Figure 1.
The Loaded Disc Culture System.
The Loaded Disc Culture System (LDCS; upper left), a single actuator (upper right) with culture chamber and reservoir, and a schematic cross-section (bottom) of a culture chamber with IVD.
Figure 2.
Mean IVD stiffness (N/mm) ±SD as calculated from disc loading and displacement measurements during the culture period in the LDCS. The gray dotted line represents IVD stiffness from the discs cultured under LDL condition. The black lines represent the SPL loaded IVD stiffness, respectively during the rest phase (triangles) and active phase (squares) of the diurnal SPL regime.
Figure 3.
Midsagittal paraffin sections of IVD specimens (anterior side of IVDs facing right) at day 0 and after 21 days of culture at unloaded, LDL and SPL loading conditions. The left column shows safranin-O stained sections for the proteoglycan distribution (red). In the right column Masson's trichrome staining for distribution of the collagen (blue) is shown.
Figure 4.
Fluorescent live/dead staining.
Representative images at 10× magnification of the nucleus and outer annulus regions of IVD. Shown is fluorescent staining on transverse cryosections of live cells with (green) and dead cells (red). The upper two images are taken from a fresh disc, stained directly after dissection from the lumbar goat spine at baseline control (day 0). The lower two images are taken from an IVD after 21 days of culture in the LDCS under unloaded conditions.
Figure 5.
Cell viability (above; live cells/total cells +SD) and cell density (below; mean total cells/mm2 +SD) as percentage of day 0 for each loading condition in the nucleus and outer annulus region after 7, 14 and 21 days of culture in the LDCS. P values comparing experimental groups with day 0 group in a linear mixed model with Bonferroni post-hoc testing: *p<0.05; #p<0.01; &p<0.001.
Table 1.
Cell Viability and Cell Density.
Figure 6.
Relative gene expression (log means ± SEM, normalized to YWHAZ) in the baseline (day 0; grey), unloaded (blue), LDL (green) and SPL (red) group. Shown in the left column is the data for the nucleus region and in the right column for the outer annulus region. Graph rows from top to bottom show respectively the anabolic, remodeling and inflammation-related genes. Brackets indicate significant statistical differences between groups when comparing in a linear fixed model with Bonferroni post-hoc testing. P values are indicated by: *p<0.05; #p<0.01; &p<0.001.
Table 2.
Mean water-content (table 2a; percentage water in tissue ± SD), mean GAG-content (table 2b; µgr GAG/mg dry weight) and mean collagen-content (table 2c; µgr hyp/mg dry weight) for day 0, unloaded, LDL and SPL group in the nucleus and outer annulus region at 7, 14 and 21 days of culture.
Table 3.
Primer sequences used for PCR.