Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction: Intervertebral disc (IVD) degeneration phenotypes involving annulus fibrosus (AF) defects and herniation can cause disabling back pain, which increases with age peaking ~50 years [1]. AF repair remains an unmet clinical challenge in need of novel thinking and deeper understanding of IVD healing. Interestingly, neonatal mice IVDs functionally regenerate from large AF injuries while skeletally mature mice IVDs heal poorly[3]. Regenerative tendon healing in neonatal mice is similarly lost with age and affected by immune cell changes[4], implicating immune cell involvement in IVD healing. Aging changes IVD cells, matrix composition, structure, and function[2] yet little is known about IVD immune cell changes with age or healing. This study determined effects of age on: (1) immune cell populations in naïve IVDs; (2) IVD immune cell responses to injury; and (3) AF reparative responses in a mouse model.

Methods: AF-herniation injury was created in coccygeal IVDs of mice at three ages: Neonatal (14 days; regenerative), Adult (4 months; skeletal maturity), and Aged (1 year; equivalent to peak human back pain disability). Single-Cell RNA Sequencing (scRNA-Seq) was performed on cells isolated from naïve IVDs, data processing included filtering, normalization, and unsupervised clustering. IVD segments were collected 14 days post injury and CD68 immunohistochemistry (IHC) was used to assess macrophages. Picrosirius Red-Alcian Blue (PR-AB) stained collagen and glycosaminoglycan (GAG).

Results: scRN-Seq revealed cell clusters at each age including native IVD cells and distinct immune cell populations that expressed macrophage markers: Cd68, Lyz2, Mpeg1 (Fig. 1). Macrophages (CD68 IHC) were present in endplates and outer AF of control neonate IVDs, with macrophage presence diminishing with age (Fig. 2A). Control IVDs had decreased NP cellularity and buckling of AF lamellae with age (Fig. 2B,C). Injury caused  macrophage immune staining localized to the outer AF and fibrotic healing response that was strongly affected by age (Fig. 3A). Specifically, Neonates exhibited a near-regenerative healing response characterized by infiltration of cellularized repair tissue within the injury, retained organization of adjacent AF layers, and no notable scarring (Fig. 3B,C). Adult and aged IVDs had a cellular/fibrotic cap surrounding the injury site which was most robust in Adult, and absent in Neonates.

Discussion: scRNA-Seq revealed distinct immune cell populations in naïve IVDs across all ages, challenging the conventional belief that the IVD is immune privileged[5]. CD68 IHC confirmed macrophage presence in uninjured IVDs, localized to outer AF and endplates, and diminishing with age. Neonatal IVDs demonstrated a controlled immune response that corresponded to more organized and scarless AF healing. Adults and Aged IVDs, exhibited dysregulated immune cell recruitment, robust fibrous cap formation, limited AF repair tissue, and less organized AF structure. Regenerative neonatal tendon healing involved  controlled type-2 macrophage responses facilitated by T-regulatory cells[4], prompting further investigation in IVDs. We conclude that aging resulted in three immune-cell modulated IVD healing responses to herniation injury: “regenerative” healing in Neonates, “robust fibrotic” healing in Adults, and “limited fibrotic” healing in Aged mice. Understanding age-related IVD healing may guide development of regenerative IVD repair akin to the neonatal mice healing.