仿生双层鱼胶原基纳米复合支架的构建及其引导牙周组织再生的效应与机制研究

The development of biomaterials which have the ability to guide periodontal tissue regeneration and segregate gingiva with good biosafety is the hotspot and difficulty in the field of regenerative medicine nowadays. In this project, we innovatively presents the ideas to solve the clinical issue in the functional reconstruction of periodontal tissue defects as follows, fish collagen is acted as an entry point to fabricate a novel guide periodontal tissue regeneration material by combining fish collagen/iron-doped bioactive glass nanoparticle and fish collagen/polyvinyl alcohol to form a double-layered nano-composite scaffold. The effects of structure, hydrophilicity and mechanical property of the nanocomposite scaffold on regulating biological behaviors of human periodontal ligament cells （HPDLCs）, human osteoblast-like cells and gingival fibroblasts（HGFs）are discussed. The macrophages which represent the immune system are used to illustrate the ability of the nanocomposite scaffold on regulating inflammation and osteogenic differentiation in cells related to periodontal tissue regeneration. Furthermore, the restorative effect of the nanocomposite scaffold in vivo is testified by dog periodontal tissue defect model. We try to illuminate the pathway and mechanism of guiding periodontal tissue regeneration induced by the nanocomposite scaffold on different levels of histology, cytobiology and molecular biology. This project is aimed to solve a series of key scientific problems such as the preparation technology of fish collagen based double-layered nanocomposite scaffold and to study its biological effect, tissue regeneration ability and regulatory mechanism. This research will give a comprehensive assessment of the potential clinical application of the nanocomposite scaffold and provide a safe and effective way to promote functional periodontal tissue regeneration.

构建兼具诱导牙周组织再生、屏蔽牙龈长入和生物安全的牙周缺损修复材料是当今再生医学领域的热点和难点。本项目围绕牙周组织缺损重建这一临床问题，创新性地提出：以鱼胶原为切入点，研制新型鱼胶原/掺铁生物玻璃纳米颗粒和鱼胶原/聚乙烯醇复合双层支架作为牙周组织再生修复材料。揭示该支架的结构、亲水性、力学性能等因素调控人牙周膜细胞、成骨细胞和牙龈成纤维细胞的生物学行为规律，并引入代表免疫体系的巨噬细胞，探讨该体系下材料的炎症调节性能及对人成牙周组织相关细胞成骨分化的调控机制，最后通过犬牙周组织缺损模型验证其对于体内牙周组织缺损的修复效果。从整体、细胞和分子生物学层面上，阐明支架诱导牙周组织再生的作用途径及其机制。项目最终试图解决双层鱼胶原基纳米复合支架的制备技术、生物学效应、牙周组织再生能力及相关的修复机制等一系列关键的科学问题，全面评估其临床应用潜能，为促进牙周缺损功能性重建提供一条安全有效的途径。

研发兼具诱导牙周组织再生和屏蔽牙龈长入功能的牙周缺损修复材料一直是口腔再生医学领域中重点发展的方向之一。本项目构建了一种鱼胶原/聚乙烯醇/掺铁介孔生物玻璃水凝胶和聚乙烯醇水凝胶组成的双层鱼胶原基纳米复合支架。探讨该双层支架材料的形貌结构、理化性能、免疫调节功能及对牙周组织相关细胞的成骨分化效应和作用机制，并通过体内牙周组织缺损模型验证其修复效果，为新一代口腔牙周组织再生修复材料的研发和应用提供科学依据。研究发现：（1）该鱼胶原基纳米复合支架有明显的双层结构，上层为致密的聚乙烯醇水凝胶，下层为具有多孔结构的胶原/聚乙烯醇/掺铁介孔生物玻璃复合水凝胶，其形貌结构和孔隙率由胶原和聚乙烯醇的比率和掺铁介孔生物玻璃的浓度调节。（2）该支架降解速率适宜，可作为稳定的牙龈屏障，并能释放具有生物活性的钙、硅和铁离子，有利于牙周缺损中骨组织修复。（3）该支架中聚乙烯醇水凝胶层能减少人牙龈细胞的粘附，而胶原/聚乙烯醇/掺铁介孔生物玻璃水凝胶层能促进人牙周膜细胞的增殖活性、成骨分化相关基因(RUNX2、OCN、COL1和CEMP1）和ALP蛋白的表达。（4）该支架具有免疫调节功能，能通过抑制巨噬细胞的炎症基因表达和促进抑炎基因表达来增强人牙周膜细胞的成骨分化能力。(5) 该支架能明显促进体内牙周组织缺损愈合，增强牙槽骨的生成和矿化，这与其结构形貌和组成成分等能有效屏蔽牙龈、促进缺损周围细胞生长和成骨分化有关。本项目制备的鱼胶原/聚乙烯醇/掺铁介孔生物玻璃和聚乙烯醇双层鱼胶原基纳米复合支架显示出良好的生物学活性、免疫调节及促牙周组织和牙槽骨再生修复能力，为牙周组织缺损功能性重建提供安全有效的途径。