Harnessing the\nBiomimetic Effect of Macromolecular\nCrowding in the Cell-Derived Model of Clubfoot Fibrosis

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