Human gingival fibroblast-mediated remodeling of three-dimensional fibrin hydrogels

Abstract

The extracellular matrix (ECM) critically regulates fibroblast behavior during tissue repair and regeneration. However, how culture dimensionality influences fibroblast-mediated ECM remodeling remains unclear. This study investigated the effects of three-dimensional (3D) fibrin hydrogels on the phenotype and remodeling activity of primary human gingival fibroblasts (GFs) compared to conventional two-dimensional (2D) monolayer cultures. Live/dead staining confirmed high GF viability in both conditions, with elongated and branched cell morphologies in 3D fibrin hydrogels, contrasting with spindle-shaped cells in 2D monolayers. Hematoxylin and Eosin, and Masson’s Trichrome staining revealed progressive fibrin degradation and de novo collagen deposition over 21 days of culturing. Gene expression analysis showed that while FN1, COL1A1, and COL3A1 levels remained relatively stable, TGFB1 expression increased significantly from day 7 to day 14 in 3D hydrogels (p < 0.05) and was higher than in 2D cultures at day 21 (p < 0.05). This coincided with a marked upregulation of ACTA2 (p < 0.01), indicating myofibroblast-like differentiation. MMP-2 activity increased significantly over time in both 2D and 3D cultures (p < 0.01 and p < 0.001, respectively). In contrast, PLAU and PLAT expression decreased significantly at days 14 and 21 (p < 0.001 and p < 0.05, respectively), reflecting a temporal shift from fibrinolytic to collagenolytic remodeling. Despite active remodeling, mechanical testing showed no significant changes in hydrogel stiffness or relaxation between day 1 and day 7, or between cell-seeded and acellular gels (p > 0.05), likely due to the small contractile forces generated by the cells relative to the gel’s bulk modulus. Together, these findings demonstrate that 3D fibrin hydrogels provide a biologically active and physiologically relevant microenvironment that supports fibroblast-mediated ECM remodeling, offering a biomimetic model for investigating the mechanobiology of periodontal and peri-implant soft tissue regeneration.