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Biodegradable, Photocrosslinked Alginate Hydrogels With Independently
Tailorable Physical Properties and Cell Adhesivity
Article in Tissue Engineering Part A · September 2010
DOI: 10.1089/ten.TEA.2010.0096 · Source: PubMed
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TISSUE ENGINEERING: Part A
Volume 16, Number 9, 2010
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2010.0096
Biodegradable, Photocrosslinked Alginate Hydrogels
with Independently Tailorable Physical Properties
and Cell Adhesivity
Oju Jeon, Ph.D.,1 Caitlin Powell,1 Shaoly M. Ahmed,1 and Eben Alsberg, Ph.D.1,2
Biocompatible polymers capable of photopolymerization are of immense interest for tissue engineering applications as they can be injected in a minimally invasive manner into a defect site and, then upon application of
ultraviolet light, rapidly form hydrogels in situ. Cell adhesion interactions with a biomaterial are known to be
important in regulating cell behaviors such as proliferation and differentiation. Therefore, we have covalently
modified photocrosslinkable alginate with cell adhesion ligands containing the Arg-Gly-Asp amino acid sequence to form biodegradable, photocrosslinked alginate hydrogels with controlled cell adhesivity. This unique
polymer system allows for independent modulation of the physical and biochemical signaling environment
presented to cells. The physical properties of the hydrogels such as elastic moduli, swelling ratios, and degradation profiles were similar at the same crosslinking density regardless of the presence of adhesion ligands.
Chondrocytes seeded on the surface of the adhesion ligand-modified hydrogels were able to attach and spread,
whereas those seeded on unmodified hydrogels exhibited minimal adherence. Importantly, the adhesion-ligandmodified hydrogels enhanced the proliferation and chondrogenic differentiated function of encapsulated
chondrocytes as demonstrated by increased DNA content and production of glycosaminoglycans compared to
unmodified control hydrogels. This new photocrosslinkable, biodegradable biomaterial system in which the
soluble (e.g., growth factors) and insoluble (e.g., cell adhesion ligands) biochemical signaling environment and
the biomaterial physical properties (e.g., the elastic moduli) can be independently controlled may be a powerful
tool for elucidating the individual and combined effects of these parameters on cell function for cartilage tissue
engineering and other regenerative medicine applications.
Introduction
H
ydrogels, which are used extensively in biomedical
applications as drug and gene delivery vehicles and
tissue engineering scaffolds, are water-insoluble threedimensional (3D) networks formed via the crosslinking of
water-soluble polymers. Hydrogels made from natural
polymers such as alginate, chitosan, collagen, hyaluronate,
and dextran are frequently used in tissue regeneration
strategies because they are either components of or have
similar macromolecular structure to constituents of natural
tissue extracellular matrix (ECM). For example, ionically
crosslinked alginate has great potential as a biomaterial for
tissue engineering because it can form highly hydrated hydrogels that present a hospitable environment for transplanted cells and cellular infiltration. Unfortunately, due to
the limited control over the loss of crosslinking ions,1 it is
difficult to manipulate many of the physical properties of
these gels, such as their mechanical properties and degra-
dation rates, which have been shown to be important parameters in regulating cell behavior and new tissue
formation.2,3 To overcome these limitations, we recently reported on the development of new methacrylated alginate
macromers that can be photopolymerized to form hydrogels
with controllable mechanical properties, swelling ratios, and
degradation rates.4 The photocrosslinked alginate hydrogels
exhibit excellent cytocompatibility, as encapsulated cells
within the material remain highly viable, and are promising
as cell carriers for tissue engineering applications since
macromer solutions containing cells can be injected minimally invasively into the target tissue defect and, then upon
application of light, rapidly transformed into hydrogels
in situ. However, due to the hydrophilic nature of alginate
and resultant low protein adsorption, cells are unable to interact with the alginate via cell surface receptors.5
Since cell adhesion interactions with a biomaterial are important in directing cell behaviors (e.g., proliferation, migration, differentiation) and the development of new tissue,6,7
Departments of 1Biomedical Engineering and 2Orthopaedic Surgery, Case Western Reserve University, Cleveland, Ohio.
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cell adhesion ligand-modified photopolymerized biomaterials have been widely used for regulating cell function in tissue engineering applications. A variety of synthetic [e.g.,
poly(ethylene glycol), poly(vinyl alcohol)], natural (e.g.,
alginate, hyaluronic acid, and chitosan), and hybrid photopolymerizable biomaterials have been modified with ArgGly-Asp (RGD)-containing peptide sequences and others to
regulate cell–biomaterial interactions.8–19 There is a paucity of
photopolymerizable biomaterial systems, however, that permit independent regulation of biopolymer physical and biochemical properties. In this study, photocrosslinkable alginate
was covalently modified with a peptide containing the RGD
sequence using standard carbodiimide chemistry to influence
chondrocyte adhesion, spreading, proliferation, and differentiated function. To our knowledge this is the first report of a
photocrosslinkable alginate hydrogel system with tunable
biodegradation and mechanical properties that offers the
additional capacity to indepently regulate its cell adhesive
properties. The ultimate goal of this work was to engineer a
photocrosslinkable, biodegradable biomaterial system in
which the soluble (e.g., growth factors) and insoluble (e.g., cell
adhesion ligands) biochemical signaling environment and the
biomaterial physical properties (e.g., the elastic moduli) can
be separately controlled so that their individual and combined
effects on cell function may be elucidated.
Materials and Methods
Preparation of RGD-modified methacrylated alginate
RGD-modified methacrylated alginate was synthesized
in a two-step reaction utilizing standard carbodiimide chemistry (Fig. 1). Low-molecular-weight sodium alginate
(37,000 g/mol) was prepared by irradiating Protanal LF 20/
40 (196,000 g/mol; FMC Biopolymer, Philadelphia, PA) at a
gamma dose of 5 Mrad. Twenty-five percent actual methacrylation of alginate carboxylic acid groups was performed
as described previously.4 Methacrylated alginate solutions
(1%, w/v) were prepared with 50 mM of 2-(N-morpholino)ethanesulfonic acid hydrate (Sigma, St. Louis, MO) buffer
solution containing 0.5 M NaCl (Sigma) at pH 6.5, and
sequentially mixed with N-hydroxysuccinimide (Sigma) and
1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC;
Sigma). The molar ratio of N-hydroxysuccinimide to EDC
was 0.5:1.0, and the weight ratio of EDC to methacrylated
alginate was 1.0:20.7. The Gly-Arg-Gly-Asp-Ser-Pro (Commonwealth Biotechnologies, Richmond, VA) amino acid
peptide sequence was added to the methacrylated alginate
solution at a weight ratio of 10 mg/g methacrylated alginate.
After reacting for 24 h at 48C, the reaction was stopped by
addition of hydroxylamine (0.18 mg/mL; Sigma), and the
solution was purified by dialysis against ultrapure deionized
water (diH2O) (MWCO 3500; Spectrum Laboratories, Rancho Dominguez, CA) for 3 days, treated with activated
charcoal (0.5 mg/100 mL, 50–200 mesh; Fisher, Pittsburgh,
PA) for 30 min, filtered (0.22 mm filter), and lyophilized.
Control methacrylated alginate was prepared in the same
manner but without the presence of peptide.
Characterization of RGD-modified methacrylated alginate
To verify the RGD modification of the methacrylated
alginate, an 1H-nuclear magnetic resonance (1H-NMR)
JEON ET AL.
spectra of RGD-modified methacrylated alginate was recorded. RGD-modified methacrylated alginate was dissolved in deuterium oxide (D2O Sigma) and placed in an
NMR tube. The 1H-NMR spectra of the RGD-modified
methacrylated alginate was recorded on a Varian Unity-300
(300 MHz) NMR spectrometer (Varian, Palo Alto, CA) using
tetramethylsilane as an internal standard. To analyze the
degree of peptide modification, a ninhydrin assay was
performed. Briefly, 100 mg of RGD-modified methacrylated
alginate was dissolved in 5 mL of 1 M sodium acetate buffer
(pH 5), and 5 mg of ninhydrin reagent was added. The
mixture was kept in boiling water for 20 min. After incubation, 75 mL of a diH2O/absolute ethanol mixture (1/1, v/
v) was added, and the reaction mixture was cooled to room
temperature for 2 h in complete darkness. Ninhydrin reacted with free amino groups and created a water-soluble
blue compound. The amount of free amino groups in the
RGD-modified methacrylated alginate was determined by
measuring the ultraviolet (UV) absorbance of the supernatant at 570 nm. Methacrylated alginate and glycine (Fisher)
were used as the control and the standard, respectively.
Photocrosslinking
To fabricate RGD-modified or unmodified photocrosslinked alginate hydrogels, RGD-modified methacrylated alginate (0.2 g) or unmodified methacrylated alginate (0.2 g)
was dissolved in Dulbecco’s modified Eagle’s medium with
high glucose (DMEM; Sigma) or diH2O (10 mL) with 0.05%
(w/v) photoinitiator (Irgacure D-2959; Sigma) for ultimate
placement in DMEM or diH2O, respectively. The alginate
solutions were injected between two glass plates separated
by 0.75 mm spacers and photocrosslinked with 365 nm UV
light (Model ENF-260C; Spectroline, Westbury, NY) at
*1 mW/cm2 for 10 min to form the hydrogels. Photocrosslinked hydrogel disks were created using a 6-mmdiameter biopsy punch and placed in DMEM or diH2O for
swelling and degradation studies, mechanical testing, and
culture of cells on the hydrogel surfaces.
Swelling and degradation of hydrogels
The RGD-modified or unmodified photocrosslinked alginate hydrogels were lyophilized, and dry weights (Wi) were
measured. Dried hydrogel samples were immersed in 50 mL
of DMEM or diH20 and incubated at 378C to reach an
equilibrium swelling state. The DMEM or diH20 was replaced every 3 days. Over the course of 8 weeks, samples
were removed from the DMEM or diH20, and the swollen
(Ws) hydrogel sample weights were measured. The swelling
ratio (Q) was calculated as follows: Q ¼ Ws/Wi (N ¼ 3 for
each time point). After weighing the swollen hydrogels, the
samples were lyophilized and weighed (Wd). The percent
mass loss was calculated as follows: [(Wi Wd)/Wi]100
(N ¼ 3 for each time point).
Mechanical testing
The elastic moduli of the RGD-modified and unmodified
photocrosslinked alginate hydrogels were determined by
performing constant strain rate compression tests using a
Rheometrics Solid Analyzer (RSAII; Rheometrics, Piscataway, NJ) equipped with a 10-N load cell. The RGD-
PHOTOCROSSLINKED ALGINATE HYDROGEL WITH CONTROLLED CELL ADHESIVITY
2917
FIG. 1. (A) Schematic illustration for the preparation and photocrosslinking of RGD-modified methacrylated alginate. (B)
1
H-NMR spectra of RGD-modified methacrylated alginate in D2O. Proton peaks of the methacrylate and coupled peptide are
labeled a, b, and c and identified in the text. Morphology of RGD-modified and unmodified photocrosslinked alginate
hydrogels after 24-h equilibration in (C) DMEM and (D) deionized water. RGD, Arg-Gly-Asp; DMEM, Dulbecco’s modified
Eagle’s medium; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide; UV, ultraviolet;
GRGDSP, Gly-Arg-Gly-Asp-Ser-Pro. Color images available online at www.liebertonline.com/ten.
modified and unmodified photocrosslinked alginate hydrogel disks were prepared as described in the photocrosslinking section and maintained in DMEM or diH2O at
378C. After 24-h incubation, swollen alginate hydrogel
disks were punched once again to form 6mm diameter
disks, their thickness was measured using calipers, and
uniaxial, unconfined compression tests were performed on
the hydrogel disks at room temperature using a constant
crosshead speed of 5%/s. Elastic moduli of photocrosslinked alginate hydrogels were determined from the
slope of stress versus strain plots, and limited to the first
5% of strain (N ¼ 3).
Cell culture on the alginate hydrogels
Chondrocytes (passage number 2) isolated from bovine
articular cartilage as previously reported20 were seeded on
RGD-modified or unmodified photocrosslinked alginate
hydrogel disks in DMEM (1 mL) containing 10% fetal bovine serum (FBS) at a seeding density of 1104 cells/cm2 in
24-well tissue culture plates and allowed to adhere for 4 h in
a humidified incubator at 378C with 5% CO2. The RGDmodified or unmodified photocrosslinked alginate disks
were then transferred to new plates containing fresh media (1 mL), and cultured. The viability and morphology of
2918
JEON ET AL.
FIG. 2. (A) Representative stress–strain curves and (B) elastic moduli in compression of RGD-modified and unmodified
photocrosslinked alginate hydrogels after 24-h equilibration in DMEM. (C) Swelling ratios and (D) in vitro degradation of
RGD-modified and unmodified photocrosslinked alginate hydrogels in DMEM. (E) Swelling ratios and (F) in vitro degradation of RGD-modified and unmodified photocrosslinked alginate hydrogels in deionized water. *p < 0.05 compared to
unmodified control.
adhered cells on the alginate disks were examined using a
live/dead assay comprised of fluorescein diacetate (FDA;
Sigma) and ethidium bromide (EB; Sigma). FDA stains the
cytoplasm of viable cells green, whereas EB stains the nuclei
of nonviable cells orange-red. The staining solution was
freshly prepared by mixing 1 mL of FDA solution (1.5 mg/
mL of FDA in dimethyl sulfoxide; Research Organics, Cle-
veland, OH) and 0.5 mL of EB solution (1 mg/mL of EB in
phosphate-buffered saline [PBS]) with 0.3 mL of PBS (pH 8).
At predetermined time points, 20 mL of staining solution
was added into each well and incubated for 3–5 min at
room temperature, and then stained hydrogel–cell constructs were imaged using a fluorescence microscope
(ECLIPSE TE 300; Nikon, Tokyo, Japan) equipped with
PHOTOCROSSLINKED ALGINATE HYDROGEL WITH CONTROLLED CELL ADHESIVITY
2919
FIG. 3. (A) Fluorescence photomicrographs of live (FDA, green) and dead (EB, orange-red) chondrocytes cultured in vitro
for 2, 5, and 7 days on the surface of RGD-modified and unmodified photocrosslinked alginate hydrogels. (B) Fluorescence
photomicrographs of live (FDA, green) and dead (EB, orange-red) bovine chondrocytes encapsulated and cultured in RGDmodified and unmodified photocrosslinked alginate hydrogels in vitro for 2, 4, and 6 weeks, and (C) quantification of DNA/
dried hydrogel weight and (D) GAG/DNA content in the constructs. The scale bar indicates 200 mm and all photographs
were taken at the same magnification. *p < 0.05. FDA, fluorescein diacetate; EB, ethidium bromide; GAG, glycosaminoglycan.
Color images available online at www.liebertonline.com/ten.
2920
a digital camera (Retiga-SRV; Qimaging, Burnaby, BC,
Canada).
Encapsulation of chondrocytes
Chondrocytes (passage number 2) were photoencapsulated in RGD-modified or unmodified alginate hydrogels
by suspension in RGD-modified or unmodified methacrylated alginate solution (2% [w/v] in DMEM) with 0.05% (w/
v) photoinitiator. The cell/macromer solutions (300 mL,
1107 cells/mL) were pipetted into 96-well tissue culture
plates) and photocrosslinked with UV light for 10 min. The
resulting hydrogel–cell constructs were removed from the
wells, placed in new 24-well tissue culture plates with 1 mL
of fresh DMEM containing 10% FBS, and cultured in a humidified incubator at 378C with 5% CO2 for 6 weeks. The
viability of encapsulated chondrocytes in the photocrosslinked RGD-modified alginate hydrogels was investigated using the live/dead assay (N ¼ 3 for each time point).
Images were obtained at a depth halfway into the hydrogels
using a fluorescence microscope.
Biochemical assays for DNA content
and glycosaminoglycan production
At each time point, hydrogel–cell constructs were removed from media, homogenized, and digested in 1 mL
papain buffer solution (25 mg/mL papain [Sigma], 2 mM
cysteine [Sigma], 50 mM sodium phosphate [Fisher], and
2 mM ethylenediaminetetraacetic acid [Fisher], pH 6.5, in
nuclease-free water) at 658C for 3 h. Hoechst 33258 dye
(0.1 mg/mL in nuclease-free water; Acros Organics, Morris
Plains, NJ) was used for the DNA assay as previously described.21 Calf thymus DNA standards (Rockland
Immunochemicals, Gilbertsville, PA) were prepared with 0–
4 mg/mL DNA in nuclease-free water. After the centrifugation
of papain-digested samples, 100 mL of supernatant was mixed
with 100 mL of the prepared dye solution. Fluorescence intensity of the dye-conjugated DNA solution was measured in
96-well plates on a plate reader (358 nm excitation and 452 nm
emission; Safire, Tecan, Austria), and the DNA content was
calculated from a standard curve generated with the calf
thymus DNA. Glycosaminoglycan (GAG) content was measured using the standard dimethylmethylene blue (Sigma)
assay in 96-well plates.22 In each well, 50 mL of supernatant
was mixed with 250 mL of dye containing 16 mg/L dimethylmethylene blue and 3.04 g/L glycine (pH 1.5). The
absorbance was read at 595 nm using the plate reader.
Chondroitin-6-sulfate (Sigma) from shark cartilage was used
to construct the standard curve.
Encapsulation of chondrocytes and transforming
growth factor-beta 1
An in vitro transforming growth factor-beta 1 (TGF-b1)
release study was performed to examine the time course of
JEON ET AL.
TGF-b1 release from photocrosslinked RGD-modified alginate hydrogels. TGF-b1 (0.75 mg; PeproTech, Rochy Hill, NJ)
was added to RGD-modified methacrylated alginate solution
(1.5 mL, 2% [w/v] in diH2O) with 0.05% (w/v) photoinitiator. After gently mixing for 5 min, aliquots (300 mL) of
solution were placed in 96-well tissue culture plates and
photocrosslinked as before for 10 min. Each photocrosslinked
hydrogel (150 ng TGF-b1/hydrogel) was immerged in a 15mL centrifuge tube containing 10 mL PBS and incubated at
378C (N ¼ 5). At predetermined time points, the supernatant
was withdrawn and fresh buffer was replenished. The
amount of TGF-b1 in the supernatants was determined using
an enzyme-linked immunosorption assay kit (Human TGFb1 Duoset; R&D Systems, Minnepolis, MN). TGF-b1-loaded
unmodified photocrosslinked alginate hydrogels were used
as a comparative control (N ¼ 5).
RGD-modified and unmodified photocrosslinked alginate
hydrogel–cell constructs containing TGF-b1 (100 ng/
hydrogel) were prepared in 96-well tissue culture plates as
described above (300 mL, 1107 cells/mL), removed from the
wells, placed in new 24-well tissue culture plates with 1 mL
of fresh DMEM containing 10% FBS, and cultured in a humidified incubator at 378C with 5% CO2 for 6 weeks (N ¼ 3).
As a control, hydrogel–cell constructs without TGF-b1 were
cultured in DMEM containing 10% FBS and 10 ng/mL TGFb1 (N ¼ 3). The medium was changed every 3 days. At predetermined time points, live/dead, GAG, and DNA assays
were performed as described above.
Statistical analysis
All quantitative data are expressed as mean standard
deviation. Statistical analysis was performed with one-way
analysis of variance with Tukey honestly significant difference
post hoc test using Origin software (OriginLab, Northampton,
MA). A value of p < 0.05 was considered statistically significant.
Results and Discussion
Preparation and characterization of cell adhesion
peptide-modified methacrylated alginate
While alginate hydrogels provide space and mechanical
support for tissue regeneration, in their native form they do
not provide a mechanism for encapsulated cells to interact
and receive important signaling information via adhesion.
To partially mimic the cell adhesion capacity of native ECM,
specific cell adhesion ligands such as the ubiquitous cell
adhesion peptide sequence RGD,5,23,24 which is present in
numerous ECM molecules such as fibronectin, collagen, and
laminin,25,26 may be chemically incorporated onto substrate
surfaces and into biomaterial systems to promote cell–
biomaterial interactions. In fact, ionically crosslinked alginate
hydrogels covalently modified with RGD-containing peptides have been shown to promote proliferation of cells such
‰
FIG. 4. (A) Release profiles of TGF-b1 from RGD-modified and unmodified photocrosslinked alginate hydrogels over 3 weeks.
(B) Fluorescence photomicrographs of live (FDA, green) and dead (EB, orange-red) cells and (C) GAG/DNA content of bovine
chondrocytes encapsulated with TGF-b1 in RGD-modified and unmodified photocrosslinked alginate hydrogels cultured in
DMEM containing 10% fetal bovine serum and encapsulated bovine chondrocytes without TGF-b1 in RGD-modified and
unmodified photocrosslinked alginate hydrogels cultured in DMEM containing 10% fetal bovine serum and TGF-b1 for 2, 4, and
6 weeks. The scale bar indicates 200 mm and all photographs were taken at the same magnification. *p < 0.05 compared to 6
weeks in same group. TGF-b1, transforming growth factor-beta 1. Color images available online at www.liebertonline.com/ten.
2921
2922
as osteoblasts and chondrocytes and the formation of new
bone,7,27 cartilage,27 and blood vessels.28 In this study, peptides containing the RGD sequence were covalently coupled
onto the methacrylated alginate main chains to prepare the
first reported photocrosslinkable and biodegradable alginate
hydrogels with controlled cell adhesion as shown in Figure
1A. After alginate methacrylation, the remaining carboxylic
acid functional groups along the alginate polymer backbone
offered the potential for covalent modification with RGDcontaining cell adhesion ligands. 1H-NMR spectra of RGDmodified methacrylated alginate macromers (Fig. 1B) exhibit
proton peaks that were newly formed by the reaction with
the peptide Gly-Arg-Gly-Asp-Ser-Pro, which are located at
d2.75 (from aspartic acid) and 1.7 (from arginine)29 (Fig. 1Bc).
The proton peaks of vinyl methylene (Fig. 1Ba) and methyl
(Fig. 1Bb) protons of 2-aminoethyl methacrylate (AEMA) are
located at d6.2 and d5.7, and d1.9, respectively. The conjugated RGD concentration was 3.76 0.24 mg/g methacrylated alginate as measured by ninhydrin assay, which makes
the peptide coupling reaction efficiency 37.6%. The gross
morphologies of DMEM-equilibrated RGD-modified and
unmodified photocrosslinked alginate hydrogel disks are
exhibited in Figure 1C, and their mean diameters are
6.6 0.2 mm and 6.6 0.1 mm, respectively. There are no
significant differences in gross morphology or size between
the groups after 24 h equilibration. The gross morphologies
of the diH2O-equilibrated RGD-modified and unmodified
photocrosslinked alginate hydrogel disks are shown in Figure 1D, and their mean diameters are 10.1 0.6 mm and
8.9 0.5 mm, respectively. While there are no significant
differences in gross morphology or size between the diH2Oequilibrated groups after 24 h equilibration, these hydrogels
were significantly larger than the DMEM-equilibrated
groups. This difference in size may be attributable to
increased osmosis driven swelling in diH2O.
Elastic moduli, swelling kinetics, and degradation
of the RGD-modified and unmodified photocrosslinked
alginate hydrogels
To use this system to study the effects of controlled biomaterial cell adhesion on cell behavior as an independent
variable, it is important to demonstrate that the adhesion
ligand coupling modification minimally alters the biomaterial physical properties. To examine whether peptide modification has an effect on photocrosslinked hydrogel
mechanical properties, constant strain-rate compression tests
were performed after 24 h equilibration in DMEM. Representative stress–strain curves of the RGD-modified and
unmodified photocrosslinked alginate hydrogels (Fig. 2A)
are similar in shape. There was no significant difference in
compressive modulus between the two groups (Fig. 2B).
These results provide evidence that adhesion peptide modification of methacrylated alginate does not substantially
affect the crosslinked structure of photocrosslinked alginate
hydrogels because the compressive mechanical response of
the hydrogels was unaltered. In addition, the swelling ratio
changes and degradation profiles in DMEM and diH2O were
measured to further evaluate whether RGD modification has
an effect on the crosslinked structure of the alginate hydrogels. In DMEM, both RGD-modified and unmodified photocrosslinked alginate hydrogels showed rapid swelling for
JEON ET AL.
the first day (Fig. 2C). The swelling of both groups then
gradually increased over the course of 8 weeks. The hydrogels exhibited similar swelling kinetics for 4 weeks, and the
swelling ratio of the RGD-modified photocrosslinked alginate hydrogels was only slightly higher than that of unmodified alginate hydrogels at 8 weeks. These results also
indicate that RGD modification of methacrylated alginate
does not substantially affect the macromolecular structure of
photocrosslinked alginate hydrogel over time. The mass loss
(%) of alginate hydrogels in DMEM over time was determined as a measure of degradation (Fig. 2D). The degradation of photocrosslinked RGD-modified alginate hydrogels
was slightly faster than that of unmodified alginate hydrogels at 2 and 4 weeks, but there was no difference between
the two groups at 8 weeks. In diH2O, both RGD-modified
and unmodified photocrosslinked alginate hydrogels exhibited rapid swelling for the first day (Fig. 2E), increased up
to 1 week, and then gradually decreased. Compared to the
unmodified hydrogels, RGD-modified hydrogels exhibited
greater swelling at 1 day and 1 week. The unmodified hydrogels swelled to a greater extent at 2 and 4 weeks. The
RGD-modified hydrogels exhibited greater degradation than
the unmodified hydrogels after 2 and 4 weeks in diH2O
(Fig. 2F). However, both RGD-modified and unmodified
alginate hydrogels were completely degraded by 6 weeks.
Characterization of 2D and 3D chondrocyte cultures
Bovine chondrocytes were seeded onto RGD-modified
and unmodified photocrosslinked alginate hydrogels to
evaluate if the peptide modification would enhance chondrocyte adhesion and spreading. Chondrocytes adhered to
the surfaces of RGD-modified hydrogels by 4 h (data not
shown) and exhibited substantial spreading at 5 and 7 days
(Fig. 3A). Few chondrocytes seeded on the surfaces of unmodified hydrogels were able to adhere, and those that did
remained rounded through 7 days. These results indicate
that the chondrocyte cell adhesion and spreading were promoted by the immobilized adhesion ligands.
Chondrocytes were then photoencapsulated within RGDmodified or unmodified photocrosslinked alginate hydrogels
to provide a 3D culture environment that more closely resembles native cartilage tissue. To examine cell survival
during the photocrosslinking process and culture, the viability of the photoencapsulated chondrocytes in the alginate
hydrogels was evaluated using a live/dead assay. High cell
viability was observed throughout both alginate hydrogel
compositions for 6 weeks (Fig. 3B). Chondrocytes in native
cartilage are located in lacunae surrounded by ECM.30
ECM–cell interactions promote chondrocyte aggregation,31
reduce the level of chondrocyte apoptosis,32 and are essential
for chondrocyte proliferation, differentiation and survival.33,34 The DNA content of RGD-modified hydrogels was
signficantly greater than that of unmodified hydrogels at 2
and 6 weeks, indicating that RGD modification promoted
chondrocyte proliferation in alginate hydrogels (Fig. 3C).
DNA content also significantly increased over time in both
groups. The capacity of the new biomaterial system to enhance specific chondrogenic differentiated function, such as
production of cartilage ECM, was then investigated. After 2
and 4 weeks of culture, chondrocytes that were photoencapsulated in the RGD-modified alginate hydrogels pro-
PHOTOCROSSLINKED ALGINATE HYDROGEL WITH CONTROLLED CELL ADHESIVITY
duced significantly more GAG, one of the major constituents
of cartilage ECM,35 normalized to DNA content than cells in
the unmodified alginate hydrogels (Fig. 3D). No difference
was present at 6 weeks. This result demonstrates that regulating chondrocyte–ECM interactions through controlled integrin adhesion ligand signaling promotes and accelerates
the chondrogenic activity of chondrocytes encapsulated in
the photocrosslinked alginate hydrogels. Importantly, this
positive effect on chondrogenesis occurred in the absence of
any specific soluble chondrogenic factors other than those
present in the serum used. This finding corroborates other
reports where hydrogels modified with RGD-containing ligands enhanced the chondrogenic activity of encapsulated
chondrocytes without the exogenous addition of specific
chondrogenic growth factors.27,36
Encapsulation of chondrocytes and TGF-b1
Growth factors, such as TGF-b1, a member of the TGF-b
superfamily, are an important part of the soluble biochemical
signaling environment that regulates chondrogenesis during
development and promotes chondrocyte-specific cellular
function and cartilaginous ECM production.37 When chondrocytes are cultured in a 3D environment (e.g., aggregate
culture or in a hydrogel), TGF-b1 can stimulate synthesis of
cartilaginous ECM components such as GAG and collagen
type II.38,39 Therefore, the effect of adhesion ligand modification on the responsiveness of photoencapsulated chondrocytes to TGF-b1 delivered either exogenously in the cell
culture media or released from the alginate hydrogel itself
was investigated. TGF-b1 release profiles over 21 days were
similar from both peptide-modified and unmodified hydrogels with most TGF-b1 released within the first 4 days (Fig.
4A). This demonstrates that the peptide modification did not
affect TGF-b1 release. The photoencapsulated chondrocytes
exposed to TGF-b1 in the alginate hydrogels exhibited high
cell viability as observed throughout all construct compositions for 6 weeks using the live/dead assay (Fig. 4B).
Chondrogenic activity of the chondrocytes as measured by
GAG/DNA content revealed that TGF-b1, delivered in the
media or from the hydrogels, promoted GAG production
per cell (Fig. 4C). No significant difference in chondrogenic
activity was revealed between cells encapsulated in unmodified or RGD-modified alginate hydrogels that were
exposed to TGF-b1 either in the media or from the hydrogels at all time points. These results indicate that for the
specific conditions examined in this study (i.e., peptide type
and concentration, and TGF-b1 concentration), chondrogenic activity of the chondrocytes in the hydrogels was
more strongly influenced by the presence of TGF-b1 than
controlled ECM–cell interactions by RGD modification.
Importantly, however, similar levels of GAG production
were measured when growth factor was delivered via
either manner at all time points, even though release of
TGF-b1 from the alginate hydrogels is relatively rapid and
the total amount released was less than that presented exogenously. This biomaterial system will permit future
studies aimed at elucidating the relative roles of these
insoluble and soluble biochemical signaling parameters
individually and together on promoting chondrogenic
specific differentiated function.
2923
Conclusions
Herein we have engineered biodegradable, photocrosslinked alginate hydrogels with controlled cell adhesivity
properties. Cell adhesive and nonadhesive photocrosslinked
alginate hydrogels exhibited similar elastic moduli and
swelling ratios over time, whereas the peptide-modified
hydrogels degraded slightly faster than nonmodified hydrogels. Since peptides vary greatly in their chemical properties (e.g., hydrophilicity, charge, and secondary structure),
future studies will determine if the hydrogel physical
characterization results in this current report may be generalizable to other peptides. RGD modification promoted
chondrocyte adhesion and spreading on the surface of the
hydrogels, whereas minimal adhesion was supported on the
unmodified hydrogels. In addition, chondrocytes encapsulated in these hydrogels retained high viability with RGDmodified alginate, significantly enhancing production of
GAG for 2 and 4 weeks in the absence of soluble chondrogenic growth factors. Delivery of TGF-b1 from within the
hydrogels or the surrounding media to chondrocytes promoted GAG production to similar levels irrespective of
growth factor delivery route or presence of peptide modification. While in this study only a single type and concentration of adhesion peptide and growth factor were
examined, the novel biomaterial system described is highly
modular, and will allow for future studies using high
throughput systems to examine the effects of multiple adhesion peptide types and concentrations, growth factor types
and concentrations, and the degree of alginate methacrylation both separately and in combination on encapsulated cell
behavior. This new photocrosslinkable biomaterial with
controlled degradation and cell adhesive properties may find
great utility in cartilage tissue engineering and other regenerative medicine applications.
Acknowledgments
The authors thank Rachel Manthe for technical assistance
and acknowledge funding support from a New Scholar in
Aging Award from the Ellison Medical Foundation, the
Muskuloskeletal Transplant Foundation, and Biomedical
Research and Technology Transfer Grant 08-081 from the
Ohio Department of Development.
Disclosure Statement
No competing financial interests exist.
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2925
Address correspondence to:
Eben Alsberg, Ph.D.
Departments of Biomedical Engineering
and Orthopaedic Surgery
Case Western Reserve University
10900 Euclid Ave.
Cleveland, OH 44106
E-mail: eben.alsberg@case.edu
Received: February 12, 2010
Accepted: April 23, 2010
Online Publication Date: June 21, 2010
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