Experimental Studies On Mesenchymal Stem Cells Combined With SF/PLCL Nanofibrous Scaffolds For Bone Regeneration

ObjectivesIn order to develop more efficient methods for cell-based therapies for bone regeneration,this study will investigate the effect of microRNA-26a(miR-26a)on human adipose-derived stem cells(hADSCs)osteogenesis and the involved mechanism,and the roles of human ethmoid sinus mucosa mesenchymal stem cells(hESMSCs)cell sheet and the hybrid silk fibroin(SF)/poly(lactide-co-e-caprolactone)(PLCL)nanofibrous scffolds in bone regeneration will also be explored in vitro and in vivoMaterials and Methods1.Construction of lentiviral vectors encoding miR-26a or inhibitor.After hADSCs were tracsfected by lentiviral vectors,quantitative real time PCR(qPCR),western blot and alkaline phosphatse were used to determine the effecs of miR-26a on osteogenic differentiation of hADSCs.Dual luciferase reporter assay and choromatin immunoprcipitation were used to detect the regulation mechanisms of miR-26a' s effect on hADSCs osteogenesis2.hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation.In particular,hESMSCs displayed strong osteogenic differentiation potential,and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells(rBMSCs)in vitro.Next,hESMSCs were prepared into a cell sheet and combined with a poly(sebacoyl diglyceride)(PSeD)scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats,which showed excellent reparative effects3.hADSCs were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion,proliferation,cytotoxicity and osteogenic differentiation;the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated.Results1.Overexpression of miR-26a promoted hADSC osteogenesis,miR-26a directly targeted the 3'UTR of the GSK3?,suppressing the expression of GSK3? protein.GSK3? influences the expression of its downstream target C/EBPa.In turn,C/EBPa transcriptionally regulated the expression of miR-26a by physically binding to the CTDSPL promoter region.2.hESMSCs displayed strong osteogenic differentiation potential,and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells(rBMSCs)in vitro.hESMSCs cell-sheet combined with a PSeD scaffold seeded with rBMSCs showed excellent effects on repairing critical-sized calvarial defects in rats.3.The SF/PLCL(50/50)scaffold facilitated cell adhesion and proliferation,and promoted the osteogenic differentiation of hADSCs.In vivo assays showed that SF/PLCL(50/50)scaffold improved the repair of the critical-sized calvarial defect(CSD)in rats.Conculsions1.A novel feedback regulatory circuitry composed of miR-26a,GSK3? and C/EBPa contribute to the regulation of hADSC osteogenesis.2.hESMSCs possessed strong osteogenic potential and paracrine capability,and the cell sheet of hESMSCs could remarkably promote new bone regeneration,indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.3.The SF/PLCL(50/50)nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo,suggesting that the SF/PLCL(50/50)nanofibrous scaffold holds great potential in bone tissue regeneration.