构建新型共轴式大体积血管化组织工程骨的实验研究

The major progresses were done in the field of bone regenerative medicine during the years, and tissue-engineered bone grafts (TEBG) had offered a promising alternative approach to treat large bone defects. However,the creation of large TEGB with full viability and functional activity still presents a major challenge. An important prerequisite for the large TEBG is vascularization of the centre of large scaffold, so as to allow cells in the deep space of the scaffold to survive. In our previous studies, we found that the induction of vascularization by an axial vascular bundle can create a stable and perfect vascular network in the center of TEGB. However, vascularization of the implant proceeded slowly within the biomaterial and a few vessels reached the periphery of the scaffold. Interestingly, the newborn vascular network was likely to progress quickly towards the in vivo environment through the side groove which connecting the central tunnel of the scaffold. In addition, despite the successful outcome of microsurgery approaches by implanting an axial vascular bundle one can not disregard its drawbacks such as the inconvenience of two surgical interventions and donor-site morbidity. Therefore,we try to develop a novel protocol for generating large prevascularized TEBG by fabricating a co-axial scaffold to overcome the limitations described above. First, the co-culture system of endothelial colony-forming cells (ECFCs) and bone marrow mesenchymal stem cells (BMSCs) in a 3D hydrogel was used, and the vascularization process of this system was investigated in vitro and in vivo. Second, a novel scaffold with a hollow tubular structure is designed and obtained. The outer wall of this cylinder scaffold is made by highly porous β-TCP and has several “windows” connecting the central tunnel, which is filled by 3D hydrogel contained ECFCs and BMSCs. The hydrogel could directly intertconnect with the in vivo environment through the “windows”, and the newborn vascular network in the hydrogel could quickly anastomose with the host vessels. In order to find out the best design for the novel co-axial scaffolds, the influence of different kinds of “windows” in scaffold on the process of vascularization was also evaluated. Furthermore, we try to demonstrate that the newborn vascular network inside and outside of the scaffold may act as “sandwich” which can facilitate the vascularization in the outer wall zone. Third, the osteogenesis and angiogenesis of this novel co-axial TEGB is evaluated for bone repair in vivo in a femoral critical-sized segmental defect in rabbit. We hypothesize this co-axial TEBG by the novel prefabricated protocol will be able to provide adequate and timely vascularization in centre of the scaffold, leading to the satisfied healing results.

骨组织工程技术为骨缺损的修复提供了新思路，但大体积组织工程骨的构建仍充满挑战，究其原因是无法实现其轴心区充分血管化。我们前期研究发现，轴向血管束植入可改善组织工程骨内的血供，但其再血管化速度缓慢，操作复杂，供区选择严格受限，影响了该技术的推广应用。为了改进上述方法，实现组织工程骨轴心区早期、及时血管化，本研究拟建立一种新型共轴式组织工程骨构建方案。首先，以晚期血管内皮祖细胞与骨髓间充质干细胞为血管化种子细胞，建立基于温敏水凝胶的三维共培养体系，明确其在体外、体内环境中快速构建微血管网的能力；其次，设计中空圆柱状共轴式支架，其外壁具有特定的开窗结构，保证轴心区内填充的水凝胶三维共培养体系可与体内环境直接接触，早期促进宿主血管长入和轴心区的微血管网形成，及时实现支架内外血管网的吻合，从而形成微血管“夹心效应”，共同保障支架外周区的血供。最后，借助兔大体积股骨骨缺损修复模型，验证该方案的可行性。

骨组织工程技术为骨缺损的修复提供了新思路，但大体积组织工程骨的构建仍充满挑战，究其原因是无法实现其轴心区充分血管化。我们前期研究发现，轴向血管束植入可改善组织工程骨内的血供，但其再血管化速度缓慢，操作复杂，供区选择严格受限，影响了该技术的推广应用。为了改进上述方法，实现组织工程骨轴心区早期、及时血管化，本研究拟建立一种新型共轴式组织工程骨构建方案。首先，以晚期血管内皮祖细胞与骨髓间充质干细胞为血管化种子细胞，建立基于温敏水凝胶的三维共培养体系，明确其在体外、体内环境中能够快速构建具有功能的微血管网，而且植入的细胞能在体内与宿主细胞整合，直接参与新生血管网的形成；其次，设计中空圆柱状共轴式支架，其外壁具有特定的侧槽结构，保证轴心区内填充的水凝胶三维共培养体系可与体内环境直接接触，早期促进宿主血管长入和轴心区的微血管网形成，及时实现支架内外血管网的吻合，从而形成微血管“夹心效应”，共同保障支架外周区的血供。最后，借助兔大段股骨干骨缺损修复模型，再次验证了该方案的可行性，为共轴式组织工程骨的构建建立了实验基础。