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

Abstract

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

Fig. 1

Scheme of the stimulation device for the application of high hydrostatic pressure (2.5 MPa) and photo of a sterile bag with cell-seeded collagen gels

Fig. 2

Comparison of hydrostatic pressure effects on matrix protein expression of human nucleus cells induced by different pressure magnitudes. Relative changes of mRNA expression normalized to the endogenous control (GAPDH) of stimulated cell-collagen constructs (0.25 and 2.5 MPa) compared to the respective unstimulated controls (n = 14 for 0.25 MPa, n = 9 for 2.5 MPa) are shown. (*) indicates significant pressure-induced differences (P < 0.007, Wilcoxon signed-rank test) and (*[) indicates significant differences between the two loading groups (Aggrecan: P = 0.0028, Collagen-I: P = 0.0062, nonparametric Wilcoxon 2-sample test)

Fig. 3

Comparison of hydrostatic pressure effects on matrix metalloproteinase expression of nucleus cells induced by different pressure magnitudes. Relative changes of mRNA expression normalized to the endogenous control (GAPDH) of stimulated cell-collagen constructs (0.25 and 2.5 MPa) compared to the respective unstimulated controls (n = 14 for 0.25 MPa, n = 9 for 2.5 MPa) are shown

Fig. 4

Influence of high hydrostatic pressure (2.5 MPa) on gene expression of bovine nucleus cells. Relative changes of mRNA expression normalized to the endogenous control (GAPDH) of stimulated cell-collagen constructs (2.5 MPa) compared to the respective unstimulated controls from four bovine subjects are shown. (*) indicates pressure-induced differences with P < 0.02 (Aggrecan: P = 0.008, Collagen-II: P = 0.023, MMP3: P = 0.008, Wilcoxon signed-rank test)