A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Bovine annulus fibrosus hydration affects rate-dependent failure mechanics in tension

The high water content of the intervertebral disc is essential to its load bearing function and viscoelastic mechanical behavior. One of the primary biochemical changes associated with disc degeneration is the loss of proteoglycans, which leads to tissue dehydration. While previous studies have reported the effects of in vivo degeneration on annulus fibrosus (AF) failure mechanics, the independent role of water remains unclear, as does the tissue’s rate-dependent failure response. Our first objective was to determine the effect of loading rate on AF failure properties in tension; our second objective was to quantify the effect of water content on failure properties. Water content was altered through enzymatic digestion of glycosaminoglycans (GAGs) and through osmotic loading. Bovine AF specimens were tested monotonically to failure along the circumferential direction at 0.00697%/s or 6.97%/s. Increased loading rate resulted in a ∼50% increase in linear-region modulus, failure stress, and strain energy density across all treatment groups (p < 0.001). Decreased GAG and water contents resulted in decreased modulus, failure stress, and strain energy density; however, these differences were only observed at the low loading rate (p < 0.05; no changes at high rate). Osmotic loading was used to evaluate the effect of hydration independently from GAG composition, resulting in similar decreases in water content, modulus, and strain energy density. This suggests that hydration is essential for maintaining tissue stiffness and energy absorption capacity, rather than strength, and that GAGs contribute to tissue strength independently from mediating water content.     

Temporal changes of mechanical signals and extracellular composition in human intervertebral disc during degenerative progression

In this study, a three-dimensional finite element model was used to investigate the changes in tissue composition and mechanical signals within human lumbar intervertebral disc during the degenerative progression. This model was developed based on the cell-activity coupled mechano-electrochemical mixture theory. The disc degeneration was simulated by lowering nutrition levels at disc boundaries, and the temporal and spatial distributions of the fixed charge density, water content, fluid pressure, Von Mises stress, and disc deformation were analyzed. Results showed that fixed charge density, fluid pressure, and water content decreased significantly in the nucleus pulposus (NP) and the inner to middle annulus fibrosus (AF) regions of the degenerative disc. It was found that, with degenerative progression, the Von Mises stress (relative to that at healthy state) increased within the disc, with a larger increase in the outer AF region. Both the disc volume and height decreased with the degenerative progression. The predicted results of fluid pressure change in the NP were consistent with experimental findings in the literature. The knowledge of the variations of temporal and spatial distributions of composition and mechanical signals within the human IVDs provide a better understanding of the progression of disc degeneration.     

Three-dimensional inhomogeneous triphasic finite-element analysis of physical signals and solute transport in human intervertebral disc under axial compression

A 3D inhomogeneous finite-element model for charged hydrated soft tissues containing charged/uncharged solutes was developed and applied to analyze the mechanical, chemical, and electrical signals within the human intervertebral disc during an axial unconfined compression. The effects of tissue properties and boundary conditions on the physical signals and the transport of fluid and solute were investigated. The numerical simulation showed that, during disc compression, the fluid pressurization and the effective (von Misses) solid stress were more pronounced in the annulus fibrosus (AF) region near the interface between AF and nucleus pulposus (NP). In NP, the distributions of the fluid pressure, effective stress, and electrical potential were more uniform than those in AF. The electrical signals were very sensitive to fixed charge density. Changes in material properties of NP (water content, fixed charge density, and modulus) affected fluid pressure, electrical potential, effective stress, and solute transport in the disc. This study is important for understanding disc biomechanics, disc nutrition, and disc mechanobiology.     

Streaming potential of human lumbar anulus fibrosus is anisotropic and affected by disc degeneration.

The streaming potential responses of non-degenerate and degenerate human anulus fibrosus were measured in a one-dimensional permeation configuration under static and dynamic loading conditions. The goal of this study was to investigate the influence of the changes in tissue structure and composition on the electrokinetic behavior of intervertebral disc tissues. It was found that the static streaming potential of the anulus fibrosus depended on the degenerative grade of the discs (p = 0.0001) and on the specimen orientation in which the fluid flows (p = 0.0001). For a statically applied pressure of 0.07 MPa, the ratio of streaming potential to applied pressure ranged from 5.3 to 6.9 mV/MPa and was largest for Grade I tissue with axial orientation and lowest for Grade III tissue with circumferential orientation. The dynamic streaming potential responses of anulus fibrosus were sensitive to the degeneration of the disc: the total harmonic distortion factor increased by 108%, from 3.92 +/- 0.66% (mean +/- SD) for Grade I specimens to 8.15 +/- 3.05% for Grades II and III specimens. The alteration of streaming potential reflects the changes in tissue composition and structure with degeneration. To our knowledge, this is the first reported data for the streaming potential of human intervertebral disc tissues. Knowledge of the streaming potential response of the intervertebral disc provides an understanding of potentially important signal transduction mechanisms in the disc and of the etiology of intervertebral disc degeneration.     

Annulus Fibrosus Cell Characteristics Are a Potential Source of Intervertebral Disc Pathogenesis

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.     

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell–matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.     

Elastic,permeability and swelling properties of human intervertebral disc tissues: A benchmark for tissue engineering

The aim of functional tissue engineering is to repair and replace tissues that have a biomechanical function, i.e., connective orthopaedic tissues. To do this, it is necessary to have accurate benchmarks for the elastic, permeability, and swelling (i.e., biphasic-swelling) properties of native tissues. However, in the case of the intervertebral disc, the biphasic-swelling properties of individual tissues reported in the literature exhibit great variation and even span several orders of magnitude. This variation is probably caused by differences in the testing protocols and the constitutive models used to analyze the data. Therefore, the objective of this study was to measure the human lumbar disc annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates (CEP) biphasic-swelling properties using a consistent experimental protocol and analyses. The testing protocol was composed of a swelling period followed by multiple confined compression ramps. To analyze the confined compression data, the tissues were modeled using a biphasic-swelling model, which augments the standard biphasic model through the addition of a deformation-dependent osmotic pressure term. This model allows considering the swelling deformations and the contribution of osmotic pressure in the analysis of the experimental data. The swelling stretch was not different between the disc regions (AF: 1.28±0.16; NP: 1.73±0.74; CEP: 1.29±0.26), with a total average of 1.42. The aggregate modulus (Ha) of the extra-fibrillar matrix was higher in the CEP (390 kPa) compared to the NP (100 kPa) or AF (30 kPa). The permeability was very different across tissue regions, with the AF permeability (64 E⁻¹⁶ m⁴/N s) higher than the NP and CEP (~5.5 E⁻¹⁶ m⁴/N s). Additionally, a normalized time-constant (3000 s) for the stress relaxation was similar for all the disc tissues. The properties measured in this study are important as benchmarks for tissue engineering and for modeling the disc's mechanical behavior and transport.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.     

Confocal microscopy demonstrates association of LTBP-2 in fibrillin-1 microfibrils and colocalisation with perlecan in the disc cell pericellular matrix

Comparative immunolocalisations of latent transforming growth factor-beta-1 binding protein (LTBP)-2, fibrillin-1, versican and perlecan were undertaken in foetal human and wild type C57BL/6 mouse and Hspg2 exon 3 null HS deficient mouse intervertebral discs (IVDs). LTBP-2 was a prominent pericellular component of annular fibrochondrocytes in the posterior annulus fibrosus (AF), interstitial matrix adjacent to nucleus pulposus (NP) cells and to fibrillar and cell associated material in the anterior AF of the human foetal IVD and also displayed a pericellular localisation pattern in murine IVDs. Perlecan and LTBP-2 displayed strong pericellular colocalisation patterns in the posterior AF and to fibrillar material in the outer anterior AF in the foetal human IVD. Versican was a prominent fibril-associated component in the posterior and anterior AF, localised in close proximity to fibrillin-1 in fibrillar arrangements in the cartilaginous vertebral rudiments around paraspinal blood vessels, to major collagen fibre bundles in the anterior and posterior AF and shorter fibres in the NP. Fibrillin-1 was prominent in the outer anterior AF of the human foetal IVD and in fibres extending from the AF into the cartilaginous vertebral rudiments. LTBP-2 was prominently associated with annular fibrils containing fibrillin-1, versican was localised in close proximity to these but not specifically with LTBP-2. The similar deposition levels of LTBP-2 observed in the AF of the Hspg2 exon 3 null and wild type murine IVDs indicated that perlecan HS was not essential for LTBP-2 deposition but colocalisation of LTBP-2 with perlecan in the foetal human IVD was consistent with HS mediated interactions which have already been demonstrated in-vitro.     

Validation and application of an intervertebral disc finite element model utilizing independently constructed tissue-level constitutive formulations that are nonlinear,anisotropic, and time-dependent

Finite element (FE) models are advantageous in the study of intervertebral disc mechanics as the stress–strain distributions can be determined throughout the tissue and the applied loading and material properties can be controlled and modified. However, the complicated nature of the disc presents a challenge in developing an accurate and predictive disc model, which has led to limitations in FE geometry, material constitutive models and properties, and model validation. The objective of this study was to develop a new FE model of the intervertebral disc, to validate the model?s nonlinear and time-dependent responses without tuning or calibration, and to evaluate the effect of changes in nucleus pulposus (NP), cartilaginous endplate (CEP), and annulus fibrosus (AF) material properties on the disc mechanical response. The new FE disc model utilized an analytically-based geometry. The model was created from the mean shape of human L4/L5 discs, measured from high-resolution 3D MR images and averaged using signed distance functions. Structural hyperelastic constitutive models were used in conjunction with biphasic-swelling theory to obtain material properties from recent tissue tests in confined compression and uniaxial tension. The FE disc model predictions fit within the experimental range (mean±95% confidence interval) of the disc?s nonlinear response for compressive slow loading ramp, creep, and stress-relaxation simulations. Changes in NP and CEP properties affected the neutral-zone displacement but had little effect on the final stiffness during slow-ramp compression loading. These results highlight the need to validate FE models using the disc?s full nonlinear response in multiple loading scenarios.     

An anisotropic multiphysics damage model with application to annulus fibrosus

An anisotropic multiphysics damage model is developed to characterize the couplings among multiple physical fields within soft tissues and the tissue damage based on thermodynamic principles. This anisotropic multiphysics damage model integrates the continuum mixture theory and a continuum damage model, and the anisotropic damage is considered by evolution of internal damage variables governing the anisotropic mechanical behaviors of tissues. The energy dissipation associated with the transport of fluid and ions is generally related to tissue damage. The anisotropic multiphysics damage model is applied to simulate a case of annulus fibrosus (AF) damage in an isolated intervertebral disc under compression, to understand the damage initiation and propagation. It is found that, for this case (with 1000 N/s of compression rate and neglected ground matrix damage), the damage initiated in the outer and middle posterior regions of AF at about 700 N of axial compression. The region-dependent yield stretch ratio predicted by this model is consistent with experimental findings. A sensitive study on the damage parameters is also presented. This study provides an additional insight into AF damage in the isolated disc under mechanical compression.     

Aquaporin expression in the human intervertebral disc

The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.     

Empirical measurements of biomechanical anisotropy of the human vocal fold lamina propria

The vocal folds are known to be mechanically anisotropic due to the microstructural arrangement of fibrous proteins such as collagen and elastin in the lamina propria. Even though this has been known for many years, the biomechanical anisotropic properties have rarely been experimentally studied. We propose that an indentation procedure can be used with uniaxial tension in order to obtain an estimate of the biomechanical anisotropy within a single specimen. Experiments were performed on the lamina propria of three male and three female human vocal folds dissected from excised larynges. Two experiments were conducted: each specimen was subjected to cyclic uniaxial tensile loading in the longitudinal (i.e., anterior–posterior) direction, and then to cyclic indentation loading in the transverse (i.e., medial–lateral) direction. The indentation experiment was modeled as contact on a transversely isotropic half-space using the Barnett–Lothe tensors. The longitudinal elastic modulus E _L was computed from the tensile test, and the transverse elastic modulus E _T and longitudinal shear modulus G _L were obtained by inverse analysis of the indentation force-displacement response. It was discovered that the average of E _L /E _T was 14 for the vocal ligament and 39 for the vocal fold cover specimens. Also, the average of E _L /G _L , a parameter important for models of phonation, was 28 for the vocal ligament and 54 for the vocal fold cover specimens. These measurements of anisotropy could contribute to more accurate models of fundamental frequency regulation and provide potentially better insights into the mechanics of vocal fold vibration.     

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.     

Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration.     

Numerical analysis of the influence of nucleus pulposus removal on the biomechanical behavior of a lumbar motion segment

Nucleus replacement was deemed to have therapeutic potential for patients with intervertebral disc herniation. However, whether a patient would benefit from nucleus replacement is technically unclear. This study aimed to investigate the influence of nucleus pulposus (NP) removal on the biomechanical behavior of a lumbar motion segment and to further explore a computational method of biomechanical characteristics of NP removal, which can evaluate the mechanical stability of pulposus replacement. We, respectively, reconstructed three types of models for a mildly herniated disc and three types of models for a severely herniated disc based on a L4–L5 segment finite element model with computed tomography image data from a healthy adult. First, the NP was removed from the herniated disc models, and the biomechanical behavior of NP removal was simulated. Second, the NP cavities were filled with an experimental material (Poisson's ratio = 0.3; elastic modulus = 3 MPa), and the biomechanical behavior of pulposus replacement was simulated. The simulations were carried out under the five loadings of axial compression, flexion, lateral bending, extension, and axial rotation. The changes of the four biomechanical characteristics, i.e. the rotation degree, the maximum stress in the annulus fibrosus (AF), joint facet contact forces, and the maximum disc deformation, were computed for all models. Experimental results showed that the rotation range, the maximum AF stress, and joint facet contact forces increased, and the maximum disc deformation decreased after NP removal, while they changed in the opposite way after the nucleus cavities were filled with the experimental material.     

The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P?<?0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.