| Objective The aim of this study is to observe the osteogenic potential of ASCs, which were induced for 7days(i ASCs), infulened by co-cultured with different types of MSCs, such as bone marrow-mesenchymal stem cells(BMSCs), umbilical cord-derived mesenchymal stem cells(UC-MSCs) and placenta-derived mesenchymal stem cells(PDMSCs), under condition of directly or indirectly contact, respectively.Materials and methods 1. Isolation of ASCs Adipose tissue obtained from liposuction was washed and digested by 0.075% type I collagenase. Cells was resuspended and cultured in incubator with complete dulbecco's modified eagle medium/Ham's F-12(DMEM/F-12) under the condition of 37?, 5% CO2 and saturated humidity. 2. Proliferative curve of proliferation of ASCs?BMSCs?UC-MSCs and PDMSCs were tested by cck-8 kits. 3. Grouping of the experiment: directly co-cultured group: ASCs at P3 were induced in osteogenic differentiation medium(ODM) for 7 days. Induced ASCs(i ASCs) were digested and adjusted the cell density to 2×104/m L. BMSCs, UC-MSCs and PDMSCs were adjusted to 1×104/m L, respectively. 100?L i ASCs suspension and undifferentiated MSCs suspension, respectively, were plated in 96-well plate, as d-BMSCs, d-UC-MSCs and d-PDMSCs experiment group. 3×103 i ASCs were cultured alone as control group(d-i ASCs). Indirectly co-cultured group: 100?L i ASCs was plated in 96-well plate and cultured with condition medium of BMSCs?UC-MSCs and PDMSCs, respectively, as ind-BMSCs, ind-UC-MSCs and ind-PDMSCs group. 100?L i ASCs was setted as control group(ind-i ASCs). 4. Test and analysis At 3, 5, 7, 10, 12 and 14-day, 6 wells of each group were tested the activity and concentration of ALP. At 7, 10, 12 and 14-day, all samples were stained by Alizarin red S. For quantitative analysis of mineralized extracellular matrix(ECM), Alizarin red S was redissoluted by cetylpyridinium chloride(CPC). 5. Data analysis was processed by SPSS 20.0 software, and P<0.05 was considered as statistically significant different.Results 1. The isolated ASCs showed fibroblastic-like shaped, MSCs-associated cell markers and multipotent of differentiation. BMSCs, UC-MSCs, PDMSCs showed as similar pattern as ASCs. As proliferation curve, UC-MSCs>PDMSCs>ASCs> BMSCs. 2. In regard of influence of MSCs on ALP, BMSCs exhibited strongest effect on ALP expressed by i ASCs under directly contacts. UC-MSCs and PDMSCs showed similar effect on expression of ALP. Under indirectly co-cultured condition, UC-MSCs and PDMSCs showed stronger effects on expression of ALP than BMSCs. All experiment group showed higher concentration of ALP than control group. 3. MSCs from different tissue showed various effects. Mineralized ECM in directly co-cultured groups were higher than control group. PDMSCs group showed the strongest induced effect on forming mineralized ECM and UC-MSCs was higher than BMSCs. In indirectly co-cultured group, UC-MSCs showed higher effect on regulation of forming mineralized ECM than PDMSCs and BMSCs.Conclusion 1. ASCs exhibited typical morphology and cell markers of MSCs. 2. ASCs showed similar morlogy, growth kinetics and charactoristics of proliferation with BMSCs, UC-MSCs and PDMSCs. 3. ASCs could mimic osteoprogenitor cell and provide an ideal cell source for investigation, after inducing for 7 days. 4. MSCs showed positive promotion on osteogenic differentiation of ASCs. Tissue-specific could influence effect of MSCs. The ability of BMSCs to promote expression of ALP was stronger than formation of mineralized ECM. UC-MSCs and PDMSCs had shown similar characteristic on induction of osteogenesis. In regard of mineralized ECM, PDMSCs was better than UC-MSCs. 5. BMSCs, UC-MSCs and PDMSCs could regulate osteobastic differentiation of i ASCs. The effect of regulation of direct contact was higher than indirect contact. 6. MSCs displayed an important effect on process of differentiation of osteogenitor. |
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