Department of Trauma Surgery, Johann-Wolfgang-Goethe University, Frankfurt/Main, Germany.
Cell Transplant. 2012;21(8):1667-77. doi: 10.3727/096368912X638937. Epub 2012 Apr 10.
Early vascularization of a composite in a critical bone defect is a prerequisite for ingrowth of osteogenic reparative cells to regenerate bone, since lack of vessels does not ensure a sufficient nutritional support of the bone graft. The innovation of this study was to investigate the direct and indirect effects of endothelial progenitor cells (EPCs) and cotransplanted mesenchymal stem cells (MSCs) on the in vivo neovascularization activity in a critical size defect at the early phase of endochondral ossification. Cultivated human EPCs and MSCs were loaded onto β-TCP in vitro. A critical-sized bone defect (5 mm) was created surgically in the femoral diaphysis of adult athymic rat and stabilized with an external fixateur. The bone defects were filled with β-TCP, MSCs seeded on β-TCP, EPCs seeded on β-TCP, and coculture of MSCs and EPCs seeded on β-TCP or autologous bone of rat. After 1 week, the rats were sacrificed. Using quantitative CD34 immunohistochemistry as well as qualitative analysis of vascularization (staining of MHC and VEGF) in decalcified serial sections were performed by means of an image analysis system. Fluorescence microscopy analyzed the direct effects and indirect effects of human implanted EPCs for vessel formation at bone regeneration site. Formation of a primitive vascular plexus was also detectable in the β-TCP, MSC, or autologous bone group, but on a significantly higher level if EPCs alone or combined with MSCs were transplanted. Moreover, highest amount of vascularization were detected when EPCs and MSCs together were implanted. Early vascularization is improved by transplanted EPCs, which formed new vessels directly. Indeed the indirect effect of EPCs to vascularization is much higher. Transplanted EPC release chemotactic factors (VEGF) to recruit EPCs of the host and stimulate vascularization in the bone defect. Transplantation of human EPCs displays a promising approach to improve early vascularization of a scaffold in a critical bone defect. Moreover, coculture of EPCs and MSCs demonstrate also a synergistic effect on new vessel formation and seems to be a potential osteogenic construct for in vivo application.
在临界骨缺损中，复合组织的早期血管化是成骨修复细胞向内生长以再生骨骼的前提，因为缺乏血管不能确保骨移植物得到足够的营养支持。本研究的创新之处在于研究内皮祖细胞（EPCs）和共移植间充质干细胞（MSCs）对体外成软骨骨化早期临界尺寸缺陷中体内新生血管化活性的直接和间接影响。体外培养人 EPCs 和 MSCs 加载到β-TCP 上。在成年无胸腺大鼠股骨干上通过手术创建临界尺寸的骨缺损（5mm），并用外固定器稳定。β-TCP 填充骨缺损，MSCs 接种于β-TCP 上，EPCs 接种于β-TCP 上，MSCs 和 EPCs 共培养物接种于β-TCP 或大鼠自体骨上。1 周后处死大鼠。通过定量 CD34 免疫组化以及对脱钙连续切片进行血管化的定性分析（MHC 和 VEGF 染色），使用图像分析系统进行分析。荧光显微镜分析了人植入 EPCs 在骨再生部位形成血管的直接和间接作用。在β-TCP、MSC 或自体骨组中也可检测到原始血管丛的形成，但如果单独移植 EPCs 或与 MSCs 联合移植，则水平明显更高。此外，当共移植 EPCs 和 MSCs 时，检测到最高量的血管化。移植的 EPCs 可通过直接形成新血管来改善早期血管化。事实上，EPCs 对血管化的间接作用要高得多。移植的 EPC 释放趋化因子（VEGF）募集宿主的 EPCs 并刺激骨缺损中的血管化。移植人 EPCs 显示出改善临界骨缺损中支架早期血管化的有前途的方法。此外，EPCs 和 MSCs 的共培养也显示出对新血管形成的协同作用，并且似乎是用于体内应用的潜在成骨构建体。
