Kadomatsu H, Matsuyama T, Yoshimoto T, Negishi Y, Sekiya H, Yamamoto M, Izumi Y
Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
J Periodontal Res. 2008 Oct;43(5):483-9. doi: 10.1111/j.1600-0765.2007.01023.x. Epub 2008 Jul 4.
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.
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.
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.
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.
目前作为生物材料使用的胶原蛋白类型是从动物组织中纯化而来的。然而，一个日益严重的主要问题是其安全性，因为存在病毒和朊病毒污染以及未知的潜在人畜共患传染病风险。本研究旨在通过免疫组织化学评估重组人生长/分化因子-5（rhGDF-5）与重组人胶原蛋白I（rhCI）联合应用对小鼠颅骨骨形成的影响。
将复合rhGDF-5-rhCI或单独的rhCI皮下注射到小鼠颅骨中。3、7或14天后，对组织进行放射学、组织学和免疫组织化学检查。还评估了从新生小鼠颅骨中酶法分离的原代成骨细胞、骨膜细胞和结缔组织成纤维细胞产生血管内皮生长因子（VEGF）的情况。
在注射rhGDF-5/rhCI复合物的部位，颅骨上观察到一个突起。通过二维微型计算机断层扫描显示其矿物质密度与现有骨骼不同。II型胶原阳性染色仅限于新形成的组织。因此，新形成的组织似乎是骨样和软骨样组织。在新形成的组织中检测到许多对血管性血友病因子呈阳性染色的细胞的血管。rhGDF-5增强了培养的结缔组织成纤维细胞中VEGF的产生。在肥厚的骨膜中检测到Sry相关的HMG盒9（Sox9）阳性细胞，并渗透到新形成的组织中。
这些结果表明，rhCI似乎允许rhGDF-5释放，并且rhGDF-5-rhCI复合物通过Sox9表达和血管生成在小鼠颅骨中诱导软骨内成骨。
