梯次构建大块血管化组织工程骨及其修复骨缺损的实验研究

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.Since the seed cells are unable to survive at a distance of more than 200 μm from a capillary,it is imperative for TEBG to be well perfused by a rich vascular network.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 TEGB.However,it's still difficult to guarantee that most of TEGB succeed if the typical ways are used for the construction of a vascular network and seeding cells on the scaffold simultaneously.Therefore,we try to develop a novel protocol,which is fabricating a prevascularized scaffold before the process of osteogenesis,for generating large vascularized TEBG and hope it will be able to overcome the limitations associated with the lack of capillary in?ltration.Firstly,a novel scaffold with a hollow tubular structure similar to the rabbit femoral shaft is designed and obtained. This cylinder scaffold has a side groove connecting the central tunnel,which passed through the scaffold along its long axis.The wall of this scaffold is made by β-TCP and is highly porous.The pores are well intertconnected with each other and open into the central tunnel and the outer surface of the scaffold.The central tunnel of scaffold is filled with fibronectin-collagen gel suitable for vascular ingrowth.Secondly,the scaffold is prevascularized by elevating the femoral vascular bundle from the muscular layer and inserting into the central tunnel through the side groove.Afterward,the solution of gel loaded with BMSCs and ECs is filled in and encapsulated the vascular bundle.Subsequently,the gel mixture warms to allow polymerization to form the cavity of TEGB.After that the scaffold is wrapped by a silica bag to prevent surrounding tissue to occupy the pores of the β-TCP and is implanted into the muscular layer to incubate until the vascular networks form and reach the wall of scaffold.Thirdly, when the vascular network is fully incorporated with the scaffold, the osteogenically differentiated BMSCs harvested and loaded onto the wall of the scaffolds act as a source of osteogenic cells.Meanwhile,a slight vacuum is applied to ensure the cells filled into the pores in the β-TCP.The TEGB is implanted into the muscular layer to continue culture.Fourthly,when the structure of prevascularized TEBG is stable,the osteogenesis and angiogenesis of TEGB is evaluated for bone repair in vivo in a femoral critical-sized segmental defect in rabbit.We hypothesize this fully vascularized TEBG fabricated by the novel protocol will be able to provide adequate and timely nutrients to cells seeded in the scaffold and to lead to the satisfied healing results.

骨组织工程技术为解决骨缺损修复提供了一条令人鼓舞的新思路，但如何保证植入材料内种子细胞的存活是目前的难点。近年研究证实，及时充分的血液供应是解决这一问题的关键，也是构建大块组织工程骨的前提。我们前期研究表明，将轴向动静脉血管束植入材料中心可促进材料内的新生血管网的形成和分布，在此基础上，本课题拟建立一种新的血管化组织工程骨构建方案。以兔作为实验动物，首先分离股骨部轴向动静脉血管束并植入特制的仿股骨干结构支架材料的管腔内，管腔由载荷有血管化种子细胞的胶原/纤连蛋白凝胶填充并包绕血管束构成，然后回植体内预构血管化支架材料，明确材料血管化的最佳时间点。其次将成骨细胞植入充分预血管化支架材料的β-TCP管壁结构中，实现成骨化支架的构建，明确该方法能否解决成骨细胞早期植入后无法及时获得充分氧供而凋亡的问题。最后待支架材料结构稳定，用获得的大块血管化组织工程骨修复股骨缺损，探讨该梯次构建方法的可行性。

创伤、感染、肿瘤切除等各种原因造成的大体积骨缺损一直是临床治疗的难点。组织工程骨技术借助种子细胞，生长因子，支架材料已成功构建出具有良好成骨活性的小体积组织工程骨，这为骨缺损的修复提供了新的思路。但是在现有基础上，构建大体积的组织工程骨仍充满挑战，制约了组织工程骨由基础研究向临床应用过渡。.要获得大体积组织工程骨，首先要实现其充分再血管化，这是由骨组织自身特点决定的。事实上，组织工程骨在与宿主建立充分血供之前，其只能依靠周围组织液的浸润和渗透来维持营养供应，而这种范围极其有限，只有100到200微米。特别在组织工程骨的中心区，血管长入更是困难。由此可见，早期建立及时、充分的血供，是影响组织工程骨构建成功与否的关键。.本研究针对如何实现组织工程骨内的早期血管化构建这一科学问题开展了相关研究。我们在前期建立的以轴向血管束植入为技术核心的血管化组织工程骨构建模型基础上，拟借助基于水凝胶载荷骨髓间充质干细胞（BMSCs）和血管内皮祖细胞（EPCs）构建三维血管网，利用水凝胶可任意塑形的特点，将其填充于材料中心区与植入的轴向血管束间，以此完善支架中心区的微血管构建。其后，利用构建的血管化组织工程骨进行兔股骨大段骨缺损的修复，以此进一步评价该构建方案的可行性。.我们研究发现，利用Ι型胶原和纤维蛋白可以成功构建出适合血管化种子细胞生长的三维培养微环境。该微环境具有温敏效应，即4℃条件下为液态，37℃可固化成胶冻状，而且我们进一步通过体内与体外实验比较了不同比例的BMSCs与EPCs共培养体系，明确了当BMSCs：EPCs为1:4时，二者在水凝胶构建的三维培养体系中成血管网最丰富，在裸鼠体内植入后5天便能形成新生血管网。另外，骨缺损修复模型进一步证实，利用血管束植入联合水凝胶构建支架的中央区微血管网可实现其内部的早期血管化，micro-CT和微血管灌注，以及组织学切片检测进一步证实，水凝胶植入可促进植入轴向血管束与体内血管早期吻合，远期可实现大段负重骨缺损的修复，并且在术后3个月时，四点弯曲实验提示修复后股骨的刚度可达正常兔股骨的83.2%。.本研究的科学意义在于，目前大体积组织工程骨的构建仍充满挑战，主要是由于其中心区无法实现早期充分血管化，而本研究提供的血管化组织工程骨构建方案能够很好的解决中心区早期充分血管化这一难题，在血管化组织工程骨的构建及骨缺损的修复中有良好的应用前景。