Experimental Studies On BMSCs Modified With HBMP-2 Gene Combined With NHAC/PLA Scaffold To Construct Tissue Engineering Bone In Vitro

Objective To construct tissue engineering bone via bone marrow stromal cells (BMSCs) which were transferred by lentiviral vector with bone morphogenetic protein- 2(BMP-2) gene were planted into the scaffold of nano-hydroxyapatite/collagen /PLA(nHAC/PLA )material , then to observe the capacity of BMSCs bone formation in vitro.Methods First, BMSCs were isolated from SD rat bone marrow cells, cultured, purified, expanded and passaged in vitro. Second, BMSCs were divided into transfection group (A), empty vector group (B) and control group(C). A: BMSCs transfected with a lentiviral vector containing both the BMP-2 gene and the enhanced green fluorescent protein (eGFP) gene (MOI = 25pfu); B:BMSCs transfected with a lentiviral vector,but not containing the BMP-2 gene;C:BMSC alone. Third, the BMSCs proliferation and transfection efficiency were confirmed by fluorescence microscope; the proliferation and cell cycle analysis of gene-modified BMSCs were also assessed by MTT method and FCM after transfected with LV-BMP-2 and LV-eGFP gene. The methods of RT-PCR,immunohistoehemical staining and enzyme-linked immunosorbent assay (ELISA) were used to test the expression of BMP-2 protein, BMSCs were induced to differentiate into osteoblast phenotype and the new born cells demonstrated positive staining for alizarin red and alkaline phosphatase (ALP). Scanning electronic microscoping (SEM) was used to observer the adhesion, growth and cell-matrix interaction.Results①Mononuclear cells collected by density gradient centrifugation were cultured and passaged and morphological obsevation suggested these cells were rat BMSCs,and BMSCs can grow quickly when cultured in vitro in low abundance.②No significant changes in morphology of infected BMSCs were observed .There was no significant difference in proliferation and cell cycle between infected and non-infected BMSCs.③The expression of BMP-2 was confirmed by RT-PCR,immunohistoehemical staining analysis and ELISA test after the gene transfection in different time, the expression had significant difference in the transfection group compared with the empty vector group and control group (P < 0. 05).④After the gene transfection the BMSCs gained the structural and morphological characters which were similar to those of osteoblasts. The ALP activity in transfected BMSCs were increased significantly compared with the empty vector group and control group (P<0.05).⑤BMSCs modified by BMP-2 gene adhered to the surface and hole of the nHAC/PLA scaffold and grew well by SEM observation.The expression of BMP-2 was confirmed by Western blot analysis.Conclusions①LV-BMP-2 could infect BMSCs with high efficiency, and BMP-2 could be expressed stably in high level in BMSCs.②After transfection with BMP-2 gene, BMSCs differentiate efficiency towards osteoblast and showed typical morphological characteristics of osteoblast and strong biological activity.③BMSCs modified with BMP-2 gene grow well on nHAC/PLA scaffold, and may still express BMP-2 highly; Tissue engineering bone used to regional gene therapy was constructed successfully;④nHAC/PLA should be a good scaffold for tissue engineering bone.