Annulus fibrosus cell phenotypes in homeostasis and injury: implications for regenerative strategies

Abstract

Despite considerable efforts to develop cellular, molecular, and structural repair strategies and restore intervertebral disk function after injury, the basic biology underlying intervertebral disk healing remains poorly understood. Remarkably, little is known about the origins of cell populations residing within the annulus fibrosus, or their phenotypes, heterogeneity, and roles during healing. This review focuses on recent literature highlighting the intrinsic and extrinsic cell types of the annulus fibrosus in the context of the injury and healing environment. Spatial, morphological, functional, and transcriptional signatures of annulus fibrosus cells are reviewed, including inner and outer annulus fibrosus cells, which we propose to be referred to as annulocytes. The annulus also contains peripheral cells, interlamellar cells, and potential resident stem/progenitor cells, as well as macrophages, T lymphocytes, and mast cells following injury. Phases of annulus fibrosus healing include inflammation and recruitment of immune cells, cell proliferation, granulation tissue formation, and matrix remodeling. However, annulus fibrosus healing commonly involves limited remodeling, with granulation tissues remaining, and the development of chronic inflammatory states. Identifying annulus fibrosus cell phenotypes during health, injury, and degeneration will inform reparative regeneration strategies aimed at improving annulus fibrosus healing.

Keywords: annulus fibrosus; cell phenotype; healing; intervertebral disk; intervertebral disk injury models; regeneration.

Figure 1.

Schematics and representative microscopic images of the different cell types and their locations in the healthy adult AF. (A) Peripheral cells. (B) Outer annulocytes. (C) Inner annulocytes. (D) Interlamellar cells.

Figure 2.

The AF injury response in a mouse caudal IVD eight weeks following injury. Picrosirius red/alcian blue staining reveals uninjured control IVDs have an intact and proteoglycan-rich NP (A) and highly organized, collagen-rich AF as visualized under polarized light (C). Injury response 8 weeks after puncture consists of NP fibrosis and incomplete healing of puncture tract, with evidence of AF buckling, delamination, and fissures throughout the IVD, as shown by picrosirius red/alcian blue staining (B). Loss of AF lamellae organization is shown by decreased birefringence in polarized light imaging (D). 5 μm thick paraffin sections, 10X magnification.

Figure 3.

Recruitment of unidentified and irregular cells in a punctured rat lumbar IVD six weeks following injury. Safranin-O staining reveals a dense, cellular cap surrounding the injury site on the dorsal aspect of a lumbar IVD (A). Rounded, unidentified cells that are potentially inflammatory cells remaining in the repair region over time are observed adjacent to the puncture tract (inset A’ shows higher magnification of the corresponding region in panel A). 5 μm thick paraffin sections, 10X magnification.