The Evolutionary Importance of Cell Ratio between Notochordal and Nucleus Pulposus Cells An Experimental 3D Co-Culture Study
1
+1Gantenbein-Ritter B; 1Chan S CW
ARTORG Center, Spine Research, Institute for Surgical Technology and Biomechanics, University of Bern, Bern
[email protected]
Introduction
Notochordal cells (NC) are remnant cells originating from the notochord
present in all chordates in early embryogenesis and therefore these cells
are located in the center of intervertebral disc (IVD) (1). With ageing
these presumably progenitor-like cells disappear in some species and in
other species they persist up to adulthood (2). In human they disappear
early in childhood. Strikingly, these cells co-exist with nucleus pulposus
cells (NPCs) at different ratios among different vertebrate species.
Rodents (rats and mice) and lagomorphs (e.g. rabbits) maintain a high
number of NC cells throughout their lifetime whereas in other animals
such as cattle, goat and sheep these cells disappear early in lifetime (3).
Previous work on co-culture of non-chondrodystrophoid dog cells (i.e.
Greyhound) with bovine NPCs seems to point towards regulatory
mechanism and cell-cell interaction (4, 5). Here, we hypothesized
whether there is a ratio of NC relative to NPC, which is most favorable
for both cell populations in terms of cell activity and extracellular matrix
(ECM) production. Thus, we co-cultured porcine coccygeal NCs (in fact
a NCs+NPCs mix) and bovine coccygeal NPCs at different ratios, i.e.
0%, 25%, 50%, 75% and 100%, respectively.
Methods
Porcine notochordal cells (pNCs) were isolated from the nucleus
pulposus (NP) tissue of 4 to 5-month old porcine tails that were obtained
from the local abattoir. The high percentage of NCs in pig NP tissue was
confirmed by size using brightfield microscopy (~80%). Bovine nucleus
pulposus cells (bNPCs) were harvested from the NP tissue of ~1 yr old
bovine tails obtained from the abattoir. Both cells were separated from
native ECM by combined pronase/collagenase type 2 (Roche /
Worthington) digestion overnight and primary culture. The NCs from
pig NP tissue were expanded in monolayer up to Passage 2, which has
been previously described as non-problematic concerning
dedifferentiation (5). The cells were encapsulated at a density of 4M/ml
into 1.2% alginate by application of a syringe and 22G needle and
dropping into a 102mM CaCl salt solution (6). Assuming porcine NP
tissue to be 100% notochordal, the cells were kept in coculture of pNC :
bNPC ratios of 0%, 25%, 50%, 75% and 100% in serum-free defined
medium, containing antibiotics, ascorbic acid and ITS+ (Gibco + Sigma,
Switzerland). All bead-bead co-cultures were conducted in duplicate in
12-well plates, using 0.4-µm pore size, high pore density, polyethylene
terephthalate (PET) track-etched culture inserts (Becton, Dickinson and
Company). The co-cultures were tracked on day 0, day 7 and day 14.
DNA content (Hoechst staining), cell activity (Alamar Blue, Invitrogen),
Glycosaminoglycan content (GAG) (DMMB assay), and relative gene
expression (ACAN, Col 1, Col 2 and r18S) were monitored for both,
pNCs and bNPCs. All data are given as relative to the pure cell
population of the same culture day. Statistical significance was tested
using Wilcoxon signed rank test and Dunn’s post-hoc pairwise test.
2
Results
In both, pNCs and bNPCs, DNA content of beads was stable over
culture time or even increased slightly relative to day 0 control (data not
shown). As for the cell metabolism (Fig 1A-B) the bNPCs were more
strongly activated by the presence of pNCs (Fig. 1A) (p = 0.035) than
vice versa (Fig. 1B) but this effect was strongest in 75% pNC coculture
(Fig. 1A). We found a significant increase in GAG/DNA ratio for the
50% pNC group after 14 days of co-culture (Fig. 1C) but no effect for
pNC cocultures (Fig. 1D).
For bNPCs, relative gene expression revealed upregulation of ACAN by
~2-8 times and slight upregulation of Col 2. It was most strongly
upregulated in the 50% pNC coculture group (data not shown). For
pNCs ACAN was found to be up-regulated by about 3-5 times, however,
this was not significant. Remarkably, for pNCs, Col 2 was found to be
upregulated by a factor of ~100 for all of the three co-culture ratios
containing bNPCs, indicating progenitor-like cell status of pNCs.
Figure 1. A, B) Cell metabolism per DNA (cell) in bNPCs and pNCs
respectively. C, D) GAG/DNA ratio. N = 4 co-cultures, mean ± SEM. *
indicates p < 0.05, ** p < 0.01.
Discussion & Conclusions
Our results clearly indicate indirect cell-cell interaction by soluble
secreted factors from both cell types proven in serum-free defined
medium. Our findings are in congruence with Boyd et al.’s findings who
also used serum-free conditions but did not check for different cell ratio
(5). bNPCs were stimulated by the presence of pNCs and vice versa.
bNPCs were activated by mainly expressing Col 2 and increased cell
activity and GAG/DNA ratio. This finding is in agreement with previous
results on co-culture between dog non-chondrodystrophoid vs
chondrodystrophoid cells (7) and vs bNPCs, respectively (4). The fact
that the 50% pNC group showed strongest effects (Fig. 1C) for
GAG/DNA ratio and gene expression might have been in reality at the
pNCs : bNPCs ratio of ~30% : 70%. The assumption of a 100%
notochordal porcine NP tissue likely is not true and thus, experimental
ratios are biased towards fewer pNCs. Future trials should split up the
porcine cell population by FACS using size and antibodies against
reliable markers (8, 9). Furthermore, future trials should also consider
hypoxic conditions, which might also increase stimulating effects in
both cell populations (10). The debate whether in the IVD co-exist two
distinct cell lineages or cells from two closely related cell populations
has been reinitiated (11) but our data confirm stimulating interaction
between these two populations in vitro.
Acknowledgements
This work was supported by the ARTORG Center and the Insel
Hospital, Bern, Orthopedic Department.
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Poster No. 718 • ORS 2011 Annual Meeting