Pro-osteogenic Effects of DPSC-derived Extracellular Vesicles on Jaw Bone-derived MSCs

Objectives: Dental pulp stem cells (DPSCs) represent a unique subpopulation of mesenchymal stem cells (MSCs) with potential for bone regeneration with its osteogenic propensity and pro-bone regeneration potentials. The use of extracellular vesicles (EVs) by MSCs have several practical advantages over MSC-based therapy, including easiness for production, storage, less potential regulatory and ethical issues for translational application in clinical setting. Hence, we aim to explore the effect of DPSC-derived EVs on osteogenic differentiation of human jaw bone-derived mesenchymal stem cells (JB-MSCs).
Methods: DPSCs were isolated from human dental pulp tissues of extracted wisdom teeth. The conditioned media of cultured DPSCs were harvested and DPSC-released EVs were purified. The concentration and size distribution of DPSC-exosomes were determined using Nanosight NS300 instrument. The expression of EV surface markers CD63 and CD9 were determined by Western blot. Ex vivo expanded jaw bone-MSCs (JB-MSCs) were cultured under osteogenic differentiation medium in the presence of different doses of DPSC-derived EVs (10, 20, 40μg/mL), while JB-MSCs cultured under normal conditions were used as negative control. After culturing for different time periods (1, 2, 3, 4, and 6 weeks), each group of cells were collected for RNA and protein extractions to quantify expression of osteogenic genes by qRT-PCR and Western blot. Meanwhile, mineralization of differentiated cells was evaluated by using Alizarin Red S staining.
Results: Treatment with DPSC-derived EVs led to a dose-dependent increase in the expression of osteogenic genes, including RUNX2, alkaline phosphatase (ALP), type I collagen (COL1A1), and osteocalcin (OCN), at both mRNA and protein levels in JB-MSCs as determined by qRT-PCR and Western blot, respectively. Alizarin Red S staining showed that DPSC-derived EVs increased the mineralized deposits of calcium in JB-MSCs following osteogenic induction for 4 and 6 weeks, respectively.
Conclusions: DPSC-derived EVs possess pro-osteogenic effects on JB-MSCs, thus supporting the notion that DPSC-derived EVs might be used as a novel cell-free product for translational application in clinic for regeneration and reconstruction of craniofacial bone defects.