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    Modulating RhoA signaling can restore nucleus pulposus (NP) cell phenotype and gene expression. Optimal RhoA pathway modulation for disc regeneration depends on the specific cell microenvironment.

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    Area of Science:

    • Biomedical Engineering
    • Cell Biology
    • Regenerative Medicine

    Background:

    • Intervertebral disc degeneration involves loss of nucleus pulposus (NP) cell phenotype and extracellular matrix.
    • Changes in cytoskeletal contractility and cell shape are linked to NP degeneration.

    Purpose of the Study:

    • To investigate if microenvironment-specific modulation of RhoA signaling can restore NP cell morphology and gene expression.
    • To assess the impact of RhoA pathway perturbation in 2D and 3D cell culture models.

    Main Methods:

    • Utilized 2D monolayer and 3D alginate cultures of NP cells.
    • Employed pharmacologic RhoA inhibition (CT04) and activation (CN03).
    • Analyzed cell morphology, actomyosin contractility markers, and gene expression via bulk RNA sequencing.

    Main Results:

    • In 2D culture, RhoA inhibition promoted a more circular NP cell phenotype and increased NP-related gene expression (e.g., ACAN, GDF5).
    • In 3D alginate culture, RhoA activation enhanced NP markers (e.g., ACAN, KRT19) and promoted a compact morphology with repeated dosing.
    • Cell roundness correlated with increased ACAN expression across culture conditions.

    Conclusions:

    • RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D cultures.
    • The optimal direction of RhoA modulation is context-dependent on the microenvironment.
    • RhoA signaling represents a potential, context-specific therapeutic target for disc regeneration.