Directing the Chondro-Fibro Axis via Early Microenvironmental Interactions to Enable Precise and Volumetric Cartilage Repair

<jats:p>Successful cartilage repair remains one of the most significant challenges in the musculoskeletal field. Microfracture (MFx), a form of marrow stimulation, remains the predominant repair technique, but it exhibits routine failure due to inadequate defect fill and inferior fibrotic tissue formation. Whereas current strategies focus on augmenting MFx with scaffolds and bioactive factors, the potential to target the MFx clot itself and use the capabilities of this dynamic environment to guide MFx repair remains largely unexplored. We verified that MFx contraction and fibrosis hinder repair success in minipigs and become evident as early as one week in multiple animal models. Therefore, our objective was to investigate and direct microenvironmental interactions in the MFx clot to promote volumetric maintenance and reprogram cells from a fibrotic to more chondrogenic phenotype. Extracellular control of cell-environment interactions, through fibrinogen augmentation or anti-fibrinolytic treatment, limited contraction but had no effect on or even exacerbated the fibrotic susceptibility of marrow-derived cells (MDCs). Intracellular control of microenvironmental interactions, through modulation of the Rho-ROCK pathway, drove TGF-B3 activity of MDCs along a chondro-fibro axis. In particular, treatment with the ROCK inhibitor Fasudil drove TGF-B3-treated cells away from a myofibroblast phenotype and towards chondrogenesis. Short-term Fasudil treatment prevented TGF-B3-driven macroscale clot contraction and enhanced cartilage-specific matrix deposition in vitro. In a pilot rat study, this combination treatment improved GAG deposition and better protected surrounding cartilage. These findings suggest that Rho-ROCK modulates TGF signaling along this chondro-fibro axis and its precise control could be the key to promoting precise and volumetric cartilage repair through microenvironmental interactions.</jats:p>