Our recent study suggested that cyclic compressive loading may promote chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures through the transforming growth factor (TGF)-β signaling pathway. It has been shown that the activating protein 1 (AP-1) (JunFos) complex mediated autoinduction of TGF-ß1 and its binding activity was essential for promoting chondrogenesis of mesenchymal cells, whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c-Fos, c-Jun, and TGF-ß type Ι and II receptors) and induction of their corresponding proteins in agarose culture of rabbit BM-MSCs subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were made by suspending BM-MSCs in 2% agarose gel (10 7 cells/ml) for cyclic, unconfined compression tests performed in a custommade bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, cJun, and both TGF-ß receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c-Fos was detected only in the samples subjected to 1-hour dynamic compressive loading. These findings suggest that the TGF-ß signal transduction and activities of AP-1 and Sox9 are involved in the early stage of BM-MSC chondrogenesis promoted by dynamic compressive loading.

Calcium-containing crystals such as basic calcium phosphate (BCP) 1 and calcium pyrophosphate dihydrate (CPPD) are two of the most common forms of pathologic articular materials that are associated with destructive arthropathies involving cartilage degeneration (1, 2). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors like platelet-derived growth factor, epidermal growth factor, and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro (3); stimulate the production of prostaglandin via the phospholipase A 2 /cyclo-oxygenase pathway (4); activate phospholipase C and inositol phospholipid hydrolysis (5); induce the expression of the proto-oncogenes, c-fos and c-myc (6, 7); and induce the synthesis and secretion of metalloproteinases (MMPs) 1, 3, 8, and 13 (8 -12).In contrast to other mitogenic and growth factors, BCP crystal-elicited signal transduction pathways have not been completely studied. However, we have identified some of the component molecules involved in calcium-containing crystal signal transduction mechanisms. One pathway activated upon crystal stimulation of human fibroblasts (HF) is the p44 and p42 mitogen-activated protein kinase (p44/42 MAPK) pathway, also known as extracellular signal-related mitogen protein kinases 1 and 2 (ERK1 and ERK2), respectively. The MAPK cascade can be blocked by the selective inhibitors, PD98059 (13) and U0126 (14), which hinder the activation and phosphorylation of MEK (MAPK/ERK kinase). Co-treatment of HF with BCP crystals and PD98059 blocks crystal-induced p44/42 MAPK activation and mitogenesis (15) in addition to crystalinduced up-regulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover, phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals (17), also blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF (15).Another messenger with an apparent role in crystal-activated signal transduction is calcium. We have previously shown that treatment of HF with BCP crystals induces a rapid transient rise of intracellular calcium levels in seconds due to calcium influx from outside the cell, followed by a slow and sustained increase of intracellular calcium within 60 min after stimulation, due to crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP crystal in-

Matrix metalloproteinases (MMPs) are a family of structurally and functionally related zinc-containing endopeptidases that are capable of degrading almost all of the components of the extracellular matrix (ECM). Under physiological and pathological conditions, the MMPs play a significant role in the efficient tissue turnover and remodeling. Specific MMPs are responsible for the matrix degradation and remodeling. Maintenance of the equilibrium between deposition and degradation of the extracellular matrix is essential to the normal tissue development. Therefore, synthesis and breakdown of the MMPs are tightly controlled by protein kinases which mediate a host of other cellular processes. The MMPs are often induced by several agents and any uncontrolled expression of the MMPs can contribute to the pathogenesis of many human diseases. This review focuses on the regulation of the MMPs by the protein kinases at the level of gene expression and their signaling pathways.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) can differentiate into chondrogenic cells for the potential treatment of injured articular cartilage. To evaluate agarose gels as a supportive material for chondrogenesis of hBM-MSCs, this study examined chondrogenesis of hBM-MSCs in the agarose cultures. Pellet cultures were employed to confirm the chondrogenic potential of the hBM-MSCs that were used in agarose cultures. The hBM-MSCs were seeded in 2% agarose constructs at the initial cell-seeding densities of 3, 6, and 9 ϫ 10 6 cells/ml while each of pellets was formed using 2.5 ϫ 10 5 cells. Chondrogenesis of hBM-MSCs was induced by culturing cell-agarose constructs and pellets for 21 days in the presence of a defined medium containing transforming growth factor ␤3 (TGF-␤3). The analysis of reverse transcription-polymerase chain reaction showed that hBM-MSCs of agarose and pellet cultures expressed the chondrogenic markers of collagen type II and aggrecan in the presence of TGF-␤3. The deposition of cartilage-specific macromolecules was detected in both agarose and pellet cultures by histological and immunohistochemical assessments. Chondrogenesis of hBM-MSCs in agarose gels directly correlated with the initial cell-seeding density, with the cell-agarose constructs of higher initial cellseeding density exhibiting more cartilage-specific gene expressions. This study establishes a basic model for future studies on chondrogenesis of hBM-MSCs using the agarose cultures.

Although matrix metalloproteinase-8 (MMP-8) was regarded as the exclusive product of the neutrophils, recent studies have shown that it is also expressed in articular chondrocytes, rheumatoid synovial fibroblasts and endothelial cells. Our aim was to determine the expression of MMP-8 in human fibroblasts (HF) by reverse transcription/polymerase chain reaction (RT/PCR). Northern and Western blotting methods and MMP-8 activity assay. We have shown the expression of MMP-8 in HF and its dose-dependent upregulation by basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPPD) crystals which are markers of severe joint degeneration in osteoarthritis. These effects require new protein synthesis and are reversed by phosphocitrate (PC). The results also show that this fibroblast MMP-8 is distinct from the neutrophil MMP-8 and from the fibroblast MMP-1. These results indicate that MMP-8 may play a significant role in the pathogenic effects of the crystals in osteoarthritis.

Although basic calcium phosphate (BCP) crystals are common in osteoarthritis, the crystal-induced signal transduction pathways in human fibroblasts have not been fully comprehended. We have previously demonstrated that the induction of matrix metalloproteinases (MMP) 1 and 3 by BCP crystals follows both the calciumdependent protein kinase C (PKC) pathway and the calcium-independent p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. Although we showed that the calcium-dependent PKC pathway was characterized by calcium-dependent PKC␣, here we show that the calcium-independent p44/42 MAPK pathway is mediated by calcium-independent PKC . Inhibition of PKC synthesis and activity by antisense oligodeoxynucleotides and H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide), respectively, results in the inhibition of p44/42 MAPK activation, thus demonstrating that p44/42 MAPK activity is dependent upon PKC . Reverse transcription-polymerase chain reaction and Western blotting also show that inhibition of PKC results in the inhibition of MMP-1 and MMP-3 mRNA and protein expression as a result of p44/42 MAPK inhibition. These results now lead us to the conclusion that BCP crystal activation of human fibroblasts follows two pathways: 1) the calcium-dependent PKC pathway characterized by PKC␣ and 2) the calcium-independent p44/42 MAPK pathway mediated by PKC , which operate independently leading to an increase in mitogenesis and MMP synthesis and ultimately complementing each other for the efficient regulation of cellular responses to BCP crystal stimulation of human fibroblasts.Basic calcium phosphate (BCP) 1 crystals are among the most common forms of pathologic articular minerals. They frequently occur in the joints of osteoarthritis patients and can be phlogistic (1, 2). There is compelling evidence that these crystals engender multiple biological effects that promote joint degeneration. The presence of BCP crystals correlates strongly with radiographic evidence of cartilaginous degeneration and synovial thickening and is associated with larger joint effusions when compared with joint fluid from osteoarthritis knees where BCP crystals are absent (3, 4). Conversely, osteoarthritis is both more common and more severe in patients with calciumcontaining crystals. We have demonstrated that BCP crystals stimulate the proliferation and synthesis of matrix metalloproteinases (MMPs) in cultured human foreskin and synovial fibroblasts (5-9). The addition of BCP crystals to the growth medium yielded an immediate 10-fold rise in the intracellular calcium level and a second rise starting at 60 min and lasting for 3 h. This second rise in the intracellular calcium level is probably because of the intracellular dissolution of phagocytosed crystals, which may activate a variety of calcium-dependent signals and induce sustained cell proliferation and MMP synthesis (10).To date, BCP crystal-induced signal transduction has not been fully comprehended. We have previously demonstrated that the induction of human MMP-...

Phosphocitrate [PC] is a powerful inhibitor of biological crystallization and a potential disease modifying drug for crystal associated diseases such as crystals associated osteoarthritis [OA]. Recently, it has been reported that a new PC complex salt, calcium sodium PC [CaNaPC], is much more powerful than its precursor, sodium PC [NaPC], in reducing the size of chemically-induced calcified plaques in rat when examined using a calcergy assay (1). The molecular mechanisms underlying such a superior activity as a calcification inhibitor over its precursor NaPC are currently unknown. In order to evaluate the potential of CaNaPC as a disease modifying drug for crystals associated OA, we examined and compared CaNaPC and its precusor NaPC using several cell- based assays. CaNaPC was found to have an inhibitory potency similar to that of NaPC toward preventing the stimulating effects of basic calcium phosphate [BCP] crystals on the induction of MMP1, thymidine uptake and endocytosis. However, CaNaPC proved much more powerful than NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death. These results suggest that the superior anti-calcification activity of NaCaPC over NaPC observed in rat is probably due to its superior activity in the inhibition of the effects associated with amorphous calcium phosphate clusters/aggregates/precipitates but not the effects associated with BCP crystals. Since amorphous calcium phosphate clusters/aggregates/precipitates are precursors of BCP crystals and coexist with calcium-containing crystals in calcified tissues (2-6), these amorphous clusters/aggregates/precipitates, similar to BCP crystals, may have played a significant role in pathological calcifications and in the development of crystals associated diseases such as crystals associated OA. The superior activity of CaNaPC over its precursor NaPC in the inhibition of amorphous calcium phosphate-DNA coprecipitates-induced cell death may, at least in part, explain its powerful anti-calcification activity in vivo. The findings suggest that CaNaPC through a dual action of inhibiting both the detrimental biological effects of formed BCP crystals and preforming amorphous calcium phosphate clusters/aggregates/precipitates, could present as a better disease-modifying drug for crystals associated OA than its parent NaPC.

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