牛骨由来骨形成因子添加骨代用材の異種移植に関する実験的研究

Abstract

Fresh autogenous bone graft is generally considered to be the most effective procedure for the repair of bone defects. However, there are some problems such as donor site morbidity and limited quantities. For these reasons, extensive research on bone substitutes has been done. Bone morphogenetic protein (BMP) induces differentiation of mesenchymal-type cells into cartilage and bone even in xenogeneic extraskeletal sites. However, an appropriate carrier is thought to be required when applied clinically because BMP alone does not induce large amounts of bone formation because of its rapid diffusion. This preliminary report presents xenogeneic osteogenesis in mice using bone substitutes which consisted of BMP and atelocollagen (AC) derived from bovine bones alone. The bovine BMP was purified by utilizing extraction from the demineralized bone matrix (DBM) with 4M guanidine/HCl, followed by differential precipitation, gel filtration and heparin affinity chromatography. The finally obtained heparin-bound fraction (H-B) consisted mainly of Mr 22,000 and 18,500 components. Bone AC and pepsinized bone matrix residue gelatin (BMG) were prepared from the guanidine/HCl-extracted DBM by pepsin digestion and differential precipitation. Intramuscular injection of the crude BMP with bone AC solution resulted in reproducible bone induction, and its increase was dose-dependent on the concentration of the AC solution between 0% to 0.5%, followed by a plateau up to 1.0%. However, intermuscular implantation of H-B and bone AC resulted in bone formation only around the periphery of the implant. Moreover, an implant of H-B and BMG induced only slight bone formation resulting from possibly rapid diffusion of BMP. In contrast, an implant of H-B and a carrier consisting of 20% AC and 80% BMG induced the largest deposits of bone formation, the degree of which was superior to allogeneic DBM. Injection of BMP and AC solution was the most reproducible bioassay system and implantation method. An osteoinductive bone substitute derived entirely from unlimitedly available bovine bones induced bone formation, even in xenogeneic species. The obtained results suggest a possibility of clinical applications of xenogeneic bone substitutes combined with BMP in osseous reconstruction and augmentation.