Full Paper

A Collagen Peptide-Based Physical Hydrogel for

Cell Encapsulationa
Charles M. Rubert Perez, Alyssa Panitch, Jean Chmielewski*

Collagen peptide-based hydrogels are prepared and characterized for application in 3D cell
growth. Physical hydrogels are formed by covalently linking a collagen-based peptide to an
8-arm poly(ethylene glycol) star polymer. The resulting viscoelastic hydrogels have the ability
to melt into a liquid-like state near the melting temperature of the collagen triple helix and
reform back into an elastic-state at room
temperature, adding a thermoresponsive feature to the material. In addition,
the hydrogels possess desirable stiffness,
as well as a highly cross-linked network
of pores where cells are found to reside,
making the hydrogels promising scaffolds for the culture of hMSCs.

Introduction
Collagen is the most abundant protein in mammals, found
in connective tissue, ligaments, skin, bone, and cartilage as
well as being one of the main components of the
extracellular matrix (ECM).[1] The most common primary
structure of collagen consist of repeating units of an XaaYaa-Gly tripeptide sequence, where Xaa and Yaa are
occupied by L-proline (Pro) and 4(R)-hydroxy-L-proline
(Hyp) residues, respectively.[2] Single collagen strands
adopt a polyproline type II (PPII) helical conformation that
assemble into super-coiled right handed triple helices the
major structural motif exhibited by collagen.[3] This triple-

C. M. Rubert Perez, J. Chmielewski

Department of Chemistry, Purdue University, 560 Oval Drive,
West Lafayette, IN 47907, USA
E-mail: chml@purdue.edu
A. Panitch
Weldon School of Biomedical Engineering, Purdue University, 206
S. Martin Jischke Drive, West Lafayette, IN 47907, USA
a

1426

Supporting Information for this article is available from the Wiley

Online Library or from the author.

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

helical structure forms the basis for higher order assemblies

of collagen into fibers and fibrous networks.[4]
Natural collagen derived from animal sources has been
used to construct collagen-based scaffolds for applications
such as drug delivery,[5] bone repair,[6] cell growth,[7] and
tissue engineering.[8] However, a number of disadvantages
of using naturally derived collagen has limited potential
use, including the heterogeneity of the materials, the
possibility of immunogenic response due to the transfer of
toxic agents,[9] and difficulties in further modifying the
collagen sequence and structure to alter its physical
properties. In order to overcome these disadvantages,
chemically synthesized collagen peptides have provided a
new alternative for the formation of improved biomaterials. When using synthetic collagen peptides, one is able to
chemically control the amino acid sequence by rational
design and alter the overall self-assembly of collagen
peptide triple helices into high order structures to provide
more controllable materials for matrix engineering.[10]
In this study, we focused on the conjugation of a
synthetic collagen peptide sequence to a multiarm star
polymer in an effort to form a collagen-polymer hydrogel
with tunable properties for cell encapsulation and growth.
Hydrogels are highly hydrated materials that are able to

wileyonlinelibrary.com

DOI: 10.1002/mabi.201100230

A Collagen Peptide-Based Physical Hydrogel for Cell Encapsulation

www.mbs-journal.de

Figure 1. Schematic representation of hydrogel formation with a collagen triple helical peptide, CGG-POG8, and an 8-arm PEG-MAL star
polymer.

form fibrous 3D networks, therefore hydrogel materials are

some of the most used scaffolds for mimicking the ECM.[11]
Collagen peptide hydrogels have been used for thermoresponsive drug delivery,[12] cell encapsulation,[13] and
particle tracking analysis.[14] Our hydrogel design consisted
of an 8-arm 40 kDa poly(ethylene glycol) star polymer with
terminal maleimide functionality (8-arm PEG-MAL), and a
collagen peptide consisting of eight repeating units of the
tripeptide sequence Pro-Hyp-Gly, with an N-terminal GlyGly-Cys triad [CGG-(POG)8]. The collagen peptide was
designed to maintain a stable triple helical structure under
physiologically relevant conditions,[10b,15] whereas the
cysteine residue provides the point of reactivity with the
maleimide group of the star PEG polymer. This strategy
would lead to arms of the polymer that are functionalized
with the collagen peptide (Figure 1). Hydrogel formation is
proposed to form, therefore, through physical crosslinks
between the collagen peptide triple helices that are
attached to the star polymer arms. This architecture would
allow for multiple sites of attachment for each PEG star unit
to potentially form a highly ordered network.

Experimental Section
Materials
1

H-Rink Amide-ChemMatrix resin was purchased from BioMatrix

Inc. (Quebec, Canada). Fmoc-Gly-OH and Fmoc-Pro-OH amino acids
were purchased from Anaspec Inc. (Fremont, CA). Fmoc-Hyp(tbutyl)-OH amino acid and O-(benzotriazol-1-yl)-N,N,N,0 N0 -tetramethyluroniumhexafluorophosphate (HBTU) were purchased
from Aapptec Inc. (Louisville, KY). Fmoc-Cys(Trt)-OH amino acid
was purchased from SynPep Corp. (Dublin, CA). N-hydroxybenzotriazole hydrate (HOBT) was purchased from Oakwood Products,
Inc. (West Columbia, SC). 2,4,6-Trimethylpyridine or 2,4,6-collidine
(TMP) was purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MI) CellTiter 96 AQeous One Solution Cell Proliferation Assay was

www.MaterialsViews.com

purchased from Promega (Madison, WI). 8-arm PEG-MAL (MW

40 kDa) was purchased from Nektar Inc. (Huntsville, AL). Tris(2carboxyethyl)phosphine (TCEP) was purchased from TCI America
Inc. (Portland, OR). Live/dead cell viability/toxicity kit was
purchased from Invitrogen (Carlsbad, CA). BD Falcon cell culture
inserts (0.3 cm2 growth area) were purchased from BD Biosciences
(West Chester, PA). All other chemicals were purchased from Sigma
Chemical Co. (St. Louis, MI).

Peptide Synthesis and Purification

Peptides were synthesized using a solid-phase fluorenylmethoxycarbonyl (Fmoc)-based approach on the ChemMatrix resin with
HBTU as the coupling reagent. For cysteine coupling, a combination
of HBTU and HOBT (1 equiv. each) were used for couplings.[16] After
the final Fmoc deprotection the resin was treated with acetic
anhydride to acetylate the amino-terminus. The peptide was
cleaved from the resin using a trifluoroacetic acid (TFA) cocktail
solution [90% TFA, 1% triisopropylsilane (TIPS), 1% thioanisole, 2.5%
ethanedithiol, 2.5% anisole]. The resulting mixture was filtered, the
resin was washed with additional TFA and concentrated in vacuo.
The residue was triturated with cold diethyl ether, and the
precipitate was collected by centrifugation. For purification via
high-performance liquid chromatography (HPLC), the crude peptide was dissolved in containing 0.01 M TCEP and purged with N2 for
2 h for disulfide bond reduction. Peptide solution was then purified
by reverse phase HPLC using a Phenomenex Luna C18
, 5 mm) with an eluent consisting of solvent
(50
21.20 mm, 100 A
A (CH3CN/0.1% TFA) and solvent B (H2O/0.1% TFA) with a 230%
solvent-A gradient over 60 min and a flow rate of 10.00 mL  min1
(l214nm and l260nm). Purity of the peptides was verified by analytical
reverse-phase HPLC (see Supporting Information) using a Phenom , 5 mm) with an eluent
enex Luna C18 column (250
4.6 mm, 100 A
consisting of solvent A (CH3CN/0.05% TFA) and solvent B (H2O/
0.05% TFA) with a 250% solvent-A gradient over 30 min and a flow
rate of 1.20 mL  min1 (l214nm). The structure of the peptide was
confirmed by mass spectrometry {matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of CGG(POG)8 [MH]: calcd. 2 412.06; found 2 411.94}.

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1427

C. M. Rubert Perez, A. Panitch, J. Chmielewski

www.mbs-journal.de

Circular Dichroism (CD)

CD wavelength spectra scans were performed on a JASCO Model
J810 CD spectropolarimeter (Easton, MD) equipped with a PFD-425S
Peltier temperature control unit at 4 8C using a 0.1-cm path-length
quartz cell. The spectra was averaged over three scans taken from
300 to 210 nm with a data pitch of 0.1 nm with a bandwidth of
1 nm. The scan rate of was 100 nm  min1 with a response time 1 s.
The CD data obtained was processed into from degrees of rotation
to mean residue ellipticity. CD melting curves were determined by
measuring the mean residue ellipticity at 225 nm, while running a
temperature slope between 4 and 90 8C at 6 8C  h1 with a data
pitch of 0.2 8C, bandwidth of 4 nm and response time of 4 s. The
peptide sample was prepared by making a 2
104 M CGG-(POG)8
solution in 0.01 M phosphate buffer pH 7.4 and 5
103 M TCEP.
The polymer/peptide sample was prepared by heating a 4% PSPPOG8 hydrogel and aliquoting the required volume to make a
2
104 M PSP-POG8 solution in 0.01 M phosphate buffer pH 7.4.

Rheology
All rheological analyses were performed on a TA instrument ARG2
rheometer (New Castle, DE) using a 20-mm cone and plate
geometry with a 18 angle and a sample gap of 200 mm. For
rheological experiments, an 8-arm PEG-MAL solution in phosphate
buffer at pH 7.4 (5% w/v, 50 mL) was mixed with a CGG-(POG)8
peptide solution also in phosphate buffer at pH 7.4 (0.01 M, 50 mL)
on the rheometer plate to produce the 4% PSP-POG8 hydrogel
(100 mL). The 8% PSP-POG8 hydrogel was prepared in the same way,
but with double the concentration of the star polymer and peptide.
To avoid evaporation, a solvent trap was placed around the sample.
For each sample, three different measurements were performed.
First the storage and loss moduli was monitored while applying an
oscillation stress between 0 and 500 Pa with a constant frequency
of 5 Hz. Then, a frequency sweep was performed between 1 and
30 Hz with an constant oscillation stress of 5 Pa. Lastly, the
temperature sweep was monitored between 2560 8C and 6025 8C
with a constant oscillation stress of 5 Pa and a frequency of 5 Hz
with a gradient of 5 8C  min1.

Encapsulation Studies
For cell encapsulation studies, an 8-arm PEG-MAL solution (5 or
10 wt%, 35 mL) in PBS buffer pH 7.4 was mixed in a Falcon cell
culture insert with a CGG-(POG)8 peptide solution made by
dissolving the solid peptide (0.6 or 1.2 mg) in 35 mL of cell
suspension (1.5
106 hMSCs  mL1 in PBS buffer pH 7.4). After
hydrogel formation occurred, inserts were placed in a 24-well plate
with 700 mL of mesenchymal stem cell basal medium (MSCBM)
supplemented with the correspondent growth supplements
(MCGS, Wakersville, MD). For the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) cell viability assay, cells were incubated for 24 h and cells
growing in 2D and cells encapsulated within the 4% PSP-POG8 and
8% PSP-POG8 hydrogels were treated with 20 mL of the CellTiter 96
Aqeous One solution and incubated at 37 8C for 4 h. Following
incubation, each of the wells containing the corresponding sample
was removed from the insert and dissolved with 700 mL of media.

1428

Absorbance at 490 nm was measured using the TECAN SpectraFluorplus microplate reader (Durham, NC). Percent cell viability
was calculated by comparing the absorbance of cells growing in 2D
versus the cells growing inside of the hydrogel. For the Calcein-AM
cell viability assay, 2D cells and cells encapsulated within the 4%
PSP-POG8 hydrogel were incubated with Calcein AM (106 M) for
30 min after 24 h of cell culture. Cells were then visualized for green
fluorescence using a Optical Microscope Olympus BX51 equipped
with a CCD camera (Center Valley, PA). Calcein-AM was excited
using a U-MWB2 filter with excitation of 460495 nm and the
fluorescence emission was collected using a 520 nm filter.

Cryo-Scanning Electron Microscopy (Cryo-SEM)

Imaging
Hydrogel samples were prepared in situ on the Cryo-SEM slit
sample holder as described in the previous section. Samples were
frozen with liquid nitrogen and transferred to the Gatan Alto 2500
pre-chamber (cooled to 170 8C). The surface of the sample was
fractured in various locations using a scalpel to produce free-break
surfaces before being sublimated for 20 min at 85 8C. Pt sputter
coating followed for 120 min and then sample was transferred to
the microscopes cryo stage (130 8C) for imaging. Samples were
imaged with a FEI NOVA nanoSEM field emission (FEI Company,
Hillsboro, Oregon) using the through-the-lens (TLD) or EverhartThornley (ET) detector at 5 kV accelerating voltage and a working
distance (WD) of 5 mm at different magnifications.

Results and Discussion

Synthesis of CGG-(POG)8 and Hydrogel Formation
The CGG-(POG)8 peptide was synthesized using solid phase
methods on the Rink amide ChemMatrix resin, purified to
homogeneity by reverse phase HPLC and analyzed by
MALDI mass spectrometry. The purified CGG-(POG)8 peptide was tested for its ability to induce hydrogel formation
when conjugated to the 8-arm PEG-MAL. To this end, star
polymer solutions (5 and 10 wt%) were added to a solution
of peptide both in phosphate-buffered saline (PBS) at
pH 7.4 to achieve a ratio of 1:12, respectively. Gelation of
the samples was observed within 1 min in each case,
yielding hydrogels that were 4 and 8% in the peptide/PEG
star polymer (PSP-POG8).
Physical Characterization of the PSP-POG8 Hydrogels
CD was used to evaluate if the peptides within the hydrogel
maintained their ability to adopt a collagen triple helical
conformation, as compared to the peptide in free solution.
Both the peptide in solution and peptide in the 4% hydrogel
displayed a maximum in the CD spectrum at approximately
225 nm, a feature that is characteristic of a collagen triple
helix (See Supporting Information).[10b,17] To determine
what role the hydrogel architecture plays on triple helix
stability, we performed thermal denaturation studies by CD

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.MaterialsViews.com

A Collagen Peptide-Based Physical Hydrogel for Cell Encapsulation

www.mbs-journal.de

Table 1. Thermal denaturation (Tm) and storage modulus (G0 ) data

for PSP-POG8 hydrogels.

Sample

Tm
[-C]

CGG-(POG)8a)
4% PSP-POG8 hydrogel
8% PSP-POG8 hydrogel

53
b)

G( [Pa]
After
Before
thermal
thermal
annealing annealing
ND

ND

56

685

798

n.d.c)

1 337

1 714

2
104 M peptide solution in 0.01 M phosphate buffer at

pH 7.4 with 5
103 M TCEP; b)An aliquot was taken from the
hydrogel to make a solution that was 2
104 M in peptide in
0.01 M phosphate buffer at pH 7.4; c)Not determined.
a)

(Table 1). The collagen peptide alone, CGG-(POG)8, exhibited

a Tm of 53 8C (Table 1), a value that is close to that reported
for the (POG)8 peptide.[18] The triple helix of the polymerconjugated peptide was found to melt at a slightly higher
temperature, most likely due to intramolecular scaffold
stabilization of the helical structure, as has been observed
for collagen sequences tethered to dendrimer scaffolds.[19]
These data demonstrate that the multiarm polymer allows
for the formation of a stable triple helix within the context
of the hydrogel matrix.
Rheology experiments were performed to probe the
viscoelastic properties of the peptide/PEG star polymer
hydrogels (PSP-POG8). Oscillation stress and frequency
sweep experiments were carried out to determine the linear
dynamic range of the two hydrogels by measuring the
storage (G0 ) and loss (G00 ) moduli. For instance, at a constant
oscillation stress of 5 Pa, average G0 values of 685 and
1 337 Pa were obtained for the 4 and 8% PSP-POG8

hydrogels, respectively, with a frequency sweep of

130 Hz (See Table 1 and Supporting Information). The G0
values for both hydrogels dominated over the G00 values
over the course of the analysis. These data demonstrate that
CGG-(POG)8 crosslinks the polymer to form hydrogel whose
stiffness can be modulated through changes in the
concentration of the starting materials, with the 8%
hydrogel showing on approximately a twofold increase
in storage modulus over the 4% hydrogel.
Studies above investigated the thermal denaturation of
the collagen triple helix within the hydrogel by CD. Since
the collagen peptide is the major physical crosslinking
component of the hydrogel, we performed rheometry
temperature sweep experiments to determine the effect of
increasing temperature on the properties of the hydrogels.
Using a constant oscillation stress of 5 Pa and a frequency of
5 Hz, the hydrogels were submitted to increasing temperature. In these experiments, the storage modulus was found
to decrease as the 4 and 8% hydrogels were heated, with the
materials completely changing into a liquid-like state at
approximately 60 8C (Figure 2), a value that is in the same
range as the collagen triple helix melting temperatures
determined by CD. These data confirm that as the collagen
triple helix melts, the physical properties of the hydrogels
change significantly from a polymer network to a liquidlike state. These data also provide supporting evidence that
the collagen peptide triple helix is a major factor in hydrogel
formation within the PEG star polymer.
These melted materials were found to reform a
gelatinous material when cooled to room temperature,
and the cooled hydrogels were found to have higher storage
modulus values as compared to their starting, unheated
states (Table 1). The increase in the storage modulus was
somewhat larger for the 8% PSP-POG8 hydrogel, perhaps
indicating a change in the morphology of the hydrogel
through the thermal annealing cycle. The annealed

Figure 2. Temperature sweep experiments were performed by the rheometry for (a) 4% PSP-POG8 and (b) 8% PSP-POG8. Filled symbols
represent the first analysis of the hydrogels and open symbols represent analysis after recooling the samples from the first analysis. G0
storage modulus (^), G00 loss modulus (D).

www.MaterialsViews.com

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1429

C. M. Rubert Perez, A. Panitch, J. Chmielewski

www.mbs-journal.de

hydrogels were also submitted to a

second temperature sweep analysis,
and, although the starting G0 values were
higher this time; liquid-like states were
obtained again at a temperature of
60 8C (Figure 2).
Cryo-SEM was used to visualize the
internal morphology of the hydrogels
and any changes due to thermal annealing. Both the 4 and 8% PSP-POG8 hydrogels were analyzed before thermal
annealing, and the morphology of the
hydrogels was found to consist of a
fibrous, honeycomb-like structure, with
round compartments or pores that were
525 mm in size (Figure 3ac). Within
some pores, the fibrous sheets seemed to
be broken or incomplete. Upon thermal
annealing the cryo-SEM of the 4% PSPPOG8 hydrogel remained essentially
unchanged, whereas the 8% hydrogel
had the same overall morphology, but
now with significantly smaller pore sizes
of 2 mm and with more complete pores
(Figure 3d). Interestingly, this more concentrated material was found to respond
to the annealing and a more crosslinked
structure resulted.

Figure 3. Internal hydrogel morphology was investigated by cryo-SEM. (a) and (b) 4%
PSP-POG8 at two different magnifications, (c) and (d) 8% PSP-POG8 at same magnification before and after thermal annealing, respectively.

Cell Encapsulation Experiments

with the PSP-POG8 Hydrogels
For the PSP-POG8 hydrogels to have
applications in tissue engineering and
regenerative medicine, it was essential to
determine if cells could be encapsulated
within the hydrogel and remain viable.
To test this, a suspension of human
mesenchymal stem cells (hMSCs) in a
Figure 4. (a) Fluorescence images of viable hMSCs stained with Calcein AM encapsulated
CGG-(POG)8 solution in PBS buffer
within the 4% PSP-POG8 hydrogel. (b) Cryo-SEM images of hMSCs occupying the porous
pH 7.4 was treated with 5 or 10% of
structure of the 4% PSP-POG8 hydrogel.
the PEG-MAL star polymer to successfully
provide the 4 or 8% PSP-POG8 hydrogels.
A colorimetric MTS analysis was used to determine cell
were able to be transferred to a 2D plates to continue their
viability of encapsulated hMSCs within the hydrogel. It was
growth (Data not shown). Cumulatively these data provide
determined that the cells were 95% viable after 1 d in both
evidence that the hydrogel has low toxicity on the
4 and 8% hydrogels, favorably comparable to the viability
encapsulated cells.
found for hMSCs that were grown in 2D cell culture plates.
The internal morphology of the above 4% hydrogel with
Fluorescence microscopy was also used to provide visual
the encapsulated hMSCs was also visualized using cryoevidence for cell viability (Figure 4a). hMSCs that were
SEM (Figure 4b). The cells were observed to occupy the pores
encapsulated within the 4% hydrogel for 24 h were treated
of the hydrogel, thereby providing structural support for the
with the viability stain Calcein AM, and green fluorescence
cells within the matrix. At a higher magnification the
was observed for live cells throughout the hydrogel.
boundary between the hydrogel network and the cell
Encapsulated cells remained viable for more than 5 d and
membrane could be distinguished, providing evidence of

1430

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.MaterialsViews.com

A Collagen Peptide-Based Physical Hydrogel for Cell Encapsulation

www.mbs-journal.de

the physical contacts between cell and collagen-based

scaffold (see Supporting information).

Conclusion
In summary, we have designed a collagen peptide-based
hydrogel using an 8-arm-PEG star polymer to generate a 3D
cell culture matrix with appropriate viscoelastic properties.
This material has the ability to melt into a liquid-like state
at increased temperatures and reverts back into a gel upon
cooling, presumably due to the denaturation and refolding
of the collagen peptide triple helix. The internal morphology of the hydrogel exhibited a pore size that is reasonable
for cell encapsulation as shown by cryo-SEM, and the
hydrogel demonstrates low cell toxicity. Since the collagen
triple helix plays a significant role in the properties of the
hydrogel, a range of materials for cell culture can be
envisioned through judicious changes to the collagen
peptides used to form the hydrogels, including peptides
containing cell adhesion signals or collagenase sequences.

Acknowledgements: We are grateful to the NSF (0848325-CHE) for

support of this research, to D. Sherman for assistance with SEM,
and to J. Paderi for assistance with rheology.

Received: June 8, 2011; Published online: August 9, 2011; DOI:

10.1002/mabi.201100230
Keywords: biomaterials; cell encapsulation; collagen peptide;
hydrogels; star PEG polymers

[1] a) J. Brinckmann, Top. Curr. Chem. 2005, 247, 1; b) T. Koide,

K. Nagata, Top. Curr. Chem. 2005, 247, 85; c) M. K. Gordon, R. A.
Hahn, Cell Tissue Res. 2010, 339, 247.
[2] a) P. P. Fietzek, K. Ku
hn, Mol. Cell. Biochem. 1975, 8, 141; b) J. A.
Ramshaw, N. K. Shah, B. Brodsky, J. Struct. Biol. 1998, 122, 86.
[3] a) J. Josse, W. F. Harrington, J. Mol. Biol. 1964, 9, 10262; b) C. L.
Jenkins, R. T. Raines, Nat. Prod. Rep. 2002, 19, 49; c) J. Engel,
H. P. Bachinger, Top. Curr. Chem. 2005, 247, 7.

www.MaterialsViews.com

[4] a) D. J. Hulmes, Essays Biochem. 1992, 27, 49; b) S. RicardBlum, F. Ruggiero, M. Van der Rest, Top. Curr. Chem. 2005, 247,
35; c) D. J. Prockop, K. I. Kivirikki, Annu. Rev. Biochem. 1995,
64, 403; d) P. Martin, Science 1997, 276, 75.
[5] D. G. Wallace, J. Rosenblat, Adv. Drug. Deliv. Rev. 2003, 55,
1631.
[6] M. M. Stevens, J. H. George, Science 2005, 310, 1135.
[7] A. Alavi, D. G. Stupack, Methods Enzymol. 2007, 426, 85.
[8] J. Glowacki, S. Mizuno, Biopolymers 2008, 89, 338.
[9] A. K. Lynn, I. V. Yanmas, W. Bonfield, J. Biomed. Mater. Res.
2004, 71, 343.
[10] a) D. E. Przybyla, J. Chmielewski, Biochemistry 2010, 21, 4411;
b) M. D. Shoulders, R. T. Raines, Annu. Rev. Biochem. 2009, 78,
929; c) T. Koide, Phil. Trans. R. Soc. B 2007, 362, 1281.
[11] a) N. C. Hunt, L. M. Grover, Biotechnol. Lett. 2010, 32, 733; b)
K. Y. Lee, D. J. Mooney, Chem. Rev. 2001, 101, 1869; c) H. Geckil,
F. Xu, X. Zhang, S. Moon, U. Demirci, Nanomedicine 2010, 5,
469; d) X. Jia, K. L. Kiick, Macromol. Biosci., 2009, 9, 140; e) G. D.
Nicodemus, S. J. Bryant, Tissue Eng. Part B 2008, 14, 149; f)
T. Lu
hmann, H. Hall, Materials 2009, 2, 1058.
[12] C. Kojima, S. Tsumura, A. Harada, K. Kono, J. Am. Chem. Soc.
2009, 131, 6052.
[13] a) H. J. Lee, J. S. Lee, T. Chansakul, C. Yu, J. H. Elisseeff, S. M. Yu,
Biomaterials 2006, 27, 5268; b) J. Lee, C. Yu, T. Chansakul,
N. Hwang, S. Varghese, S. Yu, J. Elisseeff, Tissue Eng. Part A
2008, 14, 1843; c) G. Bayramoglu, N. Kayaman-Apohan,
H. Akcakaya, M. V. Kahraman, S. E. Kuruca, A. Gu
ngor,
J. Mater. Sci. Mater. Med. 2010, 21, 761; d) S. Q. Liu, Q. Tian,
J. L. Hendrick, J. H. P. Hui, P. L. R. Ee, Y. Y. Yang, Biomaterials
2010, 31, 7298.
[14] P. J. Stahl, N. H. Romano, D. Wirtz, S. M. Yu, Biomacromolecules
2010, 11, 2336.
[15] a) J. A. M. Ramshaw, N. K. Shah, B. Brodsky, J. Struct. Biol. 1998,
122, 86; b) B. Brodsky, G. Thiagarajan, B. Madhan, K. Kar,
Biopolymers 2008, 89, 345; c) R. Improta, C. Benzi, V. Barone,
J. Am. Chem. Soc. 2001, 123, 12568.
[16] a) Y. M. Angell, J. Alsina, F. Albericio, G. Barany, J. Peptide Res.
2002, 60, 292; b) Y. Han, F. Albericio, G. Barany, J. Org. Chem.
1997, 62, 4307.
[17] a) N. K. Shah, J. A. M. Ramshaw, A. Kirkpatrick, C. Shah,
B. Brodsky, Biochemistry 1996, 35, 10262; b) N. J. Greenfield,
Nat. Protoc. 2006, 1, 2876; c) B. Ranjbar, P. Gill, Chem. Biol.
Drug. Des. 2009, 74, 101; d) J. Bella, B. Brodsky, H. M. Berman,
Structure 1995, 3, 893.
[18] a) A. Y. Wang, C. A. Foss, S. Leong, X. Mo, M. G. Pomper, S. M.
Yu, Biomacromolecules 2008, 9, 1755; b) C. Chen, W. Hsu,
T. Kao, J. Horng, Biochem. 2011, 50, 2381.
[19] G. A. Kinberger, W. Cai, M. Goodman, J. Am. Chem. Soc. 2002,
124, 15162.

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1431