Changes of osteodifferentiation potential of MSC-AT during in vitro co-cultivation with heparin

Abstract

The aim of this study was to investigate the effect of heparin at a concentration of 1 IU/mL on changes in the osteodifferentiation potential of MSC from human adipose tissue under in vitro cocultivation.Materials and methods. Assessment of the phenotypic profile of MSC from human adipose tissue during cultivation in the presence/absence of heparin was performed by the flow cytometry method using the appropriate dyes according to the manufacturer’s protocol on a MACS Quant flow cytometer after 14 days of cultivation. To evaluate the migration and proliferation potential of MSCs in the presence of heparin, we were using a continuous monitoring electrode system, xCELLigence ® RTCA DP. After cultivation MSCs with heparin for 14 days, the intracellular expression of osteodifferentiation genes was evaluated by real-time PCR. In addition, the differentiation profile of MSCs from human adipose tissue cultured with heparin was evaluated by cytological staining with alizarin red to detect islands of mineralization after 21 days of cultivation. In addition, the amount of growth factors, chemokines, molecules with pro- and anti-inflammatory activity was estimated in the supernatants of the 14-day cultures.Results. There was a significant decrease ( compared with the control group of the study) in the number of cells with stem markers (CD73, CD90, CD105) on the cell surface of the culture in the MSC + heparin model; increase in proliferative and decrease in migratory activity of MSCs during co-cultivation with heparin; increased levels of relative mRNA expression of genes for osteodifferentiation (ALPL, RUNX2, BMP2, BMP6) and cell adhesion (CD49d); increase in mineralization area in the study model in the presence of heparin after 21 days of cultivation. There was a tendency to increase secretion of growth factor VEGF and pro-inflammatory factor IL -6 in the MSC + heparin model.Conclusion. The obtained results may serve as a basis for the development of new therapeutic tactics for the treatment of surgical patients undergoing osteosynthesis operations with a high risk of thrombosis.