Human Adipose And Synovial Mesenchymal Stem Cells Synergistically Inhibit The Degeneration Of Inflammatory Chondrocytes

Background and PurposeChondrocytes in the early course of osteoarthritis are inflammatory chondrocytes.Inhibition of cartilage inflammation and regeneration of chondrocytes are the two main mechanisms underlying the cartilage degeneration of early phase of osteoarthritis.Both inhibiting the inflammation and improving the regeneration of chondrocytes will slow down the process of cartilage degeneration.However,the connection between the inflammation and regeneration of OA chondrocytes is not understood as well as its effect on cartilage degeneration.Our previous studies showed that adipose derived mesenchymal stem cell(ADMSCs)can inhibit the inflammation response and synovium mesenchymal derived stem cells(SDMSCs)have a very strong potential to differentiate into Chondrocytes in vitro.In the present study,we hypothesized that combined both ADMSCs and SDMSCs will yield the synergistic inhibiting effect of OA chondrocytes degeneration in vitro.MethodPrimary culture of adipose and synovial derived mesenchymal stem cells.In vitro two-dimensional culture conditions,MTS(cell proliferation assay)to detect the proliferation of three kinds of cells.The differences of chondrogenic markers between the three adherent cell genes and protein levels were analyzed by quantitative PCR and immunofluorescence.In vitro three-dimensional mixed culture conditions,divided into three groups: 1.ADMSCs + inflammatory chondrocytes(A + C group)2.SDMSCs + inflammatory chondrocytes(S + C group)3.ADMSCs-SDMSCs +inflammatory chondrocytes group A + S + C group).Alcian blue,Safranin O staining and type II collagen immunohistochemistry staining were performed on the mixed-cultured cell mass paraffin sections and quantitatively analyzed,Three groups of cells were collected for quantitative PCR detection of markers of cartilage differentiation,while digestion of cell clumps for cell counting,and for statistical analysis.Culture supernatants of different time periods were collected to detect the secretion of proinflammatory and anti-inflammatory factors by enzyme-linked immunosorbent assay.the resulting mean ± standard deviation(sx ±)that the application of SPSS 16.0 statistical software analysis,datas were compared before and after surgery were statistically significant by t test for quantitative data analysis,longitudinal group in this diversity were applied to compare the number of single-factor analysis of variance.ResultsThe proliferative rate of adipose-derived mesenchymal stem cells> inflammatory chondrocytes and synovial mesenchymal stem cells were found to be statistically significant(P <0.05)by MTS cell proliferation assay.Quantitative PCR assay showed that the expression of cartilage marker type II collagen and proteoglycan in inflammatory chondrocytes was significantly higher than that in the other two cells(P<0.01).Quantitative analysis of inflammatory chondrocytes by immunofluorescence with collagen type II collagen in the cell slide was significantly higher than the other two groups(P <0.01).The cartilage differentiation specific staining of ADMSCs-SDMSCs + inflammatory chondrocytes were significantly higher than that of the other two groups(Alcian blue staining 6.2 ± 0.1 in A + C group,36.2 ± 0.21 in S + C group and 54.7 ± 0.43 in A+S + C group;safranin O staining,14.2 ± 0.15 in A + C group,37.7 ± 0.4 in S + C group and 58.8 ± 0.13 in A + S + C group;Collagen type II by IHC 13.6 ± 0.24 in A + C group,30.1 ± 0.35 in S + C group and 54.6 ± 0.27 in A +S + C group)(P <0.05).The supernatant of 3D culture was collected and tested by ELISA.The levels of IL-1,IL-6 and TNFα in S + C group were significantly higher than those in A + C group and A + S + C group.The level of Anti-inflammatory facter IL-10 in A+C group and A+S+C group was significantly higher than the S + C group and had significant statistical difference(P <0.05).The expression of proteoglycan and type II collagen,which were markers of cartilage differentiation,was analyzed by Q-RT-PCR.The expression of collagen type II and collagen was significantly higher in A + S + C group than the other two groups(P <0.01).After digestion and counting of cell pellets,the results showed that A + S + C group> S + C group> A + C group after 14 days and had significant statistical difference(P <0.05).Conclusion1.In vitro two-dimensional culture conditions,synovial mesenchymal stem cells and adipose derived mesenchymal stem cells have poor chondrogenic differentiation.2.In vitro 3D culture conditions,synovial mesenchymal stem cells have a strong chondrogenic differentiation ability,adipose derived mesenchymal stem cells have a strong anti-inflammatory effect.3.Combined both ADMSCs and SDMSCs had a synergistic inhibitory effect on the degeneration of inflammatory chondrocytes.