Preparation Of PEI/pOPG-PLGA Core-shell Gene Activated Matrix And Related Research

Periodontal disease is a common and multiple oral chronic disease.In the complete removal of the infected,the use of membrane-guided tissue regeneration technology is currently used to treat periodontitis the most commonly.But the reconstruction of bone tissue is a complex process,how to make the missing bone tissue regenerate and recover is still a very challenging problem in the world.The traditional guidance of bone tissue regeneration technology can only play a physical barrier role in the induction of periodontal tissue repair and regeneration.The gene activated matrix（GAM）,as a new tissue engineering method,combines the biological scaffold material with the plasmid vector to form a gene local release system.Compared with the direct use of exogenous growth factor,GAM stent gene expression of the protein produced by the relatively low cost and long-term effects,so its related preparation and research more and more attention to the hot.Reconstruction of bone repair is a complex process,is the formation and absorption of bone at the same time the existence of the dynamic process.Osteoprotegerin（OPG）plays a central role in the development and differentiation of osteoclasts.OPG can inhibit osteoblast differentiation and activity to inhibit the absorption process of bone tissue,so as to promote the role of bone repair and reconstruction,OPG can also be induced by alkaline phosphatase and calcification nodules to promote the formation of bone.The biological scaffold prepared by electrospinning has a natural morphology similar to that of the extracellular matrix.It is beneficial for cell migration and tissue growth,intake of nutrients and metabolic wastes,with ultrafine diameter and large specific surface area.Coaxial electrospinning technology can prepare core –shell fiber,to avoid the shell of organic matter and the core of the bio-active substances in direct contact with the preservation of biological activity.Coaxial electrospinning is therefore widely used for the preparation of bioactive scaffolds.Purposes: The core-shell GAM of PLGA（poly（lactic-co-glycolic acid））and OPG（osteoprotegerin）plasmid microspheres were prepared by coaxial electrospinning technology.The microstructure and mechanical properties of the fibers were measured.Biocompatibility,transfection efficiency,OPG Expression and osteogenesis were detected by cell experiments and animal experiments.Lay the foundation for the next large animal experiment,the construction of tissue engineering stent,the treatment of per iodontal disease and other bone defects.Methods: 1.Double digestion and preparation of the vector,to capture and amplify the purpose of fragments,to construct p Ds Red2-N1-h OPG recombinant plasmid,identified by PCR and gene sequencing.Finally,a large number of amplified recombinant plasmid were extracted.2.The PEI（polyethylenimine）/p OPG-PLGA core-shell type GAM was prepared by coaxial electrospinning.The microstructure,internal structure and mechanical properties of the core-shell fiber were measured.3.The periodontal ligament stem cells cultured with PEI/p OPG-PLGA core-shell GAM.MTT assay for biocompatibility of GAM.Laser confocal microscopy and flow cytometry were used to detect transfection.The expression of OPG was detected by RT-PCR andWestern Blotting.The osteoblast differentiation inhibition assay was used to detect the activity of OPG.4.Construction of skull limit defect model in SD rats.Implanted PEI/p OPG-PLGA core-shell GAM.Continue to keep eight weeks and then executed.The osteogenesis of GAM was investigated by general observation,Micro-CT analysis and histological analysis.Result: 1.A vector of about 4.7 kb was obtained by double digestion with Xho I and Kpn I.PCR capture to obtain 1245 bp of the target gene fragment.The vector was ligated with the gene fragment of interest.PCR amplification got a 1479 bp fragment.Genetically sequenced and compared with the Gene Bank in OPG m RNA sequence（NM₀02546）,exact match.The recombinant plasmid p Ds Red2-N1-h OPG was constructed successfully.2.This experiment prepared a core-shell p OPG/PEI-PLGA GAM by coaxial electrostatic spinning as tissue engineering membrane.The p OPG was encapsulated in the PEI phase as a core and PLGA was employed to control p OPG release as a shell.We have detected the GAM has suitable surface characteristic,mechanical property and a continuous release behavior.3.PEI/p OPG-PLGA core-shell GAM was cultured with periodontal ligament stem cells.The membrane has a high biosafety performance by MTT test,and transfection efficiency up to 90%.RT-PCR and WB detection showed that OPG plasmid could be introduced into the target cells after being released by PEI/p OPG-PLGA core-shell GAM and could be expressed in cells.After the osteoclast inhibition assay of RAW264.7,we found that the OPG protein expressed by the PEI / p OPG-PLGA core-shell GAM could significantly inhibit the differentiation of osteoclast-like cells.4.The PEI / p OPG-PLGA core-shell GAM was implanted into SD rat skull limit defect model.Feed for 8 weeks and then sacrifice the material.After micro-CT and histological analysis,we found that coaxial GAM can greatly improve the bone repair speed of rat skull defect.Conclusion:In this study,a nanometer core-shell GAM was successfully constructed by coaxial electrospinning.The OPG plasmid was encapsulated with PEI as the core and PLGA was the shell.The GAM has a suitable morphology,mechanical properties and stable sustained release properties.GAM showed high biosecurity and transfection efficiency,and can continuously express protein with biological activity in the target cell.GAM in SD rats can significantly increase the speed of reconstruction of bone defects.These provide the theoretical basis and basis for the next step in the use of this technique for large animals,clinical trials,and ultimately for the treatment of periodontal disease.