Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

Two‐photon polymerization (2PP) is a lithography‐based 3D printing method allowing the fabrication of 3D structures with sub‐micrometer resolution. This work focuses on the characterization of gelatin–norbornene (Gel–NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol‐ene system allows 2PP processing of cell‐containing materials at remarkably high scanning speeds (1000 mm s−1) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2–0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel–NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki‐67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high‐definition bioprinting of well‐defined constructs for long‐term cell culture studies.

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