SHED Aggregate Derived Exosomes-shuttled miR-222 Promotes the Regenerative Properties of Periodontal Ligament Stem Cells

Abstract Background: Periodontal ligament stem cells (PDLSCs) aggregate is still limited in clinical application for lack of angiogenesis. This study aimed to investigate the effects and underlying mechanism of exosomes derived from stem cells from human exfoliated deciduous teeth (SHED) aggregate (SA-Exo) on the aggregate formation and angiogenic properties of PDLSCs.Methods: SA-Exo were isolated by ultracentrifugation. The effect of SA-Exo on the aggregate formation and angiogenic differentiation of PDLSCs were evaluated by investigating extracellular matrix (ECM) deposition and tube formation assay. MicroRNA (miRNA) sequencing was employed to screen different miRNA expression. The effect of targeting miRNA on ECM deposition and angiogenesis of PDLSCs aggregate was investigated after overexpression and inhibition of miRNA. Periodontal bone defect rat models were established to evaluate the effect of the PDLSCs aggregate and SA-Exo combination on periodontal bone regeneration. Results: SA-Exo could significantly enhance the ECM deposition and angiogenic ability of PDLSCs. The expression of ECM-associated proteins (COL-I, integrinβ1, and fibronectin), angiogenesis-related proteins (PDGF, ANG, TGFβRII), and related pathway (p-SMAD1/5 and p-SMAD2/3) were upregulated in PDLSCs aggregate with SA-Exo. Mechanistically, miR-222 was found relatively abundant in SA-Exo, which promoted ECM deposition and angiogenesis of PDLSCs. In vivo experiment further validated that combinational use of PDLSCs aggregate and SA-Exo promote more bone formation and neovascularization in rat’s periodontal bone defect.Conclusions: SA-Exo-shuttled miR-222 contributes to PDLSCs aggregate engineering by promoting aggregate formation and angiogenesis, which might through activate the TGF-β/SMAD signaling pathway.