Development of a hybrid gelatin hydrogel platform for tissue engineering and protein delivery applications

Article abstract of DOI:10.1039/c5tb00645g

In this study, to improve the cellular interaction and protein release of gelatin hydrogels, we reported the development of a new hybrid hydrogel platform as a promising tissue engineering scaffold and drug delivery carrier. The biodegradable, biocompatib

Full text of DOI:10.1039/c5tb00645g

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Materials Chemistry B
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Development of a hybrid gelatin hydrogel
platform for tissue engineering and protein
Cite this:DOI: 10.1039/c5tb00645g
delivery applications†
ab
c
d
ab
e
Xiaodi Sun,‡ Xin Zhao,‡* Lili Zhao, Qing Li, Mathew D’Ortenzio,
e
ab
ab
Brandon Nguyen, Xin Xu* and Yong Wen*
In this study, to improve the cellular interaction and protein release of gelatin hydrogels, we reported
the development of a new hybrid hydrogel platform as a promising tissue engineering scaffold and drug
delivery carrier. The biodegradable, biocompatible hybrid hydrogel platform was fabricated from gelatin
methacrylamide (Gel-MA) and arginine based unsaturated non-peptide polycations (Arg-UPEA) through UV
photo-crosslinking, combining the favorable properties of gelatin and arginine. The hydrogels were
systematically characterized based on their mechanical properties, swelling mechanics, interior morphology,
and biodegradation capability. The in vitro biocompatibility study showed that the hybrid hydrogels show
better performance than GelMA hydrogels, in terms of cell attachment and proliferation. Therapeutic
proteins were loaded into the hydrogels and their release behavior was investigated. The loading and release
profiles indicated that the new cationic gelatin hydrogels could significantly improve the protein loading
capabilities, and release the proteins in vitro in a sustained manner. The structure-function study indicated
that the material composition has a large effect on the properties of the hydrogels.
Received 9th April 2015,
Accepted 3rd July 2015
DOI: 10.1039/c5tb00645g
www.rsc.org/MaterialsB
Among the reported hydrogel systems, natural material
based hydrogels, such as dextran, chitosan, hyaluronic acid,
Introduction
With the ability to swell and hold large amounts of water in the alginate and collagen/gelatin, have received plenty of attention
1
–3
18–26
wet state,
hydrogels generally consist of three-dimensional due to their high biocompatibility and natural abundance.
(
3D) material networks that are cross-linked chemically and/or Gelatin, the denatured form of collagen, has been widely utilized
4
,5
physically. Due to their significant water content, hydrogels and considered as the one of the gold standards in terms of
also possess a degree of flexibility and softness similar to biocompatibility and assisting cell attachment and prolifera-
natural tissues. Different from other formulations,
1–3
6–12
27–30
hydrogels tion.
Because gelatin itself will dissolve below 37 1C, solid
have shown significant advantages and attracted strong interest in gelatin scaffolds need to be fabricated via physical or chemical
2
7–31
the fields of tissue engineering and drug delivery because of their means.
high water content, 3D microporous structure, biocompatibility, chemical strategy,
organic solvent free environment, permeability for oxygen and material. For photochemical applications, the gelatin need to be
The glutaraldehyde crosslinking method is the main
2
9,30
however, it may cause the toxicity of the
1–5,12–17
nutrients, and tissue-like elastic properties.
first modified into photo-crosslinkable materials, such as gelatin
methacrylamide (Gel-MA).
2
7,28
However, due to the extremely
complicated mechanism of tissue or organ regeneration, the
currently available hydrogel scaffolds may not meet the high
a
School of Stomatology, Shandong University, Wenhuaxi Road 44-1, Jinan, 250012,
China. E-mail: xinxu@sdu.edu.cn, wenyong@sdu.edu.cn
requirements or demands of tissue engineering under many
b
c
Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan, 250012, China
Center for Biomedical Engineering, Department of Medicine, Brigham and
Women’s Hospital, Harvard Medical School, Cambridge, MA, 02139, USA.
E-mail: xinzhao02@fas.harvard.edu
27,28,32
circumstances.
For example, one of the major limitations is
the unsatisfactory cellular attachment and proliferation performance
2
7,28,32
of scaffolds, even for the gelatin based platforms.
The
d
e
Department of Endoscopy, The First Affiliated Hospital of Nanjing Medical
University, Jiangsu Province Hospital, Nanjing, Jiangsu, 210029, China
University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1,
Canada
inadequate cellular interactions greatly limit biomedical applica-
tions since cellular interactions are critical at the start of tissue/
organ regeneration. Another limitation is the low efficacy of
delivering and releasing large biomacromolecules, such as proteins
and nucleic acids, in a spatiotemporal manner. Due to the large
interior pore size, hydrophilicity, 3D microporous structure and fast
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5tb00645g
Co-first author.
‡
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diffusion of drug molecules in aqueous environments, hydrogel otherwise specified, were purchased from Sigma-Aldrich
14
releasing rates of drugs are normally rapid. Other key limitations (St. Louis, MO).
include unacceptable and uncontrollable biodegradation rates and
27,28,32
Synthesis of hydrogel precursors
mechanical properties.
Here one important question arises:
without decreasing their biocompatibility, could the gold standard The Gel-MA precursor was synthesized following a previously
2
7,28
hydrogel platforms show enhanced cellular interactions and drug reported method.
release performance, along with tuneable mechanical properties/ was added into 100 mL of 10 wt% gelatin phosphate buffer
biodegradation rates? solutions (PBS) at 60 1C, and was vigorously stirred for 3 h. After
Briefly, 6.0 mL of methacrylic anhydride
For the above question, a few strategies have been investigated purification, Gel-MA was obtained with a degree of methacryla-
for plenty of hydrogel platforms, including physically mixing new tion of around 50–60%. The Arg-UPEAs were synthesized by the
33–36
37,40
components/systems and chemical modifications.
Among them, polycondensation method reported before.
Briefly, the
fabricating hybrid hydrogels containing two or more components unsaturated di-p-nitrophenyl ester of dicarboxylic acid (I) and
26,37–39
(precursors) have aroused strong interests.
The chemical tetra-p-toluenesulfonic acid salts of bis(L-arginine), a,o-alkylene
introduction of new functional precursors could help to tune or diesters (II) were prepared first following previous reports. After
bring new properties/functionalities to hydrogels, these may include that, the Arg-UPEA (III) was prepared via solution polyconden-
charge properties, mechanical properties, tuneable degradation sation of (I) and (II). The molecular weight (M
W
) of Arg-UPEA is
À1
rates, hydrophobic/hydrophilic properties, pH and temperature around 10.0–15.0 kg mol . The Gel-MA or Arg-UPEA precur-
2
6,33–39
responsive properties, and functional groups.
sors were purified first by dissolving the precursors in distilled
In this study, aiming to obtain new gelatin based hydrogel water and were dialyzed against deionized water (MW cut off
platforms with better biological and drug release performance 4000) under a dark environment for 5 days. Next, the solutions
than the collagen/gelatin hydrogels, we proposed the following were lyophilized for 5 days using a freeze-drier at À45 1C.
hypothesis: the chemical introduction of cationic arginine
Preparation of hydrogels
unsaturated poly(ester amides) into gelatin hydrogels could
significantly improve their cellular interactions, tune the degradation Gel-MA/Arg-UPEA hybrid hydrogels were prepared by the photo-
rate and mechanical property, while keeping excellent bio- polymerization of two precursors (Gel-MA and Arg-UPEA) at
compatibility because of their inherent strong cationic property different weight ratios in aqueous solution with an initiator.
and biocompatibility. Gelatin was chemically modified here An example of the fabrication protocol is given below: 0.40 g of
into photo-crosslinkable gelatin methacrylamide (Gel-MA). The Gel-MA, 0.10 g of 2-UArg-2-S and 5.0 mg of photoinitiator
second components, a family of arginine based unsaturated Irgacure 2959 (1.0 wt% of total amount of precursors) were
poly(ester amide)s (Arg-UPEA), were developed due to their added into a glass vial and dissolved in 3.5 mL of distilled water
strong cationic properties, tunable properties, very low cyto- to form a clear homogeneous solution. The solution mixture
3
7
toxicity and muted inflammatory response. The hydrogels was then transferred into a 20-well Teflon mold (diameter
were fabricated by UV photo-crosslinking, from the mixture of 12 mm and thickness E 4 mm for each well). Then, the precursor
Gel-MA and Arg-UPEA aqueous solutions. The physicochemical solution in the mold was irradiated using a long-wavelength UV
properties, biocompatibility and biological performance of the lamp (365 nm, 100 W) for a specific time (normally 1–2 min) at
hydrogels were then systematically evaluated. In addition, to room temperature. The irradiation distance was 5–10 cm. After
evaluate the capability for sustainable release of proteins, bone fabrication, the resultant hydrogels were removed from the mold
morphogenetic protein (BMP) was selected as a model protein and soaked for 24 h in distilled water or buffer to remove the toxic
drug in this study, and was pre-loaded into the hydrogels before residues, then dried in a vacuum at room temperature for 24 h
photo-crosslinking. The various hydrogel formulations were prior to further characterization. For protein loaded hydrogels, a
then characterized in terms of in vitro release kinetics.
pre-determined amount of proteins was mixed with the precursor
solution before photo-crosslinking. All other fabrication condi-
tions were same.
Experimental
Equilibrium swelling ratio and swelling kinetics of hydrogels
Materials
The equilibrium swelling ratio (Qeq) of the hydrogel is calcu-
Type A porcine skin gelatin (gel strength 300, cat No: G2500, lated by the following equation: Qeq = [(W
around 330 kD), 2-hydroxy-l-[4-(hydroxyethoxy)phenyl]-2- where W is the weight of a swollen hydrogel at equilibrium,
methyl-l-propanone (Irgacure 2959), L-Arginine (L-Arg), p-toluene- and W is the weight of the corresponding dry hydrogel at t = 0.
sulfonic acid monohydrate, fumaryl chloride, ethylene glycol, All swelling ratio results were obtained from triplicate samples
e
À W
d d
)/W ] Â 100%,
M
W
e
d
1
,4-butanediol, 1,6-hexanediol, p-nitrophenol, triethylamine and and data were expressed as the mean Æ standard deviation.
lipopolysaccharide (LPS, from E. coli 0111:B4) were purchased The swelling kinetics of the Gel-MA/Arg-UPEA hydrogels was
from Sigma-Aldrich (St. Louis, MO) and used without further measured over a period of 48 h at room temperature. Each dry
purification. BMP-2 and BMP-2 ELISA kits were purchased Gel-MA/Arg-UPEA hydrogel sample was weighed and immersed
from R&D systems. The TNF-a ELISA kit was purchased from in 20 mL of solutions with different parameters for predeter-
Invitrogen (Carlsbad, CA). Other chemicals and reagents, if not mined periods. Before weighing, the samples were taken from
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the solutions and blotted with filter papers to remove excess surface sterilized under UV light (in the cell culture hood) for at least 2 h
water. The swelling ratio (Q) of the hydrogels, at time t, is calculated before being put into the 24-well cell culture plates. After that,
using the following equation: Q = [(W À W )/W ] Â 100%.
the hydrogels were washed twice by PBS buffer and cell culture
media. Then, the hydrogels were placed in the wells of the cell
culture plate and fixed by a sterilized rubber ring, which had the
t
d
d
Compressive modulus of hydrogels
The mechanical properties of the Gel-MA/Arg-UPEA hydrogels same diameter as the well of cell culture plate. The cells were
were measured using a 2980 Dynamic Mechanical Analyzer seeded at an appropriate cell density (40 000 cells per well) and
(DMA) (TA Instruments Inc., New Castle, DE) in ‘‘controlled incubated overnight. The media was changed after 12 h to wash
force’’ (CF-mode) mode. The swollen hydrogel samples, as the unattached cells. After 48 h of incubation, the cell attach-
circular discs, were submerged in distilled water and mounted ment and proliferation on the hydrogel surface was recorded
between the movable compression clamp (diameter 30 mm) using an optical microscope. For cellular interaction study inside
and the fluid cup with a 0.1 N preloading force. A force ramp hydrogels, precursors/initiators were pre-dissolved in serum free
À1
from 0.1 N at a rate of 0.3 or 0.5 N min was applied. All medium (5.0 wt%). Then the solution was quickly mixed with
measurements were carried out at room temperature. The predetermined amount of freshly collected NIH 3T3 fibroblasts
compression elastic modulus (E) of the swollen hydrogel was (cell concentration 1 million per mL gel). The mixed solution was
extracted by plotting the compressive stress versus strain. All then transferred into 96 well cell culture plates (100 mL per well),
compression elastic modulus data in this study were obtained irradiated using the 100 W UV lamp for 1 min at room tempera-
from triplicate samples and data were expressed as mean Æ ture. After that, 100 mL 20 wt% FBS media was added into each
standard deviation.
well and the cells encapsulated in hydrogels were cultured at
7 1C in 5% CO . Medium was replaced everyday. Cell prolifera-
3
2
In vitro enzymatic biodegradation of hydrogels
s
tion was enumerated using PicoGreen DNA quantification
À1
The in vitro biodegradation of the Gel-MA/Arg-UPEA hybrid assay. 500 mL of 50 mg mL proteinase K solution was added
hydrogels was evaluated using collagenase type II. Briefly, a to each sample followed by incubation at 37 1C overnight and
4
1
freshly prepared and purified hydrogel was placed in a glass vial subsequent centrifugation at 4 1C at 14000 rpm for 10 min. In
À1
containing 10 mL of PBS buffer (pH 7.4, 0.067 M) or 2 U mL
the dark, 100 mL of supernatant was mixed with 100 mL of
collagenase in 10.0 mL of PBS buffer. The hydrogel (dry weight PicoGreen solution (1: 200 dilution) for 5 min. Using 485 and
around 0.10–0.20 g) was then incubated at 37 1C with constant 520 nm as excitation and emission wavelengths respectively,
reciprocal shaking (ca. 100 rpm). At the end of a predetermined fluorescence of the samples was measured using a microplate
period, the hydrogels were removed (or collected by filtration if reader (BioTex, Inc. TX).
broken into small parts), then washed with distilled water 3 times,
and dried in a freeze drying machine for 24 h to completely
In vitro inflammatory response of hydrogels
remove the residue water. The collagenase solution was refreshed The in vitro inflammatory response of the Gel-MA/Arg-UPEA
2
3
every other day to maintain enzyme activity.
hydrogel was studied according to the reported protocol. J774
macrophages (ATCC, Manassas, VA) were seeded onto the
hydrogel surface in 24-well tissue culture plates at a cell
Interior morphology of hydrogels
Interior morphology of Gel-MA/Arg-UPEA hydrogels was investigated concentration of 10 000 cells per well. Positive controls were
using a scanning electron microscope (SEM). The swollen hydrogel glass coverslips in media containing LPS at final concentrations
À1
À1
samples, after reaching their maximum swelling ratio in distilled of 1.25 mg mL and 5.00 mg mL . A cell free medium alone
water at room temperature, were quickly frozen in liquid nitrogen was used as a cell-free negative control. Cells cultured without
and then freeze-dried under vacuum at À48 1C for 2 days until all hydrogels and Gel-MA hydrogels were also used as controls.
water inside the hydrogel was sublimed. The freeze-dried hydrogel Macrophage activation after 48 h of incubation was measured
samples were then cut, fixed on aluminum stubs, and coated with using an ELISA kit, to measure released mouse TNF-a according
gold for 30 seconds for interior morphology observation with a SEM to the manufacturer’s protocol. Sample TNF-a concentrations
instrument (Leica S440, Germany).
were calculated from a standard calibration curve using a
-parameter standard curve-fitting algorithm (Gen5 software,
BioTek Instruments, Winooski, VT). All testing samples were
4
Cell attachment and proliferation on the hydrogel surface and
inside the hydrogel
run in triplicate (N = 3). All samples and standards were read in
The cellular interaction of the Gel-MA/Arg-UPEA hybrid hydrogel duplicate on a 96-well plate reader at 450 nm and referenced
surface and inside hydrogel was evaluated in terms of the cell against a chromogen blank.
attachment and proliferation performance. For cellular inter-
Controlled release of proteins via hydrogels
action on hydrogel surface, pure Gel-MA and PEG-DA (MW 8000)
hydrogels were selected as the hydrogel controls. The cells used The release of BMP-2 from the Gel-MA/Arg-UPEA hydrogels was
for this study were HeLa cells (ATCC, Manassas, VA). The carried out in a PBS buffer solution at 37 1C. Proteins were
purified swollen hydrogels were cut into round shapes with a preloaded into the hydrogel samples according to the following
diameter that just filled the well of a 24-well cell culture plate, protocol: pre-determined amounts of protein were directly
and incubated at 37 1C in cell culture media. The hydrogels were mixed with Gel-MA first, then mixed with Arg-UPEA precursors
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2
and photo-initiators in distilled water, and finally the solution in aqueous solutions due to the different CH segment in the
mixture was UV irradiated for 1–2 min to form protein loaded repeating unit, indicating different hydrophobicity.
hydrogels. The protein loaded hydrogels were then placed
inside small vials containing 10.0 mL PBS solution (one piece Fabrication of hydrogels
of hydrogel per vial). The vial was incubated at 37 1C with
In this report, Gel-MA and Arg-UPEA were photo-crosslinked by
UV treatment, with Irgacure 2959 as the photo initiator in an
aqueous system. For easy comparison, the amount of Gel-MA in
solution was fixed at 10 wt%. To keep the gelatin as the
majority component of hydrogel, the weight ratio of Gel-MA
to Arg-UPEA was chosen at 9 : 1, 4 : 1, or 3 : 2 in this report. In
this study, the hybrid hydrogel was named: Gel-MA/Arg-UPEA
constant reciprocal shaking (ca. 100 rmp). The protein contents
were then analyzed by an ELISA assay. The released BMP-2
proteins were also collected and compared with the untreated
BMP-2 proteins (same concentration) using ELISA assay. All
protein release tests, at each time point, were carried out in
triplicate and the variation was expressed as a standard error of
the mean.
(m/n, w/w), where m/n is the weight ratio of Gel-MA to Arg-UPEA.
Table S1 (ESI†) is the summary of the developed hydrogels. The
photo-crosslinking time was suggested to be as short as possible
so that the bioactivity of the proteins would not be affected.
Meanwhile, to maximally reduce the side crosslinking of Gel-MA
Results and discussion
It has been reported that arginine-based material could improve the and Arg-UPEA, the precursor should be completely mixed well
cellular interactions of synthetic and natural hydrogels. However, and the crosslinking rate should also be as quick as possible.
these improvements are based on the poor or normal cellular Therefore, a 100 W UV light is utilized so that the photo-
performance of the original hydrogels. For those ‘‘gold standard’’ crosslinking could be finished within minutes (typically 1–2)
hydrogels with superior cellular interaction performance, depending on the composition of the hydrogel. An increased
whether Arg-UPEA could help the improvements has not yet amount of Arg-UPEA inside the hydrogel would need a longer
been investigated. To achieve excellent cellular interactions, time due to the relative weak reactivity of the double bonds of
especially for the gelatin based hydrogel platforms, we developed a Arg-UPEA. Fig. 1 showed the chemical structure of Gel-MA, Arg-
novel biocompatible and biodegradable hybrid hydrogel platform UPEA and the image of a Gel-MA/2-UArg-2-S hydrogel (swollen
utilizing a new simple fabrication strategy. This would maximally (left) and dry (right)). All the fabricated GEL-MA/Arg-UPEA
keep the original properties of hydrogels and be suitable for tissue hydrogels in this report were light white or colorless after
engineering and drug delivery applications. Subsequently, the hybrid reaching their swelling equilibrium. The yields of formed hydro-
hydrogel system combines the favorable properties of Gel-MA and gels were evaluated in terms of the mass conversion percentage.
arginine polymer (high pK ). The variety of the hydrogel component Table S1 (ESI†) indicates that the percentage is high (around or
a
types and parameters (molecular weight, component ratio, cross- above 90 wt%), which may be due to the very high UV irradiation
linking density) can allow us to engineer the hydrogel platforms for a power, causing the crosslinking to be finished within seconds or
wide range of tissue engineering and drug delivery needs.
minutes. The successful formation of hydrogels was confirmed
by the formation of stable 3D scaffolds and the other characteristics
including swelling capability, mechanical properties, interior 3D
morphology (SEM), enzyme biodegradation and other properties.
Synthesis of precursors
In this report, Gel-MA was chosen as a precursor component
due to its excellent biocompatibility, water solubility, low
toxicity, low immunogenicity and photocrosslinkability. The
synthesis and characterization details of the Gel-MA could be
found in the ESI.†
For Arg-UPEAs, the synthesis and characterization details
can also be found in the ESI.† The unsaturated di-p-nitrophenyl
ester of dicarboxylic acid (Monomer I, di-p-nitrophenyl fuma-
rate (NF), x = 2) was prepared by reacting fumaryl chloride with
p-nitrophenol as previously reported. Moreover, three types of
p-toluenesulfonic acid salt of L-arginine diesters (Monomer II)
were prepared in this study: tetra-p-toluenesulfonic acid salt of
bis(L-arginine)ethane diesters Arg-2-S ( y = 2); tetra-p-toluenesulfonic
acid salt of bis(L-arginine)butane diesters, Arg-4-S ( y = 4); tetra-p-
toluenesulfonic acid salt of bis(L-arginine)hexane diesters, Arg-6-S
(
y = 6). Arg-UPEAs were prepared by the solution polycondensation
of (I) and (II) monomers at different combinations. The Arg-UPEAs
synthesized here with different combinations of diacids and diols
building blocks were: 2-UArg-2-S, 2-UArg-4-S and 2-UArg-6-S. The
Fig. 1 Illustration of the chemical structure of hydrogel precursors (Gel-MA
(
left) and Arg-UPEA (right)) and the photo image of Gel-MA/2-UArg-2-S
three Arg-UPEAs synthesized have significantly different solubility (4 : 1, w : w) hydrogel (swollen (left) and dry (right)).
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Fig. 2 Equilibrated swelling ratio of Gel-MA/Arg-UPEA hydrogels in PBS
(
pH = 7.4) solution and DI water at room temperature. (a) Gel-MA; (b) Gel-MA/2-
UArg-2-S (9/1, w/w); (c) Gel-MA/2-UArg-2-S (4/1, w/w); (d) Gel-MA/2-UArg-2-S
3/2, w/w); (e) Gel-MA/2-UArg-4-S (9/1, w/w); (f) Gel-MA/2-UArg-4-S (4/1,
w/w); (g) Gel-MA/2-UArg-4-S (3/2, w/w); (h) Gel-MA/2-UArg-6-S (9/1, w/w);
i) Gel-MA/2-UArg-6-S (4/1, w/w); (j) Gel-MA/2-UArg-6-S (3/2, w/w).
(
(
Equilibrated swelling ratio and swelling kinetics of hydrogels
The swelling activity of the developed hydrogels was systematically
investigated in terms of the equilibrated swelling ratio and the
swelling kinetics as a function of hydrogel compositions (the weight
feed ratio of Gel-MA to Arg-UPEA) and y value of Arg-UPEA.
For the swelling ratios at equilibrium (SRE), they were tested
in both 1X PBS buffer solutions and deionized water (DI water).
Fig. 3 Swelling kinetics of the Gel-MA/Arg-UPEA hydrogel in DI water at
room temperature: (a) effect of Arg-UPEA types; (b) effect of Arg-UPEA
Fig. 2 shows the SRE at room temperature for the Gel-MA weight percentage.
hydrogels. Due to the strong cationic property of Arg-UPEA,
the SRE of Gel-MA/Arg-UPEA hydrogels in DI water was always
Gel-MA hydrogel, the modulus decreases, but within the same
magnitude. In this platform, the types of Arg-UPEA may not
show significant difference.
slightly higher than that of the corresponding hydrogels in PBS
solution. Additionally, the SRE of all Gel-MA/Arg-UPEA hybrid
hydrogels were higher than that of pure Gel-MA hydrogel both
in DI water and PBS, respectively. These results implied that the
introduction of Arg-UPEA moiety did enhance the SRE of the Interior morphology (SEM) of Arg-UPEA/hydrogels
hybrid hydrogels. The SRE of the hybrid hydrogels generally
increase with the increasing of weight feed ratio of Arg-UPEA to
GEL-MA/Arg-UPEA hydrogel was examined by SEM to understand
Gel-MA either in PBS buffer or DI water. Furthermore, the SRE
of the hybrid hydrogels increase with an increase of the y value
structure/morphology of hydrogel after freeze-drying would be
of the Arg-UPEA either in DI water or PBS solution at a fixed
weight ratio of Arg-UPEA to Gel-MA.
The swelling kinetics of the Gel-MA/Arg-UPEA hydrogel was
studied over a period of 3 days in deionized (DI) water at room
hydrogels, the GEL-MA/Arg-UPEA hydrogel has a similar average
temperature. Fig. 3 shows that the hydrogels with various
The cross-sectional interior morphology of the freeze-dried
the 3D structure of hybrid hydrogel. Even though the interior
different from the natural state of the swelling hydrogel, it is still
helpful for understanding the detail of the hydrogel 3D interior
micro-structure. As shown in Fig. 5, compared with Gel-MA
pore size (5–10 mm) and cell wall thickness, but with a slight
compositions (different types of Arg-UPEAs or feed weight ratio
of Gel-MA to Arg-UPEA) had a high swelling rate during the
initial 2 h. After the initial 2 h, the swelling rate leveled off, and
finally reached their swelling equilibrium within 14–18 h.
Compared with the pure Gel-MA hydrogel, the corresponding
Gel-MA/Arg-UPEA hydrogel did not show an obvious swelling
rate difference.
Compressive modulus of hydrogels
Fig. 4 shows a comparison of the mechanical properties of the
swollen Gel-MA/Arg-UPEA hydrogels in terms of the compression
Fig. 4 Compressive modulus of swollen hydrogels: (a) Gel-MA; (b) Gel-MA/2-
UArg-2-S (9/1, w/w); (c) Gel-MA/2-UArg-2-S (4/1, w/w); (d) Gel-MA/2-UArg-2-S
(
elastic modulus. Compared with Gel-MA (control), a small
3/2, w/w); (e) Gel-MA/2-UArg-4-S (9/1, w/w); (f) Gel-MA/2-UArg-4-S
4/1, w/w); (g) Gel-MA/2-UArg-4-S (3/2, w/w); (h) Gel-MA/2-UArg-6-S
modulus. After chemically introducing more Arg-UPEA into the (9/1, w/w); (i) Gel-MA/2-UArg-6-S (4/1, w/w); (j) Gel-MA/2-UArg-6-S (3/2, w/w).
amount of Arg-UPEA may slightly increase the compression
(
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relative slower degradation than the Gel-MA. This indicates that
the introduction of Arg-UPEA would cause a relatively slower
degradation, possibly due to the charge interaction with
enzymes and the more complicated 3D structure. The degradation
curves for enzyme treated groups ended before 100 wt% because
the degraded hydrogel debris were difficult to collect.
Cell attachment and proliferation on the hydrogel surface and
inside hydrogels
An excellent cellular interaction for the hydrogel is normally
4
3
considered as a prerequisite for its potential application.
Subsequently, one of the main goals of this report is to
investigate the possibility to enhance the cellular interaction
performance of the Gel-MA hydrogel, a type of collagen-based
hydrogel. Here the cellular interaction with Gel-MA/Arg-UPEA
hybrid hydrogels was studied in terms of the cell attachment
and proliferation on hydrogel surface and inside hydrogels.
For the cellular interaction on surface, the HeLa cells were
cultured with a high density on the surface of Arg-UPEA hybrid
hydrogels to investigate the cell attachment and proliferation
performance. High cell density is used to obtain the cell density
difference in terms of the attached cells. After 12 h, the medium
was changed and the cells were slightly washed to remove those
that are unattached or weakly attached. Fig. 7 showed the example
of the HeLa cells cultured for 48 h on the surface of pure the
Gel-MA hydrogel, the PEG-DA hydrogel, and the Gel-MA/2-UArg-2-S
hybrid hydrogel with different weight ratios, respectively. Com-
pared with the pure PEG-DA hydrogel control (Fig. 7b), the pure
Gel-MA hydrogel (Fig. 7a) demonstrated a better cell morphology
Fig. 5 SEM images of freeze dried Gel-MA and Gel-MA/2-UArg-2-S
hybrid hydrogel (A: Gel-MA; B: Gel-MA/2-UArg-2-S (9/1, w/w); C: Gel-MA/
2-UArg-2-S (4/1, w/w); D: Gel-MA/2-UArg-2-S (3/2, w/w)). Scale bar is 20 mm.
higher number of nanofibers entangled with the hydrogel cells.
0 wt% or 20 wt% of Arg-UPEA may not bring many nanofibers,
1
but 40 wt% of Arg-UPEA does cause obvious fibrous nanostructures.
These fibrous nanostructures may be helpful to assist the formation
of vascular structures or improve the sustained release of proteins.
Biodegradation of hydrogels
Fig. 6 shows the biodegradation results of Gel-MA/Arg-UPEA
hydrogels. Before biodegradation, all the hydrogels were soaked
in buffers for 24 h to remove the residues. In PBS buffer,
without collagenase type II enzyme, all the hydrogels showed
a slow degradation within 5 weeks. The types of Arg-UPEA did
(fully attached and expanded) and higher amounts of attached/
proliferated (high density) HeLa cells after 48 h; which was normal
and reasonable for Gel-MA hydrogels. Furthermore, after the
introduction of Arg-UPEA, the result (Fig. 7c–e) indicated that the
Arg-UPEA did not change the morphology of the attached cells, but
significantly enhance the density of attached cells. Fig. 7c–e
illustrated the example of the HeLa cells cultured for 48 h on the
surface of the Gel-MA/2-UArg-2-S hybrid hydrogel with different
weigh ratios. Therefore, the cell culture results indicated that the
À1
not show obvious changes. However, the addition of 2 U mL
of collagenase enzyme helps to degrade the pure Gel-MA
À1
hydrogel completely within two weeks. The 2 U mL collage-
nase concentration was around the concentration found during
4
2
wound healing. For the effects of Arg-UPEA types, the results
indicated that Gel-MA/Arg-UPEA (1 : 4, w/w) hydrogels showed
2Arg-UPEA could support and enhance the cell attachment and
proliferation.
Fig. 7 Representative micrographs of HeLa cells after 48 h culture on the
hydrogel surface, 10Â. Cells cultured on the hydrogel surface: (a) Gel-MA;
(b) PEG-DA; (c) Gel-MA/2-UArg-2-S (9/1, w/w); (d) Gel-MA/2-UArg-2-S
(4/1, w/w); (e) Gel-MA/2-UArg-2-S (3/2, w/w).
Fig. 6 Biodegradation of the Gel-MA/Arg-UPEA hydrogels at 37 1C in PBS
buffer or enzyme solution.
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Furthermore, the normal cells, such fibroblasts, were encap-
sulated into hydrogels to compare the cellular spreading and
proliferation inside the hydrogels. After 1 day, the live-dead
assay results (Fig. 8) indicated that the majority of the encap-
sulated cells were alive (green) for Gel-MA and Gel-MA/2-UArg-
2-S (4/1, w/w) groups. The different size and intensity of the
light dots confirmed that the cells were in the different sites of
the 3D hydrogel scaffolds. Comparing with Gel-MA group, Gel-
MA/2-UArg-2-S (4/1, w/w) group exhibited comparable cell sur-
vival to the control Gel-MA but improved cell spreading (spread-
ing of cells encapsulated in Gel-MA/2-UArg-2-S could be
observed after 1 day culture). After 3 days, the encapsulated
cells all spread and proliferated (Fig. 9). Both groups showed
high amount of living cells with complete attachment and the
Gel-MA/2-UArg-2-S (4/1, w/w) group shows obvious higher Fig. 10 Proliferation of NIH 3T3 fibroblasts after 7 days culture inside the
hydrogels (Gel-MA and Gel-MA/2-UArg-2-S (4/1, w/w)).
degree of cell spreading (compare Fig. 9a and b), possibly due
to the positive charges of Gel-MA/2-UArg-2-S, enhancing cell
spreading. Moreover, cell proliferation was enhanced when
2-UArg-2-S was incorporated into the Gel-MA hydrogels
(Fig. 10) as suggested by the picogreen DNA quantification assay.
All these results indicated that this new hybrid hydrogel
platform has great potential to act as a new type of scaffold for
tissue engineering and drug delivery applications.
In vitro inflammatory response of hydrogels
In this report, to evaluate the in vitro inflammatory response
from mouse J774 macrophage activation after being cultured
on hydrogel surface for 48 h, an ELISA assay was utilized to
measure the TNF-a produced from the macrophage. The in vitro
inflammatory results (Fig. 11) showed that both Gel-MA and
Fig. 11 In vitro inflammation assay of hydrogels.
Gel-MA/Arg-UPEA hydrogels exhibited a low level inflammatory
response. The increasing amount of Arg-UPEA component
percentage did not show an obvious change in terms of
TNF-a production. Therefore, the introduction of Arg-UPEA into
Gel-MA hydrogel did not increase the inflammation response of
Gel-MA. Other hydrogels were not used here for comparison due
to the low macrophage attachment on their surfaces.
Fig. 8 Viability of NIH 3T3 fibroblasts after 1 day culture inside the
hydrogel, 10Â. (a) Gel-MA; (b) Gel-MA/2-UArg-2-S (4/1, w/w). Green:
living cells; red: dead cells.
Controlled release of BMP-2 via Gel-MA/Arg-UPEA hybrid
hydrogels
The sustained release of protein drugs is still a challenge for
most of the hydrogel system. With the introduction of a strong
cationic moiety, the new platform is expected to be able to
release the proteins in a more controllable manner. With a
loaded BMP of 5 mg per hydrogel pellet, Fig. 12 demonstrates
the protein release profiles from hydrogels in PBS buffer at
37 1C. The BMP content was then analyzed by an ELISA assay.
Subsequently, the release data from pure Gel-MA hydrogel
showed that the free BMP-2 release was completed around
Fig. 9 Representative fluorescent micrographs of NIH 3T3 fibroblasts
after 3 day culture inside the hydrogel, 10Â. (a) Gel-MA; (b) Gel-MA/2-
UArg-2-S (4/1, w/w). Phalloidin stains cell filament green and DAPI stains
cell nuclei blue.
2–3 days. For the Gel-MA/2-UArg-2-S hydrogel, the protein
release was completed around 5–10 days due to the strong
electrostatic interaction between Arg-UPEA and BMP-2. An
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Journal of Materials Chemistry B
4
5
A. S. Hoffman, Adv. Drug Delivery Rev., 2002, 54, 3–12.
K. Y. Lee and D. J. Mooney, Chem. Rev., 2001, 101,
1869–1879.
6
7
8
9
N. Bertrand, J. Wu, X. Xu, N. Kamaly and O. C. Farokhzad,
Adv. Drug Delivery Rev., 2014, 66, 2–25.
J. Panyam and V. Labhasetwar, Adv. Drug Delivery Rev., 2003,
55, 329–347.
Z. Zhao, J. Wang, H.-Q. Mao and K. W. Leong, Adv. Drug
Delivery Rev., 2003, 55, 483–499.
K. W. Leong and R. Langer, Adv. Drug Delivery Rev., 1988, 1,
1
99–233.
0 Y. Pan, X. Du, F. Zhao and B. Xu, Chem. Soc. Rev., 2012, 41,
912–2942.
1 J. Wu, N. Kamaly, J. Shi, L. Zhao, Z. Xiao, G. Hollett, R. John,
S. Ray, X. Xu, X. Zhang, P. W. Kantoff and O. C. Farokhzad,
Angew. Chem., Int. Ed., 2014, 53, 8975–8979.
1
1
2
Fig. 12 Controlled release of BMP-2 from hydrogels.
increase of percentage of the Arg-UPEA component would cause 12 Y. Wen, W. Liu, C. Bagia, S. Zhang, M. Bai, J. M. Janjic,
slower protein release. The bioactivity comparison (Fig. S10,
ESI†) indicated there is no obvious bioactivity change for BMP-2
after being released from the Gel-MA/2-UArg-2-S hydrogel.
N. Giannoukakis, E. S. Gawalt and W. S. Meng, Acta Biomater.,
2014, 10, 4759–4767.
13 D.-Q. Wu, J. Wu and C.-C. Chu, Soft Matter, 2013, 9,
3
965–3975.
4 J. Wu, X. Zhao, D. Wu and C.-C. Chu, J. Mater. Chem. B,
014, 2, 6660–6668.
1
1
Conclusions
2
5 S. K. Seidlits, C. T. Drinnan, R. R. Petersen, J. B. Shear,
A new biocompatible and biodegradable Gel-MA/Arg-UPEA
hybrid hydrogel platform was successfully developed via UV
photo-crosslinking as a promising tissue engineering scaffold
and drug delivery carrier. The physicochemical, swelling,
mechanical, and morphological properties were systematically
investigated. Conclusively, cellular tests indicated that Gel-MA/
Arg-UPEA hydrogels show excellent cell attachment and prolif-
eration on its hydrogel surface and inside the hydrogel, better
than the Gel-MA itself. A drug release study of BMP-2 also
showed that the controlled and sustained release of protein
drugs could be achieved for a few days, depending on the
hydrogel composition. The excellent biocompatibility and
interesting controllable drug release profiles of these hybrid
hydrogels reveal that the hybrid cationic hydrogel system
developed in this study would have the great potential to act
as a tissue engineering scaffold and drug carrier.
L. J. Suggs and C. E. Schmidt, Acta Biomater., 2011, 7,
2
401–2409.
6 D.-Q. Wu, J. Wu, X.-H. Qin and C.-C. Chu, J. Mater. Chem. B,
015, 3, 2286–2294.
1
1
2
7 Y. Wen, H. R. Kolonich, K. M. Kruszewski, N. Giannoukakis,
E. S. Gawalt and W. S. Meng, Mol. Pharmaceutics, 2013, 10,
1035–1044.
1
1
2
8 T. Xu, P. Molnar, C. Gregory, M. Das, T. Boland and
J. J. Hickman, Biomaterials, 2009, 30, 4377–4383.
9 C. Zhong, J. Wu, C. Reinhart-King and C. Chu, Acta Biomater.,
2010, 6, 3908–3918.
0 G. Sun, X. Zhang, Y.-I. Shen, R. Sebastian, L. E. Dickinson,
K. Fox-Talbot, M. Reinblatt, C. Steenbergen, J. W. Harmon
and S. Gerecht, Proc. Natl. Acad. Sci. U. S. A., 2011, 108,
20976–20981.
2
2
2
2
1 Y.-C. Chen, R.-Z. Lin, H. Qi, Y. Yang, H. Bae, J. M. Melero-
Martin and A. Khademhosseini, Adv. Funct. Mater., 2012, 22,
2027–2039.
2 J. Wu, X. Wang, J. K. Keum, H. Zhou, M. Gelfer, C. A. Avila-
Orta, H. Pan, W. Chen, S. M. Chiao and B. S. Hsiao,
J. Biomed. Mater. Res., Part A, 2007, 80, 800–812.
3 S. Gerecht, J. A. Burdick, L. S. Ferreira, S. A. Townsend,
R. Langer and G. Vunjak-Novakovic, Proc. Natl. Acad. Sci.
U. S. A., 2007, 104, 11298–11303.
Acknowledgements
Project is supported by the National Natural Science Foundation
of China (Grant No. 81300885) and Shandong Provincial Natural
Science Foundation Grant No: (ZR2013HQ052; ZR2013HM086).
Yong Wen is supported by Young Scholars Program of Shandong
University (YSPSDU).
4 K. Obara, M. Ishihara, T. Ishizuka, M. Fujita, Y. Ozeki,
T. Maehara, Y. Saito, H. Yura, T. Matsui and H. Hattori,
Biomaterials, 2003, 24, 3437–3444.
Notes and references
1
N. A. Peppas, J. Z. Hilt, A. Khademhosseini and R. Langer, 25 J. A. Rowley, G. Madlambayan and D. J. Mooney, Biomaterials,
Adv. Mater., 2006, 18, 1345–1360.
1999, 20, 45–53.
K. Y. Lee and S. H. Yuk, Prog. Polym. Sci., 2007, 32, 669–697. 26 A. Gaowa, T. Horibe, M. Kohno, K. Sato, H. Harada,
2
3
S. Varghese and J. H. Elisseeff, Adv. Polym. Sci., 2006, 203,
5–144.
M. Hiraoka, Y. Tabata and K. Kawakami, J. Controlled Release,
2014, 176, 1–7.
9
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2015

View Article Online
Journal of Materials Chemistry B
Paper
2
7 S. A. Oh, H. Y. Lee, J. H. Lee, T. H. Kim, J. H. Jang, H. W. Kim 36 X.-Z. Zhang, P. Jo Lewis and C.-C. Chu, Biomaterials, 2005,
and I. Wall, Tissue Eng., Part A, 2012, 18, 1087–1100. 26, 3299–3309.
8 C. B. Hutson, J. W. Nichol, H. Aubin, H. Bae, S. Yamanlar, 37 J. Wu, D. Wu, M. A. Mutschler and C. C. Chu, Adv. Funct.
2
S. Al-Haque, S. T. Koshy and A. Khademhosseini, Tissue
Eng., Part A, 2011, 17, 1713–1723.
9 S.-M. Lien, L.-Y. Ko and T.-J. Huang, Acta Biomater., 2009, 5,
Mater., 2012, 22, 3815–3823.
38 L. A. Reis, L. L. Y. Chiu, Y. Liang, K. Hyunh, A. Momen and
M. Radisic, Acta Biomater., 2012, 8, 1022–1036.
39 A. S. Hoffman, Adv. Drug Delivery Rev., 2012, 64(supple-
ment), 18–23.
40 J. Wu, M. A. Mutschler and C.-C. Chu, J. Mater. Sci.: Mater.
Med., 2011, 22, 469–479.
2
3
3
3
3
670–679.
0 Y.-H. Lin, H.-F. Liang, C.-K. Chung, M.-C. Chen and H.-W.
Sung, Biomaterials, 2005, 26, 2105–2113.
1 A. Bigi, G. Cojazzi, S. Panzavolta, N. Roveri and K. Rubini,
Biomaterials, 2002, 23, 4827–4832.
41 J. A. Benton, C. A. DeForest, V. Vivekanandan and K. S. Anseth,
Tissue Eng., Part A, 2009, 15, 3221–3230.
2 J. L. Ifkovits and J. A. Burdick, Tissue Eng., 2007, 13,
2
369–2385.
42 H. Shin, B. D. Olsen and A. Khademhosseini, Biomaterials,
2012, 33, 3143–3152.
43 X. Zhao, Q. Lang, L. Yildirimer, Z. Y. Lin, W. G. Cui, N. Annabi,
K. W. Ng, M. R. Dokmeci, A. M. Ghaemmaghami and
A. Khademhosseini, Adv. Healthcare Mater., 2015, DOI:
10.1002/adhm.201500005.
3 R. M. K. Ramanan, P. Chellamuthu, L. Tang and K. T. Nguyen,
Biotechnol. Prog., 2006, 22, 118–125.
3
4 N. Wang, X. S. Wu and J. K. Li, Pharm. Res., 1999, 16, 1430–1435.
5 J. D. Kretlow, L. Klouda and A. G. Mikos, Adv. Drug Delivery
Rev., 2007, 59, 263–273.
3
This journal is ©The Royal Society of Chemistry 2015
J. Mater. Chem. B