Bioengineered Tricomposite Hydrogel Enhances Chondrogenic Phenotype and Hyaline Matrix Formation in Human Chondrocytes

Fibrin hydrogels are biocompatible but often lack instructive cues needed to sustain chondrocyte phenotype and cartilage-like matrix formation; therefore, we investigated whether a tricomposite fibrin hydrogel incorporating decellularized articular cartilage matrix (dACM) and decellularized amniotic membrane matrix (dAMM) enhances human articular chondrocyte performance in vitro. Human articular chondrocytes were encapsulated in tricomposite or fibrin-only hydrogels and cultured for 28 days, evaluating degradation kinetics, viability and cell density, histological remodeling (H&E, Masson's trichrome, Safranin O), immunohistochemistry for type II collagen, aggrecan, and type I collagen, and qPCR of SOX9, COL2A1, ACAN, RUNX2, COL1A2, and COL10A1. The tricomposite remained cytocompatible (~99% viability), supported marked cell expansion (~250% by day 28), and degraded more slowly than fibrin controls. It increased chondrogenic gene expression (SOX9 >3-fold vs. control by day 28; sustained COL2A1 at 1.5-2-fold; early ACAN at 3-5-fold) while attenuating off-target transcriptional programs (RUNX2 ~50% of control, reduced COL1A2, and negligible COL10A1). Consistently, histology showed progressive lacuna-like morphology and proteoglycan-rich matrix accumulation, accompanied by strong type II collagen and aggrecan immunoreactivity and reduced type I collagen. Overall, adding dACM and dAMM to fibrin improved hydrogel biofunctionality and promoted hyaline-like extracellular matrix assembly, supporting further evaluation of this cell-instructive platform for focal articular cartilage repair.