Exogenous ATP increases the collagen and proteoglycan synthesis of engineered human chondrocyte constructs.
1,2,3
Bow, J.K., 2,3Harrison, M.M. , 1,3,4Waldman, S.D.
Department of Mechanical and Materials Engineering, Queen’s University, Kingston, Ontario, Canada; 2 Division of Orthopaedic Surgery, Kingston
General Hospital, Kingston, Ontario, Canada; 3 Human Mobility Research Centre, Kingston General Hospital and Queen’s University, Kingston, Ontario,
Canada; 4 Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada
Senior author: [email protected]
1
INTRODUCTION: ATP has been implicated as an autocrine/paracrine
signal in the mechanotransduction pathway of chondrocytes.1,2
Exogenous ATP has also been used to stimulate tissue engineered
bovine cartilage to improve the collagen and proteoglycan content of the
resulting construct.3 In this study, human chondrocytes in a 3D agarose
scaffold were stimulated with ATP in varying concentrations to
determine whether human chondrocytes respond to ATP stimuli.
Further experimentation was performed to determine how ATP exerts its
effects on chondrocytes in terms of basal ATP release, ATP degradation
and expression of connexin 43, as well as the P2Y1 and P2Y2
purinoreceptors on the cultured cells.
METHODS: Chondrocytes - Cartilage was obtained from the
uninvolved portions of joints of osteoarthritis patients (ages 48-69yrs)
undergoing total knee arthroplasty or resurfacing hip arthroplasty at the
authors’ institution with the patients’ informed consent. The protocol
was approved by the ethics review board at our institution.
Chondrocytes were isolated by trypsin and collagenase digestion with
viability confirmed and cells counted. Flow cytometry – 106
cells/sample were incubated with primary antibody or appropriate
isotype controls for 1hr at 4oC, followed by incubation with secondary
antibodies in the dark for 20min at 4oC. The following antibodies were
used: mouse monoclonal anti-connexin 43, rabbit polyclonal anti-P2Y1,
goat polyclonal anti-P2Y2, donkey anti-mouse IgG1 FITC, donkey antirabbit IgG PE, donkey anti-goat IgG PE-Cy5 (all Santa Cruz Biotech).
The cells were then suspended in 1% paraformaldehyde and kept in the
dark at 4oC prior to cell counting. Cell culture – Chondrocytes were
seeded in 2% agarose gels to form 3D constructs. The cells were then
cultured at 37oC and 5% CO2 in DMEM/F12 media supplemented with
20% FBS and 100mcg/mL ascorbic acid, with media changed every
48hrs. Basal ATP Release – The serum concentration supplemented to
the cultures was first reduced from 20% to 1% over a 48 hr period.
After an 18 hour culture period in 1% FBS media, aliquots of media
were taken and the ATP concentration determined using the Adenosine
5’-Triphosphate Bioluminescent Assay Kit (Sigma Aldrich). ATP
degradation – The ATP-lite assay (PerkinElmer) was used to determine
the background level of ATP in the media as well as the ATP
concentration in the media following an initial injection of 1μM ATP
into the culture medium over a 6-hour period. ATP stimulation – After 1
week of culture, ATP was added to the cultures in concentrations
varying from 50nM-1mM in the presence of 4mcCi [3H]-proline and
4mcCi [35S]-sulfate to act as markers of collagen and proteoglycan
synthesis, respectively. The chondrocytes were cultured a further 24 hrs,
then digested in papain (40 μg/mL in 20 mM ammonium acetate, 1 mM
EDTA, and 2 mM DTT) at 65oC for 72hrs. Aliquots of the digest were
assayed for the contents of DNA (Hoechst 33258 dye binding assay and
fluorometry) and the [3H]-proline and [35S]-sulfate content determined
by scintillation counting. Statistics – Results for ATP stimulation were
compared with a single-tailed paired Student’s t-test; R2 and linear
correlation was performed in MS Excel.
RESULTS: Flow Cytometry – None of the samples showed significant
expression of connexin 43 (range 0-5.8%). As expected, expression of
P2Y1 and P2Y2 receptors varied widely between individuals, with a
range of 11-76% expression and of 3-67% expression for P2Y1 and
P2Y2 receptors, respectively. Almost all cells expressing P2Y2
receptors also expressed P2Y1 receptors, and 4/8 patients also had
significant cell populations expressing P2Y1 but not P2Y2 receptors
(range of 4-17% of cells). Basal ATP Release – Of the 8 patients
studied, only 1 patient had measurable ATP within the culture media,
with a concentration of 1.8E-10 M. This was reproducible on repeat
experimentation, and with longer culture times prior to sampling the
media. ATP Degradation – Background ATP levels were undetectable
in all culture media prior to adding ATP to the cultures. ATP
degradation within the culture media was measured, with the measured
ATP half-life and elimination rate constants displayed in Table 1.
Chondrocyte Response to Exogenous ATP - Cartilage was obtained and
cultured from 22 patients. We identified 2 separate groups of patients:
responders to ATP (16/22) and non-responders to ATP (6/22). Patients’
demographics, co-morbidities and medications were reviewed and no
correlating characteristics were identified. The average increase in [3H]proline incorporation was 242% the control (range 115%-388%, p<0.02)
and the average increase in [35S]-sulfate incorporation was 238% (range
124%-711%, p<0.02). Correlation of ATP Degradation to Receptor
Expression – A graph of ATP elimination rate constant values versus
P2Y receptor expression shows good correlation, with an R value of
0.994 for P2Y1 receptor expression (Figure 1). The low expression of
connexin 43 prevented the correlation between expression and ATP
degradation. Correlation of ATP Stimulation to Receptor Expression –
No correlation was seen between the expression of P2YR1, P2YR2 or
connexin 43 and the response to ATP stimulation.
Table 1: Elimination rate constant and ATP half-life for various
chondrocyte cultures. (n=6)
Blank
Blank w/agarose
O
P
Q
I
L
N
Elimination Rate Constant
(min-1)
0.00421
0.00412
0.00948
0.011
0.0108
0.00503
0.00594
0.00679
Half-life of ATP
(min)
165
168
73
63
64
138
117
102
Figure 1: Correlation of ATP Elimination Rate Constants versus
Expression of P2Y1 and P2Y2 Receptors on Chondrocytes.
DISCUSSION: Adding exogenous ATP to the culture media of human
chondrocytes in agarose gel 3D culture reproducibly increased the
collagen and proteoglycan synthesis only within a subset of the
osteoarthritic population. Determining the population who is likely to
respond to this form of stimulation would be useful both for the tissue
engineering of cartilage, as well as for targeting mechanisms to optimize
the healing of diseased cartilage in vivo. Our attempts to determine
which patients would respond to the ATP stimulation, based on basal
ATP release, degradation of exogenous ATP, and the expression of ATP
receptors or connexin 43 did not yield a suitable method. We did,
however, show expression of P2Y1 and P2Y2 receptors on a significant
proportion of chondrocytes from patients with osteoarthritis and a
significant correlation of the expression of these receptors to the ATP
elimination rate constants. It is presently not clear why little expression
of connexin 43 was found on these cells, as other studies have shown
connexin 43 expression by articular chondrocytes by
immunohistochemistry.2 The mechanisms by which ATP acts in the
mechanotransduction pathway of osteoarthritic chondrocytes is still
unknown; however, the addition of exogenous ATP to developing
engineered cartilage constructs appears to be a promising technique to
improve the extracellular matrix production in these constructs.
REFERENCES:
1 Garcia M, Knight MM. Cyclic loading opens hemichannels to release ATP as part of a chondrocyte
mechanotransduction pathway. J Orthop Res. 2010 Apr;28(4):510-5.
2 Knight MM, McGlashan SR, Garcia M, Jensen CG, Poole CA. Articular chondrocytes express connexin 43
hemichannels and P2 receptors - a putative mechanoreceptor complex involving the primary cilium? J Anat. 2009
Feb;214(2):275-83.
3 Waldman SD, Usprech J, Flynn LE, Khan AA. Harnessing the purinergic receptor pathway to develop functional
engineered cartilage constructs. Osteoarthritis Cartilage. 2010 Jun;18(6):864-72.
ACKNOWLEDGEMENTS: This research is supported by the Natural Sciences and
Engineering Research Council of Canada.
Poster No. 1808 • ORS 2011 Annual Meeting