gms | German Medical Science

Objectives: Cell communication and epigenetic regulation are critically dependent on various contents that extracellular vesicles (EVs) deliver, including miRNA, mRNA, lipids, and proteins¹. Recently, emerging studies^2,3 showed that EVs isolated from human bone marrow stem cells (hBMSC) in mid stage of osteogenic differentiation could regulate osteogenesis and promote bone regeneration, but the late stage is still unclear. Therefore, we investigated the effects of EVs derived from hBMSC in different stages of osteogenic differentiation and their effects on osteogenic differentiation capacity of hBMSC.

Methods: The use of hBMSC was approved by the local ethics committee (No. 14-101-0189). EVs were isolated from conditioned media collected from different time points (day = D0, D21, D28 and D35) of hBMSC undergoing osteogenic differentiation (i.e., EVs_D0= naïve hBMSC derived EVs, EVs_D21= EVs isolation of hBMSC undergoing osteogenic differentiation for 21 days, EVs_D28= EVs isolation after 28 days, EVs_D35= EVs isolation after 35 days).

The different EV groups (5μ g/ml) and control group (no EVs) were evaluated for osteogenic activity by Alizarin Red Staining and Alkaline Phosphatase (ALP) assay. Proliferation, vitality and apoptosis of hBMSC after stimulation with the different EV groups (5μ g/ml) or control group were measured with BrdU assay, CellTiter-Blue Cell Viability(CTB) assay and Caspase 3/7 assay, respectively. One way Anova with multiple comparisons test or One sample t test was used for comparisons of results between EV groups and control group.

Results and Conclusion: hBMSC were kept in osteogenic differentiation medium for 21 days and were treated the last 15 days with EVs from the experimental groups or the controls, as shown in Figure 1a and 1b [Fig. 1]. We observed that EVs_D35 promoted osteogenic differentiation of hBMSC correlated to Alizerin Red staining quantity indicating significantly enhanced calcium deposition. Moreover, after 7 days of osteogenic differentiation of hBMSC, treated the last 5 days with the exp. EV groups, EVs_D0 treatment induced significantly increased ALP activity. BrdU assay and Caspase 3/7 assay results showed that proliferation ability of hBMSC was upregulated and apoptosis was inhibited after treatment with EVs_D0, EVs_D21, EVs_D28 and EVs_D35 (Figure 1d and 1e [Fig. 1]). In CTB assay, vitality of hBMSC increased significantly when cultured with EVs_D0, EVs_D21 and EVs_D35 (Figure 1f [Fig. 1]).

Taken together, these results suggest EVs derived from hBMSC in the late stage of osteogenic differentiation (day 35) could promote bone regeneration potential of BMSC through the increase of their osteogenic differentiation capability, proliferation and vitality, and inhibition of apoptosis.