海藻酸钠-玉米醇溶蛋白/纳米羟基磷灰石复合支架的仿生构建和性能研究

In recent years, researches on hydroxyapatite/biopolymers bone scaffolds have made significant progress, but their mechanical properties and biological performance are still difficult to meet clinical needs. To improve the structural stability and biological properties of sodium alginate gel, gel modified and bio-mineralization are employed to prepare sodium alginate -Zein/nano-hydroxyapatite (SA-Zein/HA) porous bone scaffold. Firstly, zein will be used to improve the surface property and mechanical property of sodium alginate gel based on interactions between protein and polysaccharide, at the same time the active groups of zein will increase nucleation sites during the biomineralization of HA. Secondly, HA crystals are biomineralized in SA-Zein composite gel with evenly dispersion and ordered arrangement. At last, SA-Zein/HA porous scaffolds with high mechanical property and biological performance will be fabricated through the thermal induced phase separation and freeze drying technique. The growth mechanism of nano-hydroxyapatite crystal in this gel system will be studied from the influence of gel composition, spatial structure and the biomineralization conditions on hydroxyapatite nucleation, growth and arrangement. Material formula will be optimized by the test of mechanical properties, surface properties and degradation behavior. In vitro biocompatibility of the SA-Zein/HA porous scaffolds will be assessed in terms of attachment, differentiation and proliferation of eosteoblastic cells or mesenchymal stem cells on and in scaffolds. SA-Zein/HA porous scaffolds based on biomineralization under the gel template could mimic the structure, composition, and biological function of bone extracellular matrix. The results of this research will provide the scientific experimental basis for the application of SA-Zein/HA porous scaffolds in bone tissue engineering and the inspiration for novel bone repair materials with high mechanical property with light mass.

纳米羟基磷灰石/高分子仿骨支架材料的研究取得了显著进展，但材料的力学性能和生物学性能仍难满足临床需求。针对如何提高海藻酸钠凝胶结构稳定性和生物学性能，本项目联合凝胶复合和仿生矿化两种手段制备海藻酸钠-玉米醇溶蛋白/纳米羟基磷灰石(SA-Zein/HA)仿骨支架材料。首先基于蛋白质-多糖相互作用，利用玉米醇溶蛋白改善海藻酸钠凝胶的表面和力学性能并增加矿化位点；然后对复合凝胶进行仿生矿化诱导羟基磷灰石在凝胶体系中均匀生长和有序排列，最后通过冷冻干燥技术构建SA-Zein/HA多孔支架。研究复合凝胶体系的组成、结构对性能的影响，结合矿化条件探讨羟基磷灰石晶体在复合凝胶中的生长机制；通过力学性能、表面性能和降解性能等测试优化材料制备工艺；考察细胞在支架上的生长增殖情况，评价材料的生物相容性，为SA-Zein/HA多孔支架最终应用于组织工程仿骨支架及新型轻质高强骨修复材料的设计提供科学实验依据。

本项目针对提高海藻酸钠凝胶结构稳定性和生物学性能的科学问题，联合凝胶复合和仿生矿化两种手段制备了海藻酸钠-玉米醇溶蛋白/纳米羟基磷灰石(SA-Zein/HA)复合仿骨支架材料。首先利用绿色植物蛋白-玉米醇溶蛋白来改善海藻酸钠凝胶的表面性能、力学性能并为仿生矿化增加矿化位点，采用复合凝胶的方法在SDS促溶和碱性促溶条件下构建了海藻酸钠-玉米醇溶蛋白复合凝胶(SA-Zein)。采用扫描电镜、接触角测试、力学测试、降解性能测试和细胞相容性测试对SA-Zein复合凝胶进行了研究，同时优化凝胶复合条件和制备工艺。结果发现SA-Zein复合凝胶比SA凝胶具有更稳定的三维多孔形貌，玉米醇溶蛋白纳米粒分布于多孔表面；SA-Zein复合凝胶的弹性强度较SA凝胶有提高2.9倍，复合凝胶的膨胀率和降解率均有不同程度的降低，人间充质干细胞（hMSCs）在SA-Zein复合凝胶上的生长状态较好，复合凝胶更有利于细胞的贴壁生长。然后我们重点开展了SA-Zein复合凝胶的仿生矿化生成SA-Zein/HA复合支架材料的研究，比较了SBF、10SBF和钙、磷原料分开三种矿化的效果，最终我们确定采用磷酸钠和氯化钙作为矿化原料，分开放置于透析袋内外进行仿生矿化的方法。借助XRD、SEM等手段考察了复合凝胶矿化24h后矿化产物的晶型和生长排列情况；材料表面性能、力学性能、降解性和细胞相容性等测试研究表明SA-Zein/HA复合材料具有友好的表面环境，hMSCs细胞存活率比矿化前提高1.9倍，力学强度比矿化前提高3.1倍，降解性降低。最后将SA-Zein/HA复合支架进行皮下植入和体内骨修复实验，其中皮下植入实验早期有轻微炎症反应，随后形成薄层纤维包膜，炎症反应消失；体内骨修复实验是在20mm长完全性骨缺损模型上进行的，实验发现SA-Zein/HA复合支架外观上能有效支撑缺损部位的生理功能，在3个月骨修复期间该支架材料与周边骨组织形成骨性结合，能有效促进新骨形成。在植入1个月后发现，在支架材料和骨组织界面处以及骨缺损区有新骨生成、成骨细胞生长活跃；3个月时在材料接触区域发现大量新骨形成，骨缺损区基本被新骨修复完成，长期植入情况仍需进一步考察。本研究为该新型轻质高强骨修复材料的设计和后期应用提供了全面的科学实验依据。