体外构建人源性血管-骨微组织中内皮细胞的侵入过程和调控机制研究

Bone tissue engineering represents an appealing strategy to improve osseous defects. Despite the clear potential of engineered constructs, translation into the clinic remains difficult. One of the major limitations of this approach for a large bone defect is the inability to provide sufficient blood vessel supply during the early stages of bone regeneration. Lead to limited survival of the osteogenic cells early after implantation, result in failure of fracture healing.Improve vascularization in a bone tissue-engineered construct remains an important challenge in bone tissue engineering.However, no advanced in vitro models can be used for analyzing interactions between blood vessel and a bone tissue-engineered construct. The motivation for our research and the focus on neovascularization of the construct occurred by invading blood vessels deriving from the surrounding host vasculature. We mimicking the process of invasion of blood vessels into the tissue-engineered bone based on "human-derived blood vessel -bone microtissue".To overcome this problem, several methods have been proposed to improve the structure relations of vessel and bone in experimental models. The nanofiber scaffold with different geometric character was builded as vascular networks. Tissue-engineered bone was derived form 3D bioactive porous scaffold. And according to bottom-up tissue engineering the "human-derived blood vessel -bone microtissue" was assembled. Various microscale technologies and modular approaches were developed for the complex microtissue. The formation mechanism of new blood vessels from pre-existing ones in the porous scaffold, the sprouting of endothelial cells and the regulation mechanism will be explored. In the end, more new details of angiogenesis in bone defect repair which treated by tissue engineering will be clarified.

组织工程骨在骨缺损治疗方面具有广泛应用前景。但组织工程骨治疗大面积骨缺损时因缺乏功能性血供，植入后存活差、修复效果不理想，成为阻碍其临床应用的关键问题。因缺乏理想的“血管侵入组织工程骨”体外模型，目前血管侵入组织工程骨过程中内皮细胞的行为及其调控机制尚不明确。本项目拟构建“人源性血管-骨微组织” 并深入研究血管侵入机制。针对血管和组织工程骨的结构关系、以及对材料的不同要求，我们设计具有特定微结构的纳米纤维材料以模拟血管，制备三维多孔矿化支架以诱导干细胞成骨分化，再根据bottom-up组织工程将两者组合构建成“人源性血管-骨微组织”。研究多种组合形式、多种组装方式对微组织结构关系的影响，阐明复杂微组织的bottom-up构建原理。并采用分子生物学、micro-CT等技术研究内皮细胞向组织工程骨侵入的规律及其对成骨分化的影响。为微组织体外构建和骨血管化研究提供新的理论和实验依据。

研究表明骨再生修复中血管长入是实现骨组织快速高效修复的必备条件。骨修复支架的结构，孔隙，成分都会影响内皮细胞的迁移和长入。骨缺损修复时，将支架材料植入后，周围中的血管是否能及时长入到支架内部，向内部输送营养物质、氧气和细胞对骨缺损整体修复至关重要。但目前研究对骨缺损治疗过程内皮细胞逐渐侵入修复支架的过程和调控机制尚不明确。本项目针对内皮细胞与骨再生过程中支架材料的调控作用进行深入研究，分别设计了模拟血管的纳米纤维和模拟骨组织的多孔支架材料，从不同角度去探索成血管、成骨的相关联机制。研究设计PCL纳米纤维模拟血管细胞外基质，并进行表面修饰以促进对内皮细胞的调控作用。单宁-铁修饰有利于内皮细胞铺展，与单纯PCL纳米纤维表面的细胞相比，细胞显著铺展，面积大，形成伪足紧密粘附纳米纤维，细胞与细胞之间相互联通，部分逐渐融合，内皮化趋势明显。PLGA微球可烧结形成多孔支架，经过修饰后含铁微球支架相较于不含铁微球支架，BMSCs在表面种植粘附后，更容易向支架内部空隙迁移。通过兔股骨缺损动物实验分析HE染色发现含铁和含BMP-2支架修复效果较为理想，因为铁元素大大提高了支架材料促进细胞归巢能力，更多的干细胞达到支架的中心部位，继而在BMP-2的作用下形成新生骨。借助Pickering乳液法，我们成功制备了孔结构均匀、可持续释放Mg2+的GelMA-co-PEG大孔复合水凝胶。凝胶孔径范围在50-200µm，随着MgO含量的增高，凝胶孔径减小，压缩模量增大，由27.34 kPa增大至152.37 kPa。体外实验0.5 wt%的组干细胞增殖与铺展情况最好，且表现出明显的定向成骨分化趋势，大鼠颅骨缺损模型实验结果表明，0.5 wt%的组可实现有效的骨再生修复。3D打印生物活性玻璃-丝素/酪胺改性明胶（Cu-BG/SF/Tyr-Gel）水凝胶复合支架，设计铜离子和硅离子协同促进血管再生和BG促进成骨分化、矿化和骨组织再生。通过探究铜离子对内皮细胞的激活作用，确定最适宜的局部铜离子浓度，实现了体外促进人脐静脉内皮细胞的迁移分化成管。