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Orthopaedic Biomechanics
Using notochordal cells to regenerate
the degenerated intervertebral disc
S.A.H. de Vries, I.T.M. Arkesteijn, E. Potier, M.A. Tryfonidou, K. Ito
Introduction
Disc degeneration is one of the main causes of low back
pain and is characterized by the inability of the resident
cells to maintain a healthy tissue, due to a decreasing
number and/or a change of phenotype. Current treatments
are only symptomatic and new approaches addressing the
underlying cause of disc degeneration are needed, like cellbased regenerative treatments. In fact, mesenchymal stem
cells (MSCs) have already been shown to stop but not
reverse this degenerative process [1]. Innovative
approaches are therefore needed to further promote disc
matrix production by resident and/or regenerative cells.
In human adults, the core of the disc is mainly populated by
chondrocyte-like nucleus pulposus cells (NPCs). During
growth, when the largest amount of matrix is produced,
large vacuolated notochordal cells (NCs) are, however, also
present. As the disappearance of these cells coincides with
the onset of disc degeneration, they are believed to be
involved in disc homeostasis.
We thus hypothesize that NCs can increase the synthesis
of functional matrix by resident cells, the NPCs, or by
regenerative cells, the MSCs.
and GAG deposition location; and gene expression
(qPCR) of osteogenic- and chondrogenic differentiation
markers and matrix and remodeling proteins.
Results
At day 1, the characteristic vacuoles of the NCs were
clearly visible, as were the surrounding MSCs and NPCs in
the co-culture groups (figure 1).
A
B
C
Figure 1: 40x confocal
images of A: NCs alone,
B: NCs + MSCs, C: NCs
+ NPCs. Arrows indicate
NCs, white arrowheads
indicate MSCs and red
arrowheads
indicate
NPCs.
Methods
As certain dog breeds (chondrodystrophoid) develop disc
degeneration similar to the human condition [2], a canine
model will be used in this study.
Tissue material will be sampled from healthy dogs used in
unrelated experiments. Canine NPCs and MSCs will be
retrieved from chondrodystrophoid beagles, and NCs from
mixed breed dogs. The following groups of cells will be cocultured up to 28 days in 1.2% alginate beads:
1. NCs
5. NCs + MSCs
2. MSCs
6. NCs + NPCs
3. MSCs + TGFb
7. MSCs + NPCs
4. NPCs
The cell density is 3 millions cells/ml for the single cultures
and 6 millions cells/ml for the co-cultures using a ratio of
1:1. The cells will be cultured in 5% O2 at 37oC, and culture
medium consists of DMEM supplemented with 5% ITS.
At day 1, 14 and 28 beads will be analyzed: using confocal
imaging for cell viability and morphology (Calcein AM,
Propidium Iodide); GAG (DMMB assay) and DNA (Hoechst
assay) content; histology (Alcian Blue, HE) for morphology
/ Department of Biomedical Engineering
Culture (n=5) is ongoing and samples will be analysed at
day 14 and 28.
Future Research
If co-culture with NCs up-regulates matrix production, the
most successful strategy, stimulating either resident cells
(NPCs) or regenerative cells (MSCs), will be tested for its
therapeutic potential on NP tissue explants [3].
Furthermore, the mechanism behind NC’s up-regulation of
matrix production by NPCs/MSCs (i.e. cell-cell contact or
molecular interactions) will be deduced, and in case of
molecular interactions, the factor(s) responsible for the
NC’s up-regulating effect will be identified.
Ultimately, this study will provide not only knowledge on the
role of NCs in disc regeneration, but also an innovative
treatment for patients suffering from disc degeneration.
[1] F. Yang V.Y.L. Leung, K.D.K Luk, Mesenchymal stem cells arrest intervertebral disc degeneration
through chondrocytic differentiation and stimulation of endogenous cells., Mol Ther, 2009
[2] J. Lotz, Animal models of intervertebral disc degeneration: lessons learned, Spine, 2004
[3] B.G.M. van Dijk, E. Potier, K. Ito. Culturing bovine nucleus pulposus explants by balancing medium
osmolarity, Tissue Eng Part C, 2011.
This work was supported by an AOSpine
International Hans Joerg Wyss Foundation
Research Award grant N HJW2011-SU12.