3B's Research Group, I3Bs - Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal.
Biomater Sci. 2021 Jan 5;9(1):186-198. doi: 10.1039/d0bm01272f.
Decellularized matrices are attractive substrates, being able to retain growth factors and proteins present in the native tissue. Several biomaterials can be produced by processing these matrices. However, new substrates capable of being injected that reverse local kidney injuries are currently scarce. Herein, we hypothesized that the decellularized particulate kidney porcine ECM (pKECM) could support renal progenitor cell cultures for posterior implantation. Briefly, kidneys are cut into pieces, decellularized by immersion on detergent solutions, lyophilized and reduced into particles. Then, ECM particles are analyzed for nuclear material remaining by DNA quantification and histological examination, molecular conformation by FITR and structural morphology by SEM. Protein extraction is also optimized for posterior identification and quantification by mass spectrometry. The results obtained confirm the collagenous structure and composition of the ECM, the effective removal of nucleic material and the preservation of ECM proteins with great similarity to human kidneys. Human renal progenitor cells (hRPCs) are seeded in different ratios with pKECM, on 3D suspensions. The conducted assays for cell viability, proliferation and distribution over 7 days of culture suggest that these matrices as biocompatible and bioactive substrates for hRPCs. Also, by analyzing CD133 expression, an optimal ratio for specific phenotypic expression is revealed, demonstrating the potential of these substrates to modulate cellular behavior. The initial hypothesis of developing and characterizing a particulate ECM biomaterial as a consistent substrate for 3D cultures is successfully validated. The findings in this manuscript suggest these particles as valuable tools for regenerative nephrology by minimizing surgeries and locally reversing small injuries which can lead to chronic renal disfunction.
去细胞化基质是一种很有吸引力的基质，能够保留天然组织中存在的生长因子和蛋白质。通过处理这些基质可以生产出几种生物材料。然而，目前能够注射的、可逆转局部肾脏损伤的新基质仍然很少。在这里，我们假设去细胞化的颗粒状猪肾脏细胞外基质（pKECM）可以支持肾祖细胞的培养，以备后续植入。简要地说，肾脏被切成小块，用去污剂溶液浸泡进行去细胞化，冻干后再还原成颗粒。然后，通过 DNA 定量和组织学检查分析 ECM 颗粒中残留的核材料，通过傅里叶变换红外光谱（FTIR）分析分子构象，通过扫描电子显微镜（SEM）分析结构形态。还优化了蛋白质提取方法，以便通过质谱法进行后续鉴定和定量。所得结果证实了 ECM 的胶原结构和组成，有效去除了核酸材料，并保留了与人类肾脏非常相似的 ECM 蛋白。将人肾祖细胞（hRPCs）以不同比例与 pKECM 接种在 3D 悬浮液中。进行了细胞活力、增殖和培养 7 天分布的测定，表明这些基质是 hRPCs 的生物相容性和生物活性基质。此外，通过分析 CD133 表达，揭示了一种用于特定表型表达的最佳比例，证明了这些基质在调节细胞行为方面的潜力。开发和表征颗粒状 ECM 生物材料作为 3D 培养一致基质的初始假设得到了成功验证。本文的研究结果表明，这些颗粒作为再生肾脏学的有价值工具，通过减少手术和局部逆转小的损伤，可以减少慢性肾功能衰竭的发生。
