gms | German Medical Science

Objectives: Cell communication and epigenetic regulation critically dependent on the type of cargo that extracellular vesicles (EVs) deliver, including miRNA, mRNA, lipids, and proteins [1]. Recently, our emerging study [2] showed that EV isolation from human bone marrow derived stem cells (hBMSC) in late stage of osteogenic differentiation can regulate osteogenesis and promote bone regeneration under normoxia. Contrary, many studies [3] reported that hypoxia suppresses osteogenic differentiation of BMSC.

Therefore, we investigated whether EVs derived from hBMSC in late stages of osteogenic differentiation, cultured under normoxic conditions, can rescue osteogenic differentiation capacity of hBMSC, which was inhibited by hypoxia.

Methods: The use of hBMSC was approved by the local ethics committee (No. 14-101-0189). EVs were isolated from conditioned media collected at different time points (day = D0, D28-35) of hBMSC undergoing osteogenic differentiation under normoxia (i.e.,EVs_D0= naïve hBMSC derived EVs; EVs_D28-35= EV isolation of hBMSC undergoing osteogenic differentiation for 28-35 days).

The different EV treated groups (10μg/ml) and control group (no EVs) under hypoxia were evaluated for osteogenic activity by Alizarin Red staining and Alkaline Phosphatase (ALP) assay. Control group (no EVs) kept under hypoxia served as negative control group, and that under normoxia served as positive control group. Expression of osteogenic differentiation stage markers such as OPN (osteoprotegerin) and RUNX2 were analysed via qPCR, and the hypoxia marker Hif-1alpha was analysed via Western blotting. One-way Anova was used as statistical test.

Results and conclusion: hBMSC were kept in osteogenic differentiation medium for 21 days under hypoxia, and were treated for the last 16 days with EVs_D0 or EVs_D28-35. We observed that EVs_D28-35 promoted osteogenic differentiation of hBMSC, seen in the Alizerin Red staining quantity, that displays a significantly enhanced calcium deposition under hypoxia (Figure 1a, b). In addition, after 14 days of osteogenic differentiation of hBMSC under hypoxic condition, when treated for the last 6 days with EVs_D0 or EVs_D28-35, these groups induced significantly ALP activity under hypoxia (Figure 1c). qPCR results showed that OPN and RUNX2 expression was upregulated after treatment with the EVs_D28-35 group under hypoxia (Figure 1d). Using Western blotting, we observed that Hif-1alpha protein expression decreased when EVs_D28-35 were applied under hypoxia (Figure 1e).

Taken together, these results suggest that EVs derived from hBMSC in the late stage of osteogenic differentiation (day 28-35) can promote bone regeneration potential of hBMSC which were kept under hypoxic condition presumably through increasing of OPN and RUNX2 expression.