Cell-Friendly 3D Bioprinting of Gelatin-Norbornene Bioink via Thiol-Ene Crosslinking Enabled by Interfacial DTT Delivery from Alternating Support Layers.

Abstract

Thiol-ene gelatin-based hydrogels, particularly gelatin-norbornene (GelNB)-based hydrogels, are promising for extrusion-based three-dimensional (3D) bioprinting owing to their rapid oxygen-insensitive crosslinking and excellent biocompatibility. Dithiothreitol (DTT) is commonly used as a thiol crosslinker because of its efficient and well-characterized reaction kinetics and ability to fine-tune the mechanical properties of the resulting hydrogel with minimal changes to its composition. However, application of DTT in cell-laden constructs is limited by cytotoxicity arising from direct cellular exposure to its strong reducing activity. To address this limitation, we introduced an indirect DTT delivery approach, in which DTT was incorporated into a starch-based support material alternately deposited with a GelNB bioink, enabling interfacial diffusion-mediated thiol-ene crosslinking upon light irradiation while minimizing direct cellular exposure to free DTT. Incorporation of 64.8 mM DTT into a 16.7 w/w% starch-based support material produced constructs (10 w/v% GelNB) with mechanical properties, shape fidelity, and stability comparable to those fabricated using direct DTT premixing (6.5 mM). Fibroblasts encapsulated within the constructs prepared via indirect DTT supply exhibited enhanced cytocompatibility (up to a 47% increase in the cell viability), a significantly higher mitochondrial activity, and reduced reactive oxygen species generation compared with those in the constructs prepared via direct DTT premixing in the bioink. These improvements were consistently observed across multiple cell types. The proposed extrusion-based 3D bioprinting strategy offers a practical and cell-friendly approach for expanding the application scope of a DTT-mediated thiol-ene reaction for GelNB-based bioprinting and may be adapted to other bioink systems relying on cytotoxic crosslinkers.