Establishing a Bioink Assessment Protocol: Gelma and Collagen in the Bioprinting of a Potential In Vitro Intestinal Model.

Abstract

Collagen and gelatin methacryloyl (GelMA) are widely studied biomaterials for extrusion-based bioprinting (EBB) due to their excellent biological properties and ability to mimic the extracellular matrix of native tissues. This study aims to establish a preliminary workflow for approaching EBB by assessing collagen and GelMA printability and biological performance. GelMA was selected for its cost-effectiveness and ease of synthesis, while our collagen formulation was specifically optimized for printability, which is a challenging aspect of bioprinting. A parallel evaluation of their printability and biological performance is provided to develop a preliminary 3D intestinal model replicating the submucosa, lamina propria, and epithelial layer. Rheological analyses demonstrated that both materials exhibit a shear-thinning behavior. Collagen (u-CI) displayed a shear-thinning parameter p = 0.1 and a consistency index C = 80.62 Pa·s, while GelMA (u-GI) exhibited a more pronounced shear-thinning effect and enhanced shape retention (p = 0.06, C = 286.6 Pa·s). Postextrusion recovery was higher for collagen (85%), compared to GelMA (45%), indicating its greater mechanical resilience. Photo crosslinking improved hydrogel stability, with an increase in storage modulus G' for both materials. Printing tests confirmed the suitability of both hydrogels for bioprinting, with GelMA demonstrating higher print fidelity than collagen. Dimensional stability assessments under incubating conditions revealed that collagen constructs maintained their shape for 14 days before degradation, whereas GelMA constructs exhibited a gradual decrease in diameter over 21 days. Cell culture studies showed that human skin fibroblasts (HSFs) and human colon adenocarcinoma cells (HCT-8) could be successfully cocultured in an optimized RPMI 1640-based medium. AlamarBlue assays and Live/Dead staining confirmed high cell viability and proliferation within both hydrogel matrices. Notably, HSFs in GelMA exhibited more elongated morphologies, likely due to the material's lower stiffness (380 Pa) compared to collagen (585 Pa). HCT-8 cells adhered more rapidly to GelMA constructs, forming colonies within 7 days, whereas on collagen, colony formation was delayed to 14 days. Finally, a layered intestinal model was fabricated, and immunostaining confirmed the expression of tight junction (ZO-1) and adhesion (E-cadherin) proteins, validating the epithelial monolayer integrity. These findings highlight the potential of collagen and GelMA in 3D bioprinting applications for gut tissue engineering and pave the way for future developments of in vitro intestinal models.