基于丝素蛋白-高支化多糖血管化骨组织生物材料的构造及性能

Vascularization is a pivotal technology to avoid necrosis in the centre of artificial bone tissue. Nutrients and oxygen can be supplied continuously, and metabolic wastes can be removed immediately in the vascularizing bone tissue biomaterials. Bone tissue engineering aims at creating or inducing the formation and long-term survival of blood vessels within a specific bone tissue in a specific location through the selection and manipulation of cells, matrices and biologic stimuli. Major efforts are directed toward the vascularization of scaffold materials that can mimic native bone tissues with respect to both architecture and functionality. In this project, we attempt to fabricate a new three-dimensional hydrogel scaffold composite based on cross linking of silk fibroin and carboxylate hyperbranched polysaccharide due to the abundant of terminal carboxyl or hydroxyl groups, in which vascular growth factor can be conjugated specifically to induce the formation of vascularization, and cells can be capsulated in the scaffold during the formation of hydrogel. The vascular growth factor may induce the formation of capillary-like blood vessels through the proliferation and differitiation of the cells. Osteoblast cells will be co-cultured in the obtained scaffolds with capliary-like blood vessels, which can provide homogeneous microenvironments, which enhance the transportation of nutrients/metabolic wastes and prevent pore occlusion during tissue formation. The favorable microenvironments can allow the in-growth of osteoblasts for generating a homogeneous tissue with high viability and facilitate vascularization. The fabricated bone-mimicing scaffolds will be assessed for surface morphology, tensile modulus, water-absorbality, hydrophilic properties, biodegradation, and biological capabilities. The proliferation and differentiation of vascular cells and osteoblast cells will be assessed, and the density, length, and degree of branch of the capalary-like blood veseels will be characterization. The relationship between physicochemical structures and biologic properties of the scaffolds will be clarified. This may support a crucial technology which will be applied to mimic native bone tissue architecturally and functionally.The accomplishment of this proposal will accumulate valuable and scientific data in the fields of biomaterials and tissue engineering.

血管化是抑制人工骨组织内部坏死的关键技术。血管化骨组织材料可维持营养物质、氧气持续供应及代谢产物及时排出。基于基质、生长因子及细胞的选择和调控，本项目拟利用羧甲基高支化多糖生物相容好及外围大量羧基和羟基的特点,在适合细胞存活条件下特异性键合血管生长因子、包埋内皮细胞、交联丝素蛋白形成水凝胶，以期该水凝胶支架材料诱导内皮细胞体外增殖、分化成毛细血管网络；同时共培养成骨细胞，以期新生的毛细血管网络为成骨细胞生长、分化成均匀的骨组织提供营养物质、氧气且及时排出代谢产物；从而实现结构上和功能上仿生骨组织工程用生物材料的制备及应用。并表征该水凝胶支架材料的结构、力学性能及生物降解性；评价血管细胞和成骨细胞在水凝胶支架材料中生长、分化，及新生血管密度、长度和支化结构特征；建立水凝胶支架材料物性与生物性能之间的相互关系。为实现人工骨组织血管化提供关键技术，为其在生物材料和医学领域的应用提供科学依据。

本研究基于虎奶菇高支化多糖和丝素蛋白制备血管化骨组织用水凝胶，用热水从虎奶菇菌核中提取出的一种高支化多糖TM3a，用重沉淀分级法将TM3a分级成不同分子量的级分，采用氯乙酸对TM3a级分进行化学修饰制备羧甲基化多糖（CTM3a）；13 C核磁共振光谱及元素分析结果显示C3，C4及C6处的羟基被部分取代；用粘度计、静态激光光散射和动态光散射仪测定它们在pH=7.4的磷酸缓冲盐溶液（PBS）中37 ℃下的特性粘数、分子量、均方根旋转半径、第二维利系数和流体力学半径，由此建立CTM3a的Mark-Houwink等方程；基于高分子溶液理论，成功计算出它的分形维数及链构象参数；由此表明：CTM3a高支化多糖羧甲基化衍生物在溶液中呈伸展的球形链构象；将不同分子量的CTM3a溶解在PBS中，分别用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）活化，在37 ℃下与丝素蛋白交联制备高支化多糖-丝素水凝胶，用去离子水清洗且冷冻干燥后得高支化多糖-丝素支架。采用红外光谱、扫描电镜、Instron、溶胀法研究高支化多糖-丝素多孔支架的化学结构、形貌，压缩模量、压缩屈服伸长率及在PBS介质中的溶胀率，以牛血清蛋白作为模型分子考察细胞生长因子或药物分子在该水凝胶支架中的释放行为。结果表明：在EDC和NHS的活化下，CTM3a羧甲基化高支化多糖中的羧基与丝素蛋白中的氨基交联成功制备出高支化多糖-丝素三维贯通多孔支架。CTM3a的分子量对水凝胶支架的力学性能及溶胀率有一定的影响，在所研究分子量范围内的高支化多糖-丝素水凝胶的压缩模量为1.20～4.84 KPa，压缩屈服伸长率为9.45%～25.1%，质量溶胀率及体积溶胀率分别为：10.04～15.22和1.24～1.96。模型分子牛血清蛋白在该高支化多糖-丝素多孔支架中包埋量大，且具有良好的可控释放行为。通过上述基础研究确定了虎奶菇高支化多糖羧甲基化衍生物的结构、分子尺寸及其在溶液中的链构象，并且研究了羧甲基化衍生物的分子量对高支化多糖-丝素水凝胶的多孔结构、力学性能、溶胀率及对小分子蛋白可控释放的影响。本项目为菌类多糖生物大分子及生物材领领域的研究提供了有价值的科学数据，具有重要学术意义。同时，也为实现结构上和功能上仿生骨组织工程用生物材料的制备及进一步的研究和开发应用提供科学依据，具有应用前景。