Purpose: Differentiating mesenchymal stem cells are often used as an alternative supply of chondrocytes that allow observations not only into the molecular signalling events that occur during chondrogenesis, but also in mature cartilage tissue. The resulting tissue can then also be used to model various cartilage pathologies or to attempt cartilage repair. It is difficult to generate the required phenotype of the chondrocytes/tissue produced and models may therefore not accurately reflect the appropriate type of cartilage tissue required, which has different functions and properties depending on the location within the body. Since osteoarthritis is a disease that degrades articular cartilage, the purpose of this study was to identify a culturing model with physiological relevance that specifically produces articular cartilage-like tissue. Methods: ATDC5 chondrogenic precursor cells were grown in three-dimensional, high-density micromass, stimulated with a combination of insulin (which induces differentiation in ATDC5 cells) and ascorbic acid over time, and directly compared to traditional ATDC5 differentiation in monolayer by insulin stimulation alone. Quantitative (q)RT-PCR was used to identify regulation of various genes related to chondrogenesis, chondrocyte maturation/hypertrophy (reminiscent of growth plate cartilage) and markers of the articular cartilage phenotype. Confirmation of some genes was observed at the protein level by immunofluorescence and histological staining. Results: ATDC5 cells induced to differentiate in the traditional monolayer culture developed heterogeneously, forming aggregates termed "cartilage nodules", which stained for Alcian blue and increased in number with ascorbic acid stimulation. Areas between nodules did not stain with Alcian blue, indicating a heterogeneous mixture of chondrogenic and non-chondrogenic areas, confirmed by Type II Collagen immunofluorescence. In contrast, central micromasses remained as one large nodule, with noticeably extensive extra-cellular matrix deposition, staining deeply and uniformly with Alcian blue, indicating more homogenous differentiation. By qRT-PCR, differentiating micromasses stimulated with ascorbic acid for three weeks had a 231-fold increase in Col2a1 expression (Type II Collagen) compared to traditional differentiating monolayer cultures with stimulation by insulin alone. Frzb, a secreted Wnt antagonist previously described as a marker for articular cartilage, was increased 35-fold in the same comparison. Col10a1 (Type X Collagen), a hypertrophic region marker, was increased with micromass culture, but protein was immunolocalised to a small region within the central micromasses surrounded by extensive Type II Collagen staining. Other markers of hypertrophic differentiation, which would resemble a more growth plate-like phenotype, were either not expressed (Mmp13) or did not demonstrate altered expression (Runx2). Taken together, these results suggest that a marked difference in gene expression is seen when ATDC5 cells are cultured in micromass with ascorbic acid. Conclusions: Using the traditional culturing method in monolayer with insulin stimulation alone, ATDC5 chondrogenesis occurs heterogeneously, forming separate cartilage nodules, which we showed can be increased in number with ascorbic acid stimulation. Using a three-dimensional culturing technique, which is more physiologically relevant, and combining this with ascorbic acid stimulation, we showed that chondrogenesis was both more homogenous in nature and also highly enhanced expression of markers of chondrogenesis and of articular cartilage. We therefore suggest that this modified culture system for ATDC5 cells provides a good model for studying articular cartilage.

Purpose: Damaged cartilage is increasingly recognised as a permissive environment for neurovascular infiltration. Neuropeptides involved in pain transmission in osteoarthritis, have recently been implicated in cartilage breakdown and also in chondrocyte proliferation and survival. We took an unbiased approach to explore the effect of substance P and bradykinin on expression 95 genes which may be important in cartilage breakdown including all members of the MMP, ADAM and ADAMTS families of metalloproteinases their inhibitors the TIMPs, all CCN growth factor family members, certain FGFs and neuropeptide precursors and receptors in immortalised human chondrocytes, C28/I2. Methods: We compared the response of C28/I2 cells cultured in monolayer (2D) or on polyhema (3D; preventing cell adhesion to the culture plastic) which is known to maintain chondrocyte phenotype, exploring expression of 95 genes analysed by Taqman Low Density Array. Ten genes showed significant changes (p < 0.05) of at least 2-fold. Results: In 2D cultures Interleukin 6 (IL6) was up-regulated 3-fold by both bradykinin and Substance P. IL6 is up-regulated in OA joint fluid and elevated IL6 is associated with enhanced sensitivity to pain. Although in 3D culture no significant modulation of IL6 was observed by neuropeptides we did observe up-regulation of IL6 expression in response to IL1/Oncostatin M (used as a positive control). In 3D culture expression of six genes was significantly repressed (at least 2-fold) by both bradykinin and substance P and no genes were significantly induced. Amongst these genes it was interesting to note suppression of inhibitors TIMP2 (inhibitor of all MMPs) and TIMP3 (inhibits all MMPs, certain ADAMs and ADAMTS proteinases). Expression of three metalloproteinases, ADAMTS9 (an aggrecanase), MMP2 and MMP15 was also significantly suppressed although expression of these enzymes was still high. FGF18 was also suppressed by both neuropeptides in 3D only and this growth factor has been shown to promote cartilage repair. WISP3/CCN6 was repressed 2-fold in 3D culture by both neuropeptides. We have previously demonstrated that WISP3/CCN6 is up-regulated in end stage OA cartilage and regulates MMP and ADAMTS4 and 5 in chondrocytes. Whilst neurotrophin BDNF expression was repressed by both neuropeptides, tachykinin 3 (the precursor to neurokinin B) was also repressed fold by bradykinin but interestingly not by the tachykinin substance P. We also explored expression of two of the genes regulated in C28/I2 cells by qPCR analysis in primary human chondrocytes and confirmed that expression of both WISP3/CCN6 and TIMP3 was also repressed by the two neuropeptides. Conclusions: Overall our data suggest that the effects of substance P and bradykinin on chondrocyte gene expression and matrix metabolism are environment-dependent.

A number of studies have shown elevated matrix metalloproteinase expression in chronic wound fluid compared to an acute wound; however, little has been done to characterize animal models in a similar manner and thus determine their usefulness. The diabetes mouse is an animal model of type II diabetes that shows impaired dermal wound healing and has been proposed as a model of human impaired wound healing. In this study we have determined the mRNA and protein expression profiles of matrix metalloproteinases 2, 3, and 9 during the first 10 d of dermal healing for the diabetes mouse and its normally healing littermate. Additionally, human wound fluid from diabetic chronic wounds and acute surgical wounds were studied to enable a comparison of the model to the human condition. We show that during the early stages of wound healing the diabetes mouse possesses significantly reduced protein levels of pro-matrix metalloproteinases 2 and 9 within the wound tissue and active matrix metalloproteinase 3 within the fluid. Pro-matrix metalloproteinase 3 levels are also significantly reduced in the diabetes mouse during the later stages of healing. These differences may be contributing to the impaired healing of the diabetes mouse; however, they differ from the human data presented here, which show elevated matrix metalloproteinase 2 and reduced matrix metalloproteinase 9 in human diabetic chronic wound fluid compared to acute wound fluid. Therefore, although clearly showing the importance of appropriate matrix metalloproteinase regulation for normal acute wound healing to occur, the diabetes mouse may not be an ideal model for study of matrix metalloproteinase involvement in human chronic wound healing.