Development and Degeneration of the Intervertebral Disc-Insights from Across Species

Abstract

Back pain caused by intervertebral disc (IVD) degeneration has a major socio-economic impact in humans, yet historically has received minimal attention in species other than humans, mice and dogs. However, a general growing interest in this unique organ prompted the expansion of IVD research in rats, rabbits, cats, horses, monkeys, and cows, further illuminating the complex nature of the organ in both healthy and degenerative states. Application of recent biotechnological advancements, including single cell RNA sequencing and complex data analysis methods has begun to explain the shifting inflammatory signaling, variation in cellular subpopulations, differential gene expression, mechanical loading, and metabolic stresses which contribute to age and stress related degeneration of the IVD. This increase in IVD research across species introduces a need for chronicling IVD advancements and tissue biomarkers both within and between species. Here we provide a comprehensive review of recent single cell RNA sequencing data alongside existing case reports and histo/morphological data to highlight the cellular complexity and metabolic challenges of this unique organ that is of structural importance for all vertebrates.

Keywords: annulus fibrosus; biomarkers; development; inflammation; intervertebral disc; intervertebral disc degeneration; notochord; nucleus pulposus; species.

Figure 2

Illustrates the timeline of embryonic development from the onset of gastrulation to birth across different species such as mice, rats, rabbits, non-chondrodystrophic (NCD) dogs, chondrodystrophic (CD) dogs, humans, cows, and horses, in addition to the reported disappearance of notochord (NC)-like cells in the NP. Embryonic development data in humans is compiled from [40] and all other animals from [41] while NC-like cell disappearance is compiled from mice [37], rats [26,42], rabbits [26,42], dogs [26,37], humans [30,42,43], cows (personal observation) and [14], horses [11]. Information on NC or NC-like cells disappearance varied across sources and was based on histological or morphological data. Advancements in scRNA seq analysis can provide further clarification and identified NC-like cells into adulthood in humans.

Figure 3

Mallory’s tetrachrome histological staining on formaldehyde preserved tissues was performed as previously described [14] to visualize the AF and NP tissue across species. All but the human sample were coccygeal IVDs. Ages were generally from adult organisms but varied across species. The dog IVD was from a 1-day old non-chondrodystrophic boxer docked breed. Scale bar reflects 50 μm. Staining represents: nuclei (red), collagen fibrils, ground substance, cartilage, mucin and amyloid (blues), erythrocytes and myelin (yellow) or elastic fibrils (pale pink, pale yellow or unstained) [54].

Figure 1

Simplified illustration of IVD formation adapted from [18,19,20,21,22,23,24]. (A) Specification of the embryonic axial skeleton, shortly after the formation of metameric somites is initiated by signals from the NC. (B) Ventromedial cells undergo an epithelial to mesenchymal transformation (EMT), taking on a mesenchymal sclerotome fate and migrate to surround the NC and neural tube. (C/D) Resegmentation of the axial sclerotome: The cell-dense caudal portion of one somite (C in (C)→ dark gray in (D)) will combine with the more loosely organized rostral portion of the adjacent somite (R in (C)→ light gray in (D)). (E) Following this resegmentation, the anterior layer of the cell dense section will give rise to the AF of the IVD (green), while the NP is derived from the NC (pink). Chondrogenesis enables the formation of the CEP and VB. (F) The reorganization and shift influenced by complex signaling events will enable innervation (light blue) of the myotome derived skeletal musculature (purple). (G) Transverse depiction of an isolated IVD illustrating only the caudal CEP for simplicity. (H) Depicts a sagittal section of the VB and IVD. The simplified illustration is not drawn to scale. AS: axial sclerotome; C: caudal; CEP: cartilage end plates; iAF: inner annulus fibrosus; IVD: intervertebral disc; LS: lateral sclerotome; NC: notochord; oAF: outer annulus fibrosus; R: rostral; VB: vertebral body.

Figure 4

Fluorescent microscopy of primary adult coccygeal bovine IVD (NP and AF) and adipose (FAT) cells from the same animal and low passage fetal NP cells cultured in low glucose DMEM with 10% FBS. Cells were stained with MitoView^TM (green), which quickly accumulates in mitochondria. DAPI (blue) labels nuclei. All cells were stained and imaged the same day at the same magnification. Scale bar reflects 50 μm for all images. All tissue was collected as waste from local abattoirs.

Figure 5

Top image shows a composite image created from overlayed sequential brightfield images of 7 μm paraffin sections of an adult bovine coccygeal IVD taken across the diameter of the disc from oAF to NP to oAF. The composite fluorescence image shows the DAPI labeled nuclei of each corresponding image. Scale bar represents 100 μm.