DEVELOPMENT OF A NEW THERAPEUTC APPROCH TO BONE REGENERATION FOR GENE THERAPY

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We conducted the following experiments.1. Characterization of mesenchymal stem cells during bone regenerationWe first made a round hole, 2.2 mm in a diameter, at diaphysis offemurs of growing mice. To investigate the proliferation activity of mesenchymal cells during the bone repair, we explored incorporation of BrdU. These studies demonstrated that proliferating mesenchymal cells incorporating BrdU increased 3 days after injury in our experimental system.2. Changes in gene expression during bone regenerationUsing above described experimental model, we investigated the changes in expression patterns of bone-related genes by Northern blot analysis. ALP mRNA increased on day 4 and osteocalcin mRNA increased on day 5 after bone injury. Histochemical and immunohistochemical studies indicated similar expression patterns of these molecules.3. Above experiments suggested that mesenchymal stem cells appeared during days 4 and 5 after injury in our experimental model. We are now exploring the genes related to mesenchymal stem cells by CDNA chips.4. To develop new therapeutic method, we transplanted skin fibroblasts in bone defects (2.2 mm in a diameter) in calvariae of growing mice with carrier (PGS). Transplantation of the fibroblasts over-expressing BMP-2 CDNA induced more accelerated bone regeneration than that transplanted with the fibroblasts expression empty vector. In contrast, the fibroblasts over-expressing Cbfa l gene failed to stimulate bone regeneration, compared with that transplanted with the fibroblasts expression empty vector.