The intervertebral disk (IVD) is a composite structure essential for spine stabilization, load bearing, and movement. Biomechanical factors are important contributors to the IVD microenvironment regulating joint homeostasis; however, the cell type-specific effectors of mechanotransduction in the IVD are not fully understood. The current study aimed to determine the effects of cyclic tensile strain (CTS) on annulus fibrosus (AF) cells and identify mechano-sensitive pathways. Using a cell-type specific reporter mouse to differentiation NP and AF cells from the murine IVD, we characterized AF cells in dynamic culture exposed to CTS (6% strain) at specific frequencies (0.1 Hz, 1.0 Hz, or 2.0 Hz). We demonstrate that our culture model maintains the phenotype of primary AF cells and that the bioreactor system delivers uniform biaxial strain across the cell culture surface. We show that exposure of AF cells to CTS induces cytoskeleton reorganization resulting in stress fiber formation, with acute exposure to CTS at 2.0 Hz inducing a significant yet transient increase ERK1/2 pathway activation. Using SYBPR-based qPCR to assess the expression of extracellular matrix (ECM) genes, ECM-remodeling genes, candidate mechano-sensitive genes, inflammatory cytokines and cell surface receptors, we demonstrated that exposure of AF cells to CTS at 0.1 Hz increased Acan, Prg4, Col1a1 and Mmp3 expression. AF cells exposed to CTS at 1.0 Hz showed a significant increase in the expression of Acan, Myc, and Tnfα. Exposure of AF cells to CTS at 2.0 Hz induced a significant increase in Acan, Prg4, Cox2, Myc, Fos, and Tnfα expression. Among the cell surface receptors assessed, AF cells exposed to CTS at 2.0 Hz showed a significant increase in Itgβ1, Itgα5, and Trpv4 expression. Our findings demonstrate that the response of AF cells to CTS is frequency dependent and suggest that mechanical loading may directly contribute to matrix remodeling and the onset of local tissue inflammation in the murine IVD.
Objective: Low back pain (LBP) is the leading cause of global disability and is thought to be driven primarily by intervertebral disc (IVD) degeneration (DD). Persistent upregulation of catabolic enzymes and inflammatory mediators have been associated with severe cases of DD. Nuclear factor kappa B (NF-κB) is a master transcription regulator of immune responses and is over expressed during inflammatory-driven musculoskeletal diseases, including DD. However, its role in triggering DD is unknown. Therefore, this study investigated the effect of NF-κB pathway over-activation on IVD integrity and DD pathology. Methods: Using skeletally mature mouse model, we genetically targeted IVD cells for canonical NF-κB pathway activation via expression of a constitutively active form of inhibitor of κB kinase B (IKKβ), and assessed changes in IVD cellularity, structural integrity including histology, disc height, and extracellular matrix (ECM) biochemistry, biomechanics, expression of inflammatory, catabolic, and neurotropic mediators, and changes in macrophage subsets, longitudinally up to 6-months post activation. Results: Prolonged NF-κB activation led to severe structural degeneration, with a loss of glycosaminoglycan (GAG) content and complete loss of nucleus pulposus (NP) cellularity. Structural and compositional changes decreased IVD height and compressive mechanical properties with prolonged NF-κB activation. These alterations were accompanied by increases in gene expression of inflammatory molecules (Il1b, Il6, Nos2), chemokines (Mcp1, Mif), catabolic enzymes (Mmp3, Mmp9, Adamts4), and neurotrophic factors (Bdnf, Ngf) within IVD tissue. Increased recruitment of activated F4/80+ macrophages exhibited a greater abundance of pro-inflammatory (CD38+) over inflammatory-resolving (CD206+) macrophage subsets in the IVD, with temporal changes in the relative abundance of macrophage subsets over time, providing evidence for temporal regulation of macrophage polarization in DD in vivo, where macrophages participate in resolving the inflammatory cascade but promote fibrotic transformation of the IVD matrix. We further show that NF-κB driven secretory factors from IVD cells increase macrophage migration and inflammatory activation, and that the secretome of inflammatory-resolving macrophages mitigates effects of NF-κB overactivation. Conclusion: Overall the observed results suggest prolonged NF-κB activation can induce severe DD, acting through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and inflammatory activation of a macrophage cell populations, that can be mitigated with inflammatory-resolving macrophage secretome.
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