Extracellular matrix of cartilaginous endplate: composition, structure and function
The healthy endplate is comprised of an osseous and a cartilaginous part (Fig. 2B–C); when referring to the latter, the term cartilaginous endplate (CEP) is generally used. Similar to the other IVD tissues (NP and AF) and to articular cartilage, the main component of the CEP is water (close to 80% after birth, but below 70% after 15 years of age), followed by type II collagen and proteoglycans. The ratio between proteoglycans and collagens (measured as GAG to hydroxyproline ratio) within the CEP is 2 : 1, which is similar to that of articular cartilage and much lower than that of NP (Mwale et al. 2004). As for the AF and NP, other smaller types of proteoglycans (e.g. decorin, biglycan) have also been described recently for CEP (Hayes et al. 2011b).
The thickness of the human CEP is 0.5–1 mm at the periphery and diminishes toward the centre. The CEP is organised in a highly hydrated proteoglycan matrix reinforced by collagen fibrils. The orientation of the collagen fibrils changes across the CEP with collagen fibrils orientated parallel to the vertebral bodies in the centre of the CEP (corresponding to the NP location) but are curved closer to the inner AF region, where they merge with the AF collagen fibres (Maroudas et al. 1975; Humzah & Soames, 1988). Elastic fibres run parallel to collagen fibrils in the inner AF region that connects to the CEP, and it has been observed that elastic fibres contribute to anchor the NP to the adjacent CEPs (Yu et al. 2002).
The CEP has a structural, semi-permeable barrier and load-bearing functions. The structural function of the CEP is to separate the intervertebral disc from the adjacent vertebrae and to contain the NP tissue. Concerning the barrier function, it has to be noted that although a blood supply is present in the outer AF, the healthy NP is avascular. Indeed, during first decade of life, the NP has vascular supply both from the CEP and the AF, but the blood vessels recede with age and thus are not present within the adult NP. The diffusion distance from the blood supply to cells in the central portion of the NP can reach 8 mm (Benneker et al. 2005). Therefore, the CEP represents the main route by which small solutes diffuse into the NP. The exchange of solutes is ensured by capillaries found in the calcified part of the CEP that form buds in the proximity of the CEP. The central zone of the end-plate allows for the highest diffusion of small molecules (Maroudas et al. 1975). The outer AF also allows for small molecule diffusion, but the inner AF (region of interconnection between AF and CEP collagen fibres) is almost impermeable. However, it should be noted that the diffusion process is not only influenced by the CEP permeability but also by the molecule size and ionic charge (Urban et al. 1977). Indeed, small molecules such as glucose and oxygen can easily migrate through the IVD, whereas larger molecules (e.g. enzymes) or charged molecules have difficulties migrating through the disc. In terms of mechanical perspective, the CEP contributes towards evenly distributing the compressive load originating from the IVD on to the vertebral body (Broberg, 1983). The ability of the CEP to bear load is governed by the balance between collagen, proteoglycan and water content, in addition to the structural integrity of the matrix (Antoniou et al. 1996b).
Changes in the endplate with ageing have been summarised by Moore (2006) and are often due to or accompanied by changes in the NP and AF tissues. During IVD degeneration, the cartilage endplate becomes thinner, and fissures and sclerosis of the subchondral bone may be observed (Roberts et al. 2006). The first defects found in endplate are transversal fissures, which may be accompanied by blood vessel invasion and endplate ossification. Several studies, both theoretical (Natarajan et al. 1994) and experimental (Tanaka et al. 1993; Moore et al. 1996), have shown that the point of failure of the endplate is generally located in the vicinity of the subchondral bone. Additionally, disc protrusion in the vertebral body through a small opening in the CEP (also called ‘Schmorl’s nodes’) is commonly observed, both in younger and older spines (Moore, 2000). This defect causes reduction in disc height and eventually formation of cartilage and new bone around the prolapsed region. It has been hypothesised that, in the absence of trauma or disease, these defects may originate in highly vascularised regions, as the scar tissue formed after the closure of the blood channels is weaker than the rest of the CEP matrix (Moore, 2006).
The permeability of the CEP has been shown to decrease with IVD degeneration (Humzah & Soames, 1988; Roberts et al. 1996). The vascular supply to the CEP diminishes and calcification of the CEP increases, starting from the second decade of life, concomitantly with the onset of NP breakdown. At later stages, the calcified part is completely replaced by bone and nutrients canals are partially to completely occluded (Lee et al. 2001). Benneker et al. (2005) found a strong correlation between opening density in the range of 20–50 μm in the human endplate and grade of disc degeneration. In an in vivo ovine model, Van der Werf et al. (2007) showed that inhibiting perfusion through the endplate resulted in a nine-fold decrease in transport rate. Similar results were also obtained in a canine model, where the blockage of the endplate affected solute diffusion to the NP more than blood vessel disruption within the AF (Ogata & Whiteside, 1981). However, recent in vitro and in vivo studies using magnetic resonance imaging (MRI) and X-ray microtomography techniques have demonstrated that endplate permeability and porosity increased with age (Rajasekaran et al. 2004; Rodriguez et al. 2011, 2012). Therefore, changes in cell function and capillary density may be the primary reason for disc degeneration rather than inhibited disc nutrition via the endplate (Rajasekaran et al. 2004; Rodriguez et al. 2012).
At the cellular level, higher degrees of senescence and matrix metalloproteinase production were observed in CEP cells from herniated discs and those affected by spondylolisthesis had very long processes. In the case of other spinal disorders such as scoliosis, ectopic calcification in the CEP, and even in the IVD, has been observed (Roberts et al. 2006). In a mouse model, Ariga et al. (2001) found that cell apoptosis increased with ageing. Apoptotic cells were found in the NP, AF and CEP, although most of the apoptotic cells were localised in the CEP. Endplate ossification followed this apoptotic process and preceded IVD degeneration (Ariga et al. 2001). Abnormal mechanical stress has been suggested as a possible cause of CEP cell apoptosis but other factors linked to ageing are likely involved (Roberts et al. 2006).
Phenotype of cartilaginous endplate cells
Cells of the CEP have a rounded morphology, similarly to articular chondrocytes. While slight local variations in the cell distribution of the CEP are found, no distinct layers are observed as in articular cartilage (Moore, 2000). Compared with the AF and NP, the CEP is a region with a higher cell density of approximately 15 × 106 cells cm−3 (Maroudas et al. 1975; Roughley, 2004); a similar cell density is also found in articular cartilage.
Several studies have addressed the phenotype of NP and AF cells and have suggested markers to distinguish them from articular chondrocytes. However, few studies have investigated the phenotype of CEP cells and potential changes with ageing, and most of the available literature on CEP phenotype is based on immunostaining of matrix proteins.
Antoniou et al. (1996b) investigated the endplates from 121 human lumbar segments of different age and grades of degeneration. Based on extensive extracellular matrix investigations, they have identified three distinct phases of matrix turnover. In the first phase (‘growth’), an active synthesis of matrix molecules and active denaturation of type II collagen take place. In the second phase (‘aging and maturation’), a drop in synthetic activity and reduction in denaturation of type II collagen was observed. In the third phase (‘degenerative’), an increase in type II collagen denaturation and type I procollagen synthesis was detected. Aggrecan and collagen are the main components of both the CEP and the other disc tissues, but their synthetic profiles are very different. The epitope levels of type I and II procollagen in the younger groups (< 5 years of age) are two to three times lower in CEP than in NP and AF, whereas aggrecan chondroitin sulphate 846 epitope (CS-846) levels (indicating aggrecan synthesis) are two to three times higher in CEP (Antoniou et al. 1996b). In another study, the expression of type X collagen was investigated, a well known marker for chondrocyte hypertrophy. It was found that type X collagen content in the CEP of beagle dogs increased with age, although it was also present in some dogs within the AF and NP (Lammi et al. 1998).