Introduction: Loss of nutrient supply, seen in disc degeneration, leads to low concentrations of oxygen and glucose in the centre of the disc. Here we investigate the effect of low nutrient concentrations on the metabolism and viability of the nucleus cells.

Methods: Isolated bovine nucleus pulposus cells were cultured for 24–72 hrs over a range of pH levels and glucose and oxygen concentrations. Changes in metabolite concentrations with time were measured in a purpose-built chamber using embedded electrodes, or biochemically; and metabolic rates determined. On completion, cell numbers were counted and viability assessed.

Results: Metabolic rates varied both with oxygen concentration and with pH. At low oxygen (2% pO₂) and low pH (pH 6.2) for example, oxygen consumption rates and lactic acid production rates were 10–30% those in air at pH 7.4. Low pH in air saturated medium, or low pO2 in neutral medium, reduced metabolism but not as drastically. Glucose concentrations in the range 0.5–5mM in contrast did not affect cellular metabolism. Cells could survive with zero oxygen, although metabolism was seriously dimished; but after 24 hours at low (< 0.5mM) glucose, cell death was observed.

Discussion: Regulation of the concentrations of nutrients in vivo is complex, and depends on both supply and demand. Little is known about cellular demand, and studies such as this could give insight into the situation in the disc in vivo and help determine the cellular consequences of a fall in nutrient supply.

Our results, apart from showing the deleterious effects of low nutrient concentrations, also indicate that isolated cells may metabolise differently from cells in the tissue; at low pO₂ we observed a fall in lactate production, the opposite effect to that seen in tissue previously. The mechanism for this difference is as yet unknown.

Introduction: The intervertebral disc is a significant contributor to back pain, and is thus a tissue that is often examined postmortem. Tissue preservation during storage is of importance both experimentally, for research and teaching purposes, and clinically, for possible use in transplantation. The biomechanical function of the disc after storage has been investigated. However, to our knowledge the biological and metabolic consequences of storage have not been studied. Here we have investigated the effects of storage in the intervertebral disc on glucose, lactate, and cell viability.

Method: A total of 53 discs from 14 bovine tails were obtained within 24 hours of slaughter. Discs were either removed immediately and wrapped in clingfilm or kept in situ, surrounded by muscle. Tissue was stored at 4_C, and samples were taken at 2 hours to 9 days. Disc tissue was analysed for lactate, glucose, and cell viability. Muscle was analysed for lactate. Statistical analysis of data was performed using Student’s t test.

Results: Lactate concentrations in discs stored in tails increased with time of storage, being significantly higher even after 24 hours (p< 0.01). In contrast, lactate levels in isolated discs remained constant. Glucose levels were undetectable in discs, irrespective of storage. Muscle lactate was always significantly higher than disc (p< 0.01). The percentage of live cells fell significantly with storage in situ (p< 0.01).

Discussion: The increase in lactate observed in discs remaining in situ appears to arise from lactate diffusing in from surrounding muscle, as no increase was noted in isolated discs. As would be expected, this high concentration of lactate and low glucose appears to affect cell viability adversely, possibly as a consequence of lowered pH. This change in metabolite concentration and hence cell viability is important to note when considering human postmortem tissue, as it may affect the biological function of the disc.

Introduction: Although the cell density of the intervertebral disc is low, cells perform a vital role, being responsible for maintaining and remodelling the extracellular matrix. In animal models of scoliosis, cell viability of epiphyseal chondrocytes was found to be adversely affected. Here we examine cell density and viability of surgical disc specimens.

Method: A total of 41 discs were removed from 13 consenting patients (3M, 9F, 5–40 yrs) during corrective surgery for scoliosis. Control samples were obtained from 3 non-scoliotic discs. These were further dissected to compare the outer annulus of the disc from the more concave and more convex sides of the quadrant removed at surgery. Cell density was measured using a modified Hoechst’s method. Cell viability was determined microscopically in sections using intracellular fluorescent probes.

Results: Cell density was found to be lowest in apical discs, independent of absolute disc level (p< 0.01, Student’s t test). A significantly lower percentage of live cells was found in samples taken from the convex side of the scoliotic curve (p< 0.01, Student’s t test). No significant differences in cell viability were found in either side of control discs.

Discussion: Cell viability was seen to be lower on the convex side of the scoliotic curve, suggesting that it is more difficult for cells to survive under the conditions on the convexity compared with the concavity. This may be due to differences in the mechanical conditions or the diffusion distances across the disc. Cell numbers were lowest in the apical disc, where stresses are thought to be maximal. Fewer viable cells may decrease production of matrix macromolecules, and thus compromise matrix integrity. A delicate balance exists between production and breakdown of matrix macromolecules, and any factor that interrupts this equilibrium state has the potential to affect the structure and function of the intervertebral disc.

Intervertebral disc cells exsist in a precarious nutritional environment. Local concentrations depend on both nutritional supply and demand. Little is known about the metabolism of disc cells; existing data focuses on intact tissue, where the local metabolic environment is unknown. We have thus developed a closed chamber to study the metabolism of isolated cells under controlled conditions.

Bovine disc cells were isolated from coccygeal discs and transferred to the sealed chamber, in which embedded electrodes measured pH, pO2 and glucose concentration, and a port allowed sampling and addition of metabolic reagents. Metabolic rates were assessed from concentration changes. Cell viability was assessed and intracellular ATP measured at completion of each experiment.

Under standard conditions, metabolic rates were similar to those measured in tissue, with a glucose:lactic acid ratio of approximately one to two. We have also examined the effect of extracellular pH on nucleus pulposus cell metabolism. Between pH 7.4–6.8, metabolism is insensitive to extracellular pH, and lactic acid production agrees with the literature 1, 2. Below pH 6.8, lactic acid production fell linearly with decreased pH. At pH 6.4, lactic acid production had fallen by 60%, and intracellular ATP by 80%.

These results show a fall in lactic acid production with extracellular acidification, which in vivo arises mainly from lactic acid produced by the cells. This may be protective. However the decrease in metabolism, and hence loss of ATP, may have a detrimental effect on the cells. There is thus a complex interplay between different components of the nutritional environment. Investigating these in combination should give valuable information about disc cell metabolism, as changes in cells metabolism can affect nutrient availability and hence cellular activity and viability.