Crosstalk between osteoblasts and adipocytes has impact on bone repair, since these cells coexist and interact in bone marrow. In a recent study, we observed that adipocytes differentiated from mesenchymal stem cells (MSCs) derived from adipose tissue inhibit osteoblast differentiation of MSCs derived from bone marrow by synthesizing and secreting tumor necrosis factor alpha (TNF-±), an inflammatory cytokine. Considering the importance of the interaction between osteoblasts and adipocytes to osteogenesis, we hypothesized that adipocytes, in addition to inhibit osteoblast differentiation through TNF-±, also regulate the synthesis of inflammatory cytokines by osteoblasts, which may affect the process of bone repair. Thus, the aim of this study is to investigate the effect of adipocytes differentiated from MSCs of adipose tissue on inflammatory cytokine expression by osteoblasts differentiated from MSCs of bone marrow in a model of indirect coculture. MSCs will be obtained from bone marrow and inguinal adipose tissue of rats and cultured in expansion medium until subconfluence. Then, MSCs from bone marrow will be cultured in osteogenic medium and MSCs from adipose tissue, in adipogenic medium for 7 days to induce osteoblast and adipocyte differentiation, respectively. From day 7 to 10, osteoblasts will be cocultured with adipocytes using transwells in osteogenic medium and, as control, osteoblasts will be cultured in the absence of adipocytes during the same period. On day 10, the gene expression of the proinflammatory cytokines, TNF-±, interferon gamma (INF-³), interleukin 1 beta (IL1-²), IL17a, anti-inflammatory cytokines, IL4 e IL10 , and of the osteoblast markers RUNX2, osterix, alkaline phosphatase and osteocalcin, these ones to confirm the inhibitory effect of adipocytes on osteoblast differentiation, will be evaluated by real-time PCR. The data will be obtained in triplicate (n=3) and submitted to the Mann-Whitney non-parametric test for independent data and two samples to compare osteoblasts grown in presence and absence of adipocytes (pd0.05).
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