Introduction

Elastic fibres are constructed of a central core of elastin surrounded by microfibrils that are composed mainly of fibrillin-1 and fibrillin-2. Patients with mutations in the gene encoding fibrillin-1 or fibrillin-2 develop Marfan syndrome or Beals syndrome (congenital contractural arachnodactyly), respectively. Scoliosis is one of the clinical manifestations in these patients, but how a defect in the elastic proteins could lead to a spinal deformity is not clear. On the one hand, the mutations could induce scoliosis via mechanical means as they could lead to alterations in the biomechanics of the elastic fibre system. On the other hand, elastic fibres also bind growth factors such as transforming growth factor β (TGFβ) and bone morphogenic proteins (BMPs), and the mutations could hence change patterns of spinal growth.

This study aims to investigate femoral blood flow during Metal-on-Metal Hip Resurfacing (MMHR) by monitoring oxygen concentration during the operative procedure.

Patients undergoing MMHR using the posterior approach were evaluated. Following division of fascia lata, a calibrated gas-measuring electrode was inserted into the femoral neck, aiming for the supero-lateral quadrant of the head. Baseline oxygen concentration levels were detected after electrode insertion 2-3cm below the femoral head surface and all intra-operative measures were referenced against these. Oxygen levels were continuously monitored throughout the operation. Data from ten patients are presented.

Oxygen concentration dropped most noticeably during the surgical approach and was reduced by 62% (Std.dev +/-26%) following dislocation and capsulectomy. Insertion of implants resulted in a further oxygenation decrease by 18% (Std.dev +/-28%). The last obtained measure before wound closure detected 22% (Std.dev +/-31%) of initial baseline oxygen levels. Variation between subjects was observed and three patients demonstrated a limited recovery of oxygen levels during implant insertion and hip relocation.

Intra-operative measurement of oxygen concentration in blood perfusing the femoral head is feasible. Results in ten patients undergoing MMHR showed a dramatic effect on the oxygenation in the femoral head during surgical approach and implant fixation. This may increase the risk of avascular necrosis and subsequent femoral neck fracture. Future experiments will determine if less invasive procedures or specific positioning of the limb can protect the blood supply to femoral neck and head.

Intervertebral discs (IVDs) are fibrocartilagenous ovoids located between the vertebral bodies of the spine that provide the sole source of flexibility in that structure. IVDs are clinically very important as degeneration has been shown to be strongly associated with lower back pain, sciatica, and disc herniation: potentially disabling conditions that affect a very large section of the UK population.

The aetiology of disc degeneration is poorly understood although upregulation of matrix metalloproteinase (MMP) activity is thought to be involved. Degradation products of the extra-cellular matrix are known to increase MMP production and activity in other tissues. This project concentrated on examining the effects of degredation products of elastin. Elastin fragments (κ-elastin peptides) have been shown to upregulate mRNA levels and increase expression of pro-MMP-1 in human skin fibroblasts, cells that are thought to be similar to those residing in the annulus fibrosus of intervertebral discs. This study examined their effect on disc cells and on skin fibroblasts.

Total MMP-2 and -7 activity produced by cells extracted from the annulus fibrosus of bovine intervertebral disc cells and cultured for 24 hours with 0–300μg/ml κ-elastin was determined using fluorimetric and zymographic analyses. κ-elastin was prepared from bovine ligamentum nuchae or bovine intervertebral discs.

Culture with κ-elastin prepared from bovine ligamentum nuchae caused skin and disc cell potential pro-MMP-2 activity to increase in a dose-dependent manner; the potential pro-MMP-2 activity of both cell types is more than doubled when cultured with 300μg/ml κ-elastin.

These findings suggest that in the bovine disc, matrix breakdown may cause a feedback loop with degraded elastin stimulating disc cells to increase production of pro-MMP-2, with the possibility of further degrading elastin and other proteins and contributing to IVD breakdown.

Introduction: Avascular necrosis of the femoral head after resurfacing hip replacement is an important complication which may lead to fracture or failure. We compared the changes in femoral head oxygenation resulting from the anterolateral approach to those resulting from the posterior approach.

Methods: In 22 patients undergoing hip resurfacing surgery, a calibrated gas-sensitive electrode was inserted supero-laterally in the femoral head via the femoral neck following division of the fascia lata. Inter-operative X-ray confirmed correct electrode placement. Baseline oxygen concentration levels were recorded immediately after electrode insertion. All results were expressed relative to this baseline, which was considered as 100% relative oxygen concentration. Oxygen levels were monitored continuously throughout the operation. 10 patients underwent surgery through the posterior approach, 12 patients through the antero-lateral approach.

Results: During the operation patterns were similar for both groups, except following joint relocation and soft tissue reconstruction; oxygen concentration recovered significantly in the anterolateral group only. The posterior approach resulted in significantly lower (p< 0.01) oxygen concentration at the end of the procedure (22%, SD 31) than the antero-lateral approach (123%, SD 99).

Discussion and Conclusion: The anterolateral approach disrupts the femoral head blood supply significantly less than the posterior approach in patients undergoing resurfacing. The incidence of complications related to avascular necrosis might be decreased by adopting blood supply conserving surgical approaches.

Introduction: Loads acting on scoliotic spines are thought to be asymmetrical and involved in progression of the scoliotic deformity. Abnormal loading patterns could lead to changes in bone and disc cell and activity and hence to vertebral body and disc wedging. At present however there are no direct measurements of intradiscal stresses or pressures in scoliotic spines.

Methods: Stress profilometry was used to measure horizontal and vertical stresses at 5mm intervals across 25 intervertebral discs of 7 scoliotic patients during anterior reconstructive surgery. Identical horizontal and vertical stresses for at least two consecutive readings defined a region of hydrostatic pressure. Results were compared with similar stress profiles measured during surgery across 10 discs of 4 spines with no lateral curvature and with data from the literature.

Results: Profiles across scoliotic discs were very different from those measured across normal discs of a similar age. Hydrostatic pressure regions were only seen in 16/25 discs, extended only over a short distance and were displaced towards the convexity. Mean pressures were significantly greater (0.24MPa) than those measured in other anaesthetised patients (< 0.06 MPa). A stress peak in the concave annulus was a common feature (13/25) in scoliotic discs. In 21/25 discs, stresses in the concave annulus were greater than in the convex annulus, indicating asymmetric loading in these anaesthetised, recumbent patients.

Conclusions: Intradiscal pressures and stresses in scoliotic discs are abnormal even in the absence of significant applied load. Disc cells respond to changes in pressure, hydration and deformation by altering matrix synthesis and turnover in vivo and in vitro. Hence, whatever the cause of the abnormal pressures and stresses in the scoliotic discs, if present during daily life, these could lead to disc matrix changes and especially if asymmetrical, to disc wedging and progression of the scoliotic deformity.

Work supported by Fondation Cotrel

Background: The intervertebral disc and spinal ligaments contain extensive and well organised elastic fibre networks which provide these tissues with elasticity. Morphologically elastic fibres are composed of an amorphous central core consisting mainly of elastin surrounded by a microfibrillar mesh. The importance of the microfibrils has been emphasised by the clinical manifestations of Marfan Syndrome (MFS) and congenital contractual arachnodactyly (CCA) which are caused respectively by mutations of Fibrillin-1 and Fibrillin-2, the main protein components of the microfibrillar mesh. Both patients of MFS and CCA can develop a spinal deformity. Recent studies on genetically modified mice suggested that minor components of the microfibrillar mesh can also play an important role in spine development; knockout mice containing no fibulin-5, microfibrillar associated glycoprotein-2 (MAGP-2), or latent TGF-b protein 3 (LTBP-3) can all develop spinal deformity. Our aim in this study was to understand the involvement of elastic fibre system in pathogenesis of scoliosis.

Methods: Tissue from Marfan patients and adolescent idiopathic kyphoscoliotic human intervertebral discs were removed during routine surgery with consent and ethical permission. Here we report on examination of disc tissue from three Marfan’s syndrome and three AIS patients (with ethical approval), age range 13–33 years. Tissues were dissected and then snap frozen within 4 hours after surgical excision and kept in −80 OC till used. Tissue sections of 20 micron were cut with a cryostat microtome and fixed with 10% formalin before immunostaining. Microfibrils and elastin fibre network were studied by immunostaining fibrillin-1 and elastin. The collagen network was examined by using fluores-cent microscopy with a polarised light system. Spines from transgenic mice, producing no elastin or fibulin-5, were paraffin embedded and sections were stained with Haematoxylin & Eosin or Alcien Blue. The morphology of cells, vertebral body and disc matrix were studied at light microscopic level.

Results and Discussion: Our histological studies on IVD tissues from MFS and AIS patients found that the elastic fibre and collagen networks were disorganised compared to that of normal controls. Studies on spines from fibulin-5 null or elastin null mice indicated delayed ossification of the vertebral body, lower expression of proteoglycans and an abnormal growth plate. Our initial results thus indicate that the elastic fibre system has an effect on matrix synthesis in connective tissue and plays a part in regulating bone growth. They are in agreement with reports that kypho-scoliosis occurs in transgenic mice deficient in other matrix components e.g. collagen-II and perlecan. Matrix-generated regulation of spine development and vertebral body growth thus appears to play an important role in the development of scoliosis.

The aim of this study was to determine cell viability in different stages of rotator cuff tendon tears using a cell viability molecular probe. Surgical biopsies taken from the edge of the Supraspinatus tendon tear from12 patients, 5 women and 7 men, mean age of 61 years were subjected to a cell viability assay using Molecular Probes Live/Dead cell viability assay. Specimens were then incubated with Calcein-AM and Ethidium Homodimer-1 and following snap freezing, sections were viewed under fluorescent microscopy. Cells which remained metabolically active fluoresced green, whereas dead cells were red. Populations of live and dead cells were counted for each specimen on ten high powered (x400 magnification) fields of view. The results show that the percentage of live cells is reduced in large chronic degenerate tears but greatest in acute traumatic tears. In addition, for those cases where tissue was assayed from the edge of the tear and 1 cm more proximally, there was a considerable increase in the percentage of viable cells in more proximal tissue. Use of this simple assay demonstrates high cell viability and consequently good quality tissue in traumatic tears, but lower quality tissue in larger more degenerate tears. This suggests that traumatic lesions have a high propensity to heal while larger more degenerate tears are less likely to heal but have better quality tissue more proximally.

Purpose: The inflammatory response around herniated tissue in the epidural space is believed to play a major role in the spontaneous regression of herniated lumbar disc. Numerous macrophages invade the herniated tissue along with newly formed blood vessels which influence oxygen gradient. Inflammatory cytokines such as interleukin-1 are produced by macrophages. These chemical mediators could stimulate disc cells to produce proteases such as MMPs which degrade the intervertebral disc matrix and could hence influence regression of the herniation. Here we have examined the influence of IL-1β and oxygen tension on proteoglycan turnover using a three-dimensional disc-cell culture system.

Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs by enzyme digestion. They were initially cultured for 14 days in alginate beads in DMEM containing 6% FBS at 4.10⁶ cells/ml under 21% oxygen to accumulate matrix. They were then cultured for 6 days under 0% or 21% oxygen and with or without IL-1β. Glycosaminoglycan (GAG) accumulation (as a measure of proteoglycan content) was measured using a DMB assay. Lactate and glucose production were measured using a standard enzymatic method. Rates of sulfated GAG synthesis was measured from rates of ³⁵S-sulfate accumulation. MMP activity was measured using coumarin fluorescent assay.

Results: The results showed that IL-1β had a significant effect on GAG accumulation and production and that its effect was dependent on oxygen tension. GAG production and sulfate incorporation rates decreased in the presence of IL-1β at high oxygen but low oxygen inhibited the effects of this cytokine. MMP activity increased with IL-1β under 21% oxygen, but not at low oxygen.

Conclusion: Exogenous IL-1β can activate MMP activity and digest the extracellular matrix of the disc but only at high oxygen tensions. Angiogenesis as well as inflammation is thus required for resorption of herniations.

Purpose: Proteoglycan loss is one of the first signs of disc degeneration. There is increasing interest in developing biological methods for its replacement both by in vivo repair and through tissue engineered constructs. Many factors influence the rate of proteoglycan accumulation. In this study, we examine how physiological levels of extracellular osmolality and oxygen tension influence proteoglycan accumulation in nucleus pulposus cells in a three-dimensional culture system.

Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 0%, 5% and 21% O₂, Medium osmolality was altered by NaCl addition over the range 270–570 mOsm. Cell viability was determined by manual counting using trypan blue. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a DMB assay.

Results: There was no difference in the cell viability. Lactate production decreased under hypo- (270 mOsm) after 6 days in culture. After 6 days GAG accumulation was maximal in beads cultured at 5% O₂ in 370 mOsm where GAG accumulation was 86.1% greater than at 21% O₂ and DMEM at standard Osmolarity (270 mOsm).

CONCLUSION: In our model the prevailing osmolality was a powerful regulator of GAG accumulation by cultured nucleus cells. In vivo prevailing osmolality is governed by GAG concentration. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of degeneration and for tissue engineering.

Purpose: To investigate the effect of timed incubations and osmolarity on the cellular protein profile between nucleus pulpous cells and articular chondrocytes to identify possible cellular markers. Both cell types exists in a constantly interchanging environment in which osmolality changes significantly during disc and joint loading.

Methods: Bovine nucleus pulpous and articular chondrocyte cells were isolated and digested with collagenase. The cells were resuspended in alginate beads and incubated in DME medium. DMEM was prepared with increasing osmolarity (280–580mOsm). At T=0,1,3 & 5 cells were collected by dissolving the alginate beads and then washed. Cellular proteins were analysed by large SDS-PAGE, scanned and analysed by computer package. Bands of proteins of interest were cut out for mass spec analyses.

Results: Analysis of whole cells from the nucleus pulpous and articular chondrocytes by SDS-PAGE at T=0 revealed very similar protein patterns. Over 5 days a peak at around 26kDa that appeared in both cell groups. Differences occurred when the cells were incubated with increasing osmolarity. Nucleus pulpous cells showed a loss of peak intensity around 60kDa and 32kDa. Chondrocyte cells showed increased peak intensity around 140kDa, as well two peaks flanking a major peak at around 55kDa.

Conclusion: Incubation of both cell types in alginate beads caused the appearance of a new peak on SDS-PAGE. When both cell types were incubated with increasing osmolarity new protein peaks appeared which may assist in deciphering between the two cell types. Mass spec will identify the protein peaks.

Introduction: Autologous chondrocyte implantation is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogenous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.

Methods: Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into poly vinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM+10% FBS within 15ml conical tubes rotated at 37°C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H& E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X, and to the glycosaminoglycans, chondroitin-4- and -6-sulphate and keratan sulphate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein, and small leucine-rich PGs.

Results: H& E staining of 10μm-thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining revealed the presence of GAGs, increasing with time. Ki-67 revealed approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X and the glycosaminoglycans, chondroitin-4-, -6 and keratan sulphate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.

Conclusions: Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.

Purpose This study aims to investigate blood flow in the femoral head during Metal-on-Metal Hip Resurfacing (MMHR) through the posterior approach by monitoring oxygen concentration during the operative procedure.

Methods Following division of fascia lata, a calibrated gas-measuring electrode was inserted into the femoral neck, aiming for the anterolateral quadrant of the head. Baseline oxygen concentration levels were detected after electrode insertion 2–3cm below the femoral head surface and all intra-operative measures were referenced against these. Oxygen levels were continuously monitored throughout the operation. Results of measurements from ten patients are presented.

Results Oxygen concentration was reduced during the surgical approach and average oxygen concentration following dislocation and circumferential capsulotomy dropped to 43% of baseline (Std.dev +/−37%), this was a highly significant reduction (p< 0.005). Insertion of implants resulted in a further significant drop in oxygen concentration (p< 0.02) to 16% of baseline (Std. dev +/−27%). Oxygen concentration rose slightly after relocation of the resurfaced joint and reconstruction of posterior soft tissues, reaching 22% (Std.dev +/−31%) of initial baseline oxygen levels. Considerable variation between subjects was observed. Three subjects had no remaining oxygen concentration at the end of surgery.

Conclusion Intra-operative measurement of oxygen concentration in blood perfusing the femoral head is feasible. During MMHR there is a dramatic decrease in femoral oxygenation during surgical approach and implant fixation. This may increase the risk of avascular necrosis and subsequent femoral neck fracture. Future experiments will determine if less invasive procedures or a different approach can protect the blood supply to femoral neck and head.

Introduction: The incidence of femoral neck fracture is approximately 2% after resurfacing hip replacement. Avascular necrosis is thought to be a contributory factor. The aim of this study was to investigate oxygen concentration in the femoral head during metal-on-metal hip resurfacing (MMHR).

Materials and Methods: In ten patients, following division of the fascia lata, a calibrated gas-measuring electrode was inserted into the supero-lateral quadrant of the femoral head via the femoral neck. Xsrays confirmed placement of the electrode 2-3 cms below the femoral head surface. Baseline oxygen concentration levels were recorded immediately upon electrode insertion and used as a reference for all intra-operative measures. Oxygen levels were monitored continuously throughout the operation.

Results: Oxygen concentration was reduced during the surgical approach and average oxygen concentration following dislocation and circumferential capsulotomy dropped significantly (p< 0.005) to 38% of baseline (SD=26%). Insertion of implants resulted in a further significant drop in oxygen concentration (p< 0.04) to 21% of baseline (SD=28%). Oxygen concentration rose slightly, but not significantly after relocation of the resurfaced joint and reconstruction of posterior soft tissues, reaching 22% of initial baseline oxygen levels. Considerable variation between subjects was observed.

Discussion: Intra-operative measurement of oxygen concentration in blood perfusing the femoral head is possible. During MMHR there is a dramatic decrease in femoral head oxygenation during surgical approach and implant fixation. This may increase the risk of avascular necrosis and subsequent femoral neck fracture. Future experiments will determine if a different surgical approach can protect the blood supply to the femoral head and neck. Measurements of femoral head oxygenation during metal-on-metal hip resurfacing demonstrated a significant concentration decrease during surgical approach and implant fixation.

Introduction Metal-on-Metal Hip Resurfacing (MMHR) has been established as a successful alternative to Total Hip Replacement (THR). However, several series report a 2 % incidence of early femoral neck fractures. Avascular necrosis (AVN) was considered to be responsible for the majority of observed fractures, raising concerns about the femoral head blood supply during MMHR. This study aims to further understand the mechanisms of femoral blood flow restriction by monitoring gas levels (O2) during the operative procedure.

Methods Patients undergoing MMHR using the posterior approach were evaluated. Following division of fascia lata, a guide wire was introduced up the femoral neck, aiming for the anterosuperior quadrant of the head. It was then removed. A calibrated gas-measuring electrode was inserted in the created bone channel. X-ray confirmation was obtained to ensure that the active measurement area of the electrode was 2–3cm below the femoral surface. O2 and N2O levels were then continuously monitored throughout the operation.

Results A preliminary analysis of four patients is presented: Stable N2O- measurements throughout the procedure confirmed valid electrode measurements. Baseline oxygen concentration levels of 40%– 60% were detected before division of short rotators. After hip dislocation oxygen concentration dropped in all patients to levels ranging between 0% and 5%. Oxygen concentration was found to remain depressed at these levels throughout the entire operation in three patients. Recovery of O2 concentration to baseline levels was observed in one patient 15 minutes after dislocation.

Discussion In three patients the extended posterior approach and joint dislocation had a dramatic effect on the perfusion in the femoral head. These patients have a high risk for development of AVN and potential femoral neck fracture. Whilst the results require further verification, subsequent experiments will determine if less invasive procedures or specific positioning of the limb can protect the femoral blood supply.

Aim To determine if tissue metabolism varies in supraspinatus tendons with distance from the edge of the rotator cuff tendon tear and also with differing size of tear.

Background Tissue metabolism can be assessed by measuring oxygen and nitrous oxide concentrations within the tissue. Viable tendon tissue consumes oxygen and contains nitrous oxide (used in the general anaesthetic) from the blood stream. Non-viable tendon tissue will not consume oxygen but will contain nitrous oxide.

Methods Oxygen and Nitrous Oxide concentrations were measured amperometrically using silver needle microelectrodes.

The needle was inserted into the supraspinatus tendon of patients with massive, large, medium and small full thickness rotator cuff tears and patients with partial thickness and no tears. Patients undergoing open stabilisation were used as controls. Measurements were made at a number of quantifiable points from the tendon edge to allow the creation of a topographical map of tissue metabolism. Oxygen consumption was calculated using measured oxygen and nitrous oxide levels at each point.

Results In patients with rotator cuff tears oxygen consumption was significantly lower near to the edge of the tear.

Patients with impingement syndrome but no evidence of a rotator cuff tear also showed a decreased level of oxygen consumption in the anterior part of supraspinatus, but this was significantly higher than the levels seen in the torn tendon.

The control group showed no significant alteration in oxygen levels

Conclusion Patients with rotator cuff tendon tear demonstrate significantly reduced levels of tissue metabolism. This reduction in tissue viability is significantly greater at the edge of the tear and in larger tears. Patients with intact tendons and impingement syndrome also demonstrate minor reduction in tissue tendon viability compared with controls.

Background and Purpose: Intervertebral discs are the largest avascular tissue sources in the human body. The transport of vital nutrients and oxygen into and metabolic waste products out of the disc, relies mainly on the diffusion through the disc matrix. The health or degree of degeneration of the disc is thought to be directly related to the transport properties of the disc. The diffusivity of nutrients and metabolites varies with matrix composition and especially with matrix hydration. The hydration of the disc varies by approximately 25% in the normal 24hour loading cycle of human beings. This work addresses the question of the effect of hydration of the disc tissue on the solute diffusivity.

Methods: Measurements of the diffusion of solutes were performed in ca. 2year bovine caudal discs. Diffusivity of dissolved oxygen and nitrous oxide was monitored electrochemically. Diffusivity of 0.05 to 70kDa solute species was determined by measuring concentration gradients using either fluorescent or radiotracers. Hydration was controlled by either mechanical static load or by osmotic equilibration.

Results: Diffusion rates varied with solute molecular weight (MW), decreasing steeply with an increase in MW. For small solutes, the diffusivity was greater in the nucleus than the outer annulus, but this difference was insignificant for the larger solutes. Diffusivity changed by a significant amount with hydration changes, which were significantly affected by loading. Application of a 0.2MPa mechanical load led to a drop in hydration of the outer annulus and nucleus of 33.3% and 42.1% and corresponding falls in diffusivity of glucose of 34.0% and 81.3% respectively.

Conclusions: The large changes in hydration experienced during normal loading of the spine have a marked effect on nutrient and metabolite diffusivity. This effect has not been considered previously but could significantly influence supply of nutrients to the disc cells.

Introduction: The intervertebral disc consists of three regions; the nucleus pulposus and the inner and outer annulus contain cells with individual phenotypes. However, no molecular markers are known to discriminate between the various cell types. Molecular markers would help identify the cell types in development, ageing, localisation and pathological states. Here we reveal major differences between the cell types using SDS-PAGE and mass spectrometry, which will help to develop molecular markers.

Methods: Intervertebral discs were isolated from bovine tails and separated into three distinct regions; the nucleus, inner and outer annulus. Cells were separated by enzyme digestion overnight and then washed extensively. The cells were then separated into membrane and supernatant fractions followed by SDS-PAGE analysis; various fractions revealed bands of interest that were then cut from the gel, digested with trypsin and analysed by mass spectrometry.

Results: Analysis of the membrane and supernatant fractions of the cells by SDS-PAGE revealed unique protein patterns between the regions and fractions. A broad band from the membrane of nucleus cells was analysed by mass spectrometry and revealed strong matches for myosin and clathrin. A control band from the outer annulus also revealed a strong match for myosin. A strong band from the membrane fraction of the outer annulus revealed a mass spectrometry match to actin. Analysis of the corresponding supernatant fraction revealed a strong match to actin, whereas a band of similar molecular weight from the inner annulus revealed another myosin chain.

Conclusion: The differences revealed in the protein profile of cells from the three regions of disc and the identification of prominent proteins demonstrates that such differences can be used to identify molecular markers. Specific molecular markers for cells from different disc regions will aid in both understanding disc pathology and in development of new biological treatments.

Introduction: Degeneration of the intervertebral disc is characterised by loss of normal cell activity, disc matrix and loss of disc height. There is currently much interest in using cells to effect a biological repair in connective tissues, eg autologous chondrocyte implantation for cartilage repair. Intervertebral discs have a low cell density, with those cells present often being unhealthy and necrotic. Hence, identification of an alternative source of cells for autologous disc repair could be beneficial. Thus we have investigated other types of connective tissue cells to determine if they may be encouraged to undertake a disc cell phenotype.

Materials and Methods: Cells were enzymatically/mechanically extracted from bovine coccygeal discs (annulus and nucleus), skin, bone marrow, periosteum and tendon and the efficiency and proliferation rates assessed. Dermal fibroblasts and bone marrow cells were also grown in a 3D alginate system and compared to disc nucleus pulposus cells for phenotypic expression from 0–28 days. Cell phenotype was assessed via morphology, immunohistochemistry, Western blotting and RT-PCR for mRNA expression.

Results: All cell types could be extracted and proliferated in monolayer, with a flattened and fibroblast-like morphology. Proliferation was slowest for bone marrow cells (4 times slower than nucleus pulposus cells). Cells cultured in alginate became rounded with chondrocyte-like morphology. They remained viable for 4 weeks, but with little replication. Expression or production of proteoglycans, both aggrecan and the small proteoglycans (especially fibromodulin) and collagen types I, II and X was demonstrated for all cell types. There was, however, a difference in the timescale of production between some cell types.

Conclusions: Plasticity of different cell types is well known and the connective tissue cells investigated in this study are capable of responding to the environment in which they are cultured. They can synthesise matrix molecules typically produced by disc cells in vivo and hence warrant further investigation as a potential source of cells for biological repair of the intervertebral disc.

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.

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.