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.

The aim of this study was to measure diffusion coefficients of solutes through the disc in relation to molecular weight.

The intervertebral disc is avascular thus nutrients and other factors from the blood supply are transported into the intervertebral disc by diffusive and convective flow. For small solutes such as lactate and glucose and oxygen, diffusion appears to predominate however convection may aid transport of larger molecules such as growth factors.

At present there however, there is virtually no information on diffusion of solutes of different molecular weights through the disc; this information is necessary for assessing and modelling transport pathways.

Diffusion coefficients were measured in nucleus and annulus sections of bovine intervertebral discs by a novel method which prevented tissue swelling and proteoglycan loss. Briefly strips of fluorescent or radiolabelled solute-saturated filter-paper were placed adjacent to the disc and the resulting concentration gradients measured at appropriate times. Solute sizes from 0.01 to 70 kDa were investigated. All results are reported as mean + s.e.m (n=6).

Diffusion coefficients (D) fell steeply with increase in molecular weight following a log-log relationship as predicted by theory. For small solutes (lactate) D for the outer annulus was 3.4 ± 1.1.10⁻⁶ cm²/sec while for 70 kDa dextran, D was 1.4 ± 0.6.10⁻⁷. There was no significant difference between values of D for nucleus and outer annulus for any solute.

Diffusion coefficients through the disc follow relationships seen in other cartilages and are dependant on tissue properties and molecular weight. The similarities between values for nucleus and outer annulus demonstrate the conflicting roles of proteoglycan and water contents in governing diffusion through the matrix with D decreasing both with increase in proteoglycan and decrease in water content.

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.

Scoliosis is a disease characterised by vertebral rotation, lateral curvature and changes in sagittal profile. The role of mechanical forces in producing this deformity is not clear. It is thought that abnormal loading deforms the disc, which becomes permanently wedged. Modelling and in vitro studies suggest that such deformations should increase intradiscal pressure. Intradiscal pressure has been measured previously in a variety of clinical environments. The aim of this study is to measure pressure profiles across scoliotic discs to provide further information on the role of mechanical forces in scoliosis.

Pressure readings were obtained in consented patients with ethical approval using a needle-mounted sterilised pressure transducer (Gaeltec, Dunvegan, Isle of Skye) calibrated as described previously. The transducer needle was introduced into the disc of an anaesthetised patient during routine anterior scoliosis surgery and pressure profiles measured. Signals were collected, amplified and analysed using Power-lab and a laptop computer.

Pressure profiles across 10 human scoliotic discs from 3 patients have been measured to date. Pressures varied from 0.1 to 1.2 MPa.

Annular pressures showed high pressure, non-isotropic regions on the concave but not convex side of these discs.

Nuclear pressures recorded from the discs of these scoliotic patients were higher than those recorded previously in non-scoliotic recumbent individuals.

The aim of this study was to identify potential inflammatory mediators in herniated and non-herniated intervertebral disc. It has been suggested that inflammation of the nerve root is a pre-requisite for disc herniations to be symptomatic. What leads to this inflammation is a matter of conjecture; one possible cause may be inflammatory mediators released from the herniated disc tissue itself. In this study we have examined discs from individuals with and without disc herniations to determine if there is a different degree of occurrence.

Twenty two discs from 21 patients with disc herniation were examined together with four discs from patients with other disc disorders and five age-matched discs from individuals obtained at autopsy. Samples were studied for the presence of blood vessels and inflammatory cytokines: IL-1α and β, IL-6, INOS, MCP1, TNFα, TSG-6 and thromboxane.

Of the herniated discs 10 were protrusions, six extrusions and six sequestrations. There was less of all the cytokines in the non-herniated discs than found in the herniated, with very little immunostaining for iNOS or IL-1α in any samples. Staining was seen in all herniated samples for IL-1β, but in fewer for IL-six and MCP1 (86%), thromboxane (68%), TNFα (64%) and TSG-6 (59%). The presence of cytokines was strongly associated with the presence of blood vessels. Protruded discs had less TNFα and thromboxane than sequestrated or extruded discs.

Cytokines appear to play an active role in the aetiopathogenesis of disc herniations. Some may be involved in the stimulation of degradative enzymes and hence resorption of, for example, sequestrations, whereas others may be responsible for an inflammatory response in the surrounding tissues such as nerve roots.

Glycosaminoglycans (GAGs) govern the osmotic environment of cartilaginous tissues and hence determine their ability to resist the large compressive forces encountered during normal activity. In degeneration GAGs are lost and there is now much interest in biological repair processes where cells from cartilaginous tissues synthesise replacement GAGs and other matrix components in situ. In addition, cells can be grown in tissue engineered constructs. Unfortunately, GAG synthesis is slow.

The aim of this study was to determine whether GAG accumulation could be hastened by increasing cell density in a construct using articular cartilage and intervertebral disc cells cultured in alginate beads.

Bovine chondrocytes and intervertebral disc cells were placed in alginate bead suspension at varying cell densities. GAG synthesis rates, total GAG accumulation and lactate production rates were determined by standard methods. The cell viability profile across intact beads was determined using fluorescent probes.

Increasing cell density causes a reduction in lactate production and sulphate incorporation per million live cells. At greater than 20 million cells per ml, cell death is increased compared with lower densities. GAG produced per bead is not increased in proportion to increasing cell density.

These results show that there is a limit to the rate at which matrix per volume of tissue can be produced and accumulated. At high cell densities cellular activity is limited by toxicity arising from low pH and hypoxia.

Articular cartilage from the femoral heads of 27 patients having an arthroplasty for subcapital fracture was studied, and its mechanical and chemical properties compared to those of a group of 33 age-matched macroscopically normal autopsy specimens. Water and proteoglycan contents were measured, as were swelling ability, compressive and tensile strength of the cartilage, and the density of the underlying bone. Cartilage from the fracture specimens had a significantly reduced proteoglycan content, as measured by fixed charge density, and increased swelling ability. These results indicate that this group differs from the "normal" population and care should be taken before they are accepted as control material for studies on osteoarthritic cartilage. Another finding was that bone density was much the same in the fracture and the normal group. This casts some doubt upon the concept that patients who sustain subcapital fractures are more osteoporotic than the average for the same age range.

We studied the mechanical and biochemical properties of articular cartilage from 22 osteoarthritic femoral heads obtained at operation and 97 femoral heads obtained at autopsy. Cartilage from the zenith and from the antero-inferior aspect of each head was tested both in tension and in compression. Water content, swelling ability and proteoglycan content were measured, the cartilage was examined histologically and the density of the underlying bone was assessed. Fifty-five of the autopsy specimens were defined as macroscopically normal because they exhibited no progressive fibrillation patterns on staining with Indian ink; but significant changes in water content, bone density and tensile strength related to age were seen in this group. In 20 pairs of femoral heads which were both macroscopically normal, we found, surprisingly, that cartilage from the left and right sides of the same patient was sometimes very different. Compared with the normal autopsy specimens the osteoarthritic specimens had a significantly increased swelling ability, a lower proteoglycan content and impaired mechanical properties, being both weaker in tension and softer in compression. Abnormal autopsy specimens had values intermediate between those of osteoarthritic and normal groups. Results from this abnormal group suggest that there is no primary loss of proteoglycan in early osteoarthritis.