【作者】 丁珊；

【导师】 周长忍；

【作者基本信息】 暨南大学 ， 生物医学工程， 2012， 博士

【摘要】 壳聚糖(CS)及其衍生物在组织修复中的应用研究报道很多，也非常系统，其中壳聚糖的生物功能化是目前国内外研究的热点。本研究以壳聚糖为基材，创新性的利用功能特异的胆固醇对其进行仿生功能化，并系统研究改性壳聚糖的生物相容性和矿化性能，为开辟一类新型的骨组织修复材料奠定了基础。本研究首先将胆固醇与卵磷脂掺入到壳聚糖和羟基磷灰石的水溶液中，经过浇铸成膜，碱液中和、陈化处理后，考察胆固醇和卵磷脂含量对羟基磷灰石微结构的影响。结果表明，卵磷脂与胆固醇的比例增加，羟基磷灰石可由球状转变为棒状，当卵磷脂/胆固醇为5:1时，体系中的羟基磷灰石形貌与尺寸与天然骨组织接近；而无胆固醇与卵磷脂作用的壳聚糖模板中主要以无定形的片状为主。此结果表明胆固醇、卵磷脂具有改变羟基磷灰石微结构的功能。本研究还借助琥珀酸酐将胆固醇接枝到壳聚糖氨基上，合成了具有不同接枝率的胆固醇-g-壳聚糖(CHCS)。研究表明，胆固醇在壳聚糖上的接枝率随着胆固醇投料量的提高略有增加，并呈非线性关系。当投料摩尔比为15:1，10:1和5:1时(壳聚糖糖环数比胆固醇分子)，接枝率(胆固醇分子数/100个壳聚糖糖环)分别为3.71，4.02与5.94。胆固醇改性壳聚糖的疏水性随着接枝率的增加而增大，壳聚糖膜接触角为72.1°，而接枝率为3.71，4.02与5.94的CHCS膜接触角依次为76.6°，81°与85.8°。白蛋白在膜材上的吸附也是随着接枝率的增加而增大，壳聚糖膜上的蛋白吸附量为0.354μg，而接枝率为3.71，4.02与5.94的CHCS膜上的蛋白吸附量依次为7.664μg，8.248μg，16.815μg。同时，研究结果还表明改性后的壳聚糖具有促进成纤维3T3细胞黏附、增殖的效果。3T3细胞在壳聚糖及不同改性程度的胆固醇-g-壳聚糖膜上培养一天后，改性后材料上细胞的铺展明显比纯壳聚糖膜上充分，且有较多的板状伪足与丝状伪足伸出。在支架材料上培养三天后，CHCS材料上的细胞分泌较多的细胞外基质，且随着改性程度的提高，纤维状细胞外基质越明显。模拟体液仿生矿化表明，本研究研制的CHCS所沉积的钙磷盐稳定性更好。支架材料的快速矿化结果表明，24h内钙磷盐在改性后的材料上呈球状，在壳聚糖材料上呈无定形状。48h后，壳聚糖材料上所沉积的钙磷盐为颗粒状与无定形状，而低接枝率的CHCS支架上钙磷盐呈现海岛针状，高接枝率的CHCS材料上钙磷盐呈现有序化片状堆积。与成骨细胞的相互作用研究表明，改性后材料比纯壳聚糖具有更好的促进细胞黏附、增殖的效果。细胞在改性后的材料上伸出的伪足更为丰富，成骨细胞的特征形态维持得更好。细胞在改性后的材料上更容易地呈有序性黏附与增殖。特别是高接枝率的CHCS在一定时间内具有促进成骨细胞增殖和抑制分化的趋势，而7天后则是促进成骨细胞的分化。更多还原

【Abstract】 Chitosan and its derivates have been studying systematically as tissue repairmaterials, especially the biological functionality are focused in current chitosan research.A series of novel chitosan derivates were prepared by innovative cholesterol modificationin this study. The biocompatibility and mineralization of the cholesterol-g-chitosan(CHCS) were explored in detail. The obtained data and theories are hoped to be used forR&P of new bone repair materials.To investigate the effects of the cholesterol and lecithin contents on themicrostructure of hydroxyapatite, cholesterol and lecithin with certain ratio were added tochitosan and hydroxyapatite mix water solution. Then membranes were prepared by thecommon solution cast method and ageing in the lye. The microscopic structure ofhydroxyapatite in the composite membrane was investigated by SEM and TEM. Theresults show that with the increasing proportions of cholesterol and lecithin in thecomposites, the hydroxyapatite morphology changed from the spherical to the rod-likeform. As the mass ratio of lecithin and cholesterol was5:1, the morphology and size ofthe hydroxyapatite in composite materials were close to nature bone tissue. But in thecontrol system without lecithin and cholesterol, the microscopic structures ofhydroxyapatite were almost lamellate. The results revealed that the cholesterol andlecithin would affect the microscopic structure of hydroxyapatite.Cholesterol-g-chitosan (CHCS) was synthesized with succinyl linkages and thechemical composition was verified by Fourier transforms infrared (FTIR) and protonnuclear magnetic resonance (1~H NMR) spectra. The grafting ratio of cholesterol wascharacterized by elemental analysis. The results show that the grafting ratio ofcholesterol on chitosan chain was increased slightly with the quantity raw material ofcholesteryl hemisuccinate (CHS) increasing, but there were no linear correlation.When the raw molar ratio (the number of chitosan sugar ring compare to the numberof cholesterol molecule) were15:1,10:1and5:1, the grafting ratio (the number ofcholesterol molecule connected on100chitosan sugar ring) were3.71,4.02and5.94.The hydrophobility of CHCS was increased with the grafting ratio of cholesterol increasing as well. The contact angle of chitosan membrane was72.1°, but thecontact angles of the CHCS membranes were76.7°,81°and85.8°respective withthe grafting ratio increasing. The mass of BSA absorption on membrane materialsincreased with the grafting ratio increasing. The mass of BSA absorption on chitosanmembrane was0.354μg, but the value increased from7.664μg to16.815μg on CHCSmembrane with the grafting ratio increasing. The results also demonstrate that3T3cells adhesion and proliferation on CHCS were quicker than that on chitosanmembrane. Furthermore lamellipodia and filopodia of fibroblast3T3cells on CHCSmembrane spread out more fully than that on chitosan membrane after cultured1day.Extracellular matrix express on CHCS scaffold more than on chitosan scaffold after3days and the threadiness extracellular matrix increasingly with the graft ratioincreasing.Two biomimetic mineralization systems were performed in this study tocharacterize the mineralize ability of the products: in simulated body fluids (SBF) andrapid mineralization. In the first system, the calcium phosphate apatite precipitated onCHCS membrane was more stable than that on chitosan membrane. In the latter system,spherical granulates were observed on different graft ratio CHCS scaffold at24h bySEM, and on chitosan scaffold was amorphous. After rapid mineralization48h, theshape of calcium phosphate apatite were island needle-like on low graft ratio CHCSscaffold, and ordering lamellar accumulate on high graft ratio CHCS scaffolds. Theresults of the interaction between CHCS and osteoblast show that osteoblast attach andproliferation more ordering, easily and quickly on CHCS membrane than that onchitosan membrane. The feature morphology of osteoblast on CHCS membranemaintain better, and there are more osteoblast pseudopodia stretch out on CHCSmembrane than on chitosan membrane. Especially, high graft ratio CHCS have thetendency of promoting osteoblast proliferation and restraining osteoblast differentiationin certain time, and facilitating osteoblast differentiation after7days.更多还原