Research Paper
Osteobiology
The use of tissue-engineered bone with human bone morphogenetic protein-4-modified bone-marrow stromal cells in repairing mandibular defects in rabbits

https://doi.org/10.1016/j.ijom.2006.07.005Get rights and content

Abstract

In this study, the capacity of hBMP-4 gene therapy combined with tissue-engineering techniques to improve the repair of mandibular osseous defects in rabbits was explored. A mammalian plasmid vector expressing enhanced green fluorescent protein–human bone morphogenetic protein-4 (pEGFP-hBMP-4) was initially constructed through subcloning techniques. Bone-marrow stromal cells (bMSCs) from New Zealand White rabbits were cultured and either transfected with pEGFP-hBMP-4 or pEGFP, or left untransfected in vitro. Once the transfer efficiency was determined through the expression of EGFP, cells from the three groups were combined with natural non-organic bone (NNB) at a concentration of 50 × 106 cells/ml and placed in 15 mm × 6 mm bilateral, full-thickness, mandibular defects surgically made in 12 rabbits. Together with NNB control, there were six samples per group. Four weeks after surgery, the implants were harvested and evaluated histomorphologically. Under optimal experimental conditions, gene transfer efficiency reached a maximum of 38.2 ± 9.4%. While the percentage of new bone area in the NNB control group was 8.8 ± 3.1%, in the untransfected bMSC group 22.5 ± 8.2%, and in the pEGFP group 18.1 ± 9.0%, a significantly higher amount of 32.5 ± 6.1% was observed in the pEGFP-hBMP-4 group. These results suggest that transfection of bMSCs with hBMP-4 enhances their inherent osteogenic capacity for maxillofacial bone tissue-engineering applications.

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.

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