Human Adipose-derived Stromal Cell secretome beneficial potential in contrasting osteoarthritis

OBJECTIVE: Adipose-derived Stromal Cells (ASCs) possess a strong anti-inflammatory potential, which is mediated by a wide array of released bioactive factors. The use of conditioned medium (CM) instead of cells presents substantial advantages especially in terms of handling and safety. The purpose of this study is to investigate the potential of hASC secretome in contrasting osteoarthritis (OA), an inflammatory disease characterized by hypertrophic differentiation of chondrocytes (CHs) and extracellular-matrix-degrading proteases production. MATERIALS AND METHODS: CM was collected from hASCs derived from subcutaneous adipose tissue, cultured for 72 hours in starving conditions. The CM was then concentrated trough Amicon Ultra-15 Centrifugal Filter Unit (Merck-Millipore) of about 46±10-folds (n=26). Hypertrophy was induced in vitro in human primary articular chondrocytes with 10ng/ml TNFα. CM (ratio 5:1, hASCs:hCHs) effect on cell proliferation (up to 9 days) was assessed by AlamarBlue. Gene and protein expression of MMPs and other hypertrophic markers were evaluated (24 and 72 hours) by RT-PCR, Western blot and multiplex immunoassay. RESULTS: hCH proliferation was prompted (+40%) by a 9-day-treatment with 10 ng/ml TNFα, suggesting the induction of a hypertrophic growth status. Despite the lack of CM effect on CH proliferation, the conditioned medium treatment reverted the TNFα–induced significant increase in MMP3 and MMP13 expression (-50% and -30%, respectively), after 24 hours. The reduction in MMP13 protein expression was also evident after both 24 and 72 hours. Moreover, hASC conditioned medium inhibited TNFα-mediated osteocalcin release. The effect of CM on other OA and hypertrophic markers, in TNFα-inflamed CHs, is currently under investigation. CONCLUSION: Our data reinforce the idea that ASC secretome might be considered a promising source of factors for future therapeutic applications in the OA treatment. The analysis of CM sub-components (EVs and soluble factors) and the comparison of ASC secretome with CM from cells lacking this therapeutic potential, might allow us to reveal the factors responsible for secretome beneficial action.