双重3D打印超弹性组织工程血管化骨及其促骨促血管形成协同作用与机制研究

In spite of the wide application of three-dimensional (3D) printing technology in tissue engineering, the mechanical properties of scaffold and vascularization of engineered tissue still cannot meet the demands of clinical application. In this project, the compound liquid ink containing strontium or silicon replaced hydroxyapatite whiskers and polylactide was designed and used to fabricate the honeycomb shaped porous scaffold with the structure of hexagon micropore inlayed large vascular channels via 3D printing. Afterwards, by means of thermally induced phase separation technique, chitosan nano-fibers were introduced into the porous scaffold to construct micro-nano fiber interpenetrating dual network structure, which with good mechanical strength and hyperelasticity. Then, dual growth factors were synergistically immobilized onto the scaffold to promote osteogenesis and angiogenesis based on the dopamine. In order to promote the formation of large blood vessels, another composite liquid ink comprised of chitin whiskers liquid crystal, endothelial cells and vascular endothelial growth factor (VEGF) was further designed and used to engineer the liquid crystal elastomer vascular channel inside the scaffold via the secondary 3D printing. This project innovatively combined multiple concept designs concerning the reinforcing and toughening of the whiskers, the implementation of 3D printing fused seamlessly, the structural fabrication of the micro-nano fiber dual network, hexagon micropore honeycomb and liquid crystal elastomer vascular channel, from which to endow the porous scaffold with favorable mechanical strength and hyperelasticity, as well as the formation of vascular network. The core of this project is to clarify the internal mechanism and synergic effects of the multilevel composition and structure of the scaffold on its mechanical properties, surface cell response behavior and the formation of new vascular network and bone. The findings of this project will provide scientific support for the preparation of 3D printing vascularized tissue engineering bone.

3D打印技术在骨组织工程领域的应用日趋广泛，但支架材料的力学性能和组织的血管化至今无法满足临床应用的要求。本项目采用含锶或硅羟基磷灰石晶须/聚乳酸复合墨水，3D打印镶嵌大血管通道的六边形微孔蜂巢状多孔支架，并在支架中引入壳聚糖纳米纤维，构建微-纳纤维“贯穿”的具有良好力学强度和超弹性的双网络新型支架；同时借助多巴胺在支架上协同固定成骨成血管化生长因子。为了促进大血管的形成，利用甲壳素晶须液晶、内皮细胞和VEGF复合墨水，在支架中二次3D打印液晶弹性体通道。通过创新性地利用晶须增强增韧、无缝粘结3D打印、以及六边形微孔蜂巢、微-纳纤维双网络和液晶弹性体通道分级结构的多重设计，赋予支架良好的力学强度和超弹性，并促进血管网的形成。项目的核心是探明多孔支架的多级组成与多级结构对其力学性能以及细胞响应行为、血管网络和骨组织形成的协同作用与内在机制，为3D打印血管化组织工程骨的构建提供科学依据。

3D打印技术在骨组织工程领域的应用日趋广泛，但支架材料的力学性能和组织的血管化至今无法满足临床实际应用的要求。针对目前制约3D打印组织工程骨临床应用的关键问题，本项目创新性地利用晶须增强增韧、微-纳纤维双网络和液晶弹性体通道结构的构筑以及引入生物活性分子等多重设计，构建了一类具有良好的力学性能、独特的贯通性孔洞结构和优异的成骨和成血管化能力的骨组织工程支架材料。围绕这一研究目的，主要研究工作如下：首先，制备了一系列的纳米晶须（甲壳素晶须、羟基磷灰石晶须、含锶羟基磷灰石晶须），然后制备晶须/聚乳酸复合墨水，并基于3D打印技术构建具有六边形微孔结构和哈弗氏管通道的蜂巢状多孔支架；晶须的引入可以显著提高3D打印聚乳酸多孔支架的力学性能，独特的多孔结构有利于成骨和血管网络的生成。在此基础上，我们进一步利用热致相分离法在多孔支架中引入壳聚糖纳米纤维，构建微-纳纤维“贯穿”的双网络结构，并基于多巴胺在支架上协同固定成骨成血管化生物活性分子，构建了一类具有优异的成骨活性和血管化能力的复合多孔支架材料。此外，我们还基于甲壳素晶须制备了含交联剂和成血管化活性药物的甲壳素晶须液晶凝胶前驱液，并通过二次3D打印在聚乳酸多孔支架中构筑仿天然骨ECM微环境的载药液晶弹性体通道。最后，深入研究了复合多孔支架的六边形微孔蜂巢、微-纳纤维双网络和仿天然骨ECM液晶弹性体通道结构的多重设计，以及引入的不同的生物活性分子对复合多孔支架表面的细胞响应行为、成骨和成血管化能力的影响规律，阐明了不同因素促进成骨和成血管化的分子机制和协同作用。通过本项目的研究，最终构建了一类具有良好的力学性能，优异的成骨和成血管化能力的3D打印聚乳酸基骨组织工程支架材料，为血管化骨组织工程支架材料的设计及其应用提供了借鉴。