血管化与成骨细胞膜片复合构建组织工程骨膜的研究

The large bone defects caused by trauma, tumor and fracture remains a clinical problem in orthopedic surgery. Periosteum has the ability of promoting the bone formation, which plays a pivotal role in repairing and regenerating bone defects. However, current tissue-engineered periosteum has the limitations of low cell viability, insufficient vascularization and deficient bone formation, and the same disadvantages as bone grafts. Our previous study indicated that combination of bone marrow mesenchymal stem cell sheet(multiple cell layers and intact extracellular matrix) produced by our modified technique with porous ceramic scaffold promoted new bone formation and that cell sheet activated the bioactivity of inorganic bone substitutes as a cell delivery carrier. Nevertheless, the bone formation within the scaffold remained deficient and the bone healing efficacy of scaffold was not well developed due to the insufficient blood supply. Therefore, we propose that the biomimetic tissue-engineered periosteum with prevascularized microvessels networks developed by the combination of prevascularized cell sheets and osteogenic cell sheets could effectively provide not only osteoblasts but also the ability of rapid anastomoses with host vascular, significantly promoting the new bone formation. In this project, our aims are at investigating the feasibility of developing a scaffold-free tissue-engineered periosteum with three-dimension biomimetic periosteum-like structure based cell sheet engineering technique, and further uncovering the mechanism in which prevascularized tissue-engineered periosteum enhances the bone formation when combined with scaffold. Our goals are also to develop a new strategy of using a biomimetic tissue-engineered periosteum promotes the bone formation and to bring a new promising way for clinic treatment of large bone defects.

各种原因导致的骨缺损是临床常见问题。骨膜在骨组织再生中发挥重要作用，组织工程骨膜有望促进骨修复重建, 但目前构建的工程化骨膜仍存在许多不足。我们前期研究表明：改良方法获得的骨髓基质干细胞膜片（有多层细胞组成且保留了细胞外基质等结构）可作为细胞释放载体赋予骨替代材料生物活性并促进骨再生。然而由于缺乏血管供给，材料内部成骨仍较少。我们设想：将预血管化与成骨预分化的成体干细胞膜片复合，体外构建具有微血管网的仿生骨膜，既能提供成骨细胞又能促进与宿主血管的快速吻合加速骨修复再生。因此，本项目拟通过体内外研究探索基于细胞膜片技术构建三维仿生的无支架组织工程骨膜的可行性，进一步揭示预血管化组织工程骨膜与支架材料组合促进骨再生的机理，以期探索构建组织工程骨膜的新策略。研究成果有望为临床大型骨缺损修复治疗提供新思路。

骨膜在骨组织再生修复中发挥重要作用。本项目拟通过体内外研究探索基于细胞膜片技术构建三维仿生无支架组织工程骨膜的可行性, 为临床实现骨缺损修复提供新的思路。本项目自2013年立项以来,按照计划书,主要完成了以下几部分的研究:1、基于人BMSCs和脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)构建三维结构预血管化细胞膜片的实验研究 ;2、兼具成血管成骨特征的仿生骨膜的构建及体内外成骨效能研究。3、基于兔骨髓间充质干细胞 (Bone marrow mesenchymal stem cells, BMSCs)预血管化细胞膜片的体外构建。4. 完全基于大鼠BMSCs构建无支架组织工程血管化骨并修复大鼠颅骨临界骨缺损的实验研究；5. 完全基于大鼠BMSCs构建的仿生骨膜在体内成骨效能研究；6.内皮细胞与干细胞膜片共培养成血管机理的研究。通过以上研究,获得如下结果:1、内皮细胞在未分化骨髓间充质干细胞膜片上共培养, 可以形成预血管化网络结构的细胞膜片。将此膜片折叠形成三维结构组织后植入体内,发现植入组织内血管密度增加,且体外预成血管可以与宿主血管在体内发生吻合,形成功能化血管网。此结果为工程化血管组织构建提供了新方法;2、内皮细胞接种于未分化MSC膜片共培养,再和成骨分化MSC膜片组合,可以构建兼具成血管、成骨特征的组织工程骨膜。该仿生骨膜与外源性骨替代材料 羟基磷灰石复合,植入体内发现体外构建的仿生骨膜不仅可以促进支架材料的血管化, 同时也提高了其成骨潜能,有望构建同时具有成血管、成骨特征的组织工程骨。3.骨髓间充质干细胞诱导形成的内皮样细胞与干细胞膜片共培养能够形成血管网络结构，与成骨膜片复合构建的仿生骨膜在体内的成骨效能与自体骨膜相类似。此结果为临床骨缺损修复提供了新的研究思路。4. 不同的基质微环境对内皮细胞成血管具有不同的作用，成骨膜片更有利于内皮细胞的迁移并形成血管网络结构；膜型基质金属蛋白酶1（MT1-MMP）在预血管化膜片形成过程中，对细胞外基质的重建和细胞外基质调节血管网形成中起到了关键的作用。