BMP-2 Gene Modified Canine bMSCs Promote Ectopic Bone Formation Mediated by a Nonviral PEI Derivative

摘要

The study was to explore the effects of BMP-2 gene modified canine bone marrow stromal cells (bMSCs) mediated by a nonviral PEI derivative (GenEscortTM II) in promoting bone formation in vitro and in vivo. Canine bMSCs were cultured and transfected with plasmids containing bone morphogenetic protein-2 gene (pBMP-2) or enhanced green fluorescent protein gene (pEGFP). Gene transfection conditions were initially optimized by varying GenEscort? II/plasmid ratios. Osteogenic differentiation of gene modified bMSCs was investigated via alkaline phos-phatase (ALP) activity analysis and real-time quantitative PCR (RT-qPCR) analysis in vitro. The bone formation ability of pBMP-2 transfected bMSCs combined with apatite-coated silk scaffolds (mSS) was explored and compared with pEGFP transfected bMSCs/mSS or untreated bMSCs/ mSS at 8, 12 weeks after operation. Results showed that gene transfection efficiency reached up to 36.67 ± 4.12% as demonstrated by EGFP expression. ALP staining and activity assay were stronger with pBMP-2 gene transfection, and the mRNA expression of BMP-2, bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx-2), and osteopontin (OPN) up-regulated in bMSCs 3, 6, 9 days in pBMP-2 group. Besides, the tissueengineered bone complex with pBMP-2 modified bMSCs achieved significantly increased de novo bone formation compared with control groups (p<0.01). We conclude that pBMP-2 transfection mediated by Gen Escort? II could enhance the osteogenic differentiation of canine bMSCs and promote the ectopic new bone formation in nude mice. GenEscort? II mediated pBMP-2 gene transfer appears to be a safe and effective nonviral method for gene enhanced bone tissue engineering.