脱细胞真皮基质制备及其生物相容性研究

发布时间：2018-02-13 03:28

  本文关键词： 软骨组织工程 脱细胞真皮基质 支架材料 Ⅱ型胶原　出处：《中国修复重建外科杂志》2014年06期 　论文类型：期刊论文

【摘要】：目的制备脱细胞真皮基质,检测其细胞及组织相容性,探讨其作为软骨组织工程支架材料的可行性。方法取新生小牛背部真皮组织,以0.5%SDS溶液水平振荡脱细胞,0.5%胰蛋白酶行胶原纤维结构重塑,甲醛浸泡进行交联,0.5%硫酸软骨素溶液中超声振荡进行表面修饰,制备脱细胞真皮基质支架材料。乙醇排除法检测结构重塑前后材料孔隙率变化;MTT法检测材料细胞毒性;将材料植入成年SD大鼠背部皮下,评价组织相容性。采用以3∶7比例共培养的第2代新西兰大白兔软骨细胞与骨髓基质细胞作为种子细胞,种植于脱细胞真皮基质支架材料(实验组),48 h后扫描电镜观察细胞黏附情况;RT-PCR和Western blot检测接种于支架的种子细胞Ⅱ型胶原mRNA和蛋白的表达,并与单纯培养的种子细胞(对照组)进行比较。结果经0.5%胰蛋白酶溶液结构重塑后的脱细胞真皮基质支架材料表面光滑、孔隙均匀;孔隙率达85.4%±2.8%,显著高于重塑前的支架(72.8%±5.8%)(t=—4.384,P=0.005);细胞毒性检测为1级,合格;埋植于大鼠背部皮下后,随时间延长,组织炎性细胞数呈减少趋势(P0.05)。种子细胞接种后,扫描电镜示大量细胞贴附于支架上,细胞形态饱满,可见表面微绒毛和分泌现象。RT-PCR与Western blot检测结果示,实验组和对照组Ⅱ型胶原mRNA和蛋白表达量比较,差异均无统计学意义(t=1.265,P=0.235;t=0.935,P=0.372)。结论经脱细胞-结构重塑-交联-表面修饰制备的脱细胞真皮基质结构良好,种子细胞能大量黏附于支架材料上,细胞Ⅱ型胶原表达水平无明显改变,有望作为软骨组织工程支架材料。
[Abstract]:Objective to prepare acellular dermal matrix, detect its cell and histocompatibility, and explore its feasibility as scaffold material for cartilage tissue engineering. The collagen fibers were reconstructed by 0.5% acellular trypsin in 0.5% SDS solution, and the surface was modified by ultrasonic oscillation in 0.5% chondroitin sulfate solution immersed in formaldehyde. Acellular dermal matrix scaffolds were prepared. The changes of porosity before and after structural remodeling were detected by ethanol exclusion and cytotoxicity was detected by MTT method. The materials were implanted subcutaneously into the back of adult Sprague-Dawley rats. To evaluate the histocompatibility, chondrocytes and bone marrow stromal cells of the second passage of New Zealand rabbits co-cultured at the ratio of 3: 7 were used as seed cells. The adhesion of acellular dermal matrix scaffolds was observed by scanning electron microscope 48 hours after implantation. RT-PCR and Western blot were used to detect the expression of type 鈪，

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