Department of Bioengineering, University of California San Diego, La Jolla, California.
Sanford Consortium for Regenerative Medicine, University of California San Diego, La Jolla, California.
Tissue Eng Part C Methods. 2018 Dec;24(12):697-708. doi: 10.1089/ten.TEC.2018.0180.
Different approaches have investigated the effects of different extracellular matrices (ECMs) and three-dimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM. Impact Statement The article compares different methodologies for the generation of a pancreas-derived hydrogel for tissue engineering applications. The biochemical characterization of the newly generated hydrogel shows that the material retains all the extracellular molecules of the native tissue and is capable of sustaining functionality of the encapsulated beta-cells.
不同的方法研究了不同的细胞外基质（ECM）和三维（3D）培养对胰岛功能的影响，结果令人鼓舞。理想情况下，合适的支架应模拟天然组织的生化组成，因为它可以驱动参与组织稳态和功能的许多信号通路。组织衍生的去细胞生物材料可以保留天然组织的 ECM 组成，使其成为 3D 组织工程应用的理想支架。然而，去细胞化过程可能会影响特定成分的保留，选择合适的去污剂对于保留天然 ECM 组成至关重要。在这项研究中，我们评估了不同去细胞化方案对胰腺细胞外基质（pECM）水凝胶的机械性能和生化组成的影响。新鲜猪胰腺组织被采集、切成小块，在水中冲洗，然后用两种不同的去污剂（十二烷基硫酸钠[SDS]或 Triton X-100）处理 1 天，再在水中处理 3 天。通过 PicoGreen 测定、Hoescht 染色和 H&E 染色证实了有效的去细胞化，各组之间没有差异。还评估了使用蛋白酶抑制剂（PI）的效果。通过 PicoGreen 测定和苏木精和伊红（H&E）染色证实了有效的去细胞化，各组之间没有差异。在适当的条件下，Triton 处理的样品能够形成坚固的水凝胶，而 SDS 的使用对水凝胶的凝胶性质有不利影响。通过定量质谱分析对新鲜猪胰腺和所有去细胞化的 pECM 水凝胶的 ECM 生化组成进行了表征。纤维状胶原蛋白是所有组的主要 ECM 成分，所有生成的水凝胶的含量均高于新鲜胰腺。当与 Triton 组相比时，SDS 处理的水凝胶中这种效果更为明显，显示出对其他 ECM 分子的保留很少。相反，当组织用 PI 预处理并用 Triton X-100 去细胞化时，基底膜和基质细胞蛋白保留得更好，使水凝胶更类似于天然组织。总之，我们表明，研究中评估的所有方案都显示出有效的组织去细胞化，但只有当组织用 PI 预处理并用 Triton 去污剂去细胞化时，水凝胶的生化组成才更接近天然组织 ECM。影响陈述本文比较了用于组织工程应用的胰腺衍生水凝胶的生成的不同方法。新生成的水凝胶的生化特性表明，该材料保留了天然组织的所有细胞外分子，并能够维持包封的β细胞的功能。
