生物支架材料胶原膜交联前后的特性分析

目的 比较胶原膜交联前后的生物特性,为胶原膜的应用奠定基础. 方法 取市售新鲜牛尾,分离肌腱,制备胶原蛋白,以紫外分光光度计测定胶原蛋白含量;采用SDS-聚丙烯酰胺凝胶电泳(SDS-polyaerylamide gelelectrophoresis,SDS-PAGE)波长扫描,氨基酸含量检测分析胶原蛋白特性.将制备的胶原蛋白,真空冷冻干燥,制备胶原膜,采用戊二醛交联.大体观察及扫描电镜观察交联前后胶原膜形态,并测定胶原酶溶解时间、pH值、吸水性能及抗撕强度.取自愿捐赠的骨髓制备BMSCs,按2×103/100 μL浓度接种于交联前后胶原膜上培养7 d,测量细胞吸光度(A)值及扫描电镜观察. 结果 制备的胶原蛋白含量约为2 mg/mL,最大吸收波长约为230 nm.胶原蛋白氨基酸分析显示,甘氨酸含量约为21.47%,脯氨酸12.04%,羟脯氨酸10.18%,未检出色氨酸.交联前胶原膜为白色海绵状,孔径较大且不均匀,pH值为4.5～5.0;扫描电镜观察为孔网状结构,孔径大;吸水力为其重量的61倍,抗撕强度为210 g/cm3,胶原酶溶解时间为30 min.交联后胶原膜变薄,无色,半透明,致密,柔韧性好;镜下可见致密排列的胶原纤维成网状;pH值为6.5～7.0,吸水力降低,抗撕强度约为3 400 g/cm3,胶原酶溶解时间为90 min.细胞增殖实验表明,细胞在交联前后的胶原膜上均能良好生长,A值差异无统计学意义(P＞0.05).扫描电镜见BMSCs在交联后的胶原膜上生长良好. 结论 从牛肌腱中成功提取胶原蛋白,并制备胶原蛋白膜.经化学交联剂交联后,胶原膜仍具有良好的生物学特性,且理化性质优于交联前,可作为生物支架材料,用于皮肤创伤修复等研究领域.

ANALYSIS OF PROPERTIES OF COLLAGEN MEMBRANES BEFORE AND AFTER CROSSLINKED

OBJECTIVE: To compare the properties of collagen membranes before and after crosslinked and to establish the foundation of application of collagen membranes. METHODS: Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophilization. And then the biocharacteristics of the membranes before and after glutaraldehyde crosslinked were compared. BMSCs separated from volunteer's bone marrow were seeded on collagen membranes before and after crosslinked by 2 x 10(3) in 100 microL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). RESULTS: The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecular weight of main bands was more than 66.2 x 10(3), the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% praline and 10.18% hydroxyproline. No tryptophan was found. Before crosslinked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3,400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crosslinked collagen membranes or on after-crosslinked ones. CONCLUSION: As biomaterial scaffolds, after crosslinked collagen membranes were better than before-crosslinked ones.