Fibrotic changes in pediatric clubfoot provide an opportunity to improve corrective therapy and prevent relapses with targeted drugs. This study defines the parameters of clubfoot fibrosis and presents a unique analysis of a simple pseudo-3D in vitro model for disease-specific high-throughput drug screening experiments. The model combines clubfoot-derived fibroblasts with a biomimetic cultivation environment induced by the water-soluble polymers Ficoll and Polyvinylpyrrolidone, utilizing the principle of macromolecular crowding. We achieved higher conversion of soluble collagen into insoluble collagen, accelerated formation of the extracellular matrix layer and upregulated fibrosis-related genes in the mixed Ficoll environment. To test the model, we evaluated the effect of a potential antifibrotic drug, minoxidil, emphasizing collagen content and cross-linking. While the model amplified overall collagen deposition, minoxidil effectively blocked the expression of lysyl hydroxylases, which are responsible for the increased occurrence of specific collagen cross-linking in various fibrotic tissues. This limited the formation of collagen cross-link in both the model and control environments. Our findings provide a tool for expanding preclinical research for clubfoot and similar fibroproliferative conditions.

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

Department of Histology and Embryology 2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague 5 Czech Republic

Department of Orthopaedics Masaryk Hospital Socialni Pece 3316 12A 401 13 Usti nad Labem Czech Republic

Department of Orthopaedics University Hospital Bulovka Charles University Budinova 67 2 180 81 Prague 8 Czech Republic

Faculty of Science Charles University Albertov 6 128 00 Prague 2 Czech Republic

Laboratory of Biomaterials and Tissue Engineering Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic

Laboratory of Biomathematics Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic

Laboratory of Translational Metabolism Institute of Physiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic

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