Introduction: Increased cell senescence has been reported in the human intervertebral disc (IVD) and was associated with degenerative pathology, particularly herniation. Increased IVD innervation and blood vessel ingrowth is associated with disc degeneration and the development of back pain. This preliminary study examines whether there is a relationship between the prevalence of senescent IVD cells and the extent to which the tissue is innervated and/or vascularised.

Methods: Specimens of herniated IVD (n=16 patients: aged 36–71) were stained for senescence associated β-galactosidase activity (SA β-gal), then snap frozen and cryosectioned prior to immunolocalisation procedures to detect nerves (NF200) or blood vessels (CD34). Stained sections were counterstained with DAPI to reveal cell nuclei. The proportion of SA β-gal +ve cells was scored and the extent of neural and blood vessel ingrowth semi-quantitated.

Results: The proportion of SA β gal +ve IVD cells ranged from 6% – 91% (median=16%) and was significantly correlated with age. The degree of neural or blood vessel ingrowth ranged from tissue which contained numerous (i.e. ≥10) positive cells/cell processes to tissue which was completely aneural or avascular. However, there was no clear relationship between the presence of SA β-gal +ve IVD cells and IVD innervation or vascularisation.

Conclusions: Cell senescence has been associated with up-regulated expression of catabolic enzymes, e.g. MMPs and increased synthesis of trophic cytokines, e.g. VEGF. Such cellular activity might by thought to contribute to the pathological ingrowth of nerves or blood vessels into the IVD. The data presented here, however, does not support such a hypothesis.

Conflicts of Interest: None

Source of Funding: Institute of Orthopaedics, RJAH Orthopaedic Hospital

Introduction: Intervertebral disc (IVD) cell transplantation is used to treat back pain. However, IVD cell activity may also contribute to pathology, e.g. IVD cells can undergo senescence or promote nerve growth, which in the IVD is associated with discogenic back pain. Serum deprivation of bovine IVD cells results in cell senescence. We have examined the influence of oxygen supply combined with serum deprivation on human IVD cells.

Methods: Cells from herniated IVD (n=3 patients) were subjected to serum deprivation and then cultured under hypoxic (1%) or atmospheric (21%) conditions for 10 days. IVD cell growth, viability and cell senescence (via Senescence Associated β-galactosidase activity; SA β-gal) were examined. The growth and migration of HMEC-1 (endothelial) and SH-SY5Y (neuronal) cells treated with conditioned medium from the IVD cell cultures (1% versus 21% oxygen) were subsequently monitored.

Results: Hypoxia significantly decreased IVD cell proliferation, but was also found to reduce cell senescence. Hence, the proportions of SA β-gal positive IVD cells in 1% and 21% oxygen at day 10 were 18±6% and 56±10%, respectively. There was no marked difference in cell viability (> 95%). Conditioned medium from IVD cells cultured under hypoxia stimulated endothelial and neural cell growth (determined via the MTS assay) and endothelial cell migration and neurite outgrowth to an extent that was significantly greater than conditioned medium from IVD cells cultured at 21% oxygen.

Conclusions: The trophic activity of human IVD cells is responsive to oxygen supply. However, hypoxia may influence the capacity of IVD cells to reduce back pain for better or worse.

Conflicts of Interest: None

Source of Funding: Institute of Orthopaedics, RJAH Orthopaedic Hospital.

Non-union is poorly understood. It is unknown if multipotent cells are present in non-union tissue or whether the activity of such cells is dysfunctional. Clinically, this is important as it may predict the success of novel therapies such as BMP treatments and cell-transplantation. This study aimed to study the characteristics of cell types present in human fracture non-union tissue, in comparison with bone marrow stromal cells (BMSC) from the patient and other healthy patients.

Non-union tissue was harvested (n=8) from long bones. Cells were isolated enzymatically and cultured in monolayer. BMSC were isolated by density gradient centrifugation of iliac crest biopsies. Their phenotype was assessed by FACS analysis for CD34, 45 and 105 markers. Their comparative growth kinetics was examined, as was their osteogenic and adipogenic capacity following extended culture in defined medium. Cell differentiation status was evaluated using alkaline phosphatase, von Kossa and oil-red O staining. Cell senescence was assessed via cell morphology, senescence associated Beta-galactosidase (SA-Beta)-Gal) activity.

Non-union cells grew in monolayer, but showed different morphologies; many non-union cells contained stress filaments (typical of senescent cells) or were of stellate appearance. In addition, significantly more non-union cells were positive for SA-Beta-Gal activity compared to BMSC (P=0.0006). Growth kinetics showed longer doubling times for cells isolated from non-union tissue when compared to BMSC isolated from the patient. Long term culture of non-union cells showed early growth arrest at passages 3–8. FACS analysis showed isolated cells to be CD34/45 negative and CD105 positive. Both non-union cells and BMSC differentiated along osteogenic and adipogenic lineages to varying extents.

Our novel results show that cells from non-union tissue exhibit senescence in culture. Hence, cell senescence is potentially involved in the aetiopathogenesis of non-unions. Whether or not this senescence has arisen through cell division (during failed repair attempts) or via abnormal biomechanical loading warrants further study. The influence of senescent cells on the healing process also requires investigation. Clearly these cells are able to differentiate into osteoblasts in vitro but may have an aberrant influence on union in vivo.

Background: Growth and development of the intervertebral disc and its adjacent vertebrae is regulated via relative levels of cell proliferation, cell death and hypertrophy, and through extracellular matrix synthesis or degradation [1]. The synthesis of matrix molecules in the growing spine of embryonic rats has been reported in some detail [2,3]. In addition, increased levels of apoptotic disc cell death have been described in normal ageing, disc degeneration and in a murine model of disc spondylosis [4,5]. However, levels of cell proliferation in the developing spine have not been formally investigated.

Methods/Results: BALB/c mice were injected with the thymidine analogue, bromodeoxyuridine (BrdU), at weeks 1–4 postnatally and killed 1 or 24 hours later. The lumbar spines were decalcified and tissue sections immunostained for BrdU-incorporation. The intervertebral disc was fully formed at weeks 1–4, consisting of a notochordal nucleus pulposus, lamellar anulus fibrosus, and cartilaginous endplates between the disc and vertebral growth-plates. BrdU-immunopositivity was most marked in 1 week old mice, particularly in the proliferative zone of the growth-plate and the apophyseal ring. By 4 weeks, few, if any, BrdU-labelled cells were present in the disc, but some positivity remained in the apophyses. There were more paired BrdU-labelled cells at 24 hours than 1 hour post-injection in all regions, indicating likely clonal growth of these cells.

Conclusions: Cell proliferation forms an important part of the growth of the vertebrae, but also features in the early postnatal growth of the murine intervertebral disc. An understanding of how proliferation in these cell populations is regulated will help augment repair and regenerative responses in damaged adult discs or scoliosis.

Background: Increased nerve growth into degenerated intervertebral discs is associated with discogenic low back pain [1]. Many of these growing nerves are in neo-vascularised areas of the tissue [1,2] and endothelial cells that penetrate the disc express neurotrophic factors [3]. Thus, disc neovascularisation and disc innervation may be closely linked. Whilst disc aggrecan has been found to inhibit sensory nerve growth in vitro [4], the effects of disc aggrecan on endothelial cells are unknown.

Methods/Results: Adapting in vitro assays used previously [4], with HMEC-1 and EAhy-926 cell lines as models of endothelial cell growth, we found that disc aggrecan inhibited endothelial cell migration in a dose-dependent manner. Endothelial cells traversed over collagen substrates until they encountered disc aggrecan substrates (1mg/ml human aggrecan), where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the collagen:aggrecan border (Figure 1). After reaching the aggrecan border, some endothelial cells also migrated away from the disc aggrecan. At lower concentrations of disc aggrecan (0.01mg/ml), no such inhibition of endothelial cell growth was seen.

Conclusions: Loss of aggrecan, increased innervation and neovascularisation are all marked features of disc degeneration [1,2,5]. This study provides evidence that disc aggrecan inhibits endothelial migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predisposes the tissue to vascular invasion.

Introduction: Intervertebral disc degeneration occurs with ageing and is often associated with back pain. During such degeneration, gross morphological differences between the central nucleus pulposus (NP) and outer annulus fibrosus (AF) are lost and the disc loses hydration and height due to decreased proteoglycan content. The cartilage endplate may also become calcified and this blocks the passage of nutrients into the disc, causing cell death and further degeneration. A potential therapy of degeneration is “re-inflation” of the disc with the use of hydrogels seeded with autologous disc cells. In this study, we have assessed the ability of a variety of hydrogels to support intervertebral disc cell growth.

Method: Intervertebral disc cells were isolated enzymatically from bovine tails and cultured as a monolayer in 10% foetal calf serum in DMEM containing antibiotics and ascorbic acid. This stimulates the cells to proliferate and thereby produces increased cell numbers. The cells were then seeded onto various hydrogels including hyaluronic acid (HA), 2-hydroxyethyl methacrylate (HEMA), N’N’ dimethyl methacrylate (NNDMA) and polyacryloyl morpholine (AMO) before harvesting at set time points of 1, 3, 6 and 9 days for hyaluronic acid and 1, 7, 14, 21, and 28 days for the other hydrogels. Cell number, morphology, viability and adherence to or migration into the hydrogels were assessed. Cell proliferation was also determined by immunostaining for the Ki67 antigen.

Results: Disc cells became incorporated in the HA gel, adopted a spherical morphology and remained viable for up to nine days. However, after a few days, a large proportion of the cells began to migrate through the gel to form a monolayer on the bottom of the tissue culture well. These monolayered cells became fibroblastic and proliferated. NP cells appeared to proliferate to a greater extent than AF cells both in monolayer and in suspension. Ki67 antigen immunostaining confirmed cell proliferation. On the non-porous HEMA, NNDMA and AMO, both cell types adhered and adopted a fibroblast-like morphology. Cell adhesion was greatest to the HEMA. NNDMA and AMO had lower levels of cell adherence. Both cell types became incorporated into the porous materials and adopted a rounded morphology. Cell incorporation appeared to be greatest into porous HEMA.

Conclusion: These initial studies show that intervertebral disc cells will adhere to or migrate into a variety of hydrogels and remain viable. The morphology and proliferative capacity of cells derived from both the AF and NP were responsive to the structure of the hydrogel with which they were cultured. Thus, cells were able to become fibroblastic or chondrocytic. Further analyses will reveal whether matrix synthesis by disc cells is similarly responsive to the hydrogel format. The results of these experiments suggest that the hydrogels tested have potential as support matrices in intervertebral disc repair to provide relief from discogenic low-back pain.

Mature human intervertebral disc cells have generally been described as being either fibroblast-like or chondrocyte-like; i.e. appearing either elongated and bipolar or rounded/oval. Fibroblast-like cells are observed within the outer regions of the anulus fibrosus whilst chondrocyte-like cells are found in the more central regions of the disc. However, a few reports have noted that in some circumstances disc cells appear to extend more elaborate cytoplasmic processes into their surrounding extracellular matrix. In this study, we have examined healthy and pathological human intervertebral discs for the presence of the cytoskeletal elements, F-actin and vimentin.

Tissues examined included discs of no known pathology, discs with spondylolithesis, scoliosis specimens taken from the convex and concave sides, and degenerated discs. F-actin was not readily observed within discs cells but was a marked feature of vascular tissue within the disc and occasionally seen in infiltrating cells. Vimentin was more readily seen within cells of the inner anulus fibrosus and nucleus pulposus. In general, disc cell morphology was fibrocyte or chondrocyte-like; however, in spondylolisthetic discs, cells with numerous cytoplasmic projections were frequently observed.

The differential morphologies and cytoskeletal composition observed in disc cells may be indicative of variations in mechanical strains and/or pathologies, or indeed of cell function.

Although an increased and deeper innervation of painful and degenerate intervertebral discs (IVDs) has been reported, the mechanisms that regulate nerve growth into the IVD are largely unknown. In other tissues, proteoglycans have been found to act as nerve guidance molecules that, generally speaking, inhibit nerve growth. As disc degeneration is characterised by a loss of proteoglycans, we assessed the effects of IVD proteoglycans on nerve growth and guidance.

Using in vitro assays of nerve growth, we found that human disc proteoglycans inhibited nerve attachment, neurite extension and induced sensory growth cone turning in a dose-dependent manner. Digestions with chondroitinase ABC or keratinase abrogated these inhibitory effects. Proteoglycans of the anulus fibrosus were more inhibitory than those from the nucleus pulposus.

Disc proteoglycans inhibit nerve growth and this inhibitory activity may dependent on proteoglycan glycosylation and/or sulfation. A loss of proteoglycans from degenerative discs may therefore predispose the discs to nerve invasion.