Extract
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Introduction
As defects of hyaline cartilage have been known to have poor capacity of healing spontaneously, current treatments do
not result in complete regeneration of the original hyaline
architecture[1_3]. Use of the tissue engineering technique could be
a promising way of regenerating the articular lesions. This technique combines isolated cells, biomaterials, and
environmental factors in order to produce new cartilage tissue with the typical characteristics of hyaline cartilage. As far as the choice of
seeding cells is concerned, autologous cells are generally preferred to avoid the immunological rejection. Autologous
cultured chondrocytes transplantation has been used in treatment of osteochondral
defects[4,5]; however, the technique was
restricted clinically because the procurement of many
chondrocytes caused harm to the healthy cartilage. Long
time culture for expansion in vitro will induce chondrocyte
dedifferentiation[6]. As an alternative to chondrocytes, many
researches have focused on bone marrow-derived
mesenchymal stem cells (MSC) as a source of chondrogenic cells
for cartilage repair[7_10]. Unfortunately, MSC also have
problems, such as donor site morbidity, lower cell numbers,
and limited differentiation potential as the age of the donor
increases[11].
Recent studies have shown that human adipose tissue
contain multipotential cells, termed processed lipoaspirate
or human adipose derived stem cells
(hASC)[12]. Isolation of single hASC cell populations (clones) illustrates that they
have at least a trilineage potential to form bone, cartilage,
and fat. Immunofluorescence and flow cytometry analysis
demonstrated that these cells are similar to MSC, but have
distinct differences[13]. Adipose tissue is easily obtained
with relatively little discomfort, carrying a lower donor-site
morbidity and is available in larger
numbers[14,15]. Therefore, it seems that stem cells from adipose tissue can be a feasible
choice as the seeding cells in constructing tissue engineered
cartilage.
Cytokines that belong to the transforming growth factor
(TGF) beta superfamily are thought to play important roles
in the chondrogenic differentiation of stem cells. Although
data is plentiful on the contribution of TGF
beta1 to hASC
chondrogenesis[12,13,16_18], reports on the role of TGF beta2
and its expression in these cells are scarce. Previous
experimental results have strongly suggested that TGF
beta2 was involved in
chondrogenesis[19_22]. In the present study, we
sought to demonstrate the chondrogenesis of hASC induced
by human TGF (hTGF) beta2 both in vitro and
in vivo.
The goals of our study are (i) to investigate whether
chondrogenesis of hASC can be obtained by combining pellet
culture and hTGF beta2 treatment in
vitro; and (ii) to determine whether the differentiated hASC can maintain the
chondrogenic phenotype and produce neocartilage
in vivo.
Materials and methods
Harvest of hASC After informed consent from all
patients and approval from the Ethics Committee of Peking
university, Third hospital, leftover subcutaneous adipose
tissue was obtained form 7 patients with femoral neck
fracture (mean age 66 years, range 56_78) undergoing routine
total hip joint replacement. To isolate hASC, adipose tissue
was dissected and washed extensively with
phosphate-buffered saline (PBS) to remove contaminating debris and red
blood cells, then digested with 0.1% type I collagenase
(Sigma-Aldrich, St Louis, MO, USA) at 37 °C and shaken at
170 rpm for 45 min. Enzyme activity was neutralized with
Dulbecco's modified Eagle's medium (DMEM, GibcoBRL,
Grand Island, NY, USA) containing 10% fetal bovine
serum(FBS) and centrifuged at 800×g for 10 min to obtain a
high-density cell pellet and remove the mature adipocytes. The
pellet was resuspended in DMEM and filtered through a 100
µm nylon mesh to remove cellular debris. After incubation
overnight at 37 °C in 5% CO2 in DMEM supplemented with
10% FBS, 100 units/mL sodium penicillin, and 100 µg/mL
streptomycin, the plates were washed extensively with PBS
to remove residual non-adherent red blood cells. The
culture medium was changed every 2_3 d. To expand cell
numbers, dense cell plaques were trypsinized and re-plated
until they formed confluent monolayers that were then
trypsinized and passaged into duplicate flasks.
Flow cytometry analysis hASC grown for 5 passages
were characterized by flow cytometry.
1×106 cells were trypsinized and centrifuged at 1500 r/min for 5 min. The cells
were then washed with PBS and fixed in 3%
paraformaldehyde for 30 min at 4 °C for blocking. Unlabeled monoclonal
mouse anti-human CD45 and CD29 were purchased from
Pharmingen (San Diego, CA, USA). Fluorescein isothio-
cyanate (FITC)-labeled mouse anti-human CD14 and CD90
were purchased from Serotec (Oxford, UK).
The following procedure was performed for staining with
CD45 and CD29: the cells were incubated with the primary
antibodies for 60 min. After wash, the cells were incubated
with FITC-conjugated goat antimouse immunoglobulin for
30 min. CD14 and CD90 staining was performed in a single
step during which cells were incubated with FITC-labeled
CD14 and CD90 for 60 min. Cell fluorescence was evaluated
by flow cytometry (FACSCalibur, BD Biosciences,
Mountain View, CA, USA) with an excitation of 488 nm and
analyzed with CELLQuest software (BD Biosciences, San Jose,
CA, USA). Mouse isotype antibodies served as the
controls (BioLegend, San Diego, CA, USA).
Pellet culture For the preparation of each pellet, hASC
of passage 5 were trypsinized, counted and resuspended in
the chondrogenic DMEM containing 10 ng/mL hTGF beta2,
1×10-7 mol/L dexamethasone, 6.25 µg/mL insulin, and 50
µg/mL ascorbate-2-phosphate. Aliquots of
1×106 cells were spun down at
500×g in 15 mL conical tubes. Pellets were
divided into 2 experimental groups (hTGF beta2-treated and
untreated controls). The cells were cultivated at 37
°C in a humidified atmosphere with 5%
CO2 for 14 d by changing the medium every 2 d. The cells in the monolayer culture
were also used as the negative control.
Release of cells from pellets After 14 d of induction, the
cells were digested with trypsin for 5 min and then with 0.2%
crude type II collagenase (Life Technologies, Rockville,
Maryland, USA ) in DMEM for 1 h. Released cells were
reseeded onto the prepared coverslips. Twenty-four hours
after seeding, the cells were fixed in 4% paraformaldehyde
and prepared for histological and immunohistochemical
analysis.
RT-PCR analysis The cell pellets were treated with Trizol
reagent (Invitrogen, Carlsbad, CA, USA). Extracted cellular
RNA was dissolved in RNase-free water. Reverse
transcription was carried out using the Kit (SuperScriptIII,
Invitrogen, USA). 1 µg RNA was used for the first strand cDNA
synthesis in a total volume of 20 µL. The regimen for PCR was 5 min
at 94 °C followed by the appropriate number of cycles of 5 s
at 94 °C, 30 s at the proper annealing temperature for each
primer pair, and 30 s at 72 °C, with a final 7 min extension at
72 °C. The reaction was also carried out with GAPDH as the
housekeeping gene. Amplified PCR products were analyzed
by ethidium bromide staining after gel electrophoresis. The
primers for the following human cDNA are indicated in Table 1.
Western blot analysis Six pellets from variant
conditions were washed twice with PBS and lysed in the lysis
buffer [20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1
mmol/L ethylene diamine tetra acetic acid (EDTA), 1 mmol/L
ethylene glycol tetra acetic acid (EGTA), and 1 mmol/L
phenyl-methanesulfonyl fluoride (PMSF)]. After centrifugation at
18 000×g for 20 min, the supernatant was collected and
protein concentrations were determined by bicinchoninic acid
(BCA) assay (Pierce, Rockford, USA). Equal protein samples
were separated by electrophoresis in 10% polyacrylamide
gels (Bio-Rad, Hercules, CA, USA) and electrophoretically
transferred to a supported nitrocellulose membran (Amersham Pharmacia, Biotech UK Limited, UK). The
membranes were blocked in 5% non-fat milk diluted with
Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h
and incubated overnight at 4 °C with mouse monoclonal
antibodies against type II collagen, type X collagen, and
β-actin (Sigma-Aldrich, St Louis, MO, USA) at a dilution of
1:500 in blocking buffer, respectively. The membranes were
then washed 3 times for 10 min each with TBST and
incubated for 1 h in the dark with the appropriate IRDyeTM
800-conjugated secondary antibodies (L1-COR Bioscience Inc,
USA) prepared in TBST/5% non-fat milk. The signal was
detected using the Odyssey Imaging System (L1-COR
Bioscience Inc, USA).
Preparation of alginate beads The cells released from
the induced cell pellets were prepared for alginate beads.
hASC were resuspended in 1.2% low-viscosity alginate
(Sigma-Aldrich, USA) solution in 0.9% NaCl at a
concentration of 1×106,
5×106 and 1×107 cells/mL, respectively.
Alginate beads were formed by expressing 50 µL cell suspension
through a 16-gauge needle into a bath of 102 mmol/L
CaCl2. The beads were allowed to gel for 10 min at room temperature.
Once hardened, they were washed 3 times with 0.9% NaCl,
and overlaid with culture medium (DMEM containing 10%
FBS, 100 units/mL sodium penicillin, and 100 µg/mL
streptomycin). The alginate beads were prepared and
implanted 24 h later for the in vivo study.
Animal surgeries For the in vivo evaluation, 12
4_6-week-old female BALB/C nude mice (Peking University
Experimental Animal Center, Beijing, China) were used. All
animal experiments were performed in accordance with the
institutional animal guidelines. Before implantation, the mice
were anesthetized with ketamine (50 mg/kg) administered
intraperitoneally using the sterile technique. Subcutaneous
pockets were created on the dorsum of each mouse. Cell
pellets after induction were cautiously placed inside the
pockets. Each pocket received 4 alginate beads with the
same implanted cell concentration. The location of each
implant was randomized and recorded. The skin pockets
were closed using Vecryl suture.
Cell pellets were harvested 2 or 4 weeks after surgery;
alginate beads were explanted 12 weeks after in
vivo culture. The animals were sacrificed with an inflation of
CO2 atmosphere; the constructs were carefully removed and fixed
in 4% paraformaldehyde for at least 24 h.
Histological and immunohistochemistry analysis
The cell pellets and explants harvested from the nude mice were
fixed, dehydrated, and subsequently embedded in paraffin.
The sections (5 µm thick) were stained with HE.
Chondrogenesis was evaluated with toluidine blue staining and
immunohistochemical analysis for type II collagen. Briefly,
following deparaffinization and rehydration, the sections were
incubated for 10 min with 0.1% toluidine blue (Sigma-Aldrich,
St Louis, MO, USA) in 0.1 mol/L sodium acetate buffer (pH
4.0) to visualize the tissue proteoglycans. For the
immunohistochemical analysis of type II collagen, the sections were
incubated for 10 min with newly diluted 3%
H2O2 solution to destroy internal peroxidases, followed by trypsin digestion
for 10 min. After digestion, the slides were incubated for 20
min with normal goat serum for blocking. The slides were
then immunoblotted with a goat anti-human polyclonal
antibody against type II collagen (Santa Cruz, CA, USA)
overnight at 4 °C. Incubation with biotin-labeled rabbit anti-goat
IgG for 30 min was followed by a 10 min incubation with
horseradish peroxide-conjugated streptavidin (SP kit,
Vector Laboratories, Burlingame, CA, USA). After several
washes, antibody binding was visualized using
diaminoben-zidine and the nuclei were counterstained with hematoxylin.
The reaction was stopped by immersion in water. HASC
grown on the coverslips were fixed and carried the same
procedure to detect the chondrogenic phenotype.
Specimens were processed using identical protocols, but without
the primary antibodies were used as the negative control;
human articular cartilage was used as a positive control.
Results
Characterization of hASC from subcutaneous adipose
tissue Within 2_3 passages after the initial plating of the
primary culture, hASC appeared as a monolayer of large, flat
cells. They approached confluence and assumed a more
spindle-shaped, fibroblastic morphology. Flow cytometry
analysis of hASC of passage 5 demonstrated that the cells
lacked CD14 and CD45, but the majority expressed CD29 and
CD90 (Figure 1).
Chondrogenic differentiation of hTGF beta2-treated
hASC in the pellet culture in vitro Chondrogenic marker
genes were analyzed by RT-PCR; the analysis using total
RNA was performed at 7 and 14 d after induction. As shown
in Figure 2, the expression of the chondrocyte maker genes,
type II collagen, and aggrecan were detected after 7 d of
induction. The expression continued throughout the
culture period. Type X collagen mRNA transcription, which
indicates the presence of hypertrophic chondrocytes, was
not observed during the culture periods in any of the groups.
Collagen type II production was also demonstrated by
Western blot analysis at 7 and 14 d time points (Figure 3).
Two weeks after induction, positive clone staining with
toluidine blue confirmed glycosaminoglycan (GAG)
biosynthesis in the cell pellets. The presence of type II collagen
was demonstrated by immunohistochemistry staining (Figure 4). When the cell pellets were digested into single
cell suspension and reseeded in the monolayer, the ASC
displayed a polygonal and triangle morphology compared
with the primary spindle shape (Figure 5A, 5B). The pellets
with control medium and hASC cultured in monolayer
culture did not show any obvious evidence of chondrogenesis.
In all immunohistochemical analyses, specimens examined
with secondary antibody alone failed to demonstrate any
immunoreactive protein.
Predifferentiated hASC embedded in alginate gel
produced neocartilage in vivo The cell pellets retrieved at 2
weeks became much smaller than their original size.
More-over, invasions of fibrous tissues and angiogenesis were
found (Figure 6A, 6B). The specimens were weakly stained
with toluidine blue (Figure 6C) and immunohistochemistry
against type II collagen (Figure 6D) as compared with the
pellets before implantation. Four weeks after in
vivo culture, no implants were identified macroscopically at the site of
subcutaneous implantation, and neither cartilage nor bone
formation was detected at histological examination.
Cartilage formation of the predifferentiated hASC
embedded in alginate gel was demonstrated by histological and
immunohistochemistry examination. HE staining revealed
that hASC appeared to be round and encased in lacunae
(Figures 8B, 9B). Toluidine blue staining was positive and
consistent with the presence of GAG around the cells (Figures
8C, 9C). The presence of collagen type II was revealed by
positive staining of immunohistochemistry with collagen type
II specific antibody (Figures 8D, 9D). The quality of
neocartilage was obviously influenced by the implanted cell
concentration. When the implanted cell concentration was
1×106/mL, large amounts of fibrous tissues and
angiogenesis invaded into the constructs, and only scattered islands
of cartilage were observed (Figure 7A, 7B). When the cell
concentration was 5×106/mL, the integrity of the gel was
separated by relatively limited invaded tissues (Figure 8A). Little
fibrous tissues were detected when the implanted cell
concentration reached 1×107/mL and the constructs displayed a
typical cartilage structure (Figure 9A). In
immunohistochemistry staining, the control sections processed using
identical protocols, but without primary antibodies, showed
negative labeling in all cases.
Discussion
Chondrogenic potential of hASC makes them a possible
source of seeding cells for cartilage tissue engineering. In
the present study, hASC were successfully isolated from the
human subcutaneous adipose tissue. Although we could
not confirm that all harvested cells were stem cells, it is
possible that small amounts of pericytes, endothelial cells, and
smooth muscle cells were included. An examination of
expressed surface proteins revealed the presence of CD29
and CD90, but a small fraction of the cells expressed CD14
and CD45. A similar profile is found in human bone marrow
stromal cells[23].
Our study demonstrated that hASC could be induced
into chondrocyte-like phenotype combining pellet culture
system and hTGF beta2 treatment in vitro. However, this
predifferentiation did not guarantee ectopic cartilage
formation in vivo unless the alginate gel was used as the cell carry
scaffold, which is an appropriate 3-D scaffold, and certain
implanted cell concentrations were required for the
in vivo cartilage formation of the predifferentiated hASC.
TGF beta2 is regarded as an "activator"-like molecule in
chondrogenic pattern formation[22]. Researchers also
revealed that TGF beta2 treated dedifferentiated
chondro-cytes expressing GAG and collagen type II and recovered
their cartilage phenotype[24,25]. Perichondrium-derived
progenitor cells cultured in medium with insulin growth factor-I
(IGF-I) plus TGF beta2 produced a cartilage-specific matrix
and showed evidence of chondrogenic
potential[26]. Wang reported that transfection with pcDNA3.1(+)/hTGF beta2
stimulated both the short- and long-term induction of bone
marrow-derived mesenchymal progenitor cells into a
chondrogenic lineage[27]. All these findings indicate that TGF
beta2 plays important roles in chondrogenic pattern
forma-tion; however, there have been few reports on the influence
of hTGF beta2 on the chondrogenic differentiation of hASC.
In this study, chondrogenic induction of the hASC in pellet
culture induced by hTGF beta2 was confirmed by (i) the
upregulation of mature chondrocyte markers, collagen type
II, and aggrecan 7 d after induction revealed by RT-PCR
analysis; (ii) the positive staining of toluidine blue specific
for highly sulphated proteoglycans characteristic of
cartilaginous extracellular matrix (ECM); (iii) the collagen type II
production revealed by Western blot analysis and positive
staining of immunohistochemistry the principal constituents
of cartilage ECM; and (iiii) the single cell released from the
cell pellets that appeared to be polygonal and triangular
after being reseeded in monolayer. Collagen type II and
aggrecan are believed to be specific to articular cartilage
which are known to be expressed during chondrogenesis of
mesenchymal progenitor cells[28,29]. The results presented
here implicated that hTGF beta2 treatment could initiate or
enhance the chondro-lineage differentiation of hASC in
pellet culture in vitro. One interesting finding in the study was
that after culture in the monolayer with chondrogenic media,
hASC tended to aggregate and formed small spheroids, but
no signs of chondrogenic differentiation were detected.
These combined findings demonstrate that the condensed
environment of pellet culture and bioactive hTGF beta2 both
contribute to the chondrogenesis of hASC in
vitro.
Transplantation of multipotent adult stem cells implies
risks because of their uncertain differentiation ability.
Predifferentiation in vitro has been regarded as a useful step
for inducing specific lineage commitment and reducing the
risk of undesired tissue formation. To determine the stability
of chondrocyte-like phenotype of hASC obtained in
vitro, the cell pellets after induction were implanted in
subcutaneous pockets of the nude mice. The results revealed that they
rapidly lost the chondrocyte phenotype and could not resist
the fibrous tissues and angiogenesis invasion.
This was also reported by De Bary who found that
in vitro-differentiated mesenchymal stem cells from the synovial membrane
could not form ectopic stable cartilage in
vivo[30]. It was implied that predifferentiation
in vitro was not sufficient to guarantee stable lineage commitment and restriction of
differentiation; however, hASC embedded in alginate gel kept
their ability of synthesizing aggrecan and collagen type II
after several months in in vivo culture. These differences
may be explained by the biophysical environment which
alginate gel provides. Alginate itself constitutes a relatively
containing environment, and the cells in alginate hydrogel
were embedded in lacunae like chondrocytes being
surrounded by specific ECM. This 3-D environment may be
superior in keeping the chondrogenic phenotype compared
with the pellet culture system in vivo. Another significant
demonstration in this study is that certain cell density is
required for the cartilage formation ability of the
predifferen-tiated hASC in vivo. A low implanted concentration
(1×106/mL) in the alginate gel could not resist the fibrous tissues
invasion. Alginate gel integrity was separated into pieces
by large amount of fibrous tissues, and as the cell density
increased, less invasion was observed, and the structure of
the neocartilage became similar to the native cartilage. The
results implied that a specific threshold level was required
for in vivo cartilage tissue formation by the predifferentiated
hASC in alginate gel. High quality cartilage was obtained
only when the cell concentration reached a definite level.
Based on the results in our study, an implanted cell
concentration of 1×107/mL or higher was recom-mended. Alginate
hydrogels have been extensively used as a biomaterial
scaffold in chondrogenic induction of stem cells and to
regenerate cartilage
tissue[17,18,31_36]; however, as far as the future
clinical application is concerned, alginate gel alone as a cell
carrier scaffold cannot stand mechanical loading nor
surgical handling; an ideal biomaterial is required to provide
initial strength to the graft.
Chondrogenesis of stem cells may therefore be
determined by the microenvironment of the cells, including
secreted factors, integral membrane proteins, and the
extracellular matrix[37]. In this regard, subcutaneous implantation
actually is not an optimal site for cartilage formation.
Wakitani[38] et al demonstrated that osteochondral
progenitor cells from rabbit bone marrow, when transplanted into an
osteochondral defect, formed bone in the side adjacent to
the bone marrow and generated new cartilage in the side
abutting joint cavity. The author pointed out that the
diarthrodial joint provided a good microenvironment for
cartilage formation. The fate of in vitro predifferentiated
hASC seeded in alginate gel when implanted in the
diarthro-dial joint cavity is unknown; however, high quality
tissue-engineered cartilage is expected.
In summary, our study demonstrated chondrogenesis of
hASC in vitro could be induced by combining pellet culture
and hTGF beta2 treatment. Predifferentiated hASC
embedded in alginate gel had the ability of producing neocartilage
in vivo. The results implied important functional
considerations for autologous hASC in the treatment of articular
cartilage injuries. However, animal models of joint surface
defect repair are needed to evaluate potential use of hASC
and their phenotypic behavior within the articular cartilage
microenvironment.
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