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Biomaterials 25 (2004) 3211–3222
Chondrogenic differentiation of adipose-derived adult stem cells in
agarose, alginate, and gelatin scaffolds
Hani A. Awada, M. Quinn Wickhama, Holly A. Leddya, Jeffrey M. Gimbleb,Farshid Guilaka,*
aDepartment of Surgery, Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710, USAbPennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA
Received 9 September 2003; accepted 29 September 2003
Abstract
The differentiation and growth of adult stem cells within engineered tissue constructs are hypothesized to be influenced by cell-
biomaterial interactions. In this study, we compared the chondrogenic differentiation of human adipose-derived adult stem
(hADAS) cells seeded in alginate and agarose hydrogels, and porous gelatin scaffolds (Surgifoam), as well as the functional
properties of tissue engineered cartilage constructs. Chondrogenic media containing transforming growth factor beta 1 significantly
increased the rates of protein and proteoglycan synthesis as well as the content of DNA, sulfated glycosaminoglycans, and
hydroxyproline of engineered constructs as compared to control conditions. Furthermore, chondrogenic culture conditions resulted
in 86%, and 160% increases ( po0:05) in the equilibrium compressive and shear moduli of the gelatin scaffolds, although they didnot affect the mechanical properties of the hydrogels over 28 days in culture. Cells encapsulated in the hydrogels exhibited a
spherical cellular morphology, while cells in the gelatin scaffolds showed a more polygonal shape; however, this difference did not
appear to hinder the chondrogenic differentiation of the cells. Furthermore, the equilibrium compressive and shear moduli of the
gelatin scaffolds were comparable to agarose by day 28. Our results also indicated that increases in the shear moduli were
significantly associated with increases in S-GAG content (R2 ¼ 0:36; po0:05) and with the interaction between S-GAG and
hydroxyproline (R2 ¼ 0:34; po0:05). The findings of this study suggest that various biomaterials support the chondrogenicdifferentiation of hADAS cells, and that manipulating the composition of these tissue engineered constructs may have significant
effects on their mechanical properties.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Alginate; Agarose; Hydrogel; Collagen; Gelatin; Cartilage tissue engineering; Stem cell; Differentiation
1. Introduction
Tissue engineering is a promising therapeutic ap-
proach that combines cells, biomaterials, and environ-
mental factors to induce differentiation signals into
surgically transplantable formats and promote tissue
repair and/or functional restoration [1–5]. Despite many
advances, tissue engineers still face significant challenges
in repairing or replacing tissues that serve predomi-
nantly biomechanical functions such as articular carti-
lage. One obstacle has been the development of a
competent cartilage scaffold that: (a) has mechanical
properties and capability to withstand the large contact
stresses and strains of an articulating joint, (b) allows
functional tissue growth, and (c) provides for appro-
priate cell–matrix interactions to stimulate tissue growth
[6,7]. An evolving discipline termed ‘‘functional tissue
engineering’’ (FTE) seeks to address the functional
challenges of tissues such as cartilage by attempting to
define sets of criteria that must be satisfied in order to
overcome challenges associated with developing a
successful tissue-engineered graft [6].
Challenges related to the cellular component of an
engineered tissue include cell sourcing, expansion, and
differentiation as well as regulatory and production
issues, such as sterility, safety, storage, shipping, quality
control, and scale-up [8]. The use of human adipose
derived adult stem (hADAS) cells represents a feasible
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*Corresponding author. Tel.: +1-919-684-2521; fax: +1-919-681-
8490.
E-mail address: [email protected] (F. Guilak).
0142-9612/$- see front matterr 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.10.045
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approach to many of these issues [9]. Adipose tissue is
routinely available in liter quantities from liposuction
surgeries and yields an average of 400,000 cells per ml of
tissue after expansion, providing the numbers of cells
necessary for many tissue engineering applications [9].
The hADAS cells are pluripotent, expressing the
biochemical profile of adipocytes, chondrocytes, hema-topoietic supporting cells, myocytes, neurons, and
osteoblasts under appropriate culture conditions
in vitro [9].
Other challenges are associated with the biomaterial
scaffolds designed to deliver the cells and guide tissue
growth and differentiation. These biomaterials must
meet several criteria to maximize the chances of a
successful repair, including biodegradability and/or
biocompatibility, facilitating functional tissue growth,
and appropriate biomechanical properties [10–13].
Biomaterials used for cartilage tissue engineering can
have the form of either hydrogels or porous scaffolds.
Among the hydrogel biomaterials, the seaweed-derived
alginate and agarose are typically thought to be inert
because they lack native ligands that could allow
interaction with mammalian cells [14]. However, these
hydrogels provide a number of advantages for tissue
engineering including the possibility of minimally
invasive injection of hydrogel/cell constructs [15,16].
Various researchers have investigated the ability of
alginate and agarose hydrogels to act as a scaffold
material for chondrocytes to regenerate cartilage tissue
[14,17–25]. While many of these studies demonstrated
that alginate and agarose promote maintenance of
the chondrocytic phenotype in vitro, the successof tissue engineering applications using these hydrogels
may be hindered by their poor biomechanical properties
and handling characteristics. Furthermore, the ability of
these hydrogels to support chondrogenic differentiation
of adult stem cells has been less studied [26,27].
Gelatin, a porous denatured collagen scaffold, has
been recently used as a scaffolding structure for cartilage
tissue engineering [28,29]. The biological origin of
collagen-derived gelatin makes this material an attrac-
tive choice for tissue engineering [10]. However, there is
some concern that type I collagen scaffolds may not
preserve the chondrocyte phenotype of cells as well as
type II collagen scaffolds [30]. Furthermore, very little is
known about the functional (mechanical) properties of
this biomaterial although its ability to support chon-
drogenic differentiation of adult stem cells has been
demonstrated [28].
The goal of this study was to assess the functional
(biologic, biochemical, and biomechanical) properties of
alginate hydrogel, agarose hydrogel, and gelatin (Surgi-
foams, denatured porcine collagen type I) porous
sponge as scaffolding biomaterials for cartilage tissue
engineering using hADAS cells that have been shown to
possess a chondrogenic potential under defined culture
conditions [26,31]. We hypothesized that the functional
properties of tissue engineered cartilage will depend on
the choice of biomaterial scaffold, and that the construct
mechanical properties would be directly correlated to
the synthesis and accumulation of extracellular matrix
components.
2. Materials and methods
2.1. Isolation of hADAS cells
Human adipose derived adult stem (hADAS) cells
were isolated from subcutaneous adipose tissue (n ¼ 3
female donors, 29.777.2 years old (mean7standard
deviation) with a body mass index of 28.672.6 kg/m2)
as previously described [26,32,33]. Briefly, liposuction
waste tissue was digested with 0.25 mg collagenase type I
(200 units/mg) per ml of Krebs-Ringer-Bicarbonate
solution (Sigma, St Louis, MO) for 40 min at 37C with
intermittent shaking. The floating adipocytes were
separated from the precipitating stromal fraction by
centrifugation. The stromal cells were then plated in
tissue culture flasks at approximately 3500 cell/cm2 in
stromal media (DMEM/F-12 with 10% fetal bovine
serum (FBS), 100 units/ml penicillin and 100 mg/ml
streptomycin). The primary cells (P0) were cultured for
4 to 5 days, after which they were harvested by
treatment with trypsin (0.05%)/EDTA, counted, and
then frozen in liquid nitrogen in cryopreservation
medium (80% FBS, 10% dimethylsulfoxide, 10%
Dulbecco’s Modified Eagle Medium (DMEM)) untilthey were used in the following experiments.
2.2. Preparation of the biomaterial scaffolds
Cryopreserved cells were thawed and plated in
stromal media for 5 to 7 days until the cultures became
confluent. Cells were harvested using trypsin/EDTA,
counted, and then loaded onto alginate, agarose, and
gelatin scaffolds as described. For the alginate scaffolds,
cells were suspended in 2% (w/v) low viscosity alginate
(Sigma) in 0.9% NaCl at a concentration of 107 cells/ml.
The cell suspensions were cast in custom molds (25 mm
diameter and 2 mm thickness). The alginate molds were
placed into a bath of 102 mm CaCl2 and allowed to gel
for 10 min. The CaCl2 was removed and the molds
were washed three times in PBS. Similarly, cells were
suspended in 2% (w/v) low-melting point agarose
(Type VII, Sigma) at a concentration of 107 cells/ml.
The agarose molds were allowed to gel at 4C for
10 min. Smaller alginate and agarose disks were then cut
out using a 6 mm diameter biopsy punch and placed in
the appropriate culture conditions.
Porous, absorbable gelatin (Surgifoams, Ethicon,
Inc., Somerville, NJ) disks (8mm diameter) were
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pre-wetted in culture medium in flat bottom tubes.
Aliquots (200ml) of the cell suspension (107 cells/ml)
were pipetted on top of each scaffold. The disk and cell
suspension were then centrifuged at 50 g for 30s to
seed the cells into the scaffold. The tubes containing the
seeded scaffolds were then incubated on an orbital
shaker at 5% CO2 and 37
C for 2h to enhance cellinfiltration into the scaffold. The disks were then
incubated undisturbed overnight to allow for cell
attachment. The following day, all disks were removed
from the tubes and grown in the appropriate culture
media. Acellular blank scaffolds were also prepared and
incubated in identical conditions.
2.3. Culture conditions
The scaffolds were grown in control or chondrogenic
culture media in a humidified environment at 5% CO2and 37C for up to 28 days, with culture media
replenished every 3 days. Control culture media
comprised Dulbecco’s Modified Eagle Media–high
glucose (DMEM-hg), 10% fetal bovine serum,
100 units/ml penicillin, and 100 mg/ml streptomycin.
The chondrogenic culture media comprised the control
media, 1 insulin-transferrin-selenium supplement
(ITS+, Collaborative Biomedical, Becton Dickinson,
Bedford, MA), 0.15 mm ascorbate 2-phosphate (Sigma),
100nm dexamethasone (Sigma), and 10 ng/ml rh-TGF-
b1 (R & D Systems, Minneapolis, MN) [26,34,35].
A broad array of biological, biochemical, and
mechanical analyses were used to assess the functional
properties of the scaffolds (Table 1).
2.4. Biological properties
Cell viability and cellular morphology were examined
in situ on days 1, 7, 14, and 28 using a confocal laser
scanning microscope (LSM 510, Carl Zeiss Microima-
ging, Inc., Thornwood, NY ) and the fluorescent Live-
Dead probes (Calcein AM and Ethidium homodimer,
Molecular Probes, Eugene, OR). To quantify the
protein and proteoglycan biosynthetic activity, con-
structs were dual-labeled with 10mCi/ml [3H]-proline
and 5mCi/ml [35S]-sulfate for 24 h on days 1, 7, 14 and
28. Afterwards, the scaffolds were washed 4 times to
remove unincorporated free label and then digested in1ml of a 50 mg/ml papain solution in glass scintillation
counting tubes at 65C overnight. Aliquots (100ml) of
the scaffold digests were sampled from each vial, diluted
to 1 ml, and stored at –80C for later DNA quantifica-
tion as described below. To the remaining 900 ml of the
construct digests, 4.5 ml of Hionic-Fluor Scintillation
Fluid was added (Packard Instrument Company,
Meriden, CT) and the [3H]-proline and [35S]-sulfate
disintegrations per minute were measured on a Model
1900TR Liquid Scintillation Analyzer (Packard).
2.5. Biochemical composition
The DNA content in the scaffold digests was
determined using the fluorescent picoGreen dsDNA
quantification assay (Molecular Probes, Eugene, OR).
Sulfated glycosaminoglycan (S-GAG) content of scaf-
fold digests was measured using a modified dimethyl-
methylene blue (DMB) assay in 96 well plates. Alginate
disk digests were analyzed using a pH 1.5 dye, as
previously described [36], while agarose and gelatin
digests were analyzed using a pH 3.0 dye.
Hydroxyproline (OHP) content was measured using
the Ehrlich’s reaction assay previously described [37,38].
Aliquots (50ml) of scaffold digests, after proper dilu-tions, were hydrolyzed in 6n HCl (Pierce) at 110C for
18h and then lyophilized. The samples were then
reconstituted in 200 ml of the assay buffer (5 g/l citric
acid (monohydrate), 12 g/l sodium acetate (trihydrate),
3.4 g/l sodium hydroxide, and 1.2 ml/l glacial acetic acid
in distilled water, pH 6.0). The reconstituted sample
solutions were subsequently filtered through activated
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Table 1
Summary of the techniques used to assess the functional properties of the scaffold materials
Functional assay Days in culture Sample sizea
Cellular viability andmorphology
Fluorescent calcein-AM and ethidium labeling, andImaging using confocal laser scanning microscopy (CLSM)
1, 7, 14, 28 days n ¼ 3
Protein and proteoglycan
biosynthesis rates
Radioactive [3H]-proline incorporation
Radioactive [35S]-sulfate incorporation
1, 7, 14, 28 days n ¼ 9
Biochemical composition DNA content (PicoGreen assay) 1, 7, 14, 28 days n ¼ 9
Sulfated glycosaminoglycan content (DMB assay)
Collagen content (hydroxyproline assay)
Immunohistochemistry Type II collagen
Chondroitin sulfate
28 days n ¼ 9
Biomechanical properties Equilibrium compressive modulus (step-wise stress-relaxation in
unconfined compression)
0, 14, 28 days n ¼ 9
Equilibrium shear modulus (step-wise shear stress relaxation)
Rheological properties (frequency sweep in dynamic shear)
aSample size per culture condition, per time point. Scaffolds were prepared from cells of three different donors.
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charcoal. Added to 50ml of each filtered sample in a 96
well plate was 50ml of 62mm chloramine-T (Sigma). The
mixture was incubated at room temperature for 15 min
to allow oxidation reaction. Oxidized samples were then
mixed with 50 ml of 0.94m dimethylaminobenzaldehyde
(p-DMBA) colorimetric solution and incubated at 37C
for 30 min. The optical densities of the assayed sampleswere measured using a plate reader at 540 nm, and
the OHP content of the samples was computed relative
to a standard curve of trans-4-hydroxy-L-proline
(0–300mg/ml).
For immunohistochemical analysis, scaffolds were
fixed in 10% buffered formalin for 24h at room
temperature. The fixed scaffolds were dehydrated in a
gradient of alcohols and then embedded in paraffin
blocks. Sections of 7 mm thickness were obtained from
each scaffold and mounted on microscope slides.
Following deparaffinization, rehydration, and endogen-
ous peroxidase activity quenching, the sections were pre-
digested for 60 min at room temperature with 0.25 units/
ml chondroitinase ABC, or for 10 min at 37C with
pepsin to allow for the retrieval of the chondroitin
sulfate (CS) or collagen type II (Coll-II) antigens,
respectively. Sections were then incubated with the
2B6 monoclonal antibody specific to CS (kind gift from
Dr. Virginia Kraus, Duke University Medical Center)
overnight at 4C or with the II-II6B3 antibody specific
to Col II (Developmental Studies Hybridoma Bank,
Iowa City, IA) for 1 h at 37C, respectively. Immunos-
taining was detected using Histostain-Plus Kit for AEC
(Zymed Laboratories Inc., San Francisco, CA).
2.6. Biomechanical properties
The elastic compressive modulus was determined
from equilibrium stress–strain curves generated from
stepwise stress relaxation tests in unconfined compres-
sion at strains of 4%, 8%, 12%, and 16%. Similarly, the
elastic shear modulus was determined from equilibrium
shear stress–strain curves from stepwise shear stress-
relaxation (pure torsion) experiments at shear strains
of (0.03, 0.04, and 0.05 rad). Following the stress-
relaxation tests, the rheological properties of the
constructs were determined by subjecting the samples
to oscillatory shear strain gðtÞ ¼ go:sinðotÞ of afixed amplitude (go ¼ 0:05 rad) and varying frequency(1–100 rad/s). The resultant oscillatory shear stress
sðtÞ ¼ so:sinðot þ dÞ was recorded and the rheologicalproperties such as the complex shear modulus
jG ðoÞj2 ¼ ½G 0ðoÞ2 þ ½G 00ðoÞ2 and the loss angle d were
determined for each of the applied frequencies; where G 0
is the storage modulus [G 0ðoÞ ¼ so:cosðdðoÞÞ=go] and G 00
is the loss modulus [G 00ðoÞ ¼ so:sinðdðoÞÞ=go]. Biome-chanical tests were performed in a bath of DMEM at
room temperature using an ARES Rheometrics System
(Rheometric Scientific, Piscataway, NJ).
2.7. Statistical analysis
Analysis of variance with Student–Newman–Keuls
(SNK) multiple ranges tests were used to compare the
different biomaterials and culture conditions (a ¼ 0:05).Data was further examined in a multiple linear
regression context to determine which of the biochem-ical parameters (DNA, OHP, S-GAG) or combination
of parameters correlated with the biomechanical proper-
ties (E ; G ; d; |G |). Statistical analyses were performedusing Statistical Analysis Software (SASs, Cary, NC)
and S-Pluss (Insightful Corp. Seattle, WA).
3. Results
3.1. Biological properties
Cell viability and morphology in the different scaffold
materials were visualized using confocal laser scanning
microscopy and the Live-Dead fluorescent probes. All
scaffolds showed relatively uniform distributions of cells
with viability greater than 95% at all time points. Cells
in agarose (Fig. 1a) and alginate (Fig. 1b) scaffolds
displayed a spherical morphology that persisted
throughout the culture period, regardless of culture
conditions. In contrast, the cells in the gelatin scaffolds
(Fig. 1c) displayed a distinct ‘‘fibroblastic’’ morphology.
By day 28, the cells in the gelatin scaffolds proliferated
and became confluent with notable cell-to-cell contact
that was associated with the significant cell-mediated
contraction of the gelatin disks, with reduction of up to70% and 87% their initial diameters under chondro-
genic and control culture conditions, respectively
(Fig. 1d). Alginate and agarose disks containing cells
and acellular gelatin disks did not exhibit any contrac-
tion. Furthermore, culture conditions did not appear to
affect cell morphology, but cells were sparse in control
conditions compared to chondrogenic conditions.
Protein and proteoglycan biosynthesis rates were
quantified by [3H]-proline (Fig. 2a) and [35S]-sulfate
(Fig. 2b) incorporation, respectively. Biosynthesis rates
in the hydrogel (agarose and alginate) scaffolds were
significantly greater in chondrogenic conditions com-
pared to control conditions (1.2–20 fold greater; data
not shown). However, for scaffolds grown in chondro-
genic conditions, protein and proteoglycan biosynthesis
rates were significantly lower for the agarose scaffolds
than those of the alginate and gelatin scaffolds
throughout most of the culture ( po0:05). Whennormalized by the DNA content (Figs. 2c and d),
protein and proteoglycan biosynthesis rates in the
gelatin scaffolds were significantly greater than agarose
(31%) and alginate (68%), respectively, on day 1
( po0:05). However, the differences between biosynth-esis rates in the different scaffolds diminished by days 14
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and 28. Regardless of culture conditions or scaffold
biomaterial, incorporation rates decreased significantly
with time ( po0:05).
3.2. Biochemical composition
Biochemical analysis of the scaffolds was performed
to quantify the DNA, S-GAG, and OHP content of
the scaffolds at different times in culture. The DNA,
S-GAG, and OHP content of scaffolds grown in
chondrogenic conditions were significantly higher than
their control conditions counterparts on days 7, 14,
and 28. For scaffolds grown in chondrogenic conditions,
the DNA content of gelatin scaffolds was 37–51%
greater than agarose and alginate scaffolds on days 14
and 28 (Fig. 3a, po0:05). DNA content increased,reaching peak values of nearly 4.3 mg/scaffold on day 7
for the agarose and alginate and 6.5mg/scaffold on day
14 for the gelatin with insignificant declines afterwards.
For scaffolds grown in chondrogenic conditions, there
were no significant differences in S-GAG content among
the scaffold materials (Fig. 3b), whereas the OHP
content in gelatin was 28–47% greater than agarose
and alginate on days 14 and 28 (Fig. 3c, po0:05).
Furthermore, the S-GAG and OHP content increased
significantly by 2.5–9 fold between days 1 and 28 for all
scaffold materials grown in chondrogenic conditions.
When normalized by DNA content, S-GAG (Fig. 4a)
and OHP (Fig. 4b) content increased significantly
between days 1 and 28 for all scaffold materials grown
in chondrogenic conditions ( po0:05). However, ingeneral, there were no significant differences between
the different scaffold materials.
The accumulation of cartilage matrix macromolecules
was evident in the agarose and alginate hydrogel in disks
cultured under chondrogenic conditions, as demon-
strated by the positive immunohistochemical staining
against the 2B6 epitope of chondroitin sulfate and type
II collagen (Fig. 5). The staining was most intense in the
pericellular matrix, characteristics associated with cells
found in native cartilage. Positive staining against the
same antigens was also observed in the gelatin scaffolds.
In areas of sparse cell density where little contraction
had occurred, the staining was confined to regions of
neomatrix within the folds of the scaffold (Fig. 5c). By
contrast, in regions where significant contraction has
occurred, there was intense staining of both antigens in a
hyper cellular matrix (Fig. 5f ).
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Fig. 1. Cell viability and morphology in the different scaffold materials visualized using confocal laser scanning microscopy and the Live–Dead
fluorescent probes. Cells in agarose (a) and alginate (b) scaffolds displayed a spherical morphology that persisted throughout the culture period,
regardless of culture conditions. By contrast, the cells in the gelatin scaffolds (c) displayed a distinct ‘‘fibroblastic’’ morphology at day 7. By day 28,
the cells in the gelatin scaffolds proliferated and became confluent with significant cell–cell contact as they exerted considerable contraction of the
scaffolds (d).
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3.3. Biomechanical properties
The stress–strain behavior of all scaffold materials
was linear in both compression and shear stress-
relaxation experiments over the range of strains used.
There were no significant effects of culture conditions on
the mechanical properties of the hydrogel materials
(agarose and alginate), however, after 28 days gelatin
scaffolds grown in chondrogenic conditions had equili-
brium compressive and shear moduli that were 86%,
and 160%, respectively, greater than gelatin scaffolds
grown in control conditions ( po0:05; data not shown).The equilibrium compressive modulus (Fig. 6a) showed
a 48% and 53% softening between days 0 and 14 for the
agarose and alginate disks, respectively ( po0:05). Bycontrast, the equilibrium compressive modulus of
gelatin scaffolds increased by 3.9 and 4.6 fold of day
zero values by days 14 and 28, respectively ( po0:05).The equilibrium shear modulus (Fig. 6b) increased
significantly over time in chondrogenic culture by 2.6,
1.8 and 6 folds for the agarose, alginate, and gelatin
scaffolds, respectively. Likewise, the complex shear
modulus at o ¼ 10rad/s and go ¼ 0:05 (Fig. 6c)increased over time by 2.5, 1.8 and 8.3 folds for the
agarose, alginate, and gelatin scaffolds, respectively. By
the end of 28 days in chondrogenic culture, the
equilibrium shear modulus of alginate and gelatin was
22% and 67% of agarose ( po0:05). Similarly, thedynamic shear modulus at o ¼ 10 rad/s (|G (10 rad/s)|)
of alginate and gelatin was 22% and 79% of agarose,
respectively ( po0:05) (Fig. 7a). Values for the complexshear modulus jG ðoÞj showed linear trends with the
logarithm of frequency. The loss angle (d) showed no
specific trends with frequency (Fig. 7b). The loss angle
for all scaffold materials was less than 15, indicating
that all scaffolds tested behave like viscoelastic solids.
Multiple linear regression analysis suggested that
increases in G ; and jG j; were significantly associatedwith increases in SGAG content (Table 2, po0:05).Increases in E and d were associated with increases in
OHP content, though not significantly (Table 2,
p ¼ 0:09).
4. Discussion
Cellular based tissue engineering approaches have
increasingly used adult stem cells from different sources
including bone marrow [39–42], trabecular bone [43],
muscle [44], and adipose tissue [26,31–33,45–48]. Despite
the many advantages of these abundant and accessible
cells, progress in their utility in tissue engineering has
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Fig. 2. Protein and proteoglycan synthesis rates in chondrogenic culture conditions, quantified by [3H]-proline (a) and [35S]-sulfate (b) incorporation,
respectively, were significantly higher for the gelatin scaffolds compared to agarose and alginate cultured in chondrogenic conditions early in culture.
When normalized by the DNA content (c and d), the differences between biosynthesis rates in the different scaffolds diminished especially at later
times in culture. Data presented are mean7standard deviation. po0:05; po0:01:
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been limited by our ability to exercise precise control
over the cells’ differentiation potential. While much of
the stem cell based-tissue engineering research has been
focused on controlling differentiation using soluble
chemical factors (such as growth factors) and/or
manipulating the mechanical signals to which the cells
are exposed, less attention has been paid to the
importance of the biomaterial scaffold in regulating
differentiation and tissue growth [10,11]. In this study,
we corroborated our previous findings that hADAS cells
can differentiate into a chondrocytic phenotype under
defined culture conditions [26,49]. We further demon-
strated that the functional properties of tissue engi-
neered-cartilage vary with culture conditions, culture
time, and the choice of the scaffolding biomaterial.
Culturing the hADAS cell-laden constructs in chon-
drogenic media containing TGF-b1 significantly in-
creased DNA, S-GAG, and OHP content over control
conditions by nearly 3 fold in the hydrogel materials and
1.5 fold in the gelatin disks. In general, there were no
significant differences in the S-GAG content of all
scaffolds cultured in chondrogenic conditions, although
the DNA and OHP contents in the gelatin scaffolds was
greater than those in the hydrogels late in culture.
Furthermore, the biosynthesis rates of proteins and
proteoglycans were significantly higher for gelatin disks
compared to the agarose and alginate hydrogels. While
these results are similar to the previous observation that
TGF-b1 stimulates chondrogenic differentiation of
adult stem cells [49], they also imply that the cells’
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Fig. 3. Biochemical analysis of the scaffolds at different times in chondrogenic culture conditions. The DNA content (a) of gelatin scaffolds was
significantly higher than agarose and alginate on days 14 and 28. DNA content in all scaffolds increased initially reaching peak values on day 7 for
the agarose and alginate and on day 14 for the gelatin with insignificant declines afterwards. There were no significant differences in s-GAG content
among the scaffold materials (b). OHP content in gelatin was significantly higher than agarose and alginate on days 14 and 28 (c). Data presented are
mean7standard deviation. po0:05; po0:01:
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response to chondrogenic mediators may depend on the
physical and biological properties of the biomaterial
scaffold. These properties may include the diffusion
rates that regulate nutrient and metabolic waste trans-
port, the ability to regulate the cellular morphology that
is thought to affect differentiation, and the presence of
bioactive ligands that can provide anchorage sites forcell attachment.
We have recently shown that molecular diffusion
coefficients in agarose, alginate, and gelatin constructs
seeded with hADAS cells, measured by fluorescence
recovery after photobleaching (FRAP) of dextran
molecules of equal or greater size than culture nutrients
and growth factors (70 kDa), are at least twice those in
native cartilage even after 28 days in culture with no
significant differences among the constructs [50]. The
implications of these findings are two fold: (1) the
differences in molecular diffusion kinetics do not fully
explain the differences in cell and tissue growth in these
constructs and (2) the transport of nutrients and
metabolites to cells within the constructs is not hindered
in the early stages of tissue generation.
The ability of the scaffolds to biologically interact
with the cells, however, may explain some of the
differences in tissue growth within the constructs. The
entrapment of the cells in the hydrogels imposed a
spherical cellular morphology, whereas the cells seeded
in gelatin displayed various morphologies but were
predominantly of fibroblastic morphology. These find-
ings, together with the biochemical and biological
properties we measured, suggest that the beneficial
effects of spherical cellular morphology in chondrogen-esis may be hindered in the absence of bioactive cell
attachment ligands within the matrix. In addition, the
importance of providing a natural substrate for cell
attachment is manifested by the cell-mediated contrac-
tion of the gelatin scaffolds and the concomitant
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Fig. 4. S-GAG and hydroxyproline content normalized by DNA
content. The normalized s-GAG (a) and hydroxyproline (b) content
increased significantly between days 1 and 28 for all scaffold materials.
However, in general, there were no significant differences between the
different scaffold materials. Data presented are mean7standard
deviation. po0:05:
Fig. 5. Immunohistochemical sections of the agarose (a,d), alginate (b,e), and (c, f) scaffolds cultured up to 28 days in chondrogenic conditions.
Sections were stained for antibodies against the 2B6 epitope of chondroitin sulfate (CS; 20 , Scale bar 50m) and Collagen type II (Coll-II; 40 ,
Scale bar 100 m).
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increases in cell proliferation and collagen synthesis,
which supports previous findings in the literature
[51,52]. Moreover, recent studies have demonstrated
that overcoming the inert nature of the alginate
hydrogel by inserting RGD-containing peptide se-
quences promotes cell multiplication and cellular and
structural organization [53].The growth of the tissue engineered cartilage con-
structs appears to have progressed in two stages that
depend upon the biomaterial scaffold. The first stage, a
cell growth phase, was characterized by increased cell
proliferation and lasted up to 7 days in the hydrogel
materials and continued up to 14 days in the gelatin
scaffolds. The second stage can be described as a cell
differentiation and tissue growth stage, which was
characterized by decreased proliferation and increased
ARTICLE IN PRESS
Table 2
Multiple linear regression correlations between the biomechanical
properties and the biochemical composition of the tissue-engineered
cartilage constructs
Biomechanical Biochemical Slope Intercept R2 p
E OHP 30.56 8.67 0.28 p ¼ 0:08
SGAG 29.2 7.69 0.22 p ¼ 0:12SGAGOHP 109.11 9.51 0.19 p ¼ 0:16
G OHP 10.83 1.56 0.2 p ¼ 0:15SGAG 14.89 0.87 0.32 po0:05
SGAGOHP 56.55 1.67 0.28 p ¼ 0:07d OHP 21.25 5.44 0.26 p ¼ 0:09
SGAG 13.96 5.42 0.10 p ¼ 0:32SGAGOHP 94.88 5.82 0.28 p ¼ 0:08
|G | OHP 14.98 1.83 0.24 p ¼ 0:10SGAG 19.62 0.81 0.36 po0:05
SGAGOHP 76.72 1.99 0.34 po0:05
Fig. 6. Biomechanical properties of the scaffold materials at different times in chondrogenic culture. The elastic compressive modulus (a) of agarose
and alginate showed no improvements between days 0 and 28 following a significant decrease on day 14. By contrast, the elastic compressive modulus
of gelatin scaffolds increased significantly with time reaching values comparable with agarose on day 28. The elastic shear modulus (b) and the
dynamic shear modulus (c), at o ¼ 10 rad/s and go ¼ 0:05; increased significantly with time for all scaffold materials, and was significantly highest foragarose. Data points represent the mean7SEM. po0:01:
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collagen and proteoglycan deposition. An improved
understanding of the interactions between the cell and
the biomaterial scaffold (cell shape, cell–cell contact,
cytoskeletal changes, and cell–matrix interactions) may
improve the ability to control the switch between
cellular proliferation and differentiation [51].
The compressive Young’s modulus of the hydrogel
scaffolds (alginate and agarose) decreased in value
between days 0 and 14, suggesting that the hydrogels
have experienced softening, possibly due to a loss of the
cross-linking Ca2+ cations [54] in alginate and a loss in
gel stability due to thermal factors in agarose. However,
the subsequent increase in the compressive moduli in
agarose and alginate between days 14 and 28 is likely
due to increases in new matrix synthesis. The compres-
sive Young’s modulus of the gelatin scaffolds increasedsignificantly over time, reaching values comparable to
agarose by day 28. The mechanism behind the stiffening
of the gelatin scaffolds likely involves the packing of the
scaffold material as a result of the cell-mediated
contraction and the deposition of matrix macromole-
cules within the scaffold. Similarly, the equilibrium and
dynamic shear moduli increased significantly over time
for all scaffold materials, with agarose and gelatin
having shear moduli almost 3 times greater than
alginate. However, it should be noted that the compres-
sive and shear moduli of these scaffolds are on the order
of 5% or less of those of native cartilage [55,56]. These
results are not different from observations reported by
others [17,54,57], although it was recently suggested that
the mechanical properties of agarose disks can be
improved by increasing cell seeding density and the
application of a compressive loading regimen to
stimulate neotissue synthesis [17,58]. Even with the use
of large numbers of chondrocytes and prolonged culture
periods in these studies, the application of mechanical
loading improved the functional properties of the
chondrocyte-seeded agarose constructs to no more than
20% of native cartilage [58]. These studies, while
demonstrating the importance of in vitro mechanical
conditioning and cell seeding density, indirectly under-
score the importance of the inherent mechanical proper-
ties of the biomaterial scaffold and suggest that even the
use of large numbers of cells and prolonged culture
periods might not be sufficient to overcome the
mechanical deficiencies of the hydrogels.
Our results also indicate that increases in G and jG j
for all scaffolds are significantly correlated with
increases in S-GAG content and with the interaction
between S-GAG and OHP (Table 2), but not with OHP
alone. This intriguing finding is similar to previous
studies using chondrocytes in agarose disks [58] and
gives more credence to the presence of a structure–
function relationship in these tissues. Although our
analysis did not examine the structure of the developing
collagen and proteoglycan networks, our data indicatethat manipulating the composition and structure of
these tissue-engineered constructs may have important
implications on the construct’s ability to assume their
mechanical functions.
In conclusion, it is quite apparent that the biomaterial
scaffold of choice influences the growth and differentia-
tion of adult stem cells. Biologically active biopolymers,
such as gelatin, have distinct advantages stemming from
the fact that they modulate cell functions in manners
that can be exploited to create biologically functional
tissue-engineered grafts. While neither of the biomater-
ials studied approached native cartilage mechanical
properties they demonstrated significant composition-
function relationships that could provide important
clues for engineering functional tissues.
Acknowledgements
This study was supported in part by Artecel Sciences,
Inc., the North Carolina Biotechnology Center, the
Kenan Institute, and NIH grant AR49294. We would
like to thank Dr. Lori Setton and Charlene Flahiff for
ARTICLE IN PRESS
Fig. 7. Typical dynamic data for the frequency sweep (shear) response for the different scaffold materials at go ¼ 0:05 and o ¼ 12100rad/s.Scaffolds were cultured 28 days in chondrogenic conditions. Data points represent the mean7SEM.
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help with the biomechanical testing; and Julie Fuller and
Steve Johnson for help with the histology.
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