Dual Functionalization of Gelatin for Orthogonal and Dynamic Hydrogel Cross-Linking

Abstract

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 norbornenefunctionalized 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.

Editorial: Special Issue on Advanced Biomedical Hydrogels

“…Varying the degree of tetrazine or norbornene modification on gelatin and the molar ratio of the tethering groups in the hydrogel, the elastic moduli ranged from 0.5 to 5 kPa. Chien-Chi Lin and co-workers further report a methods article on the functionalization of gelatin for orthogonal and dynamic cross-linking . They focus on norbornene modified gelatin, as well as modifications that allow dimerization, hydrazone bonding, and boronate ester bonding to allow for dynamic stiffening and control over stress relaxation.…”

Bioinspired Hydrogels as Platforms for Life-Science Applications: Challenges and Opportunities

“…There were identified several general principles that guide the design of hydrogels: (a) the use of one or more approaches to obtain networks with improved mechanical and physical properties, (b) the implementation of new strategies using unconventional polymer networks, and (c) orthogonal design of hydrogels to achieve multiple combined physico-chemical, mechanical, and biological properties [30][31][32][33]. By a careful synthesis and in some cases by combining various approaches (physical, chemical, enzymatic, irradiation), biocompatible hydrogels can be obtained, with antimicrobial and biodegradable properties and mechanical characteristics adjusted to suit the targeted applications.…”

“…Inside the human body, the ECM stiffness (measured through Young's modulus, a measure of the resistance to deformation) presents different values (see Figure 2 from [157]). Thus, it varies from brain (1-3 kPa) and muscle (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) to tendon (136-820 MPa) and bone . The bone is the hardest human tissue (15-40 GPa) [157,158], whereas for the demineralized bone matrix the stiffness decreases (~0.67 MPa [157,159]); the aggregation of endothelial cells in blood vessel is superior in stiff matrices (37.7 kPa) compared to soft matrices (13 kPa) [160].…”

“…Ampholytic swelling behavior in salted solutions is influenced by pH value, influencing the elastic properties of the gelatin gels [280]. Various crosslinking strategies (including physical, chemical, or enzymatical methods [31,32,[281][282][283], or the creation of IPNs with polysaccharides [284][285][286] were considered to realize stable hydrogels at physiological temperature and to improve the mechanical properties.…”

“…[301,302]. Alternatives for overcoming the actual limitations are also given by the nature principle applied in orthogonal crosslinking or selfassembling methods, multi-component and hybrid networks, stimuli-responsive hydrogels and nanogels, 3D printed hydrogels, and electrospun nanofibers [30][31][32][33]. The final costs for biomaterials and the environmental protection cannot be neglected when trying to manufacture them on a large scale.…”