modified Eagles Medium and Ham's F12 (DMEM/F12) containing 10% heat inactivated fetal bovine serum (Gibco BRL, Gaithersburg, MD), 25 μg/ml gentamicin (Sigma Chemical Co., St. Louis, MO) and a mixture of bacterial collagenases, Type II and Type IV (Worthington Chemical, Freehold, NJ), at a final concentration of 1.0 mg/ml each. The cartilage samples were incubated for 36 h at 37°C in 5% CO2 and 100% humidity to ensure complete digestion. Chondrocytes released from matrix were filtered through nylon mesh to isolate single cells. The cells were collected by repeated centrifugation steps (450× gravity for 15 min) in which the cells were resuspended and collected in DMEM/F12 (3×50 ml) to remove collagenase. The final cell pellet was suspended in the serum-free medium and the cells were counted in a hemacytometer with viability assessed by Trypan Blue exclusion. Although total cell yield varied, the viability of the chondrocytes was greater than 95% in all samples tested. The cells were then plated in 60 mm tissue culture dishes (Nunc, Naperville, IL) at a density of 5±105 cells per plate in DMEM/F12 supplemented with 10% fetal bovine serum, 25 μg/ml ascorbate and 25 μg/ml gentamicin.
Twenty-four hours prior to loading, the confluent chondrocyte cultures were washed three times with PBS and 4 ml of serum-free medium was added to each plate. The serum-free medium consisted of a 1:1 mixture of Ham's F12/DMEM supplemented with 30 mM HEPES, selenium, ascorbate, gentamicin, and a lipid supplement
. At the time of loading, the culture medium was removed and the outside surfaces of the culture plates were sterilized using 70% ethanol. The plates were then immersed in sterile bags containing 40 ml of serum-free medium with air being evacuated by mechanical manipulation. The bags containing the culture plates and serum-free medium were then heat-sealed and transferred to a water filled stainless steel loading vessel interfaced with an MTS 858 servo-hydraulic testing system (MTS Systems Corporation, Eden Prairie, MN) (Fig. 1). Air was evacuated from the system and intermittent hydrostatic pressure was applied at a level of 10 MPa (1500 psi) and a frequency of 1 Hz. For temperature regulation, the loading vessel immersed in a large circulating water bath was maintained at 37°C.
All experiments were conducted in serum-free medium. Unloaded control cultures were tested under identical conditions with the exception that load was not applied (Fig. 1). The presence of MMP-2, MMP-9, TIMP-1, IL-6 and MCP-1 was used to determine the effects of intermittent hydrostatic pressure on chondrocyte metabolism. Medium samples were harvested after 6, 12 and 24 h periods, and stored at −20°C until assayed.
Cytokine and MMP quantification
Medium samples from cultures exposed to intermittent hydrostatic pressure and those maintained as unloaded controls were concentrated using ultrafiltration with a molecular weight cutoff of 10 kDa (Amicon, Inc., Beverly, MA). Monospecific antibodies to IL-6 and MCP-1 (R&D Systems, Minneapolis, MN) were purchased and levels of cytokine expression were measured via ELISA with the respective human recombinant proteins used as standards for the reference curves (R&D Systems). Briefly, coating antibodies were diluted to 4 μg/ml in PBS, and 100 μl was added to each well of 96 well Nunc Maxisorb plates. After 24 h, the plates were washed three times with wash buffer (0.05% Tween 20 in PBS), and blocked for 1 h with blocking buffer (1% bovine serum albumin, 5% sucrose, 0.05 M sodium azide in PBS). The plates were subsequently washed three times followed by addition of samples and standards. After 2 h, the plates were washed four times with wash buffer, and 100 μl of biotinylated secondary antibodies (concentrations: IL-6, 25 ng/ml; MCP-1, 50 ng/ml) was added per well. Following a 2-h incubation period the plates were washed four times with wash buffer, and 100 μl of streptavidin HRP (100 ng/ml in DPBS) (Zymed, So. San Francisco, CA) was added per well. After 30 min, the plates were washed four times in wash buffer, and 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma Chemical Co., St. Louis, MO) was added per well. The reaction was stopped after 25 min with the addition of 50 μl of 0.5 M sulfuric acid. The plates were subsequently read on a microplate reader (Model 3550-UV, Bio-Rad Laboratories, Hercules, CA) at 450 nm with background correction of 595 nm. Levels of MMP-2, MMP-9, and TIMP-1 were measured using commercial ELISA kits according to the manufacturer's instructions (Oncogene Research Products, Cambridge, MA).
Zymographic analysis of MMP activity
Concentrated medium samples from cultures exposed to intermittent hydrostatic pressure and those maintained as unloaded controls were activated via incubation in 1 mM p-aminophenylmercuric acetate (APMA) in 1 mM NaOH for 1 h at 37°C prior to separation in 10% SDS polyacrylamide gels impregnated with 1 mg/ml gelatin (Sigma). After electrophoretic separation, the gels were placed in 2.5% Triton X-100 for 1 h at 25°C. The gels were then incubated for 16 h in substrate buffer (50 mM Tris–HCl, 10 mM CaCl2, 0.02% Azide, pH 8) at 37°C. The gels were stained with 0.1% Coomassie Brilliant Blue R250 (Bio-Rad) in 30% ethanol and 15% formaldehyde for 1 h, then destained in water to visualize gelatinolytic activity.
Statistical analysis was carried out using Friedman's method for randomized blocks followed by Wilcoxon's signed ranks tests to compare unloaded and loaded cultures at 6, 12 and 24 h. Bonferroni's correction for multiple comparisons was applied, and P-values less than 0.05 were considered significant. Non-parametric statistical methods were applied to account for variation in basal levels of protein expressed in chondrocytes from six individual osteoarthritic cartilage donors. With these statistical methods, direction of change is an important determinant of significance levels.
In the absence of IHP, human osteoarthritic chondrocytes released MMP-2, TIMP-1, IL-6 and MCP-1 in a time-dependent manner into the culture medium (Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6). MMP-9 release was not detected in culture medium of unloaded cultures or those exposed to IHP using ELISA.
Intermittent hydrostatic pressure inhibits human osteoarthritic chondrocyte release of matrix metalloproteinase-2 but not tissue inhibitor of metalloproteinase
Release of matrix metalloproteinase-2 by chondrocytes was inhibited following exposure to IHP by 20, 39, and 80% at 6, 12 and 24 h, respectively, relative to unloaded control cultures tested at the same time period (Fig. 2). Zymographic analysis demonstrated that chondrocytes release MMP-2 (72 kDa) in a time-dependent manner (Fig. 3). Expression of MMP-2 was inhibited following exposure to IHP at 6, 12 and 24 h time periods. A faint band was evident for both active forms of MMP-9 (82 and 84 kDa), however this band did not appear to change with time or exposure to IHP. Exposure of the chondrocytes did not significantly alter TIMP-1 levels at any time period tested (Fig. 4).
Intermittent hydrostatic pressure inhibits human osteoarthritic chondrocyte release of interleukin-6
Release of interleukin-6 by chondrocytes was inhibited following exposure to IHP by 41, 43, and 20% at 6, 12 and 24 h, respectively, relative to unloaded control cultures tested at the same time period (Fig. 5).
Intermittent hydrostatic pressure inhibits human osteoarthritic chondrocyte release of monocyte chemoattractant protein-1
Release of MCP-1 by chondrocytes was inhibited following exposure to IHP by 12, 31, and 49% at 6, 12 and 24 h, respectively, relative to unloaded control cultures tested at the same time period (Fig. 6).
The physical properties of the cartilage extracellular matrix influences the chondrocyte response to mechanical loading due to the major matrix components, water, aggrecans and type II collagen
. In contrast, mechanical loads distorting cartilage would be expected to induce a thinning of the matrix. Mechanical loading of a degraded cartilage matrix leads to disruption of collagen and increased susceptibility to metalloproteinases
showed that matrix metalloproteinase levels correlate with the extent of cartilage destruction whereas levels of TIMP-1 do not vary significantly with severity of cartilage destruction. Altered expression of degradative enzymes and protease inhibitors in osteoarthritic joints may account for the constitutive expression of MMP-2 observed in chondrocytes examined in this study. In addition, the chondrocytes used here were isolated from cartilage not exhibiting visible softening or fraying; normal appearing cartilage from osteoarthritic joints expresses a degradative enzyme profile associated with matrix degeneration
. It is possible that other loading paradigms, including static loading or specific loading regimens, may either be inhibitory or stimulatory when compared to the results presented in this study.
The data presented here show that IHP applied to osteoarthritic chondrocytes in vitro decreased IL-6 and MCP-1 release. Since IL-6 inhibits proteoglycan synthesis in vitro in chondrocytes and is synergistic with IL-1, down regulation of IL-6 expression by IHP may serve as a regulatory mechanism to preserve cartilage in vivo
. In contrast to IHP, application of shear stress to chondrocytes increases release of pro-inflammatory mediators, nitric oxide, IL-6, MCP-1 and prostaglandin E2, factors implicated in cartilage degeneration
. This study utilized chondrocytes in high-density monolayer culture which may influence the cellular response to mechanical loading due to focal adhesions and alterations in intracellular elements and cellular morphology. However, similar responses were observed for GAG synthesis between normal bovine chondrocytes in monolayer culture and cartilage explants with exposure to IHP
. In addition, similarities in the normal bovine and human OA chondrocyte responses to IHP are exemplified by increased aggrecan and type II collagen mRNA expression and matrix deposition following exposure to specific regimens of IHP
As demonstrated here, IHP decreased expression of MMP-2 and the pro-inflammatory mediators, IL-6 and MCP-1. The data also demonstrated that osteoarthritic chondrocytes retain the capacity to respond to IHP in vitro. Understanding the precise mechanisms by which hydrostatic pressure alters cartilage metabolism will provide vital insights for development of approaches for the treatment of diseased joints.
Contact pressures in the human hip joint measured in vivo.