Biomedical subjects

Jill P G Urban

Publications and source records attributed to Jill P G Urban.

At least 19 recordsLinked to original sources

PubMed2007

Study of on-line monitoring of lactate based on optical fibre sensor and in-channel mixing mechanism.

A PDMS-based microfluidic device with integrated optical fibres was developed for online monitoring of lactate. The detection is based on the optical adsorption of the colour solution formed by in-channel mixing of sample and reagent solutions. Computational Fluid Dynamics (CFD) simulation and kinetic study of the colour development were conducted to determine the minimum channel length and optimum residential time for homogenous mixing and maximum optical adsorption respectively. The system was proved to be able to detect lactate with good linearity (R(2)=0.98), response time of about 130 s and estimated limitation of detection (LOD) of 0.52 mM (about 47 mg/L), which is sufficient for a general online lactate detection. Due to the miniaturization of sensing system and micro-scale mixing, higher detection sensitivity (0.15 V/mM) can be realized than the mixing in lab-scale equipments (0.08 V/mM).

Dimethylpolysiloxanes↗

PubMed2006

Monitoring of metabolite gradients in tissue-engineered constructs.

At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.

Alginates↗

PubMed2006

Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure.

Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2-sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way.

Adolescent↗

PubMed2006

Influence of perfusion on metabolism and matrix production by bovine articular chondrocytes in hydrogel scaffolds.

Articular cartilage has a limited capacity for self-repair after damage. Engineered cartilage is a promising treatment to replace or repair damaged tissue. The growth of engineered cartilage is sensitive to the extracellular culture environment. Chondrocytes were seeded into alginate beads and agarose scaffolds at 4 millions/mL, and the response to static and perfusion culture was examined over period of up to 12 days. For both types of scaffolds, the chondrocytes kept their differentiated morphology over 12 days in all culture conditions. In alginate beads, more glycosaminoglycans (GAGs) were produced in perfusion culture than in static conditions. GAG distribution in alginate constructs was more uniform in perfusion culture than in static culture. However, in agarose constructs there was no significant difference in GAG production between static culture and perfusion culture. Under perfusion culture, the retention rate of GAG in alginate was higher than in agarsoe. It is suggested that the positive effect of perfusion culture only can be achieved by an appropriate choice of other factors such as scaffold materials.

Alginates↗

PubMed2006

Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture.

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 microm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.

Animals↗

PubMed2006

Nutrient supply and intervertebral disc metabolism.

The metabolic environment of disc cells is governed by the avascular nature of the tissue. Because cellular energy metabolism occurs mainly through glycolysis, the disc cells require glucose for survival and produce lactic acid at high rates. Oxygen is also necessary for cellular activity, although not for survival; its pathway of utilization is unclear. Because the tissues are avascular, disc cells depend on the blood supply at the margins of the discs for their nutrients. The nucleus and inner anulus of the disc are supplied by capillaries that arise in the vertebral bodies, penetrate the subchondral bone, and terminate at the bone-disc junction. Small molecules such as glucose and oxygen then reach the cells by diffusion under gradients established by the balance between the rate of transport through the tissue to the cells and the rate of cellular demand. Metabolites such as lactic acid are removed by the reverse pathway. The concentrations of nutrients farthest from the source of supply can thus be low; oxygen concentrations as low as 1% have been measured in the discs of healthy animals. Although gradients cannot be measured easily in humans, they can be calculated. Measured concentrations in surgical patients are in agreement with calculated values.

Biological Transport↗

PubMed2005

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 microm) and straight in outer anulus, whereas in inner anulus, they nearly ran parallel to each other and at an angle of approximately 60 degrees or 120 degrees 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.

Adolescent↗

PubMed2005

Metabolism of the intervertebral disc: effects of low levels of oxygen, glucose, and pH on rates of energy metabolism of bovine nucleus pulposus cells.

STUDY DESIGN: In vitro measurements of metabolic rates of isolated bovine nucleus pulposus cells at varying levels of oxygen, glucose, and pH. OBJECTIVES: To obtain quantitative information on the interactions between oxygen and glucose concentrations and pH, and the rates of oxygen and glucose consumption and lactic acid production, for disc nucleus cells. SUMMARY OF BACKGROUND DATA: Disc cells depend on diffusion from blood vessels at the disc margins for supply of nutrients. Loss of supply is thought to lead to disc degeneration, but how loss of supply affects nutrient concentrations in the disc is not known; nutrient concentrations within discs can normally only be calculated, because concentration measurements are invasive. However, realistic predictions cannot be made until there are data from measurements of metabolic rates at conditions found in the disc in vivo, i.e., at low levels of oxygen, glucose, and pH. METHODS: A metabolism chamber was designed to allow simultaneous recording of oxygen and glucose concentrations and of pH. These concentrations were measured electrochemically with custom-built glucose and oxygen sensors; lactic acid was measured biochemically. Bovine nucleus pulposus cells were isolated and inserted into the chamber, and simultaneous rates of oxygen and glucose consumption and of lactic acid production were measured over a range of glucose, oxygen, and pH levels. RESULTS: There were strong interactions between rates of metabolism and oxygen consumption and pH. At atmospheric oxygen levels, oxygen consumption rate at pH 6.2 was 32% of that at pH 7.4. The rate fell by 60% as oxygen concentration was decreased from 21 to 5% at pH 7.4, but only by 20% at pH 6.2. Similar interactions were seen for lactic acid production and glucose consumption rates; we found that glycolysis rates fell at low oxygen and glucose concentrations and low pH. Equations were derived that satisfactorily predict the effect of nutrient and metabolite concentrations on rates of lactic acid production rate and oxygen consumption. CONCLUSIONS: Disc cell metabolism in air and at pH 7.4 differs markedly from that found in the disc nucleus in vivo, where low levels of oxygen, glucose, and pH all coexist.

Animals↗

PubMed2005

A SU-8/PDMS hybrid microfluidic device with integrated optical fibers for online monitoring of lactate.

A microfluidic device with integrated optical fibres was developed for online monitoring of lactate. The device consists of a SU-8 waveguide, microfluidic channels and grooves for the insertion of optic fibres. It was fabricated by one-step photolithography of SU-8 polymer resist. Different channel widths (50-300 microm) were tested in terms of detection sensitivity. A wide range of flow rates were applied to investigate the influence of flow rate on signal fluctuations. The separation between optical fibre sensor and microfluidic channel and the width of fluidic channel have been optimized to maximize the detection sensitivity. It was revealed that 250 microm of channel width is the optimum light path length for a compromise between detection sensitivity and interference of ambient light. The independence of detection signals on flow rates was demonstrated within the range of flow rate (0.5-5 ml/hr) tested. Compared with conventional lactate detection, the device is proved to have high accuracy, relatively low limit of detection (50 mg/L) and reasonably fast response time (100 sec). The fabrication of device is simple and low cost. The present work has provided some fundamental data for further system optimization to meet specific detection requirements.

Biosensing Techniques↗

PubMed2004

Nutrition of the intervertebral disc.

STUDY DESIGN: A review of the literature on disc nutrition. OBJECTIVES: To summarize the information on disc nutrition in relation to disc degeneration. SUMMARY OF THE BACKGROUND DATA: The disc is avascular, and the disc cells depend on diffusion from blood vessels at the disc's margins to supply the nutrients essential for cellular activity and viability and to remove metabolic wastes such as lactic acid. The nutrient supply can fail due to changes in blood supply, sclerosis of the subchondral bone or endplate calcification, all of which can block transport from blood supply to the disc or due to changes in cellular demand. METHODS: A review of the studies on disc blood supply, solute transport, studies of solute transport in animal and human disc in vitro, and of theoretical modeling studies that have examined factors affecting disc nutrition. RESULTS: Small nutrients such as oxygen and glucose are supplied to the disc's cells virtually entirely by diffusion; convective transport, arising from load-induced fluid movement in and out of the disc, has virtually no direct influence on transport of these nutrients. Consequently, there are steep concentration gradients of oxygen, glucose, and lactic acid across the disc; oxygen and glucose concentrations are lowest in the center of the nucleus where lactic acid concentrations are greatest. The actual levels of concentration depend on the balance between diffusive transport and cellular demand and can fall to critical levels if the endplate calcifies or nutritional demand increases. CONCLUSIONS: Loss of nutrient supply can lead to cell death, loss of matrix production, and increase in matrix degradation and hence to disc degeneration.

Animals↗

PubMed2004

Factors influencing the oxygen concentration gradient from the synovial surface of articular cartilage to the cartilage-bone interface: a modeling study.

OBJECTIVE: There is very little information on the gradients of oxygen concentration from the synovial surface to the subchondral bone in articular cartilage. Cartilage is usually regarded as hypoxic, even though cellular metabolism is inhibited at low oxygen concentrations. We therefore measured rates of cellular consumption of oxygen and used these rates to calculate oxygen tension profiles across articular cartilage. METHODS: The rate of oxygen consumption by bovine articular chondrocytes was measured in vitro, either in intact cartilage slices or in isolated chondrocytes. The oxygen tension profile across articular cartilage was predicted by solving a 1-dimensional reaction-diffusion equation. The effect of synovial fluid oxygen concentration, cell density, cartilage thickness, and influx of oxygen from the subchondral bone on the oxygen profile in the tissue was examined. RESULTS: Oxygen consumption rates were relatively independent of oxygen tension at high oxygen tensions (5-21%), where they were approximately 10 nmoles/10(6) cells/hour for both isolated chondrocytes and for cartilage slices. Below 5% oxygen, the rate fell in an oxygen tension-dependent manner. Analysis showed that the oxygen profile across cartilage fell steeply in all but the thinnest cartilage samples but only fell to approximately 1% for low oxygen tensions in synovial fluid, with no supply from the subchondral bone. CONCLUSION: The oxygen tension in normal cartilage is not likely to fall to 1% except under abnormal conditions. Oxygen tensions within cartilage are strongly affected by a number of factors, including oxygen concentrations in synovial fluid, cartilage thickness, cell density, and cellular oxygen consumption rates. Supply from the subchondral bone may be of particular importance.

Animals↗

PubMed2003

Finite element study of nutrient diffusion in the human intervertebral disc.

STUDY DESIGN: The diffusion of small nutrients in the intervertebral human disc was examined using a finite element model. OBJECTIVE: To investigate nutrient transport into the disc using a numerical approach. SUMMARY OF BACKGROUND DATA: The intervertebral disc is the largest avascular tissue in the body. Nutrients necessary for cellular survival diffuse from the blood supply around the disc margins to the cells. Limited analytical studies have been performed and compared with measurements. However, the studies have only considered supply through the center of the nucleus and have only examined single solutes. A more sophisticated model is required to investigate the solute supply. MATERIALS AND METHODS: An axisymmetric finite element model has been created to study the transport of three solutes, i.e., oxygen, glucose, and lactate, using nonlinear consumption-concentration and production-concentration rates. For each of them, data for the consumption/production rate, diffusivity, and concentration in the blood were taken from experimental measurements and used in the model. The effect of varying disc height, exchange area with the blood supply, solute consumption rates, and diffusivities was investigated. RESULTS: The model predicted that concentrations of oxygen and glucose, which are consumed by cells, fell towards the disc center. Concentration levels decreased with a decrease in fractional exchange area and diffusivity, or with an increase in disc height and consumption rate. In contrast, the concentration of lactate, produced by the cells, was highest in the center and fell towards the disc-blood vessel interface. The absolute values of concentrations were in agreement with available measurements in vivo and those computed by few available analytical models, indicating the reliability of the finite element simulations. CONCLUSIONS: Finite element methods can be used to predict concentration gradients of solutes throughout the disc in relation to changes in disc and endplate morphology, disc properties, and cellular activities. This study provides a foundation for investigating the effect of load-induced changes or effects of changes in cellular metabolism on disc nutritional supply.

Diffusion↗

PubMed2003

The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus.

It has long been known that very acidic conditions can be found in degenerate discs. The effect of these acid conditions on matrix turnover are, however, unknown. This study aimed to examine the effect of acidity on production of matrix components and on agents which break down the matrix in order to gain insight into the effect of pathological values of pH on matrix turnover. Cells were isolated from the nucleus of bovine discs and from bovine articular cartilage, embedded in alginate beads and cultured at pH levels maintained within the ranges seen in normal and pathological discs: pH 7.4-pH 6.3 for 48 h. Rates of sulphated glycosaminoglycan (GAG) and protein synthesis were measured, as well as rates of production of some agents involved in matrix breakdown, i.e. total and activated matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The results showed that acid conditions had a profound effect on cell matrix turnover; at pH 6.4, total production of most species measured was inhibited by more than 50% compared to production at pH 7.2; production of sulphated GAGs and of TIMP-1 fell by >90%. However production of active metalloproteinases by disc cells was relatively insensitive to pH, with activity at pH 6.3 not statistically different from that at pH 7.2. These findings indicate that exposure to acid conditions appears particularly deleterious for the disc matrix, as it inhibits the disc cells from synthesising functionally important molecules such as the sulphated GAGs but does not prevent the production of agents able to degrade matrix components. The low values of pH seen in some degenerate discs are thus likely to be involved in breakdown of the disc matrix.

Animals↗

PubMed2003

Degeneration of the intervertebral disc.

The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.

Animals↗

PubMed2003

Measurement of the chondrocyte membrane permeability to Me2SO, glycerol and 1,2-propanediol.

The addition of cryopreservative agents (CPAs) to chondrocytes and natural and engineered cartilage is critical to protect the cells and tissues from freezing damage during cryopreservation, but this may cause cell damage, e.g. by osmotic shock. The damage could be minimized by the control of the cell volume excursion with the knowledge of cell membrane permeability. In this study, the cell volume responses of chondrocytes to three commonly used CPAs were evaluated using a perfusion microscope stage. The osmotic response of chondrocytes was measured to the perfusion with 1.4 M dimethyl sulfoxide (Me2SO), 1,2-propanediol and glycerol at 21 degrees C. Cell volumes and their transients were determined with image analysis. The cell membrane permeability parameters, including the hydraulic conductivity (Lp), the CPA permeability (omega) and the reflection coefficients (sigma) in the Kedem-Katchalsky (K-K) model, and the Lp and omega in the two-parameter model were determined. The correlated K-K parameters at 21 degrees C were Lp=0.166 +/- 0.035, 0.149 +/- 0.061, 0.212 +/- 0.041 microm/min atm, omega=(7.630 +/- 0.174) x 10(-2), (1.428 +/- 0.627) x 10(-2), (2.744 +/- 0.775) x 10(-2) microm/s and sigma=0.91 +/- 0.09, 0.82 +/- 0.11, 0.88 +/- 0.10 for Me(2)SO, glycerol and 1,2-propanediol, respectively. For the two-parameter model, the parameter values were Lp=0.163 +/- 0.040, 0.128 +/- 0.031, 0.169 +/- 0.025 microm/min atm, omega=(7.881 +/- 0.178) x 10(-2), (1.529 +/- 0.525) x 10(-2), (3.716 +/- 0.493) x 10(-2) microm/s for Me2SO, glycerol and 1,2-propanediol, respectively. No significant difference in the predictions of cell volume excursion during CPA addition was observed when using either the K-K model or the two-parameter model and it was hence advised to adopt the simple two-parameter model in the evaluation. The measured parameters can be used to optimise the CPA addition and removal protocols to maximize the cell survival during cryopreservation.

Animals↗

PubMed2003

Adaptation of articular chondrocytes to changes in osmolality.

Articular chondrocytes are exposed to a unique osmotic environment, which varies throughout the depth of cartilage, and in response to mechanical loading or pathological conditions. In light of such osmotic variations we investigated the response of chondrocytes cultured in alginate beads to long term hypo- and hyperosmotic challenge. Following pre-incubation at 380 mOsmol, exposure to hyperosmotic conditions (550 mOsmol) initially decreased 35S-sulphate incorporation, but after 24 hours of culture, rates had recovered and surpassed their original levels. MAP kinase inhibitors abrogated this response suggesting their involvement in the adaptation mechanism. Hypo-osmotic challenge caused a decrease in 35S-sulphate incorporation throughout the culture period. These results suggest that osmolality is a powerful regulator of macromolecular synthesis, and that perturbations in the osmotic environment may alter the set point for turnover.

Adaptation, Physiological↗

PubMed2002

Cell viability in scoliotic discs in relation to disc deformity and nutrient levels.

STUDY DESIGN: Intervertebral disc tissue was analyzed during or removed at routine surgery for correction of scoliosis. Tissue was analyzed for glucose, lactate, oxygen, glycosaminoglycan, collagen concentrations, and cell viability. OBJECTIVES: To investigate the cell viability of the scoliotic disc on the concave and convex sides and in relation to curve apex, and to relate cell viability to concentrations of nutrients, metabolites, and extracellular matrix components. SUMMARY OF BACKGROUND DATA: Compositional differences have been measured in relation to the deformation of scoliotic discs. However, the causes of these in relation to cellular activity or viability are unknown. METHODS: Oxygen concentration was measured at surgery using a microelectrode. A segment of disc then was removed and sections at defined locations measured for cell viability and glucose, lactate, glycosaminoglycan, and collagen concentrations. RESULTS Cell viability was lower toward the convex side of the curve, with the greatest difference between the sides in the apical disc. The apical disc had the lowest oxygen and highest lactate concentrations, and lowest total number of cells. Glucose concentration correlated with the number of live cells. Concentrations of glycosaminoglycans and collagen per dry weight of tissue were similar on both sides of the disc. CONCLUSIONS: Differences in cell viability correlated with changes in nutrient and metabolite levels, and also with disc deformity (convex concave and distance from curve apex). Thus asymmetrical loads, tissue deformation, and nutrient supply may work separately or in combination to cause cell death. A loss of matrix macromolecules was not seen, possibly because the period between cell death and surgery was too short, as compared with long matrix turnover times. Cell death is expected eventually to have a deleterious effect on cell matrix and disc function.

Adolescent↗

PubMed2002

Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype.

STUDY DESIGN: This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production. OBJECTIVE: To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc. SUMMARY OF BACKGROUND DATA: It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus. METHODS: Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization. RESULTS: Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan. CONCLUSIONS: The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.

Animals↗