基于微型组织工程技术模拟创面生态微环境构建促血管化的快速成型真皮替代物

Angiogenesis is one of the key mechanisms in the whole process of wound repairing. Limited efficacy has been found when supplementing growth factor only. Reconstruction of local microenvironment can improve activation, migration and proliferation of vascular endothelial cells (VEC) which are vital steps in angiogenesis. We previously found that with amniotic membrane particle(mAM) as cell carrier, micro-tissue engineering technology could quickly construct skin substitute enriched with growth factors and natural extracellular matrix, thus automatically regulating the pathological microenvironment and accelerating wound healing process. Here we hypothesize: with mAM as micro-carrier for amplification of VEC and as growth factor source, adding fibrin gel as scaffold for new vessels, as delivery system of growth factor released from mAM and as gel medium which can well-distribute mAM, is able to construct a novel and rapid-gelling dermal substitute based on micro-tissue engineering technology. It is expected that this novel dermal substitute could simulate physiological micro-environment in local wounds thus enhancing the activation, proliferation and migration of VEC, hereby promoting angiogenesis. In this study, we will explore its effect on angiogenesis involving VEC in vitro, and evaluate its effect and mechanisms in vivo on accelerating angiogenesis process and wound healing. The study is expected to provide a new sight for angiogenesis-promoting strategy in tissue engineering and provide a novel dermal substitute for the clinical treatment of acute and chronic wounds.

血管化是贯穿创面修复全过程的关键机制之一，单一补充生长因子调控效果有限，重建局部微环境可改善血管内皮细胞的活化、迁移、增殖三个血管化关键环节。前期研究发现以微粒羊膜为载体的微型组织工程技术可快速构建富含生长因子和天然细胞外基质的高活性皮肤替代物，能主动调控创面生态微环境、加速创面愈合。本项目设想：以具有三维立体结构的微粒羊膜作为内皮细胞扩增微载体和促血管化生长因子释放源，复合纤维蛋白胶作为新生血管管腔支架、生长因子缓释系统和微粒均相分散凝胶介质，构建基于微型组织工程技术的快速成型高活性真皮替代物，以期模拟创面生态微环境，改善内皮细胞活化、增殖和迁移行为，加快血管化速度。本研究将通过体外实验评价其增强血管内皮细胞的促血管化行为，动物实验评价其促进创面血管化、加速创面愈合的效果及机制。该研究有望为组织工程促血管化策略提供一种新思路，为急慢性创面的临床治疗提供一种新型真皮替代物。

血管化是贯穿创面修复全过程的关键机制之一，单一补充生长因子调控效果有限，重建局部微环境可改善血管内皮细胞的活化、迁移、增殖三个血管化关键环节。本项目构建了具有三维立体结构的微粒羊膜作为内皮细胞扩增微载体和促血管化生长因子释放源，复合纤维蛋白胶作为新生血管管腔支架、生长因子缓释系统和微粒均相分散凝胶介质，形成基于微型组织工程技术的快速成型高活性真皮替代物，以模拟创面生态微环境，改善内皮细胞活化、增殖和迁移行为，加快血管化速度。本项目研究结果显示：制备的微粒羊膜保留了细胞外基质成分和支架结构和部分促血管化生长因子，去除了免疫抗原成分；采用HUVEC细胞作为对象细胞，将其与微粒羊膜按一定比例加入旋转培养体系中进行培养，内皮细胞扩增速度较快，内皮细胞部分活化，干细胞性能维持良好。体外构建纤维蛋白胶-微型组织工程化羊膜-内皮细胞复合体体外实验可对血管内皮细胞的迁移能力有明显促进作用，大鼠体内皮下埋置实验显示FAH复合体中的微粒羊膜降解速度慢，纤维蛋白胶降解速度较快，促血管化效应明显，早期仍可诱导出一定的炎症反应，后期炎症反应减弱。动物实验显示纤维蛋白胶-微型组织工程化羊膜-血管内皮细胞复合体可加速创面愈合进程，增加创面局部促血管化生长因子和炎症介质表达水平。本项目证实微型组织工程羊膜复合纤维蛋白胶构建新型真皮替代物，通过模拟创面的三种生态微环境(因子微环境、生长信号微环境、趋化信号微环境)，分别改善血管化的三个关键环节(活化、增殖、迁移)，从而加速创面血管化过程，促进创面愈合。该研究有望为组织工程促血管化策略提供一种新思路，为急慢性创面的临床治疗提供一种新型真皮替代物。