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. We have investigated the localisation of elastic proteins in different spinal tissues at different stages of curve development in mouse models and in human tissue obtained during scoliosis surgery.Introduction
Methods
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.
Work supported by Fondation Cotrel
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.
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.
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.
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
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.
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
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.