The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells

Abstract

Objective: To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells.

Design: TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively.

Results: TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001).

Conclusions: Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply.

Keywords: Cell metabolism; Glucose concentration; Oxygen level; Temporomandibular joint (TMJ) disc; Tissue nutrition.

Figure 1

WST-1 standard curve showing the linear relationship between plated porcine TMJ disc cell number and absorbance. R² = 0.9228.

Figure 2

The cell viability of porcine TMJ disc cells. The data shown were means and 95% confidence intervals. Experiments were performed in triplicate on three independent experimental days (n=9). Absorbance values were normalized to the control (25 mM glucose and 21% oxygen level). The effects of oxygen levels (1%, 5%, 10%, and 21%) and glucose concentrations (0, 1.5, 5, and 25 mM) were assessed. An increase of glucose concentration significantly increased TMJ disc cell viability (P < 0.0001). In the presence of glucose, an increase of oxygen level significantly decreased cell viability (P < 0.0001). The interaction between oxygen and glucose was significant (P < 0.0001). Significance (P < 0.05) of oxygen level effect and glucose level effect are denoted by a * and #, respectfully.

Figure 3

The measurement of ATP per viable cell of porcine TMJ disc cells. The data shown were means and 95% confidence intervals. Experiments were performed in triplicate on three independent experimental days (n=9). The total per viable cell based ATP production was calculated by normalizing to the WST-1 absorbance values. The total ATP production per viable cell was then normalized to the control (25 mM glucose and 21% oxygen level). The effects of oxygen levels (1%, 5%, 10%, and 21%) and glucose concentrations (1.5, 5, and 25 mM) were assessed. An increase of glucose concentration from 1.5 mM to 25 mM had no significant impact on the average ATP measurement (P = 0.1388). In the presence of each of the glucose concentrations, an increase of oxygen level significantly increased intra- and extracellular ATP production (P < 0.0001). The interaction between oxygen and glucose was significant (P = 0.00015). Significance (P < 0.05) of oxygen level effect is denoted by a *.

Figure 4

The [2, 3-³H] proline incorporation per viable cell (i.e. collagen synthesis per viable cell) of porcine TMJ disc cells. The data shown were means and 95% confidence intervals. Experiments were performed in triplicate on three independent experimental days (n=9). The per viable cell based proline incorporation was calculated by normalizing to the WST-1 absorbance values. The [2, 3-³H] proline incorporation per viable cell was then normalized to the control (25 mM glucose and 21% oxygen level). The effects of oxygen levels (1%, 5%, 10%, and 21%) and glucose concentrations (1.5, 5, and 25 mM) were assessed. Although the effect of glucose concentration on collagen synthesis was statistically significant (P < 0.0001), the magnitude of impact was much smaller compared to that of the oxygen level. At the 21% oxygen level, an increase in glucose concentration from 1.5 mM to 5 mM significantly increased collagen synthesis (P = 0.033), while further increase of the glucose concentration had no significant effect (P = 0.187). In the presence of glucose, a reduced oxygen level resulted in a significant reduction of collagen synthesis within all glucose concentrations (P < 0.0001). The interaction between oxygen and glucose was significant (P = 0.0002). Significance (P < 0.05) of oxygen level effect is denoted by a *.

Figure 5

The [³⁵S] sulfate incorporation per viable cell (i.e. proteoglycan synthesis per viable cell) of porcine TMJ disc cells. The data shown were means and 95% confidence intervals. Experiments were performed in triplicate on three independent experimental days (n=9). The per viable cell based sulfate incorporation was calculated by normalizing to the WST-1 absorbance values. The [³⁵S] sulfate incorporation per viable cell was then normalized to the control (25 mM glucose and 21% oxygen level). The effects of oxygen levels (1%, 5%, 10%, and 21%) and glucose concentrations (1.5, 5, and 25 mM) were assessed. An increase in glucose concentration from 1.5 mM to 25 mM had no significant effect on average proteoglycan synthesis (P = 0.0821). In the presence of all glucose mediums, reduced oxygen level resulted in a significant reduction of proteoglycan synthesis (P < 0.0001). The interaction between oxygen and glucose was significant (P < 0.0001). Significance (P < 0.05) of oxygen level effect is denoted by a *.