Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies

This study developed and validated agarose-collagen composite hydrogels as a reproducible, cell-laden 3D platform for mechanotransduction studies relevant to nucleus pulposus (NP) and cartilage-like tissues. Agarose was maintained at 2% wt/vol to preserve load-bearing stability, while collagen I was physically blended at 2 or 4.5 mg/mL to introduce adhesion motifs without cytotoxic chemical functionalization. Confocal reflection microscopy confirmed homogeneous collagen distribution in the blended network. Rheology showed that 2 mg/mL collagen largely preserved agarose-like elastic properties, whereas 4.5 mg/mL collagen reduced storage modulus and increased loss modulus after collagen fibrillogenesis. All agarose-based gels remained dimensionally stable over 21 days, and encapsulated bovine NP cells remained highly viable (>80%) with low proliferation and rounded morphology. Composite gels increased ECM-associated gene expression (COL1A2,COL2A1,ACAN) from day 1 to 7 and improved GAG/DNA versus agarose at day 7. Importantly, collagen blending enabled cell adhesion (2D assay) and increased focal adhesion kinase signaling, with substantially higher baseline pFAK/β-tubulin in agarose-collagen (2 mg/mL) versus agarose and a modestly higher pFAK/FAK response shortly after dynamic compression, supporting improved integrin-mediated mechanotransduction capacity in the composite system.