Osteogenically Induced Human Umbilical Cord Mesenchymal Stem Cell-derived Small Extracellular Vesicles Carrying Distinct microRNA Signature Improve Osteoinductivity of Tissue-engineered Bone

Abstract

Background: Clinical application of stem cell-based tissue-engineered bone (TEB) has been severely hindered by a low viability of seed cells, slow osteogenesis and biosafety. Mesenchymal stem cells-derived small extracellular vesicles (sEVs) possess similar properties to donor cells and have low immunogenicity, strong stability and favorable biosafety. It’s validated that human umbilical cord mesenchymal stem cells-derived sEVs (hUCMSCs-sEVs) exert therapeutic effects on various diseases. However, little is known whether hUCMSCs-sEVs can effectively be used to construct TEB. This study aimed to investigate whether hUCMSCs-sEVs bore positive effects on bone regeneration and elucidate the underlying mechanisms. Methods: Herein, PM-sEVs and OM-sEVs were separately harvested from proliferation medium (PM) and osteogenic medium (OM) cultured hUCMSCs and identified. The effects of PM-sEVs and OM-sEVs on the bioactivities and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) were evaluated. The differential microRNAs (miRNAs) expressions between PM-sEVs and OM-sEVs were screened by miRNA sequencing. Furthermore, PM-sEVs and OM-sEVs were separately incorporated in tricalcium phosphate (TCP) scaffolds to build TEBs, which were used to repair critical-sized calvarial bone defects of rats. Results: Both PM-sEVs and OM-sEVs had similar morphology, size and expressed specific surface markers of exosomes while didn’t have the surface marker from donor cells. Both PM-sEVs and OM-sEVs could be internalized by hBMSCs, thus significantly promoting the migration, proliferation and osteogenic differentiation of hBMSCs. Remarkably, OM-sEVs outweighed PM-sEVs in accelerating hBMSCs’ migration and osteogenic differentiation. Besides, miRNA sequencing analysis revealed that several differentially expressed miRNAs might be responsible for sEVs’ bioactivities. Furthermore, OM-sEVs could sustainably release from TCP scaffolds, and the resultant TEB had better reparative outcome than that of PM-sEVs group and pure TCP. Conclusion: sEVs isolated from osteogenically induced hUCMSCs (OM-sEVs) could prominently facilitate osteogenic differentiation of hBMSCs by unique miRNAs signature. TEB graft derived from OM-sEVs provided a promising alternative for clinical transplantation.