Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.

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Quinolones and glucocorticoids are frequently used drugs that may cause tendinopathy as a rare adverse effect. We exposed human tenocyte cultures to the steroid dexamethasone alone or in combination with either ciprofloxacin or levofloxacin at concentrations of 3 mg/L and 10 mg/L. At concentrations corresponding to peak levels in plasma and tissues during therapy (ca. 3-10 mg/L), ciprofloxacin caused a significant decrease in collagen type I and the 1 -integrin receptor. In contrast, no corresponding effect was induced by 3 mg/L levofloxacin.With both quinolones at 3 mg/L and 10 mg/L, the amount of matrix metalloproteinase-1 (MMP-1) and MMP-13 was increased. In addition, 3 mg/L ciprofloxacin and 10 mg/L levofloxacin activated caspase-3. Apoptotic changes were confirmed by electron microscopy. Incubation of human tenocytes with dexamethasone decreased the main matrix protein collagen type I, the

Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor kappaB (NF-kappaB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha in osteoarthritis are regulated by the activation of NF-kappaB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL-1beta. Human articular chondrocytes were prestimulated with 10 ng/mL IL-1beta for 0, 4, 8, 12, or 24 h and then cotreated with 50 microM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage-specific collagen type II and matrix-degrading enzyme matrix metalloproteinase-3 (MMP-3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF-kappaB were observed by immunofluorescence microscopy. IL-1beta induced a decrease in collagen type II and upregulation of MMP-3 in a time-dependent manner. Upregulation of MMP-3 was inhibited by curcumin in a time-dependent manner. In addition, IL-1beta-induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL-1beta, NF-kappaB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL-1beta-mediated upregulation of proinflammatory MMP-3 in chondrocytes via an NF-kappaB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL-1beta, such as MMP-3 upregulation, and interestingly also relieved cytokine-induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL-1beta signaling that are known to contribute to the pathogenesis of osteoarthritis.