Hasson, M. | bioRxiv Science

bioRxiv2026

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

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-{beta}3 activity of MDCs along a "chondro-fibro axis". In particular, treatment with the ROCK inhibitor Fasudil drove TGF-{beta}3-treated cells away from a myofibroblast phenotype and towards chondrogenesis. Short-term Fasudil treatment prevented TGF-{beta}3-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-{beta} signaling along this chondro-fibro axis and its precise control could be the key to promoting precise and volumetric cartilage repair through microenvironmental interactions.

bioengineering↗

bioRxiv2024

Revealing Early Spatial Patterns of Cellular Responsivity in Fiber-Reinforced Microenvironments

Fiber-reinforcement approaches have been utilized to replace aligned tissues with engineered constructs after injury or surgical resection, strengthening soft biomaterial scaffolds and replicating anisotropic, load-bearing properties. However, most studies focus on the macroscale aspects of these scaffolds, rarely considering the cell-biomaterial interactions that govern remodeling and ECM organization towards aligned neo-tissues. Since initial cell-biomaterial responses within fiber-reinforced microenvironments likely influence long-term efficacy of repair and regeneration strategies, here we elucidate roles of spatial orientation, substrate stiffness, and matrix remodeling on early cell-fiber interactions. Bovine mesenchymal stromal cells (MSCs) were cultured in soft fibrin gels reinforced with a stiff 100 {micro}m polyglycolide-co-caprolactone fiber. Gel stiffness and remodeling capacity were modulated by fibrinogen concentration and aprotinin treatment, respectively. MSCs were imaged at 3 days and evaluated for morphology, mechanoresponsiveness (nuclear YAP localization), and spatial features including distance and angle deviation from fiber. Within these constructs, morphological conformity decreased as a function of distance from fiber. However, these correlations were weak (R2 = 0.01043 for conformity and R2 = 0.05542 for nuclear YAP localization), illustrating cellular heterogeneity within fiber-enforced microenvironments. To better assess cell-fiber interactions, we applied machine-learning strategies to our heterogeneous dataset of cell shape and mechanoresponsive parameters. Principal component analysis (PCA) was used to project 23 input parameters (not including distance) onto 5 principal components (PCs), followed by Agglomerative Hierarchical Clustering (AHC) to classify cells into 3 groups. These clusters exhibited distinct levels of morpho-mechanoresponse (combination of morphological conformity and YAP signaling) and were classified as High Response (HR), Medium Response (MR), and Low Response (LR) clusters. Cluster distribution varied spatially, with most cells (61%) closest to the fiber (0 - 75 {micro}m) belonging to the HR cluster, and most cells (55%) furthest from the fiber (225 - 300 {micro}m) belonging to the LR cluster. Modulation of gel stiffness and fibrin remodeling showed differential effects for HR cells, with stiffness influencing the level of mechanoresponse, and remodeling capacity influencing the location of responding cells. Overall, clustering of individual cells in stiff-soft microenvironments revealed spatial trends in cellular responsivity not seen by evaluating individual cell parameters as a distance from fiber alone. Impact StatementThis study used PCA-AHC based clustering to identify MSC sub-groups from a heterogeneous population with distinct responses to stiff-soft microenvironments. Cell responsivity within a soft, fiber-reinforced fibrin gel microenvironment was influenced by the spatial localization of individual cells around a stiffer polyglycolide-co-caprolactone fiber. Additionally, modulation of gel substrate stiffness and matrix remodeling capacity further influenced the level of responsiveness and localization of responsive cell clusters around the fiber, which may contribute to scaffold design at the cellular level and foreshadow longer-term aligned tissue deposition.

bioengineering↗