Amine-Selective Crosslinking of Collagen via Pre-Activated L-Glutamic Acid for Maintaining Ionic Interactions and Enhancing Mechanical and Biological Performance.

Collagen-based biomaterials possess many advantages, such as low immunogenicity, biodegradability, biocompatibility, hydrophilicity, and ease of processability. Nevertheless, natural collagen has inherent limitations as an in vivo scaffold, including insufficient mechanical strength, low thermal stability, and low resistance to enzymatic degradation. To overcome these drawbacks, various approaches have been studied, such as mixing collagen with other biopolymers or inducing physical and chemical crosslinking. However, using non-biologically derived polymers or crosslinking agents carries the risk of persistence in the body, potentially causing cytotoxicity. Considering this, recent studies have reported that the molecular flexibility of collagen networks can be improved by activating the carboxyl groups of collagen chains using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide and then crosslinking them through amide bonding with the amino groups present in the collagen chains, or by adding free L-lysine to induce a crosslinking reaction. When the carboxyl groups of collagen are activated and form covalent bonds with amino groups, native ionic interactions (e.g., salt bridges) may be reduced, which can potentially influence the stability of its inherent higher-order structure. In this study, we proposed a selective amine-targeted cross-linking strategy designed to minimize modification of collagen carboxyl groups while enhancing mechanical properties and cellular compatibility. First, free L-glutamic acid was pre-activated to cross-link collagen chains through amide bonds with the amino groups of L-lysine residues, thereby providing a cross-linking pathway intended to reduce the involvement of collagen carboxyl groups in the reaction. By controlling the concentration of L-glutamic acid, the cross-linking rate of the collagen could be controlled within a range of 10.26% to 25.02%. All cross-linked collagen scaffolds exhibited higher tensile strength compared to non-cross-linked scaffolds. Although the scaffolds with a high cross-linking rate (25.02%) displayed excellent mechanical properties, their cellular compatibility was relatively low. Conversely, collagen scaffolds with cross-linking rates of 10.26% and 14.43% demonstrated excellent mechanical properties and very high cellular compatibility, suggesting potential applications in the fields of biomedicine and tissue engineering. The present findings are consistent with the proposed selective cross-linking strategy; however, direct experimental verification of collagen carboxyl-group preservation will require complementary analytical studies.