促尿道修复细菌纤维素基双层仿生支架的构建及评价

Vascularization and epithelialization of grafts are the key issues to achieve physiologically urethral repair using tissue engineering technology. In previous studies, we constructed a biomimetic scaffold in terms of the structure and component to promote vascularization. And we have achieved preliminary results for urethral repair. But the regeneration of the mucosal layer was limited, which affected the long-term stability of the urethral reconstruction. Therefore, inspired from the structure and function of native urethra, this project is to construct a bilayered bacterial cellulose based scaffold. We choose bacterial cellulose as "skeleton" and bladder acellular matrix as "active ingredient" to construct a porous scaffold which mimics urethral spongiosum. Effects of physical properties (pore size, stiffness) of porous scaffold on vascularization is to be studied. Then we construct sodium alginate hydrogel that mimics the urethral mucosa on the porous layer and study the effect and mechanism of hydrogel microenvironment (stiffness, stress relaxation, patterning) on epithelialization. Vascularization and epithelialization of scaffold can be promoted by regulating the physical properties of the porous layer and the hydrogel, and physiologically urethral repair will be promoted through the synergistic effect of vascularization and epithelialization. This project will provide the theoretical support and experimental basis for clinical application of the tissue engineered scaffold.

移植物血管化、上皮化是以组织工程技术实现尿道生理性修复的关键问题。在前期研究中，我们通过构建结构、成分双重仿生尿道海绵体的支架促进血管化，尿道修复取得了初步成果，但尿道粘膜层再生较差，影响重建尿道的远期稳定效果。因此，本项目通过仿生天然尿道结构和功能构建细菌纤维素基双层仿生支架，以细菌纤维素为“骨架”和膀胱脱细胞基质为“活性成分”构建仿生尿道海绵体的多孔材料，深入研究多孔材料物理特性（孔径、硬度）对血管化的影响；并在多孔层上构建仿生尿道黏膜的海藻酸钠水凝胶，研究水凝胶微环境（硬度、应力松弛、图案化）对尿道上皮细胞的影响与作用机制，通过调控多孔层及水凝胶的物理特性促进支架血管化、上皮化，以期通过支架血管化、上皮化的协同作用促进尿道生理性修复，为开发具有临床应用前景的组织工程尿道支架提供实验基础和理论依据。

组织工程技术为尿道狭窄的修复带来希望，但移植物血管化、上皮化是以组织工程技术实现尿道生理性修复的关键问题。本项目以细菌纤维素（Bacterial cellulose, BC）、丝素蛋白（Silk fibroin, SF）及明胶（Gelatin, Gel）采用定向冷冻与原位培养技术构建具有定向通道与纳米纤维网络结构的仿生SF/Gel/BC材料，定向通道支架有利于氧气和营养物质的传输，促进人脐静脉内皮细胞（HUVECs）在材料上的粘附、增殖和功能性蛋白的表达。进一步以离子交联法在SF/Gel/BC材料上构建仿生尿道黏膜的BC/海藻酸钠（Alginate, Alg）水凝胶，通过研究水凝胶微环境对尿道上皮细胞的影响，发现硬度高的水凝胶（弹性模量为32.3 kPa）比低硬度水凝胶（弹性模量为3.2 kPa）更利于上皮细胞的迁移和增殖。基于定向通道材料与水凝胶构建的双层仿生尿道支架，能够有效促进材料的血管生成，诱导上皮细胞爬行并覆盖缺损部位，加快缺损部位上皮化，成功修复了2 cm兔尿道缺损。本项目通过调控材料物理特性构建双层仿生尿道支架，通过支架血管化、上皮化的协同作用促进尿道生理性修复，为开发具有临床应用前景的组织工程尿道支架提供了实验基础和理论依据。