Anabolic and catabolic responses of human articular chondrocytes to varying oxygen percentages

15 Pages·2010·3.21 MB·English

Ströbeletal.ArthritisResearch&Therapy2010,12:R34
http://arthritis-research.com/content/12/2/R34
RESEARCH ARTICLE Open Access
Anabolic and catabolic responses of human
articular chondrocytes to varying oxygen
percentages
Simon Ströbel1, Marko Loparic1,2, David Wendt1, Andreas D Schenk2, Christian Candrian1,3, Raija LP Lindberg4,
Florina Moldovan5, Andrea Barbero1*, Ivan Martin1
Abstract
Introduction: Oxygen is a critical parameter proposed to modulate the functions of chondrocytes ex-vivo as well
as in damaged joints. This article investigates the effect of low (more physiological) oxygen percentage on the
biosynthetic and catabolic activity of human articular chondrocytes (HAC) at different phases of in vitro culture.
Methods: HAC expanded in monolayer were cultured in pellets for two weeks (Phase I) or up to an additional two
weeks (Phase II). In each Phase, cells were exposed to 19% or 5% oxygen. Resulting tissues and culture media were
assessed to determine amounts of produced/released proteoglycans and collagens, metalloproteinases (MMPs),
collagen degradation products and collagen fibril organization using biochemical, (immuno)-histochemical, gene
expression and scanning electron microscopy analyses. In specific experiments, the hypoxia-inducible factor-1a
(HIF-1a) inhibitor cadmium chloride was supplemented in the culture medium to assess the involvement of this
pathway.
Results: Independent from the oxygen percentage during expansion, HAC cultured at 5% O (vs 19% O ) during
2 2
Phase I accumulated higher amounts of glycosaminoglycans and type II collagen and expressed reduced levels of
MMP-1 and MMP-13 mRNA and protein. Switching to 19% oxygen during Phase II resulted in reduced synthesis of
proteoglycan and collagen, increased release of MMPs, accumulation of type II collagen fragments and higher
branching of collagen fibrils. In contrast, reducing O during Phase II resulted in increased proteoglycan and type II
2
collagen synthesis and reduced expression and release of MMP-13 mRNA and protein. Supplementation of
cadmium chloride during differentiation culture at 5% O drastically reduced the up-regulation of type II collagen
2
and the down-regulation of MMP-1 mRNA.
Conclusions: The application of more physiologic oxygen percentage during specific phases of differentiation
culture enhanced the biosynthetic activity and reduced the activity of catabolic enzymes implicated in cartilage
breakdown. Modulation of the oxygen percentage during HAC culture may be used to study pathophysiological
events occurring in osteoarthritis and to enhance properties of in vitro engineered cartilaginous tissues.
Introduction chondrocytes become less resistant to extrinsic stress.
Homeostasis of normal cartilage in adults represents a This in turn causes a disturbance of tissue homeostasis
delicate balance between the synthesis and the degrada- and thus the risk of degenerative pathologies of osteoar-
tion of extracellular matrix components to maintain the thritic nature [2]. In particular the oxidative stress is
functional integrity of the joint. In elderly individuals, proposed to play a key role in cartilage degeneration.
together with changes in proliferation activity, energy Oxygen is a critical parameter proposed to modulate
metabolism and response to growth factors [1], chondrocyte metabolic activity [3]. Indeed, articular car-
tilage is generally exposed to a finely regulated gradient
of relatively low oxygen percentages (from about 10% at
*Correspondence:[email protected]
1DepartmentsofSurgeryandofBiomedicine,UniversityHospitalBasel, the surface to about 1% in the deepest layers) [4], which
Hebelstrasse20,Basel,4031,Switzerland
©2010Ströbeletal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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is essential for maintenance of specialized tissue func- percentage influences the structural organization of the
tion [5]. During the onset of cartilage degeneration, pos- collagen fibrils produced by HAC and whether those
sibly due to surface fibrillation and/or microfractures of features have a pathophysiological counterpart in
the subchondral bone, such gradients have been pro- healthy and osteoarthritic cartilage tissue. Finally, in
posed to break down [6], thus contributing to the pro- order to address whether the metabolic effects of HAC
gression of the disease. culture at low oxygen percentage involve signaling
The influence of various oxygen percentages on chon- through the hypoxia-inducible factor-1a (HIF-1a) path-
drocyte function has been investigated in a broad variety way, some cultures were supplemented with the specific
of models, differing with respect to (i) the cell source inhibitor cadmium chloride.
used (species: bovine, chicken, rodents, human, and ana-
tomical locations of cell harvesting: knee, hip, interpha- Materials and methods
langeal joint, nose), (ii) the characteristic of the donor Cartilage samples collection
(age, stage of cartilage degeneration), (iii) the oxygen Macroscopically normal human articular cartilage sam-
percentage applied (from less then 1% to more than ples (Mankin Score: 2 to 3) were obtained post mortem
60%), (iv) the hydrodynamic culture conditions (static (within 24 hours after death) from the knee joints of a
culture or mixing within bioreactors), and (v) the stage total of six donors with no clinical history of joint disor-
of cell differentiation (cells in native tissue, de-differen- ders (mean age: 56 years, range: 43 to 65 years), after
tiated cells, re-differentiating expanded cells in pellets, informed consent by relatives and in accordance with
alginate gels, or different types of porous scaffolds). It is the local ethics committee (University Hospital Basel,
thus not surprising that the data reported in literature Switzerland). Cells from different donors were used for
on the influence of oxygen percentage on chondrocyte independent experimental runs. Osteoarthritic cartilage
behavior are rather controversial [3]. For instance, as tissues (Mankin Score: 6 to 7) harvested from three
compared to culture under normoxic conditions (18 to patients undergoing total or partial knee replacement
21% oxygen), culture at more physiological, low oxygen (female:male = 2:1, mean age: 67 years, range 65 to 71
percentages (1 to 8%) has been reported to increase years) were used as controls for degenerated structural
[7-10], decrease [11,12] or have no effect on the chon- organization of collagen fibrils.
drocyte proliferation rate [6,13-15]. Moreover, the
expression of cartilage specific genes and/or the extent Chondrocyte isolation and expansion
of matrix protein synthesis/deposition was reported to Cartilage tissues were minced in small pieces and
be up-regulated [6-9,12,15-22], down-regulated digestedwith0.15%typeIIcollagenase(10mlsolution/g
[10,23-26] or not modulated at all [6,9] by culture under tissue)for22hours.Theisolatedhuman articularchon-
more physiological oxygen percentages. drocytes (HAC) were expanded for two passages with
Importantly, in addition to the still controversial find- Dulbecco’sEagle’sMedium(DMEM)containing4.5mg/
ings, in the above mentioned studies the effect of oxy- ml D-glucose, 0.1 mM nonessential amino acids, 1 mM
gen percentage on chondrocytes has mainly been sodiumpyruvate,100mMHEPESbuffer,100U/mlpeni-
investigated with regard to the cell biosynthetic activity, cillin, 100 μg/ml streptomycin and 0.29 mg/ml
without considering and exploring chondrocyte catabolic L-glutamate supplemented with 10% of foetal bovine
processes. We thus aimed our study at investigating the serum(completemedium) and 1ng/ml of Transforming
effect of a low (more physiological) oxygen percentage Growth Factor b1 (TGFb-1), 5 ng/ml of Fibroblast
both on the cartilage tissue forming capacity of human Growth Factor 2, and 10 ng/mL of Platelet-Derived
articular chondrocytes (HAC), and on their pro-cata- GrowthFactor-BB(all fromR&DSystems,Minneapolis,
bolic, matrix degradative activity. In particular, we MN, USA) (expansion medium) [27] in a humidified
hypothesized that culture at a more physiological oxygen incubator (37°C/5% CO ) at either normoxic condition
2
percentage has a dual role in the chondrocyte metabo- (19% O )orlow,morephysiologicaloxygen tension (5%
2
lism, by enhancing their biosynthetic activity and at the O ). Expansion medium was equilibrated under 5% and
2
same time reducing the expression of matrix degradative 19% O for at least six hours before each media change.
2
enzymes. To test these hypotheses, HAC were exposed Expandedcellsweresubsequentlycultivatedinpelletsas
to normoxic conditions (19%) or to a low oxygen per- describedbelow.
centage (5%) during culture in two simple and widely
used model systems (that is, monolayer expansion or 3D pellet cultures
differentiation in micromass pellets), as well as at differ- The chondrogenic capacity of expanded HAC was inves-
ent phases of tissue development (that is., during de- tigated in pellet cultures under the two oxygen condi-
novo tissue formation or in pre-formed tissues). We tions (19% O and 5% O ) used for the expansion.
2 2
further investigated whether the applied oxygen Chondrocytes were re-suspended in complete medium
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supplemented with 10 μg/ml insulin (ACTRAPID HM), Resulting tissues were analyzed histologically, immu-
0.1 mM ascorbic acid 2-phosphate (SIGMA, San Gallen, nohistochemically, biochemically and via scanning elec-
Switzerland), 10 ng/mL Transforming Growth Factor-b3 tronic microscopy to determine the quality of generated
(Novartis, Basel, Switzerland) (chondrogenic medium) tissue, anabolic and catabolic cell functions and collagen
[27]. Chondrogenic medium was equilibrated under 5% fibril organization.
and 19% O for at least six hours before each media
2
change. Pellet characterization
Pellets generated by cells from two donors after two Biochemical analyses
weeks of culture under the two oxygen percentages For the determination of the glycosaminoglycan (GAG)
(19% O or 5% O ) (Phase I) were further cultured for and DNA contents, pellets were digested with protease
2 2
up to two weeks (Phase II) in chondrogenic medium at K (0.5 ml of 1 mg/ml protease K in 50 mM Tris with 1
the same or at interchanged oxygen percentages (that is, mM EDTA, 1 mM iodoacetamide, and 10 μg/ml pepsta-
from 5% to 19% O or from 19% to 5% O ) (Figure 1). tin-A for 15 hours at 56°C) as previously described [29].
2 2
For the HIF-1a inhibition experiments, pellets generated GAG contents of pellets were measured spectrophoto-
by cells from three donors after two weeks of culture at metrically using the dimethylmethylene blue (DMMB)
19% O were subsequently exposed to 5% O and cul- assay [30]. The DNA amount was measured spectro-
2 2
tured for six hours or three days in chondrogenic med- fluorometrically using the CyQUANT® Kit (Molecular
ium supplemented with 5 μM cadmium chloride (CdCl , Probes, Eugene, OR, USA) following the kit’s instruc-
2
SIGMA) [28]. tion. GAG contents were reported as μg GAG/μg DNA.
19%O 5%O
2 2
Expansion
(2 - 3 weeks)
19%O 5%O 19%O 5%O Differentiation
2 2 2 2
Phase I
(2 weeks)
19%O 5%O 5%O 19%O
2 2 2 2 Differentiation
Phase II
(4 days - 2 weeks)
Figure1Experimentaldesign.Humanarticularcartilagewereculturedinmonolayer(Expansion)under5%and19%oxygenpercentages.Cells
werethenculturedfortwoweeksagainunderthetwooxygenpercentages(DifferentiationPhaseI).Pelletsgeneratedat5%and19%oxygen
werefurtherculturedatthesameconditionsoratinterchangedoxygenpercentages(DifferentiationPhaseII).
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Measurement of [35S]SO and [3H]proline incorporation applied to identify the collagen fibrils. The skeletonized
4
The proteoglycan and collagen synthesis of pellets were data were subjected to an algorithm identifying the end
measured by assessing the incorporation of (35S)SO points and intersections of the skeleton. Using this
4
and (3H)proline for a period of 24 h as described pre- information, the individual line segments were identified
viously [31]. Briefly, pellets were incubated in the pre- and analyzed. Finally, the following parameters were
sence of both (35S)SO (1 μCi/culture) to label determined from each pellet condition: (i) the bending
4
proteoglycans and (3H)proline (1.5 μCi/culture) to label ratio, calculated as the mean-squared end-to-end dis-
collagen. For the assessment of the released ECM frac- tance divided by the mean-squared contour length and
tion, radiolabeled proteoglycan and collagen were preci- (ii) the persistence length, calculated using a previously
pitated overnight at 4°C using respectively 100% ethanol described model [38]. Both these parameters were
and 70% ammonium sulphate and subsequently, resus- required to correlate the linearity of the fibrils and
pended in 4 M guanidine hydrochloride or 10% sodium length before branching of each individual fibril to its
dodecyl sulphate in Tris buffer (0.1 M, pH 7.0) respec- mechanical properties, respectively [39].
tively for proteoglycan and collagen. For the assessment Total RNA extraction and cDNA synthesis
of the incorporated ECM fraction, tissue pellets were Total RNA of pellets was extracted using Trizol (Life
digested with protease K as previously described. The Technologies, Basel, Switzerland) and the standard sin-
incorporation of (35S)SO and (3H)proline in culture gle-step acid-phenol guanidinium method. RNA was
4
™
pellet and in conditioned medium was measured in a treated with DNAseI using the DNA-free Kit (Ambion,
Packard b-liquid scintillation counter with scintillation Austin, Texas) and quantified spectrometrically. cDNA
fluid (Ultima Gold, Perkin Elmer, Schwerzenbach, Swit- was generated from 3 μg of RNA by using 500 μg/ml
zerland). The amount of synthesised molecules was nor- random hexamers (Promega AG Dübendorf, Switzer-
malized to the DNA content of the tissue. land) and 1 μl of 50 U/ml Stratascript™ reverse tran-
Histological and immunohistochemical analyses scriptase (Stratagene, Amsterdam, NL), in the presence
Pellets were fixed in 4% formalin, embedded in paraffin of dNTPs. Real-time RT-PCR reactions were performed
and cross-sectioned (5 μm thick sections). The sections and monitored using the ABI Prism 7700 Sequence
were stained with Safranin O for sulfated GAG and pro- Detection System (Perkin-Elmer/Applied Biosystems,
cessed for immunohistochemistry to visualize type II Rotkreuz, Switzerland). Cycle temperatures and times as
collagen (II-II6B3, Hybridoma Bank, University of Iowa, well as primers and probes used for the reference gene
Iowa City, IA, USA), as described in Grogan et al. [32] (18-S rRNA) and the genes of interest (collagen type II
and type II collagen fragments according to Roy-Beau- and aggrecan) were as previously described [40]. Assays
dry et al. [33]. on-Demand (Applied Biosystem) were used to measure
Electronic microscopy (SEM) the expression of MMP-1 (Hs00233958_m1), MMP-2
Imagesobtainedfrombothscanningelectronmicroscopy (Hs00234422_m1), MMP-9 (Hs00234579_m1) and
(SEM) and transmission electron microscopy (TEM) MMP-13 (Hs00233992_m1). For each cDNA sample,
were used for the structural analysis of collagen fibrils. the threshold cycle (Ct) value of each target sequence
PelletsamplesweregluedontoaTeflondiscwithafive- was subtracted to the Ct value of 18-S rRNA, to derive
minute curing epoxy glue (Devcon Epoxy, ITW Brands, ΔCt. The level of gene expression was calculated as
Wood Dale, IL, USA). After which, the mounted speci- 2ΔCt. Each sample was assessed at least in duplicate for
menswere placedina vibratorymicrotome (VT1000E, each gene of interest.
Leica, Heidelberg, Germany) to trim off the outermost, Quantification of released matrix metalloproteinases
approximately150μmthickcartilagelayerparalleltothe Matrix metalloproteinases (MMP) were quantified in
support surface to minimize inhomogenities across the media collected from cultured pellets by using the Mul-
surface among samples. The surface layer of the adult tiAnalyte Profiling MMP base Kit (Fluorokine® MAP:
healthy and OA cartilage was examined without any LMP000) complemented with the specific MMPs
modification. The samples were then prepared for SEM (MMP-1: LMP901; MMP-3: LMP513; MMP-9: LMP911;
andTEManalysisaspreviouslydescribed[34].ForTEM MMP-13: LMP511, R&D Systems, Minneapolis, MN,
™
analysis, the samples were further homogenised into USA). The assay was performed on a Luminex 100
smallpiecesinordertoisolatesinglecollagenfibrils. analyzer (Austin, Texas, USA) following the manufac-
Image analysis turer’s instructions. The amount of released MMPs was
Quantitative data on the collagen fibril organization normalized to the DNA content of the tissue.
were obtained using the Image Processing Library &
Toolbox (IPLT) image analysis software package (Basel, Statistical analysis
Switzerland) [35]. A Canny edge detection algorithm For each analysis, triplicate pellets for each condition
[36], followed by a skeletonization algorithm [37] was and donor were assessed. Statistical evaluation was
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performed using SPSS software version 7.5 software The protein levels of MMP-1, -2, -9, -13 were assessed
(SPSS, Sigma Stat, Erkrath, Germany). Values are pre- in the supernatant of pellet cultures at the end of Phase
sented as mean ± standard deviation (SD). Differences I. Consistent with the mRNA results, the amounts of
between groups were assessed by Mann Whitney tests. MMP-1 and -13 released were reduced in the pellets
Differences in the persistence length and bending ratio cultured at 5% O as compared to those cultured at 19%
2
of collagen fibrils from different conditions were O (8.2- and 11.3-fold respectively). The protein expres-
2
assessed by one-way analysis of variance (ANOVA) with sion levels of MMP-2 and -9 remained similar at the dif-
Bonferroni post hoc test. Values of P < 0.05 were con- ferent oxygen percentages (Figure 3B).
sidered statistically significant.
Effect of oxygen percentage on HAC anabolic and
Results catabolic activity in pre-formed cartilaginous tissues
Chondrogenic differentiation of HAC cultured under We next investigated the influence of oxygen in anabolic
different oxygen percentages (synthesis and accumulation of cartilaginous matrix pro-
HACwereinitiallyculturedinmonolayerwithexpansion teins) and catabolic (MMPs expression, activity and
medium at 5% or 19% O and subsequently re-differen- degradation products) processes of pre-formed tissues.
2
tiated in three-dimensional pellets at the two different Pellets generated after two weeks of culture at 19% O
2
oxygenpercentages(PhaseI)(SeeFigure1fortheexperi- or 5% O (Phase I) were subsequently cultured up to an
2
mental design). HAC proliferated at comparable rates additional two weeks (Phase II) at the same or at inter-
(less than 5% variation in the number of doublings/day; changed oxygen percentages (Figure 1).
data not shown) at the two oxygen conditions. Cells Accumulation and synthesis of cartilaginous matrix proteins
expanded at either oxygen percentage and subsequently In agreement with the above described results, pellets
differentiatedat19%O producedtissuesfaintly stained cultured for four weeks (two weeks of Phase I and two
2
for GAG and type II collagen (Figures 2A, I and 2II and weeks of Phase II) at 5% O were more strongly stained
2
2B,I and 2II).Instead, reducing oxygen percentage dur- for Safranin O and type II collagen, and accumulated
ingdifferentiationenhancedtheamount ofcartilaginous larger amounts of GAG (4.0-fold) as compared to those
matrix accumulation, as evidenced by a qualitative cultured for the same time at 19% O (Figure 4A, B, C).
2
increased size of the generated tissues (Figure 2A, low Reducing oxygen percentage during Phase II for pellets
magnification), an increased intensity of Safranin O and cultured at 19% during Phase I resulted in an improved
typeIIcollagenstain(Figure2A,B)andastatisticallysig- quality of the cartilaginous tissues, as assessed by an
nificant higher amount of GAG (3.4- and 3.1-fold for increased accumulation of cartilaginous matrix positive
HAC expanded at 19% or 5% O respectively) (Figure for GAG and type II collagen (Figure 4A, B) and by a
2
2C). Due to the fact that expansion at 5% O did not higher GAG content (3.3-fold) (Figure 4C). Conversely,
2
influence the extent of HAC differentiation, further increasing oxygen percentage during Phase II for pellets
assessmentswereonlyperformedwithcellsexpandedat cultured at 5% during Phase I resulted in a reduced
19%O .Inagreementwiththehistologicalandbiochem- accumulation of cartilaginous matrix (Figure 4A, B) and
2
ical results, the RT-PCR analysis confirmed statistically GAG content (1.9-fold) (Figure 4C).
significant higher expression of the cartilage specific Results from the radiolabelling experiments indicated
genes typeIIcollagen(86.6-fold)andaggrecan(8.5-fold) that similar amounts of total collagen and proteoglycan
at5%O thanat19%O afterthePhaseIdifferentiation (that is, released + accumulated) were synthesized by
2 2
culture(Figure2D,E). pellets cultured for 18 days (two weeks of Phase I and
four days of Phase II) at the two oxygen percentages.
Expression of catabolic mediators However, as compared to 19% oxygen (Phase I and
We then investigated the possible role of oxygen Phase II), the released fractions of these newly synthe-
percentage in modulating the expression of catabolic sized macromolecules by pellets cultured at 5% O
2
mediators. Analysis of specific matrix metalloproteinases (Phase I and Phase II) were markedly and statistically
(that is, MMP-1, MMP-2, MMP-9 and MMP-13) by significantly lower (2.0- and 2.9-fold respectively for col-
RT-PCR indicated that low oxygen percentage applied lagen and proteoglycan), while the accumulated fractions
during the Phase I differentiation culture selectively were higher (2.1- and 6.6-fold respectively for collagen
down-regulated MMP-1 and MMP-13 mRNA expres- and proteoglycan). Consistent with the biochemical
sion (7.7- and 3.5-fold, respectively). MMP-2 mRNA results, the culture at 5% O during Phase II of tissues
2
was highly expressed and not modulated by the oxygen pre-formed at 19% O during Phase I resulted in an
2
percentage. The expression of MMP-9 mRNA remained augmented synthesis of collagen and proteoglycan
unaffected and was at the limit of detection at both (respectively by 2.7- and 1.4-fold). In particular, the
oxygen percentages (Figure 3A). increased synthesis of the newly synthesized
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A B
Safranin-O Type II collagen
Expansion Expansion
19%O 5%O 19%O 5%O
2 2 2 2
I II I II
2
O
%
9
n 1
o
ati
nti
e
er III IV III IV
Diff
2
O
%
5
C
GAG accumulation
10
*
g) 8 *
g/ 6
(
A 4
N
D 2
G/
A 0
G
2D0if%f 1 D9i%ff 5D%iff D5i%ff 2D0if%f 1 D9i%ff 5D%iff D5i%ff
2E0x%pa enxspioann s1io9n% 5E%xp eaxnpsaionnsi o5n%
D E
Type II collagen mRNA Aggrecan mRNA
*
s 1.0E-03 s 1.0E-03
e * e
c c
nS nS
e81.0E-04 e8 1.0E-04
er1 er1
diffom1.0E-05 diffom 1.0E-05
dfr dfr
Fol 1.0E-06 Fol 1.0E-06
DDiiffff 1290%% DDiiffff 55%% DDififff 1290%% DDififff 55%%
Figure2AnabolicresponseofHACtodifferentoxygenpercentagesduringtheexpansionanddifferentiationPhaseI.(A-B)Safranin
OandtypeIIcollagenimmunohistochemicalstainingsofrepresentativetissuesgeneratedbyhumanarticularchondrocytes(HAC)expandedat
19%(IandIII)or5%(IIandIV)oxygenandfurtherculturedinpelletsat19%(IandII)or5%(IIIandIV)oxygen.Bar=100μm.(C)
Quantificationofglycosaminoglycans(GAG)accumulatednormalizedtotheamountofDNA.(D-E)Realtimereversetranscription-polymerase
chainreactionanalysisoftheexpressionoftypeIIcollagenandaggrecanmRNAbyHACculturedinpelletsat19%and5%O.Levelsare
2
expressedasfoldofdifferencefromribosomal18S.Forthegeneexpressionanalysisonlyexpansionat19%O wasconsidered.Valuesaremean
2
±SDofmeasurementsobtainedfromthreeindependentexperiments.*=significantlydifferentfromthe19%O.
2
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A
MMPs mRNA expression
1.0E-01
S
8
1 1.0E-02 *
m
o
r
f 1.0E-03
s
e
c
n
1.0E-04
e
r *
e
f
f
di 1.0E-05
d
ol
F 1.0E-06
DDiiffff 2190%% DDiiffff 55%% DDiiffff 2190%% DDiiffff 55%% DDiiffff 2190%% DDiiffff 55%% DDiiffff 2109%% DDiiffff 55%%
MMMMPP--11 MMMMPP--22 MMMMPP--99 MMMMPP--1133
B
MMPs protein release
30
)
g 25
/
g
n 20
(

A
N 15
D
/
n 10 * *
ei
t
o
5
r
P
0
DDiiffff 2109%% DDiiffff 55%% DDiiffff 1290%% DDiiffff 55%% DDiiffff 1290%% DDiiffff 55%% DDiiffff 1290%% DDiiffff 55%%
MMMMPP--11 MMMMPP--22 MMMMPP--99 MMMMPP--1133
Figure3QuantificationofMMPsproducedbyHACculturedatdifferentoxygenpercentagesduringthePhaseI.(A)Realtimereverse
transcription-polymerasechainreactionanalysisoftheexpressionofMMP-1,-2,-9,-13mRNAbyhumanarticularchondrocytes(HAC)cultured
inpelletsat19%and5%O.Levelsareexpressedasfoldofdifferencefromribosomal18S.(B)QuantificationofMMP-1,-2,-9,-13releasedin
2
theculturemedium.LevelsarenormalizedtotheamountofDNAmeasuredinrelativepellets.Valuesaremean±SDofmeasurementsobtained
fromthreeindependentexperiments.*=significantlydifferentfromthe19%O.
2
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A B
Safranin-O Type II collagen
Phase I: 19%O Phase I: 19%O
2 2
Phase II: 19%O Phase II: 5%O Phase II: 19%O Phase II: 5%O
2 2 2 2
I II I II
Phase I: 5%O Phase I: 5%O
2 2
Phase II: 5%O Phase II: 19%O Phase II: 5%O Phase II: 19%O
2 2 2 2
III IV III IV
C GAG accumulation
°
10
g) *
8
g/
6 *
(
A 4
N
D 2
G/
A 0
G
Phase II: 19% Phase II: 5% Phase II: 5% Phase II: 19%
Phase I: 19% Phase I: 5%
D
E
Collagen synthesis Proteoglycan synthesis
g)m/ 40000 *a raeclceuamseudlated m/g) 40000 raeclceuamseudlated
A (cp 2300000000 °a, r *a, r A (cp 2300000000 * a °a, r *a, r
N 10000 N 10000
ne/D 0 PPhhaassee IIII:: PPhhaassee IIII:: PPhhaassee IIII:: PPhhaassee IIII:: G/D 0 PPhhaassee III:I: PPhhaassee III:I: PPhhaassee III:I: PPhhaassee III:I:
oli 1290%% 55%% 55%% 1290%% -P 2109%% 55%% 55%% 1209%%
r S
H-p PPhhaassee II:: 2109%% PPhhaassee II:: 55%% 35 PPhhaassee I:I :2 109%% PPhhaassee II: :5 5%%
3
Figure4AnabolicresponseofHACtodifferentoxygenpercentagesduringdifferentiationPhaseIandII.(A-B)SafraninOandtypeII
collagenstainingsofrepresentativetissuesgeneratedbyhumanarticularchondrocytes(HAC)culturedinpelletsfortwoweeks(PhaseI)at19%
(IandII)or5%(IIIandIV)oxygenandfurtherculturedfortwoadditionallyweeks(PhaseII)at19%(IandIII)or5%(IIandIV)oxygen.Bar=
100μm.(C)Quantificationofglycosaminoglycans(GAG)accumulatedinpelletsculturedasdescribedin(A-B)normalizedtotheamountof
DNA.(D-E)Amountsofnewlysynthesizedcollagen(D)andproteoglycan(E)measuredinpelletsculturedfor18days(twoweeksofPhaseI
andfourdaysofPhaseII).Theupperandlowerpartsofthecolumnsrepresentthereleasedandaccumulatedfractionsrespectively.Valuesare
mean±SDofmeasurementsobtainedfromtwoindependentexperiments.*=significantlydifferentfromthegroupculturedwiththesame
oxygenpercentageinPhaseIbutwithdifferentoxygentensioninPhaseII;°=significantlydifferentfromthegroupculturedentirelyat19%O;
2
a=accumulated,r=released.
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macromolecules was mainly reflected by an augmented and bending ratio). Considerable decrease in persistence
accumulation (up to 5.9-fold). Instead, the culture at length and bending ratio would indicate softening and
19% O during Phase II of tissues pre-formed at 5% O gradual deterioration of cartilage physiological function
2 2
during Phase I differently modulated the synthesis of [39].
the two extracellular matrix molecules: while a Response to low oxygen under CdCl -treatment
2
decreased accumulation (2.3-fold) and an increased To determine whether the observed pro-anabolic and
released (2.6-fold) was measured for collagen, only a anti-catabolic effects of low oxygen percentage are
reduction of the accumulated fraction was demonstrated mediated by HIF-1a, HAC from three donors were pre-
for proteoglycan (8.6-fold) (Figure 4D, E). cultured in pellets during Phase I at 19% O . During the
2
MMPs production and activity subsequent culture Phase II, the pre-cultured pellets
Pellets cultured for four weeks (two weeks of Phase I were maintained at 19% O or exposed to 5% O , with
2 2
and two weeks of Phase II) at 5% O released lower or without treatment with CdCl for six hours or three
2 2
amounts of MMP-1 and -13 (6.1- and 10.1-fold respec- days (Figure 7A). Following culture at low oxygen per-
tively) as compared to those cultured for the same time centage, type II collagen mRNA was up-regulated to a
at 19% O . Culture at 5% O during Phase II of tissues higher extent after six hours (up to 33.0-fold; Figure 7B)
2 2
pre-formed at 19% O during Phase I resulted in than after three days (data not shown), while MMP-1
2
reduced production of both MMPs, though only MMP- mRNA was down-regulated to a higher extent after
13 by statistically significant levels (by 1.8-fold). Instead, three days (up to 65.5-fold; Figure 7C) than after six
culture at 19% O during Phase II of pellets pre-formed hours (data not shown). Supplementation of CdCl dur-
2 2
at 5% O during Phase I resulted in increased release of ing this culture phase almost abrogated the aforemen-
2
both MMP-1 and MMP-13 (4.0- and 6.2-fold respec- tioned low O -mediated effects, so that the expression
2
tively) (Figure 5A, B). of type II collagen and MMP-1 mRNA reached levels
Inordertoassesswhethertheobservedincreasedpro- comparable to those of cells cultured at 19% O for the
2
duction of MMPs corresponded to an increased protei- corresponding times (Figure 7B, C).
nase activity, pellets culturedfora total offour weeksat
thedifferentoxygenpercentageswereassessedimmuno- Discussion
histochemicallytodetectthepresenceoftypeIIcollagen In this study we found that culture at low, more physio-
C-telopeptides,derivedbyMMP-1and-13collagenolytic logical (5%) oxygen percentage has a dual role in HAC
activity [33]. Analyses indicated that only the pellets metabolism, namely to enhance the proteoglycan and
formed at 5% O during Phase I and subsequently cul- collagen synthesis and at the same time to reduce the
2
tured at 19% O during Phase II were intensely stained activity of two key catabolic enzymes involved in carti-
2
forthetypeIIcollagenfragments(Figure5C). lage breakdown (that is, MMP-1 and MMP-13). As a
Collagen fibril organization consequence, HAC exposure to 19% oxygen reduced the
To determine whether increasing oxygen percentage de novo formation of cartilage tissue and induced degra-
during cultivation Phase II of tissues pre-formed at 5% dation of pre-deposited collagen fibrils, leading to struc-
O would change the structure and arrangement of the tural features similar to those found in osteoarthritic
2
collagen fibril network, pellets were qualitatively and tissue. Interestingly, HAC appeared to be highly sensi-
quantitatively assessed via EM. Images indicated that the tive to the oxygen percentage applied during differentia-
collagen fibrils of pellets cultured at 5% O during tion culture in pellets, but not during expansion in
2
Phase I and then for two weeks at 19% O during Phase monolayers. The anti-anabolic and pro-catabolic effects
2
II were less linear than those of pellets cultured for four mediated by low oxygen percentage were HIF1a-depen-
weeks at 5% O . Interestingly, a similar trend was also dent, as assessed by specific inhibition of this factor by
2
observed in the OA cartilage as compared to healthy CdCl treatment.
2
cartilage samples (Figure 6A, B). In pellets, the collagen The application of 5% oxygen percentage during the
network was comprised of single fibrils with diameters HAC monolayer expansion did not influence the prolif-
ranging from 20 to 30 nm. In healthy adult cartilage, eration rate and chondrogenic capacity of HAC. This is
the network contained bundled and twisted collagen in contrast with results reported by Egli et al. [7], indi-
fibrils three- to four-fold larger in diameter. Quantitative cating that bovine articular chondrocytes expanded
image analysis indicated that increasing the oxygen per- under hypoxic conditions generated tissues with higher
centage during Phase II resulted in a significant reduc- amounts of cartilaginous matrix as compared to those
tion of persistence length as well as bending ratio expanded under normoxic conditions. The discrepancy
(47.9% and 10.5% respectively). Interestingly, both para- between our results and those generated by Egli et al.
meters were higher in healthy as compared to OA tis- [7] can be related to the different type of cells used
sues (30.0% and 6.6% respectively for persistence length (human vs bovine), the stage of cell de-differentiation
Ströbeletal.ArthritisResearch&Therapy2010,12:R34 Page10of15
http://arthritis-research.com/content/12/2/R34
A B
MMP-1 protein release MMP-13 protein release
) )
g g
30 20
g/ 25 g/
n * n 15
( 20 ( * *
A 15 A 10
N N
10
D ° D 5
/ 5 / °
n n
ei 0 ei 0
t t
o Phase II: Phase II: Phase II: Phase II: o Phase II: Phase II: Phase II: Phase II:
r r
P P
2109%% 5% 5% 2109%% 1290%% 5% 5% 1290%%
PPhhaassee II:: 2109%% PPhhaassee II:: 55%% PPhhaassee II:: 2109%% PPhhaassee II:: 55%%
C
Type II collagen fragments
Phase I: 19%O
2
Phase II: 19%O Phase II: 5%O
2 2
I II
Phase I: 5%O
2
Phase II: 5%O Phase II: 19%O
2 2
III IV
Figure5CatabolicresponseofHACtodifferentoxygenpercentagesduringdifferentiationPhaseIandII.(A-B)QuantificationofMMP-
1(A)andMMP-13(B)releasedinthemediumbyhumanarticularchondrocytes(HAC)culturedinpelletsforfourweeks(twoweeksofPhaseI
andtwoweeksofPhaseII).LevelsarenormalizedtotheamountofDNAmeasuredinrelativepellets.Valuesaremean±SDofmeasurements
obtainedfromtwoindependentexperiments.*=significantlydifferentfromthegroupculturedwiththesameoxygenpercentageinPhaseI
butwithdifferentoxygentensioninPhaseII;°=significantlydifferentfromthegroupculturedentirelyat19%O (PhaseIandPhaseII).(C)
2
ImmunohistochemicaldetectionoftypeIIcollagenfragmentsofpelletsculturedunderconditionsdescribedin(A-B).Bar=100μm.