| Bone defect caused by diseases or accident is a common disease,the healing of bone defect is affected by various factors containing age,general condition,the location and area of defect,resulting in non-union,which seriously affect the life of patients.Autologous bone graft,allogeneic and xenograft bone graft could be used to repair bone defect,but they are limited by immune rejection or little volume.Tissue engineering bone provides a good choice for the complete repair of bone defect,and the classical basic tissue engineering bone is generally composed of biological scaffold,seed cells and biological factor.Seed cells are the core of tissue engineering bone owing to the ability of formation of various functional cells involved in the repair of defects.Because of the wide range of sources,easy access,strong ability to proliferate and multiply differentiation ability,ASCs are more and more concerned among seed cells.However,osteogenic differentiation of ASCs is weaker than that of BMSCs and tend to differentiate into adipocytes.In order to better use ASCs for bone defect,we explored the use of Sema3 A which indicates a significant expression reduction in ASCs and could promote osteoblast differentiation,inhibit osteoclast differentiation.In this study,we constructed the lentiviral vector of Sema3 A gene and verified the infection effect in ASCs by flow cytometry,q-PCR and Western blot.By osteogenic and adipogenic differentiation induction,quantification and q-PCR analysis of osteogenic and adipogenic related genes expression in differentiation process,the regulation effect of Sema3 A on ASCs differentiation was analyzed.The effect of Sema3 A on the proliferation and migration of ASCs was studied using cck-8 and tranwell research.In order to further clarify the roles of Sema3 A,we made a biocompatible porous PLGA scaffold and compounded with Sema3A-modified ASCs.The surface morphology of composite scaffold was observed under electron microscope.Then we made the critical bone defects in SD rats and implanted with the ASCs-PLGA scaffold complex.The rat skull was removed after a period of healing,Micro-CT and histological methods were used to reconstruct the bone defect area,quantify the bone parameters and evaluate the healing of bone defect.At last,we also explored the activation of Wnt/β-catenin signaling pathway which is closely related with Sema3 A and osteogenic,adipogenic differentiation.Conducting immunofluorescence,q-PCR and Top/Fop experiments,we are hoping to find the possible mechanism by which Sema3 A regulates the differentiation of ASCs.We identified the efficient infection of Sema3 A gene in ASCs,and obtained ASCs with enhanced osteogenic differentiation ability and inhibited adipogenic differentiation ability.The expression of osteogenesis-related genes of Sema3A-modified ASCs were significantly enhanced,and the expression of adipogenesis related genes were significantly inhibited,and Sema3 A promoted the migration of ASCs without affecting its proliferation.The PLGA scaffolds showed porous structure under the electron microscope,and ASCs attached and stretched well on the scaffolds surface.It was found that the bone defect receiving Sema3A-modified ASCs was significantly smaller than that of normal ASCs,the histological sections further confirmed it.Finally,multi-directional detection showed enhanced activation of Wnt/β-catenin signaling pathway in Sema3A-modified ASCs.The overexpression of Sema3 A,which is inhibited in normal ASCs,can significantly enhance its osteogenic differentiation and migration ability while inhibiting the tendency of adipogenic differentiation,but not affect its proliferation.Combined with PLGA scaffold,Sema3A-modified ASCs can repair bone defect well.The effect of Sema3 A on the differentiation of ASCs may be related to activation of the Wnt/β-catenin signaling pathway. |
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