| Titanium alloys have been commonly utilized in bone repairing due to their good biocompatibility,excellent corrosion resistance and mechanical properties.But the surface bio-inert may induce clinical problems.The surface functionalization was needed.MicroRNAs involved in the regulation of every bone repair process.Delivery of osteogenesis-related microRNAs has been demonstrated to be an efficient way for bone healing.Thus the aim of this study was to develop a microRNA delivery platform for stable and effective microRNA transfection and titanium surface modification for promoting bone formation in vivo.By using the commercial reagent,the microRNA-21(miR-21)mimics(m-miR-21)and the antisense miR-21(as-miR-21)were firstly transfected to human umbilical cord mesenchymal stem cells(hUMSCs)respectively.The function of miR-21 in the osteogenic differentiation of hUMSCs and the mechanism were investigated.The results indicated that the overexpression of miR-21 significantly elevated the expression of osteogenic markers encoding alkaline phosphatase(ALP),runt-related gene-2(RUNX-2)and osteocalcin(OCN).Alizarin Red S staining demonstrated that miR-21 can promote the osteogenic differentiation of hUMSCs.We also proved that the PI3K-AKT signaling pathway activity had an increasing tendency responding to miR-21 up-regulation.This enhancement promoted the phosphorylation of GSK-3?,leading to the stabilization and high concentration accumulation of ?-catenin in cytoplasm to activate the transcription of RUNX-2,and finally increased the osteogenesis of hUMSCs.Secondly,miRNAs were encapsulated within nanocapsules by in situ polymerization method.The miRNA nanocapsules were further entrapped into an O-carboxymethyl chitosan(CMCS)network via electrostatic interactions,forming miRNA-activated matrix.The matrix can avoid leakage of miRNA and offer better stability for cell transfection.Besides,the miRNA-activated matrix has good biocompatibility.Delivery of miR-21 by matrix significantly promoted the mRNA expression level of osteogenic markers such as ALP and RUNX-2,which indicated that the miR-21-activated matrix can induce the osteogenic differentiation of hUMSCs.After injection of the matrix in tibial plateau defects created in SD rats,we studied the in vivo bone regeneration efficacy of CMCS/n(miR-21)at 2 and 4 weeks.The in vivo study revealed that the expression level of miR-21-related target proteins PTEN was down-rugulated and ?-catenin was up-regulated after the injection of matrix.And the bone regeneration effect was much better than controls.Thirdly,we used miR-29b-activated matrix to modify the surface of titanium.This coating was not only favorable for cell adhesion and growth but also provided sufficient microRNA transfection efficacy.Besides,it can regulate the miR-29 b target HDAC-4 and induce the osteogenic differentiation of hUMSCs in vitro.By implanting miR-29b-functionalized titanium rod in tibial plateau defects,the degree of new bone generation was assessed at 2,4,6 and 8 weeks after implantation.The results showed that the miR-29b-activated matrix coating significantly enhanced the healing of bone defects on titanium surface.Besides,the expression level of osteogenic markers RUNX-2 and OCN was evaluated and the expression of miR-29 b target HDAC-4 was down-regulated,which further proved the great bone regeneration ability on the surface of titanium by functional miR-29 b modification. |
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