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The preservation of the structure of articular cartilage depends on the availability of inhibitors of matrix-degrading enzymes. Tissue inhibitor of metalloproteinases-1 is thought to be an important contributor to the integrity of the matrix of articular cartilage, but the mechanisms that regulate its availability within the tissue are poorly understood. These studies elucidate the contributions of diffusion, fluid flow, and electrical migration to the transport of iodinated recombinant human tissue inhibitor of metalloproteinases-1 through explants of adult bovine articular cartilage under conditions relevant to the loading of cartilage. With use of measured partition coefficients of the cartilage explants, the diffusivity of the inhibitor was 0.5-1.6 x 10(-7) cm2/sec. Fluid velocities that were induced by applying an electrical current across the cartilage disks increased the flux of the inhibitor by approximately 20 to more than 150-fold compared with the effect of diffusion alone for the range of current densities that were applied. We examined the contribution of electrophoretic migration by titrating the charge on the inhibitor during measurements of flux and found that flux in the presence of the applied current decreased as the inhibitor became more negatively charged. Enhancements in the flux of the inhibitor were observed relative to the flux during diffusion alone even under conditions in which electrophoretic migration opposed the flux due to fluid flow, suggesting that of the transport mechanisms tested, fluid flow was dominant. These results suggest that the physical phenomena present during physiologic loading conditions (e.g., fluid flows and streaming currents) can affect the transport of tissue inhibitor of metalloproteinases-1 through the matrix of cartilage.

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of human plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2.

Mandibular condyles in organ culture commonly have been used as a model system for examination of the factors that influence skeletal growth and development. The work reported here complements previously published histological studies by providing quantitative temporal information on growth and matrix accumulation. Condyles maintained for as long as 5 weeks in serum-free and 1% serum-supplemented culture media were found to remain viable and metabolically active as demonstrated by continued dimensional growth as well as cell and matrix accumulation. Growth occurred by a combination of cell proliferation, matrix synthesis and accumulation, and cell hypertrophy (with the latter two mechanisms dominating). Increases in tissue volume correlated directly with increased glycosaminoglycan content; both increased 7-fold over 5 weeks. In comparison with serum-free culture, after 35 days in medium containing 1% serum, glycosaminoglycan content was 24% lower and collagen content was 36% higher, whereas dry weight, condyle length, and DNA content were not significantly different; in addition, histological observation suggested that, for samples cultured with serum, chondrogenic phenotype had been lost from some regions. The temporal behavior for all growth parameters exhibited a transient phase 1-2 weeks in duration followed by a steady-state period in which dimensions and tissue constituents or content increased at a constant or near constant rate. Comparison of the rates of incorporation of [35S]sulfate with glycosaminoglycan content in serum-free cultures suggests that the loss of glycosaminoglycan occurs only initially or is negligible; therefore, under these baseline conditions, cartilage glycosaminoglycan content reflects the biosynthetic rate. The high degree of reproducibility seen during steady-state growth suggests that these data provide reliable baseline information and further supports the notion that this model system is useful for investigation of the effects of specific physical factors on in vitro growth and development.

Cartilage growth and remodeling are known to be influenced by the biochemical and mechanical environment of the tissue. Previous investigators have shown that chemical factors that are relevant to mechanical loading, such as osmotic pressure and pH, induce changes in cartilage metabolism in vitro. Using a neonatal rat mandibular condyle culture system, the objectives of the work reported here were to determine (1) how the growth is influenced by osmotically applied mechanical loads; and (2) whether changes in intratissue osmotic pressure or pH cause metabolic changes in the cartilage which are then reflected by altered growth behavior. High molecular weight (MW) uncharged macromolecules polyvinylpyrrolidone (PVP) and Ficoll (presumed unable to penetrate the tissue matrix) were used to examine the effect of osmotic loading on tissue growth; concentrations corresponding to osmotic pressures of up to 100 kPa resulted in a dose-dependent depression in growth and matrix accumulation. Raffinose (which can penetrate the matrix but not the cells) had no significant effect on growth for osmotic pressures of up to 87 kPa, suggesting that compression-induced changes in intratissue osmotic pressure are unlikely to provide a signal by which cells sense and respond to mechanical compression. By contrast, changes in medium pH resulted in dose-dependent changes in growth behavior. Specifically, slight alkalinity (acidity) greatly enhanced (diminished) growth and matrix accumulation; the sensitivity to pH suggests that intratissue pH could provide a mechanism for cells to sense local glycosaminoglycan concentration and mechanical compression.(ABSTRACT TRUNCATED AT 250 WORDS)