Study of bone-forming factors and their clinical evaluation

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1.Serum-free bone-forming organ culture system using twenty-day fetal rat parietal bones was established. Bones were cultured on grids and placed on a rocking platform under the aerobic condition. This condition increased the bone-forming activities of cultured rat parietal bones. The calcium content, dry weight of bone, and lactate produced by bone tissue in the medium were measured to study the precise bone-forming process of vital bones.2.Using this improved bone-forming organ culture system, the effects of insulin on bone formation were studied. Insulin increased calcium content and dry weight of bone. These effects were maximum in bones treated with insulin at 10^<-6>M.Histomorphometrical analysis showed that the areas of mineralized bone and bone matrix increased, and that many osteoblasts and few osteoclasts appeared in bones treated with insulin at 10^<-6>Mor higher.Insulin inhibited the release of ^<45>Ca into the medium. From these results, it was suggested that insulin has a stimulatory effect on bone formation enhancing both bone mineralization and bone matrix formation.3.Cloning of bone morphogenetic protein (BMP) cDNA by PCR method was performed. Ectopic bone formation induced by BMP,was investigated, and also the molecular cloning of BMP type I receptor was done.4.In situ hybridization of the rat tibial bone marrow cvity, in which bone formation and resorption were indluced by colchicine, demonstrated that osteopontin mRNA was expressed during bone resorption process.5.Clinicostatistical and long-term follow-up studies of the pediatric fracture cases were performed.