Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.

Abstract

Photocrosslinked and biodegradable alginate hydrogels were engineered for biomedical applications. Photocrosslinkable alginate macromers were prepared by reacting sodium alginate and 2-aminoethyl methacrylate in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide. Methacrylated alginates were photocrosslinked using ultraviolet light with 0.05% photoinitiator. The swelling behavior, elastic moduli, and degradation rates of photocrosslinked alginate hydrogels were quantified and could be controlled by varying the degree of alginate methacrylation. The methacrylated alginate macromer and photocrosslinked alginate hydrogels exhibited low cytotoxicity when cultured with primary bovine chondrocytes. In addition, chondrocytes encapsulated in these hydrogels remained viable and metabolically active as demonstrated by Live/Dead cell staining and MTS assay. These photocrosslinked alginate hydrogels, with tailorable mechanical properties and degradation rates, may find great utility as therapeutic materials in regenerative medicine and bioactive factor delivery.

Full text

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Photocrosslinked alginate hydrogels with tunable biodegradation rates
and mechanical properties
Oju Jeona, Kamal H. Bouhadirb, Joseph M. Mansourc,d, Eben Alsberga,d,*
aDepartment of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA
bDepartment of Chemistry, American University of Beirut, Beirut, Lebanon
cDepartment of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA
dDepartment of Orthopaedic Surgery, Case Western Reserve University, Cleveland, OH 44106, USA
a r t i c l e i n f o
Article history:
Received 6 November 2008
Accepted 19 January 2009
Available online xxx
Keywords:
Alginate
Biodegradable
Biomaterial
Hydrogel
Photopolymerization
Tissue engineering
a b s t r a c t
Photocrosslinked and biodegradable alginate hydrogels were engineered for biomedical applications.
Photocrosslinkable alginate macromers were prepared by reacting sodium alginate and 2-aminoethyl
methacrylate in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and
N-hydroxysuccinimide. Methacrylated alginates were photocrosslinked using ultraviolet light with 0.05%
photoinitiator. The swelling behavior, elastic moduli, and degradation rates of photocrosslinked alginate
hydrogels were quantified and could be controlled by varying the degree of alginate methacrylation. The
methacrylated alginate macromer and photocrosslinked alginate hydrogels exhibited low cytotoxicity
when cultured with primary bovine chondrocytes. In addition, chondrocytes encapsulated in these
hydrogels remained viable and metabolically active as demonstrated by Live/Dead cell staining and MTS
assay. These photocrosslinked alginate hydrogels, with tailorable mechanical properties and degradation
rates, may find great utility as therapeutic materials in regenerative medicine and bioactive factor
delivery.
? 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Hydrogels are widely used in biomedical applications as drug
delivery vehicles [1], encapsulation materials for immunoisolation-
based cell therapeutics [2], wound dressings [3], anti-adhesion
materials [4], and tissue engineering scaffolds [5] because many of
their physical properties are similar to natural tissue [6]. Hydrogels
are insoluble 3-D networks of crosslinked hydrophilic polymers
which exhibit a high degree of swelling in aqueous environments
[7]. When cells are incorporated into hydrogels, the highly swollen
state of these biomaterials facilitates transport of nutrients into and
cellular waste out of the hydrogels [8]. The crosslinked 3-D
hydrogel structure can provide space and mechanical stability for
new tissue formation [9]. These properties make hydrogels great
potential candidate biomaterials for tissue engineering.
Alginate, a linear unbranched polysaccharide derived from
seaweed that contains the repeating units of 1,4-linked b-D-man-
nuronic acid anda-L-guluronic acid [10], is one of the most versatile
natural materials known to form hydrogels [11–16]. Alginates have
reversible gelling properties in aqueous solutions related to the
ionic interactions between divalent cations, such as calcium,
barium, and magnesium and carboxylic acid moieties on the
guluronic acid residues [17]. Since ionically crosslinked alginate
does not damage cells during the gelling process and is generally
considered biocompatible, there has been a great deal of interest in
the use of alginate hydrogels for drug delivery, cell encapsulation,
and tissue regeneration [17–25]. Although previous studies have
reported promising results, there is still limited control over the
mechanical properties, swelling ratios, and degradation profiles of
ionically crosslinked alginate hydrogels [26], which is likely due to
the uncontrollable loss of divalent cations into the surrounding
environment [27]. Several reports in the literature describe
approaches to chemically crosslink alginate microcapsules or
macroscopic hydrogels by utilizing intermolecular covalent cross-
linking ratherthan ionic crosslinking in order to synthesize alginate
hydrogels with a wide range of mechanical properties [28–31].
However, the reagents and reaction conditions used with these
* Corresponding author. Department of Biomedical Engineering, Case Western
Reserve University, Cleveland, OH 44106, USA. Tel.: þ1 216 368 6425; fax: þ1 216
368 4969.
E-mail address: eben.alsberg@case.edu (E. Alsberg).
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
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approaches sometimes prove unfavorable or toxic to the encapsu-
lated cells or growth factors [28].
Photocrosslinked hydrogels have recently gained increased
attention in biomedical applications because aqueous macromer
solutions containing cells and/or bioactive factors can be delivered
in a minimally invasive manner and then rapidly crosslinked in
physiological conditions in situ following brief exposure to ultra-
violet (UV) light [32,33]. Following exposure to UV or visible light,
chemicals called photoinitiators generate free radicals that can
initiate the polymerization process. The free radicals that are
created convert aqueous macromer solutions into hydrogels [34].
Short exposure to low-intensity light and the appropriate choice of
a photoinitiator allow for photopolymerization to occur with
limited deleterious effects to cells and bioactive molecules [35]. The
mechanical properties and swelling behavior of photocrosslinked
hydrogels may be readily controlled by varying the photo-curable
moiety [36,37].
The purpose of this study was to develop photocrosslinked
alginate hydrogels that are biodegradable for tissue engineering
applications. Degradability was a key design criteria in this study as
the ability to specifically tailor the degradation rate of biomaterial
carriers to that of new tissue formation by transplanted and host
cells has been shown to greatly enhance the rate and extent of new
tissue formation in tissue engineering [38]. We have engineered
alginate hydrogels with hydrolytically degradable ester linkages
incorporated during the photopolymerization process. The effect of
changing the biopolymer crosslinking density by varying the
degree of alginate methacrylation was quantified through exami-
nation of hydrogel swelling behavior, elastic moduli, and degra-
dation profiles. The viability of cells cultured with synthesized
macromer and also both in the presence of and inside of photo-
crosslinked hydrogels was examined in vitro. While there has been
a previous report describing a photocrosslinked alginate hydrogel
system, these hydrogels remained stable in aqueous solution for
over one year [37], and thus are not biodegradable on a time scale
optimally compatible with tissue regeneration. In this paper we
describe a photocrosslinked alginate hydrogel system with
controllable mechanical properties and degradation rates. The
photocrosslinked and biodegradable alginate hydrogels developed
in this study may have broad biomedical applications.
2. Materials and methods
2.1. Synthesis of methacrylated alginate
The methacrylated alginate was prepared by reacting low molecular weight
alginate with 2-aminoethyl methacrylate (AEMA, Sigma, St. Louis, MO, USA). Low
molecular weight of sodium alginate (37,000 g/mol) was prepared by irradiating
Protanal LF 20/40 (190,600 g/mol, FMC Biopolymer, Philadelphia, PA, USA) at
a gamma dose of 5 Mrad. To synthesize the methacrylated alginate with 45.26%
theoretical methacrylation of uronic acid carboxylate groups, low molecular weight
sodium alginate (2 g) was dissolved in a buffer solution (1% w/v, pH 6.5) of 50 mM
2-morpholinoethanesulfonic acid (MES, Sigma) containing 0.5 M NaCl. N-hydroxy-
succinimide (NHS, 0.53 g; Sigma) and 1-ethyl-3-(3-dimethylaminopropyl)-carbo-
diimide hydrochloride (EDC, 1.75 g; Sigma) (molar ratio of NHS:EDC ¼1:2) were
added to the mixture to activate the carboxylic acid groups of the alginate. After
5 min, AEMA (0.76 g) (molar ratio of NHS:EDC:AEMA¼1:2:1) was added to the
product and the reaction was maintained at room temperature for 24 h. Less AEMA
was used to prepare alginates with lower degrees of theoretical methacrylation
while keeping the NHS:EDC:AEMA molar ratio constant. The mixture was precipi-
tated with the addition of excess of acetone, dried under reduced pressure, and
rehydrated to a 1% w/v solution in ultrapure deionized water (diH2O) for further
purification. The methacrylated alginate was purified by dialysis against diH2O
(MWCO 3500; Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) for 3 days,
filtered (0.22 mm filter), and lyophilized.
2.2. Photocrosslinking
To fabricate photocrosslinked alginate hydrogels, methacrylated alginate (0.2 g)
was dissolved in diH2O (10 ml) with 0.05% w/v photoinitiator (Irgacure D-2959,
Sigma). The alginate solutions were injected between two glass plates separated by
0.75 mm spacers and photocrosslinked with 365 nm UV light at w8–20 mW/cm2for
8 min to form the hydrogels. Photocrosslinked hydrogel disks were created using
a 6 mm diameter biopsy punch and placed in diH2O.
2.3.
1H NMR characterization
Methacrylated alginate was dissolved in deuterium oxide with 0.05% w/v pho-
toinitiator, placed in an NMR tube, and photocrosslinked at 365 nm UV light at
w8–20 mW/cm2for 8 min to form a hydrogel. The1H NMR spectra of the photo-
crosslinked alginate hydrogel were recorded on a Varian Unity-300 (300 MHz) NMR
spectrometer (Varian Inc., Palo Alto, CA, USA) using tetramethylsilane (TMS) as
internal standard. To analyze the methacrylation efficiency of alginate,1H NMR
spectra were also recorded for uncrosslinked, unmodified alginate and uncros-
slinked, methacrylated alginate. The efficiency of alginate methacrylation was
calculated from1H NMR spectra based on the ratio of the integrals for the photo-
initiator protons to the methylene protons of methacrylate. The actual meth-
acrylation % was determined as the product of the theoretical methacrylation % and
the methacrylation efficiency, and it was used in a naming code for the different
alginate formulations (see Table 1).
2.4. Swelling and in vitro degradation of photocrosslinked alginate hydrogel
The photocrosslinked alginate hydrogels were lyophilized and dry weights (Wd)
were measured. Dried hydrogel samples were immersed in 50 ml of diH2O and
incubated at 37?C toreachequilibrium swelling state. The diH2O was replacedevery
three days. Over the course of 5 weeks, samples were removed, rinsed with diH2O,
and the swollen (Ws) hydrogel sample weights were measured. The swelling ratio
(Q) was calculated by Q¼Ws/Wd(N¼3 for each time point).
The dried hydrogelsamples (N¼3) wereweighed (Wi) and incubatedin 50 mlof
diH2O at 37?C for in vitro degradation testing. The diH2O was replaced every three
days. At predetermined time points, the samples were removed, rinsed with diH2O,
lyophilized and weighed (Wd). The percent mass loss was calculated by
(Wi?Wd)/Wi?100 (N¼3 for each time point).
2.5. Mechanical testing
The elastic moduli of the photocrosslinked alginate hydrogels were determined
by performing constant strain-rate compression tests using a Rheometrics Solid
Analyzer (RSAII, Rheometrics Inc., Piscataway, NJ, USA) equipped with a 10 N load
cell. The photocrosslinked alginate hydrogel disks were prepared as described in
Section 2.2 and maintained in diH2O at 37?C. At predetermined time points, swollen
alginate hydrogel disks were punched once again toform 6 mm diameterdisks, their
thickness was measured using calipers, and uniaxial, unconfined compression tests
were performed on the hydrogel disks at room temperature using a constant strain
rate of 5%/s. Elastic moduli of photocrosslinked alginate hydrogels were determined
from the slope of stressvs. strainplots, limited tothe linear first 5% strain of the plots
(n ¼3 for each time point).
The number average molecular weight between crosslinks (Mc) was calculated
according to following equation: Mc¼3rRT/E [39,40]. T is the temperature (298 K) at
which the modulus was measured, r is the concentration of methacrylated alginate
Table 1
Methacrylation efficiency (%) and actual methacrylation (%) of alginates, and elastic
modulus and molecular weight between crosslinks of photocrosslinked alginate
hydrogels.
CodeTheoretical
methacrylation
(%)a
Methacrylation
efficiency (%)b
Actual
methacrylation
(%)
Elastic
modulus
(kPa)c
–d
Mc
(g/mol)
MAALG-
4
MAALG-
8
MAALG-
14
MAALG-
25
5.66 73.294.15
–
11.3167.237.6034.29?9.224335
22.6361.0813.82 143.54 ?4.84 1036
45.2655.74 25.23174.77?14.88 851
aTheoretical methacrylation of the carboxylic acid reactive groups was calculated
based on the mass of alginate in 1% (w/v) alginate solution and molecular weight of
the repeat unit (M0¼198).
bMethacrylation efficiency was calculated from1H NMR data.
cElastic modulus was measure after incubation in diH2O at 37?C for 24 h.
dIt was not possible to perform a compression test on the MAALG-4.
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(g/m3) in the crosslinkingsolution, R is the gas constant (8.3145 Jmol?1K?1), and E is
the elastic modulus.
2.6. Cytotoxicity tests of methacrylated alginate and photocrosslinked alginate
hydrogel
To evaluate cytotoxicities of methacrylated alginate and photocrosslinked
alginate hydrogels, an indirect contact methodology was employed. Briefly, chon-
drocytes isolated from bovine articular cartilage as previously reported [41] were
plated in 6-well plates at 1?106cells/well in 3 ml of Dulbecco’s modified eagle
medium (DMEM) containing 10 % fetal bovine serum (FBS, Gibco BRL, Gaithersburg,
MD) and cultured at 37?C, 95% humidity and 5% CO2for 24 h. Cell culture inserts
(25 mm in diameter, 8 mm pore size on PET track-etched membrane; Becton Dick-
inson Labware Europe, Le Pont De Claix, France) were placed into each well. Sterile
methacrylated alginate solution (1 ml, 2% w/v in diH2O) or a photocrosslinked algi-
nate hydrogel (1 ml) was added into each culture insert (N¼3). Chondrocytes
cultured with a cell culture insert but without the presence of any macromer or
hydrogel material was maintained as a comparative group (cellsþinsert). A control
group, notexposed toanychemicals or inserts, was maintained inparallel. After48 h
incubation, media, inserts, and hydrogels were removed. Each well was rinsed with
phosphatebufferedsaline (PBS), and 3 mlof a 20%CellTiter96 AqueousOne Solution
(Promega Corp., Madison, WI, USA), which contains 3-[4,5-dimethylthiazol-2-yl]-5-
[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS-tetrazolium),
O
O
O
O
OH
H
H
H
H
OH
O-
O-
O-
O-
O
H
H
H
H
H
OH
O
H
OH
OH
OH
H
H
H
HO
HO
O
O
O
O
O
H
H
H
H
H
H
O
O
Alginate
EDC/NHS
2-aminoethyl methacrylate
O
O
NH2
O
OO
NH
OO
O
HN
O
O
O
O
O
O
HN
NH
O
OO
NH
OO
O
HN
Biodegradable linkages
(ester and amide bonds)
Alginate
UVPhotoinitiator
OH
OH
O
OH
OH
OH
OH
HO
O
HO
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
HH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O-
O-
Fig. 1. Schematic illustration for the preparation of the methacrylated alginate and photocrosslinking of methacrylated alginate.
O. Jeon et al. / Biomaterials xxx (2009) 1–113
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in PBS was added to each well. The MTS-tetrazolium compound can be metabolized
by mitochondria in living cells into a colored formazan product that is soluble in cell
culturemedium.Afterincubatingat37?Cfor90 min,theabsorbanceof thesolutions
was determined at 490 nm using a 96-well plate reader (SAFIRE, Tecan, Austria).
The viability of chondrocytes in the presence of methacrylated alginate and
photocrosslinked alginate hydrogels was also investigated by a Live/Dead assay
system (Sigma) composed of fluorescein diacetate(FDA) and ethidium bromide (EB).
FDA stains the cytoplasm of viable cells green, while EB stains the nuclei of non-
viable cells orange-red. The staining solutionwas freshly prepared by mixing 1 ml of
FDA solution (1.5 mg/ml of FDA in dimethyl sulfoxide) (Research Organics Inc.,
Cleveland, OH) and 0.5 ml of EB solution (1 mg/ml of EB in PBS) with 0.3 ml of PBS
(pH 8). After 48 h incubation of the alginate with the cells, inserts and hydrogels
were removed. Then, 60 ml of staining solution was added into each well and
incubated for 3–5 min at room temperature. After staining, samples were examined
on an inverted fluorescence microscope (ECLIPSE TE 300, Nikon, Tokyo, Japan)
equipped with a digital camera (Retiga-SRV, QImaging, Burnaby, BC, Canada). Three
images of each well (N¼3 wells per condition) were randomly selected for counting
live and dead cells.
2.7. Viability assay of cells photoencapsulated in alginate hydrogels
Chondrocytes were photoencapsulated in alginate hydrogels by suspending the
cells in methacrylated alginate solution (2% w/v in DMEM) with 0.05% w/v photo-
initiator (N¼3). The cell/macromer solutions (500 ml) were thenpipetted into 24-well
tissue culture plates (1?107cells/ml) and photocrosslinked with UV for 10 min. The
resultinghydrogel–cellconstructswereremovedfromthewells,placedinnew24-well
tissue culture plates with 500 ml of fresh DMEM containing 10% FBS (changed every 3
days) and cultured in a humidified incubator at 37?C with 5% CO2for 1 week. To
determine the viabilityof the cellsafterencapsulation, MTS and Live/Dead assays were
performed. Immediately following encapsulation or after 1 week culture, the culture
medium was discarded and the hydrogel–cell constructs were washed with PBS and
transferredintonewwells.500 mlofa20%CellTiter96AqueousOneSolutioninPBSwas
addedinto eachwell. Afterincubatingat37?C for 90 min,the hydrogel–cellconstructs
were homogenized and the absorbance of the solutions was determined at 490 nm
using a 96-well plate reader. For the Live/Dead assay, 20 ml of staining solution was
addedinto eachwelland incubatedfor 3–5 minatroomtemperature and thenstained
hydrogel–cell constructs were examined using fluorescence microscopy.
O
O
H
H
OH
OH
O
H
H
H
NH
O
H
O
H
H
O
H
HO
O
H
H
O
OH
H
H
O
H
O
O
O-
O
H
HO
H
H
O
NH
H
OH
H
OH
O-
O
O
O
O
O
a
b
c
a
b
c
MAALG-4
MAALG-8
MAALG-14
MAALG-25
6543210ppm
Fig. 2.1H NMR spectra of methacrylated alginate with various degrees of methacrylation in D2O.
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2.8. Cell culture on photocrosslinked alginate hydrogels
Chondrocytes were seeded onto photocrosslinked alginate disks (6 mm
diameter) in24-welltissue culture
1 ?104cells/cm2and allowed to adhere for 4 h (N¼3). The photocrosslinked
alginate disks were then transferred to new plates containing fresh media and
cultured in an incubator. After 48 h incubation, alginate disks were removed
from culture and stained using the Live/Dead assay to examine the viability and
platesataseeding density of
morphology of adherent cells. The numbers of adhered cells were counted in
three different fields of view on the disks using fluorescence microscopy at
a magnification of 100?.
2.9. Statistical analysis
All quantitative data is expressed as mean?standard deviation. Statistical
analysis was performed with one-way analysis of variance (ANOVA) with Tukey
O
O
H
a
H
OH
OH
O H
H
H
O-
O
H
O
H
H
O
H
HO
O
H
H
O
OH
H
H
O
H
OH
O
O-
O
H
HO
H
a
H
b
O O-
H
OH
H
OH
O-
O
a
a
b
b
b
b
a
O
O
H
a
H
OH
OH
O H
H
H
NH
O
H
O
H
H
O
H
HO
O
H
H
b
O
OH
H
H
O
H
OH
O
O-
O
H
HO
H
a
H
O
NH
H
OH
H
OH
O-
O
O
O
O
O
b
a
a
a
b
c
d
c
d
O
8 6 4 2 0 ppm
ppm
N
H
O
O
Alginate
O
H
N
O
O
Alginate
d
d
f
d
f
A
B
C
e
e
e
e
Photoinitiator
Photoinitiator
8 7 6 5 4 3 2 1 0
ppm 8 7 6 5 4 3 2 1 0
Fig. 3.1H NMR spectra of (A) alginate, (B) MAALG-25, and (C) photocrosslinked MAALG-25 hydrogel in D2O.
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honestly significant difference post hoc test using Origin software (OriginLab Co.,
Northampton, MA). A value of p<0.05 was considered statistically significant.
3. Results
3.1. Synthesis and characterization of macromer and
photocrosslinked hydrogel
To prepare photocrosslinked biodegradable alginate hydrogels,
methacryl groups were introduced into the alginate main chains as
shown in Fig. 1. Alginate was covalently reacted with various
amounts of AEMA. The theoretical methacrylation of the carboxyl
groups of alginate was varied from 5.66% to 45.26%. Theoretical
methacrylation was calculated on the basis of the concentration of
AEMA added to the alginate solution (Table 1). Increasing the
theoretical methacrylation of alginate from 5.66% to 45.26% resul-
ted in a decrease in the methacrylation efficiency of alginate from
73% to 56%. The elastic modulus of photocrosslinked alginate
hydrogels increased with increasing theoretical methacrylation,
while Mcof photocrosslinked alginate hydrogels decreased. The
experimental efficiency of alginate methacrylation was calculated
from1H NMR spectra. The1H NMR spectra of methacrylated algi-
nates are shown in Figs. 2 and 3(B). The intensities of proton peaks
of AMEA (Fig. 2; a, b, and c) increased as the actual methacrylation
of the alginate increased from 4.13% to 25.35%. The peaks at d2.9
and 1.1 were proton peaks of residual EDC–urea.
The1H NMR spectra of alginate, methacrylated alginate, and
photocrosslinked alginatehydrogelare shown in Fig. 3. The1H NMR
spectrum of methacrylated alginate exhibits peaks of vinyl meth-
ylene (Fig. 3(B); c) and methyl (Fig. 3(B); d) protonsthatwere newly
formed by the reaction with AEMA at d6.2 and 5.7, and 1.9,
respectively. The completeness of photocrosslinking was also
1 day 1 week2 weeks
Time (weeks)
0123456
Swelling ratio (Ws/Wd)
0
100
200
300
400
500
600
2
cm
345678
MAALG-8
MAALG-14
MAALG-25
B
A
Fig. 4. (A) Morphology and size change of MAALG-25 after 1 day,1 week, and 2 weeks
equilibration in water. (B) Swelling ratios of photocrosslinked alginate hydrogels over
time. Values represent mean and standard deviation (N ¼3).
Time (weeks)
0123456
Mass loss (%)
0
20
40
60
80
100
MAALG-8
MAALG-14
MAALG-25
**
*
*
Fig. 5. In vitro degradation of photocrosslinked alginate hydrogels. Mass loss
(%)¼(Wi?Wd)/Wi? 100, where Wi¼ initial weight and Wd¼weight after degrada-
tion. Values represent mean and standard deviation (N ¼3). *p <0.05 compared with
MAALG-14 and MAALG-25, and **p < 0.05 compared with MAALG-14.
Strain (%)
02468 10
Stress (kPa)
0
5
10
15
20
25
1 day
1 week
2 weeks
3 weeks
Modulus (kPa)
0
50
100
150
200
1 day1 week 2 weeks3 weeks
*
*
*
A
B
Fig. 6. (A) Stress–strain curves and (B) elastic moduli in compression of photo-
crosslinked MAALG-25 hydrogels during degradation. *p<0.05.
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A
Cells+Insert
Photocrosslinked
MAALG-25 hydrogel
FDA
EB
Merged
MAALG-25
Dead Cellsx103/cm2
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Live Cellsx103/cm2
10
20
30
40
50
60
70
80
90
Cells+Insert
MAALG-25
*
**
*
Photocrosslinked
MAALG-25hydrogel
B
Fig. 7. (A) Fluorescent photomicrographs of chondrocytes in monolayer culture exposed to only a cell culture insert, methacrylated alginate or a photocrosslinked alginate hydrogel
for 48 h and (B) quantified cell density of live and dead cells. Cells were stained by FDA/EB. Green and orange-red colors indicate viable and dead cells, respectively. Values represent
mean and standard deviation (N ¼ 3). The scale bar indicates 200 mm. *p<0.05, **p>0.05.
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verified with1H NMR. After photocrosslinking of methacrylated
alginate, the disappearance of the peaks of vinyl methylene and
shift of methyl peak to d1.2 (Fig. 3(C); d) in the1H NMR spectrum
indicate the complete reaction of the methacrylate group. The peak
of newly formed methylene proton (Fig. 3(C); f) following photo-
crosslinking appears at d2.2.
3.2. Swelling kinetics, degradation, and elastic moduli of the
photocrosslinked alginate hydrogel
The size change of a photocrosslinked alginate hydrogel
(MAALG-25) during two weeks of swelling in diH2O is exhibited in
Fig. 4(A). The size of photocrosslinked alginate hydrogel increased
substantially over the course of two weeks.
Photocrosslinkedalginatehydrogels
different degrees of alginate methacrylation. The swelling ratio
were preparedwith
change of the hydrogels measured over time reflects changes in
their physical and chemical structure. Swelling ratios of these
hydrogels in diH2O are shown in Fig. 4(B). All hydrogels displayed
rapid swelling. The swelling of MAALG-14 and MAALG-25 reached
equilibrium stage within 48 h, increased up to 1 and 2 weeks,
respectively, and then gradually decreased. Compared to the
MAALG-14 and MAALG-25, MAALG-8 exhibited much faster
swelling kinetics. The swelling of MAALG-8 reached a maximum by
24 h and then rapidly decreased.
The mass loss (%) of alginate hydrogels over time was deter-
mined as a measure of degradation (Fig. 5). MAALG-8 displayed the
fastest degradation with a mass loss of 95.67% at 1 week, while
MAALG-25 showed the slowest degradation. As the actual meth-
acrylation of the alginate increased from 7.60% to 25.23%, the
degradation rate of the crosslinked hydrogels decreased (Fig. 5).
However, photocrosslinkedalginate hydrogelswithactual
Fig. 8. Fluorescence micrographs of live (FDA, green) and dead (EB, orange-red) encapsulated bovine chondrocytes in various photocrosslinked alginate hydrogels cultured in vitro
for 7 days. The scale bar indicates 200 mm.
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methacrylations of 13.82% and 25.23% had relatively similar
degradation rates, with complete degradation of both conditions
occurring by 5 weeks.
To examine the correlation between mechanical property
changes and degradation, constant strain-rate compression tests
were performed on the alginate hydrogels during the degradation
study in order to determine their elastic moduli. Representative
stress–strain curves of the MAALG-25 alginate hydrogels during the
course of degradation are presented in Fig. 6(A). The compressive
modulus of alginate hydrogels decreased with degradation time
(Fig. 6(B)).
3.3. Cytotoxicity of macromer and photocrosslinked alginate
hydrogel
The cytotoxicities
crosslinked alginate hydrogels were evaluated by measuring the
mitochondrial metabolic activity of chondrocytes cultured on
tissue culture plastic in the presence of the biomaterial using
a standard MTS assay. The cell viability was calculated by normal-
izing the absorbance of samples at 490 nm to that of the control
without any macromer or insert in the medium. The viability of
cells cultured in the presence of macromer (90.93 ? 4.70%) was
slightly lower than that of cells in the cells þ insert group
(97.13 ? 4.05%). There was no significant difference in cell viability
after 48 h culture between the photocrosslinked MAALG-25
hydrogels(95.73 ? 2.80%)andthemacromerorcells þ insertgroup.
Thecytotoxicityof macromer
crosslinked MAALG-25 hydrogel was also examined by fluores-
cence staining with a Live/Dead assay, where live cells and dead
cells were fluorescently labeled green and red, respectively
(Fig. 7A). Almost all of the chondrocytes were alive after 48 h
exposure to the macromer and photocrosslinked MAALG-25
hydrogel. Quantification of stained images demonstrated that
there was no significant difference in the number of live cells
between the cellsþinsert group and the macromer or photo-
crosslinked MAALG-25 hydrogel group (Fig. 7B). Although the
number of dead cells in the MAALG-25 macromer and photo-
crosslinked hydrogel exposed groups was larger than that of
cellsþinsert group, the number of dead cells was minimal
compared with the number of live cells.
of methacrylated alginate and photo-
(MAALG-25) andphoto-
3.4. Chondrocyte encapsulation
Bovine chondrocytes were photoencapsulated within MAALG-8,
MAALG-14, and MAALG-25 hydrogels. The viability of the photo-
encapsulated chondrocytes in the alginate hydrogels was evaluated
by MTS and Live/Dead assays to examine cell survival during the
photocrosslinking process and in culture. There were no significant
differences in absorbances for the MTS assay between the MAALG-
8, MAALG-14, and MAALG-25 conditions immediately following
photoencapsulation (0.495?0.017, 0.499?0.019, 0.496 ?0.023,
respectively) or after 7 days in 3-D culture (0.525?0.006,
0.511?0.026, 0.502?0.026, respectively). In addition, a high cell
viability (>w80%) was observed throughout all alginate hydrogel
compositions at 7 days (Fig. 8) using the Live/Dead assay.
3.5. Cell attachment and morphology
Few chondrocytes seeded on the surface of MAALG-25 hydro-
gels were able to adhere (0.06 ?0.022%), and those that did adhere
showed a viability of 48?4.7% after 2 days of culture. As shown in
Fig. 9, all of the adherent cells exhibited a spherical morphology
and none spread on the surface of the hydrogels.
4. Discussion
In this study, we developed a cytocompatible alginate bioma-
terial system capable of forming homogeneous, photocrosslinked
hydrogels in situ with controlled mechanical properties and
degradation rates for tissue engineering applications. Control of
hydrogel mechanical properties and degradation rates is very
Fig. 9. Fluorescence micrographs of live (FDA, green) and dead (EB, orange-red)
chondrocytes cultured in vitro for 48 h on the surface of a photocrosslinked alginate
hydrogel (MAALG-25). The scale bar indicates 200 mm.
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important for their use in regenerative medicine [9]. The
mechanical properties of hydrogels can affect cell behaviors such as
proliferation, migration, and differentiation [42–44]. The ability to
control the rate of hydrogel degradation provides the opportunity
to match tissue regeneration rates, to provide appropriate space for
tissue ingrowth, and to control the release of bioactive molecules
[5,8]. The results of this study demonstrate that the swelling
behavior, elastic moduli, and degradation rates of photocrosslinked
alginate hydrogels can be controlled by changing the percentage of
alginate methacrylation.
Previously, Smeds et al. prepared photocrosslinked alginate
hydrogels with controllable mechanical properties [37]. However,
their hydrogels were not biodegradable on the time scale of new
tissue formation. Therefore, we utilized AEMA, which has biode-
gradable ester groups, as a crosslinker. We have shown that the
hydrogel swelling ratio change and degradation rate correlated to
the degree of alginate methacrylation (Figs. 4B and 5). The change
in swelling ratio of alginate hydrogels exhibited a similar trend as
observed for hydrogel mass loss. Since the swelling ratio of
hydrogels reflects the macromolecular structure of the crosslinked
network [45], the increased alginate hydrogel swelling over time is
due to the hydrogel degradation likely resulting from hydrolysis of
ester linkages of AEMA. This hydrolytic degradation can create
space for cell growth and deposition of a new extracellular matrix
to replace the hydrogel.
Hydrogels for biomedical applications such as tissue engi-
neering must also be cytocompatible. As determined by the MTS
assay, the viability of chondrocytes in monolayer culture following
exposure to macromer and photocrosslinked alginate hydrogels
indicates the cytocompatible and non-toxic nature of the hydrogel.
Cell viabilities in the presence of macromer or photocrosslinked
alginate hydrogel were at least 90% compared to controls, sug-
gesting that the biomaterial and the photocrosslinked alginate
hydrogel degradation products are relatively non-toxic. Results
from the FDA/EB staining corroborated the findings of the MTS
assay. Cells exposed to macromer or photocrosslinked alginate
hydrogel were highly viable, with over 99% of the cells live (Fig. 7).
These assays also confirmed that high cell viability was achieved
when the chondrocytes were photoencapsulated and cultured
within the 3-D hydrogels. In this study, a small amount of residual
EDC–urea, which could adversely affect the cell viability, may have
remained on the methacrylated alginate and in the photo-
crosslinked alginate hydrogels. However, the cytotoxicity results
indicate that the residual EDC–urea had minimal affect on the cell
viability in this study. It should also be noted that the alginate used
for the cells studies was reconstituted and cultured in DMEM. From
gross observation, thesehydrogels
decreased swelling compared to the hydrogels reconstituted and
assayed in diH2O. DMEM contains low concentrations of positively
charged cations, and a small degree of ionic crosslinking may occur
resulting in decreased hydrogel swelling.
Photocrosslinked, biodegradable alginate hydrogels provide
many advantages over chemically crosslinked alginate hydrogels.
The photoinitiated reaction permits rapid polymerization rates in
cytocompatible conditions, as evidenced by high cell viability of
encapsulated cells after culture in vitro (Fig. 8). Since transdermal
UV illumination has been previously shown to be feasible for in situ
hydrogel photocrosslinking [46], photocrosslinked alginate hydro-
gels could be implanted easily via injection through a syringe or
similar minimally invasive procedures, which could obviate the
need for open surgical treatment and reduce patients’ pain.
Furthermore, cells could be homogeneously encapsulated within
alginate hydrogels by suspending them in aqueous alginate solu-
tion prior to photocrosslinking, and the photocrosslinkable alginate
solution could then be easily injected or placed in tissue defects of
exhibited substantially
complex shapes and subsequently crosslinked to form a hydrogel of
exactly the required dimensions [47].
The adhesion of chondrocytes cultured on photocrosslinked
alginate hydrogels was limited because native alginate hydrogels
do not support cell adhesion [48]. Alginate presents an environ-
ment absent of cell adhesion molecules and it exhibits minimal
protein absorption due to its hydrophilicity. Therefore,cell adhesive
amino acid sequences, such as the RGD sequence, have been
covalently coupled to the alginate polymer backbone to promote
cell attachment, proliferation, differentiation, and tissue formation
[15,16,18,20,21,23]. Cell attachment and spreading on the surface of
alginate hydrogels were significantly improved when the alginates
were modified with adhesion peptides, and implementation of this
strategy with photocrosslinked alginate hydrogels would be an
additional potential approach for regulating cell interactions with
the biomaterials and function within photocrosslinked alginate
hydrogels.
5. Conclusions
In this study we have engineered a photocrosslinked and
biodegradable hydrogel using methacrylated alginate. To our
knowledge, this is the first report of photocrosslinked and biode-
gradable alginate hydrogels with controllable swelling ratios,
elastic moduli, and degradation rates. These physical properties
could be tuned by varying the extent of alginate methacrylation.
The photocrosslinked alginate hydrogels showed low cytotoxicity
and excellent cytocompatibility. Cells can be encapsulated within
the alginate hydrogel via suspension followed by in situ photo-
crosslinking in aqueous solutions of methacrylated alginate and
remain viable. The photocrosslinked alginate hydrogel developed
in this study may have broad biomedical applications, including use
in cell transplantation and drug delivery.
Acknowledgements
The authors gratefully acknowledge funding support from
Biomedical Research and Technology Transfer Grant 05-065 from
the Ohio Department of Development (EA) and a New Scholar in
Aging Award from the Ellison Medical Foundation (EA), and thank
Loran D. Solorio for supplying the chondrocytes.
Appendix
Figures with essential colour discrimination. Certain figures in
this article, inparticular parts of Figs. 7–9 are difficult tointerpret in
black and white. The full colour images can be found in the on-line
version, at doi:10.1016/j.biomaterials.2009.01.034.
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