3D Bioprintable hydrogels via enzymatic crosslinking of hyaluronic acid and phenol-functionalized gelatin to mimic extracellular (ECM) network

• Properties of ECM mimics based on phenol-functionalized gelatin and hyaluronic acid can be tuned varying content and crosslinking gradient. • Enzymatic crosslinking using HRP allow to easily control the gradient of the biopolymers employed and the stiffness of the final hybrid hydrogel. • Differential dosage of hyaluronic acid and gelatin can be applied taking inspiration from the ECM of interest and induce variation of hBM-MSCs morphologies and organization. Tissues regeneration remains a complex challenge due to the articulated structure and composition of their extracellular matrix (ECM). 3D bioprinting offers a promising approach to in vitro replicating these articulated structures, but it requires the development of bioinks with suitable biochemical and viscoelastic properties. This study investigates various hydrogel formulations based on gelatin and hyaluronic acid, aiming to replicate the cellular and biochemical gradients found in physiological tissues. The crosslinking was performed enzymatically exploiting horseradish peroxidase (HRP) and H 2 O 2 , once both gelatin and hyaluronic acid were functionalized with tyramine. Following the successful formation of the hydrogels, different formulations were assessed for their morphological, physical and viscoelastic properties. The two most promising formulations were selected as candidates for tissue engineering, and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) tests showed effective cell interactions. 3D Bioprinting of the two formulations, each with different concentrations of gelatin and hyaluronic acid, resulted in constructs with distinct cell morphologies, demonstrating their suitability for mimicking the biochemical gradient found in microenvironmental extracellular matrix (ECM).