| Background:Periodontal disease is one of the most common human oral diseases.Periodontitis is a chronic infectious disease caused by periodontal pathogens,and attachment loss and alveolar bone resorption is the main clinical manifestations.Ultimate goal of periodontal treatment is to repair alveolar bone and restore the periodontal physiological structure and function.Though the current periodontal treatment can effectively control periodontal inflammation,and periodontal surgery or other therapies can not effectively repair the periodontal tissue structure and function.So,an effective method is needed to realize the regeneration of the periodontal tissue.The key factors of periodontal tissue engeneering are stem cells,scaffold material and growth factor.The tissue engeering technology is a new method for periodontal regeneration.Dental follicle stem cells derived from the dental follicle tissues around tooth germ,and could differentiate into alveolar bone,periodontal ligament and cementum.The dental follicle stem cells have self-renewal and differentiation ability.in a specific environment the cells could differentiate into osteoblasts,adipocytes,neurons.Bone morphogenetic protein 9(Bone morphogenetic 9 protein,BMP9),as one family member of BMP(Bone morphogenetic proteins,BMPs)has the strongest osteogenic ability.BMP9 as a growth factor was used in periodontal tissue engineering,and it has great research potential and significance.CHA(coralline hydroxyapatite CHA)was from marine coral,with natural porous structure and good biocompatibility.Stem cells could grow and proliferate on the surface of the material.Therefore,in this study,the compound of BMP9,rDFCs,CHA were used to treat the rat periodontal bone defect effect.Objective:1.rDFCs were cultured in vitro by enzyme digestion and gradient centrifugation.The adenovirus carrying BMP9 gene was stably and efficiently transfected into rDFCs.2.to investigate the biocompatibility of CHA and explore the effect of CHA on osteogenic differentiation of rDFCs.3.to study the therapeutic effect of BMP9,rDFCs and CHA as the three elements of tissue engineering on alveolar bone defect in rats.4.To study the role of Smad1/5/8 signaling pathway in osteogenesis of BMP9-induced rDFCs.Methods:1.enzyme digestion method was used to obtain the primary rDFCs and the purified rDFCs was obtained by gradient centrifugation method.RDFCs.2.Electron microscope was used to observe the surface morphology of CHA and the adhesion and growth of rDFCs on the surface of CHA.The effect of CHA on the expression of osteogenic factor and osteogenic differentiation of rDFCs was explored by RT-q PCR,and the biocompatibility of CHA and rDFCs was explored.3.The compound of BMP9,CHA and rDFCs was implanted to the acute periodontal bone defect.After 6 weeks,the samples were observed by Micro CT and staining(rDFCs).4.The role of Smad1/5/8 signaling pathway in osteogenesis of BMP9-induced rDFCs was detected by Western Blot,RT-q PCR,staining.Results:1.Primary dental follicle cells had different shapes,like long spindle shaped and polygonal,and the third passage rDFCs were main spindle shape,1-3 nuclei and high density particle could be found in cells.After transfection of Ad-BMP9 into rDFCs,the stable expression of green fluorescence was observed under microscope.2.The structure of CHA was observed by scanning electron microscope.CHA showed a porous structure,and the pores were interconnected.The pore size was 100~600 ?m.rDFCs can stably grow,proliferate and secrete extracellular matrix on the surface of CHA.The results of PR-q PCR showed that CHA could promote the expression of OPN,ALP,Osterix and promote the osteogenic differentiation of rDFCs.3.Micro CT data analysis showed that BMP9 transfected rDFCs and CHA could achieve more bone tissue regeneration compared with the pure CHA implantation and rDFCs/CHA implantation group.4.BMP9 could promote the osteogenic differentiation of rDFCs by activating the Smad 1/5/8 signaling pathway.Conclusion:Dental follicle stem could be obtained by enzyme-tissue digestion,cells showed long fusiform cytoplasm.1-3 nuclei and high density could be found in cells.CHA has good biocompatibility and rDFCs could stably and firm Ly adhere to the surface of CHA,proliferate and secrete extracellular matrix.CHA can promote the osteogenic differentiation of rDFCs and promote the expression of osteogenic factors.BMP9,rDFCs and CHA,as the three elements of tissue engineering,could promote the formation of periodontal alveolar bone.This study provides a theoretical basis for the application of BMP9 and rDFCs in periodontal tissue regeneration,and provides support for the application of tissue engineering technology in periodontal tissue regeneration. |
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