Impact of transient versus continuous IL−1β-driven inflammatory 3D models on annulus fibrosus cells

Chronic inflammation plays a key role in intervertebral disc (IVD) herniation contributing to extracellular matrix (ECM) disorganization, annulus fibrosus (AF) tissue fibrosis, and consequent failure. Despite its importance, the impact of chronic inflammatory environment has been poorly explored in 3Dmodels. This study establishes a relevant 3D human-derived model to investigate the progression of inflammation in human AF cells. For that, cells from herniated IVD of patients with low back pain were cultured in 3D collagen type I hydrogels under basal and inflammatory (IL-1β) conditions for 7 days, in a transient (2 days) or continuous (7 days) periods. In parallel, 2D cultures were conducted in the same conditions. The 3D model recapitulated key pathological features of AF degeneration/failure, as actin filament alterations and reduced collagen type I deposition under continuous inflammation, which led to increased fibronectin production. Continuous inflammation also increased pro-inflammatory mediators in 3D cultures and enhanced immune cell infiltration in vivo. These findings validate collagen type I hydrogels with continuous inflammation, as a valuable model for replicating in vitro the AF pathomechanisms, offering insights into potential biomarkers and therapeutic targets for IVD herniation prognosis. STATEMENT OF SIGNIFICANCE: This study evaluates the effects of continuous versus transient inflammation in a 3D model of annulus fibrosus (AF) cells, providing insights on how sustained inflammatory conditions impact tissue components and inflammatory response in intervertebral disc (IVD) herniation. While previous research has focused on transient inflammation, this work uses a collagen-based 3D model to replicate prolonged inflammatory states, evaluating extracellular matrix alterations, inflammatory mediators' production and immune response. This work is of broad scientific interest, presenting a valuable approach for studying AF tissue pathology and inflammation in human IVD disorders.