RESUMEN
BACKGROUND AND OBJECTIVE: The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor-5 (rhGDF-5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae. MATERIAL AND METHODS: Composite rhGDF-5-rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed. RESULTS: A protrusion was observed on the calvariae at the site injected with rhGDF-5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two-dimensional microcomputed tomography. Type II collagen-positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone- and cartilage-like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF-5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry-related HMG box 9 (Sox9)-positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues. CONCLUSIONS: These results suggest that rhCI seems to allow the release of rhGDF-5 and that rhGDF-5-rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.