Objective: To manufacture fish swim bladder membrane material by crosslinking techniques, and to explore its physical and chemical properties and cytotoxicity.
Methods: After decellularization, the swim bladders were randomly divided into two groups. The swim bladders were treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) crosslinking method, surface hole making, and freeze-drying in crosslinking group, and only surface hole making and freeze-drying in non-crosslinking group. The physical and chemical properties of the materials were observed, including microstructure by scanning electron microscopy (SEM), mechanical properties (tensile strength and breaking elongation) by universal tensile machine, hydrophilicity by contact angle measuring instrument, porosity by ethanol infiltration method, degradation performance in vitro and thermal stability test, and the components of materials by infrared spectrum analysis. Mouse fibroblasts (L929) were cultured with the extracts of two groups of materials in order to determine the cytotoxicity of materials by using cell counting kit 8 (CCK-8) method.
Results: The porous structure and rough surface of materials were observed by SEM. Compared with the non-crosslinking group, the tensile stress of the crosslinking group was higher, the breaking elongation was lower, and the porosity increased, showing significant differences ( P<0.05). There was no significant difference in contact angle between the two groups ( P>0.05). The degradation was faster within the first 7 days and then tended to be smooth in the two groups. But the degradation rates of crosslinking group were significantly lower than those of non-crosslinking group ( P<0.05). Differential scanning calorimeter showed that the denaturation temperature of the crosslinking group was (75.2±1.3)℃, which was significantly higher than that of the non-crosslinking group [(68.5±0.4)℃] ( t=4.586, P=0.002). Compared with the non-crosslinking group, the crosslinking group produced new C=O bond and N-H bond, and no other new groups were introduced into the cross-linking group. CCK-8 method showed that the absorbance values of the crosslinking group and the non-crosslinking group were not significant when compared with the positive control group ( P>0.05).
Conclusion: The fish swim bladder membrane obtained by crosslinking treatment with EDC/NHS method has good physical and chemical properties, no cytotoxicity, and is expected to be used as a dura mater repair material.
目的: 采用交联技术制备鱼鳔膜材料，检测其理化性能及细胞毒性。.
方法: 取鱼鳔经脱细胞处理后随机分为两组，交联组采用碳二亚胺［1-ethyl-3-（3-dimethylaminopropyl）carbodiimide，EDC］/N-羟基琥珀酰亚胺（N-hydroxysuccinimide，NHS）法交联处理后，行表面制孔及冻干处理；未交联组仅行表面制孔及冻干处理。观测两组材料理化性能，包括扫描电镜观察微观结构，电子万能材料试验机测试力学性能（拉伸强度及断裂伸长率），接触角测量仪检测材料亲水性，乙醇浸润法测定材料孔隙率，体外降解性能及热稳定性检测，以及红外光谱分析支架成分。取小鼠成纤维细胞 L929 与两组材料浸提液培养，细胞检测试剂盒 8（cell counting kit 8，CCK-8）测定细胞毒性。.
结果: 扫描电镜观测两组支架均具有多孔结构，且表面粗糙。与未交联组相比，交联组材料拉伸强度明显增高、断裂伸长率降低、孔隙率增大，差异有统计学意义（ P<0.05）；接触角差异无统计学意义（ P>0.05）。两组材料降解趋势一致，最初 7 d 内降解较快，之后趋于平缓；各时间点交联组降解率低于未交联组，差异均有统计学意义（ P<0.05）。差示扫描量热法检测显示，交联组材料变性温度为（75.2±1.3）℃，高于未交联组的（68.5±0.4）℃，差异有统计学意义（ t=4.586， P=0.002）。红外光谱分析，与未交联组相比，交联组生成了酰胺键中新的 C=O 键和 N-H 键，并且未引入其他新的基团。CCK-8 法检测示，交联组及未交联组 A 值与阳性对照组比较，差异均无统计学意义（ P>0.05）。.
结论: 经 EDC/NHS 法交联处理获得的鱼鳔膜材料具有优良的理化性能，无细胞毒性，有望作为硬膜修复材料。.
Keywords: Fish swim bladder membrane; crosslinking; dura mater repair; physical and chemical properties.