Background Tissue-engineering techniques combined with gene therapy have beenrecently reported to improve osteogenesis. In this study, tissue-engineered bone constructed byhuman Bone Morphogenetic Protein 4 (hBMP-4) g...Background Tissue-engineering techniques combined with gene therapy have beenrecently reported to improve osteogenesis. In this study, tissue-engineered bone constructed byhuman Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) wasexplored in an ectopic bone formation model in rabbits. Methods A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtainedfrom rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected invitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the targetgene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted toexplore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined withNatural Non-organic Bone ( NNB) , a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x3 mm, at a concentration of 5 x 10~7 cells/ml. They were subsequently implanted into 6 rabbitssubcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery,the implants were evaluated with histological staining and computerized analysis of new boneformation. Results pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, genetransfer efficiency reached more than 30% , Target gene transfer could strengthen the transcriptionof BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In theectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in thebMSCs group was (17.2 ± 7.1)%, and pEGFP group was (14.7 ± 6.1) % , while pEGFP-hBMP-4 group was(29.5 ± 8.2) % , which was the highest among the groups (F = 7.295, P < 0. 01). Conclusions Themammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transferefficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoteddifferentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection ofbMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bonetissue engineering applications.展开更多
文摘Background Tissue-engineering techniques combined with gene therapy have beenrecently reported to improve osteogenesis. In this study, tissue-engineered bone constructed byhuman Bone Morphogenetic Protein 4 (hBMP-4) gene-modified bone marrow stromal cells (bMSCs) wasexplored in an ectopic bone formation model in rabbits. Methods A pEGFP-hBMP-4 mammalian plasmid (EGFP: Enhanced Green Fluorescent Protein) was constructed by subcloning techniques. bMSCs obtainedfrom rabbits were cultured and transfected with either pEGFP-hBMP-4, pEGFP or left uninfected invitro. Transfer efficiency was detected through the expression of EGFP. Transcription of the targetgene was detected by RT-PCR. Alkaline phosphatase (ALP) and Von Kossa tests were also conducted toexplore the phenotypes of osteoblasts. The autologous bMSCs of the 3 groups were then combined withNatural Non-organic Bone ( NNB) , a porous hydroxyapatite implant with a dimension of 6 mm x 6 mm x3 mm, at a concentration of 5 x 10~7 cells/ml. They were subsequently implanted into 6 rabbitssubcutaneously using NNB alone as a blank control (6 implants per group). Four weeks after surgery,the implants were evaluated with histological staining and computerized analysis of new boneformation. Results pEGFP-hBMP-4 expression plasmid was constructed. Under optimal conditions, genetransfer efficiency reached more than 30% , Target gene transfer could strengthen the transcriptionof BMP-4, and increase the expression of ALP as well as the number of calcium nodules. In theectopic animal model, NNB alone could not induce new bone formation. The new bone area formed in thebMSCs group was (17.2 ± 7.1)%, and pEGFP group was (14.7 ± 6.1) % , while pEGFP-hBMP-4 group was(29.5 ± 8.2) % , which was the highest among the groups (F = 7.295, P < 0. 01). Conclusions Themammalian hBMP-4 expression plasmid was successfully constructed and a comparatively high transferefficiency was achieved. The gene transfer technique enhanced the expression of BMP-4 and promoteddifferentiation from bMSCs to osteoblasts. These in vivo results suggested that transfection ofbMSCs with hBMP-4 might be a suitable method to enhance their inherent osteogenic capacity for bonetissue engineering applications.
