Chondrocyte Primary Cilia are Mechanosensitive in a Duration-dependent Manner
1Poole, CA, 2Chowdhury, TT, 3Joshi, P, 3Jensen, CG, 3Kennedy SA, 2Knight MM, +3 McGlashan, SR.
University of Otago, Dunedin, New Zealand, 2Queen Mary University of London, London, UK, 3University of Auckland, Auckland, New Zealand.
Senior author: [email protected]
1
INTRODUCTION
During joint loading, several forms of mechanical stimuli are active,
including cell deformation and fluid flow. These stimuli activate
intracellular signalling cascades that regulate cell proliferation and ECM
production. Although it is clear that chondrocytes are mechanosensory,
the underlying mechanisms are only partly understood.
One potential mechanosensory route may occur through the primary
cilium. Primary cilia are single cytoplasmic organelles with a 9+0
microtubular architecture found in virtually all vertebrate cells. They are
known to function as mechanosensors in several cell types other than
chondrocytes via the activation of intracellular Ca2+ following their
stimulation. Our previous studies suggest that similar mechanosensory
processes could occur in articular cartilage via the chondrocyte primary
cilium, since chondrocyte cilia project from the cell surface into the
ECM and interact with collagen and glycoproteins via integrins.
However, while there is compelling structural evidence supporting a
mechanosensory role for the chondrocyte primary cilium, there are
currently no functional data to support this hypothesis. Therefore, in this
study, we used a well-established cell straining apparatus to test the
hypothesis that chondrocyte primary cilia are mechanically-regulated.
METHODS
3D culture and mechanical loading: Bovine articular chondrocytes were
isolated by sequential enzyme digestion and seeded in 4% agarose gel
(Type IX, Sigma) at 10 x 106 cell.ml-1 and cultured for up to 6 days
(144h). After 24h in culture, constructs were subjected to cyclic
compressive strain ranging from 0% and 15% in a sinusoidal waveform
at 1Hz for 0.5, 6, 24 and 48h. Control constructs remained as
uncompressed, free-swelling cultures. In a separate experiment,
following 24h of culture, constructs were subjected to 48h of cyclic
compression, followed by 72h in free-swelling culture conditions (total
time in culture = 144h). These constructs were termed
‘compression+recovery’ constructs.
Immunohistochemistry and image analysis: All constructs were fixed
in 4% paraformaldehyde followed by standard histological processing
into paraffin wax. 12µm-thick sections underwent microwave antigen
retrieval followed by incubation with antibodies raised against either
acetylated α-tubulin (6-11-B; 1:500: Sigma) and/or proliferating cell
nuclear antigen (PCNA; 1:500: Dako). Negative controls comprised
sections not incubated with primary antibody.
The percentage of cells expressing a primary cilium was calculated
for each condition from 30 fields of view (n ≥ 3 constructs), using
fluorescence microscopy. Cilia length was measured from confocal
images using Image J software (NIH Image). The mean % of PCNApositive nuclei was also calculated. Statistical analysis was performed
using ANOVA and post-hoc t-tests with a 5% level of significance.
swelling conditions, cilia incidence increased 3-fold compared to cells
compressed for 48h alone, although incidence did not return to levels
observed for cells in free-swelling conditions at 144 h.
Table 1. Mean (± SEM) cilia incidence (%) and cilia length (µm) in
free-swelling and compressed chondrocyte/agarose constructs.
*represents the 'compression + recovery' group.
Time in
Cilia Incidence (%)
Cilia Length (µm)
Culture
Free-swelling Compressed Free-swelling Compressed
(h)
24
23.7 (± 2.8)
-
1.3 (± 0.1)
-
24.5
17.0 (± 4.5)
20.3 (± 0.5)
1.4 (± 0.1)
1.6 (± 0.1)
30
27.3 (± 3.6)
24.2 (± 2.9)
1.8 (± 0.2)
1.9 (± 0.2)
48
36.5 (± 4.7)
26.2 (± 1.6)
2.1 (± 0.2)
1.95 (± 0.2)
72
34.6 (± 8.5)
7.0 (± 2.4)
2.2 (± 0.2)
1.54 (± 0.2)
*144
47.1 (± 4.6)
22.7 (± 2.3)
2.1 (± 0.2)
2.61 (± 0.3)
Cilia length: There was a small increase in cilia length following 0.5h
and 6h of strain; however this difference was not significant. Following
48h of strain, there was a 30% reduction compared to free-swelling
controls (p < 0.01), which returned to significantly above free-swelling
control length in the ‘compression +recovery’ group (p < 0.05).
Cell proliferation: Cells in free-swelling conditions showed a
significant increase in the % of PCNA+ cells with time in culture (Fig.
2). In compressed cells, proliferation was maximal after 24h of
compression with a decrease to below free-swelling controls after 48h of
compression (p < 0.01; Fig. 2). A similar reduction was also present in
the ‘compression+recovery’ condition (Fig. 2; p < 0.05).
70
Free swelling
Compressed
60
50
40
30
20
10
0
24
48
72
144*
Time in culture (hours)
RESULTS
Cilia in 3D culture: In free-swelling 3D cultures, primary cilia were
present on chondrocytes (Fig.1 A-C; arrows). Cilia incidence increased
with time in culture, with a 2-fold increase between 24 and 144 h (Table
1). Similarly, chondrocyte cilia length also increased with culture time
up to 48 h, where it reached steady state (Fig. 1; Table 1).
Figure 1. Chondrocyte primary cilia (arrows) in free-swelling 3D
agarose constructs at 24, 72 and 144 hours of culture.
Cilia incidence: Following 0.5 and 6 h of compression, cilia
incidence remained similar to uncompressed controls. In contrast,
following 48 h of compression, there was a 5-fold reduction in cilia
incidence (p < 0.01; Table 1). However, following recovery in free-
Figure 2. Mean (±SD) percentage of PCNA+ chondrocytes in freeswelling and compressed constructs. Compression was applied between
24-72 h of culture. *represents the 'compression + recovery' group.
DISCUSSION.
In this study, we have shown, for the first time, culturing chondrocytes
in agarose is a suitable model to study chondrocyte primary cilia
function, with similar incidence and length values similar to bovine
articular cartilage. Furthermore, we have shown that chondrocyte
primary cilia are mechanosensitive in a duration-dependent manner. We
believe that these data support the hypothesis that cilia are utilised by
chondrocytes as ‘fine tuners’ of sensory information within the local
cellular microenvironment. When mechanical forces over prolonged
periods are present, the cilium is no longer required by the cell and is
resorbed. We have also shown that the reduction in cilia incidence is
independent of the cilia resorption process that occurs during cell
division. Further studies will reveal which part of the cilia length
mechanism is mechanosensitive and will investigate if the process of
intraflagellar transport (the process whereby cilia are assembled and
disassembled) is also influenced by mechanical loading.
Paper No. 36 • 55th Annual Meeting of the Orthopaedic Research Society