Bone marrow stromal cells transferred with lentiviral-mediated bone morphogenetic protein-7 gene cultured in vitro in nano-hydroxyapatite/collagen scaffold

Abstract

Objective To observe osteogenous differentiation of modified bone marrow stromal cells (BMSCs)which can express bone morphogenetic protein-7(BMP-7)stably and are cultured in vitro in the scaffold of nanohvdroxyapatite/collagen(nHAC)material. Methods BMP-7 and eGFP genes were transferred through lentiviruses into the BMSCs of SD rats.The modified BMSCs were selected out by G418 and combined with the nHAC scaffold to culture in vitro.The transfection efficiency and proliferation of BMSCs were evaluated bv fluorescence microscopy and MTT method after transfection with LVBMP-7+eGFP gene.The expression of BMP-7 was tested by RTPCR and ELISA.ALP testing and staining of Gomori were used to test osteogenous differentiation.The adhesion and growth of BMSCs on the nHAC scaffold were observed by scanning electronic microscopy(SEM).Results The transfection efficiency was about 90%in the first 24 hours.There was no significant difference in proliferation between the infected and non-infected BMSCs(P>0.05).RTPCR suggested that BMP-7 gene was located at 1 300 bp in the transfeetion group hut not in the other grouDs.ELISA revealed that the BMP-7 expression was(150.2±18.3)pg/mL The strongest AIP activity was observed nearlv on 16th dav.The adhesion and growth were fine on the nHAC scaffold.ConclusionsBMP-7 gene can be stably expressed in BMSCs,and Can enhance the capacity of osteogenous differentiation.The BMSCs modified by BMP-7 can be used in combination with nHAC to construct a tissue engineered bone. Key words: Bone marrow cells; Bone morphogenetic proteins; Transfection; Tissue engineering