Mechanical stimulation alters pleiotrophin and aggrecan expression by human intervertebral disc cells and influences their capacity to stimulate endothelial migration

Study Design. The influence of mechanical load on pleiotrophin (PTM) and aggrecan expression by intervertebral disc (IVD) cells, and the effects of disc cell conditioned medium on endothelial cell migration was investigated. Objective. To examine possible interactions of mechanical loads and known pro- and antiangiogenic factors, which may regulate disc angiogenesis during degeneration. Summary of Background Data. Pleiotrophin expression can be influenced by mechanical stimulation and has been associated with disc vascularization. Disc aggrecan inhibits endothelial cell migration, suggesting an antiangiogenic role. A possible interplay between these factors is unknown. Methods. The influence of the respective predominant load (cyclic strain for anulus fibrosus and hydrostatic pressure for nucleus pulposus cells) on PTN and aggrecan expression by IVD cells was determined by real-time RT-PCR and Western blotting (PTN only). The effects of IVD cell conditioned medium on endothelial cell migration were analyzed in a bioassay using human microvascular endothelial (HMEC-1) cells. Results. Application of both mechanical loads resulted in significant alterations of gene expression of PTN (+67%, P = 0.004 in anulus cells; +29%, P = 0.03 in nucleus cells) and aggrecan (+42%, P = 0.03 in anulus cells, -25%, P = 0.03 in nucleus cells). These effects depended on the cell type, the applied load, and timescale. Conditioned media of nucleus pulposus cells enhanced HMEC-1 migration, but this effect was diminished after 2.5 MPa hydrostatic pressure, when aggrecan expression was diminished, but not 0.25 MPa, when expression levels were unchanged. Conclusion. Mechanical loading influences PTN expression by human IVD cells. Conditioned media from nucleus pulposus cell cultures stimulated HMEC-1 endothelial cell migration. This study demonstrates that the influence of mechanical loads on vascularization of the human IVD is likely to be complex and does not correlate simply with altered expression of known pro- and antiangiogenic factors.

Topographical guidance of intervertebral disc cell growth in vitro:towards the development of tissue repair strategies for the anulus fibrosus

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan

Study Design. Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. Objective. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. Summary of Background Data. Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Methods. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. Results. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. Conclusions. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Human intervertebral disc aggrecan inhibits nerve growth in vitro

OBJECTIVE: To assess the effects of human intervertebral disc aggrecan on nerve growth and guidance, using in vitro techniques. METHODS: Aggrecan extracted from human lumbar intervertebral discs was incorporated into tissue culture substrata for the culture of the human neuronal cell line, SH-SY5Y, or explants of chick dorsal root ganglia. The effects on nerve growth of different concentrations of aggrecan extracted from the anulus fibrosus and nucleus pulposus, and of these aggrecan preparations following enzymic deglycosylation, were compared. RESULTS: Disc aggrecan inhibited the growth of neurites from SH-SY5Y cells and induced growth cone turning of chick sensory neurites in a concentration-dependent manner. Aggrecan isolated from the anulus fibrosus was more inhibitory than that isolated from the nucleus pulposus, but enzymic pretreatments to reduce the glycosylation of both types of disc aggrecan partially abrogated their inhibitory effects. CONCLUSION: Nerve growth into degenerate intervertebral discs has been linked with the development of low back pain, but little is known about factors affecting disc innervation. The finding that disc aggrecan inhibits nerve growth in vitro, and that this inhibitory activity depends on aggrecan glycosylation, has important implications for our understanding of mechanisms that may regulate disc innervation in health and disease.

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 × 10(5)-10(6) cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro:implications for degenerative disc disease

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.

Intervertebral disc

The invention relates to the use of an aqueous gel in the repair of or prevention of damage to soft tissue, the gel comprising an aqueous gel obtainable by polymerizing one or more olefinically unsaturated monomers comprising one or more phosphate, phosphonate, borate, sulphate and/or sulphonate groups. This is preferably used for intervertebral discs or a lens of an eye. Kits and syringes containing the components are also provided.