控释瘦蛋白的仿生纳米纤维膜构建及促进口腔黏膜血管化和重建研究

Large oral mucosa defect is still a clinical problem to be resolved. Tissue engineering for repair of soft tissue defects in oral and maxillofacial regions has been a hot research topic of stomatology, which is considered to be the ideal solution to the above problem. However, tissue engineered oral mucosa based on collagen gel has an urgent problem that collagen gel has disadvantages of poor mechanical properties, easy to shrink and degradation. To solve this problem, we proposed to repair large oral mucosa defect through in vitro construction of tissue engineered oral mucosa using a biomimetic silk fibroin nanofiber contained leptin which played the role in promoting vascularization. Firstly, leptin was loaded in gelatin nanosphere, and then were combined with silk fibroin nanofibers in order to realized leptin loading and release. Secondly, silk fibroin scaffold with biomimetic 3D microstructure was used to regulate cell behavior to form lamina propria, and then was employed to promote oral keratinized cell to form epithelial tissues that is tissue engineered oral mucosa with double layer. Thirdly, the release of leptin was used to promote angiogenesis, and the underling vascularization mechanism induced by leptin was analyzed. Finally, tissue engineered oral mucosa is used to repair large oral mucosa defect, and study its integration mechanism in situ. This project will not only provide new ways for large oral mucosal defect repair, but also provide important theoretical and experimental basis for the clinical application of tissue engineering.

大面积口腔黏膜缺损仍然是临床医学亟待解决的难题。以材料为基础的组织工程技术是解决该问题的理想方法，相关研究已成为当前口腔组织工程领域的重点课题。本项目针对目前基于胶原凝胶构建组织工程口腔黏膜存在力学性能差、易收缩降解和修复面积小的缺点，提出采用丝蛋白纳米纤维通过控释瘦蛋白促进血管生成进而实现体内大面积黏膜缺损功能性修复的新方案。拟首先利用明胶纳米球装载瘦蛋白，并将其与丝蛋白纳米纤维复合，研究瘦蛋白的有效装载与可控释放；其次利用支架三维微结构作为调控口腔成纤维细胞生物学行为的信号途径，预构丝蛋白基类固有层，并以此促进口腔角质细胞上皮化形成双层组织工程化口腔粘膜；再次通过瘦蛋白控释促进血管发生，研究瘦蛋白在血管发生中的机理；最后利用组织工程化口腔粘膜修复大面积口腔黏膜缺损，并探讨其原位融合机制。本项目研究将不仅为大面积口腔粘膜缺损修复提供新途径，更为组织工程技术的临床应用提供理论和实验依据。

目的：将瘦蛋白负载到丝素纤维构建能够促进血管生成的纤维膜，研究其促进口腔黏膜再生的能力。方法：采用静电纺丝技术制备丝素纤维，通过薄膜分散法制备瘦蛋白脂质体并表面氨基化，以聚多巴胺为桥梁将脂质体接枝丝素纤维表面构建可血管化的纤维膜。透射电子显微镜（TEM），扫描电子显微镜（SEM），原子力显微镜（AFM）对脂质体和纤维的形貌进行表征。水接触角仪（WCA）以及力学测试仪表征纤维膜的理化性质。将大鼠成纤维细胞种植在纤维膜上，以CCK-8法和细胞荧光染色，考察纤维的细胞相容性。用纤维膜的浸出液培养人脐静脉内皮细胞（HUVECs），考察纤维成血管的能力。构建兔口腔黏膜缺损模型评价纤维促进黏膜的再生能力。结果：丝素纤维经过聚多巴胺修饰和脂质体接枝后形貌发生微小改变，但是亲水性和机械性能都明显提高。细胞实验发现纤维膜对细胞的增殖和形态没有明显的影响。HUVECs在负载瘦蛋白的纤维浸出液培养5小时后分化排列成明显的网状结构。在兔口腔黏膜大面积缺损模型中，负载瘦蛋白的纤维治疗的缺损愈合率高于其他组，再生的黏膜下CD34的信号最明显。结论：丝素纤维通过聚多巴胺表面接枝包裹了瘦蛋白的脂质体，表面粗糙度提高了，但是机械性能和亲水性也提高了。负载瘦蛋白的丝素纤维具有良好的细胞相容性。脂质体释放的瘦蛋白能够有效地促进血管再生，提高纤维膜修复口腔黏膜缺损速率。