Effects of small extracellular vesicles derived from 1a,25-dihydroxyvitamin D3 and hypoxia preconditioned hPDLSCs on osteogenic differentiation of hBMSCs

Objective Human periodontal stem cells (hPDLSCs) were preconditioned with 1α,25-dihydroxyvitamin D3 (1,25D3) to screen and extract osteogenically optimized hPDLSC-derived small extracellular vesicles (sEVs), which were subsequently used on human bone marrow mesenchymal stem cells (hBMSCs). This study aimed to obtain an optimized type of sEVs to enhance the osteogenic functionality of hBMSCs. Methods hPDLSC-sEVs were extracted using the Polyethylene glycol (PEG) precipitation combined with ultracentrifugation (PEG-ultracentrifugation) and Exosome Isolation Kit, then characterized by transmission electron microscopy (TEM), Nanopartice tracking analysis (NTA), and Western blot (WB). Alkaline phosphatase (ALP) staining and activity assays were performed to screen the concentrations of 1,25D3 for promoting osteogenic differentiation of hPDLSCs. To determine the time point and concentration of osteoinductive sEVs derived from 1,25D3 treated hPDLSCs, the hBMSCs were exposed to different sEVs treatments. After 7 days of induction, ALP staining was performed. The effects of osteogenic optimized sEVs on the proliferation and migration of hBMSCs were evaluated using the CCK-8 method and Transwell chamber assay. The effects of osteogenic optimized sEVs on the osteogenic differentiation of hBMSCs were evaluated by ALP staining, ALP activity assay, alizarin red staining, and Western blot analysis. Results In comparison to sEVs obtained by the Exosome Isolation Kit, those acquired through the PEG-ultracentrifugation method exhibited a particle size that more closely aligns with the typical range of extracellular vesicle diameters, alongside a higher concentration. The concentration of 10 nM of 1,25D3 was screened for osteogenic differentiation of hPDLSCs. 50 μg/mL sEVs isolated from the conditioned medium of hPDLSCs after 7 days of osteoinduction were selected as the working concentration. sEVs derived from 1,25D3 treated hPDLSCs (sEVs VD3 ) and 1,25D3 in combination with hypoxic cultured hPDLSCs (sEVs VD3+Hypo ) both significantly enhanced the proliferation, migration and osteogenic differentiation of hBMSCs, while sEVs VD3+Hypo exhibited even greater osteogenic efficacy. Conclusion PEG-ultracentrifugation is an effective method to extract hPDLSCs-derived sEVs and our selected sEVs derived from hPDLSCs show beneficial effects in promoting the proliferation, migration, and osteogenic differentiation of hBMSCs, which provides a promising strategy for in vitro osteogenesis and bone regeneration.