Gelatin-based hydrogels are widely used in biomedical fields because of their abundance of bioactive motifs that support cell adhesion and matrix remodeling. Although inherently bioactive, unmodified gelatin exhibits temperature-dependent rheology and solubilizes at body temperature, making it unstable for three-dimensional (3D) cell culture. Therefore, the addition of chemically reactive motifs is required to render gelatin-based hydrogels with highly controllable cross-linking kinetics and tunable mechanical properties that are critical for 3D cell culture. This article provides a series of methods toward establishing orthogonally cross-linked gelatin-based hydrogels for dynamic 3D cell culture. In particular, we prepared dually functionalized gelatin macromers amenable for sequential, orthogonal covalent cross-linking. Central to this material platform is the synthesis of norbornene-functionalized gelatin (GelNB), which forms covalently cross-linked hydrogels via orthogonal thiol-norbornene click cross-linking. Using GelNB as the starting material, we further detail the methods for synthesizing gelatin macromers susceptible to hydroxyphenylacetic acid (HPA) dimerization (i.e., GelNB-HPA) and hydrazone bonding (i.e., GelNB-CH) for on-demand matrix stiffening. Finally, we outline the protocol for synthesizing a gelatin macromer capable of adjusting hydrogel stress relaxation via boronate ester bonding (i.e., GelNB-BA). The combination of these orthogonal chemistries affords a wide range of gelatin-based hydrogels as biomimetic matrices in tissue engineering and regenerative medicine applications.

中文翻译：

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用于正交和动态水凝胶交联的明胶双功能化

明胶基水凝胶因其丰富的支持细胞粘附和基质重塑的生物活性基序而广泛应用于生物医学领域。尽管具有固有的生物活性，但未改性的明胶表现出温度依赖性流变性并在体温下溶解，使其对于三维 (3D) 细胞培养不稳定。因此，需要添加化学反应基序以使明胶水凝胶具有高度可控的交联动力学和可调的机械性能，这对于 3D 细胞培养至关重要。本文提供了一系列建立用于动态 3D 细胞培养的正交交联明胶基水凝胶的方法。特别是，我们制备了适合顺序、正交共价交联的双功能化明胶大分子单体。该材料平台的核心是降冰片烯功能化明胶（GelNB）的合成，它通过正交硫醇-降冰片烯点击交联形成共价交联的水凝胶。使用 GelNB 作为起始材料，我们进一步详细介绍了合成明胶大分子单体的方法，该明胶大分子单体易受羟苯乙酸（HPA）二聚（即 GelNB-HPA）和腙键合（即 GelNB-CH）的影响，用于按需基质硬化。最后，我们概述了合成明胶大分子单体的方案，该明胶大分子单体能够通过硼酸酯键合调节水凝胶应力松弛（即 GelNB-BA）。这些正交化学的组合提供了广泛的明胶基水凝胶作为组织工程和再生医学应用中的仿生基质。