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.

• MeSH
• Biomimetics methods   MeSH
• Extracellular Matrix metabolism   drug effects   MeSH
• Fibroblasts * metabolism   drug effects   MeSH
• Fibrosis * drug therapy   MeSH
• Collagen * metabolism   chemistry   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Clubfoot * metabolism   drug therapy   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Collagen * MeSH

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.

• Keywords
• beta-aminopropionitrile (BAPN), collagen, congenital idiopathic Talipes equinovarus, contraction, crosslinking, fibrosis, relapsed clubfoot,
• MeSH
• Aminopropionitrile pharmacology   MeSH
• Fibroblasts drug effects   MeSH
• Collagen chemistry   MeSH
• Humans MeSH
• Protein-Lysine 6-Oxidase antagonists & inhibitors   MeSH
• Clubfoot drug therapy   metabolism   pathology   MeSH
• Child, Preschool MeSH
• Cross-Linking Reagents pharmacology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aminopropionitrile MeSH
• Collagen MeSH
• LOX protein, human MeSH Browser
• Protein-Lysine 6-Oxidase MeSH
• Cross-Linking Reagents MeSH

OBJECTIVE: The aim of this study was to verify whether the Pirani and Dimeglio clinical scoring systems could predict results of Ponseti therapy. METHODS: Forty-seven patients with clubfoot deformities treated with the Ponseti method were enrolled in the study. Clinical evaluation with the Pirani and Dimeglio scoring systems was performed before the treatment and after the second cast fixation. The number of fixations, necessity for achillotomy, and recurrence of the deformity were determined as parameters of the therapy results. The patients were divided into three groups according to the severity of their deformities, and the groups were compared with one another. RESULTS: Clubfoot correction required an average of 6.8 casts. Five patients developed a recurrence. Comparing the therapy outcomes among the groups, we found statistically significant differences in the Pirani classification after the second fixation (the number of casts [p =.003] and necessity to perform an achillotomy [p =.014]) and in the Dimeglio scores before therapy (number of casts [p =.034]) and after the second fixation (number of relapses [p =.032]). CONCLUSION: Although clinical scoring systems showed some dependence on the parameters of treatment outcomes, their predictive function can be used in only a limited way. Level of evidence II, Prospective comparative study.

OBJETIVO: O objetivo deste estudo foi verificar se os sistemas de pontuação clínica de Pirani e Dimeglio poderiam servir para prever os resultados do tratamento com o método de Ponseti. MÉTODOS: Quarenta e sete pacientes com diagnóstico de pé torto equinovaro tratados pelo método de Ponseti foram incluídos no estudo. A avaliação clínica com os sistemas de pontuação de Pirani e Dimeglio foi realizada antes do tratamento e depois da segunda fixação de gesso. O número de fixações com gesso, a necessidade de realização de aquilotomia e a recorrência da deformidade foram determinadas como parâmetros dos resultados do tratamento. Os pacientes foram divididos em três grupos, de acordo com a gravidade das deformidades, e esses grupos foram comparados entre si. RESULTADOS: A correção do pé torto exigiu uma média de 6,8 gessos e cinco pacientes apresentaram recidiva. Ao comparar os resultados do tratamento entre os grupos, verificou-se diferença estatisticamente significante na classificação de Pirani após a segunda fixação (número de gessos [p = 0,003], necessidade de realizar aquilotomia [p = 0,014]) e pontuação de Dimeglio antes do tratamento (número de gessos [p = 0,034]) e depois da segunda fixação (número de recidivas [p = 0,032]). CONCLUSÃO: Embora os sistemas de pontuação clínica tenham mostrado alguma dependência dos parâmetros dos resultados do tratamento, sua função preditiva pode ser usada de maneira limitada. Nível de evidência II, Estudo comparativo prospectivo.

• Keywords
• Club foot, Foot, Foot deformities, congenital,
• Publication type
• Journal Article MeSH

Idiopathic pes equinovarus is a congenital deformity of the foot and lower leg defined as a fixation of the foot in adduction, supination, and varus. Although the pathogenesis of clubfoot remains unclear, it has been suggested that fibroblasts and growth factors are involved. To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated with the Ponseti method were included in the present study. Tissue samples were obtained from patients undergoing surgery for relapsed clubfeet. Contracted tissues were obtained from the medial aspect of the talonavicular joint. Protein was extracted after digestion and delipidation using zip-tip C18. Individual collagenous fractions were detected using a chemiluminescent assay. Amino acid analysis of tissue samples revealed a predominance of collagens, namely collagen types I, III, and VI. The high content of glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins were identified. The major result of the present study was the observation that the extracellular matrix in clubfoot is composed of an additional 16 proteins, including collagens V, VI, and XII, as well as the previously described collagen types I and III and transforming growth factor β. The characterization of the general protein composition of the extracellular matrix in various regions of clubfoot may help in understanding the pathogenesis of this anomaly and, thus, contribute to the development of more efficacious therapeutic approaches.

• MeSH
• Amino Acids analysis   MeSH
• Extracellular Matrix Proteins metabolism   MeSH
• Infant MeSH
• Collagen metabolism   MeSH
• Humans MeSH
• Clubfoot metabolism   pathology   therapy   MeSH
• Proteomics methods   MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Extracellular Matrix Proteins MeSH
• Collagen MeSH
• Transforming Growth Factor beta MeSH