Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.
Biomaterials. 2011 Jan;32(3):706-13. doi: 10.1016/j.biomaterials.2010.09.017. Epub 2010 Oct 8.
Porcine small intestinal submucosa (SIS) has been widely used in repairing various tissues and organs. Despite this, some SIS products have the capacity to cause variable inflammatory responses after implantation resulting in severe adverse effects due to porcine cell existence. In this study, we described a multi-step method including mechanical disassociation, degrease, enzyme digestion, detergent treatment, freeze-drying and sterilization by irradiation for preparation of SIS. The efficacy of acellularization was evaluated by histological observation and the content of porcine immunoreceptor DAP12 gene. The change of growth factors contents within SIS accompanying with decellularization was quantitatively assessed by ELISA. Inflammatory reaction of SIS implanted subcutaneously in a rat was investigated. The histological examination revealed no remaining cells after enzyme digestion. Moreover, qPCR analysis demonstrated that the content of a porcine immunoreceptor gene DAP12 DNA in final SIS product (SISv) was only 1.05% of that in SIS samples (SISi) prepared by mechanical disassociation. Degrease with methanol/chloroform dramatically reduced the contents of VEGF, b-FGF, TGF-β, and TNF-α within SISii, but further treatment could not significantly reduced the contents of growth factors. SIS implanted into rats showed that inflammatory cells was more accumulated surrounded to SISi at 1, and 2 weeks, but reduced in SISv samples. The degree of inflammatory reaction for SISv was significantly less than that of SISi.
猪小肠黏膜下层（SIS）已广泛用于修复各种组织和器官。尽管如此，一些 SIS 产品在植入后仍具有引起不同炎症反应的能力，这是由于猪细胞的存在而导致严重的不良反应。在本研究中，我们描述了一种多步方法，包括机械分离、脱脂、酶消化、去污剂处理、冷冻干燥和辐照灭菌，以制备 SIS。通过组织学观察和猪免疫受体 DAP12 基因的含量评估脱细胞的效果。通过 ELISA 定量评估 SIS 伴随脱细胞化过程中生长因子含量的变化。研究了 SIS 皮下植入大鼠后的炎症反应。酶消化后组织学检查未见残留细胞。此外，qPCR 分析表明，最终 SIS 产物（SISv）中猪免疫受体基因 DAP12 DNA 的含量仅为机械分离制备的 SIS 样品（SISi）的 1.05%。甲醇/氯仿脱脂可显著降低 SISii 中 VEGF、b-FGF、TGF-β和 TNF-α的含量，但进一步处理不能显著降低生长因子的含量。SIS 植入大鼠后发现，SISi 在 1 周和 2 周时周围炎性细胞更多，但 SISv 样品中则减少。SISv 的炎症反应程度明显低于 SISi。
