Jill Urban

Notochordal Intervertebral Disc Cells : Sensitivity to Nutrient Deprivation

OBJECTIVEnThe nucleus pulposus (NP) of the intervertebral disc develops from the notochord. Humans and other species in which notochordal cells (NCs) disappear to be replaced by chondrocyte-like mature NP cells (MNPCs) frequently develop disc degeneration, unlike other species that retain NCs. The reasons for NC disappearance are unknown. In humans, the change in cell phenotype (to MNPCs) coincides with changes that decrease nutrient supply to the avascular disc. We undertook this study to test the hypothesis that the consequent nutrient stress could be associated with NC disappearance.nnnMETHODSnWe measured cell densities and metabolic rates in 3-dimensional cultures of porcine NCs and bovine MNPCs, and we determined survival rates under conditions of nutrient deprivation. We used scanning electron microscopy to examine end plate porosity of discs with NCs and those with MNPCs. Nutrient-metabolite profiles and cell viability were calculated as a function of cell density and disc size in a consumption/diffusion mathematical model.nnnRESULTSnNCs were more active metabolically and more susceptible to nutrient deprivation than were MNPCs. Hypoxia increased rates of glycolysis in NCs but not in MNPCs. Higher end plate porosity in discs with NCs suggested greater nutrient supply in keeping with higher nutritional demands. Mathematical simulations and experiments using an analog disc diffusion chamber indicated that a fall in nutrient concentrations resulting from increased diffusion distance during growth and/or a fall in blood supply through end plate changes could instigate NC disappearance.nnnCONCLUSIONnNCs demand more energy and are less resistant to nutritional stress than MNPCs, which may shed light on the fate of NCs in humans. This provides important information about prospective NC tissue engineering approaches.

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

The distribution of microfibrils was studied immunohistochemically in intervertebral discs taken from young normal human surgical cases and from the bovine tail. Co‐localization of microfibrils and elastin fibres was examined by dual immunostaining of fibrillin‐1 and elastin. Collagen fibre network orientation was studied by using polarized filters. A similar microfibrillar network was seen in both bovine and human discs with network organization being completely different from region to region. In the outer annulus fibrosus (OAF), abundant microfibrils organized in bundles were mainly distributed in the interterritorial matrix. In addition, the microfibril bundles were orientated parallel to each other and co‐localized highly with elastin fibres. Within each lamella, co‐localized microfibrils and elastin fibres were aligned in the same direction as the collagen fibres. In the interlamellar space, a dense co‐localized network, staining for both microfibrils and elastin fibres, was apparent; immunostaining for both molecules was noticeably stronger than within lamellae. In the inner annulus fibrosus, the microfibrils were predominantly visible as a filamentous mesh network, both in the interterritorial matrix and also around the cells. The microfibrils in this region co‐localized with elastin fibres far less than in the OAF. In nucleus pulposus, filamentous microfibrils were organized mainly around the cells where elastin fibres were hardly detected. By contrast, sparse elastin fibres, with a few of microfibrils, were visible in the interterritorial matrix. The results of this study suggest the microfibrillar network of the annulus may play a mechanical role while that around the cells of the nucleus may be more involved in regulating cell function.

The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc.

Study Design. Immunohistochemical study of elastic fibers in human intervertebral discs (IVD) collected at surgery from patients with scoliosis. Objectives. To compare the elastic fiber network in scoliotic discs (idiopathic scoliosis or neuromuscular scoliosis) to that of control (normal) discs. To study whether the change in elastic fiber organization could contribute to the progression of spinal deformity. Summary of Background Data. Elastin and elastic fibers have been identified previously in human IVD but were believed to contribute little to the tissue’s mechanical properties. However, a recent immunohistochemical study has revealed an abundant and organized elastic fiber network in bovine IVD, indicating that elastic fibers could play an important mechanical role. This article reports the organization of elastic fibers in human IVD and the changes of elastic fiber organization in scoliosis. Methods. Intact wedges of IVD were obtained from patients undergoing surgery for scoliosis (aged 12−22 years). Control discs were obtained from a patient (aged 12 years) with a spinal tumor and a trauma patient (aged 17 years). The discs were dissected to give radial slices and were snap frozen. Frozen sections were cut and digested with hyaluronidase to remove glycosaminoglycans. Micrographs of the sections were examined by polarized light to visualize collagen organization. The elastic fiber network was visualized immunohistochemically or by histochemical staining with orcein. Results. A highly organized elastic fiber network, similar to that described in bovine discs, was revealed in the control human discs. In the anulus fibrosus of control discs, dense elastic fibers were located between adjacent lamellae, with fibers also present within individual lamellae. Elastic fibers appeared to be long (>200 &mgr;m) and straight in outer anulus, whereas in inner anulus, they nearly ran parallel to each other and at an angle of approximately 60° or 120° to those in adjacent lamellae. However, in scoliotic discs, elastic fibers were sparse, and the collagen and elastic fiber networks were disorganized with loss of lamellar structure. Cell clusters, one of typical degenerative feature, were seen in scoliotic discs but not in age-matched control discs. Conclusions. Our results reveal an abundant and organized network of elastic fibers in the adolescent (12 and 17-year-olds) human IVD, and suggest that elastic fiber network plays a significant biomechanical role. This network is sparse and disrupted in scoliotic discs, and could be involved in the progression of the spinal deformity.

Electrochemical measurement of transport into scoliotic intervertebral discs in vivo using nitrous oxide as a tracer.

Study Design. An in vivo study measuring nitrous oxide concentrations in scoliotic intervertebral discs during surgery. Objectives. To determine pathways for nutrient transport into scoliotic human discs in vivo. Summary of Background Data. The intervertebral disc is the largest avascular structure in the body. Disc cells in the nucleus rely on the blood supply from the vertebral bodies for supply of nutrients and removal of waste. Loss of nutrient supply is thought to lead to disc degeneration, but solute transport has not been measured in vivo in humans. Methods. We measured solute transport into the disc using N2O as a tracer, in 19 human discs from five patients with neuromuscular scoliosis (6–19 years of age) during surgery for correction of scoliotic deformities. During anesthesia N2O diffuses into the disc at a rate governed by effective permeability of the vertebral body–disc interface. Intradiscal N2O concentrations were measured amperometrically using silver needle microelectrodes, which were inserted into the discs once they were exposed by an anterior approach. Results. For all spines N2O concentrations were very low in the disc at the curve apex (6% those expected from unimpeded diffusion) and, although still low, were significantly higher 2 discs below or above the apex. Conclusions. Because flux into the apical disc is most restricted, the decrease in solute transport is possibly induced by changes in mechanical stress on the disc; microfocal radiographs of a scoliotic spine suggest that increased endplate calcification could be partly responsible for limiting solute diffusion.

Effect of Extracellular pH on Matrix Synthesis by Chondrocytes in 3D Agarose Gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3–6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7‐day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH‐dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

Intervertebral Disc Composition in Neuromuscular Scoliosis : Changes in Cell Density and Glycosaminoglycan Concentration at the Curve Apex

Study Design. An analysis of the variation in glycosaminoglycan, water content, and cell density with disc level in patients with neuromuscular scoliosis. Objectives. To determine whether the composition of the apical disc differed from that of adjacent discs in the same spine. Summary of Background Data. Compositional differences between the convex and concave sides of scoliotic discs have been noted and are thought to be secondary to altered loading. However, there is little information on changes relative to the apex. Methods. Intact wedges of disc obtained during anterior fusion procedures were taken from 23 discs of 6 patients with neuromuscular scoliosis. Radial profiles of glycosaminoglycan, water content, and cell density were measured. Concentrations were compared at a standard distance (5 mm) into the disc and plotted versus spinal level. Results. Glycosaminoglycan and water content were lowest in the outer annulus and increased steadily toward the disc center, whereas the cell density was highest in the outer 2 mm, fell steeply and then remained constant. At 5 mm from the annulus edge, cell density was lowest in apical discs and, in most cases, was noticeably higher in adjacent discs of the same spine. At the same point, there was no consistent change in glycosaminoglycan/dry weight from disc to disc, indicating no significant proteoglycan loss. However, glycosaminoglycan/tissue water, and therefore swelling pressure, was highest in the apical discs, suggesting that these discs were the most heavily loaded. Conclusions. The loss of cells from the disc at the curve apex probably arose because this disc experiences greater mechanical stress or is more deformed than its neighbors. The decrease in cell density was not associated with major changes in tissue composition, possibly because rates of degradation and of synthesis were reduced, leaving the matrix largely unchanged.

In vivo measurement of tissue metabolism in tendons of the rotator cuff: implications for surgical management.

We have undertaken an in vivo assessment of the tissue metabolism and cellular activity in torn tendons of the rotator cuff. Cellular oxygen consumption was measured in 13 patients undergoing mini-open repair of small, medium, large and massive full-thickness tears. Measurements were also taken from three control patients who were undergoing open stabilisation of the shoulder with grossly normal tendons. The level of oxygen and nitrous oxide was measured amperometrically using silver needle microelectrodes at the apex of the tear and 1.5 cm from its edge. With nitrous oxide indicating the degree of perfusion, oxygen consumption was calculated at each location to reflect cellular activity. All of the torn tendons had lower levels of cellular activity than the control group. This activity was lower still in the tissue nearest to the edge of the tear with the larger tears showing the lowest activity. This indicated reduced levels of tissue metabolism and infers a reduction in tendon viability. Our findings suggest that surgical repair of torn tendons of the rotator-cuff should include the more proximal, viable tissue, and may help to explain the high rate of re-rupture seen in larger tears.

Elastic fibres are broadly distributed in tendon and highly localized around tenocytes.

Elastic fibres have the unique ability to withstand large deformations and are found in numerous tissues, but their organization and structure have not been well defined in tendon. The objective of this study was to characterize the organization of elastic fibres in tendon to understand their function. Immunohistochemistry was used to visualize elastic fibres in bovine flexor tendon with fibrillin‐1, fibrillin‐2 and elastin antibodies. Elastic fibres were broadly distributed throughout tendon, and highly localized longitudinally around groups of cells and transversely between collagen fascicles. The close interaction of elastic fibres and cells suggests that elastic fibres are part of the pericellular matrix and therefore affect the mechanical environment of tenocytes. Fibres present between fascicles are likely part of the endotenon sheath, which enhances sliding between adjacent collagen bundles. These results demonstrate that elastic fibres are highly localized in tendon and may play an important role in cellular function and contribute to the tissue mechanics of the endotenon sheath.

Osmolarity effects on bovine articular chondrocytes during three-dimensional culture in alginate beads

OBJECTIVEnWith the development of engineered cartilage, the determination of the appropriate culture conditions is vital in order to maximize extracellular matrix synthesis. As osmolarity could affect the fate of chondrocytes, the purpose of this study was to determine the effects of osmolarity on chondrocytes during relatively long-term culture.nnnDESIGNnBovine articular chondrocytes were cultured in alginate beads in a biocarbonate free system at 280, 380 and 550 mOsm at pH 7.4 for up to 12 days, respectively. Cell volume, intracellular pH (pH(i)), cell number, glucosaminoglycan (GAG) and collagen retention were measured at day 5 and 12. Cell viability and volume were monitored over the 12 days of culture.nnnRESULTSnBy day 5 and 12, compared to the cell volume at 380 mOsm, around 20% (P<0.01) swelling and 15% (P<0.05) shrinkage were observed when the cells were cultured at 280 and 550 mOsm. The pH(i) over the 12 days of culture varied with osmolarity of the culture medium. In comparison with fresh cells, pH(i) became slightly more acidic by 0.15 pH units at 280 mOsm at day 5. However, by day 12, an alkalization of pH(i), by 0.2 pH units, was noted. A higher proliferation rate was seen at 280 mOsm than at other osmolarities while less GAG was produced.nnnCONCLUSIONSnChronic exposure to anisotonic conditions results in cell swelling at 280 mOsm and shrinkage at 550 mOsm. The osmolarity of 280 mOsm appears to encourage proliferation of chondrocytes, but inhibits matrix production.

The internal pressure and stress environment of the scoliotic intervertebral disc--a review.

Abstract The aetiology, in terms of both initiation and progression, of the deformity in idiopathic scoliosis is at present unclear. Even in neuromuscular cases, the mechanisms underlying progression are not fully elucidated. It is thought, however, that asymmetrical loading is involved in the progression of the disease, with evidence mainly from animal studies and modelling. There is, however, very little direct information as to the origin or mechanism of action of these forces in the scoliotic spine. This review describes the concept of intervertebral disc pressure or stress and examines possible measurement techniques. The biological and mechanical consequences of abnormalities in these parameters are described. Future possible studies and their clinical significance are also briefly discussed. Techniques of pressure measurement have culminated in the development of ‘pressure profilometry’, which provides stress profiles across the disc in mutually perpendicular axes. A hydrated intervertebral disc exhibits mainly hydrostatic behaviour. However, in pathological states such as degeneration and scoliosis, non-hydrostatic behaviour predominates and annular peaks of stress occur. Recent studies have shown that, in scoliosis, high hydrostatic pressures are seen with asymmetrical stresses from concave to convex sides. These abnormalities could influence both disc and endplate cellular activity directly, causing asymmetrical growth and matrix changes. In addition, disc cells could be influenced via nutritional changes consequent to end-plate calcification. Evidence suggests that the stress environment of the scoliotic disc is abnormal, probably generated by high and asymmetrical loading of non-muscular origin. If present in the scoliotic spine during daily activities, this could generate a positive feedback of cellular changes, resulting in curve progression. Future advances in understanding may rely on the development of computer models owing to the difficulties of in-vivo invasive measurements.