gms | German Medical Science

Objectives: The treatment of bone defects with mesenchymal stem cells is becoming increasingly important. Particularly, multipotent stem cells of adipose tissue (ASCs) are gaining increasing importance in regenerative medicine. A challenge in this context is the reduced osteogenic differentiation potential of cells obtained from aged patients. Regarding osteogenic differentiation, oxidative stress, which is mediated by reactive oxygen species (ROS), might exert inhibiting effects at elevated concentrations and promoting effects at physiological concentrations. In the present work we have examined ASC cultures, which have lost their osteogenic differentiation potential (non-responder cells; NR-ASC), as well as responder cells, which showed a regular differentiation potential (R-ASC). With both cell types we have evaluated the impact of pro- and anti-oxidative approaches on their osteogenic differentiation potential.

Methods: ASCs were isolated from human fat tissue. Mesenchymal stem cell identity was proved by plastic adherence, antigen-phenotype-characterization, and the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes. Osteogenic differentiation (OD) was induced by dexamethasone (500 nM), ascorbate (50 µM), and beta-glycerophosphate (10 mM) and was characterized by the quantification of calcified matrix, alkaline phosphatase-expression and the expression of osteoblast-specific proteins. Oxidative stress was quantified by Dichlorodihydrofluorescein Diacetate (DCFDA). As antioxidants we used glutathione (1 mM), N-Acetyl-Cysteine (NAC, 1 mM), catalase (500 U), the synthetic superoxide dismutase and catalase mimetic EUK134 (50 µM), ascorbate (0,5 mM) and the water soluble vitamin E derivate Trolox (1 mM).

Results and Conclusion: In approx. 30% of our ASC preparations we found osteogenically dysfunctional NR-ASC cultures. As compared to R-ASCs these non-responder cell cultures did not show any significant differences in ROS production during osteogenic differentiation (OD). Interestingly, in R-ASC cultures exogenously applied H2O2 enhanced the factor-induced OD at low concentrations and reduced it at higher concentrations. In NR-ASCs this effect was also present but negligible low. Co-incubation of osteogenically activated ASCs with the mentioned antioxidants only in the case of ascorbate and catalase led to significant alterations in induced OD. Whereas ascorbate significantly decreased OD of R-ASCs, exogenously applied catalase significantly enhanced OD of R-ASCs and nearly completely restores the impaired osteogenic potential of NR-ASCs to a level comparable to that of R-ASC cultures. This effect could be significantly enhanced by further addition of H2O2 into the catalase-containing medium, even at H2O2 concentrations that were shown to inhibit OD when applied alone. The results of our study reveal that Catalase and H2O2 metabolisms might represent pivotal molecular targets to bridge over impaired osteogenic potential of mesenchymal stem cell.