Introduction: Elastin is a structural protein forming a highly organised network in the annulus and nucleus of the intervertebral disc (IVD). It appears important in maintaining annulus structure as it is densely located in the interlamellar space and forms cross-bridges between lamellae. Here we have investigated elastin fibre organisation in degenerate discs and compared it to that seen in normal human and bovine discs.

Methods: Human lumbar IVD were obtained from consented patients undergoing surgery either for disc degeneration, tumour or trauma. The disc segments were collected from operating theatre and graded. A radial profile of the specimen was dissected and snap-frozen. Sections of 20μm in thickness were cut with a cryostat microtome and mounted on slides. To visualize elastin fibres, sections were digested with hyaluronidase after fixation with 10% of formalin. Elastin fibres were immunostained and fibre organisation mapped.

Results: In degenerate disc, the elastin fibre network appeared sparse and disorganised in comparison to that seen in non-degenerate human or in bovine discs in which elastin fibres are well organised. In addition, in degenerate discs the elastin fibres appear fragmented. Fragmentation of the elastin network within lamellae of the annulus in particular increased with both degeneration grade and with age.

Discussion: The loss of elastic network integrity observed in degenerate discs could contribute to loss of annulus integrity and affect disc mechanical properties adversely. Furthermore, our initial results have suggested fragmented elastin degradation products could upregulate MMP expression by disc cells thus stimulating a degenerative cascade.

Purpose: High levels of the chitinase 3-like protein HC-gp39 (human cartilage glycoprotein 39) have been found in the synovial fluid and sera of patients with arthritis. Although the function of this protein is unclear, in chondrocytes at least it appears to limit catabolic responses to cytokines such as Il-1b. Here we have investigated secretion of this protein by intervertebral disc cells and determined if its production is influenced by extracellular osmolarity.

Methods: Cells were isolated from bovine caudal discs by enzyme digestion and cultured in DMEM in alginate beads for 6 days. Medium osmolarity was increased in the physiological range by sodium/potassium addition. Supernatants were collected every 2 days and replaced with fresh media. At the end of experiment the supernatants were used for lactate determination and for detection of GP-39 by western blotting. Beads were assayed for glycosaminoglycans, cell viability and cell density.

Results: GP-39 was a major protein secreted by disc cells. It was evident on day 2 at low osmolarities. By day 4 concentrations in the medium had increased significantly and the protein was present mainly in fragmented form, particularly at high osmolarities. Osmolarity had no effect on cell density or viability. Rates of lactate production and GAG accumulation were greatest at high osmolarities.

Discussion: Changes in osmolarity, equivalent to those experienced by disc cells during the diurnal loss and regain of fluid content, had significant effects on cell metabolism and influenced production of GP-39. Osmotic changes might thus influence responses of disc cells to inflammatory signals.

Objective: To determine if (a) inflammatory mediators are present in herniated intervertebral discs and (b) if their presence correlates with inflammation of nerve roots or symptoms.

Design: Inflammation was assessed with gadolinium enhancement of MRI. Neurological compromise was measured. Disc tissue was examined for inflammatory mediators IL-1α and β, IL-6, MCP-1, TSG-6, iNOS, TNFα and thromboxane.

Patients: Sixty-five discs were removed from 64 patients undergoing surgery for disc prolapse.

Outcome measures: We developed (i) an MRI score to assess inflammation radiologically prior to surgery (n=28, mean 4.9±6.8 days), (ii) a Surgical Score to assess inflammation of the nerve roots at surgery (n=44), (iii) a Clinical Score to determine pain, disability and neurological compromise (n=17) and (iv) a Mediator Score to reflect the number and amount of inflammatory mediators present (n=20).

Results: Thirty percent of the prolapses in this study were extrusions, 19% sequestrations and 51% protrusions. Sixteen of the 28 patients with gadolinium had nerve root enhancement (86% of the extrusions, 57% of sequestrations, and 43% of protrusions), whilst 19 had enhancement of or around the disc herniation itself (71% of the extrusions, 86% of sequestrations and 57% of protrusions). The Mediator Scores were highest for the sequestrations (as was the Surgical Score) and lowest for the protrusions, but extruded discs had most IL-1α and β, IL-6, TNFα and thromboxane. Extruded discs had the highest Clinical Score and sequestrated the lowest.

Conclusions: Mediators produced in prolapsed disc appear to play an important role in inflammation of adjacent tissue and nerve roots. The type of mediator present and proximity of the prolapse to the nerve root may be the important factors in determining which pro-lapses are the most painful.

Objective: To develop and use a closed chamber to study the metabolism of isolated disc cells under controlled conditions such as reduced pH.

Design: Disc cells were incubated in the chamber for four hours, while embedded electrodes measured pH and pO2. A port allowed sampling.

Subjects: Nucleus pulposus cells were isolated from the coccygeal discs of 33 steers (18–24 months old), within three hours of slaughter.

Outcome measures: Metabolic rates were calculated from concentration changes. Cell viability was assesed on completion.

Results: At pH 7.4, metabolic rates were similar to those measured in tissue [1, 2] with lactic acid production and oxygen consumption rates of 157 and 12 nmol/million cells/hour respectively, and a 1: 2 ratio of glucose consumption: lactic acid production. Lactic acid production and oxygen consumption fell with extracellular pH, to 89 and 65 nmol/million cells/hour (lactate) and 8 and 5 nmol/million cells/hour (oxygen), at pH 6.7 and pH 6.2 respectively.

Conclusions: These results show a fall in lactic acid production and oxygen consumption with extracellular acid-ification. There is a complex interplay between different components of the nutritional environment. Investigating these in combination should give valuable information about disc cell metabolism, as changes can affect nutrient availability and hence cellular activity, viability, and matrix production rates.

Objective: To measure intradiscal pressures in scoliotic spines to further understand the role of mechanical forces in the development of scoliosis.

Design: Pressure readings were obtained in consented patients with ethical approval. A needle mounted pressure transducer was introduced into the disc during routine anterior scoliosis surgery.

Subjects: Ten human scoliotic discs from three patients.

Outcome measures: Intradiscal pressure profiles.

Results: Nuclear hydrostatic pressures varied from 0.2 to 0.6 MPa. The mean nuclear pressures for the three spines were 0.27+0.12, 0.35+0.06 and 0.47+0.12 MPa.

High stress, non- hydrostatic regions were consistently recorded in the concave annulus.

Conclusions: Nuclear pressures in these scoliotic patients were significantly higher than the 0.12 and 0.15 MPa recorded previously in non-scoliotic recumbent individuals^1;2 suggesting that spinal loading is abnormal in scoliosis.