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 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...

The effects of dynamic loading on the intervertebral disc

“…Another study using an in vivo rat tail model showed a higher amount of apoptotic cells in the NP when the disc was subjected to dynamic compression of greater than 1.3 MPa [122], whereas 0.9 MPa [122] Response to amplitude modulation: hydrostatic pressure Similar to the findings on axial compression, applying hydrostatic pressure of greater than 2.5 MPa magnitude resulted in a degenerative response of the IVD cells. Decreased anabolic gene expression and increased catabolic gene expression were found on isolated porcine NP cells [31] and AF cells [124], bovine NP cells and human IVD cells [86] using a hydrostatic pressure loading system. Overloading of disc cells even increased the chemokine synthesis, which caused an enhanced migration of endothelial cells [84].…”

“…It was suggested that cyclic compression at a moderate level simulates physical activities and that this physiological loading might be able to promote repair or postpone disc degeneration. The duration response of IHP on the IVD cells has not been specifically reported in the literature, but an increased anabolic gene expression by human disc cells was demonstrated either after a single 30-min loading [86] or 30-min daily for 4 weeks [85].…”

“…It has been shown in various in vivo and in vitro models that a low magnitude of dynamic compression [70,122] and static/dynamic hydrostatic pressure [31,36,43,46,50,65,86,98,124] at a physiological frequency could increase anabolic response, but a high magnitude of dynamic loading causes early signs of disc damage and catabolic remodelling (Tables 2, 3). …”

Recent advances in annular pathobiology provide insights into rim-lesion mediated intervertebral disc degeneration and potential new approaches to annular repair strategies

“…These include the presence of growth factors, changes in local biomechanical stresses and in the osmotic environment of the disc cell [44,47,72,98,143,144,157]. Stimulation of endogenous disc cell populations by administered growth factors and other biomimetics in slow delivery carriers within bioscaffolds aim to address some of these questions [53,85].…”

Hydrostatic Pressure Differentially Regulates Outer and Inner Annulus Fibrosus Cell Matrix Production in 3D Scaffolds

“…Material properties of the fibrous scaffold, such as stiffness and permeability, may influence the cellular response to mechanical stimulation. In addition, the osmolarity of the culture medium was not adjusted to approximate conditions found in the disc, which may also modulate the ability of cells to respond to hydrostatic pressurization 14,15,24,25 . In our study, the stated pressure was applied at 0.5 Hz, 4 hours/day, from day 3 of culture to day 14.…”