Construction And Evaluation Of Angiogenin Gene Activated PLGAm/CCS Dermal Scaffold

Objective:To develop a gene activated dermal scaffold by introducing plasmid DNA encoding angiogenin into a well structured dermal substitute via a non viral gene delivery system,and evaluate its effect on early stage angiogenesis of tissue engineered skin.Methods:Construct plasmid DNA encoding angiogenin(pAng)using vector pUCm-T which expressed both angiogenin(Ang)and enhanced green fluorescence protein(EGFP),as well as plasmid DNA encoding EGFP(pEGFP)as control,and evaluate their efficacy by gene sequencing and SDS-PAGE.In in-vitro study,pAng activated dermal scaffolds combining gene therapy and a dermal substitute were constructed.After generating nanoparticles using N,N,N-trimethyl chitosan chloride(TMC),a cationic gene vector,and pAng,we incorporated the complexes into a polylactic-co-glycolic acid knitted mesh reinforcedcollagen chitosan dermal scaffold(PLGAm/CCS)to construct a TMC/pAng-PLGAm/CCS.In vitro evaluations of the biocompatibility and transfection rates were conducted using in vitro static culture.Papain digestion and fluorescence spectrometer detection were used to determine the actual loading amounts of pAng in scaffolds.In vitro transfection of TMC/pAng-PLGAm/CCS was conducted using human embryonic kidney cells(HEK).The transfection rates were calculated after detection of EGFP expressed cells by flow cytometry.Then human umbilical vein endothelial cells(HUVEC)were implanted in the sterilized scaffolds for detection of proliferation and Ang expression using MTT and ELISA kit,respectively.PLGAm/CCS with TMC/pEGFP,naked pAng and blank scaffolds were used as controls.In in-vivo study,a series of TMC/pAng loaded scaffolds with 0,1,5,10,15 ?g pAng were constructed and implanted subcutaneously in rats to determine the rational range of loading amounts of pAng in a scaffold.Samples were collected at planned time points,and evaluations of tissue reaction and angiogenesis were made via pathological,immunohistochemical and biomolecular methods.Then,TMC/pAng-PLGAm/CCS dermal scaffolds,with a rational pAng gene loading amount and a polyurethane membrane as a layer of temporary epidermis,were transplanted in full-thickness wounds in rats,using PLGAm/CCS with TMC/pEGFP,naked pAng and blank scaffolds as controls.At planned time points,samples were collected and examined by histopathological,immunohistochemical,electronic microscopic and biomolecular methods to determine the wound reaction and angiogenesis.Key signal proteins in potential signaling pathway,such as Erk1/2 pathway,were examined by Real Time qPCR(RT-qPCR)and western blotting to further investigate the mechanisms of early stage angiogenesis mediated by pAng-PLGAm/CCS.Conclusions:A gene activated TMC/pAng-PLGAm/CCS dermal scaffold with good biocompatibility was successfully constructed.It efficiently promoted early stage angiogenesis both in vitro and in vivo,providing a promising dermal substitute for skin repair and regeneration.Erk1/2 pathway and ?-actinin system are potential and important pathways for inducing vascularization of the angiogenin gene activated dermal scaffold.