Decalcified bone matrix and lentivirus-mediated silencing of p75 neurotrophin receptor transfected bone marrow mesenchymal stem cells to construct tissue-engineered bone / 中国组织工程研究

ABSTRACT

BACKGROUND: P75 neurotrophin receptor (P75NTR) has a bidirectional role in promoting and inhibiting bone formation in bone tissue. Overexpression of P75NTR can inhibit the osteogenic mineralization of bone marrow mesenchymal stem cells. P75NTR can inhibit the degradation of fibrin and the formation of new blood vessels around the fracture, leading to fracture nonunion. Its inhibition of bone tissue repair is related to its mediating apoptosis channels. OBJECTIVE: To study the effect of silencing P75NTR on the growth activity and alkaline phosphatase activity of bone marrow mesenchymal stem cells, and to study the ectopic osteogenesis ability implanted in demineralized bone matrix to construct tissue engineering bone complex. METHODS: Lentivirus-mediated silencing P75NTR was transfected into bone marrow mesenchymal stem cells. The expression of fluorescent protein and P75NTR protein was detected by fluorescence inverted phase contrast microscope and western blot assay. Two or three days after transfection, CCK-8 assay was used to detect cell activity. After osteogenic induction solution induced differentiation culture for 7 and 14 days, alkaline phosphatase activity was detected by enzyme labeling method. Lentivirus-mediated silencing P75NTR transfected bone marrow mesenchymal stem cells were cultured with compound demineralized bone matrix. Optical inverted phase contrast microscope and scanning electron microscope were used to observe the adhesion of demineralized bone matrix and bone marrow mesenchymal stem cells. After further osteogenic differentiation for 7 days, tissue engineered bone complex was subcutaneously implanted in the rat back for 4 weeks. The osteogenesis was observed by hematoxylin-eosin staining and alkaline phosphatase staining. RESULTS AND CONCLUSION: (1) Lentiviral-mediated silencing P75NTR transfection rate was approximately 70%. The expression of P75NTR target protein was significantly lower than that of untransfected group and negative virus control group (P < 0.05). (2) Compared with untransfected group and negative virus control group, the cell activity of silencing P75NTR transfected group increased significantly at 5 and 6 days (P < 0.05). Compared with the untransfected group and the negative virus control group, the alkaline phosphatase activity of silencing P75NTR transfected group increased significantly at 7 and 14 days after osteogenic induction (P < 0.05). (3) Silencing P75NTR transfected bone marrow mesenchymal stem cells and demineralized bone matrix formed a good adhesion; the tissue engineered bone complex significantly enhanced the ability of ectopic bone formation. (4) The results showed that bone marrow mesenchymal stem cells transfected with silencing P75NTR mediated by lentivirus could be implanted in decalcified bone matrix to form a good tissue engineered bone complex, which has a certain heterotopic osteogenesis effect and provides important theoretical basis for bone tissue engineering in the treatment of bone defects.