Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering

Abstract The simplicity and versatility of hydrazone crosslinking has made it a strategy of choice for the conjugation of bioactive molecules. However, the labile nature of hydrazone linkages and reversibility of this coupling reaction restricts its full potential. Based on the fundamental understanding of hydrazone stability, this problem is circumvented by resonance‐stabilization of a developing N 2 positive charge in a hydrazone bond. A novel chemistry is presented to develop a resilient hydrazone bond that is stable and non‐ reversible under physiological conditions. A carbodihydrazide (CDH) type hydrazide derivative of the biomolecule forms intrinsically stabilized hydrazone‐linkages that are nearly 15‐fold more stable at pH 5 than conventional hydrazone. This chemoselective coupling reaction is catalyst‐free, instantaneous, and virtually non‐cleavable under physiological conditions, therefore can serve as a catalyst‐free alternative to click chemistry. This novel crosslinking reaction is used to tailor a hyaluronan hydrogel, which delivered exceptional hydrolytic stability, mechanical properties, low swelling, and controlled enzymatic degradation. These desired characteristics are achieved without increasing the chemical crosslinking. The in vivo evaluation of this hydrogel revealed neo‐bone with highly ordered collagen matrix mimicking natural bone regeneration. The proximity ligation assay or PLA is used to detect blood vessels, which highlighted the quality of engineered tissue.