Desenvolvimento de modelo de derme humana bioimpressa em 3D: Estabelecimento de protocolo in vitro e perfil metabólico do uso de bioestimuladores de colágeno

Abstract

Gelatin-alginate-based bioinks for 3D bioprinting are widely used due to their inherent advantages, such as biocompatibility, biodegradability, and cell-adhesion properties. While the alginate incorporation addresses the risks that thermal instability poses to post-bioprinting structural fidelity, sustaining long-term hydrogel integrity under cell culture conditions remains a critical challenge. This demands a precise balance between effective crosslinking and the preservation of cell viability. We aimed to enhance the stability of fibroblast-laden gelatin-alginate in vitro using a dual-crosslinking approach, thereby establishing a 3D simplified dermis model to profile the metabolomic signatures of common collagen biostimulators. For this purpose, fibroblast (HFF-1) viability and adhesion were evaluated to determine an optimal, biocompatible crosslinking protocol. Subsequently, metabolites were extracted from both the 3D bioprinted construct and its supernatant. As a result, an effective dual-crosslinking strategy was stablished, consisting of 15 minutes of calcium chloride (CaCl2) 500 mM followed by 15 minutes of 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) 100 mM / N-hydroxysuccinimide (NHS) 20 mM. Construct stability and sustained cell viability were confirmed, enabling accurate metabolomic profiling at 48h in vitro. Notably, calcium hydroxylapatite (CaHA) induced metabolic reprogramming toward a regenerative state, whereas poly(D,L-lactic acid) (PDLA) drove adaptations characterized by sphingolipid remodeling and altered cellmicroenvironment communication. Overall, our dual-crosslinked dermis model demonstrates in vitro stability, providing a validated and effective platform for evaluating the metabolic impact of biomaterials in tissue engineering.