纤维束表面拓扑结构介导EMD调控Hippo通路对拔牙窝“位点保存”的作用效果及机制

Alveolar bone defect is the main difficulty in dental implant therapy. There are some limits in traditional bone transportation; so many studies are aimed to develop new bone regenerative biomaterials. Electrospinning technique is widely studies in the researches of tissue engineering scaffolds because of its flexibility, cost-effective in preparing nanofiberous scaffolds. While the traditional fibrous scaffold has small pores that limiting the cell infiltration. It is not enough in thickness and has unstable structure. We invented alcohol bath ‘one-step crosslinking’ technique and alcohol bath-rolling mandrel technique, which can prepare large pore sized, thick, stable 3D electrospinning fibrous scaffolds. Preosteoblast were seeded on them and were found infiltrating through the full thickness of the scaffolds in several days, growing and distributing equally in the scaffolds and showed increased proliferation and osteogenic differentiation ability in 3D scaffolds. Electrospinning aligned fibers were studied in the researches of bone regeneration because of its biomimetic structure of bone, but it is not clear about the cellular and molecular mechanisms of fiber arrangement regulated osteogenic differentiation in 3D environment. We can get both aligned and random 3D fibers by our invention. So we are going to use the above fibrous scaffolds as a platform to observe the effect on osteogenic differentiation caused by differently arranged fibers, suspect and verify the emerin which play an important role in mechanotransduction of the nucleus and Hippo signal path play important roles in it, provide reference and theoretical foundation for design of the scaffolds for bone regeneration.

牙槽骨缺损是牙种植修复的主要难题，对牙槽窝行“位点保存”术，可有效地减少牙槽骨的吸收，为后期种植修复打下基础。临床上常用的“位点保存”材料效果有待提高且不易降解吸收，经济高效的新型材料急待开发。项目组发明的乙醇浴“一步交联”及乙醇浴滚筒收集法，能制备出大孔3D纳米纤维束，表面具有纳米沟槽拓扑结构，能促进前成骨细胞长轴方向的伸展、细胞的扩散（数天扩散支架全层）、增殖与成骨分化。研究认为外力能直接改变细胞核的形态，引起核膜上机械信号感受分子伊默菌素（EMD）反应，调控Hippo信号通路，防止核膜过度形变，同时影响细胞分化。项目组猜想有序纤维束是通过表面纳米沟槽结构影响细胞成骨分化，其作用机制可能与外力相同。因此，项目组欲研发胶原蛋白有序纤维束用于“位点保存”，探究其影响细胞分化的机制，观察其减少犬牙槽窝吸收的效果，为临床“位点保存”提供新的材料选择。

牙槽骨缺损是牙种植修复的主要难题，减少牙槽骨的吸收可对牙槽窝行“位点保存”术。提高“位点保存”效果需要合适的生物材料及细胞因子。项目组发明的乙醇浴“一步交联”及乙醇浴滚筒收集法，能制备出大孔3D纳米纤维束，表面具有纳米沟槽拓扑结构，能促进前成骨细胞长轴方向的伸展、细胞的扩散（数天扩散支架全层）、增殖与成骨分化。本项目制备的丝素蛋白/胶原蛋白有序纳米纤维同样促进了成骨分化。项目组随后研究发现有序纤维束是通过表面纳米沟槽结构影响细胞成骨分化的，其机制之一是通过机械信号感受分子伊默菌素（EMD），调控Hippo信号通路，从而促进BMSC的成骨分化。虽然丝素蛋白/胶原蛋白有序纳米纤维在“位点保存”中尚未找到有效应用方式，但本研究还发现，利用乙醇浴制备的无序纳米纤维支架的蓬松结构可减少细胞长期3D培养的氧化应激，提高其细胞稳定性和活力。在以上实验结果的基础上，项目组检测了纳米纤维支架在药物长期毒性测试与骨类器官形成中的作用，发现其能有效的用于药物测试，对促进骨器官形成也有良好的应用前景。这对于临床测试药物对骨组织的影响、提供研究减少骨吸收、促进骨再生等问题一种新的体外研究平台。