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Objectives: This study addressed the hypothesis that specific mechanical loading regimens will function as a stimulus for the restoration of normal phenotypic expression of injured articular cartilage cells. The experimental aims examined the effects of intermittent hydrostatic pressure on the expression of three major categories of articular cartilage cell products, extracellular matrix proteins, proinflammatory mediators and cartilage degrading enzymes.
Clinical Relevance: The results presented in this study confirm that alteration of mechanical loading within rehabilitation medicine protocols will contribute to the success in treatment and alleviation of early manifestations of osteoarthritis in the aging veteran population.
Methods: Articular chondrocytes were isolated from human osteoarthritic joint cartilage using collagenase treatment. The isolated cells were allowed to attach in DMEM/F12 medium containing 10% FBS for five days. For loading, the culture plates were immersed in 45 ml of serum-free medium in sterile, heat-sealed plastic bags. Control cultures were maintained under identical conditions. Hydrostatic pressure was applied intermittently at 1 Hz at a level of 10 MPa for periods of (A) 2, 4, 8 12, and 24 hours or (B) 4 hours per day for 4 days. Each experimental time point was tested using triplicate and the experiment loading protocol was carried out in five trials. Total nucleic acid was extracted by the phenol/chloroform method and converted to cDNA using reverse transcription. PCR was carried out within a single reaction using beta-actin as control. The amplified products were fractionated on agarose gels and 32P-signal levels were determined. Release of matrix proteins into the culture medium was determined by concentration of all samples using membrane concentrators with MW cutoff of 10 kD and subsequent SDS-PAGE in 10% acrylamide gels with protein visualization following silver staining. IL-6 and MCP-1 protein levels were measured by ELISA.
Results: Applying intermittent hydrostatic pressure using an interval loading pattern that included a 4 hour period of stimulation followed by a 20 hour period of inactivity increased the levels of type II collagen and aggrecan mRNA signal levels, relative to unloaded control cells. Continuous application of intermittent hydrostatic pressure inhibited human osteoarthritic chondrocyte release of MMP-2 at 6, 12 and 24 hour, relative to unloaded control cultures. Zymographic analysis did not demonstrate an effect of intermittent hydrostatic pressure on chondrocyte release of MMP-9 at any time period tested. APMA activation also revealed inhibition of both active forms of MMP-2 (62 and 68 kD). Intermittent hydrostatic pressure inhibited human osteoarthritic chondrocyte release of IL-6 by 41, 43, and 20% at 6, 12 and 24 hour, respectively, relative to unloaded control cultures. Intermittent hydrostatic pressure inhibited human osteoarthritic chondrocyte release of MCP-1 by 12, 31, and 49% at 6, 12, and 24 hour, respectively, relative to unloaded control cultures.
Conclusions: Application of intermittent hydrostatic pressure effectively modulated the metabolic products of isolated osteoarthritic articular chondrocytes in a manner that would be conducive to induction of extracellular matrix repair and regeneration. The levels of proinflammatory cytokines and the matrix degrading enzymes were suppressed by the loading whereas mRNA signal levels for the major matrix molecules, aggrecan and type II collagen, were increased.
Acknowledgments: This work was supported VA Rehabilitation R&D Merit Review Project A857-RC.