Poster O8 - Multifunctional Gelatin-dithiolane Hydrogels for Tissue Engineering

Introduction: Biomaterials that integrate multiple functionalities, mimic the extracellular matrix (ECM) microenvironment to support cellular growth, and adhere robustly to damaged tissues are highly needed to advance tissue engineering. Protein-based biomaterials are promising due to their inherent biocompatibility, biomimicry, biodegradation, and cell-supportive properties. Herein, by leveraging the unique ability of dithiolanes to generate on-demand in-situ thiols, we developed a new class of dithiolane-modified, protein-based biomaterial that combines unique, seemingly opposing functions for tissue engineering. Dithiolane-modified gelatin, a model protein used herein, enabled photoinitiator-free photo-crosslinking to form multi-functional gelatin-dithiolane (GelDT) hydrogels, which displayed exceptional long-term stability in cell culture media (>28 days) to support the growth of both surface-seeded and encapsulated cells. GelDT hydrogels allowed post-gelation tuning of matrix stress-relaxation rate, via responding to exogenous thiols, independently of other parameters. Furthermore, GelDT supported efficient 3D bioprinting due to its shear-thinning ability and demonstrated robust tissue adhesion in various contexts (bare skin, ex-vivo) due to covalent disulfide coupling with endogenous tissue thiols. Together, this study presents a novel multi-responsive and multi-functional protein-based biomaterial, anticipated to advance the field of tissue engineering and regeneration.

Materials and

Methods: Gelatin was modified with lipoic acid (dithiolane) to prepare GelDT precursor Lyophilized GelDT was dissolved in PBS at 50 °C at different concentrations, followed by exposure to UV light (365nm) to form hydrogels. The mechanical properties of the hydrogels were quantified using a mechanical tester. Stimuli responsiveness of GelDT was highlighted by post-gelation tuning of mechanical properties using PEG-4SH (stimuli). The biocompatibility of hydrogels in both 2D and 3D was evaluated using multiple cell lines including 3T3 fibroblasts, b4g12 corneal endothelial, and human cholangiocarcinoma (HuCCT-1). Quantification of 3T3 cell adhesion, spreading, and morphometric analysis of b4g12 corneal endothelial monolayer in 2D was done by analyzing fluorescently stained images. Comparison of HuCCT-1 tumoroid size in GelDT and GelMA (control) was done manually using Image J while the metabolic activity was measured using AlamarBlue assay. The applicability of GelDT as bioink was highlighted using extrusion bioprinting with fusion and filament collapse tests. The bio-adhesive nature of GelDT, ex-vivo, was assessed and compared to commercial Tisseel using rabbit corneas with GelDT directly crosslinked on the epithelial side both with and without an epithelial wound. All the experiments were run in triplicates and all the data was presented as mean ± SD

Results, Conclusions, and Discussions: Lipoic acid (dithiolane) conjugation to gelatin resulted in the fabrication of GelDT hydrogels via initiator-free photopolymerization alleviating the concerns related to cytotoxicity of photoinitiators. The resulting hydrogels demonstrated tunable mechanical properties, pre-gelation, which could be controlled by varying gelatin content. Increasing gelatin content significantly increased the compressive modulus (Fig.1a) of GelDT hydrogels but the stress relaxation was significantly reduced (Fig.1b) likely due to the restricted ability of chains to relax under stress. Considering the dynamic nature of disulfide bonds in GelDT, hydrogels did not disintegrate over 30 days, regardless of the initial solvent conditions, suggesting their suitability for long-term culture applications (Fig.1c). Additionally, GelDT also demonstrated post-gelation tunability of matrix stress-relaxation rate in response to exogenous thiols, independently of its compressive modulus (Fig.1d & e) likely due to associative dynamic interactions. Assessment of biocompatibility with 3T3 fibroblast cells cultured on 15% GelDT and GelMA (control) hydrogels revealed statistically similar adhesion density (Fig.1f and g) but a notable and significant increase in spreading was observed for cells on 15% GelDT surface (Fig.1h). Further, GelDT supported growth of corneal endothelial cell monolayer which expressed ZO-1 tight junction protein (Fig.1i). Morphometric analysis showed statistically similar cell circularity (Fig.1j) and area (Fig.1k) compared to polystyrene control. Furthermore, Phase-contrast images (Fig. 1l) at day 7 showed significantly larger tumoroids (Fig.1m) in GelDT than in traditional GelMA with the same prepolymer content. This could be attributed to the presence of dynamic disulfide crosslinks in GelDT, highlighting its excellence as a 3D scaffold. Besides, GelDT proved to be an excellent candidate as shear thinning, photoinitiator-free bioink for commonly employed extrusion bioprinting by demonstrating extrudability at various concentrations (Fig.1n) thus allowing bioprinting of 3D complex shapes (i.e., a crown structure) using embedded bioprinting (Fig.1o). In addition, interaction between photo-induced thiols in GelDT and native thiols in tissue (Fig.1p) lead to robust adhesion to both skin (Fig.1q) and cornea tissue (Fig.1r and s) due to formation of disulfide bridges. Overall, GelDT represents a novel, multi-responsive, and multi-functional protein-based biomaterial with anticipated applications that extends across various domains, significantly advancing the fields of tissue engineering and regeneration.

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