溶菌酶交联晶体复合水凝胶促进骨缺损修复的作用及机制研究

Due to the good biocompatibility, hydrogels have developed rapidly in the field of bone repair in recent years. However, at present, there is no hydrogel that has a combination of properties including good biocompatibility, high strength and osteoinductivity. Therefore, it is of great clinical significance to develop a new type of degradable hydrogel with both mechanical strength and osteoinductivity. This project proposes to combine hydrogel with lysozyme crystals, a degradable double-network hydrogel (with covalent and physical interactions) could be obtained by using modified sodium alginate and polyacrylamide under ultraviolet light. As a transport carrier, this kind of hydrogel has good biocompatibility and mechanical properties (high stiffness and high toughness), cross-linked lysozyme crystals loaded with magnesium/zinc ions (promote bone regeneration) could be added in hydrogels, which will provide good compatibility, better mechanical properties, sustained osteoinduction and antibacterial effects. Next, we will optimize the composite hydrogel to obtain a good mechanical strength, evaluate the effect of promoting osteoblast proliferation and differentiation in vitro, and the effect of promoting bone defect repair and bone regeneration; and reveal the mechanism of physical properties (mechanical properties, nanostructures) and biological properties (bone regeneration, antibacterial and anti-inflammatory) synergistically promote bone repair. This project will provide a new direction for the development of bone repair materials

水凝胶因良好的生物相容性，近年来在骨修复领域发展迅速。然而目前，还没有一种水凝胶可同时具备良好的生物相容性、高强度及骨诱导性。因此，开发一种新型的兼具力学强度及骨诱导性的可降解水凝胶具有重要的临床意义。本项目提出将水凝胶和溶菌酶晶体相结合，改性的海藻酸钠和聚丙烯酰胺，在紫外作用下形成共价和非共价交联的双网络可降解水凝胶，该水凝胶作为输送载体，具有良好的生物相容性和力学性能（高刚度及高韧性）；溶菌酶交联晶体可携带镁/锌离子（具促骨再生作用）复合在水凝胶中，不仅生物相容性好，还可增强水凝胶力学性能，并提供持续性骨诱导和杀菌消炎的效果。优化各组分配比，获得具良好机械性能的复合水凝胶；评估该材料体外促进成骨细胞增殖和分化，体内促进骨缺损修复和再生的作用；揭示该材料的物理性能（力学性能，微纳结构等）和生物性能（促骨再生，杀菌消炎）协同促进骨修复的作用机制。本项目将为骨修复材料的研发提供新的方向。

水凝胶因其良好的生物相容性，在再生医学领域备受关注，尤其是在骨修复领域中得到快速发展。开发具有良好机械强度和有效载药能力的重组蛋白交联水凝胶极具吸引力，但目前报道很少。在本项目中，我们设计并合成了一种T4溶菌酶复合水凝胶智能递送系统，该复合水凝胶体系可以介导镁离子和锌离子的局部传递和协同释放，进而促进骨修复。我们设计的T4溶菌酶突变体除了其固有的抗菌性能外，表面含有丰富的游离胺基团，可以作为有效的共价交联剂，加强水凝胶网络的交联；同时，两个半胱氨酸残基被引入到T4溶菌酶的表面，使其可特异性的结合锌离子，实现精确的、可控的锌离子递送。另外，我们以天然多糖为反应原料，以甘油磷酸镁、四臂聚乙二醇琥珀酰亚胺碳酸酯、T4溶菌酶为交联剂，通过点击化学反应，得到性能可控的可注射T4溶菌酶交联复合水凝胶。固定浓度的镁离子通过甘油磷酸镁引入，最终镁离子和锌离子从水凝胶中持续释放，通过MAPK信号通路协同促进hBMSCs成骨分化，从而促进预定部位的骨再生。本项目巧妙利用了T4溶菌酶的高稳定性、生物相容性、抗菌活性和金属结合能力，该策略为开发具有增强物理和生物功能的蛋白交联智能生物材料输送系统开辟了新的途径。此外，也预示着合理设计的蛋白质交联水凝胶可能在3D细胞培养、组织工程和控制药物输送等领域具有广泛的应用。