Kazald1 attenuates chondrocyte fibrosis to potentiate hyaline cartilage regeneration by interfering with the pro-fibrotic TGF-β signaling

<b>Background:</b> Cartilage regeneration remained a significant challenge, often leading to the formation of mechanically inferior fibrocartilage instead of physiological hyaline cartilage. Currently, there were no effective treatments for cartilage fibrosis, necessitating the exploration of potential molecular targets. <b>Methods:</b> We perform single-cell sequencing of rat spontaneously formed fibrocartilage following osteochondral injury and rat normal hyaline cartilage with a comprehensive analysis of the heterogeneous cell subpopulations between two groups. Subsequently, we express and purify the full length of recombinant human Kazald1 protein (aa 31-304) with predicted tertiary and secondary structures, and determine its anti-fibrotic effect and explore its regulatory mechanisms using in vitro cultured chondrocytes, in the presence or absence of the pro-fibrotic factor TGF-β1. Finally, we evaluate the therapeutic potential of recombinant Kazald1 protein in promoting hyaline cartilage regeneration and maintenance using rat osteochondral injury models and human cartilage explants, respectively. <b>Results:</b> Through single cell sequencing of hyaline cartilage and fibrocartilage, we identified Kazald1 as a key molecule in maintaining cartilage homeostasis. During cartilage fibrosis, Kazald1 expression was significantly down-regulated and becomes imbalanced with TGF-β1. Recombinant Kazald1 protein effectively inhibited TGF-β1-induced chondrocyte fibrosis and preserves chondrocyte phenotype. Mechanistically, Kazald1 formed a dimer with TGFBR1, blocking the pro-fibrotic TGF-β1-Akt/Smad3 signaling and suppressing the expression of fibrotic genes. In rat models of cartilage injuries, the combination of Kazald1 and TGF-β1 effectively promoted hyaline cartilage regeneration with structural restoration and functional recovery. This combination also enhanced hyaline cartilage maintenance and inhibited TGF-β1-induced cartilage fibrosis in human cartilage explants. <b>Conclusion:</b> This study unveils the pivotal role of Kazald1 in the regulation of cartilage fibrosis and highlights its potential as a therapeutic agent for facilitating hyaline cartilage regeneration.