INVESTIGATION OF INTERVERTEBRAL DISC HERNIATION THROUGH A HUMAN ANULUS FIBROSUS PRO-INFLAMMATORY MODEL

Abstract

Intervertebral disc (IVD) herniation involves a chronic inflammatory process and failure of the annulus fibrosus (AF), in a process still under investigated. Inflammation can lead to disorganization of extracellular matrix (ECM) and fibrotic alterations, among others. Most of the ongoing research relies on in vivo or ex vivo models that do not truly mimic the human microenvironment. Here, we developed a 3D in vitro model aimed at recapitulating inflammation progression in human AF (hAF) to improve the understanding of AF failure.hAF of herniated samples from low back pain patients were collected and digested as previously established. hAF cells were cultured in 2D and 3D, embedded in collagen-type 1 hydrogels. hAF cells in 2D and 3D were then stimulated with IL-1β (10ng/mL), in acute (48h) and chronic (7days) conditions. After 7 days, cell morphology together with extracellular matrix (ECM) (collagen I and fibronectin) were assessed by immunofluorescence. Inflammatory cytokine secretion were analyzed by a protein array.2D experiments showed alteration of cell morphology, with cell area reduction upon inflammatory stimulation. The profile of inflammatory cytokines secreted showed that: i) IL-1α increases with time in culture, while TNF-α decreases; and ii) IL-1α and MIG/CXCL9 are increased in chronic vs acute inflammation. ECM analysis showed no differences. In 3D culture, AF cell morphologic alterations with inflammation were also observed. Concerning ECM production, collagen I production was reduced with chronic inflammation, while fibronectin remained unaltered. Collagen II and aggrecan appear to increase with chronic inflammation. Cytokine production from 3D culture is currently being assessed. ECM alterations are being complemented by characterization at biomechanical (nanoindentation) level.Our data show that chronic inflammation is crucial to understand AF failure mechanisms and improve the resemblance of human IVD herniation.