Chondrozytärer Kohlenhydratmetabolismus und Zellvitalität boviner artikulärer Knorpelexplantate in statischer Kultur zur Analyse der Bedeutung von FCS und des nutritiven Potentials von Fructose für Knorpel in vitro

By assessing chondrocyte carbohydrate metabolism and cell viability in vitro this study aimed at answering the following questions: 1. Is FCS a beneficial nutritive supplement in addition to ITS (Insulin-Transferrin-Selenium)? 2. As an alternate to glucose in the culture medium, can fructose serve as a chondrocyte energy source minimizing lactate production and pH-shift? 3. How much is the energy demand of the cultured cartilage and how does it influence cell metabolism and viability? 96 chips of bovine articular cartilage were obtained from the shoulder joints of an 18 months old animal and assigned to 16 different test groups. Static incubation was carried out in culture multiwell plates at 37° C under atmospheric pressure of O2 and CO2. The culture medium (DMEM) was exchanged every 48 hours and used in 4 different variants: M1A - 1 g/L Fructose; M1B - 1 g/L Fruct, 2 % (V/V) FCS; M2A - 1 g/L Glucose; M2B - 1 g/L Gluc, 2 % (V/V) FCS. Each type contained 2 mM glutamine and 1 % (V/V) ITS. For every variant there was one group provided with 2 mL and another one supplied with 5 mL of medium. Each of these conditions was applied both to a group being cultivated 8 days and to another with 16 days of incubation. 10 freshly excised samples were used as controls and formalin-fixed and paraffin-embedded without cultivation. The cultured cartilage was processed in the same manner after 8 or 16 days, respectively. Tissue sections were HE-stained and immunohistochemically treated (TUNEL and M30 CytoDEATH®) for assessing and quantifying chondrocyte viability with a light microscope. Glucose-/fructose-, lactate- and LDH-content was analysed in the medium by photometric methods. The pH-values were measured with a pH meter. The statistical analysis was done by applying the non-parametric Kruskal-Wallis (H) test followed by a Bonferroni corrected Mann-Whitney U post-hoc test which compared groups that differed in one experimental condition. The portion of histomorphologically viable cells averaged out at 90,8 ± 5,3 % for the controls (freshly excised, not cultivated) and overall at 66,4 ± 18,9 % for the test groups. While viability of the controls was higher than cell survival in the collective of cultivated cartilage (estimated difference ~Δ: 20,5 %; p-value < 0,05), the U test indicated no significant differences of viability between the different experimental groups. Evaluating cell survival on the basis of liberated LDH as a marker of cell death also did not show statistical differences between the test groups. Carbohydrate uptake was four or more times lower than the supplied sugar amount. The carbohydrate use in the glucose groups exceeded the consumption in the fructose groups during the first 12 days with no exception (U test: ~Δ: 0,2-0,98 mg; p-value < α’ for each comparison done, α’ = 0,05/12= 0,0042 Bonferroni corrected). During the last two intervals of incubation 25 % of the comparisons yielded the same result while the remaining comparisons showed no statistical significance. When comparing the 2-mL- with the 5-mL-supply significant results (p-value < α’, α’ = 0,05/12= 0,0042) showed that higher medium supply resulted in higher carbohydrate uptake (~Δ: 0,15-0,58 mg). Assessing the effect of FCS there were cases with significant increases of metabolized sugar as well as others in which FCS decreased carbohydrate metabolism. Chondrocyte lactate production was significantly higher in the glucose than in the fructose groups. With one exception (p > α’), this difference was found for all of the 4 comparisons done for each incubation interval. It amounts to a range of ~Δ between 0,14 mg and 0,64 mg (p-value < α’, α’ = 0,05/4= 0,0125). There was a negative correlation between lactate production and pH-value (Pearson correlation coefficient: -0,62; p-value < 0,05). The pH-value was significantly higher, i. e. closer to the physiological value of 7,4 (van den Berg, 2003), in the fructose than in the glucose groups (~Δ: 0,11-0,39). In conclusion, the questions posed at the beginning can be answered as follows: 1. Supplementing culture medium with FCS is not beneficial for chondrocyte viability and does not increase cartilage carbohydrate metabolism. In this regard it cannot be recommended as a medium additive. 2. Fructose can replace glucose in the medium. To my knowledge, this study is the first to show that chondrocytes can metabolize fructose for energy production. Without affecting cell viability fructose yields lower carbohydrate consumption with lower lactate production and a more stable pH-value in the culture medium than glucose. 3. Cartilage nutritive demand was far lower than the amount supplied and can be reduced to approximately one third (~o,33 mg/L) in the future. A high carbohydrate supply resulted in an increase in carbohydrate metabolism, lactate production and pH-shift while it did not influence cell viability.