A three-dimensional collagen matrix as a suitable culture system for the comparison of cyclic strain and hydrostatic pressure effects on intervertebral disc cells

Abstract

<jats:p content-type="fine-print"><jats:italic>Object.</jats:italic> 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.</jats:p> <jats:p content-type="fine-print"><jats:italic>Methods.</jats:italic> 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.</jats:p> <jats:p content-type="fine-print"><jats:italic>Conclusions.</jats:italic> 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.</jats:p>

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

“…They were able to define a loading threshold of 0.2 MPa, reflecting homeostasis, which preserved tissue composition and found changes in the gene expression in case of under-or overshooting this load. In our own pilot study on human cells exposed to mechanical stimulation in a 3D collagen type-I gel, we have found nucleus and annulus cells to respond to cyclically applied HP at 0.25 MPa [12]. Changes in the gene expression showed anabolic tendencies, but the effects were relatively minor.…”

“…Cell isolation and cell culture was conducted as previously described [12]. Briefly, cells were isolated by collagenase-pronase digestion from the nucleus pulposus of human disc biopsies from 18 donors (average age 39 ± 17) who underwent surgery because of disc herniation (for details of all patients see Table 1).…”

“…The final concentration of type I collagen was 3 mg/ml. Depending on the cell number available, gels for low and/or high-pressure magnitudes were produced and covered with 2 ml of serum-free culture medium [12]. Because of the partly low cell numbers, not all donors were included into each experiment (details see Table 1).…”

“…Application of low HP (0.25 MPa) was conducted as described in our pilot study [12]: cell-seeded cultures were stimulated inside a custom-made pressure chamber by air compression. Loading was applied once for 30 min with a frequency of 0.1 Hz.…”

Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model?

“…The requirements therefore are to provide a scaffold with adequate mechanical properties, which allows to culture suitable cells in it, for example, intervertebral disc cells or mesenchymal progentitor cells (MPCs). A recent study showed that a threedimensional collagen matrix might suitably serve as scaffold for cell seeding, that could be used to serve as a nucleus replacement [16]. It could be shown that these cells were able to proliferate and that cell viability was still 92% after 4 weeks.…”