Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany.
Experimental Surgery, Department of Surgery, CCM|CVK, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
Biomacromolecules. 2023 Dec 11;24(12):5620-5637. doi: 10.1021/acs.biomac.3c00602. Epub 2023 Nov 27.
Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.
已溶胀、胶凝的去细胞细胞外基质 (dECM) 广泛应用于基础研究和转化研究，由于其固有生物活性，可促进结构和功能组织重塑。动物来源的胃蛋白酶已成为几乎所有类型基于胶原蛋白的 dECM 溶解的标准蛋白水解酶。在这项研究中，与木瓜蛋白酶、α-淀粉酶和胶原酶相比，胃蛋白酶用于溶解猪肝 dECM 的潜力。生物活性成分和天然 dECM 特性的最大保留被用作进一步应用酶的决定性标准，重点是最小化蛋白质结构的破坏，并保持在中性 pH 值下物理热凝胶化的能力。溶解的 dECM 消化物和/或其物理凝胶化的水凝胶的流变特性、凝胶动力学、糖胺聚糖含量、蛋白质组组成和生长因子谱进行了表征。这项研究强调了木瓜蛋白酶作为一种植物来源的酶，可以作为动物来源的胃蛋白酶的经济有效的替代品，用于有效溶解 dECM。所得的均匀的木瓜蛋白酶消化的 dECM 保留了其类似于胃蛋白酶消化的热触发凝胶化特性，并且相应的 dECM 水凝胶在成纤维细胞的单细胞力谱实验中表现出增强的生物粘附性。人 HepaRG 细胞在 dECM 凝胶上的活力和增殖与在纯大鼠尾胶原蛋白 I 凝胶上的相似。木瓜蛋白酶不仅对 dECM 溶解非常有效且具有吸引力，而且在用于再生应用的消化人组织衍生的 dECM 时也特别有趣，因为需要避免使用动物来源的材料。
