ISSLS PRIZE in Clinical Science 2023: comparison of degenerative MRI features of the intervertebral disc between those with and without chronic low back pain. An exploratory study of two large female populations using automated annotation.

OBJECTIVES: The relationship of degeneration to symptoms has been questioned. MRI detects apparently similar disc degeneration and degenerative changes in subjects both with and without back pain. We aimed to overcome these problems by re-annotating MRIs from asymptomatic and symptomatics groups onto the same grading system. METHODS: We analysed disc degeneration in pre-existing large MRI datasets. Their MRIs were all originally annotated on different scales. We re-annotated all MRIs independent of their initial grading system, using a verified, rapid automated MRI annotation system (SpineNet) which reported degeneration on the Pfirrmann (1-5) scale, and other degenerative features (herniation, endplate defects, marrow signs, spinal stenosis) as binary present/absent. We compared prevalence of degenerative features between symptomatics and asymptomatics. RESULTS: Pfirrmann degeneration grades in relation to age and spinal level were very similar for the two independent groups of symptomatics over all ages and spinal levels. Severe degenerative changes were significantly more prevalent in discs of symptomatics than asymptomatics in the caudal but not the rostral lumbar discs in subjects < 60 years. We found high co-existence of degenerative features in both populations. Degeneration was minimal in around 30% of symptomatics < 50 years. CONCLUSIONS: We confirmed age and disc level are significant in determining imaging differences between asymptomatic and symptomatic populations and should not be ignored. Automated analysis, by rapidly combining and comparing data from existing groups with MRIs and information on LBP, provides a way in which epidemiological and 'big data' analysis could be advanced without the expense of collecting new groups. LEVEL OF EVIDENCE I: Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding.

Delivering Focused Ultrasound to Intervertebral Discs Using Time-Reversal.

Chronic low back pain causes more disability worldwide than any other condition and is thought to arise in part through loss of biomechanical function of degenerate intervertebral discs (IVDs). Current treatments can involve replacing part or all of the degenerate IVDs by invasive surgery. Our vision is to develop a minimally invasive approach in which high intensity focused ultrasound (HIFU) is used to mechanically fractionate degenerate tissue in an IVD; a fine needle is then used to first remove the fractionated tissue and then inject a biomaterial able to restore normal physiologic function. The goal of this manuscript is to demonstrate the feasibility of trans-spinal HIFU delivery using simulations of 3-D ultrasound propagation in models derived from patient computed tomography (CT) scans. The CT data were segmented into bone, fat and other soft tissue for three patients. Ultrasound arrays were placed around the waist of each patient model, and time-reversal was used to determine the source signals necessary to create a focus in the center of the disc. The simulations showed that for 0.5 MHz ultrasound, a focus could be created in most of the lumbar IVDs, with the pressure focal gain ranging from 3.2-13.7. In conclusion, it is shown that with patient-specific planning, focusing ultrasound into an IVD is possible in the majority of patients despite the complex acoustic path introduced by the bony structures of the spine.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

INTRODUCTION: Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. MATERIALS AND METHODS: NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. RESULTS: Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. CONCLUSIONS: 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.

Disc size markedly influences concentration profiles of intravenously administered solutes in the intervertebral disc: a computational study on glucosamine as a model solute.

PURPOSE: Tests on animals of different species with large differences in intervertebral disc size are commonly used to investigate the therapeutic efficacy of intravenously injected solutes in the disc. We hypothesize that disc size markedly affects outcome. METHODS: Here, using a small non-metabolized molecule, glucosamine (GL) as a model solute, we calculate the influence of disc size on transport of GL into rat, rabbit, dog and human discs for 10 h post intravenous-injection. We used transient finite element models and considered an identical GL supply for all animals. RESULTS: Huge effects of disc size on GL concentration profiles were found. Post-injection GL concentration in the rat disc reached 70% blood concentration within 15 min but remained below 10% in the human disc nucleus throughout. The GL rapidly penetrated post-injection into smaller discs resulting in homogeneous concentrations. In contrast, GL concentration, albeit at much lower levels, increased with time in the human disc with a small outward flux at the annulus periphery at longer periods. CONCLUSIONS: Changes in the disc size hugely influenced GL concentrations throughout the disc at all regions and times. Increases in administered dose can neither remedy the very low concentration levels in the disc center in larger human disc at early post-injection hours nor alter the substantial differences in concentration profiles estimated among various species. The size effect will only be exacerbated as molecular weight of the solute increases and as the endplate calcifies. Extrapolation of findings from animal to human discs on the efficacy of intravenously administered solutes must proceed with great caution.

Injectable hydrogels with high fixed charge density and swelling pressure for nucleus pulposus repair: biomimetic glycosaminoglycan analogues.

The load-bearing biomechanical role of the intervertebral disc is governed by the composition and organization of its major macromolecular components, collagen and aggrecan. The major function of aggrecan is to maintain tissue hydration, and hence disc height, under the high loads imposed by muscle activity and body weight. Key to this role is the high negative fixed charge of its glycosaminoglycan side chains, which impart a high osmotic pressure to the tissue, thus regulating and maintaining tissue hydration and hence disc height under load. In degenerate discs, aggrecan degrades and is lost from the disc, particularly centrally from the nucleus pulposus. This loss of fixed charge results in reduced hydration and loss of disc height; such changes are closely associated with low back pain. The present authors developed biomimetic glycosaminoglycan analogues based on sulphonate-containing polymers. These biomimetics are deliverable via injection into the disc where they polymerize in situ, forming a non-degradable, nuclear "implant" aimed at restoring disc height to degenerate discs, thereby relieving back pain. In vitro, these glycosaminoglycan analogues possess appropriate fixed charge density, hydration and osmotic responsiveness, thereby displaying the capacity to restore disc height and function. Preliminary biomechanical tests using a degenerate explant model showed that the implant adapts to the space into which it is injected and restores stiffness. These hydrogels mimic the role taken by glycosaminoglycans in vivo and, unlike other hydrogels, provide an intrinsic swelling pressure, which can maintain disc hydration and height under the high and variable compressive loads encountered in vivo.

Computational pharmacokinetics of solute penetration into human intervertebral discs - effects of endplate permeability, solute molecular weight and disc size.

A finite element model is developed to predict the penetration time-history of three different solutes into the human lumbar disc following intravenous injection. Antibiotics are routinely administered intravenously in spinal surgery to prevent disc infection. Successful prophylaxis requires antibiotics to reach adequate inhibitory levels. Here, the transient diffusion of cephazolin is investigated over 10h post-injection in a human disc model subject to reported concentrations in the blood stream as the prescribed boundary sources. Post-injection variation of cephazolin concentrations in the disc adjacent to supply sources closely followed the decay curve in the blood stream and fell sharply with time. Much lower concentrations were computed in the inner annulus and nucleus; much of the disc (80% at 1h and 49% at 4h) experienced concentrations below required inhibitory level of 1mg/L in agreement with measurements. Changes in endplate permeability, disc size, and solute molecular weight had profound effects on concentration profiles at all times and regions, especially in the disc centre, demonstrating their crucial roles on the adequate delivery of drugs. Larger solutes markedly slow transport into the disc. The failure to reach critical therapeutic levels in the central disc regions, especially when endplates calcify and in larger discs, raises concerns and calls for caution in attempts to extrapolate findings of studies on animals with much smaller and non degenerate discs to the human discs. The current study also demonstrates the capability of computational models in predicting the transport of intravenously injected solutes into the disc.

Analysis of cell viability in intervertebral disc: Effect of endplate permeability on cell population.

Responsible for making and maintaining the extracellular matrix, the cells of intervertebral discs are supplied with essential nutrients by diffusion from the blood supply through mainly the cartilaginous endplates (CEPs) and disc tissue. Decrease in transport rate and increase in cellular activity may adversely disturb the intricate supply-demand balance leading ultimately to cell death and disc degeneration. The present numerical study aimed to introduce for the first time cell viability criteria into nonlinear coupled nutrition transport equations thereby evaluating the dynamic nutritional processes governing viable cell population and concentrations of oxygen, glucose and lactic acid in the disc as CEP exchange area dropped from a fully permeable condition to an almost impermeable one. A uniaxial model of an in vitro cell culture analogue of the disc is first employed to examine and validate cell viability criteria. An axisymmetric model of the disc with four distinct regions was subsequently used to investigate the survival of cells at different CEP exchange areas. In agreement with measurements, predictions of the diffusion chamber model demonstrated substantial cell death as essential nutrient concentrations fell to levels too low to support cells. Cells died away from the nutrient supply and at higher cell densities. In the disc model, the nucleus region being farthest away from supply sources was most affected; cell death initiated first as CEP exchange area dropped below approximately 40% and continued exponentially thereafter to depletion as CEP calcified further. In cases with loss of endplate permeability and/or disruptions therein, as well as changes in geometry and fall in diffusivity associated with fluid outflow, the nutrient concentrations could fall to levels inadequate to maintain cellular activity or viability, resulting in cell death and disc degeneration.

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

OBJECTIVE: With 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. DESIGN: Bovine 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. RESULTS: By 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. CONCLUSIONS: Chronic 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.

Solute transport in intervertebral disc: experiments and finite element modeling.

Loss of nutrient supply to the human intervertebral disc (IVD) cells is thought to be a major cause of disc degeneration in humans. To address this issue, transport of molecules of different size have been analyzed by a combination of experimental and modeling studies. Solute transport has been compared for steady-state and transient diffusion of several different solutes with molecular masses in the range 3-70 kDa, injected into parts of the disc where degeneration is thought most likely to occur first and into the blood supply to the disc. Diffusion coefficients of fluorescently tagged dextran molecules of different molecular weights have been measured in vitro using the concentration gradient technique in thin specimens of disc outer annulus and nucleus pulposus. Diffusion coefficients were found to decrease with molecular weight following a nonlinear relationship. Diffusion coefficients changed more rapidly for solutes with molecular masses less than 10 kDa. Although unrealistic or painful, solutes injected directly into the disc achieve the largest disc coverage with concentrations that would be high enough to be of practical use. Although more practical, solutes injected into the blood supply do not penetrate to the central regions of the disc and their concentrations dissipate more rapidly. Injection into the disc would be the best method to get drugs or growth factors to regions of degeneration in IVDs quickly; else concentrations of solute must be kept at a high value for several hours in the blood supply to the discs.

Cytochrome c oxidase levels in chondrocytes during monolayer expansion and after return to three dimensional culture.

OBJECTIVE: Here we investigate whether monolayer culture or culture at 21% oxygen influences activity of cytochrome c oxidase, the terminal enzyme in the respiratory chain whose activity is essential for oxidative metabolism and whether return to three dimensional (3-D) culture restores cytochrome c oxidase activity to original levels. METHODS: Primary bovine articular chondrocytes were cultured in alginate beads (3-D) for 4 weeks or in monolayer under 1% and 21% oxygen for up to 9 days and then returned to 3-D culture for up to 4 weeks. Cells were stained to localise cytochrome c oxidase within the cells. Mitochondrial protein content and cytochrome c oxidase enzymatic activity were determined. Expression of cytochrome c oxidase subunits, COXI and COXIV, was assessed by qRT-PCR. RESULTS: Cytochrome c oxidase staining remained minimal in chondrocytes cultured in alginate for 4 weeks under 21% oxygen. Mitochondrial protein content and cytochrome c oxidase activity increased significantly during 9 days of chondrocyte expansion in monolayer, accompanied by up-regulation of the COXI mitochondrial gene but not the COXIV nuclear-encoded gene. Cytochrome c oxidase staining increased from day 5 of monolayer culture and remained high even after the cells were returned to 3-D culture for 4 weeks. CONCLUSIONS: Culture of chondrocytes in monolayer leads to a rapid increase in mitochondrial protein content and cytochrome c oxidase activity. The increase in cytochrome c oxidase activity is not reversed even after chondrocytes are returned to 3-D culture for 4 weeks; high oxygen tension alone does not appear to stimulate cytochrome c oxidase activity.

Investigation of solute concentrations in a 3D model of intervertebral disc.

As the disc is the largest avascular structure in the body, disc cells depend for their normal function on an adequate supply of nutrients (oxygen and glucose) and the removal of metabolic by-products (lactic acid) via blood vessels at the cartilaginous endplates and annulus periphery. Concentration gradients develop depending on the balance between the rates of transport and rates of cellular activity. Since consumption and production rates are coupled via extracellular pH, the gradients are interdependent. This is a novel model study which takes into account the realistic 3D geometry of a L5-S1 lumbar disc in solving the nonlinear coupled diffusion equations. Effects of perturbations (calcification, sclerosis) in endplates, increases in cell metabolic rates following growth factor injection and changes in lumbar posture (kyphotic or lordotic) on extreme values of nutrient and metabolite concentrations and their spatial locations are investigated. Solute concentrations, particularly those of glucose, substantially diminish as a consequence of disturbances in supply at the endplates, increases in cell metabolic rate and more lordotic postures. Results, when compared to those from simplified axisymmetric models, demonstrate the importance of consideration of realistic 3D disc geometry.

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

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.

Application of multiple parallel perfused microbioreactors and three-dimensional stem cell culture for toxicity testing.

In this study, a multiple parallel perfused microbioreactor platform, TissueFlex, was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.

Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells.

Intervertebral discs (IVD) have a higher extracellular osmolarity than most other tissues; moreover their osmolarity changes by around 25% during each diurnal cycle. In this study, changes in aggrecan, collagen I and collagen II expression of IVD cells were examined after exposure to osmotic environment alterations or mechanical stimulation under different osmotic conditions. Human and bovine IVD cells seeded in three-dimensional (3D) collagen type I matrices were cultured under hypo-osmotic (300 mOsm), iso-osmotic (400 mOsm), or hyperosmotic (500 mOsm) conditions. Osmolarity-induced changes in gene expression of IVD cells were measured after 5 days. Load-induced changes in gene expression under the different osmotic conditions were measured after application of hydrostatic pressure (0.25 MPa, 0.1 Hz, 30 min) or cyclic strain (4%, 1 Hz, 24 h). The results showed that IVD cells respond strongly to changes in the osmotic environment by altering mRNA expression. Human cells cultured over 5 days increased expression of aggrecan and collagen II in both nucleus and annulus cells under increasing osmolarity. In contrast, collagen I expression was inhibited at high osmolarity in both cell types. Mechanically induced alterations in gene expression appear to have only modest effects on matrix protein expression, but the same stimulus partly resulted in an inhibition or stimulation of gene expression, depending on the osmotic conditions. This study showed that the osmotic environment does not only have an appreciable effect on gene expression but also affects responses to mechanical stimuli. This suggests that the osmotic conditions cannot be ignored when examining physiological and pathological behavior of IVD cells.

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.

Computation of coupled diffusion of oxygen, glucose and lactic acid in an intervertebral disc.

The present numerical study aims to investigate the disc nutrition and factors affecting it by evaluating the concentrations of oxygen, glucose and lactic acid in the disc while accounting for the coupling between these species via the pH level in the tissue and the nonlinear concentration-consumption (for glucose and oxygen) and concentration-production (for lactate) relations. The effects of changes in the endplate exchange area (EA) adjacent to the nucleus or the inner annulus for the transport of nutrients and in the disc geometry as well as tissue diffusivities under static compression loading on species concentrations are also studied. Moreover, alterations in solute diffusion following a central endplate fracture are investigated. An axisymmetric geometry with four distinct regions is considered. Supply sources are assumed at the outer annulus periphery and disc endplates. Coupling between different solutes, pH level, endplate disruptions (calcifications and fractures) and mechanical loads substantially influenced the distribution of nutrients throughout the disc as well as the magnitude and location of critical concentrations; maximum for the lactic acid and minimum for oxygen and glucose. In cases with loss of endplate permeability and/or disruptions therein, as well as changes in geometry and fall in diffusivity associated with fluid expression, the nutrient concentrations could fall to levels inadequate to maintain cellular activity or viability, thus initiating or accelerating disc degeneration.

Influences of buffer systems on chondrocyte growth during long-term culture in alginate.

OBJECTIVE: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture. METHODS: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture. RESULTS: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture. CONCLUSION: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.

Microdialysis for monitoring the process of functional tissue culture.

Continuous monitoring is important during tissue culture. However, there are still technical difficulties in monitoring the internal status of cells or tissues. In this paper, microdialysis is adopted to monitor functional tissue growth in a bioreactor. Explanted bovine caudal intervertebral disc (IVD) was used as the test tissue. A microdialysis membrane probe of 100 kDa molecular weight cut-off was employed and in situ calibration methods with phenol red and fluorescent 40 kDa dextran were developed to measure the relative recovery of the solute of interest, and membrane fouling, respectively. Tissue metabolism was monitored successfully. At the same time soluble macromolecules were picked up by the probe and were detected and quantified by Fast Protein Liquid Chromatography (FPLC) and/or Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). These proteins were believed to be associated with biofunction of engineered tissue. Monitoring of phenol red content in the dialysate indicated that there was no significant fouling of the membrane probe during a 7-day culture period and the Relative Recovery of macromolecules of interests remained roughly 9%. We concluded that microdialysis could be used to sample a wide range of molecular species released during cell metabolism and extracellular matrix turnover, which were direct or indirect indications of cell and tissue functions. The application of the developed system could be extended to monitor tissue repair in vivo, and the development of the engineered tissue.

Diurnal fluid expression and activity of intervertebral disc cells.

The intervertebral discs are large cartilaginous structures situated between the vertebral bodies, occupying around one third of the length of the spinal column. They act as the joints of the spine and carry mechanical load arising from body weight and muscle activity. Loads change with every alteration of posture and activity and the discs thus undergo a diurnal loading pattern with high loads on the discs during the day's activity and low loads on it at night during rest. As the disc is an osmotic system, around 25% of the disc's fluid is expressed and re-imbibed during each diurnal cycle with consequent changes in the osmotic environment of the disc cells. Here, present information on the effect of osmotic changes in disc cell metabolism is reviewed; results indicate that prevailing osmolarity is a powerful regulator of disc cell activity.

Effects of rapid cooling on articular cartilage.

In order to improve the technique and protocols of cryopreservation of articular cartilage, a study was carried out to assess the effects of rapid cooling on the intact articular cartilage. Cartilage slices with a thickness ranging from 0.2 to 0.5 mm taken from bovine metacarpal-phalangeal joints were subjected to rapid cooling by immersing them in liquid nitrogen with and without treatment of the VS55 cryoprotective agent (CPA). The ultrastructure, chondrocyte viability, swelling property, and glycosaminoglycan (GAG) content were then examined before and after cryopreservation to give qualitative and quantitative evaluation on the functional state of both chondrocytes and extracellular matrix. The transmission electron microscopy study demonstrated that damage to chondrocytes without CPA was far more pronounced than those with VS55 protection while the structure of the extracellular matrix altered little in either group. The cell viability assay showed that although the exposure to VS55 led to about 36% chondrocytes losing membrane integrity, the VS55 could provide protection to chondrocytes during rapid cooling and thawing, with approximately 51% of the cells having survived rapid cooling compared to fewer than 5% in the absence of CPA. There were no significant differences in degrees of swelling or the GAG contents of cartilage slices after cryopreservation indicating rapid freezing caused little damage to the matrix. Future research activities include searching improved CPA formulation, optimising the treatment protocol and investigating the long-term effects of rapid cooling on articular cartilage.