J Appl Physiol 114: 1610 –1618, 2013.
First published February 28, 2013; doi:10.1152/japplphysiol.01132.2012.
Hyaluronan initiates chondrogenesis mainly via CD44 in human
adipose-derived stem cells
Shun-Cheng Wu,1,2,3 Chung-Hwan Chen,3,4,5,6,10,11 Je-Ken Chang,3,4,5,7 Yin-Chih Fu,3,4,5,6
Chih-Kuang Wang,1,8 Rajalakshmanan Eswaramoorthy,3 Yi-Shan Lin,2,3 Yao-Hsien Wang,3
Sung-Yen Lin,3,4,5,7 Gwo-Jaw Wang,3,9,10,11 and Mei-Ling Ho1,2,3
1
Submitted 18 September 2012; accepted in final form 20 February 2013
Wu S, Chen C, Chang J, Fu Y, Wang C, Eswaramoorthy R, Lin
Y, Wang Y, Lin S, Wang G, Ho M. Hyaluronan initiates chondrogenesis mainly via CD44 in human adipose-derived stem cells. J Appl
Physiol 114: 1610 –1618, 2013. First published February 28, 2013;
doi:10.1152/japplphysiol.01132.2012.—Cell-matrix adhesion is one
of the important interactions that regulates stem cell survival, selfrenewal, and differentiation. Our previous report (Wu SC, Chang JK,
Wang CK, Wang GJ, Ho ML. Biomaterials 31: 631– 640, 2010) indicated that a microenvironment enriched with hyaluronan (HA) initiated
and enhanced chondrogenesis in human adipose-derived stem cells
(hADSCs). We further hypothesize that HA-induced chondrogenesis
in hADSCs is mainly due to the interaction of HA and CD44
(HA-CD44), a cell surface receptor of HA. The HA-CD44 interaction
was tested by examining the mRNA expression of hyaluronidase-1
(Hyal-1) and chondrogenic marker genes (SOX-9, collagen type II,
and aggrecan) in hADSCs cultured on HA-coated wells. Cartilaginous
matrix formation, sulfated glycosaminoglycan, and collagen productions by hADSCs affected by HA-CD44 interaction were tested in a
three-dimensional fibrin hydrogel. About 99.9% of hADSCs possess
CD44. The mRNA expressions of Hyal-1 and chondrogenic marker
genes were upregulated by HA in hADSCs on HA-coated wells.
Blocking HA-CD44 interaction by anti-CD44 antibody completely
inhibited Hyal-1 expression and reduced chondrogenic marker gene
expression, which indicates that HA-induced chondrogenesis in hADSCs
mainly acts through HA-CD44 interaction. A 2-h preincubation and
coculture of cells with HA in hydrogel (HA/fibrin hydrogel) not only
assisted in hADSC survival, but also enhanced expression of Hyal-1
and chondrogenic marker genes. Higher levels of sulfated glycosaminoglycan and total collagen were also found in HA/fibrin hydrogel
group. Immunocytochemistry showed more collagen type II, but less
collagen type X, in HA/fibrin than in fibrin hydrogels. Our results
indicate that signaling triggered by HA-CD44 interaction significantly
contributes to HA-induced chondrogenesis and may be applied to
adipose-derived stem cell-based cartilage regeneration.
DAMAGED ARTICULAR CARTILAGE has a limited capacity for selfrepair (17). Cell-based tissue engineering provides a novel
strategy to treat cartilage defects (37). Due to the poor proliferation capability and de-differentiation of chondrocytes
caused by in vitro expansion, mesenchymal stem cells (MSCs)
have attracted interest for possible clinical use. The MSCs
possess self-renewal and multilineage differentiation properties, which can be induced to chondrocytic, osteoblastic, and
adipocytic lineages (6, 16, 25, 47).
Hyaluronan (HA) is one of the main components of extracellular matrix in articular cartilage (29). The surface antigen
CD44 is the main receptor of HA, and the interaction between
HA and CD44 (HA-CD44 interaction) on chondrocytes is
crucial for the maintenance of cartilage homeostasis (1, 3, 19,
20). Our laboratory’s previous study (43) showed that an
HA-enriched microenvironment initiates and enhances chondrogenesis of human adipose-derived stem cells (hADSCs).
Therefore, we further hypothesize that the HA-induced chondrogenesis of hADSCs is mainly due to the HA-CD44 interaction in hADSCs.
A traditional two-dimensional (2D) monolayer culture has
been employed to investigate cellular differentiation and extracellular matrix deposition in vitro (37). In tissue engineering, using a three-dimensional (3D) culture system may provide an appropriate niche, scaffolding, and environmental bioactive signals for the cells. Fibrin has been extensively used as
a scaffold clinically (8). Accordingly, we first examined the
effect of the HA on initiation of chondrogenesis in hADSCs by
culturing the cells in 2D HA-coated wells and further tested the
influence on cartilaginous matrix formation in a 3D fibrin
hydrogel.
human adipose-derived stem cells; hyaluronan-CD44 interaction; articular cartilage repair; tissue engineering
MATERIALS AND METHODS
Address for reprint requests and other correspondence: M.-L. Ho, Dept. of
Physiology, School of Medicine, Kaohsiung Medical Univ., No. 100, ShihChuan 1st Rd., Kaohsiung 807, Taiwan (e-mail: [email protected]).
1610
Isolation and culture of hADSCs. After obtaining informed consent
from all patients and approval from the hospital ethics committee
(KMU-IRB-970267), leftover subcutaneous adipose tissue was acquired from patients undergoing orthopedic surgery. The hADSCs
were isolated from human subcutaneous adipose tissue following a
previously described method (7, 23, 40, 41). The cells used in this
study were isolated from three different donors, including two women
8750-7587/13 Copyright © 2013 the American Physiological Society
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Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Department of
Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 3Orthopaedic Research Center, College
of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Department of Orthopedics, Kaohsiung Medical University
Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 5Department of Orthopedics, College of Medicine, Kaohsiung
Medical University, Kaohsiung, Taiwan; 6Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical
University, Kaohsiung, Taiwan; 7Department of Orthopedics, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical
University, Kaohsiung, Taiwan; 8Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung
Medical University, Kaohsiung, Taiwan; 9Department of Orthopedic Surgery, University of Virginia, Charlottesville, Virginia;
10
Medical Device Innovation Center, National Cheng-Kung University, Tainan, Taiwan; 11Skeleton-Joint Research Center,
National Cheng-Kung University, Tainan, Taiwan
CD44 in Hyaluronan Induce Chondrogenesis
Wu S-C et al.
1611
hADSCs and HA encapsulation in 3D fibrin hydrogel carrier. The
hADSCs were preincubated in 1% HA solution (5 ⫻ 106 cells/30 ␮l)
or in PBS (control) for 2 h at 37°C under 5% CO2. After preincubation, every 30 ␮l of hADSCs (5 ⫻ 106 cells) suspended in HA or PBS
were mixed with 120 ␮l of fibrin solution (100 mg/ml) and then
placed in a Teflon mold 5.5 mm in depth and 5.5 mm in diameter.
Then 40 ␮l of bovine thrombin (300 U/ml) in 40 mM CaCl2 were
added to the mold and were mixed well with the cell/fibrin solution.
This mixture was incubated at room temperature for 15 min to form
a hydrogel. After gelation, the 3D fibrin hydrogel carriers containing
hADSCs with HA (HA/fibrin hydrogel) or hADSCs with PBS (fibrin
hydrogel) were removed from the Teflon molds, transferred to a
24-well plate, and cultured with 1 ml of chondrogenic medium
[containing 10 ng/ml transforming growth factor-␤1 (R&D Systems,
Minneapolis, MN), 50 ␮M L-ascorbate-2-phosphate, and 6.25 ␮g/ml
insulin] (23, 43). The medium was changed every 2 days. At every
indicated time interval, the constructs were collected for further
experimental analysis.
Cell survival in 3D HA/fibrin hydrogel carrier. Live/dead images
of HA/fibrin and fibrin hydrogel constructs were taken 4 h after cells
were encapsulated. The medium was discarded, and the constructs
were washed twice with PBS. Cell survival was assessed based on the
integrity of the cellular membrane using a LIVE/DEAD Viability/
Cytotoxicity Kit (Molecular Probes, Eugene, OR), which contains
calcein-AM (live dye, green) and ethidium homodimer-1 (dead dye,
red). A dye solution was made with 0.5 ␮l of calcein-AM and 2 ␮l of
ethidium homodimer-1 in 1 ml of the standard medium. A slice of the
construct was incubated in 1 ml of the LIVE/DEAD dye solution in a
3.5-mm dish for 30 min. Fluorescence microscopy was performed
using a fluorescein optical filter to detect calcein-AM and a rhodamine
optical filter to detect ethidium homodimer-1.
RNA isolation and real-time PCR. At the indicated time intervals,
hADSCs were collected from wells or 3D fibrin hydrogel carriers.
TRIzol (Gibco BRL, Rockville, MD) was used to extract the total
RNA from these cells by following the manufacturer’s instructions.
Briefly, 0.5–1 ␮g of total RNA per 20 ␮l of reaction volume were
reverse transcribed into cDNA using the SuperScript First-Strand
Synthesis System (Invitrogen). Real-time PCR reactions were performed and monitored using the iQTM SYBR green supermix (BioRad Laboratories, Hercules, CA) and a quantitative real-time PCR
detection system (Bio-Rad Laboratories). The cDNA samples (2 ␮l,
for a total volume of 25 ␮l per reaction) were analyzed for the gene
of interest, and the reference gene glyceraldehyde-3-phosphate dehydrogenase. The expression level of each target gene was then calculated as previously described (24). Four readings of each experimental
sample were performed for each gene of interest, and each experiment
was repeated at least three times. The primer sequences used are
shown in Table 1.
Sulfated glycosaminoglycan synthesis. At indicated time intervals,
HA/fibrin and fibrin hydrogels were collected and digested for 18 h at
60°C using 1 ml of 300 ␮g/ml papain solution. DNA content and
sulfated glycosaminoglycan (sGAG) accumulations were quantified
with a spectrofluorometer using 33258 Hoechst dye and dimethylmethylene blue, respectively (13, 42). Standard curves for the dimethylmethylene blue assay were generated using an aqueous solution of
chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO) with concentrations ranging from 0 to 25 ␮g/␮l.
Total collagen synthesis. To measure collagen synthesis, Sirius Red
dye (Direct Red, Sigma) was used to stain total collagen (38). At the
indicated time intervals, the HA/fibrin and fibrin hydrogels were
collected and lysed using the freeze-thaw method (28). The cell
extracts (50 ␮l/well) were placed in 96-well plates and kept in a dry
incubator at 37°C for desiccation. Each well was washed with 200 ␮l
of distilled H2O three times for 1 min/wash. Then 100 ␮l of 0.1%
Sirius Red stain (0.05 g Sirius Red powder per 50 ml picric acid) were
added to each well, and the wells were kept at room temperature for
1 h. The unattached stain was removed, and the plate was washed five
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and one man. The ages of two female donors are 34 and 65 yr old. The
age of the male donor is 18 yr old. The isolated hADSCs were
cultured and grown at 37°C under 5% CO2 in a K-NAC medium
containing keratinocyte-SFM (Gibco BRL, Rockville, MD) supplemented with EGF-BPE (Gibco BRL), N-acetyl-L-cysteine, L-ascorbic
acid 2-phosphate sequimagnesium salt (Sigma, St. Louis, MO), and
5% FBS (23). Each experiment was performed using adipose-derived
stem cells (ADSCs) from individual donor and repeated at least three
times. Data merged from all the examinations were analyzed for
statistical significance.
HA receptor (CD44) detection by flow cytometry. Flow cytometry
analysis was used to examine the presence of CD44 on the cell surface
of hADSCs. Following the manufacturer’s instructions, the hADSCs
were harvested from the culture dishes by treating them with 0.25%
trypsin/EDTA in phosphate-buffered saline (PBS). One million hADSCs
were suspended in 500 ␮l of PBS containing 20 ␮g/ml of an antibody.
Fluorescein isothiocyanate (FITC)-conjugated antibodies (Becton
Dickinson, Franklin Lakes, NJ) targeted against CD44 were obtained
from Becton Dickinson. As an isotype control, FITC-conjugated
nonspecific mouse IgG (Becton Dickinson) was used. After incubation for 20 min at 4°C, the cells were washed with PBS three times
and then suspended in 1 ml of PBS for analysis. Cell fluorescence was
detected using a flow cytometer (FACS Vantage SE, Becton Dickinson) with a 525-nm filter for green FITC fluorescence, and the data
were analyzed using WINMDI software.
Cell culture on HA-coated wells. To prepare HA-coated wells,
purified HA (grade FCH-200, molecular mass ⫽ 2–2.1 MDa) (Kibun
Food Chemicals, Tokyo, Japan) dissolved in PBS was coated on
24-well plates (0.5 mg/cm2) for 48 h at 37°C, followed by two washes
with PBS (43). The hADSCs were seeded at a density of 1 ⫻ 105 cells
per 500 ␮l of a basal medium (no chondro-induction growth factor
added) made from Dulbecco’s modified Eagle’s medium containing
10% FBS, 1% nonessential amino acids, and 100 U/ml of penicillin
and streptomycin (Gibco-BRL, Grand Island, NY). The culture medium in the plates was changed every 2 days. At every indicated time
interval, cells were collected for further experimental analysis.
Receptor binding inhibition assay. To investigate the correlation
between HA-CD44 interaction and the initiation of chondrogenesis in
hADSCs in an HA-enriched microenvironment, the HA receptor
binding inhibition assay was performed by treating hADSCs with
CD44-blocking antibody. Anti-CD44 antibody (Clone 5F12, Thermo
Scientific) was used to block the binding of HA to the CD44 receptor
(22). The hADSCs were pretreated with 20 ␮g/ml of anti-CD44
antibody for 2 h at 37°C under 5% CO2 (39). After 2 h of incubation,
the pretreated cells were then resuspended in 1 ml of the basal
medium (no chondro-induction growth factor added) plus 20 ␮g/ml of
anti-CD44 antibody and were seeded in HA-coated wells. The medium was changed every 2 days. At each indicated time interval, cells
were collected for further experimental analysis.
Isolation of fibrin from rabbits. Isolation of fibrin from a rabbit was
performed following a previously described method (33). The animal
study was approved by the Animal Experiment Committee of Kaohsiung Medical University, Taiwan. Male New Zealand White rabbits
weighing 2.5–3 kg were used. The rabbits were first anesthetized with
xylazine (5 mg/kg) and ketamine (45 mg/kg) (34). The anesthesia was
supplemented with a subcutaneous injection of 0.5% lidocaine hydrochloride. After anesthesia, fresh rabbit blood was collected from the
carotid vessel, and the collected blood was immediately mixed with
10% (wt/vol) sodium citrate at a ratio of 9:1. The citrated blood was
stored on ice, and then plasma was separated by centrifugation at 4°C
at 600 g for 20 min. A cryo-precipitation method was used for fibrin
preparation (33). Briefly, the plasma was frozen for 2 h at ⫺20°C and
then thawed at 4°C, and the freeze/thaw cycle was repeated three
times. Precipitated fibrin was separated from plasma by centrifugation
at 1,600 g for 5 min. The separated fibrin was dissolved in PBS (100
mg/ml) and stored at ⫺80°C until needed.
•
1612
CD44 in Hyaluronan Induce Chondrogenesis
•
Wu S-C et al.
Table 1. Primer sequences and cycling conditions for real-time PCR
Annealing
Temperature, °C
PCR Primers Sequence (Forward and Reverse)
Type II collagen
Aggrecan
Hyaluronidase-1 (Hyal-1)
GAPDH
Cycling conditions
Forward: 5=-CCT CCG CGA CGT GGA CAT-3=
Reverse: 5=-GTT GGG CGG CAG GTA CTG-3=
Forward: 5=-CAA CAC TGC CAA CGT CCA GAT-3=
Reverse: 5=-TCT TGC AGT GGT AGG TGA TGT TCT-3=
Forward: 5=-ACA GCT GGG GAC ATT AGT GG⫺3=
Reverse: 5=-GTG GAA TGC AGA GGT GGT TT-3=
Forward: 5=-AAA ATA CAA GAA CCA AGG AAT CAT GTC⫺3=
Reverse: 5=-CGG AGC ACA GGG CTT GACT-3=
Forward: 5=-TCT CCT CTG ACT TCA ACA GCG AC-3=
Reverse: 5=-CCC TGT TGC TGT AGC CAA ATT C-3=
Denature: 95°C for 30 s, 95°C for 4 min, followed by 35 cycles of 95°C for 10 s, 58.4–61.5°C
(shown in column of Annealing Temperature) for 15 s and 72°C for 15 s
61
61
55
61
Initiation of chondrogenesis of hADSCs in HA-coated well.
To test the initiation of chondrogenesis by hADSCs in an
HA-enriched microenvironment, the mRNA expression of
chondrogenic marker genes was tested in HA-coated cultures
(0.5 mg/cm2). The mRNA expression of chondrogenic marker
genes (SOX-9, collagen type II, and aggrecan) was significantly upregulated in cultures on HA-coated wells (Fig. 2).
Compared with the control group, upregulation of chondrogenic marker gene expression in HA-coated groups was increased from day 1 to day 5 [SOX-9: day 1 (P ⫽ 0.018), day
3 (P ⫽ 0.041), and day 5 (P ⬍ 0.0001); collagen type II: day
1 (P ⫽ 0.034), day 3 (P ⫽ 0.021), and day 5 (P ⫽ 0.005); and
aggrecan: day 1 (P ⫽ 0.003), day 3 (P ⫽ 0.001), and day 5
(P ⫽ 0.002)] (Fig. 2, B, C, and D). These results indicate that
chondrogenesis in hADSCs was initiated in an HA-enriched
microenvironment.
Blockade of HA-CD44 signaling inhibits Hyal-1 expression
and chondrogenic gene expression. The Hyal-1 mRNA expression for hADSCs in HA-coated cultures was significantly
CD44 positive
Isotype control
CD44+
M1
0
Flow cytometry analysis of CD44 in hADSCs. The results
from flow cytometry analysis showed that 99.9% of the
hADSC population possess CD44 (Fig. 1).
HA-CD44 interaction of hADSCs in HA-coated cultures.
The interaction between HA-CD44 for hADSCs in an HAenriched microenvironment was tested by examining the
mRNA expression of hyaluronidase-1 (Hyal-1) in HA-coated
wells (0.5 mg/cm2 per well). The results showed that the
mRNA expression of Hyal-1 was significantly upregulated in
HA-coated cultures (Fig. 2). Compared with the control group,
which did not use HA-coated wells, Hyal-1 mRNA expression
was significantly increased in HA-coated cultures from day 1
to day 5 [day 1 (P ⫽ 0.014), day 3 (P ⫽ 0.035), and day 5 (P ⫽
0.041)]. This result indicates that HA-CD44 interaction occurs
in hADSCs cultured in an HA-enriched microenvironment
(Fig. 2A).
Events
RESULTS
100
101
102
103
104
FL1 LOG
Fig. 1. Flow cytometry analysis to confirm the presence of CD44 on the cell
surface of human adipose-derived stem cells (hADSCs). The analysis showed
that 99.9% of the hADSC population can be stained with CD44 positive (gray)
compared with the isotype control (white) stained hADSCs.
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times with 200 ␮l of 0.1 M HCl. The attached stain was extracted by
mixing well with 200 ␮l of 0.1 M NaOH for 5 min. The attached
extracted stain was placed into a second plate to read the absorbance
at 540 nm using a microplate reader.
Immunohistochemistry. The effect of HA-CD44 on the formation
of a cartilaginous matrix by hADSCs cultured in HA/fibrin and fibrin
hydrogels was evaluated after 21 days in chondrogenic medium. To
assess the presence of cartilage-specific matrix proteins, the HA/fibrin
and fibrin hydrogels were fixed overnight using 4% paraformaldehyde
in PBS (pH 7.4) at 4°C and transferred to 70% ethanol until the next
processing step. The hydrogels were embedded in paraffin and were
sliced into 2-␮m-thick sections. To detect collagen type II, sections
were labeled with specific primary antibodies for collagen type II
(diluted 1:100, Chemicon), followed by FITC anti-mouse secondary
antibodies (diluted 1:200, Molecular Probes). To detect collagen type
X, sections were also labeled with specific primary antibodies for
collagen type X (diluted 1:100, Sigma), followed by rhodamine
anti-mouse secondary antibodies (diluted 1:200, Invitrogen). The
sections were counterstained with 4=,6-diamidino-2-phenylindole
(DAPI) (diluted 1:500, Sigma) to identify cellular nuclei, which can
be counted to reflect total cell number.
Statistical analysis. Four cultures were tested for each individual
experiment. Each experiment was repeated at least three times, and
the data were expressed as means ⫾ SE from combined data from all
occasions where each experiment was repeated. Statistical significance was evaluated by a one-way ANOVA, and multiple comparisons were performed by Scheffé’s method. A P ⬍ 0.05 was considered significant.
55
128
Human gene
SOX-9
CD44 in Hyaluronan Induce Chondrogenesis
Normalized fold
expressions
10
8
6
**
Control
HA-coated
**
*
4
2
0
Control
Day 1
Day 3
Normalized fold
expressions
B
25
Control
20
HA-coated
5
**
Control+CD44 blocker
A
*
50
HA-coated
Collagen type II
**
**
40
30
5
##
*
20
B
10
Day 1
Day 3
Day 5
D
Day 1
Day 3
Day 5
**
SOX-9
40
30
##
**
20
10
0
0
#
*
Day 1
C
Day 3
Day 5
Collagen type II
160
Control
Aggrecan
**
HA-coated
100
80
60
40
20
**
100
D
Day 1
Day 3
Day 5
Fig. 2. The influence of the niche interaction of hyaluronan (HA)-CD44 on
chondrogenesis of hADSCs in an HA-coated well. Expression of hyaluronidase-1 (Hyal-1; A), SOX-9 (B), collagen type II (C), and aggrecan (D) in
hADSCs was quantified with real-time PCR by using SYBR green fluorescence from day 1 to day 5 of cell culture in HA-coated wells. The hADSCs
cultured in HA-coated wells showed upregulation of Hyal-1, SOX-9, collagen
type II, and aggrecan from day 1 to day 5. We normalized the values against
expression in controls without HA at day 1, which was stated at 1. Values are
means ⫾ SE (n ⫽ 4). Values are compared with the control culture, which has
no HA coating in the wells: *P ⬍ 0.05. **P ⬍ 0.01.
increased, but not different from that in HA-CD44 blockade
group compared with the control group (Fig. 3A). Most importantly, the results showed that the upregulated Hyal-1 mRNA
expression of hADSCs in HA-coated cultures from day 1 to
day 5 was completely inhibited when treated with CD44
receptors blocking antibody (HA vs. HA⫹CD44 blockers: day
1, P ⫽ 0.043; day 3, P ⫽ 0.015; day 5, P ⫽ 0.008) (Fig. 3A).
The upregulation of chondrogenic marker genes (SOX-9, collagen
type II, and aggrecan) for hADSCs in HA-coated cultures was
also suppressed when HA-CD44 was blocked (SOX-9: day 3,
P ⫽ 0.03 and day 5, P ⫽ 0.039; collagen type II: day 3, P ⫽ 0.043
#
80
**
60
40
#
**
20
0
**
0
**
120
Day 1
140
Normalized fold
expressions
Normalized fold
expressions
120
Normalized fold
expressions
140
140
##
#
50
Normalized fold
expressions
Normalized fold
expressions
Control
Day 5
*
*
Day 3
Day 5
Aggrecan
120
**
100
80
##
60
**
40
20
0
**
Day 1
Day 3
#
Day 5
Fig. 3. The correlation between HA-CD44 activity and initiation of chondrogenesis in hADSCs in an HA-coated well. Expression of Hyal-1 (A), SOX-9
(B), collagen type II (C), and aggrecan (D) in hADSCs was quantified with
real-time PCR by using SYBR green fluorescence from day 1 to day 5 of
culture in HA-coated wells. The hADSCs cultured in HA-coated wells (HA)
showed upregulation of Hyal-1, SOX-9, collagen type II, and aggrecan from
day 1 to day 5. A: the Hyal-1 mRNA expression in hADSCs was upregulated
in HA-coated wells from day 1 to day 5, but this upregulation was completely
inhibited when the HA-CD44 interaction was inhibited. B–D: the upregulation
of chondrogenic marker genes (SOX-9, collagen type II, and aggrecan,
respectively) in hADSCs in HA-coated cultures was also partially downregulated when the HA-CD44 interaction was inhibited. We normalized the values
against expression in controls without HA at day 1, which was stated at 1.
Values are means ⫾ SE (n ⫽ 4). *P ⬍ 0.05, **P ⬍ 0.01 compared with the
control culture (0 mg/cm2) in day 1. #P ⬍ 0.05, ##P ⬍ 0.01 compared with the
HA culture (HA-coated well) at each time point.
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60
Day 3
HA+CD44 blocker
*
0
Day 1
Hyal-1
HA
10
**
15
10
SOX-9
Day 5
0
C
1613
Wu S-C et al.
and day 5, P ⫽ 0.03, aggrecan: day 3, P ⫽ 0.038 and day 5, P ⫽
0.04) (Fig. 3, B, C, and D). The decrease of mRNA expression
levels for chondrogenic genes caused by the CD44 blockade was
as follows: SOX-9: day 3, 37% decrease, day 5, 53% decrease;
collagen type II: day 3, 38% decrease, day 5, 37.5% decrease; and
Hyal-1
Normalized fold
expressions
A
•
1614
CD44 in Hyaluronan Induce Chondrogenesis
aggrecan: day 3, 42.5% decrease, day 5, 54% decrease. These
results indicate that HA-CD44 contributes to the initiation and
enhancement of chondrogenesis for hADSCs in an HA-enriched
microenvironment.
Cell survival in 3D HA/fibrin hydrogel. After a 2-h preincubation, the survival rate of hADSCs encapsulated in 3D
HA/fibrin and fibrin hydrogels was examined (Fig. 4). Four
hours after encapsulation, most of the ADSCs were alive (Fig.
4, B and C), and no obvious dead cells were found in both the
HA/fibrin and fibrin hydrogels (Fig. 4, B–E). The amount of
aggregates was not increased, but the aggregates were larger,
more pronounced in the HA/fibrin hydrogels compared with
the fibrin hydrogels, indicating more hADSC and extracellular
matrix interactions (Fig. 4, B and C).
The HA-CD44 interaction of hADSCs cultured in 3D HA/
fibrin hydrogel. The hADSCs cultured in HA/fibrin hydrogels
showed higher Hyal-1 and chondrogenic gene (SOX-9, colla-
•
Wu S-C et al.
gen type II, and aggrecan) expression than those in fibrin
hydrogels after 1, 3, and 5 days of culturing in the chondrogenic
medium (Fig. 5A). Chondrogenic marker gene expression was
upregulated in hADSCs cultured in HA/fibrin hydrogels from day
1 to day 5 compared with fibrin hydrogels [SOX-9: day 1 (P ⫽
0.023), day 3 (P ⫽ 0.019), and day 5 (P ⫽ 0.0284); collagen type
II: day 1 (P ⫽ 0.0195), day 3 (P ⫽ 0.0239), and day 5 (P ⫽
0.0408); aggrecan: day 1 (P ⫽ 0.0157), day 3 (P ⫽ 0.0131), and
day 5 (P ⫽ 0.0476)] (Fig. 5, B, C, and D). These results confirm
that HA-CD44 contributes to promoting chondrogenesis for hADSCs
in HA/fibrin hydrogels (Fig. 5, B, C, and D).
sGAG and total intracellular collagen synthesis of hADSCs
cultured in a 3D HA/fibrin hydrogel. The sGAG and total
intracellular collagen synthesis by hADSCs in HA/fibrin and
fibrin hydrogels was quantified to support the gene expression
results. The result showed that hADSCs cultured in HA/fibrin
hydrogels produced more sGAG than that in fibrin hydrogels
Fig. 4. A: low-magnification image showing
the three-dimensional (3D) HA/fibrin (right)
and fibrin (left) hydrogel cultured within wells.
Fluorescence microscopy images show encapsulated hADSCs in 3D HA/fibrin (B and D)
and fibrin (C and E) hydrogels. The live cells
were stained with calcein-AM (green; B and
C), and the dead cells were stained with
ethidium homodimer-1 (red; D and E).
B
C
D
E
J Appl Physiol • doi:10.1152/japplphysiol.01132.2012 • www.jappl.org
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A
CD44 in Hyaluronan Induce Chondrogenesis
Hyal-1
Normalized fold
expressions
A
**
50
Fibrin hydrogel
40
HA/fibrin hydrogel
**
30
20
10
0
*
Day 1
Day 3
Day 5
B
Fibrin hydrogel
HA/fibrin hydrogel
**
**
20
*
0
Day 1
C
Collagen type II
100
Normalized fold
expression
Day 3
80
Fibrin hydrogel
HA/fibrin hydrogel
Day 5
**
**
60
40
20
*
1615
Wu S-C et al.
Immunohistochemistry-based analysis of hADSCs cultured
in 3D HA/fibrin hydrogel. Expressions of collagen type II and
type X were analyzed by immunohistological staining. Both
hADSCs cultured hydrogels showed collagen type II depositions, but the HA/fibrin hydrogels (Fig. 7, B and D) showed
more collagen type II than those in fibrin hydrogels (Fig. 7, A
and C). Conversely, less collagen type X was found in HA/
fibrin hydrogels (Fig. 7, F and H) compared with fibrin hydrogels (Fig. 7, E and G).
DISCUSSION
Cell-matrix adhesion is one of the niche interactions that
direct stem cell differentiation (11). Our laboratory’s previous
report indicated that an HA-enriched microenvironment initiates and enhances chondrogenesis in hADSCs (43). In the
present study, we further hypothesize that the HA-CD44 interaction contributes to HA-initiated chondrogenesis and the subsequent formation of a cartilaginous matrix. We found that
CD44 signaling was promoted in hADSCs cultured in both
HA-coated wells and HA/fibrin hydrogels following HA preincubation. The chondrogenesis of hADSCs was initiated, and
subsequent cartilaginous matrix formation was enhanced in
either HA-enriched environment or preincubation of cell with
HA, indicating this effect may be due to HA-CD44 interaction.
Our results further demonstrated that a blockade of HA-CD44
interaction decreased the HA-upregulated mRNA expression
levels of chondrogenic marker genes, including SOX-9, collagen type II, and aggrecan. This result indicates that the HACD44 interaction significantly contributes to both chondrogenesis of hADSCs and subsequent cartilaginous matrix formation.
0
60
50
Aggrecan
Fibrin hydrogel
HA/fibrin hydrogel
Day 5
**
**
40
30
20
*
10
0
Day 1
Day 3
Day 5
Fig. 5. Real-time polymerase chain reaction analysis illustrating the interaction
between the HA-CD44 interaction and initiation of chondrogenesis in hADSCs
cultured in HA/fibrin hydrogel constructs: Hyal-1 (A), SOX-9 (B), collagen
type II (C), and aggrecan (D). We normalized the values against expression in
controls without HA at day 1, which was stated at 1. Values are means ⫾ SE
(n ⫽ 4). *P ⬍ 0.05, **P ⬍ 0.01 compared with fibrin hydrogel at each time
point.
[day 7 (P ⫽ 0.0431), day 14 (P ⫽ 0.0368), and day 21 (P ⫽
0.0076)] (Fig. 6A). In addition, HA/fibrin hydrogel cultures
also produced higher total intracellular collagen than the fibrin
hydrogel cultures from day 7 to day 21 [day 7 (P ⫽ 0.0303),
day 14 (P ⫽ 0.0251), and day 21 (P ⫽ 0.009)] (Fig. 6B).
Overall, compared with the fibrin hydrogels, the HA-CD44
interaction resulted in higher levels of sGAG and total collagens after 21 days of cultivation.
A
sGAG/DNA
(Ratio to Fibrin hydrogel)
Normalized fold
expression
D
Day 3
sGAG synthesis
Fibrin hydrogel
3.5
**
HA/fibrin hydrogel
3
2.5
*
2
*
1.5
1
0.5
0
B
Total intracellular
collagen/DNA
(Ratio to Fibrin hydrogel)
Day 1
Day 1
Day 7
Day 14
Day 21
Total intracellular collagen synthesis
**
)LEULQK\GURJHO
+$ILEULQK\GURJHO
*
*
Day 1
Day 7
Day 14
Day 21
Fig. 6. Synthesis of sulfated glycosaminoglycan (sGAG; A) and total intracellular collagen (B) from hADSCs cultivated within HA/fibrin and fibrin hydrogel constructs after 21 days. Values are means ⫾ SE (n ⫽ 4). *P ⬍ 0.05,
**P ⬍ 0.01 compared with fibrin hydrogel at each time point.
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Normalized fold
expression
40
SOX-9
•
1616
CD44 in Hyaluronan Induce Chondrogenesis
•
Wu S-C et al.
Fibrin hydrogel
HA/fibrin hydrogel
B
A
50µm
50µm
D
C
20µm
20µm
F
E
50µm
50µm
H
G
20µm
Increasing chondrogenic activity in MSCs is important for
stem cell-based tissue engineering to repair articular cartilage.
CD44 has been reported to be the main surface receptor of HA
and is important for cartilage matrix assembly and retention (3,
10, 19). Our results showed that 99.9% of the hADSCs possesses CD44. This finding increases the possibility that extracellular HA may act through its interaction with CD44 in
hADSCs. Upregulation of Hyal-1 has been reported to reflect
the activation of CD44 signaling, which is triggered by HA
20µm
binding in MSCs (26). In the 2D HA-coated culture experiment, cells cultured in basal medium expressed high gene
levels of Hya-1, SOX-9, collagen type II, and aggrecan on
the first day, caused by HA-CD44 interaction, while blocking the HA-CD44 interaction by antibody. The gene expressions were partially downregulated during days 1–5. Therefore, we conclude that the HA-CD44 interaction contributes to
inducing and enhancing the chondrogenic differentiation of
ADSCs. In the 3D fibrin hydrogel experiment, for observing
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Fig. 7. Immunohistological staining of collagen type II for hADSCs cultivated in fibrin
(A and C) and HA/fibrin (B and D) hydrogels
after 21 days. Green color represents staining
with collagen type II antibodies, and blue
color represents counterstaining with DAPI.
Immunohistological staining is shown of collagen type X for hADSCs cultivated in fibrin
(E and G) and HA/fibrin (F and H) hydrogels
after 21 days. Red color represents staining
with collagen type X antibodies, and blue
color represents counterstaining with DAPI.
CD44 in Hyaluronan Induce Chondrogenesis
Wu S-C et al.
1617
drogel. The HA/fibrin hydrogel may be used as a scaffold for
ADSC-based cartilage regeneration.
There are two major limitations in this study. First, it is not
able to precisely quantify the cartilaginous matrix proteins of
collagen types II and X in immunohistological study. Using
Western blot analysis for the extracted proteins from fibrin/
ADSCs or HA/fibrin/ADSCs construct showed lots of background noise. Instead of testing collagen II and X individually,
we measured total collagen by Sirius red staining method, and
further used immunohistochemical to confirm that the total
collagen change is due to collagen II but not collagen X.
Second, presenting cell morphology by micrographs may benefit to confirm the finding, but it is hard to have clear pictures
from hydrogel 3D cultures.
In this study, we found cells preincubated with HA and
cultured in an HA-enriched 3D environment significantly enhanced both total collagen and sGAG synthesis. This suggests
that the enhancement of cartilaginous matrix formation in an
HA/fibrin hydrogel may be mainly due to the HA-CD44
interaction. In addition, collagen type X has been reported to be
the marker of hypertrophic chondrocytes, representing chondrocyte degeneration, and it may lead to calcification of engineered cartilage (4, 27). Our immunocytochemical staining
results showed that cultivation of hADSCs in an HA/fibrin
hydrogel increases collagen type II, but collagen type X deposition appeared to be less compared with that in a fibrin gel
without HA. Based on these results, we suggest that HA, acting
through the HA-CD44 interaction, may be used as a biomaterial base to promote chondrogenesis of hADSCs for better
cartilage formation.
GRANTS
This study was supported by grants from the National Science Council
(NSC 97-2321-B-037-001-MY2 and NSC100-2325-B-037-004), the National
Health Research Institutes (NHRI-EX99-9935EI), and the Ministry of Economic Affairs (98-EC-17-A-S1-041) in Taiwan.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.-C.W., R.E., and Y.-S.L. performed experiments;
S.-C.W., C.-H.C., J.-K.C., Y.-S.L., S.-Y.L., and M.-L.H. analyzed data;
S.-C.W., C.-H.C., J.-K.C., Y.-C.F., C.-K.W., R.E., Y.-S.L., Y.-H.W., S.-Y.L.,
and M.-L.H. interpreted results of experiments; S.-C.W. and Y.-S.L. prepared
figures; S.-C.W., C.-H.C., and R.E. drafted manuscript; S.-C.W., C.-H.C.,
J.-K.C., Y.-C.F., C.-K.W., R.E., Y.-H.W., S.-Y.L., G.-J.W., and M.-L.H.
edited and revised manuscript; S.-C.W., C.-H.C., J.-K.C., Y.-C.F., C.-K.W.,
Y.-H.W., S.-Y.L., G.-J.W., and M.-L.H. approved final version of manuscript;
C.-H.C., J.-K.C., Y.-C.F., C.-K.W., Y.-H.W., G.-J.W., and M.-L.H. conception
and design of research.
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