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Orthopaedic Biomechanics
The effects of notochordal cells on matrix
production by nucleus pulposus and
bone marrow stromal cells
I.T.M. Arkesteijn, L.A. Smolders*, S. Spillekom*, E. Potier, B.P. Meij*, K. Ito, M.A. Tryfonidou*
*Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University
Introduction
The cell viability was similar in all culture groups on day 1 and 28.
Most cases of back pain can be attributed to intervertebral disc
The GAG/DNA amount increased significantly in time in all groups,
degeneration, starting in the nucleus pulposus (NP). At birth the NP
but NCs had no additive effects on NPCs or MSCs (fig 2). Histology
contains two distinct cell types: small nucleus pulposus cells
showed that NCs and non-clustered NPCs produced more
(NPCs, fig 1A) and large, vacuolated notochordal cells (NCs, fig
proteoglycans than MSCs and clustered NPCs.
1C), which disappear with age. Since the onset of degeneration
occurs soon after the loss of NCs, it is believed that NCs play a
central role in maintaining a healthy NP. As the number of NPCs
also decreases during degeneration, it has been proposed to
complement the NP cell population with bone marrow stromal cells
(MSCs). In this study we hypothesize that NCs can upregulate
matrix production by NPCs and MSCs.
Figure 2: GAG/DNA assessing the effect of A) NCs on NPCs, B) NCs on MSCs.
ACs used as control. Red bars=day 1, blue bars=day 15, green bars=day 28.
$=p<0.05 difference in increase in time.
Furthermore, in line with the biochemical analysis aggrecan gene
Figure 1: H&E staining of canine NPCs on A) day 1 and B) day 28 (insert:
clustered cells). Canine NCs on C) day 1 and D) day 28. Scale bar=50 µm
expression (fig 3) did not increase in the NC containing co-cultures.
Study design
• Fresh NCs were isolated from mongrel dogs; NPCs, MSCs and
articular chondrocytes (ACs, to control if effects are NC-specific) were
isolated from Beagles. Cells were cultured in alginate beads (1.2%) in
400 mOsm hg-DMEM+10% FBS +1% P/S in 5% O2 for 28 days and
the following groups were compared:
Single cell cultures
3*106 cells/ml alginate
Co-cultures
6*106 cells/ml alginate
A) NC; NPC; AC
A) NPC+NC; NPC+AC
B) NC; MSC; AC
B) MSC+NC; MSC+AC
• Samples of day 15 and 28 were compared to day 1 for : Cell
Figure 3: Aggrecan expression assessing the effect of A) NCs on NPCs, B) NCs on
MSCs. ACs used as control. Red bars=day 1, blue bars=day 15, green bars=day
28. #=p<0.001 difference in increase in time. $=p<0.05 difference in increase in
time.
viability (calcein-AM, PI); cell morphology (H&E); DNA content
Collagen type II expression increased in MSC+NC co-cultures
(Hoechst dye assay); Glycosaminoglycan (GAG) content (DMMB
compared to MSCs alone. However, this effect was much smaller
assay); Matrix distribution (hematoxylin, safranin-O, fast green); Gene
than in the MSC+AC control group. Brachyury expression was only
expression (NC markers – brachyury, CK-18; ECM markers -
detected in the groups containing NCs, and increased significantly in
aggrecan, collagen type I & II; matrix degradation and remodelling –
time in the NC and MSC+NC group and decreased for NPC+NC.
MMP-13, ADAMTS5, TIMP1)
Conclusions
Results
In this study, NCs were not able to up-regulate extracellular matrix
On day 1 the NCs were large, vacuolated cells, but in time the
production or disc matrix protein gene expression by NPCs and
vacuole size decreased (fig. 1A,B). Like NPCs (fig 1C,D), MSCs and
MSCs. This could be due the loss of NC phenotype in time that could
ACs developed clusters in time.
be attributed to suboptimal NC-culture conditions.
Department of Biomedical Engineering
This work was supported by AOSpine
International through an AOSpine
Research Network grant (SRN2011_11)