Exploring the impact of volumetric additive manufacturing of photo-crosslinkable gelatin on mesenchymal stromal cell behavior and differentiation

Abstract

This study investigates photo-crosslinkable gelatin-based hydrogels - thiolated gelatin (GelSH) and gelatin norbornene (GelNB) - for volumetric additive manufacturing (VAM). GelSH was synthesized with degrees of thiol substitution (DS) of 39 %, 54 %, and 63 %, and GelNB with a DS of 60 % (with respect to primary amine content). These were combined into GelNB-GelSH photo-resins at 5, 7.5, and 10 % (w/v) and crosslinked via thiol-ene chemistry. Physico-chemical analysis showed that increasing DS and polymer concentration reduced swelling and increased moduli. VAM enabled the fabrication of high-resolution 3D hydrogel constructs from optimized formulations, demonstrating the ability to encapsulate mesenchymal stromal cells (MSCs) within a mechanically tunable, cell-supportive hydrogel environment. Film-cast hydrogels, also with embedded MSCs, served as comparative controls. VAM-printed constructs exhibited significantly higher alkaline phosphatase activity and calcium deposition, indicating enhanced osteogenesis. In contrast, chondrogenic and adipogenic differentiation were more pronounced in film-cast samples, due to their lower crosslinking density and stiffness. These findings emphasize the importance of matrix mechanics in guiding stem cell differentiation and demonstrate the potential of VAM for producing complex, functional scaffolds for tissue engineering. This work supports further development of tunable gelatin-based bioresins for applications requiring lineage-specific differentiation, including those targeting softer tissue types.