Transcript Slide 0

Orthopaedic Biomechanics
Notochordal cell-secreted factors stimulate
canine NPC matrix production
S.A.H. de Vries1, E. Potier1, M. van Doeselaar1, B.P. Meij2, M.A. Tryfonidou2, K. Ito1
1 Orthopaedic
Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands
2 Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
constant in NCCM (Fig 1a). GAG content increased more
than 2-fold in NCCM compared to BM (Fig. 1b) Histology
confirmed these findings (Fig 1c). TGFβ-1 (used as positive
control) also increased DNA and GAG content.
90.38±10.66
2
14
a
*
2
GAG/bead [µg]
DNA/bead [µg]
Introduction
Intervertebral disc degeneration (IVDD) is one of the main
causes of low back pain and is characterized by the
inability of the resident nucleus pulposus cells (NPCs) to
maintain a healthy matrix. During growth however, the main
population consists of large vacuolated notochordal cells
(NCs) The disappearance of NCs coincides with the onset
of disc degeneration. We thus hypothesized that NCs
secrete bioactive factors that stimulate matrix production by
NPCs.
1
*
1
Results
At day 0 and 28, no notable cell death was observed in any
group. DNA content decreased in BM whereas it remained
/ Department of Biomedical Engineering
10
8
6
4
BM
NCCM
BM
TGF
NCCM
TGF
c
Figure 1, a: DNA content per bead at day 0 and day 28, b: GAG content per bead at
day 28, c: Alcian Blue stainings at day 28. * p<0.05 to day 0, ** p<0.05 to BM.
Collagen 1 gene expression remained constant (Fig. 2).
Both Collagen 2 and Aggrecan gene expression increased
in NCCM compared to BM, approaching gene expression
levels found in TGF. A minor increase for SOX9 with NCCM
was observed.
100
Expression relative to 18S and
normalized to day 0 (2-Ct)
At day 0 and day 28, beads were assessed for: cell viability
with Calcein AM/Propidium Iodide staining, GAG (DMMB)
and DNA (Qubit) content, gene expression (collagen 1 and
2, aggrecan and SOX9) by realtime qPCR and GAG
deposition by Alcian Blue stained histology sections.
Statistical analysis was done with a Kruskal-Wallis test
followed by a Mann-Whitney U test with Bonferroni
correction for post-hoc testing. Statistical significance was
assumed for p < 0.05.
**
0
Day 0
• Base medium (BM): hgDMEM + 1% pen/strep + 5%
stripped fetal calf serum (FCS),
• NCCM filtrate resuspended in BM, or
• Positive control medium: BM + 10 ng/ml TGFβ-1.
b
2
0
Methods
NC conditioned medium (NCCM) was produced by
incubating
NC-rich
canine
NP
tissue
from
nonchondrodystrophic dogs (retain NCs during their life
time) in hgDMEM + 1% pen/strep (1g tissue/ 30 ml medium)
for 4 days, at 37 oC, 5% O2 and 5% CO2. Subsequently, the
medium was aspirated, filtered with a filter tube, and
material on top was resuspended in new medium. NPCs
were
retrieved
from
NC-poor
NP
tissue
of
chondrodystrophic Beagle dogs (loose their NCs similar to
humans and also develop IVDD [1]). NPCs were cultured
for 4 weeks in 1.2% alginate beads at 37 oC, 5% O2 and
5% CO2, receiving:
12
**
*
010
*
*
*
BM
NCCM
TGF
001
000
Collagen 1
Collagen 2
Aggrecan
Sox9
Figure 2: Gene expression at day 28, relative to 18s, normalized to day 0.
* p<0.05 to BM
Discussion
The current results show that NCCM increases NPC matrix
production and increases expression of genes associated
with a healthy NP phenotype. However, the concentration
of bioactive factors in NCCM is probably much lower than
the TGFβ-1 concentration used as control in this study. The
use of NCCM to stimulate NPCs is promising, and the next
step will be identification of it’s bioactive ingredients.
[1] J. Lotz, Animal models of intervertebral disc degeneration: lessons learned, Spine, 2004
This work was supported by AOSpine
International through an AOSpine Research
Network grant (SRN2011_11).