ANNULUS FIBROSUS REPAIR STRATEGIES AND THE ROLE OF IMMUNE CELLS IN INTERVERTEBRAL DISC HEALING

Annulus fibrosus (AF) defects in the intervertebral disc (IVD) directly cause disabling back pain via herniation, biomechanical instability, and chronic inflammation from poor healing. AF defects also cause progression of IVD degeneration, which increases with age. We provide a historical perspective of AF repair research involving collaborations with Dr. Mauro Alini and the AO Research Institute AF-repair-consortium. We next describe effects of age on: (1) AF healing from IVD herniation injuries; (2) immune cell populations in Naïve IVDs; and (3) immune cell changes in Injured IVD using a mouse in vivo model. We hypothesize IVD immune cell populations change with age and injury, and distinct immune cell phenotypes are responsible for the shift from regenerative to fibrotic AF healing. Naïve and Injured mouse coccygeal IVDs were evaluated using single cell RNA sequencing (scRNA-seq) and immunohistochemistry for 4 ages: 0.5-Month (Neonatal regenerative healing), 4-Month (Skeletal mature fibrotic healing), 12-Month (aged), and 24-Month (geriatric). IVD-herniation injury (∼80% IVD height & 50% IVD depth) via 26- or 30-gauge needles. scRNA-seq identified clusters of native IVD cells, immune, endothelia, and erythrocyte cells (Fig. 1A). Immune cell distributions (Fig. 1B) in Naïve IVDs changed with age showing many neutrophils in 0.5-Month mice and few in 4-, 12-, or 24-Month mice, concomitant with the shift from regenerative to fibrotic healing (Fig. 1C). Immunohistochemistry for neutrophil marker Ly6G similarly decreased with age. Injury altered IVD immune cell populations across the lifespan; most notably neutrophils decreased in 0.5-Month Injured and increased in 12-Month Injured (Fig. 2A). Neutrophils in 0.5-Month Naïve IVDs had distinct neutrophil phenotypic markers (Fig. 2B) from 12-Month Injured IVDs while neutrophils in 12-Month Injured IVD were more pro-inflammatory (Fig. 2C). Immune cells are resident in Naïve IVDs across the lifespan (0.5- 24-Months). IVD immune cells changed with age and injury. Neonatal (0.5-Month) Naive IVDs had a large portion of resident neutrophils that was depleted with injury and absent at later ages, suggesting this neutrophil population could play a role in neonatal regenerative IVD healing responses. These resident neutrophils were phenotypically different from the recruited neutrophils in 12-Month Injured. We conclude that IVD immune cell populations change with age and injury, and that neutrophils may play a role in the shift from regenerative to fibrotic AF healing, with future blocking studies ongoing to confirm their mechanistic roles.