Supplemental Digital Content 1.
Validating the use of goat PBMCs in co-culture with rabbit IVD/endplate explants
The aim of this study was to demonstrate that the biological answer of IVDs to goat PBMCs does
not differ from the answer to rabbit PBMCs (group B).
Materials and Methods
16 IVD/endplates were harvested from 2 New Zealand White rabbits from T9/10 – L5/6 (8/animal),
assigned to a test group and to an assay (qPCR) (Table 1). Specimens were cultured for 6 days
before trauma induction at 37 °C and 5% CO2. Culture medium at day 1 was DMEM/F12
supplemented with 5 % FBS, 50 µg/ml penicillin, 100 U/ml streptomycin, 25 μg/ml vitamin c,
2.5 mg/L amphotericin-B, and 20 mM Na-citrate. For the following days the medium was changed
to lymphocyte medium (RPMI-1640 supplemented with 5% FBS 50 µg/ml penicillin, 100 U/ml
streptomycin, 1% MEM Vitamins, 1% MEM NEAA, 25 μg/ml vitamin c, and 50 µM 2mercaptoethanol). Rabbit or goat PBMCs were isolated by density gradient centrifugation using
Histopaque-1077, and cultured separately in lymphocyte medium until needed. Trauma was
induced at day 0 in all specimens (impact energy ~0.8 J) with a custom-made dropped weight
(172,3 g steel ball) loading device. At days 0 and 2, CM-Dil (Invitrogen) labeled PBMCs were added
on top of the fracture site. Gene transcription in the NP and nitric oxide (NO) serum concentration
were analyzed at day 4. Since no homing of PBMCs could be seen, gene transcription analysis is
not confounded with invaded immune cells.
Table 1. Type of stimuli and assays
A
B
Endplate fracture
X
X
Goat MNCs
X
Stimuli (presence of the stimulus: X)
Rabbit MNCs
X
Assays (replicate numbers)
qPCR
6
6
Histology
2
2
qPCR data were evaluated using the comparative Ct method. 2-∆Cq values were analyzed with the
non-parametric Kruskal-Wallis test with Bonferroni correction for multiple testing. Data are
represented as mean ± standard error of the mean.
Results
No differences in catabolic, pro-apoptotic, and pro-inflammatory gene transcription were found
(Figure 2c). CCL-4 was significantly up-regulated in group A (3.33 ± 1.31).
A/B
10
1
0.1
MMP1
MMP3
TNFa
IL6
FAS
FASL
Figure 1. Gene transcription analysis of rabbit nucleus pulposus.
NO concentration
No significant differences between groups were observed (Figure 3).
Group A (goat cells)
7
Group B (rabbit cells)
NO concentration [µM]
6
5
4
3
2
1
0
-1
-2
Day 0
Day 1
Day 2
Day 3
Day 4
Days after trauma
Figure 3. NO concentration in culture medium. Column height represents the mean ± SD. Significant (p < 0.05)
differences between days within singles groups are indicated in table 3.
Homing of rabbit PBMC to fractured rabbit spinal segments
Similar to goat PBMCs, rabbit PBMCs did not home to fractured rabbit spinal segments.
Figure 2. Homing of rabbit PBMCs to fractured IVD/endplates. a) Schematic drawing indicating region where photomicrographs (b,
c) were taken. b, c) Representative superimpositions of red and blue fluorescence photomicrographs of 6 m sagittal sections. b and
c are taken from two different trauma specimens. Red: MNCs labeled with DM-Dil, blue: all cell nuclei, labeled with DAPI; original
magnification: 10x.