Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading excercises 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 individualindividual 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 inf...

Object. To study intervertebral disc cell mechanobiology, the authors developed experimental systems that allow the application of cyclic strain and intermittent hydrostatic pressure (IHP) on isolated disc cells under equal three-dimensional (3D) culture conditions. The purpose of the study was to characterize disc cell proliferation, viability, morphology, and gene expression in 3D collagen matrices. Methods. The effects of cyclic strain (1, 2, 4, and 8% strain; 1 Hz) and IHP (0.25 MPa, 0.1 Hz) on gene expression (real-time polymerase chain reaction) of anabolic and catabolic matrix proteins were investigated and compared with those derived from mechanically unstimulated controls. Intervertebral disc cells proliferated in the collagen gels (mean viability 91.6%) and expressed messenger RNA for collagen I, collagen II, aggrecan, matrix metalloproteinase (MMP)—2, and MMP-3. Morphologically, both spindle-shaped cells with longer processes and rounded cells were detected in the collagen scaffolds. Cyclic strain increased collagen II and aggrecan expression and decreased MMP-3 expression of anulus fibrosus cells. No significant difference between the four strain magnitudes was found. Intermittent hydrostatic pressure tended to increase collagen I and aggrecan expression of nucleus cells and significantly decreased MMP-2 and -3 expression of nucleus cells, whereas aggrecan expression of anulus cells tended to decrease. Conclusions. Based on these results, the collagen matrix appeared to be a suitable substrate to apply both cyclic strain and IHP to intervertebral disc cells under 3D culture conditions. Individual variations may be influenced by the extent of degeneration of the disc specimens from which the cells were isolated. This experimental setup may be suitable for studying the influence of degeneration on the disc cell response to mechanical stimuli.

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