Polypeptide-engineered physical hydrogels designed from the coiled-coil region of cartilage oligomeric matrix protein for three-dimensional cell culture

Article abstract of DOI:10.1039/c4tb00107a

Photo-cross-linkable physical hydrogels based on the coiled-coil region of the cartilage oligomeric matrix protein and polyethylene glycol diacrylate were designed and synthesized to mimic the natural extracellular matrix for three-dimensional cell culture. The engineered polypeptides (Pcys and RGDPcys) were modified with polyethylene glycol diacrylate to form photo-cross-linkable multifunctional macromers via the Michael-type addition reaction between the cysteine residues and acrylates. Gel formation was confirmed by rheological measurements. The swelling ratio and stability of 10% w/v RGDP-PEG-acrylate _6k hydrogel were 38% and 15 days, respectively. Spreading and migration of encapsulated fibroblast cells were observed in these physical hydrogels, while round cells were observed in a covalent control hydrogel. In addition, rapid self-healing of these physical hydrogels can provide a flexible way to build tissue by self-assembly and bottom-up approach. The results demonstrate that such physical hydrogels are expected to have great potential applications in tissue engineering.

Full text of DOI:10.1039/c4tb00107a

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Polypeptide-engineered physical hydrogels
designed from the coiled-coil region of cartilage
oligomeric matrix protein for three-dimensional
cell culture†
Cite this: J. Mater. Chem. B, 2014, 2,
123
3
Ming-Hao Yao, Jie Yang, Ming-Shuo Du, Ji-Tao Song, Yong Yu, Wei Chen,
Yuan-Di Zhao* and Bo Liu*
Photo-cross-linkable physical hydrogels based on the coiled-coil region of the cartilage oligomeric matrix
protein and polyethylene glycol diacrylate were designed and synthesized to mimic the natural extracellular
matrix for three-dimensional cell culture. The engineered polypeptides (Pcys and RGDPcys) were modified
with polyethylene glycol diacrylate to form photo-cross-linkable multifunctional macromers via the
Michael-type addition reaction between the cysteine residues and acrylates. Gel formation was
confirmed by rheological measurements. The swelling ratio and stability of 10% w/v RGDP-PEG-
acrylate6k hydrogel were 38% and 15 days, respectively. Spreading and migration of encapsulated
fibroblast cells were observed in these physical hydrogels, while round cells were observed in a covalent
control hydrogel. In addition, rapid self-healing of these physical hydrogels can provide a flexible way to
build tissue by self-assembly and bottom-up approach. The results demonstrate that such physical
hydrogels are expected to have great potential applications in tissue engineering.
Received 17th January 2014
Accepted 26th February 2014
DOI: 10.1039/c4tb00107a
www.rsc.org/MaterialsB
8
polyethylene glycol diacrylate (PEGDA), polyethylene glycol
1
. Introduction
7
dimethyl acrylate (PEGDMA), polyethylene glycol divinyl sul-
9
10
Hydrogels are three-dimensional (3-D) polymeric networks that phone (PEGVS), and poly(propylene fumarate) (PPF), have
can absorb large amounts of water but remain insoluble due to been synthesized and characterized. In the presence of photo-
the formation of chemical and/or physical cross-links between initiator, photo-cross-linked hydrogels can be formed upon
1
polymer chains. During the last decade, hydrogels have exposure to visible or ultraviolet (UV) light. Therefore, photo-
become increasingly attractive as an important form of scaffold polymerisable hydrogels have many promising properties:
11
in the elds of tissue engineering and biomaterials because of easily dened different sizes and shapes; high photo-sensi-
their high moisture, soness, elasticity, and good biocompati- tivity against light irradiation; ignorable release of reaction heat
2
10
bility. Hydrogels are especially suitable to mimic living organ- in the process of polymerization; fast hydrogel formation from
3
isms compared with other types of biomaterials. Hydrogels are a few seconds to several minutes at room temperature or
equivalent to the extracellular matrix (ECM) in terms of material physiological temperature; good solubility of polymer precur-
characteristics. Aer absorbing water, the slippery effect sors, which makes it suitable for injection and in situ formation
4
12
reduces frictional irritation to the surrounding tissue. There- of cross-linked hydrogels. With these unique advantages,
fore, hydrogels have been used extensively in biomedicine and photopolymerisable hydrogels have been widely applied in
5
,6
tissue engineering.
Photo-cross-linkable hydrogels for use in biomedicine and prepared by physical cross-linking and chemical cross-linking
biomedical research. Photopolymerisable hydrogels have been
13
tissue engineering have attracted considerable attention owing methods. The present photopolymerisable hydrogels are
to their rapid in situ formation of hydrogel under light irradia- almost all chemically cross-linked hydrogels. However, the
7
tion. Various photo-cross-linkable macromers, such as applications of chemically cross-linked hydrogels in 3-D cell
culture are limited because the rate of hydrolysis or enzymolysis
of chemical bonds is difficult to regulate.
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for
Nowadays, physical hydrogels have attracted much attention
Optoelectronics
Department of Biomedical Engineering, College of Life Science and Technology,
Huazhong University of Science and Technology, Wuhan 430074, P. R. China. gels include synthetic polymers (PNIPAAM, PEO–PPO–PEO,
– Hubei Bioinformatics & Molecular Imaging Key Laboratory,
14–16
as biomaterials to mimic the natural ECM.
Physical hydro-
14
E-mail: zydi@mail.hust.edu.cn; lbyang@mail.hust.edu.cn; Fax: +86 27-8779-2202
PEG–PLLA, etc.), natural polymers (agarose, collagen, hyalur-
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4tb00107a
5,15
16
onic acid, gelatin, brin, etc.), and hybrid polymer systems.
1
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 3123–3132 | 3123

View Article Online
Journal of Materials Chemistry B
Paper
Synthetic polymer materials lack the biological motifs and must from Qiagen China (Shanghai) Co., Ltd. Tri-distilled water was
be carefully screened for potential cytotoxicity, and the used for all solutions.
complicated compositions of natural polymer materials make it
17
difficult to obtain identical compositions. As an alternative, 2.2. Synthesis and purication of the polypeptide
hydrogels formed from genetically engineered polypeptides
PQE9P plasmid was a gi from Prof. David Tirrell at the Cal-
ifornia Institute of Technology Pasadena, CA. The gene encod-
have been studied as scaffolds in tissue engineering applica-
18,19
tions.
Polypeptides are chains of various amino acids
ing polypeptide Pcys was synthesized by the method of
polymerase chain reaction (PCR) which used PQE9P plasmid as
the template. The PQE9Pcys plasmid was constructed from the
Pcys segment and the PQE9P plasmid through DNA recombi-
nant manipulation. The Pcys segment and the PQE9P plasmid
were digested by BamHI to yield cohesive ends. The digested
Pcys segment and PQE9P vector were ligated with T4 DNA ligase
to construct the PQE9Pcys plasmid. The segment encoding RGD
and containing NheI and SpeI restriction sites was also acquired
by PCR. The digested RGD segment with NheI and SpeI was
inserted into the NheI restriction site of PQE9Pcys to construct
the PQE9RGDPcys plasmid. The sequences of PQE9Pcys and
PQE9RGDPcys were veried at the DNA sequencing core facility
of Sunny Institute at Shanghai. PQE9Pcys and PQE9RGDPcys
plasmids were transformed into E. coli strain M15, respectively.
through peptide bonds on the basis of a certain order, which
have excellent biocompatibility and controllable biodegrad-
ability. Compared with the synthetic materials, polypeptide-
based hydrogels can better mimic the complex and dynamic
natural ECM because proteins are major players in providing
structural support, cell adhesion, and signal regulation in
20
natural ECM. Polypeptide-based hydrogels are expected to
provide an effective way for settling the biocompatibility,
functionality, and other crucial issues of biomedical materials.
We have previously investigated a hydrophilic chain anked by
a terminal self-assembling leucine zipper domain and a
terminal photoreactive acrylate group as photo-cross-linkable
21
materials. Although this system showed excellent biocompat-
ibility and allowed reversible opening and closing of 3D cell
migration paths, the hydrogel dissolved quickly in physiological
environment due to the formation of intramolecular loops.
In this study, photo-cross-linkable physical hydrogels based
on the coiled-coil region (named P) of the cartilage oligomeric
matrix protein (COMP) and PEGDA were designed and synthe-
sized. COMP is a noncollagenic glycoprotein present in carti-
ꢀ
Bacterial culture was grown at 37 C in 1 L of 2ꢁ YT medium
ꢂ1
ꢂ1
supplemented with 50 mg L of ampicillin and 25 mg L of
kanamycin. The culture was induced with 1 mM IPTG when the
optical density at 600 nm reached 0.7–1.0. The culture was
continued for an additional 4 h. Cells were harvested by
centrifugation (6000 g, 30 min) and lysed in 8 M urea (pH ¼ 8.0).
The cell lysate was centrifuged at 12 000 g for 30 min, and the
supernatant was collected for purication. A 6ꢁ histidine tag
encoded in the pQE9 vector allows the polypeptide to be puri-
22
lage, tendons, ligaments, and osteoblasts. The engineered
polypeptides Pcys and RGDPcys (each containing a C-terminal
cysteine) were modied with PEGDA via the Michael-type
addition reaction between the thiol and acrylate to form photo-
cross-linkable macromers. The macromers with multiple acry-
late arms formed hydrogels in the presence of photoinitiator
and UV light. The dynamic ve-stranded bundles of the P
domain were expected to provide paths for the spreading and
migration of cells in the hydrogel. The photo-cross-linked
hydrogels showed rapid self-healing characteristics. In addi-
tion, the cytotoxicity of these photo-cross-linked hydrogels was
tested. These characteristics of physical hydrogels photo-cross-
linked from self-assembled polypeptides will provide unique
opportunities in tissue engineering.

ed by affinity chromatography on a Ni-NTA resin following the
denaturing protocol given by Qiagen. The eluted fractions were
dialyzed against sterile tri-distilled water for three days at room
temperature, frozen, and lyophilized. The puried polypeptides
were analyzed on a Bruker Reex III reectron MALDI-TOF mass
spectrometer. Pcys (MS: 7050.3 Da, the theoretical calculation of
molecular weight: 7053.8 Da), RGDPcys (MS: 8483.5 Da, the
theoretical calculation of molecular weight: 8487.2 Da).
2.3. Synthesis of PEGDA
PEGDA was synthesized according to previously published
8
methods. Briey, a solution of PEG in dichloromethane was
2. Materials and methods
reacted under argon with acryloyl chloride and triethylamine at
an acryloyl chloride : OH molar ratio of 4 : 1. The product was
precipitated in ice-cold diethyl ether, dried under vacuum, and
2.1. Materials
Polyethylene glycol (PEG, molecular weight: 2 kDa, 6 kDa, 10
kDa) was purchased from Sinopharm Chemical Reagent Co.,
Ltd (Shanghai, China). Acryloyl chloride was obtained from
Aladdin Inc. (Shanghai, China). Photoinitiator 2-hydroxyl-1-(4-
ꢀ
stored at ꢂ20 C under the protection of argon. The nal yields
of the three products were more than 85%. High degree of
1
substitution (>95%) was conrmed by H NMR (Varian Unity
1
ꢀ
spectrometer). H NMR (300 MHz, CDCl , 25 C, TMS): d ¼ 6.2
3
(hydroxyethoxy)phenyl)-2-methyl-1-propanone (Irgacure 2959)
(
d, 2H), 6.0 (d, 2H), 5.7 (d, 2H), 4.1 (t, 4H), 3.4 ppm (m, 539H).
was a kind gi from Ciba Inc. (Tarrytown, NY). Tris(2-carboxy-
ethyl)phosphine (TCEP), b-mercaptoethanol, isopropyl-b-D-thi-
ogalactoside (IPTG), ampicillin, kanamycin, calcein AM, and
2.4. Preparation of polypeptide–PEGDA conjugates
ethidium homodimer were purchased from Sigma-Aldrich, Inc. Pcys or RGDPcys (27 mmol) was dissolved in 2.7 mL 8 M (pH ¼ 8)
(St. Louis, MO). Restriction endonuclease BamHI, NheI, SpeI, urea buffer followed by addition of 300 mL TCEP (150 mM). The
and T4 DNA ligase were obtained from New England Biolabs mixture was incubated at room temperature. Aer incubation
Inc. (Beijing, China). Ni-NTA separation column was purchased for 1 h, 270 mmol PEGDA and 27 mL 8 M (pH ¼ 8) urea were
3124 | J. Mater. Chem. B, 2014, 2, 3123–3132
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
added. The pH of the mixture was adjusted to 8.0. The mixture tubes. M1 was the weight of an empty EP tube, and M2 was the
was stirred at room temperature for 24 h in the dark. SDS- total weight of the EP tube and hydrogel. Four hundred
polyacrylamide gel electrophoresis (SDS-PAGE, 12%) was used microliters of PBS were added to the hydrogel and renewed
to monitor the degree of reaction. The excess PEGDA was every 12 h. Aer two days, PBS was removed, and the hydrogel
removed by a Ni-NTA affinity column. The puried RGDP/P- and EP tube were weighed (M3) aer quick blotting with lter
PEG-acrylate was dialyzed against sterile tri-distilled water for paper. The swelling ratio (R) of the hydrogels was calculated
3
days, frozen, and lyophilized. The products were stored at using the following equation: R ¼ (M3 ꢂ M2)/(M2 ꢂ M1) ꢁ
ꢀ
23
ꢂ20 C under the protection of argon.
100%. The stability of hydrogels was dened as the time
needed for the hydrogel to disappear. The experiments were
performed in triplicate.
2
.5. Preparation of covalently cross-linked RGDP-PEG-
acrylate
2.9. Scanning electron microscopy (SEM)
To prepare a covalently cross-linked control hydrogel, the
primary amines on the lysine and N-terminus of the polypeptide RGDP-PEG-acrylate6k hydrogels (6% and 8% w/v) were prepared,
ꢀ
P were covalently coupled with adipic acid through 1-ethyl-3-(3- frozen overnight at ꢂ80 C, and lyophilized for 3 days until their
dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N- contained water was completely sublimed. The lyophilized
hydroxysuccinimide (NHS) chemistry. The mixture of 2 mM hydrogels were fractured carefully in liquid nitrogen. The frac-
adipic acid, 20 mM EDC, and 60 mM NHS prepared in 100 mM ture surfaces of the hydrogels were coated with gold for 30 s,
MES (pH ¼ 5.5) buffer was incubated at room temperature for and the interior morphology of the hydrogels was observed by a
15 min. The pH of the solution was adjusted to 8.0, followed by SEM (Nova NanoSEM450).
addition of 0.2 mM RGDP-PEG-acrylate. The mixture was stirred
in the dark at room temperature for 4 h. The adipic acid-treated
RGDP-PEG-acrylate was analyzed using 12% SDS-PAGE. The
molar ratios of 1 : 2.5 and 1 : 5 (RGDP-PEG-acrylate : adipic
acid) were also covalently coupled according to the same
method.
2
.10. Rheological measurements
Solutions of P-PEG-acrylate6k (8% and 10% w/v) and adipic acid-
treated P-PEG-acrylate6k (8% w/v) containing 0.2% photo-
initiator Irgacure 2959 were prepared. The pH of each solution
was adjusted to 7.4, and the solution was placed between two
glass slides separated by a 1.1 mm thick spacer, followed by
2.6. Hydrogel preparation and disassembly
ꢂ2
exposure to UV light (365 nm, 12.5 mW cm ) for 5 min to form
Solutions containing various concentrations (3–11% w/v) of hydrogels. The cover glass was removed carefully, and the
P-PEG-acrylate or RGDP-PEG-acrylate and 0.2% photoinitiator hydrogel was transported to the parallel plate of a HR-2 discovery
Irgacure 2959 were prepared in PBS. The pH of each solution hybrid rheometer to perform the rheological test. A strain sweep
ꢂ1
was adjusted to 7.4, and the solutions were exposed to UV light test (0–10%) at an oscillatory frequency of 10 rad s was per-
ꢂ2
(
365 nm, 12.5 mW cm ) for 5 min to form hydrogels. To formed to reveal the linear viscoelastic regime, followed by a
determine which type of cross-links (chemical or physical) were frequency sweep test performed at a strain value in the linear
in hydrogels, 1 mL 8% (w/v) P-PEG-acrylate solution containing regime. Correlation parameters: 20 mm parallel plate, 1000 mm
ꢀ
ꢂ1
0
.2% 2959 was prepared in two transparent glass bottles. Aer gap, 37 C, 1% strain, and 100–0.1 rad s angular frequency. All
hydrogel formation under UV, 8 M urea (pH 7.4) and 0.01 M PBS measurements were repeated three times.
pH 7.4) were each added to one of the hydrogel samples.
(
Samples were shaken at room temperature for 10 h to examine
whether the hydrogel dissolved or not. The solubility of a
covalently cross-linked RGDP-PEG-acrylate hydrogel was also
examined.
2
.11. 2-D cell adhesion assay
To observe the adhesion of broblasts (NIH 3T3) on hydrogels
photo-cross-linked from P-PEG-acrylate, RGDP-PEG-acrylate,
and adipic acid-treated RGDP-PEG-acrylate (all 10% w/v, 1.1
mm thick, prepared in pH 7.4 PBS), cells were seeded on the
2.7. Self-healing of photo-cross-linked hydrogels
surface of each hydrogel in serum-free DMEM supplemented
ꢂ1
To test whether these photo-cross-linked hydrogels have the with penicillin–streptomycin (100 units mL ) at a density of
5
ꢂ2
ꢀ
capability of self-healing, two P-PEG-acrylate hydrogels with the 2.5 ꢁ 10 cells cm and allowed to adhere for 2 h at 37 C, 5%
same size were prepared. To observe conveniently, one of the CO in a cell incubator. Hydrogels were washed with PBS three
2
hydrogel was soaked in PBS containing 0.01 M rhodamine 6G times. Each sample was stained with the calcein AM/ethidium
for 10 min. The hydrogels were brought into contact with each homodimer for 20 min and examined with a 10ꢁ objective on
other without application of any external force. The hydrogels an inverted uorescent microscope (Olympus IX71, Japan)
were clipped with tweezers at different times to determine equipped with a cool color charge-coupled device (CCD) (Pixera
whether two hydrogels have healed together.
Penguin 150CL, USA).
2.8. Swelling ratio and stability of hydrogels
2.12. 3-D encapsulation of broblast
The P-PEG-acrylate hydrogels of different concentrations The solutions of RGDP-PEG-acrylate (8% w/v) and adipic acid-
3–11% w/v) were prepared and transferred into new 1.5 mL EP treated RGDP-PEG-acrylate (8% w/v) were prepared in 100 mL
(
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 3123–3132 | 3125

View Article Online
Journal of Materials Chemistry B
Paper
serum-free DMEM containing 0.2% Irgacure 2959 and supple- should be biocompatible. The P-PEG-acrylate or RGDP-PEG-acry-
ꢂ1
mented with penicillin–streptomycin (100 units mL ). Fibro- late macromer was veried by using 12% SDS-PAGE (Fig. 1). The
4
blasts (NIH 3T3, 2.5 ꢁ 10 cells) were dispersed in RGDP-PEG- conjugation reaction between each polypeptide and PEGDA was
acrylate and adipic acid-treated RGDP-PEG-acrylate solutions performed at a 1 : 10 molar ratio. Far-UV circular dichroism (CD)
and transferred to 35 mm glass bottom culture dishes (MatTek) was employed to determine the secondary structures of poly-
followed by exposure to long-wavelength UV light for 5 min. peptides RGDPcys and RGDP-PEG-acrylate (Fig. S1†). The result
DMEM containing 1% penicillin–streptomycin antibodies and conrmed that modication of RGDPcys with PEGDA did not
1
0% fetal bovine serum was added to the surface of the alter the coiled-coil structure of the polypeptide. Compared with
ꢀ
hydrogels. Encapsulated broblasts were cultured at 37 C, 5% traditional synthetic polymers, these physical hydrogels contain-
CO in a cell incubator for 26 h. The samples were stained with ing engineered polypeptide P as the major component have not
2
the calcein AM/ethidium homodimer for 20 min and examined only the advantages of the photo-cross-linkable hydrogel, but also
with a 10ꢁ objective on an Olympus FV1000 confocal micro- similar components to natural ECM. Sequences of interest, such
scope. XYZ scanning mode was adopted for 2-D and 3-D images as binding domains and enzyme cleavage sites, can be incorpo-
of cells in hydrogels.
rated into engineered polypeptides through the exibility of
25
recombinant DNA technology. In addition, in order to promote
adhesion and spreading of cells in the hydrogel, a RGD cell-
binding domain was successfully incorporated into the
2.13. Cytotoxicity
RGDP-PEG-acrylate6k (10% w/v) photo-cross-linked hydrogels
were prepared in 96-well plates. NIH 3T3 broblasts were seeded
on the hydrogel at 5000 cells per well and cultured at 37 C and
26
N-terminus of polypeptide P. The dynamic polypeptide P in
these physical hydrogels is expected to provide paths for cell
ꢀ
24,27
spreading and migration.
2
5% CO in a cell incubator for 48 h. Tests under all conditions
were run in triplicate. Cells were stained with the calcein
AM/ethidium homodimer for 20 min and examined on an
inverted uorescent microscope (Olympus IX71, Japan). Stained
cells were trypsinized and counted using a hemocytometer.
3
.2. Covalent cross-linking of RGDP-PEG-acrylate
To prepare negative control hydrogel containing covalently
cross-linked polypeptide P, RGDP-PEG-acrylate was treated with
adipic acid through EDC/NHS coupling chemistry (Scheme 2).
The carboxylates of adipic acid reacted with NHS in the pres-
ence of EDC to synthesize activated NHS ester which subse-
quently reacted with the primary amines in the polypeptide
portion of RGDP-PEG-acrylate. Different proportions of the
coupling between NHS-activated ester of adipic acid and RGDP-
PEG-acrylate macromer were investigated, and the efficiency of
the coupling was analyzed with 12% SDS-PAGE. As shown in
Fig. 1, ve unequally intense bands of the treated RGDP-PEG-
acrylate macromer indicated the presence of pentamers, tetra-
mers, trimers, dimers, and monomer, which was consistent
with previous reports. With increasing the molar ratio of the
NHS-activated ester of adipic acid and the RGDP-PEG-acrylate
macromer (from 2.5 to 10), the ratio of pentamers increases,
and the ratio of the monomer decreases. When the ratio of the
NHS-activated ester of adipic acid and the RGDP-PEG-acrylate
macromer was 10, almost no monomers were detected. There-
fore, in this study, the ratio of 10 was chosen to prepare covalent
cross-linking of RGDP-PEG-acrylate. The covalently cross-linked
RGDP-PEG-acrylate macromer also formed hydrogels in the
presence of photoinitiator under 365 nm UV light.
3. Results and discussion
3.1. Design of the hydrogel
To prepare photo-cross-linkable physical hydrogels, coiled-coil
polypeptide P which can self-assemble into pentamers was chosen
to act as physical junctions in hydrogels. The polypeptide P as the
major component of physical hydrogels has been thoroughly
24
investigated. Acryloyl groups are usually used to prepare the
photo-cross-linked hydrogels because monomers with acryloyl
groups can photo-cross-link in the presence of long-wavelength
UV light and photoinitiator. PEG with high biocompatibility,
nonimmunogenicity, and water-solubility is oen used in tissue
engineering applications. In order to prepare photo-cross-linkable
macromers, one cysteine residue containing a free thiol was
introduced at the C-terminus of the polypeptide by genetic engi-
neering methods. Polypeptides (Pcys or RGDPcys) reacted with
excess PEGDA through the Michael-type addition reaction to
obtain the photo-cross-linkable macromers (P-PEG-acrylate or
RGDP-PEG-acrylate) having PEG anked by a photoreactive acry-
late group and a self-assembling polypeptide P. It is known that a
monomer with only one acrylate group usually forms a one-
dimensional polymer, and the formation of 3-D polymeric
21
3.3. Physical hydrogel or chemical hydrogel
networks require multiple branches or cross-linker. P-PEG-acry- To verify whether the photo-cross-linked hydrogel was a phys-
late or RGDP-PEG-acrylate macromers containing only one acry- ical gel in nature, RGDP-PEG-acrylate hydrogels were immersed
late group can form multi-branched macromers through physical in 8.0 M urea or PBS, respectively. As expected, the hydrogel
assembly of polypeptide P. Under the conditions of long-wave- completely dissolved aer 10 h shaking in the 8.0 M urea due to
length UV light and photoinitiator, the multi-branched mono- the disruption of the secondary structure of polypeptide P. In
mers cross-link and form physical hydrogels. The design and contrast, the hydrogel immersed in PBS retained its integrity
amino acid sequences of the polypeptides Pcys and RGDPcys are (Fig. 2). The result indicates that these hydrogels are physical
shown in Scheme 1. Because of the absence of toxic cross-linker gels regarding the physical bonds formed by self-assembly of
and byproduct in the preparation of these physical hydrogels, they the polypeptide P as the junctions. The hydrogel formed from
3126 | J. Mater. Chem. B, 2014, 2, 3123–3132
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
Scheme 1 (a) Illustration of the formation of photo-cross-linkable physical hydrogels from self-assembled multi-functional macromers. (a) pH
8
, 8 M urea buffer, 1.5 mM TCEP; (b) 365 nm UV light, 5 min. (b) Amino acid sequences of Pcys and RGDPcys.
Fig.
2 Disassembly of photo-cross-linked RGDP-PEG-acrylate6k
Fig. 1 SDS-PAGE of RGDP-PEG-acrylate and adipic acid-treated
RGDP-PEG-acrylate. Lane 1, RGDPcys; 2, RGDP-PEG-acrylate6k; 3–5,
RGDP-PEG-acrylate6k treated with adipic acid at various molar ratios
of adipic acid to RGDP-PEG-acrylate6k (lane 3, 10 : 1; lane 4, 5 : 1; lane
hydrogels (8% w/v) in 0.01M PBS (pH ¼ 7.4) and 8 M urea (pH ¼ 7.4)
ꢀ
at 37 C.
5
, 2.5 : 1).
3.4. Self-healing of hydrogels
To examine whether this photo-cross-linked hydrogel has the
self-healing capability, two hydrogels were put together without
any external force. For clearer observations, one of the hydro-
gels was stained yellow with rhodamine 6G. We observed that
the two hydrogels welded rapidly aer half an hour (Fig. 3), and
the rhodamine 6G in the stained hydrogel diffused rapidly into
the unstained hydrogel. The result indicates that this kind of
hydrogel not only has the self-healing capability but also
possesses appropriate permeability for transport for oxygen,
essential nutrients, and metabolic waste. Quick exchange of
nutrients and metabolites is a prerequisite of biomaterials for
tissue engineering applications. The self-healing capability of
the photo-cross-linked hydrogel may be due to the dynamic
exchange of the polypeptide P at their interface of the hydro-
Scheme 2 Preparation of adipic acid treated RGDP-PEG-acrylate.
24
gels. To further determine the effect of concentration on
adipic acid-treated RGDP-PEG-acrylate retained its integrity in 8 healing, the self-healing times of hydrogels with various
M urea over one week, further demonstrating that the untreated concentrations were investigated. The results show that the self-
photo-cross-linked gels are physical but not chemical hydrogels. healing time becomes shorter with lower concentration. The
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 3123–3132 | 3127

View Article Online
Journal of Materials Chemistry B
Paper
Fig.
3
Self-healing of photo-cross-linked RGDP-PEG-acrylate6k
ꢀ
hydrogel (8% w/v) at 25 C.
self-healing time depends strongly on the concentration of the
hydrogel, demonstrating that the dynamic exchange rate of the
self-assembled polypeptide P in the physical hydrogel relates to
the concentration of macromer. As shown in Fig. 3, the healed
hydrogel exhibits a certain mechanical strength.
Hydrogels with reversible or dynamic physical bonds will not
only manage external damage and repair themselves as self-
healing materials but also gain multi-responsive properties to
28
environmental stimuli. Thus, cells xed in these hydrogels can
be injected into the targeted positions for therapeutic and 3-D
29
bioprinting applications.
3.5. Swelling ratio and stability of hydrogels
The swelling ratios and stabilities of RGDP-PEG-acrylate6k
hydrogels with different concentrations are shown in Fig. 4. The
actual swelling ratio of a physical gel is larger than the experi-
ment result because a part of the hydrogel will dissolve in PBS
during the experiment, especially at low concentration. As
shown in Fig. 4a, the swelling ratio depends on the concentra-
tion of RGDP-PEG-acrylate . For example, the swelling ratios of
6
k
Fig. 4 Swelling ratio (a) and stability (b) of photo-cross-linked RGDP-
PEG-acrylate6k hydrogels at 37 C.
3% w/v and 11% w/v hydrogels are 19% and 38%, respectively.
ꢀ
In addition, the swelling ratios of the hydrogels have a large
variation at low concentrations and vary rarely at high concen-
trations. For instance, the swelling ratio increases by 17% when
the concentration increases from 3% w/v to 5% w/v. In contrast,
the swelling ratio increases by only 1% when the concentration
increases from 5% w/v to 11% w/v. This difference may be
attributed to the poor stability of low concentration hydrogels.
The biomaterials used in tissue engineering need the prop-
erties of high swelling ratio and excellent stability. Therefore,
the stability of the photo-cross-linked hydrogels was also tested.
As shown in Fig. 4b, the stability of the photo-cross-linked
hydrogels increases gradually from 7 days to 15 days with
increasing the concentration until the concentration reaches
stability data of physical hydrogels are closely related to the
volume of the gel and the volume of added PBS. As the stability
of these physical hydrogels is mainly decided by the dissolving
rate of gel, changing the concentration of gel will provide a
convenient method to tune the stability of the gel. In addition,
the biodegradation products of polypeptide and PEGDA can be
excluded through kidney without concentration in the human
body. The swelling ratio and stability of hydrogels were also
tuned by altering the length of PEG (the swelling ratio and
stability of the PEG2k and PEG10kare not shown).
30
9
% w/v. The stabilities are almost unchanged when the
3.6. Hydrogel morphology
concentrations are above 9% w/v. Compared with our previous
photo-cross-linked hydrogels based on leucine zipper A, The interior of photo-cross-linked RGDP-PEG-acrylate6k hydro-
hydrogels based on P domain showed signicantly improved gels is shown in Fig. 5. It can be seen that hydrogels have a
21
stability. Hydrogels based on leucine zipper A readily form uniform distribution of interior pore size. The pore sizes of 8%
intramolecular loops because the leucine zipper domain adopts hydrogel (Fig. 5a) are a little bit smaller than those of 6%
24
an antiparallel orientation. The stability of these physical hydrogel (Fig. 5b). The mentioned pores have relatively large
hydrogels can be regulated by the rate of dissolution of hydro- sizes with diameters of about 20 mm. The large pore sizes make
gel, hydrolysis of ester bonds, and degradation of polypeptide. these hydrogels act for the permeation of nutrients, exchange of
The stability of the covalently cross-linked RGDP-PEG-acrylate oxygen and carbon dioxide, discharge of metabolites and so on,
hydrogels reaches more than two months, suggesting that the which can provide a comfortable environment for cell growth
stability of these photo-cross-linked physical hydrogels is and proliferation. The rapid permeation of rhodamine 6G
mainly controlled by the rate of dissolution of the hydrogel. The (Fig. 3) further demonstrates large pores in the hydrogels. The
3128 | J. Mater. Chem. B, 2014, 2, 3123–3132
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
3
1
0
00
growth. The G and G of no adipic acid-treated RGDP-PEG-
acrylate hydrogel can crossover (transition point of gel–liquid),
0
00
while the G and G of adipic acid-treated RGDP-PEG-acrylate
hydrogels have no crossover point in 0.1–100 rad s . The result
ꢂ1
gives further evidence that RGDP-PEG-acrylate hydrogel is a
physical gel, and adipic acid-treated RGDP-PEG-acrylate
hydrogel is a chemical gel. In addition, with increasing the
concentration of macromer, the crossover point shis to the
direction of low angular frequency, indicating that the higher
Fig. 5 SEM of photo-cross-linked RGDP-PEG-acrylate6k hydrogels: concentration leads to stronger viscosity and better stability.
a) 8% w/v; (b) 6% w/v. The scale bars are 40 mm.
(
3.8. 2-D cell adhesion assay
To study 3-D cell culture in the photo-cross-linkable hydrogels,
the investigation of cell adhesion and spreading on the surface
of hydrogels (2-D cell culture) should be conducted rstly. 2-D
cell culture is a powerful tool to study the basic mechanism of
the force between cells and substrate. When the cells adhere to
the substrate, the integrins relying on the cell membrane link to
result of 6% w/v hydrogel with larger pore size than 8% w/v is
consistent with the results that the stability and mechanical
strength of 6% w/v are less than those of 8% w/v hydrogel.
3.7. Rheology of hydrogels
Hydrogel formation was further conrmed using small ampli- the ECM. The change of the connection between the integrins
tude oscillatory shear experiments. For each hydrogel, a strain and ECM leads to the realignment of cytoskeleton, spreading,
3
2–34
sweep test (0–10%) was performed at an oscillatory frequency of and migration of cells on the surface of the substrate.
1
ꢂ1
0 rad s rst. It was revealed that a strain of 1% is in the linear Previous studies have shown that the RGD sequences consisting
viscoelastic range for all tested hydrogels. The linear visco- of arginine, glycine, and aspartic acid exist in a variety of ECM.
35
elastic behavior of hydrogels was characterized by oscillatory The RGD sequence could specically combine with 11 kinds of
frequency sweep measurements. The results of the storage integrins and effectively promote cell adhesion and spreading
0
00
modulus (G ) and loss modulus (G ) are shown in Fig. 6. With in biomaterials. To promote the adhesion and spreading of cells
0
00
increasing the concentration of hydrogel, both G and G
increase gradually. The G is larger than the G at high domain was successfully incorporated into the N-terminus of
on the photo-cross-linked hydrogels, a RGD cell-binding
0
00
0
0
frequency. The G of 8% w/v hydrogel is 4300 Pa, and the G of polypeptide P. Cell adhesion on the hydrogels photo-cross-
0% w/v hydrogel is 7200 Pa at an angular frequency of 10 rad linked from P-PEG-acrylate, RGDP-PEG-acrylate, and adipic
1
ꢂ1
0
s
. The G of adipic acid-treated hydrogel changes rarely acid-treated RGDP-PEG-acrylate is shown in Fig. 7. Few NIH 3T3
compared with the untreated hydrogel at the same concentra- cells adhered on the surface of P-PEG-acrylate hydrogel (Fig. 7a),
0
00
tion. The G and G of hydrogels can be tuned not only through while signicant cell adhesion was observed on the surface of
the concentration of macromer, but also by altering the length RGDP-PEG-acrylate (Fig. 7b) and adipicacid-treated RGDP-PEG-
0
00
of PEG (data of the G and G of the PEG and PEG10k are not acrylate hydrogels (Fig. 7c). These results suggest that the
2
k
shown). Changing the stiffness of materials has proven to be a adhesion and spreading of cells on hydrogels might be medi-
useful strategy for studying 2-D cell adhesion or 3-D cell ated by the RGD sequence in the materials. In addition, the
activity of RGD was not affected aer treatment with adipic acid.
Sequences of interest, such as binding domains and enzyme
cleavage sites, can be incorporated into engineered polypeptide
P because the polypeptide P is biosynthesized by gene engi-
neering. Biological functions of fused ligands can also be
studied through 2-D cell culture.
The ability to control cell-binding ligand type and density,
and thus to study their effects on 2-D cell adhesion and 3-D cell
growth and migration, has proven to be a useful strategy for
Fig. 6 Rheological oscillatory shear measurements of photo-cross-
linked RGDP-PEG-acrylate6K hydrogels at various concentrations (10%
w/v, squares; 8% w/v, triangles) and a covalently cross-linked control
0
hydrogel at 8% w/v (circles). Storage modulus G : filled symbols; loss Fig. 7 Adhesion of NIH 3T3 fibroblasts on 10% w/v hydrogels of (a) P-
0
0
modulus G : open symbols. Measurements were performed at 1% PEG-acrylate6k; (b) RGDP-PEG-acrylate6k; and (c) adipic acid-treated
ꢀ
strain, pH 7.4, and 37 C.
RGDP-PEG-acrylate6k. The scale bars are 200 mm.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 3123–3132 | 3129

View Article Online
Journal of Materials Chemistry B
Paper
understanding specic cell–materials interactions and the basic the polypeptide P in RGDP-PEG-acrylate hydrogels forms a
36–38
mechanism.
The precursor of photo-cross-linked hydrogels dynamic construction through non-covalent self-assembly to
facilitated making hydrogels with similar mechanical proper- provide the necessary paths for the spreading and migration of
ties and tuning the RGD ligand density. Because the poly- cells. Compared with untreated physical hydrogels, the adipi-
peptide P is biosynthesized by gene engineering, the modular cacid-treated RGDP-PEG-acrylate control hydrogel is a chemical
design of our engineered polypeptide facilitates the creation of gel which would not provide fast spreading and migration
identical P that differ only in the numbers of bioactive RGD conditions. Cell fast spreading and migration (26 h) indicate
ligand. By designing different numbers of cell-adhesive RGD that cells can migrate and spread well through dynamic paths
ligands into each polypeptide P and by maintaining a constant formed by the polypeptide P, and the dynamism of pentamers is
polypeptide concentration with the engineered matrices, we are continuous and eet.
able to tune the density of cell-adhesive RGD ligand in hydro-
The migration of cells within a 3-D matrix is an important
gels with similar mechanical strength. This can provide a component of many cellular processes both in vivo and in vitro.
distinct advantage over performing such studies with natural Tissue formation during embryonic development, wound
protein-based hydrogels such as collagen and elastin.
healing, and immune responses all require the orchestrated
movement of cells in particular directions to specic locations.
However, rst of all cell migration across 3-D biomaterials has
to deal with the physical obstruction posed by the matrix itself
because the porosity of a 3-D matrix is signicantly smaller than
3.9. 3-D encapsulation of broblasts in hydrogels
Building a 3-D complex that formed from cells and biomaterials
is a technically challenging but key focus for studies of tissue
17,41
the average cell size.
Therefore, some strategies of cell
39
engineering. To investigate the situation and mechanism of
cell growth and migration in the photo-cross-linkable hydro-
gels, NIH 3T3 broblasts were encapsulated into the photo-
cross-linked hydrogels formed from RGDP-PEG-acrylate and
adipic acid-treated RGDP-PEG-acrylate (8% w/v, prepared in
DMEM containing 10% fetal bovine serum). Aer 26 h
culturing, the confocal images of encapsulated cells in the
photo-cross-linked hydrogels are shown in Fig. 8. NIH 3T3
migration within 3-D chemically cross-linked hydrogels are to
utilize the passive hydrolysis or enzymolysis of chemical bonds.
However, the hydrolysis rate is hard to control in spatial and
temporal synchrony with cellular inltration and migration
because the hydrolysis rate of the whole materials is the same.
Cell migration by the enzymolysis process only can be
8
controlled in space through the action of the protease. In the
present study, we use dynamic polypeptide P in the absence of
protease physical gel to provide the paths for cell migration
which do not depend on spatio-temporal restriction. The cells
in the proteolytically degradable hydrogels started to spread

broblasts encapsulated in the RGDP-PEG-acrylate gel spread
and migrated freely (Fig. 8a and g). NIH 3T3 broblast
spreading and migration occurred in multiple planes to the
maximum imaging depth of approximately 200 mm (Fig. 8g),
indicating that cell adhesion and spreading were not limited to
the underlying culture dish. Previous studies have demon-
strated that cells may capable of sensing on the underlying rigid
8
and migrate through the hydrogel matrix aer three days, while
cell spreading and migration appeared aer 26 h in RGDP-PEG-
acrylate hydrogel. Therefore, the key advantage of these physical
hydrogels over existing systems would be the fast migration. In
addition, the mechanical strength of the polypeptide-based
physical hydrogels will not be affected because the assembly of
coiled-coil polypeptide P is reversible.
40
substrate within several microns. Meanwhile, cells encapsu-
lated in the adipic acid-treated RGDP-PEG-acrylate gel present a
round shape (Fig. 8d and h). A possible reason for this is that
Tissue is not a simple heap of cells, but an integrated
complex with special functions formed by a certain regular
arrangement of cells. The interactions between cells and well-
organized arrangement of cells of different types are key issues
Fig. 8 Cell growth in 3-D photo-cross-linked hydrogels. Confocal
fluorescence images of NIH 3T3 fibroblast encapsulation in 8% RGDP-
PEG-acrylate6k hydrogel (a–c) and adipic acid-treated RGDP-PEG-
acrylate6k (d–f). 3-D Confocal images of NIH 3T3 fibroblast encap-
sulation in 8% RGDP-PEG-acrylate6k hydrogel (g), and adipic acid- Fig.
9
Cytotoxicity of photo-cross-linked RGDP-PEG-acrylate6k
treated RGDP-PEG-acrylate6k hydrogel (h). Scale bars in (a–f) are hydrogels (8% w/v). NIH 3T3 fibroblasts were cultured on the surface
00 mm. of hydrogels for 48 h.
2
3130 | J. Mater. Chem. B, 2014, 2, 3123–3132
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
in the research of tissue engineering. For example, the vascular
system is a well-organized arrangement of smooth muscle cells
Acknowledgements
42
and endothelial cells. Traditional biomaterials are only used This work was supported by the National Key Technology R&D
to culture a type of cells, even if they are used to culture a variety Program of China (2012BAI23B02), the National Natural Science
of cells at the same time, the exact position of cells in the Foundation of China (Grant no. 31100704, 81271616), the
hydrogel is not regulated. However, the rapid self-healing Foundation for Innovative Research Groups of the NNSFC
behavior of the photo-cross-linkable hydrogels containing the (Grant no. 61121004), and the Fundamental Research Funds for
polypeptide P made it possible that different types of cells could the Central Universities (Hust, 2013TS085). The authors thank
be organized to form tissue. Various types of cells might be Prof. David Tirrell for generously providing PQE9P plasmid. We
encapsulated into different shapes of microscale hydrogels also thank the facility support of the Center for Nanoscale
(microgels), and the cell-laden microgels with different shapes Characterization and Devices, Wuhan National Laboratory for
will form a complex by way of self-assembly and bottom-up Optoelectronics (WNLO) and Analytical and Testing Center
12,43
formation.
(HUST).
Additionally, previous studies have shown that coiled-coil
polypeptide P assembles into a pentameric cylinder-like and a
hydrophobic core that is 7.3 nm long with a diameter of 0.2–0.6
nm, and it can specically load some hydrophobic drugs, such
Notes and references
22
as vitamin D , trans-retinol (ATR), and curcumin (CCM).
1 A. S. Hoffman, Adv. Drug Delivery Rev., 2012, 64, 18–23.
2 J. Kope ˇc ek, Biomaterials, 2007, 28, 5185–5192.
3 B. V. Slaughter, S. S. Khurshid, O. Z. Fisher,
A. Khademhosseini and N. A. Peppas, Adv. Mater., 2009, 21,
3307–3329.
3
Loading hydrophobic drugs in hydrogels is difficult work in
tissue engineering, and the photo-cross-linked hydrogels con-
taining the polypeptide P can provide an easy way to load
hydrophobic drugs for controlled release.
4
5
6
7
B. Balakrishnan and R. Banerjee, Chem. Rev., 2011, 111,
453–4474.
R. Y. Tam, M. J. Cooke and M. S. Schoichet, J. Mater. Chem.,
012, 22, 19402–19411.
J. L. Drury and D. J. Mooney, Biomaterials, 2003, 24, 4337–
351.
A. Brunsen, U. Ritz, A. Mateescu, I. Hofer, P. Frank,
B. Menges, A. Hofmann, P. M. Rommens, W. Knoll and
U. Jonas, J. Mater. Chem., 2012, 22, 19590–19604.
4
3
.10. Cytotoxicity
2
To test the toxicity of the photo-cross-linked hydrogels to
mammalian cells, NIH 3T3 broblasts were cultured on the
surface of RGDP-PEG-acrylate hydrogels. Cells were seeded
directly in 96-well cell culture plates as a control group. Aer
4
4
8 h of culture, only a few dead cells (live–dead assay) were
observed. Although the number of cells in test wells was less
than those in the control wells, the amount of broblasts in
test wells increased 1.6 times aer 48 h of culture in vitro
8
9
S. Halstenberg, A. Panitch, S. Rizzi, H. Hall and J. A. Hubbell,
Biomacromolecules, 2002, 3, 710–723.
S. C. Rizzi and J. A. Hubbell, Biomacromolecules, 2005, 6,
(
Fig. 9). These results suggest that photo-cross-linked
1
226–1238.
0 J. L. Iovits and J. A. Burdick, Tissue Eng., 2007, 13, 2369–
385.
hydrogels containing P domains are not toxic to mammalian
cells in vitro.
1
1
2
1 Y. H. Lee, H. J. Chung, S. Yeo, C. H. Ahn, H. Lee,
P. B. Messersmith and T. G. Park, So Matter, 2010, 6,
4
. Conclusions
9
77–983.
Polypeptide-engineered physical hydrogels photo-cross-linked 12 Y. N. Du, E. Lo, S. Ali and A. Khademhosseini, Proc. Natl.
from multi-armed photopolymerization macromers based on Acad. Sci. U. S. A., 2008, 105, 9522–9527.
the self-assembly of coiled-coil polypeptide P were designed and 13 M. Guvendiren, H. D. Lu and J. A. Burdick, So Matter, 2012,
synthesized. These hydrogels show high swelling ratio and 8, 260–272.
stability. The dynamic pentamer structures formed by the 14 H. Wei, R. X. Zhou and X. Z. Zhang, Prog. Polym. Sci., 2013,
polypeptide P in hydrogels endow the self-healing behavior. The 38, 503–535.
physical bonds formed by self-assembly of the a-helical struc- 15 D. Seliktar, Science, 2012, 336, 1124–1128.
ture of the polypeptide allow reversible opening and closing of 16 C. Wang, R. J. Stewart and J. Kope ˇc ek, Nature, 1999, 397,
3
-D migration paths for spreading and migration of cells in
these hydrogels. The self-healing characteristic of the photo- 17 A. M. Jonker, D. W. L ¨o wik and J. C. van Hest, Chem. Mater.,
cross-linkable hydrogels enables these materials to conve- 2012, 24, 759–773.
niently build tissues through self-assembly and bottom-up 18 P. C. Bessa, R. Machado, S. N u¨ rnberger, D. Dopler,
417–420.
construction. In addition, the main components of these
physical hydrogels are biosynthetic polypeptides which are
similar to natural tissue. Therefore, this kind of hydrogel is
A. Banerjee, A. M. Cunha, J. Carlos Rodr ´ı guez-Cabello,
H. Redl and M. V. Griensven, J. Controlled Release, 2010,
142, 312–318.
expected to have favorable prospects in tissue engineering as 19 E. F. Banwell, E. S. Abelardo, D. J. Adams, M. A. Birchall,
articial scaffolds to mimic the natural ECM.
A. Corrigan, A. M. Donald, M. Kirkland, L. C. Serpell,
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 3123–3132 | 3131

View Article Online
Journal of Materials Chemistry B
Paper
M. F. Butler and D. N. Woolfson, Nat. Mater., 2009, 8, 596– 31 G. A. Silva, C. Czeisler, K. L. Niece, E. Benaish,
6
00.
D. A. Harrington, J. A. Kessler and S. I. Stupp, Science,
2004, 303, 1352–1355.
2
2
2
0 H. Geckil, F. Xu, X. H. Zhang, S. Moon and U. Demirci,
Nanomedicine, 2010, 5, 469–484.
1 B. Liu, A. K. Lewis and W. Shen, Biomacromolecules, 2009, 10,
32 A. K. Howe, B. P. Hogan and R. L. Juliano, J. Biol. Chem.,
2002, 277, 38121–38126.
33 M. A. Schwartz and M. H. Ginsberg, Nat. Cell Biol., 2002, 4,
E65–E68.
3182–3187.
2 S. K. Gunasekar, M. Asnani, C. Limbad, J. S. Haghpanah,
W. Hom, H. Barra, S. Nanda, M. Lu and J. K. Montclare, 34 C. Ballestrem, B. Hinz, B. A. Imhof and B. Wehrle-Haller,
Biochemistry, 2009, 48, 8559–8567. J. Cell Biol., 2001, 155, 1319–1332.
3 L. Zhang, Z. Wang, C. Xu, Y. Li, J. Gao, W. Wang and Y. Liu, 35 U. Hersel, C. Dahmen and H. Kessler, Biomaterials, 2003, 24,
J. Mater. Chem., 2011, 21, 10399–10406. 4385–4415.
4 W. Shen, K. C. Zhang, J. A. Korneld and D. A. Tirrell, Nat. 36 K. J. Lampe, A. L. Antaris and S. C. Heilshorn, Acta Biomater.,
Mater., 2006, 5, 153–158. 2013, 9, 5590–5599.
5 W. Shen, in Biomaterials for Tissue Engineering Applications, 37 J. P. Jung, J. V. Moyano and J. H. Collier, Integr. Biol., 2011, 3,
2
2
2
ed. A. B. Jason and L. M. Robert, Springer, New York, 2011,
pp. 244–269.
6 S. E. Fischer, X. Y. Liu, H. Q. Mao and J. L. Harden,
185–196.
38 E. Du, H. He, N. Zeng, C. Liu, Y. Guo, R. Liao, M. Sun,
Y. H. He and H. Ma, J. Innovative Opt. Health Sci., 2014, 07,
13500281–13500289.
2
2
2
2
3
Biomaterials, 2007, 28, 3325–3337.
7 B. D. Olsen, J. A. Korneld and D. A. Tirrell, Macromolecules, 39 G. D. Nicodemus and S. J. Bryant, Tissue Eng. Part B: Rev.,
010, 43, 9094–9099. 2008, 14, 149–165.
8 R. J. Wojtecki, M. A. Meador and S. J. Rowan, Nat. Mater., 40 S. Sen, A. J. Engler and D. E. Discher, Cell. Mol. Bioeng., 2009,
011, 10, 14–27. 2, 39–48.
9 Y. L. Zhang, L. Tao, S. X. Li and Y. Wei, Biomacromolecules, 41 J. C. Schense and J. A. Hubbell, J. Biol. Chem., 2000, 275,
011, 12, 2894–2901. 6813–6818.
0 T. Yamaoka, Y. Tabada and Y. Ikada, J. Pharm. Sci., 1994, 83, 42 M. Hellstr ¨o m, M. Kal ´e n, P. Lindahl, A. Abramsson and
2
2
2
601–606.
C. Betsholtz, Development, 1999, 126, 3047–3055.
43 B. Liu, Y. Liu, A. K. Lewis and W. Shen, Biomaterials, 2010,
31, 4918–4925.
3132 | J. Mater. Chem. B, 2014, 2, 3123–3132
This journal is © The Royal Society of Chemistry 2014