基于琼脂糖/壳聚糖共混凝胶模型的壳聚糖生物相容性的机理研究

目的 以琼脂糖/壳聚糖共混凝胶为模型,研究壳聚糖材料生物相容性的可能机理.方法 通过共混法,制备出一系列不同壳聚糖含量的琼脂糖/壳聚糖共混凝胶.利用傅里叶变换红外光谱(FTIR)分析共混凝胶的化学基团,利用荧光素-4-异硫氰酸酯(FITC)标记法观察琼脂糖和壳聚糖之间的可共混性.通过Zeta电势测量共混凝胶的电荷,利用二喹啉甲酸(BCA)法分别测定胎牛血清(FBS)总蛋白和牛血清白蛋白(RSA)在共混凝胶上的吸附,利用酶联免疫吸附(ELISA)法测定纤黏连蛋白(FN)在共混凝胶上的吸附.细胞实验以人微血管内皮细胞系(HMEC-1)为模型,通过观测细胞的黏附、增殖和形态来评价共混凝胶的细胞相容性.结果 琼脂糖/壳聚糖共混凝胶含有壳聚糖特征性的化学基团.琼脂糖和壳聚糖之间存在着良好的可共混性,壳聚糖的氨基基团在共混凝胶中呈均匀分布.在pH酸性条件下(pH 3.0)共混凝胶带有较强的正电荷,然而在pH中性条件下(pH 7.4)所有共混凝胶的Zeta电势均降低至0 mV附近.各组共混凝胶之间对FBS总蛋白以及BSA的吸附差异无统计学意义,但是共混凝胶对FN的吸附却随着壳聚糖含量的升高而显著升高.细胞实验结果显示:随着壳聚糖含量的提高,共混凝胶的细胞相容性有明显改善,HMECs在壳聚糖含量较高的凝胶上表现出良好的黏附、铺展和增殖.结论 相对于血清中的其他蛋白,壳聚糖组分对FN存在优先吸附,从而能够促进细胞在共混凝胶表面的黏附铺展.与传统观点不同,本研究发现壳聚糖的生物相容性与其所携带的正电荷无关.

Investigation on the mechanisms for biocompatibility of chitosan using agarose/chitosan blended hydrogels as a model

Objective To investigate the possible mechanisms for biocompatibility of chitosan material using agarose/chitosan blended hydrogels as a model. Methods A series of agarose/chitosan blended hydrogels with different chitosan content were prepared by the blending method. The chemical groups of the blended hydrogels were analyzed by the Fourier transform infrared （FTIR） spectroscopy. The blending compatibility between the agarose and chitosan was evaluated with the fluorescein-4-isothiocyanate （FITC） staining method. The charge of the blended hydrogels was determined by the zeta potential measurement. The adsorption of total fetal bovine serum （FBS） proteins and bovine serum albumin （BSA） on the blended hydrogels was measured by the bicinchoninic acid （BCA） method. The adsorption of fibronectin （FN） on the blended hydrogels was measured with ELISA. Cell culture experiment adopted human microvascular endothehal cell hne （HMEC-1） as the model. The cytocompatibility was studied by evaluating adhesion, proliferation, and morphology of the cells on the blended hydrogels. Results Characteristic chemical groups of chitosan could be detected in the agarose/chitosan blended hydrogels. The chitosan had a good blending compatibility with the agarose. The amino groups of chitosan were uniformly distributed in the blended hydrogels. The blended hydrogels were strongly positively charged at acidic pH （pH 3.0）, however, the zeta potentials of all the hydrogels were reduced to nearly 0 mV at neutral pH （pH 7.4）. There were no significant differences in the adsorption of total FBS proteins and BSA between the blended hydrogel groups. However, the adsorption of FN on the hydrogels significantly increased with the increase of chitosan content. Cell culture experiment indicated that the eytoeompatibility of the blended hydrogels was significantly improved with the increase of chitosan content. The HMECs exhibited higher levels of adhesion, spreading, and proliferation on the hydrogels with higher chitosan content. Conclusion Results in this study indicated that the chitosan component preferentially adsorbed FN compared to the other serum proteins, leading to adhesion and spreading of the cells on the blended hydrogels. In contrast to prevailing views, it was found in the present study that the bioeompatibility of chitosan did not relate to its positive charge.