Jarmila Knitlova1, Martina Doubkova1,2, Martin Plencner1, David Vondrasek1,3, Adam Eckhardt1, Martin Ostadal4, Jana Musilkova1, Lucie Bacakova1, Tomas Novotny2,5. 1. Department of Biomaterials and Tissue Engineering, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic. 2. Second Faculty of Medicine, Charles University, Prague, Czech Republic. 3. Faculty of Physical Education and Sport, Charles University, Prague, Czech Republic. 4. Department of Orthopaedics, First Faculty of Medicine, Charles University and Na Bulovce Hospital, Prague, Czech Republic. 5. Department of Orthopaedics, Masaryk Hospital, Usti Nad Labem, Czech Republic.
Abstract
AIM: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. METHODS: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. RESULTS: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). CONCLUSIONS: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.
AIM: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. METHODS: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. RESULTS: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). CONCLUSIONS: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.
Authors: Jarmila Knitlova; Martina Doubkova; Adam Eckhardt; Martin Ostadal; Jana Musilkova; Lucie Bacakova; Tomas Novotny Journal: Int J Mol Sci Date: 2021-11-02 Impact factor: 5.923
Authors: Tomas Novotny; Adam Eckhardt; Martina Doubkova; Jarmila Knitlova; David Vondrasek; Eliska Vanaskova; Martin Ostadal; Jiri Uhlik; Lucie Bacakova; Jana Musilkova Journal: Sci Rep Date: 2022-03-15 Impact factor: 4.379