The effect of conditioned medium from human Adipose-derived Stem Cells on Chondrocytes in vitro

Adipose-derived Stem Cells (ASCs) are multipotent progenitors able to participate in tissue regeneration and to modulate immune response. Their regenerative and protective action seems to be mainly mediated by the release of soluble factors and/or extracellular vesicles (EVs). In a recent work we demonstrated that ASC-derived conditioned medium (CM) counteracts neuropathic pain symptoms in different preclinical mouse models (Brini et al., under revision to Scientific Report 2017). Since ASC secretome represents a promising cell-free approach in the treatment of inflammatory pathologies affecting the musculoskeletal tissue, here we investigate its in vitro effects on primary human chondrocytes. ASCs, derived from subcutaneous adipose tissue, were cultured for 72 hours in starving conditions to collect CM, which was then concentrated through Amicon Ultra-15 Centrifugal Filter Unit (Merck-Millipore). CM was concentrated 46±10-folds (n=26) and its trophic effect was tested on primary human chondrocytes (CHs). 9 days of treatment with CM slightly improved CH viability (increase of +10% respect to untreated CHs). In order to mimic articular inflammation in vitro, we treated CHs with 1 or 10ng/ml TNFα and then we evaluated CH proliferation up to day 9. Just the higher TNFα concentration determined a clear increase (+40% of control) in CH proliferation, suggesting the induction of a hypertrophic growth status. To determine a possible therapeutic effect of CM administration, we investigated cartilage-specific (ACAN and SOX9), inflammatory (COX2, MMP3 and MMP13) and hypertrophic (type X Collagen) markers. As expected, the inflammatory status induced by 10ng/ml TNFα was characterized by a strong increase in MMP3 and MMP13 gene expression at 24 hours (more than 50 and 10 folds the baseline values, respectively) that was considerably diminished by CM treatment (-50% and -30%, respectively). At later time points (48 and 72 hours), the effects of 10ng/ml TNFα±CM on gene expression became less straightforward. Furthermore, TNFα induced an increase in type X Collagen transcription at 24 hours that was partially counteracted by simultaneous CM administration. We did not observe major differences in the expression of cartilage-specific genes after treatment with TNFα in the presence or absence of CM. Taken together, our in vitro data show the trophic action and the anti-inflammatory potential of ASCs-derived CM and we strongly believe that concentrated CM represents a powerful source of factors with promising therapeutic potential.