Based On The Angiogenesis Effect Of EPCs And VEGF165 To Promote Tissue Engineered Nerve Repairing Large Nerve Defect

Objective:Clinical medicine has urgent needs for large tissue engineering nerve defects regeneration;however,the vascularization problem of current tissue engineered nerve has not solved,thus limiting the progress of large nerve defect repairing.Previously,we successfully constructed tissue engineered nerve.In this project,we think the early vascularization play an important role in surviving of tissue engineered nerve in vivo,and propose a new method for large nerve defect repairing by vascularized tissue engineered peripheral nerve.We use recombinant adenovirus carrying VEGF165 gene to transfect autologous endothelial progenitor cells,and obtain Schwann-like cells induced by autologous adipose-derived stem cells;the two kinds of cells were used as the seed cells together.The allogeneic acellular nerve scaffolds were prepared used improved method by our research group.Then,the vascularized tissue engineered peripheral nerves were constructed.The SD rat large sciatic nerve defect was repaired by them.General observation,nerve electrophysiology and histological observation were used to evaluate the repairing effect.Investigate the repairing effect on vascularized tissue engineered peripheral nerve based on the angiogenesis effect of endothelial progenitor cell and VEGF165.Methods:1.EPCs and ADSCs of SD rats were isolated,cultured and identified.ADSCs were induced to SCs.EPCs and SCs were co-cultured at different inoculation ratios(1:2,1:1 and 2:1)to determine the influence of EPCs on the biological behavior of SCs induced by ADSCs at different ratios.2.EPCs was transfected with Ad-VEGF165 gene.CCK-8 was used to detect the effect of adenovirus transfection on EPCs survival.The effect of adenovirus transfection on EPCs migration was detected by Transwell chamber.Cell immunofluorescence,Matrigel extracellular angiogenesis assay,QRT-PCR and western-blot were used to detect the effect of adenovirus transfection on the functional expression of EPCs.3.SCs and Ad-VEGF165-EPCs were used as seed cells to construct vascularized tissue-engineered peripheral nerves.HE staining,SEM and Live/Dead fluorescence staining were used to observe its growth in vitro.4.Using the vascularized tissue engineering peripheral nerves to repair the sciatic nerve defects in rats,the degree of graft vascularization and nerve repair in each group were compared by gross observation,NEP,histological observation and so on.Results:1.By co-culturing EPCs and SCs,we obtained the optimal ratio of co-culture of the above two kinds of cells(1:1).At this ratio,EPCs can promote SCs biological behaviors such as survival and functional expression,which may be mediated by the Wnt/β-catenin signal pathway.2.At MOI=200,VEGF165 gene was transfected into rat EPCs.The transfection of VEGF165 gene could promote EPCs proliferation,migration and differentiation into endothelial cells,and improve the ability of EPCs to form blood vessels,which may be mediated by Notch/Jagged signaling pathway.3.Ad-VEGF165-EPCs and SCs were used as seed cells,and the modified acellular nerve scaffold materials prepared by our research group were used as carriers to successfully construct vascularized tissue engineering peripheral nerves,which grew well in vitro.4.The comprehensive results of pathology,gross observation,ultrasound and NEP showed that the pre-vascularization strategy based on EPCs and VEGF165 genes could promote the early vascularization of tissue engineered nerves and restore the blood supply in the nerve transplantation area.Conclusion:The pre-angiogenic effect of EPCs and VEGF165 genes can promote the tissue repair of early vascularization and nerve defects of tissue engineering peripheral nerves in vivo,accelerate the recovery of normal functions and morphological structures of peripheral nerves,and promote the repair and reconstruction of peripheral nerve defects.