The Study Of Gene Enhanced Tissue-engineered Bone For Promoting Maxillofacial Bone Regeneration

Objective: Gene enhanced tissue engineered bone combines the superiority of both gene therapy and tissue engineering technique in correcting bone defect.However,the selection of gene delivery pathway,and the upcoming decisions over proper vector design,stem cell optimization,target gene selection remain the key point in the advancement of biosafety and efficiency of gene enhanced tissue engineered bone.This study dividing into three parts,will focus on the above problems,and try to systematically evaluate the regenerative capability of gene enhanced tissue engineered bone in the treatment of oral and maxillofacial bone defects on different animal models,which may offer certain extent of experiences and suggestions for its future application in clinic.Material and Methods: 1.Isolates the rabbit adipose derived stem cells(ASCs),using in vitro gene delivery pathway,transfects the ASCs with bone morphogenetic protein 2(BMP-2)by adenovirus carrier(Ad),and evaluates the osteogenic differentiation ability of the target cells via western blot and osteopontin(OPN)immunofluorescence staining.Loads the modified ASCs onto tricalcium phosphate beta(β-TCP)scaffolds to construct the gene enhanced tissue engineered bone.Analyze the early osseointegration capability of this complex by means of sequential fluorescence labeling and hard tissue histological analysis on an immediate implant model in rabbit.2.Isolates the canine ASCs,using in vitro gene delivery pathway,transfects the ASCs with BMP-2 gene by adenovirus carrier,and evaluates the osteogenic differentiation ability of the target cells via Real-time PCR,alkaline phosphatase(ALP)and Von Kossa staining.Loads the modified ASCs onto β-TCP scaffolds to construct the gene enhanced tissue engineered bone.Analyze the re-osseointegration capability of this complex by means of clinical methods,like probing depth(PD),clinical attachment level(CAL),and X-ray,as well as sequential fluorescent labeling and hard tissue histological analysis on a ligature induced peri-implantitis model in canine.3.Transfer the target gene BMP-2 in vitro into bone marrow stem cells(BMSCs)by non-virus carrier PEI-LA.Detect the osteogenic differentiation ability of modified BMSCs via Real-time PCR and alizarin red staining.Load the BMP-2 overexpressing ASCs to gelatin scaffold to construct the gene enhanced tissue engineered bone;prepare the plasmid-carrier complex(BMP-2/PEI-LA)and directly load it onto the gelatin to construct the gene enhanced tissue engineering bone via in vivo gene delivery pathway.Implant the two types of gene enhanced tissue engineered bones in rat cranial critical-sized bone defect.Exam their capabilities in realizing the rapid regeneration of maxillofacial bone defect using micro-CT scanning,hard tissue histological analysis,and immunohistochemical staining.Results: 1.Isolated the rabbit ASCs as the target cell,adenovirus vector successfully transferred the BMP-2 gene to ASCs in vitro,so that BMP-2 overexpressing ASCs showed lifted osteogenic differentiation capacity.The prepared gene enhanced tissue engineered bone of modified ASCs and β-TCP scaffolds significantly promoted the early formation of osseointegration on an immediate implant model in rabbit model.2.Isolated the canine ASCs as the target cells,adenovirus vector successfully transferred the BMP-2 gene to ASCs in vitro,so that BMP-2 overexpressing ASCs showed enhanced osteogenic differentiation capacity.The prepared gene enhanced tissue engineered bone of modified ASCs and β-TCP scaffolds significantly promoted the formation of re-osseointegration in a ligature induced peri-implantitis model in canine.3.Isolated the rat BMSCs as the seed / target cells,which presented enhanced osteogenic differentiation ability after BMP-2 gene delivery by non-viral vector PEI-LA in vitro;on the other hand,successfully prepare the plasmid-carrier complex BMP2/PEI-LA.Modified BMSCs and complex were loaded onto gelatin scaffold to construct two type of gene enhanced tissue engineered bone,which demonstrated increased capability in promoting bone repair in a critical sized defect model on rat cranium suggesting that gene delivery via in vitro or in vivo pathway could both be effective for bone regeneration.Conclusions: Virus vector could successfully deliver BMP-2 gene in in vitro pathway,and realize the formation of early osseointegration in an immediate implant model and re-osseointegration in a peri-implantitis model;non-viral vector PEI-LA could effectively deliver BMP-2 gene by via both vivo / in vitro methods,and achieve rapid reconstruction of maxillofacial bone defect.Non-viral vector’s effectiveness in in vivo delivery of target gene suggests a new direction and strategy for the clinical translation of gene enhanced tissue engineered bone in the treatment of maxillofacial bone defect.