Mechanism Of Small Extracellular Vesicles Of Adipose Mesenchymal Stem Cells Promoting Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By Transduction Of Osterix MRNA

Objective:It is well-known that bone marrow-derived stem cells(BMSCs)play a crucial role in improving the process of bone regeneration,but the role of BMSCs recruited at injury sites could be influenced by tissue microenvironment factors,including growth factors,cytokines,and small extracellular vesicles(sEVs)derived from other cells.A recent study has investigated that sEVs from adipose mesenchymal stem cells(ADSCs)after being osteogenically inducted,called ADSCs-sEVs+,could induce BMSCs to differentiate into osteoblasts,but few studies have been conducted to clarify the underlying mechanism in the effects of ADSCs-sEVs+.This study aimed to explain why ADSCs-sEVs+could promote osteoblast differentiation of BMSCs.Methods:ADSCs and BMSCs from New Zealand white rabbits were isolated and cultured to obtain sEVs by differential ultracentrifugation.These cells were characterized using flow cytometry,and ADSCs-sEVs were characterized by transmission electron microscopy(TEM),nano tracking analysis(NTA),and western blotting.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to measure Osterix expression.ALP and ARS staining were used to evaluate the osteogenic differentiation efficiency.Immunofluorescence was performed to determine the uptake of ADSCs-sEVs by the BMSC s.siRNA transfection was used to knock down the expression of Osterix mRNA,whereas recombination human bone morphogenetic protein 2(rhBMP2)was administered to strengthen the expression of Osterix mRNA.Results:The specific mRNA of Osterix was determined by qRT-PCR to be abundantly encapsulated in ADSCs-sEVs+,which could significantly enhance ALP staining of BMSCs,as well as remarkably upregulate the levels of downstream osteoblastic markers such as Runx2,OPN,and OCN.Overexpression of Osterix mRNA in ADSCs-sEVs+promoted the osteogenic differentiation of BMSCs,while downregulation of Osterix mRNA inhibited BMSCs’ differentiation into osteoblasts.Conclusion:ADSCs-sEVs+ promoted the differentiation of BMSCs into osteoblasts by transferring Osterix mRNA.This study provides a new target for the mechanism of ADSCs-sEVs promoting osteogenic differentiation of BMSCs.