International Journal of Oral and Maxillofacial Surgery
Research Paper
OsteobiologyThe use of tissue-engineered bone with human bone morphogenetic protein-4-modified bone-marrow stromal cells in repairing mandibular defects in rabbits
Osteobiology
Section snippets
Construction of hBMP-4 mammalian expression plasmid
The construction of hBMP-4 plasmid was completed by subcloning techniques as previously described10. Briefly, human BMP-4 cDNA was amplified from hBMP-4 λgt10phage clone (ATCC no.40342) using a polymerase chain reaction (PCR). The PCR product was then ligated into a pGEM T-easy vector (Promega, Madison, WI, USA) for DNA sequencing, and was then further subcloned into a mammalian expression vector, pEGFP (enhanced green fluorescence protein, Invitrogen, Carlsbad, CA, USA). The constructed
Results
The human BMP-4 gene was amplified from λgt10 phage (Fig. 1A) and ligated into a pGEM T-easy vector. After being confirmed by DNA sequencing (Genbank M22490), the amplified 1.3-kb hBMP-4 gene was then inserted into pEGFP to construct a pEGFP-hBMP-4 mammalian expression vector. This process was confirmed through double enzymatic digestion. The expected 1.2-kb band resulting from Sac I- and EcoR I-mediated enzymatic digestion was observed following gel electrophoresis (Fig. 1B).
Quantification of
Discussion
Within the realm of tissue engineering, gene therapy is a novel approach for oral and maxillofacial skeletal reconstruction. Studies evaluating the repair of mandibular bony defects using BMP proteins have shown that such an approach is effective in different animal models1, 24, 29. While the large quantity of exogenous protein required to yield such biological effects increases the risks of unwanted general side effects4, an ideal carrier to mitigate, if not negate, these effects remains
Acknowledgements
We acknowledge the help of numerous individuals: Dr. Jianguo Chen (Life Science Center at the Peking University, PR China) offered guidance in the molecular biology experiments; Dr. Guixiang Ma (Orthopaedic Research Institute at the Beijing Jishuitan Hospital, PR China) generously provided the NNB; Dr. Yilin Cao (Shanghai Institute of Tissue Engineering) was consulted for establishing the bMSC culture protocol and Dr. Hasan Uludag (Department of Chemical Engineering, University of Alberta,
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These authors contributed equally to this work.