紫外线与戊二醛双重交联法构建真皮支架的研究

Construction of dermal skeleton by double cross-linking with giutaraldehyde and ultraviolet radiation

目的比较不同预冻温度及交联方法对胶原膜内部超微结构及成纤维细胞增殖的影响。方法将牛Ⅰ型胶原溶液（10g／L）分别在-20℃和-80℃预冻12h后，放入-70℃冻干机内冷冻干燥48h。利用扫描电镜测量2种预冻温度下胶原膜的内部孔径，比较戊二醛交联法及紫外线＋戊二醛双重交联法对胶原孔径的影响，通过噻唑蓝（MTT）法检测人成纤维细胞在不同交联法制备的胶原膜中的增殖情况。结果-20℃预冻胶原膜的孔径为（172±37）μm，-80℃预冻胶原膜孔径为（99±24）μm，选择后者进行后续实验。经戊二醛交联后胶原膜孔径缩小，种植后第8天，成纤维细胞的吸光度值为1．534±0．013；紫外线联合戊二醛双重交联后胶原膜原有孔径不变，种植后第8天成纤维细胞的吸光度值为3．778±0．010，与前者比较，差异有统计学意义（P〈0．05）。结论-80℃预冻后经紫外线＋戊二醛双重交联法构建的胶原膜，可作为体外真皮支架替代物。

Objective To investigate the effects of preemptive freezing with different temperature and cross-linking methods on the uhrastructure of collagen membrane and its influence on human fibroblast proliferation. Methods Bovine collagen type [ solution in concentration of 10 g/L was preliminarily frozen at - 20 ℃ or - 80 ℃ for 12 hours,and lyophilized at - 70 ℃ for 48 hours. The diameter of apertures in collagen membranes prepared with two different preliminary temperatures were observed by scanning electron microscope （SEM）and compared. The preliminary freezing temperature of -80℃ was used for the following study. The apertures of collagen membrane performed with cross-linking glutaraldehyde and ultraviolet （UV） radiation cross-linking with glutaraldehyde （ double cross - linking ） after preliminary freezing were also compared . The proliferation of human fibroblasts inoculated in above cross-linking collagens were assessed by MTT assay,in terms of absorption value. Results The mean diameter of apertures of collagen membrane pre-frozen at -20 ℃ was （ 172 ±37） μm,while that at -80 ℃ was（99 ±24） μm. The apertures of collagen membrane were reduced in size after glutaraldehyde cross-linking,while those of double cross-linking showed no change in size. There was obvious difference in absorption value of fibroblasts 8 days after seeding between above two cross-linking methods （ 1. 534 ± 0. 013 for glutaraldehyde cross-linking,3. 778 ± 0. 010 for double cross-linking, P 〈 0. 05 ）. Conclusion The collagen membrane after preliminary freezing at -80 ℃ and double cross-linking with UVradiation and glutaraldehyde may be used as a dermal skeleton substitute.