Glucose utilisation of chondrocytes, with or without loading. Report of external placement

Transcription

1 Glucose utilisation of chondrocytes, with or without loading. Report of external placement Petra Dijkman July 2005 BMTE Supervised by: Prof. Dr. D.L. Bader Prof. Dr. D.A. Lee Dr. T.T. Chowdhury Dr. H.K. Heywood

2 Abstract A tissue engineering strategy for the repair of articular cartilage has often been proposed, because it is considered to be a good alternative for treatment of articular cartilage defects. However, a lack of matrix development and loss of cell viability are frequently observed in the centre of such tissue-engineered constructs of realistic dimensions. In order to obtain more knowledge about the optimal culture-environment of chondrocytes, two different experiments were designed to study the effects of the glucose concentration in medium on chondrocytes. The aim of the first experiment was to study the glucose consumption of superficial and deep chondrocytes in medium with different glucose concentrations. These results were compared with the oxygen consumption of chondrocytes, measured in same conditions by Heywood 1, in order to examine how glucose consumption is related to oxygen consumption. For this purpose, chondrocytes were cultured for 6 hours as free suspension cells in medium with six different glucose concentrations. Glucose consumption and lactate production were measured after the culture period. The second experiment was performed to test the effect of the glucose concentration in the medium on chondrocytes in agarose constructs. Besides using different glucose concentrations, the constructs were cultured strained, unstrained or were allowed to swell freely, to observe the influence of loading on the chondrocytes. Chondrocytes were cultured for 48 hours. Metabolic activity was examined by measuring glucose consumption, lactate production, cell-proliferation, GAG-production and nitric oxide release. From the results of the first experiment it can be concluded that chondrocytes preferentially utilise glucose, and thereby minimising their oxygen consumption. Although Heywood 1 found a significant difference between superficial and deep chondrocytes with respect to oxygen consumption, the present study indicates no significant difference between superficial and deep chondrocytes with respect to glucose consumption or lactate production. The second experiment indicates that when the glucose concentration in the medium increases from 1 g/l to 4 g/l, there is an associated four-fold increase in the glucose consumption of chondrocytes. However, this additional glucose consumption is not used for direct production of ATP and/or GAG. The strained constructs show a significant decrease in nitric oxide release compared to the unstrained constructs. Comparison of unstrained constructs with free-swelling constructs suggests that the chondrocytes in unstrained constructs are restricted in their metabolic activity compared to the chondrocytes in free-swelling constructs. Therefore, the use of unstrained constructs as controls to observe the influence of loading can be misleading. 9/21/2005 page 2

3 Table of contents 1 Introduction Articular Cartilage Loading of agarose constructs Aim of work Metabolism of chondrocyte sub-populations in unloaded cultures The effects of dynamic stimulation of chondrocyte seeded agarose constructs Materials and methods Metabolism of chondrocyte sub-populations in unloaded cultures Isolation of bovine chondrocytes Incubation with different glucose concentrations Biochemical analysis Statistical analysis The effects of dynamic stimulation of chondrocyte seeded agarose constructs Preparation of chondrocyte/agarose constructs Application of mechanical compression Biochemical analysis Statistical analysis Results Metabolism of chondrocyte sub-populations in unloaded cultures The effects of dynamic stimulation of chondrocyte seeded agarose constructs Glucose concentration of the medium: 4 g/l versus 1 g/l Strained versus unstrained Unstrained versus free-swelling Effect of cell density Discussion Metabolism of chondrocyte sub-populations in unloaded cultures The effects of dynamic stimulation of chondrocyte seeded agarose constructs Glucose concentrations of the medium: 4 g/l versus 1 g/l Strained versus unstrained Unstrained versus free-swelling Increasing cell amount Conclusion and recommendations for future research Acknowledgements References Appendices Appendix A; non-loading experiment Appendix B; loading experiment Free-swelling constructs Strained and unstrained constructs /21/2005 page 3

4 1 Introduction Injuries and degenerative diseases to load-bearing soft tissues are extremely common in hospital clinics and involve all ages of the population. 2 In case of cartilage defects this means that if left untreated, they will not only fail to heal, but will expand with time and, as a result, degenerative diseases such as osteoarthritis can develop. 3 Because of the limitations to both the normal physiological repair process of articular cartilage, and the current strategies which do not yield long term satisfactory results, tissue engineering is being considered as an alternative for the treatment of articular cartilage defects. 3;4 However, a lack of matrix development and loss of cell viability are frequently observed in the centre of such 3D tissue-engineered constructs of physiological dimensions. 4 To overcome these problems, more knowledge about the optimal culture-environment for chondrocytes is needed. It is evident that on implantation, the tissue engineered constructs will be subjected to normal physiological forces. It is important, therefore, to understand the effects of mechanical conditioning of cells within implant systems to predict their response and ultimate success in vivo. 1.1 Articular Cartilage Articular cartilage contains only one type of cell, the highly specialised chondrocyte. In humans, these chondrocytes represent only between 1-10% of the volume of hyaline cartilage, but are essential because they replace degraded matrix molecules, to maintain the appropriate physical dimensions and mechanical properties of the tissue. Some chondrocytes have cilia that extend from the cell into the ECM and are believed to play a role in sensing the mechanical environment of the cell, since chondrocytes are known to modify matrix properties in response to loading. 5 Articular cartilage can be divided in three zones; the superficial, the transitional and the deep zone. The distinction between each of these zones is based on differences in matrix and chondrocyte morphology and activity. The superficial zone is the thinnest zone and is made of two distinct layers. An acellular sheet of predominantly collagen fibers covers the joint. Deeper into the cartilage, the second layer is composed of flattened chondrocytes with their long axes parallel to the articular surface. The ECM in this area has more collagen and less proteoglycan than the other zones. The transitional zone occupies more volume than the superficial zone and includes chondrocytes that are spherical in morphology. The ECM in this area has larger collagen fibrils, more proteoglycan and less collagen and water, than in the superficial zone. The interterritorial fibrils are aligned obliquely or randomly to the articular surface, as opposed to parallel in the superficial zone. The deep zone is usually the largest in volume and has the largest diameter collagen fibrils, the most proteoglycan, and the least water. The cells are rounded, like in the transitional zone, but are stacked in columns of between 4 and 9 cells perpendicular to the articulating surface. The orientation of the interterritorial fibers changes again, so that they are now aligned perpendicular to the joint surface. Beneath this zone is a calcified layer overlying the subchondral bone. 5;6 Articular cartilage is an avascular, non-insulin-sensitive tissue that utilises glucose as its main energy source. 7 The adequate provision of glucose to the chondrocytes is essential to sustain their predominantly anaerobic metabolism. In addition glucose is a precursor 9/21/2005 page 4

5 for the ECM macromolecules, which are synthesised by the chondrocytes. Impaired glucose uptake would compromise cell function and potentially result in an imbalance of matrix synthesis and degradation which, in turn, could result in osteoarthritis. 8 Given the avascularity of cartilage, the diffusion pathways between capillaries and the cells are long, such that glucose is scarce and the partial pressure of oxygen within the matrix is low. Significant gradients in both oxygen and glucose concentration have been reported in cartilage, decreasing from the articular surface towards the subchondral bone. Calculations show that the relatively few chondrocytes that reside in a unit volume of cartilage form a sufficient sink to deplete the O 2 concentration from 6-10% at the synovial surface to 1-6%, or perhaps almost to zero, in the deepest layers. 9 Studies of chondrocyte metabolism have shown that the cells have low levels of oxygen consumption and exhibit rapid synthesis of anaerobic lactate, indicating that ATP is generated predominantly by substrate-level phosphorylation in the glycolytic pathway. 10 Chondrocytes in vitro respond to decreasing oxygen tension by increasing their glycolytic rate. Under anoxia, glycolysis is suppressed and ATP levels fall, even though this pathway does not require molecular oxygen. In situ, anaerobic glycolysis accounts for 95% of the chondrocyte glucose metabolism, which results in high levels of lactic acid production. By contrast, in the absence of oxygen, one molecule of glucose delivers 2 ATP by the glycolytic pathway, whereas, when oxygen is available, one molecule of glucose delivers 2 ATP by the citric acid cycle of the mitochondria and another 36 ATP by the electron transport chain. 4 Windhaber et al. 8 studied the glucose transport in bovine articular chondrocytes, using 2- deoxy-d-[ 3 H]-glucose. The rate of glucose uptake by chondrocytes was shown to be time-dependent, up to a saturation point when the uptake of glucose reached a steady state value. This repsons was found to be temperature-dependent and ph insensitive. 8 Heywood 4 measured oxygen consumption of isolated chondrocytes in a range of glucose concentrations. The oxygen consumption of cartilage is inhibited by glucose, consistent with the definition of the 5 Crabtree phenomena. The 4 Superficial subpopulation concentration of glucose in high glucose medium cultures did not Deep subpopulation significantly influence the oxygen uptake. However, primary chondrocytes were capable of doubling their oxygen consumption rate when glucose is lower than 2.7 mm. Heywood concluded that oxygen itself might become a limiting factor, Glucose (mm) if the glucose concentration of the medium was low. The rate of Figure 1: Oxygen consumption of chondrocytes in medium lactate release correlated with a range of glucose concentrations. 1 positively with the rate of glucose uptake. At a low glucose concentration almost all glucose was converted to lactate. In contrast, the greater glucose Oxygen consumption mol.hr -1.cell -1 9/21/2005 page 5

6 uptake in high glucose medium could not account for the increase in lactate production alone. It has been proposed that the glucose-mediated oxygen consumption represents a survival mechanism by which chondrocytes initiate oxidative energy metabolism in order to supply their basal ATP requirements from a critically limited glucose supply. The study of Heywood 1 indicates that there was a difference between the oxygen consumption of superficial and deep chondrocytes (see figure 1). As an example, superficial chondrocytes showed significant higher oxygen consumption than the deep chondrocytes. 1.2 Loading of agarose constructs The rate of diffusive transport is inadequate for the metabolic demands of human cartilage. Movement of the joints is critical to cartilage integrity. It is proposed that these movements induce mixing of the synovial fluid, which effectively reduces the diffusion distance. Most transport, involving metabolites such as glucose, occurs by diffusion, driven by the concentration gradient. However, transport of larger molecules is a result of fluid convection, due to cyclic compression. 4 Mechanical compression is known to modulate the metabolic activity and the biomechanical properties of articular cartilage. 11 Indeed, in vitro studies have demonstrated that dynamic compressive strain influenced proteoglycan synthesis and cell proliferation in a distinct and frequency dependent manner. 12 A resent study of Chowdhury et al. 11 investigated the stimulatory effects of dynamic compression using a range of compression cycles, applied in a continuous or intermittent manner. Three major markers of chondrocyte metabolism were employed, namely, proteoglycan synthesis, cell proliferation, and nitric oxide release. A conclusion drawn from the results, is that frequent bursts of intermittent compression for extended periods favoured proteoglycan synthesis, whereas shorter bursts of intermittent compression tended to favour cell proliferation. Continuous dynamic compressive strain (at an amplitude of 15 %) for 48 hours resulted in 60% more proteoglycan synthesis compared to the unstrained situation. Cell proliferation was enhanced to a maximum of 73% at 1,5 hour. However, after 48 hours only a 40% increase in cell proliferation was evident, suggesting some inhibition with continued stimulation. The release of nitric oxide, an intracellular signalling molecule involved in cartilage degradation, was independent of both the length and type of compressive regime applied Aim of work Two separate experiments were performed to study the influence of glucose concentrations in the medium on chondrocytes. The differences between these experiments will be explained below Metabolism of chondrocyte sub-populations in unloaded cultures The aim of the first experiment was to determine the glucose consumption of superficial and deep chondrocytes in medium with different glucose concentrations (0-22mM), to observe how the glucose uptake is related to the amount of oxygen used. This is performed in such a way that the results are comparable with earlier measurements in the 9/21/2005 page 6

7 host laboratory of oxygen consumption, carried out by Heywood 1. Therefore the glucose uptake (and lactate production) of superficial and deep chondrocytes, in different glucose concentrations, was measured after a culture period of six hours. As mentioned before, it was found by Heywood that there is a higher oxygen uptake at low glucose levels. Also a difference was reported between the oxygen consumption of superficial and deep chondrocytes (figure 1). It was hypothesised that the chondrocytes will breakdown glucose, as opposed to oxygen, in the presence of excess glucose. This mechanism of glucose breakdown, however, produces a 16 times fold reduction in energy compared to the breakdown of glucose with oxygen. It is interesting to note that in the case of low glucose concentrations in the medium, the chondrocytes will changes to the more effective way of releasing energy by using oxygen to breakdown glucose. The glucose consumption of chondrocytes was predicted to be less in low glucose cultures compared to high glucose cultures. Furthermore, a difference between the glucose consumption of the superficial and deep chondrocytes was predicted. The pilot study of Heywood 1 indicated that the superficial chondrocytes use more oxygen compared to the deep chondrocytes. Therefore it would be likely that the superficial chondrocytes have a lower glucose uptake than the deep chondrocytes. However, the glucose uptake is not only related to the oxygen uptake, as both storage and GAG synthesis can influence the glucose uptake of the chondrocytes The effects of dynamic stimulation of chondrocyte seeded agarose constructs To test the influence of the glucose concentration in the medium on chondrocytes in agarose constructs, a second experiment was performed. In this case, cell seeded agarose constructs were subjected to dynamic loading regimes. In particular, the effects of different glucose concentrations in the medium on the metabolic response of chondrocytes was examined, in terms of proteoglycan synthesis, cell proliferation, and nitric oxide release. It was hypothesised that the glucose concentration of the medium will affect the metabolic activities of chondrocytes which will, in turn, influence their response to mechanical loading. For this purpose, a compression experiment was devised with medium containing two glucose concentrations, namely low and high glucose medium. The low glucose concentration has been used in previous loading experiments and is comparable with that in the synovial fluid, which contains no more than 5 mm glucose (the plasma concentration). 4 The high glucose concentration corresponds with the highest concentration used in the non-loading experiment. 9/21/2005 page 7

8 2 Materials and methods 2.1 Metabolism of chondrocyte sub-populations in unloaded cultures Isolation of bovine chondrocytes Superficial and deep slices of cartilage were removed from the metacarpalphalangeal joints of month-old cattle. The superficial cartilage was defined by taking a top slice, as thin as possible with a scalpel, using a identical method to that previously described by Lee et al. 13 The remaining cartilage was defined as deep cartilage. The superficial and deep cartilage slices were kept separately during the whole process. Both slices were washed, diced finely using two scalpels, and incubated at 37 C on rollers for 1 hour in Dulbecco s minimal essential medium (DMEM) supplemented with 20% foetal calf serum (FCS), 2 µm L-glutamine, 5 µg per ml penicillin, 5 µg per ml streptomycin, 20mM Hepes buffer, and 0.85 µm L-ascorbic acid (DMEM+ 20% FCS) + 700U per ml protease. They were subsequently incubated for a further 16 h at 37 C in DMEM+20% FCS supplemented with 100U per ml collagenase type XI (All Sigma Chemical, Poole, UK). The released chondrocytes were passed through a 70 µm pore size cell sieve (Falcon, Oxford, UK), washed twice in DMEM+ 20 % FCS, and finally resuspended in medium to give a cell concentration of 50x10 6 cells per ml Incubation with different glucose concentrations In two 96-well plates (Costar, UK), 20 µl of cell-medium (cell concentration of 50x10 6 cells per ml) was added to the wells (12 wells with superficial cells and 12 wells with deep cells per plate). 290 µl of medium (DMEM+ 20 % FCS with a range of glucose concentrations) as listed in table 1 was added. One well plate was used for time zero measurement as a control, the other plate cubated for six hours at 37 C, with orbital mixing every hour. Table 1: Range of glucose concentrations, used for incubation of chondrocyte suspension. The measured glucose concentration was slightly different to nominal values. (Values measured with glucose assay as described in 2.1.3) Defined glucose concentration [mm] Measured glucose concentration [mm] Biochemical analysis Directly after the medium was added to the cells, the medium in the control was removed from the cells, after centrifuging for 5 min at 2000 rpm, and was frozen at -20 C. The other well plate was handled in a similar manner at the end of the 6 hours culture period. The glucose consumption was measured by an assay. This assay utilises the substrate specificity of enzyme catalysis. This is combined with quantitative detection of the final product, reduced nicotinamide adenine dinucleotide (NADH), by spectrophotometrically determined absorbance at 340 nm with the Fluostar Galaxy plate reader. To create 9/21/2005 page 8

9 the final product, NADH, the glucose within the sample was converted with Infinity TM glucose reagent (Thermo Trace Ltd, TR15421) whereby NAD +, from the reagent mixture, is reduced to NADH + H +. A standard curve of known glucose concentrations (0-8mM) was used. In some cases samples, for which the glucose concentration was expected to fall out of the range of the standard curve, were diluted with Phosphate Buffered Saline (PBS) before measurement. Lactate production was measured with an assay, which also uses quantitative detection of the final product NADH, by spectrophotometrically determined absorbance at 340 nm with the Fluostar Galaxy plate reader. To create the final product, NADH, the lactate within the sample is oxidised to pyruvate in a reaction catalysed by lactate dehydrogenase (LDH), which is linked to the reduction of NAD + to NADH + H +. A standard curve of known lactate concentrations (0-13.3mM) was used Statistical analysis For all glucose concentrations, duplicate experiments were performed during the incubation. Duplicate samples of medium were taken for all experiments, for glucose and lactate measurements. This experiment was repeated four times, and was therefore performed on a total of 4 separate bovine feet. 2.2 The effects of dynamic stimulation of chondrocyte seeded agarose constructs Preparation of chondrocyte/agarose constructs The isolation of bovine chondrocytes was identical to described in However, in this case, full depth slices of cartilage were removed from the metacarpalphalangeal joints of month-old cattle and the cells were finally resuspended in medium to give a cell concentration of 8x10 6 cells per ml. The chondrocyte suspension was added to an equal volume of molten 6% (w/v) agarose type VII (Sigma Chemical, Poole, UK) in Earle s balanced salt solutions (EBSS, Sigma Chemical, Poole, UK), to yield a final cell concentration of 4x10 6 cells per ml in 3% (w/v) agarose. In one of the experiments (denoted by 0501C), two cell densities were employed namely 4x10 6 and 10x10 6 cells per ml. Using a Pasteur pipette, the chondrocyte/agarose suspension was transferred into a sterile stainless steel mould, containing holes measuring 5mm in diameter and 5mm in height. The chondrocyte/agarose suspension was allowed to gel at 4 C for 20 min to yield cylindrical constructs, which were subsequently cultured in a high glucose or low glucose medium at 37 C in 5% CO 2 for 24 hour. Medium was defined high glucose or low glucose, containing DMEM+ 20% FCS with a glucose concentration of 1 g/l or 4 g/l, respectively Application of mechanical compression A well-characterised cell-straining apparatus (Zwick Testing Machines, Leominster, UK), as described by Lee and Bader 12, was used to apply dynamic compression to 9/21/2005 page 9

10 chondrocyte/agarose constructs. The constructs were transferred into a 24-well culture plate (Costar, High Wycombe, UK) and mounted within the apparatus. One ml of high glucose medium or low glucose medium supplemented with 1 µci per ml [ 3 H]thymidine + 10 µci per ml 35 SO 4 (both Amersham Pharmacia Biotech, Bucks, UK) was applied to each well. A continuous compression regime was employed over a 48 hours culture period. The unconfined compression was applied in a dynamic manner, with a compressive strain amplitude of 15% at 1 Hz. The [ 3 H]thymidine and 35 SO 4 incorporation was over a 48 hours culture period as well. Control constructs subjected to a tare strain of 0.8% were maintained in an unstrained state Figure 2: Three separate boundary conditions imposed on cell-seeded agarose within the cell-straining apparatus and another constructs. group of constructs were allowed to swell freely (figure 2). All constructs were incubated for 48 hours at 37 C and 5% CO Biochemical analysis At the end of the culture period, the constructs and the corresponding medium were removed and separately frozen at -20 C. The subsequent biochemical analysis has been previously described by Bader and Lee 12;13. In summary, chondrocyte/agarose constructs were digested with 2.8 U per ml papain and 10 U per ml agarase (both Sigma Chemical, Poole, UK). Incorporation of 35 SO 4 into newly synthesised glycosaminoglycans (GAGs) was determined in both medium and agarose/papain digests, using the Alcian Blue precipitation method 13. [ 3 H]Thymidine incorporation was measured in the agarase/papain digests by 10% trichloroacetic acid precipitation onto filters using the Millipore Multiscreen System (Millipore, Watford, UK). Total DNA, determined using the Hoechst method 14, was used as a baseline for 35 SO 4 incorporation and [ 3 H]thymidine incorporation. The number of cells per construct was assessed from the DNA content and a conversion factor of 7.7 pg DNA per chondrocyte was employed 15. Absolute concentrations of nitrite (µm), a stable end product of NO metabolism, were measured in the media of cultured cells, using a spectrophotometric method based on the Griess assay 13;16;17. Absorbance was measured at 550 nm and nitrite (µm) was determined by comparison with standard solutions of sodium nitrite. Glucose consumption and lactate production were measured with the method described earlier in section Statistical analysis The loading experiment was repeated three times, while the free-swelling condition was repeated four times. A free-swelling experiment was also performed with another celldensity, instead of 4 million cells/ml, in this case, 10 million cells/ml. For both glucose concentrations, experiments were performed with a minimum of 6 constructs for compression, 6 unstrained constructs and 6 free-swelling constructs. Duplicate samples were taken for all experiments, for all assays. Total experiments were repeated four times and involved one to three cow feet per each experiment. Statistical significance was 9/21/2005 page 10

11 assessed by analysis of variance (ANOVA) followed by the student-t test, using p<0.05 as criterion for significance. Normalised data was used as will be explained in the results section /21/2005 page 11

12 3 Results 3.1 Metabolism of chondrocyte sub-populations in unloaded cultures Complete data for these four experiments is listed in Appendix A. Both the glucose consumption and lactate production of superficial and deep chondrocytes incubated in medium with different glucose concentrations is illustrated in figures 3 and 4, respectively. Superficial cells Deep cells 6 Glucose consumption (10-4 M / hour / cell) Glucose concentration of medium (mm) Figure 3: Glucose consumption of superficial (black) and deep (grey) chondrocytes at different glucose concentrations in the medium (mean + SD; n = 4). There appears to be a general increase in glucose consumption with increased glucose concentration of the medium (figure 3). However, for each data set there was considerable variability as indicated by the error bars. There was a small associated increase in lactate production with increased glucose concentration in the medium (figure 4). Although there were differences, none have been found statistically different between the corresponding data from the two sub-populations (p>0.05). These findings are at variance with the previous pilot study by Heywood 1. 9/21/2005 page 12

13 Superficial cells Deep cells Lactate production (10-4 M / hour / cell) Glucose concentration of medium (mm) Figure 4: Lactate production of superficial (black) and deep (grey) chondrocytes at different glucose concentrations in medium (mean + SD; n = 4). The corresponding ratios of lactate/glucose for both cell sub-populations are illustrated in figure 5. It is evident that for lower glucose concentrations in the medium, the lactate Superficial cells Deep cells 6 5 Lactate/Glucose ratio Glucose concentration of medium (mm) Figure 5: Lactate/Glucose ratio for superficial (black) and deep (grey) chondrocytes (mean + SD; n = 4). Dotted line is the expected ratio for chondrocytes. production is approximately twice as high as the glucose uptake. However, at higher glucose concentrations, the ratio decreases. This latter finding is a direct result of continuing increase in glucose uptake in association with a fairly constant level of lactate production. 9/21/2005 page 13

14 Another perspective of the results is illustrated in figure 6 and 7, where the glucose consumption, oxygen consumption (by Heywood 1 ) and lactate production are illustrated in one figure, for superficial and deep chondrocytes respectively. It is evident that the glucose consumption is high when oxygen consumption is low. In addition, figure 6 en 7 Lactate Glucose Oxygen Oxygen (10-15 M / hour / cell) Glucose/Lactate (10-4 M / hour / cell) Glucose concentration of medium (mm) Figure 6: Glucose and oxygen consumption and lactate production of superficial chondrocytes (mean + SD; n = 4). illustrate that when the medium contains more glucose, there is a higher uptake of glucose by the chondrocytes, for both sub-populations. However, the uptake reaches a steady state value for medium with high glucose concentrations. Lactate Glucose Oxygen Oxygen (10-15 M / hour / cell) Glucose/Lactate (10-4 M / hour / cell) Glucose concentration of medium (mm) Figure 7: Glucose and oxygen consumption and lactate production of deep chondrocytes (mean + SD; n = 4). 9/21/2005 page 14

15 In figure 8 the oxygen consumption is plotted versus the glucose consumption for both the superficial and deep chondrocytes. In both cell sub-populations, an increase in glucose consumption rate is associated with a decrease in oxygen consumption. In figure 9 the oxygen consumption is plotted versus the lactate production for both cell subpopulations. The same general trend as illustrated for glucose in figure 8, is seen for the lactate production (figure 9). superficial chondrocytes deep chondrocytes Oxygen consumption (10-15 M / hour / cell) Glucose consumption (10-4 M / hour / cell) Figure 8: Oxygen consumption versus glucose consumption of both superficial and deep chondrocytes (mean + SD; n = 4). superficial chondrocytes deep chondrocytes Oxygen consumption (10-15 M / hour / cell) Lactate production (10-4M / hour / cell) Figure 9: Oxygen consumption versus lactate production of both superficial and deep chondrocytes. 9/21/2005 page 15

16 3.2 The effects of dynamic stimulation of chondrocyte seeded agarose constructs Complete data is provided in Appendix B Free-swelling conditions The sulphate incorporation of chondrocytes, in the free swelling state, for the four experiments is illustrated in figure 10. There is clear variability between experiments. 0501A 0501B 0501C 0501D 3,6E-04 Sulphate incorporation (um SO4 / hr / ug DNA) 3,0E-04 2,4E-04 1,8E-04 1,2E-04 6,0E-05 0,0E+00 1 g/l 4 g/l Glucose concentration of medium Figure 10: Sulphate incorporation of chondrocytes in medium with a glucose concentration of 1 g/l or 4g/l over four experiments. Similar inter-experiment variability was found for thymidine incorporation, glucose consumption, lactate production, and nitrite production (tables 10-14). This precludes the 0501A 0501B 0501C 0501D Percentage change in sulphate incorporation g/l Figure 11: Sulphate incorporation of chondrocytes in medium with a glucose concentration of 1 g/l or 4g/l, as an example of normalisation of the four experiments (mean value and SD per experiment). 9/21/2005 page 16

17 pooling data for each experiment for the two sets of glucose concentrations in the medium. Despite the large variability, each experiment yields a similar comparative value for 1 g/l and 4 g/l. In addition, the amount of DNA per sample (table 15, appendix B) is fairly constant for all experiments. Therefore the undoubted variability in metabolic parameters, for example sulphate incorporation (figure 10), can not be directly attributed to differences in cell viability. Therefore, the data of the four experiments was normalised per experiment, by taking the samples of 1g/l medium as a control (100%). The data is presented in figure 11. After the normalisation process, the data from the four experiments were pooled, which eventually gives values for 32 constructs (n=32). These final data are listed in table 2. Table 2: Biochemical analysis of free-swelling constructs; data normalised to 1 g/l; n=32, with the exception for values of lactate/glucose ratio, where n=26. (* p<0.05; *** p<0.001) Metabolic parameter Mean SD p Glucose concentration (1 vs. 4 g/l) Normalised Thymidine incorporation 1 g/l g/l Normalised Sulphate incorporation 1 g/l g/l Normalised Glucose consumption 1 g/l < *** 4 g/l Normalised Lactate production 1 g/l g/l Normalised Nitrite production 1 g/l * 4 g/l Lactate/Glucose ratio 1 g/l g/l Strained conditions With respect to the effects of dynamic compression, data for the strained group was normalised to the corresponding unstrained controls, as previous reported (Lee and Bader, ). The mean value for the three experiments (0501A, B and D) were pooled and the results summarised in table 3. 9/21/2005 page 17

18 Table 3: Biochemical analysis of strained constructs; data normalised to unstrained constructs (n=18; * p<0.05). Metabolic parameter Mean SD p Glucose concentration (1 vs. 4 g/l) Normalised Thymdine incorporation 1 g/l control 100,00 32,10 0,62 4 g/l control 100,00 39,50 1 g/l strained 99,96 55,22 0,79 4 g/l strained 83,52 33,87 Normalised Sulphate incorporation 1 g/l control 100,00 28,78 0,64 4 g/l control 100,00 27,18 1 g/l strained 106,00 22,88 0,20 4 g/l strained 87,50 24,59 Normalised Glucose consumption 1 g/l control 100,00 48,86 0,22 4 g/l control 100,00 28,84 1 g/l strained 118,08 37,02 0,04* 4 g/l strained 114,77 36,75 Normalised Lactate production 1 g/l control 100,00 50,69 0,34 4 g/l control 100,00 15,45 1 g/l strained 99,79 41,58 0,52 4 g/l strained 102,34 35,95 Normalised Nitrit Oxide production 1 g/l control 100,00 41,86 0,44 4 g/l control 100,00 25,63 1 g/l strained 83,27 41,33 0,22 4 g/l strained 71,14 19,10 Lactate/Glucose ratio 1 g/l unstrained 1,75 0,63 4 g/l unstrained 2,59 4,24 1 g/l strained 1,95 1,23 4 g/l strained 2,71 1, Glucose concentration of the medium: 4 g/l versus 1 g/l When looking at the influence of low and high glucose medium (1 g/l and 4 g/l respectively) on the free-swelling constructs (table 2) it is obvious that there is no significant difference in 35S-sulphate and 3H-thymidine incorporation. By contrast, glucose consumption is significant higher (p<0.001) in high glucose medium (table 2). However this differences is not reflected in the lactate production and thus the lactate/glucose ratio is considerably reduced in the low glucose medium (table 2). 9/21/2005 page 18

19 Nitrite production is significantly higher (p<0.05) in high glucose medium compared to the low glucose medium for free-swelling constructs. The influence of the glucose concentration on strained and unstrained constructs is summarised in table 32, Appendix B. The glucose concentration has no significant influence on the tested metabolic parameters in the unstrained constructs. For the strained constructs, there is almost no effect as well, however, the glucose consumption is significantly higher in high glucose medium. The increase in nitrite production in high glucose medium, observed in free-swelling constructs, is also observed in strained and unstrained constructs. However, this increase was only statistically significant in experiment D. The influence of the glucose concentration on nitrite production seems to increase when constructs are strained, although this was not significant for the three experiments together Strained versus unstrained The influence of compression of the constructs compared to the unstrained constructs (table 3 and Appendix B, table 32) was not significant for the 35 S-sulphate and 3 H- thymidine incorporation, glucose consumption and lactate production. The nitrite production is lower for the strained constructs, although any differences were only statistical significance from one experiment, namely 0501 B, in a glucose medium of 4 g/l (Appendix B, table 32) Unstrained versus free-swelling To examine the influence of the indenter on the constructs (see figure 2), the results of the unstrained and free-swelling constructs were also compared. Appendix B, table 33 shows that in low glucose medium (1 g/l) free-swelling leads to significantly higher 35Ssulphate incorporation, 3H-thymidine incorporation and lactate production, compared to the unstrained constructs. The same trend was observed for high glucose medium (4 g/l), where 3H-thymidine incorporation, glucose consumption and lactate production were significantly higher in free-swelling constructs. By contrast, the values for nitrite production are significantly higher in the unstrained constructs compared to the freeswelling controls. Differences were statistically significant for the glucose concentrations of 1 g/l and 4 g/l at the 5 percent and 1 percent respectively Effect of cell density In experiment 0501C, two cell densities were employed. However, the values of final DNA amount per constructs indicate that the amount of cells at the end of the culture period is approximately 5 and 7 million cells/ml instead of 4 and 10 million cells/ml (Appendix B, table 15, mean values 3.4 µg/ml and 4.9 µg/ml). The results of the biochemical analysis are listed in Appendix B, table and 18. A summary of the effect of cell density on the five metabolic parameters for two concentrations of glucose in the medium is summarised in table 4. Eight of the ten comparisons yielded differences which were statistically significant. 9/21/2005 page 19

20 Table 4: Summary of the effect of cell density on the five metabolic parameters for two concentrations of glucose in the medium. Cell density, 4x10 6 vs 10x10 6 cells/ml Metabolic parameter Glucose concentration Change and significance level Thymidine incorporation LG Decrease p<0.001 Thymidine incorporation HG Decrease p<0.001 Sulphate incorporation LG Decrease p<0.05 Glucose consumption LG Increase p<0.001 Lactate production LG Increase p<0.001 Lactate production HG Increase p<0.001 Nitrite production LG Increase p<0.001 Nitrite production HG Increase p< /21/2005 page 20

21 4 Discussion 4.1 Metabolism of chondrocyte sub-populations in unloaded cultures The results of the six hour culture period experiment (without loading) showed an increase in glucose consumption of chondrocytes when the glucose concentration of the medium increases, in a similar manner to that previously reported by Windhaber et al. 8. The lactate production appears to be twofold that of the glucose consumption at low glucose concentrations of the medium, however, the lactate/glucose ratio (figure 5) decreases at higher glucose concentrations of the medium. This was also reported by Heywood 4, who showed that the amount of lactate release could not account for the amount of glucose uptake in high glucose cultures. Indeed glucose could also been used for GAG production or for storage. When glucose consumption and lactate production of chondrocytes, found in this experiment, are compared to the results of experiments by Heywood 1 (figure 1), it is clear that the glucose consumption is a mirror image of the oxygen consumption. Thus higher glucose concentrations in the medium, lead to more glucose consumption and an associated lower oxygen consumption. It was hypothesised that the chondrocytes will breakdown glucose without oxygen when adequate levels of glucose are present. This manner of glucose breakdown, however, yields 16 times less energy than the breakdown of glucose with oxygen. In the case of low glucose concentrations in the medium, the chondrocytes will changes to the more effective way of releasing energy by using oxygen to breakdown glucose. To support this proposition, the consumption of glucose in chondrocytes is found to be less in the low glucose cultures compared to the high glucose cultures (figures 3,6 and 7). It is interesting to note that in high glucose medium, when there is less oxygen consumption, more lactate should be produced per mol glucose. In this condition the energy is released less efficiently. Therefore, the lactate production should be relatively higher in chondrocytes cultured in higher glucose medium. However, the lactate/glucose ratio of these experiments decreases towards higher glucose concentrations (figure 5). A possible explanation is that the amount of oxygen utilisation in lower glucose medium is required for the energy used by glycolysis. Furthermore it is not possible to estimate the nature of glucose breakdown (with or without oxygen) by measuring the lactate release compared to the glucose consumption. It is recognised that chondrocytes exhibit generous glycogen storage capacity and lipid accumulation, which are enhanced by high glucose culture. It is suggested that these act as sinks for the increased glucose consumption in high glucose culture. 4 Despite the significant difference in oxygen consumption between the superficial and deep chondrocytes found by Heywood 1, the present experiment does not reveal a significant difference in glucose consumption or lactate production between the two subpopulations of chondrocytes. This finding might be attributed to the method of isolating cells. In the present experiment the superficial cartilage was defined by slicing of a top 9/21/2005 page 21

22 layer, as thinly as possible with a scalpel. The remaining cartilage was defined as deep cartilage. However, a separate study could be performed to see if the cells could be separated, and hopefully more accurately, by spinning them down with different centrifuge velocities. Deep cells are supposed to be larger, and as a result of that, heavier than superficial cells. Another possible explanation for the lack of a significant difference between the two sub-populations is that the higher oxygen consumption in superficial chondrocytes is not used for breakdown of glucose, but for other metabolic activities. In that case, it is not necessary that there is a difference between glucose consumption in superficial and deep cells. Besides, the lower oxygen consumption in deep chondrocytes could also be explained by the decrease in O 2 concentration with depth in native articular cartilage 9. Although this experiment was performed with considerable care, there are still some points of improvement that could make this experiment more accurate. For example the amount of cells in this experiment was assumed to be one million cells per well, however the exact amount should have been measured, with a DNA-assay, to distinguish variations in the results due to different cell numbers. 4.2 The effects of dynamic stimulation of chondrocyte seeded agarose constructs To be able to compare the individual results of the loading experiment with chondrocytes in agarose constructs, normalisation of the data was necessary. Despite the large variability in the data of the loading experiment, the amount of DNA within the constructs was constant (table 15), which indicates that this variability is not due to cell death. It is likely that the variability is caused by differences between individual tissue samples, which have a different metabolism. Metabolic activity depends on factors like age, weight or sex of the cattle. To avoid these individual differences between experiments, it is better to obtain feet of cattle of the same age and pool the cells from at least four different specimens Glucose concentrations of the medium: 4 g/l versus 1 g/l The effect of glucose concentration in the medium on chondrocytes in agarose constructs was studied by culturing chondrocytes in high glucose medium (4 g/l = 22 mm) or low glucose medium (1 g/l = 5,5 mm). In the normalised values of the free-swelling experiments, it is clear that there is approximately four times more glucose consumption by the chondrocytes in high glucose medium compared to the chondrocytes cultured in low glucose medium (table 2). However, there were no differences between either the lactate production or the 35 S-sulphate incorporation, a marker for no additional GAG production. Thus it is strongly suggested that the extra glucose consumption in high glucose medium is not used for direct ATP or GAG-synthesis. Instead it is more likely used for storage. However, for GAG-synthesis, oxygen is required as well, therefore low oxygen concentrations could have restricted the GAG production. An interesting finding was the increased nitric oxide release, a potential marker for ECM breakdown, of the chondrocytes in high glucose medium (table 2). However, it is unlikely that a higher glucose concentration of the medium supports ECM breakdown. 9/21/2005 page 22

23 There was a difference between the effect of the glucose concentration in the medium on free-swelling, unstrained and strained constructs with respect to the glucose consumption and nitric oxide release. The glucose consumption of unstrained constructs showed no significant differences for high and low glucose medium. nitric oxide release was only found to be significantly increased in high glucose medium in one of the experiments. Both of these exceptions could be caused by non-accurate measurements of glucose and/or nitrite concentrations Strained versus unstrained The influence of loading the agarose constructs is studied by comparing strained constructs with unstrained constructs that beard only the weight of an indenter. There were no significant differences measured in metabolic activity, except for the release of nitric oxide. The nitric oxide release was lower in strained constructs compared to the unstrained constructs. Because release of nitric oxide is potential for ECM breakdown, this indicates that chondrocytes in agarose constructs prefer dynamic straining compared to the unstrained situation. In a previous study by Chowdhury et al. 11 this decrease of nitric oxide release for strained constructs was confirmed. However, they also found an increase for 35 S-sulphate and 3 H-thymidine incorporation for the strained constructs. A possible explanation for the lack of a significant increase in 3 H-thymidine incorporation in this study is the fact that Chowdhury et al. observed that cell proliferation was enhanced to a maximum after a short burst of 1,5 hour compression instead of 48 hour compression Unstrained versus free-swelling When the results of the unstrained constructs are compared with the free-swelling constructs, the differences between these two unloaded groups are remarkable. As shown in the results, the unstrained constructs have lower metabolic activities compared to the free-swelling constructs. Therefore it can be concluded that the unstrained situation reduces the metabolic activities of the chondrocytes. This could be caused by the restriction of swelling and diffusion, due to the clamping of the construct between the bottom of the well-plate and the indenter. 9/21/2005 page 23

24 Table 5: Data of Nitrite production (µm / 10^6 cells / hr) for free-swelling and strained constructs. Values are p-values of T-student-test. Experiment 1 g/l free-swelling 1 g/l strained 4 g/l free-swelling 4 g/l strained 0501 A B D Mean SD Statistics: free-swelling versus strained constructs (p) 1 g/l g/l Only nitric oxide release is significantly increased, instead of decreased, for the unstrained situation. This release is potential for ECM breakdown, and therefore indicates that constructs prefer free-swelling compared to the unstrained situation. As discussed previously the chondrocytes seem to prefer the strained situation compared to the unstrained situation. Therefore it is interesting to compare the release of nitric oxide of free-swelling and strained constructs (table 5). The mean values of the pooled experiments show no significant differences between free-swelling and strained constructs. More experiments should be done to see if chondrocytes prefer the strained situation, as suggested by Chowdhury et al. 11. With this study the strained constructs should be compared with the unstrained constructs, but also with the free-swelling constructs Increasing cell amount The results show that when the cell amount is increased the 3 H-thymidine incorporation becomes significantly lower in both low and high glucose medium. Because the results are given in concentration per µg DNA, this means that the 3 H-thymidine incorporation per cell becomes less, which means the cell proliferation is less, when the cell amount is increased. This could partly explain the difference in cell amount found at the end of the culture period, approximately 5 and 7 million cells/ml instead of 4 and 10 million cells/ml seeded. To see if cell death becomes higher when the cell amount is increased, the constructs should be analysed by staining for viability. However, this is not done in this study. 9/21/2005 page 24

25 4 million cells/ml 10 million cells/ml 0.4 Glucose consumption (mm / 10 6 cells / hr) g/l 4 g/l Glucose concentration of medium Figure 12: Glucose consumption of chondrocytes of chondrocytes in medium with a glucose concentration of 1 g/l or 4 g/l, 4 million cells/ml (black) and 10 million cell/ml (grey) The glucose consumption per cell increases significantly in the low glucose medium (figure 15) when cell amount increases. However, this extra consumption is not used for GAG production, because the 35S-sulphate incorporation significantly decreases in low glucose medium, which means less GAG production per cell when cell amount increases. The increase of glucose consumption per cell in low glucose medium indicates that the 4 million cells did not use all the present glucose of the medium. However, as a result of the low glucose concentration more oxygen should have been consumed, because glycolysis is inhibited and oxidative phosphorylation increases. 4 Because oxygen is already low in medium with 4 million cells, the 10 million cells had to use more glucose per cell to get the same amount of energy per cell. The extra low oxygen concentration in the situation with 10 million cells/ml, could, in turn, have reduced the GAG production per cell. The lack of a significant difference in glucose consumption per cell in high glucose medium is probably due to saturation of the cells. Enough glucose for the chondrocytes in high glucose medium, for even an increased amount of cells, makes the cells use less oxygen by breaking down glucose with glycolysis in stead of oxidative phosphorylation. This could explain the lack of reduction in GAG production. A rise of the lactate production per cell in both low and high glucose medium occurs when the cell amount is increased. An increase in lactate production per cell in the low glucose medium is explainable when freeing of energy is done with less oxygen, when the cell amount is increased. However, for this explanation the lactate production of cells in high glucose medium should be increased for both 4 million and 10 million cells/ml. 9/21/2005 page 25