Region specific response of intervertebral disc cells to complex dynamic loading: an organ culture study using a dynamic torsion-compression bioreactor

Abstract

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration.

Figure 1. Detailed view of the loading bioreactor and specimen chamber.

A) The bioreactor is situated inside a 37°C incubator with 5% CO₂ and 80% humidity. B) The glass walled sample chamber. IVD: intervertebral disc. C) Enlarged view of the titanium plate for holding the samples for rotation movement.

Figure 2. The 24-hour loading and resting cycles for the bovine organ culture.

Top: cyclic compression (CC), middle: cyclic torsion (CT), bottom: combined compression & torsion (CCT). Samples were loaded for 8 h and then rested for 16 hours every day; loadings and resting were continued for 14 days.

Figure 3. Percentage change in disc dimension relative to the starting of the experiment.

There was a 10% increase in disc volume in the No loading (NL) group, but increase was minor in all the other 3 groups. CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion. Plot of means ± SEM. N = 6.

Figure 4. Percentage cell viability of the disc tissue measured by fluorescence live/dead stain and confocal microscopy.

There was a significant cell death in the nucleus pulposus (NP) of the combined compression torsion (CCT) group compared to no loading (NL), cyclic compression (CC) and cyclic torsion (CT) groups. Cell viability in the annulus fibrosus (AF) maintained stable for all groups. Plot of means ± SEM. # Statistically significant different from all the other groups, p<0.0001. N = 6.

Figure 5. Activity of the cells measured by resazurin assay.

Cell activity was doubled in the NP of groups with torsion (CT and CCT) compared to when torsion was not applied (NL and CC). NL: no loading, CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion, NP: nucleus pulposus, AF: annulus fibrosus. Plot of means ± SEM. N = 6.

Figure 6. Cell apoptotic activity quantified by caspase 3/7 protein normalized by the total protein content.

No statistically significant difference in caspase 3/7 protein amount was found between groups. NL: no loading, CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion, NP: nucleus pulposus, AF: annulus fibrosus. Plot of means ± SEM. * statistically significant up-regulation p<0.05. N = 6.

Figure 7. Relative gene expression quantified by real-time PCR.

A group of anabolic genes and remodeling genes were analyzed in the NP and AF tissues. Both anabolic genes and remodeling genes were activated in the AF of both CT and CCT when torsion was applied. Plot of log means ± SEM, normalized to no loading control. * p<0.05. NL: no loading, CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion, NP: nucleus pulposus, AF: annulus fibrosus. N = 6.G.

Figure 8. Glycosaminoglycans (GAG) and hydroxyproline (HYP) contents of the tissue normalized to the fresh samples.

GAG content was maintained in all samples with a minor increase in the AF of all the 3 loading groups. HYP content was maintained without significant change. NL: no loading, CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion, NP: nucleus pulposus, AF: annulus fibrosus. Plot of means ± SEM. N = 6.

Figure 9. Histological analysis showing morphology of cells in the transition zone of the endplate and the nucleus pulposus.

MMA sections were stained with safranin O/fast green. Cells in the transition zone between the cartilaginous endplates (EP) and nucleus pulposus (NP) (separated by dotted black line) stayed as rounded chondrocyte-like cells (indicated by black arrows) as in hyaline cartilage in CC and CT groups. Some cells in the cartilaginous endplates of the CCT group stayed as chondrocyte-like cells whereas cells right after the endplates zone changed to cells with spindle-shaped nucleus and cell lacunae were lost (indicated by yellow circles). CC: cyclic compression, CT: cyclic torsion, CCT: combined compression torsion. N = 2.