Lysyl oxidase-mediated intermolecular crosslinks fine-tune collagen I molecular dynamics and regulate cell-matrix interactions through focal adhesions

Lysyl oxidase (LOX)-mediated intermolecular crosslinking is essential for collagen I fibril stability, yet its influence on collagen molecular conformation and dynamics, and the downstream consequences for cell-matrix interactions remain poorly understood. Here, we genetically modulated LOX in collagen I-producing MC3T3-E1 cells to generate matrices with elevated (overexpression, OX) or absent (knockout, KO) crosslinking. Enhanced crosslinking yielded thick, continuous, aligned fibrils, whereas reduced crosslinking produced friable, dissociated fibrils. Solid-state nuclear magnetic resonance spectroscopy (SSNMR) revealed local triple-helix unfolding and altered nanosecond- and microsecond-scale molecular motions in both OX and KO matrices, changes largely reversible upon decellularization, implicating a synergistic role of crosslinking chemistry and cell-applied forces in regulating the dynamically-accessible conformations of collagen I. Changes in the molecular structure and dynamics of collagen had a functional impact on cell adhesion and mechanotransduction. These findings identify collagen crosslinking as a tunable element of the extracellular matrix "mechanical code," integrating biochemical modification with molecular-scale mechanics to regulate cell-matrix adhesion and mechanosignalling.