Cai Guolong, Zhao Weikun, Zhu Tianhao, Oliveira Ana L, Yao Xiang, Zhang Yaopeng
State Key Laboratory of Advanced Fiber Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Materials Science and Engineering, Donghua University, Shanghai 201620, People's Republic of China.
Universidade Católica Portuguesa, CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Escola Superior de Biotecnologia, Porto 4169-005, Portugal.
Regen Biomater. 2025 Mar 20;12:rbaf019. doi: 10.1093/rb/rbaf019. eCollection 2025.
Silk fibroin (SF) hydrogels possess excellent biocompatibility and biomimetic properties of the extracellular matrix. Among them, the mild chemical crosslinked SF hydrogels show great application potential in the fields of 3D cell culture and tissue repairing and thus have attracted widespread attention. However, the mobility of hydrophobic chain segments of SF molecules in these chemical crosslinked hydrogels can easily cause the molecules to undergo a self-assembly process from random coil to -sheet conformation due to its lower energy state, thus inducing an inevitable conformational transition process. This process further leads to dynamic changes of important material features, such as the hydrogel pore size and mechanical properties, which can probably bring some non-negligible and unknown impacts on cell behaviors and their biomedical applications. In this study, a typical mild crosslinking system composed of horseradish peroxidase and hydrogen peroxide was chosen to prepare SF hydrogels. A feasible protein conformational transition rate controlling strategy based on hydrogel crosslinking density regulation was also proposed. Our results demonstrate that the lower the hydrogel crosslinking density, the faster the conformational transition rate. Subsequently, SF hydrogels with different conformational transition rates were successfully constructed to investigate the impact of the protein conformational transition rate accompanied with initial crosslinking density on the proliferation and chondrogenic differentiation of encapsulated stem cells. Results comprehensively illustrated that the conformational transition process could effectively regulate cell behavior. The hydrogel with an appropriate conformational transition rate obviously promoted the proliferation and chondrogenesis of encapsulated stem cells, while too fast or too slow transition processes slowed down these cell activities. These findings are hopefully to provide valuable guidance for the development and efficient usage of SF hydrogels in the fields of 3D cell culture and tissue engineering.
丝素蛋白（SF）水凝胶具有优异的生物相容性和细胞外基质的仿生特性。其中，温和化学交联的SF水凝胶在三维细胞培养和组织修复领域展现出巨大的应用潜力，因此受到广泛关注。然而，在这些化学交联水凝胶中，SF分子疏水链段的流动性容易导致分子因其较低的能量状态而从无规卷曲构象自组装成β-折叠构象，从而引发不可避免的构象转变过程。这一过程进一步导致重要材料特性的动态变化，如水凝胶孔径和力学性能，这可能会对细胞行为及其生物医学应用带来一些不可忽视的未知影响。在本研究中，选用了由辣根过氧化物酶和过氧化氢组成的典型温和交联体系来制备SF水凝胶。还提出了一种基于水凝胶交联密度调控的可行的蛋白质构象转变速率控制策略。我们的结果表明，水凝胶交联密度越低，构象转变速率越快。随后，成功构建了具有不同构象转变速率的SF水凝胶，以研究伴随初始交联密度的蛋白质构象转变速率对包封干细胞增殖和软骨分化的影响。结果全面表明，构象转变过程可有效调节细胞行为。具有适当构象转变速率的水凝胶明显促进了包封干细胞的增殖和软骨形成，而过快或过慢的转变过程则减缓了这些细胞活动。这些发现有望为SF水凝胶在三维细胞培养和组织工程领域的开发和高效应用提供有价值的指导。
