Department of Urology, Affiliated Drum Tower Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu, PR China.
Tissue Eng Part C Methods. 2010 Oct;16(5):1201-11. doi: 10.1089/ten.TEC.2009.0311.
In this study, we compared four decellularization protocols and finally developed an optimized one through which a porcine bladder acellular matrix (BAM) with well-preserved extracellular bioactive factors had been prepared. In this protocol, the intact bladder was treated with trypsin/ethylenediaminetetraacetic acid to remove the urothelium, then with hypotonic buffer and Triton X-100 in hypertonic buffer to remove the membranous and cytoplasmic materials, and finally with nuclease to degrade the cellular nuclear components. Bladder distention and mechanical agitation were simultaneously used to facilitate cell removal. Meanwhile, several preservative techniques, including limitation of wash time, supplement with inhibitors of proteinase, control of the pH value and temperature of the wash buffer, ethylene oxide sterilization, and lyophilization of the scaffold for storage, were used to protect the extracellular bioactive factors. This decellularization protocol had completely removed the cellular materials and well preserved the extracellular collagen, sulfated glycosaminoglycan (GAG), and bioactive factors. The preserved bioactive factors had a great potential of promoting the proliferation and migration of both human bladder smooth muscle cell and human umbilical vein endothelial cell. It was also found that the amount of two representative bioactive factors, platelet-derived growth factor BB and vascular endothelial growth factor, was positively correlated with the sulfated GAG content in the porcine BAM, implying that the amount of sulfated GAG might be a determinant for preservation of bioactive factors in the decellularized tissues. In conclusion, the porcine BAM with well-preserved extracellular bioactive factors might be a favorable scaffold for tissue engineering applications.
在这项研究中，我们比较了四种脱细胞方案，最终通过优化得到了一种方法，制备出了具有良好保留的细胞外生物活性因子的猪膀胱脱细胞基质（BAM）。在该方案中，完整的膀胱先用胰蛋白酶/乙二胺四乙酸处理以去除尿路上皮，然后用低渗缓冲液和高渗缓冲液中的 Triton X-100 去除膜和细胞质材料，最后用核酸酶降解细胞核成分。膀胱扩张和机械搅拌同时用于促进细胞去除。同时，使用了几种保存技术，包括限制洗涤时间、添加蛋白酶抑制剂、控制洗涤缓冲液的 pH 值和温度、环氧乙烷灭菌以及支架的冻干储存，以保护细胞外生物活性因子。该脱细胞方案完全去除了细胞材料，并很好地保留了细胞外胶原蛋白、硫酸化糖胺聚糖（GAG）和生物活性因子。保留的生物活性因子具有促进人膀胱平滑肌细胞和人脐静脉内皮细胞增殖和迁移的巨大潜力。还发现，两种代表性生物活性因子，血小板衍生生长因子 BB 和血管内皮生长因子的含量与猪 BAM 中的硫酸化 GAG 含量呈正相关，这意味着硫酸化 GAG 的含量可能是保存脱细胞组织中生物活性因子的决定因素。总之，具有良好保留的细胞外生物活性因子的猪 BAM 可能是组织工程应用的理想支架。
