Regenerative Therapy
Effects of small extracellular vesicles derived from 1a,25-dihydroxyvitamin D3 and hypoxia preconditioned hPDLSCs on osteogenic differentiation of hBMSCs
By Ruili Long, Qiang Wang, Sha Li and 3 others.
Abstract
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.