Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, β-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague Czech Republic

Department of Histology and Embryology 2nd Faculty of Medicine Charles University 150 06 Prague Czech Republic

Department of Orthopaedics 1st Faculty of Medicine Charles University and Na Bulovce Hospital Budinova 2 180 81 Prague Czech Republic

Department of Orthopaedics University J E Purkinje and Masaryk Hospital 401 13 Usti nad Labem Czech Republic

Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

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Harnessing the Biomimetic Effect of Macromolecular Crowding in the Cell-Derived Model of Clubfoot Fibrosis

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The possible role of hypoxia in the affected tissue of relapsed clubfoot