Changes in the Osteogenic Differentiation Potential of Adipose-Derived MSCs During In Vitro Co-Cultivation with Heparin

Objective: The aim of this study was to investigate the effect of heparin at a concentration of 1 IU/mL on the osteogenic differentiation potential of human adiposederived mesenchymal stem/stromal cells (ASCs) during in vitro co-cultivation. Materials and methods: The phenotypic profile of ASCs cultured with or without heparin for 14 days was assessed by flow cytometry using appropriate antibodies according to the manufacturer’s protocol on a MACSQuant cytometer. The migration and proliferation potential of ASCs in the presence of heparin was evaluated using a realtime cell monitoring system (xCELLigence ® RTCA DP). After 14 days of co-cultivation with heparin, the expression of osteogenic marker genes was assessed by real-time PCR. In addition, the differentiation profile of ASCs cultured with heparin was evaluated by alizarin red staining to detect mineralization nodules after 21 days of cultivation. Finally, the levels of growth factors, chemokines, and pro and antiinflammatory molecules were measured in the supernatants of 14-day cultures. Results: Compared to the control group, the MSC + heparin model showed a significant decrease in the percentage of cells expressing the stem cell markers CD73, CD90 and CD105; increased proliferative activity but decreased migratory activity of ASCs; increased relative mRNA expression of osteogenic genes (ALPL, RUNX2, BMP2, BMP6) and the adhesion gene CD49d; and a larger mineralization area after 21 days of cultivation. A trend towards increased secretion of VEGF and the proinflammatory cytokine IL6 was observed in the MSC + heparin model. Conclusion: These results may serve as a basis for developing new clinical strategies for the treatment of surgical patients undergoing osteosynthesis who are at high risk of thrombosis.