Biomaterials

Science
PAPER
Cite this: Biomater. Sci., 2013, 1, 952
Received 21st January 2013,
Accepted 22nd April 2013
DOI: 10.1039/c3bm60019j
www.rsc.org/biomaterialsscience
Hyaluronan and self-assembling peptides as building
blocks to reconstruct the extracellular environment in
skin tissue
Daniela S. Ferreira,
a,b
Alexandra P. Marques,
a,b
Rui L. Reis
a,b
and
Helena S. Azevedo*
a,b
Self-assembling bioactive membranes, incorporating hyaluronan, a structural component of the skin
extracellular matrix (ECM), and peptide amphiphiles presenting biochemical signals, are proposed in this
work for recapitulating some aspects of the skin tissue microenvironment. In the herein presented strat-
egy, the availability of cell-adhesion ligands (050% RGDS epitope) within 2D membranes is controlled
aiming at mastering the adhesion of human dermal broblasts under serum-free culture conditions. The
membranes were characterized with respect to their microstructure, by scanning electron microscopy
(SEM), epitope distribution, degradability and cell behavior, regarding adhesion, proliferation and cyto-
skeleton organization. SEM of the membrane surface showed a network of nanobers that are remark-
ably reminiscent of the lamentous structure found in the ECM. Confocal microscopy images, using a
uorescently labeled RGDS-peptide, showed that the RGDS signal is uniformly distributed on the mem-
branes. Degradation studies indicated that the membranes are susceptible to enzymatic degradation by
hyaluronidase. In the presence of the enzyme at physiological concentration, the membranes degrade
gradually over time. When grown on membranes with the cell recognition epitope RGDS, broblasts had
spread out and elongated, exhibiting extended lopodia interacting with brillar structure of the mem-
brane surface, thus showing improved adhesion to the substrate. This study demonstrates the positive
eect of the RGDS epitope, presented on a self-assembled membrane, in promoting cellmatrix
interactions.
1. Introduction
The extracellular matrix (ECM) of tissues is a dynamic and
hierarchically organized composite structure of various fibril-
lar proteins and glycosaminoglycans. This network not only
has a structural role, providing support and tensile strength
for tissues and acting as scaolds for cell adhesion and organi-
zation, but also serves as a storage site for growth factors,
chemokines and cytokines, and as a template for tissue mor-
phogenesis and cell dierentiation.
13
In skin, the ECM is the largest component of the dermal
layer, being composed of structural proteins, like elastin, that
confers skin elasticity, and collagens, primarily type I and III,
which provide structure, strength and integrity.
4
Cell-adhesive
proteins, like fibronectin, laminin and vitronectin are also
present in skin ECM. These glycoproteins have the capacity to
bind to cells, via integrins, and to other components of the
ECM, namely to glycosaminoglycans, except hyaluronan (HA),
which is one of the major ECM components in skin.
4,5
HA is
an extremely large polymer made up of N-acetylglucosamine
and glucuronic acid disaccharide repeating units (Fig. 1A).
High molecular weight HA acts as an ECM organizer which
concentrates and organizes the assembly of other proteins in
the ECM by providing a macromolecular template, thus contri-
buting to the tissue architecture and function during
homeostasis.
6,7
These properties confer on HA many unique
advantages as a starting material for skin regeneration
applications.
Cell adhesion to native ECM is mediated through the
binding of integrin proteins on the cell surface and specific
epitopes present in proteins of the ECM, creating a focal
adhesion, responsible for anchoring the cell and the com-
munication between the cell cytoskeleton and the surrounding
environment.
8
One of the ECM cell adhesive proteins,
Electronic supplementary information (ESI) available. See DOI:
10.1039/c3bm60019j
a
3Bs Research Group Biomaterials, Biodegradables and Biomimetics, University of
Minho, Headquarters of the European Institute of Excellence on Tissue Engineering
and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimares, Portugal.
E-mail: hazevedo@dep.uminho.pt; Fax: +351 253 510 909; Tel: +351 253 510 907
b
ICVS/3Bs PT Government Associate Laboratory, Braga/Guimares, Portugal
952 | Biomater. Sci., 2013, 1, 952964 This journal is The Royal Society of Chemistry 2013
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Fig. 1 Peptide design and characterization. (A) Chemical structure of the building blocks used for preparing the self-assembled membranes, hyaluronan (HA) and
dierent peptide amphiphiles (PAs): V
3
A
3
K
3
containing positively charged lysines (KKK) to bind the anionic HA, one containing the RGDS epitope (K
3
RGDS-PA), a
scrambled version (K
3
DGSR-PA). (B) Circular dichroism spectra of peptide solutions (3.3 10
5
M) at pH 5, 7 and 9 showing a predominantly -sheet secondary struc-
ture. (C) TEM images of PA nanostructures formed by the deposition of 0.1 mM solutions in water followed by air drying on a carbon-coated TEM grid.
Biomaterials Science Paper
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fibronectin, binds to integrins through a domain containing
Arg-Gly-Asp-Ser (RGDS).
9
This sequence, first proposed by
Pierschbacher and Rouslahti, functions as a general cell
adhesive sequence
10
and has been widely used in biomaterials
functionalization, including HA-based hydrogels,
1113
im-
proving cell adhesion and subsequent proliferation.
1417
Peptides represent an interesting family of building blocks
which can self-assemble to create a large number of nano-
structures, such as micelles, vesicles and nanofibers.
1820
Thus, by self-assembly, structurally simple building blocks can
be easily gathered to create functionally complex materials.
8
Bottom-up approaches based on the self-assembly of small
molecules provide a unique set of advantages to create bioma-
terials, as they oer the possibility of controlling the architec-
ture, shape and dimensions of the bioactive nanostructures, as
well as the spatial display and density of the bioactive
signals.
21
The architectural resemblance of self-assembled
nanofibers to filamentous structures found in natural
ECMs represents an additional feature to attain superior bio-
mimetic scaolds, and a clear advantage in biomaterials
engineering.
The fabrication of artificial ECMs can be used to mimic the
properties of native tissues as well as to reconstruct cellular
microenvironments in vitro. We address this challenge by com-
bining self-assembling peptides ( peptide amphiphiles) inte-
grating biochemical signals (RGDS ligand) to permit cell
adhesion and spreading, and functional molecules (HA), as
components of our matrix. These components were shown to
self-assemble in 2D membranes.
2224
Through self-assembly,
epitope spatial organization can be controlled at the
micrometer and nanometer scales to guide cellular behavior.
Materials that selectively interact with cells may be helpful in
improving our understanding of key structural and biochemi-
cal ECM components, and ultimately, harnessing the presen-
tation of specific cues to cells. This represents a simplified
approach to deconstruct the skin niche and to identify the
eect of individual niche components over cell behavior.
2. Materials and methods
Hyaluronan (HA) and fluorescein HA
The hyaluronan used in all experiments had an average molar
mass of 2 MDa and was purchased from Lifecore Biomedical,
Inc. (Chaska, USA). HA was fluorescently labeled with fluores-
ceinamine (Fig. 3B) using N-(3-dimethylaminopropyl)-N-ethyl-
carbodiimide hydrochloride (EDC) chemistry and following
the procedure described by Gajewiak et al.
25
Briefly, HA
(50 mg) was dissolved in 20 mL of water to give a 0.25% (w/v)
solution, which was then mixed with a solution of 5 mg of
fluoresceinamine (Sigma, USA) in 20 mL of dimethylform-
amide. Next, 100 mg of N-hydroxysuccinimide (NHS, Sigma,
USA) was added, and the solution pH was adjusted to 4.75
with 0.01 M HCl. Finally, 50 mg of EDC (Sigma, USA) was
added maintaining the solution pH at 4.75. After 12 h, the
solution was transferred to dialysis tubing (2000 Da MWCO,
Sigma, USA) and dialyzed exhaustively against 100 mM NaCl,
followed by dialysis against distilled water and lyophilization.
Peptide amphiphiles synthesis and purification
Three dierent peptide amphiphiles (PAs) were synthesized in
this work, consisting of a peptide segment covalently linked to
a 16-carbon alkyl chain: C
15
H
31
CO-V
3
A
3
K
3
(K
3
-PA, filler),
C
15
H
31
CO-V
3
A
3
K
3
RGDS (K
3
RGDS-PA) and C
15
H
31
CO-
V
3
A
3
K
3
DGSR (K
3
DGSR-PA, scrambled) (Fig. 1). The peptides
were synthesized in a CS Bio 136XT automated peptide synthe-
sizer (CS Bio, USA) using standard 9-fluorenylmethoxycarbonyl
(Fmoc) based solid phase chemistry and a 4-methylbenzhydryl-
amine (MBHA) rink amide resin. Amino acid couplings were
performed using 4 equivalents (4 mmol) of Fmoc protected
amino acids (Novabiochem), 4 equivalents of O-(benzotria-
zol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate
(HBTU, Novabiochem) and 6 equivalents of N,N-diisopropyl-
ethylamine (DIEA, Sigma, USA). Fmoc deprotections were per-
formed with 20% piperidine (Sigma, USA) in
dimethylformamide. A palmitic acid (C
16
H
32
O
2
, Calbiochem,
USA) tail was manually coupled under the same conditions as
the Fmoc-amino acids. Peptide cleavage from the resin and
removal of the protecting groups were carried out in a mixture
of trifluoroacetic acid (TFA, Sigma, USA)/triisopropylsilane
(TIS, Alfa Aesar)/water (95/2.5/2.5) for 3 h at room temperature.
The peptide mixture was collected and excess TFA was
removed by rotary evaporation. The resulting viscous peptide
solution was triturated with cold diethyl ether. The white pre-
cipitate was collected by filtration, washed with cold ether, and
allowed to dry under vacuum overnight. The peptide mass was
confirmed by electrospray ionization mass spectrometry
(ESI-MS, Thermo Electron Corporation Finnigan LXQ MS
Waltham, USA). Peptides were then purified on a Waters 2545
Binary Gradient high-performance liquid chromatography
(HPLC) system using a preparative reverse-phase C18 column
(Atlantis Prep OBD T3 column, Waters, USA) and a water/aceto-
nitrile (0.1% TFA) gradient. TFA counter-ions were exchanged
by sublimation from 0.01 M hydrochloric acid. Finally, the
peptides were dialyzed against ultrapure water using 500
MWCO dialysis tubing (Spectrum Labs, The Netherlands), and
lyophilized. Confirmation of mass and purity was done by
ESI-MS and HPLC (ESI, Fig. S1S3).
A fluorescent version of K
3
RGDS-PA, C
15
H
31
CO-V
3
A
3
K
3
K
rhod
-
RGDS (Fig. 3B), was also synthesized to allow examining the
availability/distribution of the RGDS motif within and on the
surface of the membranes. For that, an additional lysine
residue, with a 4-methyl trityl (Mtt) protecting group in the
amine of the lysine residue (Fmoc-Lys(Mtt)-OH), was intro-
duced in the sequence to which the rhodamine dye was
attached. The peptide was grown on the resin and after coup-
ling the palmitic tail, the Mtt protecting group was selectively
removed with a solution of TFATISDCM (4 : 4 : 92) at room
temperature. Resin was incubated with a deprotection solution
for 5 minutes and washed thoroughly with DCM. These steps
were repeated until the resin no longer turned yellow. Then,
peptide-resin (150 mg) was swollen in 1150 L of DMF and
Paper Biomaterials Science
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50 L of DIEA. Ten milligrams of 5-(and 6)-carboxytetra-
methylrhodamine succinimidyl ester (NHS-rhodamine) were
dissolved in the supernatant from the beads swelling step and
added to the resin. The reaction took place at room tempera-
ture, overnight, and protected from light. After repeated
washes with DMF and methanol, the peptide was cleaved from
the resin by following the procedure described above for the
other peptides, and purified by HPLC. Mass was confirmed by
matrix assisted laser desorption/ionization mass spectrometry
(MALDI-MS, 4800 MALDI-TOF/TOF, AbSciex) (ESI, Fig. S4).
Peptide amphiphiles characterization
Circular dichroism (CD) spectroscopy. Peptides were dis-
solved in deionized water to a final concentration of 0.033 mM
and the pH was adjusted to 5, 7 and 9. The CD measurements
were performed in a PiStar-180 spectrometer from Applied
Photophysics (Surrey, UK), under a constant flow of nitrogen
(8 L min
1
) at a constant pressure value of 0.7 MPa. Far-UV
spectra were recorded at 25 C from 190 to 300 nm in a quartz
cuvette with 1 mm path-length. All scans were performed in
the steady state with a bandwidth of 1 nm and each presented
spectrum is an average of 5 spectra.
Transmission electron microscopy (TEM). Samples for TEM
analysis were prepared by placing a drop of a 0.1 mM peptide
solution directly on the 400 mesh carbon-coated copper TEM
grid (Ted Pella, USA). For negative staining a drop of a 2%
(w/v) uranyl acetate (Electron Microscopy Sciences, USA) aqueous
solution was placed on the samples. After ca. 3 minutes, the
excess solution was wiped away with a piece of filter paper,
and the sample was allowed to dry under ambient conditions.
All images were collected with a JEOL JEM-1010 transmission
electron microscope at 100 kV (JEOL, USA).
Characterization of PAHA interactions
Quartz crystal microbalance with dissipation (QCM-D).
Measurements were performed in a quartz crystal micro-
balance with dissipation monitoring (QCM-D E4) from Q-Sense
(Gothenburg, Sweden). All the experiments were performed at
25 C with a constant flow rate of 50 L min
1
using gold
coated crystals (QSX301, Q-Sense, Gothenburg, Sweden) pre-
viously cleaned with water and H
2
O
2
for 1 h each, and then
acetone, ethanol and isopropanol for 3 minutes each at 37 C
with sonication. The system was equilibrated with a 0.15 M
sodium chloride (NaCl) solution to obtain a stable frequency
and dissipation baseline signal. Once the signal was stable,
the NaCl solution was replaced by a solution of K
3
-PA (0.2%
(w/v) in 0.15 M NaCl) during 30 minutes. To remove weakly
bound peptide, the crystals were rinsed with a NaCl solution
and then replaced by a solution of HA (0.1% (w/v) in 0.15 M
NaCl) for 30 minutes. Again, to remove weakly bound polymer,
the system was rinsed with NaCl. The QCM instrument
recorded frequencies up to the 13
th
overtone, and f and D
were monitored in real time. In the present study the results
are shown for the 7
th
overtone; the frequency of this overtone
was normalized to the fundamental resonant frequency of the
quartz crystal, by dividing it by (where = 7).
PAHA membranes preparation
PAHA membranes were prepared using a 48 well plate as a
template in a sterile environment. 150 L of a 1% (w/v) HA
solution was cast on the bottom of the wells and then 150 L
of a 2% (w/v) K
3
-PA solution was added on top of the HA solu-
tion. A membrane is immediately formed upon contact
between the two solutions. The membrane was allowed to
grow with time (overnight) as reported previously.
23
The mem-
branes were rinsed with sterile ultrapure water to ensure the
removal of unreacted HA and PA.
PAHA membrane characterization
Scanning electron microscopy (SEM). The microstructure of
the membranes was analyzed by SEM. Samples were fixed in
2% glutaraldehyde/3% sucrose in PBS for 1 h at 4 C followed
by sequential dehydration in graded ethanol concentrations
(from 20 to 100%). To remove ethanol, samples were chemi-
cally dried in hexamethyldisilazane (HMDS, Electron
Microscopy Sciences, USA) 3 times, 15 minutes each, and
excess HMDS was allowed to evaporate. Prior to observation,
the samples were coated with a gold/palladium layer and
imaged using an ultra-high resolution field emission gun scan-
ning electron microscope (Nova NanoSEM 200) from FEI
(Eindhoven, The Netherlands).
Confocal microscopy. Confocal microscopy was used to
probe the location and retention of fluorescently-labeled HA as
well as to visualize the distribution of RGDS signal on (surface)
and within (cross-section) the PAHA membrane. Membranes
were formed as previously described using a fluorescein HA
solution (1%, w/v) and a 2% (w/v) peptide mixture containing
0.1% K
3
K
rhod
RGDS-PA and 99.9% K
3
-PA. The membranes were
incubated overnight at RT protected from light. After washing,
membranes were transferred to glass microscopy slides,
covered with a glass coverslip, and sealed to prevent dehy-
dration. Membranes were imaged by a laser scanning confocal
microscope (LSCM, Olympus FluoView 1000, Japan) with
appropriate excitation and emission wavelengths. Optical
slices were captured at regular intervals to produce recon-
structed z-stacks with 100 m total thickness. Images of cross
sections were compiled from z-stack in the x-direction using
FV10-ASW software from Olympus.
In vitro enzymatic degradation. Degradation behavior of the
PAHA membranes in the absence and presence of a HA-
degrading enzyme (hyaluronidase, HAase) was analyzed in
vitro. Bovine testicular HAase (Type IV, EC 3.2.1.35) was
obtained from Sigma (USA). This enzyme has the ability to
hydrolyze (1,4) glycosidic bonds between N-acetyl-D-glucos-
amine and D-glucuronate residues producing HA fragments
with a N-acetyl-D-glucosamine at the reducing end. The
enzyme activity can thus be measured by the quantification of
these reducing ends. Degradation studies were carried out by
incubating PAHA membranes in PBS at 37 C in the absence
(control) or presence of HAase at dierent concentrations,
2.6 U mL
1
(to simulate physiological conditions in human
plasma) and 50 U mL
1
, for 14 days. The enzyme solution was
Biomaterials Science Paper
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replaced every 72 h throughout the study and stored frozen
until analysis. At predetermined time points, the membranes
were collected and their morphology examined by SEM. The
supernatants were analyzed for quantification of N-acetyl-
amino sugars using the fluorimetric MorganElson assay
method.
26
A calibration curve of N-acetyl-D-glucosamine (NAG)
standards was used. HA fragments resulting from enzymatic
hydrolysis were identified by mass spectrometry in the negative
mode (Thermo Electron Corporation Finnigan LXQ MS,
Waltham, USA).
Cell culture studies
Isolation and culture of human primary fibroblasts. Human
dermal fibroblasts (hDFb) were isolated from skin samples dis-
carded from abdominoplasty surgeries of consenting patients
at Hospital da Prelada (Porto, Portugal). Briefly, the skin tissue
was cut into pieces of 0.5 0.5 cm and digested in a dispase
solution (2.4 U mL
1
in PBS) at 4 C, overnight. After removing
the epidermis, the fibroblasts were isolated from the dermis by
overnight digestion of the dermal pieces in a collagenase IA
solution (125 U mL
1
in PBS) at 4 C. Digestion products were
poured through a 100 m cell strainer and centrifuged at 200g
for 5 minutes. The pellet was resuspended and the cells were
subsequently cultured in Dulbeccos Modified Eagle Medium
(DMEM) (Sigma, Germany) supplemented with 10% fetal
bovine serum (FBS, Gibco, UK) and 1% (v/v) antibiotic/antimy-
cotic solution (A/B) (Gibco, UK) containing 100 units per mL
penicillin and 100 mg mL
1
streptomycin, in a 37 C humidi-
fied atmosphere with 5% CO
2
.
hDFb culture on the PAHA membranes. To study the eect
of RGDS signaling on cell adhesion, PAHA membranes con-
taining dierent percentages of the RGDS motif were prepared
by mixing the filler peptide (K
3
-PA) with 1%, 10% and 50%
K
3
RGDS-PA and 10% scrambled peptide (K
3
DGSR-PA) to give a
2% (w/v) final peptide concentration. HA sterilization for cell
studies was done by dissolving the polymer, filtering the solu-
tion through a 0.22 m filter, followed by lyophilization in
sterile falcon tubes. Peptide solutions were sterilized by UV
exposure for 15 minutes. Membranes were prepared by follow-
ing the procedure previously described under sterile con-
ditions. Confluent hDFbs, at passage 4, were harvested from
monolayer cultures using trypsin-EDTA (Invitrogen, USA). Cells
were washed in PBS and centrifuged at 200g for 10 minutes in
order to remove serum residues. Cell pellets were then resus-
pended at a density of 5.0 10
4
cells per mL in serum-free
DMEM without phenol red (Sigma, Germany) supplemented
with 1% (v/v) A/B. Cells (2.5 10
4
cells per well) were cultured
on the PAHA membranes in 48 well plates at 37 C in a
humidified atmosphere of 5% CO
2
for 2, 6, 12 and 24 h.
hDFbs cultured on tissue culture polystyrene (TCPS) coverslips
were used as the control. Cells cultured on the membranes
were examined under SEM to analyze cell morphology and
cellmatrix interactions. Cell cultured membranes were fixed,
dehydrated and prepared as described for SEM analysis.
F-actin staining. Staining for the F-actin cytoskeleton fibers
of attached hDFbs was carried out after fixing cells in a 10%
formalin solution (Sigma-Aldrich, Germany) for 30 minutes at
4 C. Cells were then washed once with 0.1 M glycine in PBS
and twice with PBS and permeabilized with a 2% BSA/0.2%
Triton X-100 solution for 1 h at RT. Samples were incubated
with TRITC-conjugated phalloidin (1 U mL
1
, Sigma-Aldrich,
Germany) for 1 h at RT. Cell nuclei were counterstained with
1 mg mL
1
DAPI (1 : 1000, Sigma-Aldrich, Germany) for 1 min
and washed with PBS. Visualization was performed by CLSM
(Olympus FluoView 1000, Olympus, Japan). Background was
subtracted and images were processed using ImageJ software
(NIH, USA).
dsDNA quantification. The number of cells adherent to the
membranes was assessed at dierent culture times by quanti-
fying the amount of double-stranded DNA (dsDNA). Quantifi-
cation was performed using a Quant-iT PicoGreen dsDNA
Assay kit (Invitrogen, Molecular Probes, Oregon, USA) accord-
ing to the instructions of the manufacturer. Briefly, cells on
the dierent membranes were lysed by osmotic and thermal
shock and the supernatant was used for DNA quantification.
The fluorescent intensity of the dye was measured in a micro-
plate reader (Synergie HT, Bio-Tek, USA) with excitation at 485/
20 nm and emission at 528/20 nm. The DNA concentration for
each sample was calculated using a standard curve (DNA con-
centration ranging from 0 to 1.5 mg mL
1
) relating the quan-
tity of DNA and fluorescence intensity. Triplicates were made
for each time point and for each sample.
Data analysis and statistics
Statistical analysis for NAG and DNA quantification was per-
formed using the non-parametric KruskalWallis test, after
testing the normality of our data with the ShapiroWilk test.
Dunns post-hoc test was carried out to determine the dier-
ences between the various conditions in this study. Values of
p < 0.05 were considered to determine statistical significant
dierences between the groups.
3. Results and discussion
Peptide design and formulation in the bioactive membranes
The self-assembling peptides used in this study are amphiphi-
lic peptides consisting of a linear hydrophobic segment
coupled to a peptide sequence, which includes a -sheet
forming sequence (VVVAAA) and a domain with positively
charged amino lysines (KKK) to bind the anionic HA (K
3
-PA).
This class of self-assembling peptides, known as peptide
amphiphiles (PAs), was proposed by Stupps group for
dierent biomedical applications.
19,27,28
The fibronectin-
derived RGDS epitope was incorporated into the peptide struc-
ture (K
3
RGDS-PA, Fig. 1A) due to its known properties in
mediating cell adhesion. Dierent PA molecules have been co-
assembled, allowing for a specific bioactive molecule to be
mixed with a non-bioactive diluent molecule
2932
to vary the
epitope density on the assembled structures for optimized cell
signaling. For example, Webber and co-workers
29
have investi-
gated the co-assembly of a positively charged PA containing
Paper Biomaterials Science
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the RGDS sequence (K
3
RGDS-PA) with a negative diluent PA
(E
3
-PA) with the aim of producing mixed binary nanofibers. By
varying the RGDS composition on surfaces coated with the co-
assembled peptide nanofibers, they were able to determine the
optimal RGDS density on the adhesion of bone-marrow mono-
nuclear cells. While this co-assembly strategy has been
explored by other groups, using coated surfaces
29,31
or self-
assembled gels,
30
the co-assembly of RGDS-containing pep-
tides with HA in self-assembled membranes has been explored
by our group.
33
In this configuration, the membranes present
dierent densities of biomolecular signals designed to
enhance cell adhesion, but other biochemical functionalities
can be incorporated, like growth-factor binding sequences rele-
vant to wound healing.
Previous studies have shown that the formation of a stable
-sheet is very important and necessary for peptide self-assem-
bly.
20,34
Although the formation of -sheet secondary structure
of K
3
-PA has been demonstrated previously,
35
the inclusion of
the RGDS (or DGSR) in the PA sequence could disturb that
rearrangement and consequently aect the stability of self-
assembled membranes. Therefore, circular dichroism (CD)
spectroscopy was performed to evaluate the secondary struc-
ture of the synthesized peptides (Fig. 1B). The CD analysis of
the PAs revealed the presence of hydrogen-bonded structures,
namely -sheets or random coil, depending on the pH. At pH
9, a typical spectrum of a -sheet structure was observed for all
the molecules, with a minimum in the 210220 nm range, a
crossover from positive to negative above 190 nm, and a posi-
tive ellipticity around 200 nm. The scrambled peptide showed
this conformation in all the studied pH conditions, with stron-
ger peaks at 205 nm. Below pH 9, the K
3
RGDS-PA showed a
random coil conformation. K
3
-PA presented a -sheet structure
with a minimum at 220 nm and a maximum at 205 nm at pH
7, and a random coil conformation at pH 5. TEM analysis
showed the formation of nanofibers for all the peptides,
but K
3
DGSR-PA formed shorter fibers than the K
3
and
K
3
RGDS-PAs (Fig. 1C).
PAHA interaction
Previous studies have shown the ability of K
3
-PA to interact
with HA and form macroscopic structures, such as sacs and
membranes, by self-assembly.
2224
In the herein presented
work, we have investigated, for the first time, the interactions
between these two molecules by QCM-D. This technique
allows for marker-free measurement of specific interactions
between immobilized molecules and analytes in solution and
has been widely used for studying macromolecular inter-
actions. Due to its sensitivity, it allows detecting mass changes
of the order of ng cm
2
, and permits us to measure the visco-
elastic properties of the resulting film.
36,37
Fig. 2 shows the
normalized frequency (f

/, where is the overtone) and dis-

sipation (D) variations for the 7
th
overtone (35 MHz). The
first 15 minutes in Fig. 2 show the establishment of the base-
line with 0.15 M NaCl. The subsequent 45 minutes correspond
to the deposition of K
3
-PA, where a decrease in the frequency
(of about 23 Hz) and an increase in D were observed. After
rinsing with NaCl, the hyaluronan solution was pumped con-
tinuously for 45 minutes, when an adsorption plateau was
attained. A decrease in frequency of 38 Hz was registered, and
again an increase in dissipation has occurred. After HA depo-
sition, the film was again rinsed with NaCl. During this
washing step, the variation in both f and D was very small,
indicating the formation of a stable film and a strong inter-
action between both molecules. The frequency variation, f,
decreases with time, due to the deposition of peptide or hyal-
uronan on the quartz crystal. On the other hand, the dissipa-
tion, D, increases, revealing that the film is not rigid and
begins dissipating energy, thus exhibiting the typical visco-
elastic behavior. This behavior has been observed before when
studying the interaction between poly(L-lysine) and hyaluronan
by QCM-D.
38
The QCM-D results demonstrated and confirmed
the strong interaction between the K
3
-PA and HA and the for-
mation of a stable complex.
Membrane microstructure and degradation
In this work, we aimed to recreate some aspects of the physical
and biochemical environment of skin tissue. It was shown pre-
viously that hierarchical membranes can be formed by instant
self-assembly between a positively charged PA (K
3
-PA) and the
megadalton hyaluronan (HA) under certain conditions.
22,23
We
thought that these fibrillar membranes, containing com-
ponents of skin ECM, could provide a starting platform to
design complex biomimetic environments of skin ECM
(Fig. 3). SEM analysis of the microstructure of the membranes
revealed a very highly organized structure exhibiting two dis-
tinct surfaces, as observed previously.
22,23,32
The surface
corresponding to the HA side (Fig. 3C1) is characterized by a
rough and amorphous structure, whereas the peptide side
(Fig. 3C2) exhibits a network of organized nanostructures
(nanofibers) randomly distributed, resembling the fibrillar
structure of natural ECM. SEM micrographs of the cross
section revealed a membrane with around 14 m thickness
Fig. 2 PAHA interaction. QCM-D monitoring of frequency (f, black) and
dissipation (D, red) changes obtained at the 7
th
overtone, during deposition of
peptide (step 1) and hyaluronan (step 3) on a bare crystal (step 2 relates to
rising). The frequency of this overtone was normalized to the fundamental
resonant frequency of the quartz crystal, by dividing it by (where = 7).
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and aligned nanofiber bundles perpendicular to the interface
(Fig. 3C3). To investigate if the addition of the K
3
RGDS-PA
would aect the membrane microstructure and morphology,
SEM was used to image membranes containing 50%
K
3
RGDS-PA. Membranes containing 50% K
3
RGDS-PA pre-
sented a similar thickness and microstructure to the ones
formed with 100% K
3
-PA (Fig. S5, ESI). Fluorescent micro-
graphs obtained by confocal microscopy, using the fluores-
cently labeled K
3
K
rhod
RGDS-PA (red) and hyaluronan (green),
showed a perfect overlap of the two, in yellow, resulting from
the interaction between the peptide and hyaluronan (Fig. 3B2).
This strong yellow region is also seen in a software simulated
cross section (Fig. 3B1), surrounded by a less intense area in
red or green, that corresponds to the peptide and hyaluronan
sides, respectively. The uniform distribution of the red
signal showed that RGDS sequence was well distributed within
the membranes although the spatio control of the signal on
specific locations of the membrane is still a major challenge.
The natural ECM is an extremely dynamic network that con-
sists of protein fibers and glycosaminoglycans that support
cell fate and provide biophysical and biochemical cues to cells
through cell surface receptors, such as integrins.
1,2,39
Cells
degrade the ECM through proteases during their migration.
Synthetic ECM matrices that aim to provide an environment
for tissue regeneration should recapitulate key features of the
natural ECM such as integrin mediated cell binding, cell
migration and proliferation, while also allowing their degra-
dation, oering a platform on which cell-triggered remodeling
could occur. To mimic the turnover of natural ECM, our
matrices were designed to be sensitive to enzymes expressed
by surrounding cells (e.g. hyaluronidase). This cell-mediated
degradation is a process reminiscent of tissue remodeling. It is
well known that the degradation of hyaluronan into large
oligosaccharides is mediated by hyaluronidase.
40
Therefore,
the degradation behavior of the PAHA membranes was
studied in three dierent conditions, PBS and PBS containing
2.6 U mL
1
and 50 U mL
1
HAase. Degradation was followed
by the quantification of N-acetylglucosamine in solution and
by SEM analysis (Fig. 4). Incubation in PBS up to 14 days did
not cause degradation of the membranes since the amount of
N-acetylamino sugars in the supernatant was significantly
lower than that obtained for the membranes incubated in the
higher enzyme concentration (50 U mL
1
). Compared with the
physiological concentration (2.6 U mL
1
), only for the latest
time point a statistical dierence was observed. In addition,
no evident signs of degradation are observed in the SEM
images for the membranes incubated in PBS only. This result
indicates that the membranes are relatively stable in buer
saline solutions, but are susceptible to enzymatic degradation
by HAase. As expected, the membrane degradation was signifi-
cantly enhanced when incubated with higher enzyme concen-
tration (50 U mL
1
) than in the presence of the enzyme under
physiological conditions (2.6 U mL
1
), as demonstrated by the
higher amount of released N-acetylamino sugars (Fig. 4A).
SEM analysis of the membranes collected at dierent time
points corroborates these results, showing the appearance of
pores on the membrane surface (Fig. 4B1) and cross-section
(Fig. 4B2), as a result of HA degradation by HAase. At a higher
HAase concentration (50 U mL
1
), the degradation of the mem-
branes is more evident, but progressive over time (Fig. 4B1B2).
The presence of HA oligosaccharides as degradation pro-
ducts was confirmed by mass spectroscopy. ESI-MS analysis of
the supernatants after membrane degradation in a 2.6 U mL
1
HAase solution for 7 days showed three main peaks, corres-
ponding to [HA]
11
: [M2H]
2
m/z = 1046.25, [HA]
12
: [M2H]
2
m/z = 1142 and [HA]
14
[M2H]
2
m/z = 1332.17 (Fig. 4C).
41
The
observed mass for [HA]
11
is a fragmentation product of ESI-MS
since digestion with HAase produces even-numbered oligosac-
charides. To confirm that the observed masses are not caused
by the technique itself ESI-MS analysis of a HA solution was
performed (Fig. S6, ESI) but no known masses of HA oligosac-
charides were identified.
Fig. 3 PAHA membrane microstructure. (A) Schematic representation of
PAHA membranes functionalized with bioactive molecules (green) interacting
with cell integrins (yellow). (B) Confocal microscopy images of the membranes
prepared with 1% (w/v) uoresceinHA and a 2% (w/v) peptide mixture con-
taining 0.1% K
3
K(Rhod)RGDS-PA. Images show the localization of HA (green)
and PA (red) over the membrane surface (B2) and cross section (B1). Yellow rep-
resents the overlaping of both components. (C) SEM micrographs of self-
assembled membranes with 1% (w/v) HA and 2% (w/v) K
3
-PA, showing the
surface on the polymer (C1) and peptide (C2) sides and cross section (C3, C4).
Paper Biomaterials Science
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The membranes herein presented were shown to degrade
gradually over time in the presence of HAase at physiological
concentration. In addition, it is expected that the peptide com-
ponent of the membrane will degrade over time by hydrolytic
and enzymatic processes into amino acids, which are nontoxic
and easily cleared in the body. Peptides can be designed to be
susceptible to enzymatic cleavage by incorporating matrix
metalloproteases (MMPs)-sensitive peptide sequences,
42,43
a
strategy that is currently being explored in our lab. By having
membranes sensitive to enzymatic activities, we are pursuing
the goal of biomimetic matrix degradation. These PAHA
membranes may present an advantage over other systems,
once slow degradation will enable the migration of the
adhered cells, as well as matrix remodeling and new tissue for-
mation led by the artificial ECM template.
Fibroblast response to the PAHA self-assembled membranes
displaying dierent densities of the RGDS epitope
Preliminary studies were performed with hDFb cultured on
K
3
PAHA membranes in DMEM with 10% FBS up to 7 days.
Fig. 4 Degradation of PAHA membranes. (A) Quantication of N-acetylamino sugars released from K
3
-HA membranes in PBS and PBS containing 2.6 U mL
1
and
50 U mL
1
HAase (*p < 0.05, error bars represent standard deviation (n = 3)). (B1) SEM images showing dierences in membrane microstructure when exposed to
dierent hyaluronidase (HAase) concentrations up to 14 days. (B2) Cross section of the membranes before and after exposure to 50 U mL
1
HAase evidencing that
degradation is occurring not only on the surface but also inside the membrane. (C1) Negative ESI-MS of the supernatant after incubating the membranes in
2.6 U mL
1
HAase for 7 days showing the presence of HA fragments. (C2) Observed and theoretical molecular masses of HA oligosaccharides.
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These studies indicated that these membranes supported
the adherence of fibroblasts, without presenting cytotoxicity
(Fig. S7, ESI).
Understanding the factors that regulate cell behavior is
important in many therapeutic applications. In most studies,
cell behavior is studied in a culture medium supplemented
with serum, which is poorly defined in terms of protein
content. To investigate whether the produced PAHA mem-
branes could integrate cell-adhesive ligands and anchor
human dermal fibroblasts, serum-free culture conditions were
used to eliminate the complexity of the competing adsorption
process of serum proteins on the membrane surface.
It has been shown that fibroblast survival, proliferation and
migration are dependent on cell adhesion and that fibronectin
(FN) binding domains have significant implications for skin
wound healing.
4
These activities require fibroblast attachment
to the RGD sequence in the tenth FN type III repeat, via cell
surface membrane integrin receptors.
44
Several authors reported
that inert biomaterials benefit from the presence of the
RGDS signaling molecule for optimal cell recognition and
adhesion.
1416,31
To enhance the adhesion of cells on the de-
veloped PAHA membranes, the RGDS motif (which is a known
ligand for integrin receptors) was incorporated into the PA
structure creating adhesion points to support integrin-mediated
Fig. 5 Cell adhesion on PAHA membranes. dsDNA quantication (A) (*p < 0.05, error bars represent standard deviation (n = 3)) and confocal uorescence images
(B) of hDFb cultured on HAPA membranes containing 1, 10 and 50% K
3
RGDS-PA or 10% (w/v) K
3
DGSR-PA up to 24 hours of culture. F-actin was labeled with
TRITC-phalloidin (red) and nuclei with DAPI (blue).
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cellular adhesion and migration on the membranes. Previous
studies have shown that the density of the RGDS epitope
impacts cell recognition and adhesion.
45
Considering this,
experiments were performed varying the K
3
RGDS-PA compo-
sition on the membranes (1, 10 and 50%) to determine
the optimal RGDS density. Membranes containing 10%
K
3
DGSR-PA or only K
3
-PA were used as controls.
DNA quantification results (Fig. 5A) showed that when
seeded on 50% K
3
RGDS-PA containing membranes, hDFb
adhered at a significantly higher number than the ones with a
lower concentration of RGDS, 1 and 10%, or without RGDS, K
3
-
PA and K
3
DGSR-PA for all time points except at 12 h of culture.
Cell morphology and distribution on the membranes was
further examined by confocal microscopy analysis (Fig. 5B).
Fig. 6 Cell morphology on PAHA membranes. SEM micrographs of hDFb cultured on HAPA membranes containing 1, 10 and 50% K
3
RGDS-PA or 10% (w/v)
K
3
DGSR-PA. Cells were cultured on the PA side in medium without FBS up to 24 h. (a) and (b) show a higher magnication of cells on the surface of HAPA
membranes with 10 and 50% (w/v) RGDS, respectively. Black and white arrows indicate the presence of lopodia and lamellipodia, respectively.
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After the first two hours, fibroblasts were uniformly distributed
on the membrane surface and a large number of cells were
observed on the 50% K
3
RGDS-PA containing membranes, as
depicted by the higher density of nuclei. These results demon-
strate the positive eect of the RGDS epitope on promoting
cell adhesion, which is mediated by integrins on the cell
surface. Considering the presence of HA in the membranes
and knowing that CD44, a transmembrane receptor observed
in a number of dierent cell types,
46
mediates HA dependent
cell adhesion, the expression of this receptor by hDFbs was
assessed (ESI, Fig. S8). Although these cells showed a high
expression of CD44 (98.67%), cells cultured on HA-based hydro-
gels (ESI, Fig. S9) did not show strong adhesion on these sub-
strates. This result suggests that integrins may play a major role,
rather than CD44, on cell adhesion on PAHA membranes.
Staining of cell cytoskeleton (Fig. 5B) did not reveal strong
organized actin fibers. Most of what is known about the cell
structure and function in vitro derives from cells plated on
rigid substrates, such as plastic or glass, laminated with a thin
film of serum proteins. As a result, some prominent aspects of
cell structure, such as the elongated morphology of cultured
fibroblasts or the prevalence of large actin fibers that
are commonly studied in vitro, are rarely if ever seen
in vivo.
47,48
Furthermore, cells cultured on soft substrates show
diuse adhesion complexes with poor actin cytoskeleton
organization
49
and the PAHA membranes used in this
study were shown to have low stiness (0.9 MPa in the wet
state).
23
Previous findings have also shown that fibronectin frag-
ments containing the RGD cell-binding domain alone cannot
sustain cytoskeletal organization of human dermal fibro-
blasts,
50
which might explain our observations.
Fibroblasts morphology observed in the SEM micrographs
is similar to what has been observed previously by others on
RGD-coated glass substrates.
45
After 24 hours of culture, only
the conditions with 10% and 50% K
3
RGDS-PA showed the
majority of the cells fully adherent (Fig. 6). Under these con-
ditions, fibroblasts had spread out exhibiting extended lamelli-
podia and filopodia interacting with fibrillar structure of the
membrane surface, thus confirming strong adhesion to the
substrate (Fig. 6a and b). In contrast, cells cultured on the
surface of smooth HA hydrogel (ESI, Fig. S9) were completely
round, without showing cell protrusions (lamellipodia and
filopodia) and did not flatten upon contact with the hydrogel
surface.
This result highlights the role of the nanofibrillar structure of
the self-assembled membranes in controlling cellmatrix inter-
actions. This is in accordance with what was reported in the lit-
erature that fibroblasts in 2D cultures normally exhibit a flat
morphology with dorsalventral polarity and large lamellipodia.
51
We should highlight that the cell culture experiments were per-
formed in the absence of serum for the entire study and the
membranes are displaying the RGDS cell-adhesive domain alone.
Further experiments are yet needed to investigate cell
response to HAPA membranes with higher stiness and/or
with other fibronectin fragment domains in order to be able to
make additional considerations regarding the eect of each
one of these variables on cell morphology.
4. Conclusion
This study has demonstrated that the 2D membranes, formed
by self-assembly between hyaluronan and a positively charged
peptide amphiphile, are susceptible to enzymatic degradation
by hyaluronidase. In the presence of hyaluronidase at physio-
logical concentration, the membranes slowly degrade over
time. The gradual degradation of the membranes is important
for allowing the migration of cells and/or the controlled
release of bioactive molecules incorporated into the membrane
that can control the function of attached cells. Membranes
presenting the cell adhesive ligand RGDS showed to increase
eciently the attachment of fibroblasts. This capability to
incorporate biochemical signals into 2D self-assembled mem-
branes enables the study of cellular responses to physiologi-
cally relevant signal variations. Furthermore, these bioactive
matrices permit cell culture studies without serum for short
periods. We expect that the proposed biodegradable hybrid
membranes could oer significant potential as a biomimetic
bioactive supportive matrix for wound healing.
Acknowledgements
Funding for this study was provided by the Portuguese Foun-
dation for Science and Technology (FCT, grant PTDC/EBB-BIO/
114523/2009). D. S. Ferreira gratefully acknowledges FCT for a
PhD scholarship (SFRH/BD/44977/2008). We also thank Dr
A. M. Azevedo from Instituto Superior Tcnico (BERG, Lisbon,
Portugal) for her support in obtaining circular dichroism data,
and M. T. Cerqueira and I. M. Martins from the 3Bs Research
Group at the University of Minho (Portugal) for assistance in
the isolation of human dermal fibroblasts and FACS analysis,
respectively.
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964 | Biomater. Sci., 2013, 1, 952964 This journal is The Royal Society of Chemistry 2013
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