Effects Of Human Osteoarthritic Cartilage-derived Conditioned Medium On Donor-matched Mesenchymal Stem Cells

Osteoarthritis?OA?,especially its most common type-knee OA,is the most common disease of joints.It has been reported that knee OA has doubled in prevalence since the mid-20^th century^[1],however,there was no effective treatment and total knee replacement has to be the final resort^[2,3].Mesenchymal stem cells?MSCs?,adipose-derived stem cells in particular,are considered to be a promising source for treating knee OA and Koh et al.have shown that intra-articular injection of infrapatellar fat pad?IPFP?derived MSCs was effective for reducing pain and improving knee function in patients with knee OA^[4,5].Still,the potential mechanism for IPFP MSCs-based knee OA therapy remains unclear.As it is known that cell fate is mainly dependent on the interactions between cells and the surrounding microenvironment and a lot of study has focused on the interaction between MSCs and osteoarthritic chondrocytes.However,osteoarthritic chondrocytes would gradually lose inflammatory phenotype in vitro during change of culture medium and subculture,leading to failure of mimicking the in vivo condition.Here we used osteoarthritic cartilage fragment to make conditioned medium?CM?and cocultured with donor-matched IPFP MSCs and investigated various effects of CM from osteoarthritic cartilage on IPFP MSCs in vitro.Methods:Conditioned medium of osteoarthritic cartilage fragment was made and frozen in-80?before use.MSCs derived from donor-matched IPFP were cultured and characterized before using?passage 3?in experiments.1.IPFP MSCs were divided into CM group and Control group.To investigate the effect of CM on bioactivity and migrated activity of IPFP MSCs,Alamar blue kit for cell viability,Annexin V-FITC/PI Kit for apoptosis and Live-Dead Staining kit for live-dead staining analysis were performed for bioactivity.Wound healing assay and Transwell migration assay were performed for migrated activity.2.To determine the differentiated effect of CM on IPFP MSCs,real-time PCR and western blot were performed.And the experimental group included CM group,Control group and chondrogenic medium?Chon?group.3.To characterize the content of CM,Proteome Profiler Human Cytokine Array Kit was used for CM and control medium.Results:1.With subculturing,IPFP MSCs became more and more homogeneous and fibroblast-like from passage 0 to passage 2.Using flow cytometry,the MSCs markers were measured.The cultured IPFP MSCs at passage 3 were positive?>90%?for the MSCs markers CD44,CD73,CD90 and CD105,and negative??2%?for CD45,CD34,CD11b,CD19 and HLA-DR.2.Compared with Control group,a significant increase?P<0.01?of cell viability was observed in CM group at 24 h,48 h,and 72 h but no significant difference?P>0.05?was seen for apoptosis and live-dead staining of IPFP MSCs.3.In wound healing assay,the number of migrated IPFP MSCs in CM group was smaller than that in Control group at 8 h?P=0.016?,24 h?P=0.011?,and 36 h?P<0.001?.Similarly,in Transwell migration assay,the number of IPFP MSCs migrated from upper membrane to the other side was significantly smaller?P<.001?in CM group than in Control group.4.In gene level,chondrogenic marker genes SOX9?P<0.001?,COL2A1?P=0.014?,and ACAN?P=0.001?in CM group was higher than that in Control group.Western blot results showed that SOX9,type II collagen,and aggrecan all demonstrated a trend of upregulation in CM group in comparison with Control group.5.The real-time PCR results showed that IPFP MSCs in CM group had a higher expression of MMP13?P=0.001?and COL10A1?P=0.002?compared to Control group,but the protein expression of MMP13 and type X collagen was contrary to that at gene level.6.In gene level,IPFP MSCs treated with complete CM exhibited a chondrogenic tendency as compared to cells treated with DMEM/F-12?P=0.004 for SOX9,P=0.075 for COL2A1 and P=0.075 for ACAN?.Western blot results confirmed this trend at protein level.7.The real-time PCR results showed that IPFP MSCs treated with complete CM for 7days showed a significant higher expression of MMP13?P=0.001?,ADAMTS5?P<0.001?,COL10A1?P=0.001?,and RUNX2?P=0.003?.Western blot results further confirmed that complete CM increased the level of protein related to hypertrophy and catabolism in IPFP MSCs.8.Compared with control medium,14 cytokines in CM group were detected.Conclusion:Compared with DMEM/F12 without OA cartilage fragment,osteoarthritic cartilage-derived CM benefited chondrogenic differentiation,impeded migration,and led to the hypertrophy and catabolism of IPFP MSCs in vitro.