Full Paper

Poly(hydroxybutyrate-co-hydroxyvalerate)
Bilayer Skin Tissue Engineering Constructs
with Improved Epidermal Rearrangement

Alessandra Zonari, Mariana T. Cerqueira, Silviene Novikoff,

Alfredo M. Goes, Alexandra P. Marques, Vitor M. Correlo,* Rui L. Reis

Bilayer skin substitutes constitute an attractive strategy towards improved skin wound healing.
Therefore, solvent casting and freeze-drying methodologies are used to produce polyhydroxy-
butyrate-co-hydroxyvalerate (PHBV) thin nanoporous membranes and 3D porous scaffolds that
are combined in bilayer structures to recreate the epidermal and dermal layers, respectively. The
combination of these methodologies allow attaining a bilayer structure with a high water
retention capability and adequate mechanical properties, susceptible to enzymes degradative
action. Cultures established with human keratinocytes (hKC) and dermal fibroblasts
(hDFb) confirm the suitability of the PHBV structures to
support cell adhesion and proliferation. Nonetheless,
when co-cultured under defined conditions, hKC are able
to grow and rearrange in a multilayer structure with
proliferative cells in the basal layer, and cells expressing a
terminal differentiation marker in the upper layer.
Therefore, PHBV bilayer structures demonstrate properties
that favor skin cells performance, thus representing a
promising strategy to improve wound healing.

Dr. A. Zonari, Dr. M. T. Cerqueira, Dr. A. P. Marques, Dr. V. M.

Correlo, Prof. R. L. Reis
3B’s 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, Guimara ~es, Portugal
E-mail: vitorcorrelo@dep.uminho.pt
Dr. A. Zonari, Dr. M. T. Cerqueira, Dr. A. P. Marques, Dr. V. M.
Correlo, Prof. R. L. Reis
ICVS/3B’s - PT Government Associate Laboratory Braga/
Guimara ~es, Portugal
Dr. A. Zonari, Prof. A. M. Goes
Laboratory of Cellular and Molecular Immunology, Department of
Biochemistry and Immunology, Institute of Biological Sciences,
Federal University of Minas Gerais, Caixa Postal 486, CEP 31.270-
901, Belo Horizonte, Minas Gerais, Brazil
Dr. S. Novikoff
Department of Nephrology, Federal University of Sa ~o Paulo, CEP:
04.023-900, Sa ~o Paulo- SP, Brazil
1. Introduction
Skin, the largest organ in humans, is responsible for the
protection of the body from the external environment,
avoiding pathogens invasion, and also for the regulation of
temperature and the hydration state of an individual.[1]
Therefore, when more than 4 cm in diameter of full-
thickness skin wounds occur, closure cannot be completed
without the assistance of a graft,[2] homeostasis is
compromised[3] and the risk of bacterial infections
increases.[4,5]
Despite the secondary injury and the limited donor sites
when large skin areas are damaged, the ‘‘gold standard’’
treatment for full-thickness wounds still is split-thickness
skin autografting.[6,7] Engineered cellular skin substitutes
are clinically available since the early 1980 s,[8,9] but despite
the progress along the years, the currently available skin

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substitutes are still associated with a range of problems,
such as excessive wound contraction and scar formation,
poor engraftment resulting from deficient vascularization,
among others.[7,10]
Bilayer skin substitutes that resembles full-thickness
skin, comprising both dermal and epidermal cells, with a
defined basement membrane, stratified epidermal layer
and organized dermis are yet to be achieved. Apligraf,
composed by a membrane of bovine Type I collagen on
which foreskin human fibroblasts are cultured as the base
for the stratification of a human epidermis-like structure,
lacks dermal organization and depth. This issue has
been tackled with Orcel that combines human dermal
fibroblasts within a 3D porous side of a collagen sponge and
keratinocytes on the gel coated, non-porous side of the
collagen matrix. Although these products do not cause
immunological rejection, the use of allogeneic cells has
been associated to graft rejection and permanent wound
closure failure[11,12] due to the death of the transplanted
cells. Thus, the need to co-grafting with an autologous
epithelial source renders these substitutes as temporary
bioactive dressings. TissueTech offers an autologous
approach that comprises two independent hyaluronic
acid-based constructs, Hyalograft as a dermal substitute
and Laserskin as the epidermal substitute. However,
because they have to be independently and consecutively
applied, they cannot be considered a ‘‘true’’ dermal-
epidermal skin substitute.[13,14]
The restriction to collagen and hyaluronic acid as
the components of the cell supporting polymeric
structures in the commercial bi-layered skin substitutes
reinforces the need to provide cells with adequate
environments capable of resembling native tissue to
achieve a better wound healing.[12,15,16] Different
types of natural and synthetic materials are being
investigated, seeking the best combination of scaffold
design, mechanical properties, degradation rate, and
healing stimulation.[17–19] Poly(3-hydroxybutyrate-co-3-
hydroxyvalerate) (PHBV) is a natural based polymer
member of the polyhydroxyalkanoates (PHA) family.[20]
It became attractive in the biomedical field due to
its optical activity, biodegradability, biocompatibility
and thermoplasticity,[20,21] as well as its piezoelectric
character being capable of transforming a mechanical
stimulus into electrical charge[21–23] which is known
to direct specific cellular processes such as migration,
proliferation and differentiation.[24–29] Furthermore,
3-hydroxybutyrate, the main degradation product of
PHBV, is a component of blood and tissues and it was
already demonstrated that 3-hydroxybutyrate mono-
mers promote fibroblasts and keratinocytes proliferation
by preventing apoptotic and necrotic cell death and by
stimulating a rapid increase of cytosolic calcium ion
influx.[30–32]
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Although PHBV is being used in different tissue
engineering strategies targeting the regeneration of
different tissues,[33–36] few works have proposed PHBV
structures for skin wound healing applications. The
influence of mechanical properties of electrospun and
solvent cast PHBV matrices on the expression of extracel-
lular matrix (ECM) proteins and therefore over the re-
epithelialization and the rate of healing was demonstrated
by Kuppan et al.[19] Recently, PHBV nanofibers have been
shown not to activate the production of inflammatory
cytokine by lymphocytes.[37] Also, variation of the content
of PHBV in relation to chitosan, has been shown to affect in
vitro fibroblasts adhesion and growth, as well as the levels
of inflammation, and rate of wound closure upon
implantation,[38] which was also shown to be enhanced
by the incorporation of keratin in PHBV electrospun
mats.[39] In a tissue engineering approach, electrospun
PHBV nanofibers seeded with hair follicular dermal and/or
outer root sheath cells were shown to support wound
healing by promoting early re-epithelialization and faster
wound closure than acellular PHBV matrices. Moreover,
those PHBV nanofibers cultured in vitro for 3–5 d were able
to keep wound moist and maintain appropriate mechanical
strength during healing.[40]
To the best of our knowledge, the potential of PHBV
structures other than electrospun membranes to contribute
to better wound healing, or to create 3D skin analogues as
improved strategies to lead skin tissue regeneration,
has not been explored. Therefore this work proposes the
construction of a PHBV-based bilayer skin tissue equivalent
considering skin two main strata, epidermis and dermis.
Taking this in consideration, we chose methodologies that
would allow obtaining a thin membrane and a highly
porous structure, respectively solvent cast and freeze-
drying. Thus, the polymeric bilayer structure combines a 2D
denser nanoporous upper layer and a 3D scaffold for the
respective support of human keratinocytes and dermal
fibroblasts assuring their communication throughout the
pores (Figure 1). The mechanical properties and degradabil-
ity of the PHBV bilayer structure, tuned by the processing
methodologies independently used for each layer, together
with the cellular performance within the respective
construct counterparts, were expected to contribute to
the assembly of a skin tissue engineered construct with
potential to be used as an improved autologous graft.
2. Experimental Section
2.1. Preparation of PHBV Bilayer Structures
The PHBV polymer used in this study had a molecular weight of
approximately 425.7 kDa and was provided by PHB Industrial, Serrana,
Brazil. A 2.5% (w/v) PHBV solution was prepared in chloroform (Fisher
Scientific, UK) at 60 8C and under constant agitation.
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Poly(hydroxybutyrate-co-hydroxyvalerate) Bilayer Skin Tissue Engineering Contructs
Figure 1. Schematic representation of the construction of A) PHBV bilayer structure and of B) the followed co-culture strategy.
KSFM ¼ Keratinocyte serum free medium; MEM ¼ minimum essential medium.
The PHBV membranes were prepared by a solvent casting
method. This approach consisted of dispensing the homogenous
polymer solution in petri dishes and then allowing the solvent to
completely evaporate in the hood. The 3D porous structures were
produced by freeze-drying. After getting a homogeneous polymer
solution, acetic acid (1:1, VWR Company, UK) was added to obtain
an emulsion that was frozen for 48 hours at
80 8C. The PHBV
solvent cast membranes were placed on the surface of the frozen
polymeric emulsion and the bilayer structures were subsequently
freeze-dried (CryoDos-80, Telstar, Spain) for 94 h.
Bilayer structures were cut into cylinders of 6 mm diameter and
3 mm thickness and sterilized by ethylene oxide prior to cell culture
studies.
2.2. Micro-Computed Tomography (CT) Analysis
The microarchitecture and porosity of the bilayer structures were
evaluated by micro-CT using a SkyScan equipment (SkyScan 1072,
Belgium). The structures were analyzed using a high resolution
mode of 6.9 mm x/y/z and an exposure time of 1792 ms. The energy
parameters defined in the scanner were 50 kV and 185 mA. After
scanning, the obtained data was analyzed using NRecon and CT-An
to produce binary images. A threshold of 60–255, to distinguish the
polymeric material from pore voids, was chosen and maintained
constant for all the scanned specimens. CT Vol image software’s
(SkyScan, Belgium) was used to create a tridimensional model of
the PHBV structures and determine pore size and porosity.
2.3. Quantification of Water Uptake
The water uptake capability of the solvent cast membranes, freeze-
dried scaffolds, and bilayer structures were determined using a
simple gravimetric method. Samples (n ¼ 5) were weighted and
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incubated in phosphate buffered saline (PBS, Sigma, USA),
0.01 M, pH 7.4, at 37 8C and 60 rpm for 15 min, 30 min, 1, 2, 3, 6,
12, 24, and 48 h. After each predefined period of time, samples were
weighted in order to determine the water uptake of the scaffolds
using the following equation:
wet weight
initial weight
Water uptakeð%Þ ¼  100
initial weight
2.4. Measurement of Tensile Properties
The tensile properties of the solvent cast membranes, freeze-dried
scaffolds and bilayer structures were determined using the
universal mechanical testing equipment (Instron 5540, USA). For
tensile tests, solvent cast membranes, freeze-dried scaffolds and
bilayer structures were prepared with a width of 5 mm and a
length of 30 mm, and a thickness of 0.04 mm for the solvent cast
membrane and of a 3mm for the freeze dried scaffold and the
bilayer structure. Samples (n ¼ 5) were tested at a crosshead speed
of 5 mm min
1 and gauge length of 10 mm at room temperature
(RT). Tensile strength was determined using both dry and wet
samples previously immersed in PBS for 3 h.
2.5. In Vitro Degradation Assay
Freeze-dried scaffolds (n ¼ 5) were incubated in Lipase (150 U L
1,
Sigma, USA) and/or Lysozyme (13 mg L
1, Sigma, USA) solutions in
PBS, at concentrations similar to the ones found in human
serum.[41,42] A PBS control was also performed. Samples were
maintained at 37 8C and 60 rpm for 1, 2, 4, and 8 weeks. The
solutions were changed every 3–4 d. At the end of each degradation
period, samples were removed, rinsed twice with distilled water,
dried and weighted in order to determine the percentage of weight
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loss, using the following formula:
initial weight
final weight
Weight loss ð%Þ ¼  100
initial weight
2.6. Isolation of Human Keratinocytes (hKC) and
Human Dermal Fibroblasts (hDFb)
hKC and hDFb were isolated from discarded skin tissue obtained
from healthy patients undergoing abdominoplasty in Hospital da
Prelada, Porto, Portugal. The samples were obtained after informed
consent of the patient and under a cooperation agreement
established between the Hospital and the 3B’s Research Group.
The skin samples were collected into sterile containers with 100 ml
of PBS with 10% antibiotic–antimycotic (Gibco, USA). Firstly, the
exceeding fat tissue was removed from the dermis and the skin
samples were washed with PBS, twice. The tissue samples were
cut into small pieces of 0.5 cm2 and incubated in PBS containing
dispase (2.5 U mL
1, BD Biosciences, USA) overnight at 4 8C. The
epidermis was then mechanically separated from the dermis and
incubated in 0.5% trypsin-EDTA (EDTA ¼ ethylenediamine tetraa-
cetate; Gibco, USA) for 7 min at 37 8C to isolate hKC. The cells were
separated from the remaining tissue using a 100 mm pore size
cell strainer (BD Biosciences, USA) and the cell suspension was
centrifuged at 290 g for 5 min. hKC were seeded at a density of
100 000 cells cm
2 in Keratinocyte Serum Free Medium (KSFM)
supplemented with Epidermal growth factor and Bovine pituitary
extract (Gibco, UK).
For the isolation of hDFb, the dermis separated from the
epidermis was incubated in PBS containing collagenase IA (250 U
mL
1, Sigma, USA) for 3 h at 37 8C and 60 rpm. The hDFb were
separated from the remaining tissue using a 100 mm pore size cell
strainer, centrifuged at 290 g for 5 min and seeded at a density of
50 000 cells cm
2 in minimum essential medium alpha modifica-
tion (alpha-MEM, Invitrogen, USA) supplemented with 10% Fetal
Bovine Serum (FBS; Gibco, USA) and 1% antibiotic–antimycotic.
Media were changed every 2–3 d and hKC were used at passage
1–2 while hDFb were used at passage 2–4.
2.7. Cell Culture
2.7.1. Homotypic Cell Cultures
The suitability of the solvent cast membranes and the freeze-dried
scaffolds to respectively support the adhesion of hKC and hDFb was
independently assessed in homotypic cultures.
hKC were seeded at a density of 1.75  105 cells cm
2 and
3.5  105 cells cm
2 on the solvent cast membranes of the PHBV
bilayer structures. Cells were allowed to attach for three hours,
and then 1 mL of fresh KSFM was added.
hDFb were seeded in the freeze-dried parts of the PHBV bilayer
structures at a density of 3  106 cells cm
3. Cells were allowed to
attach for two hours and then 1 mL of fresh complete alpha-MEM
supplemented was added.
Constructs were maintained in a humidified atmosphere at
37 8C and 5% CO2 for 7 days. Media were changed every 2–3 d.
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2.7.2. Co-Culture Set-Up
To attain heterotypic epidermal/dermal-like constructs, hKC were
first seeded on the upper layer of the bilayer structure composed by
the solvent cast membrane at a concentration of 3.5  105 cells
cm
2 and cultured in KSFM medium for one day. After 24 h,
the structure was flipped around and hDFb, at a density of
3  106 cells cm
3, were then seeded in the freeze-dried layers. The
constructs were co-cultured for further three days in KSFM
medium and in a mixture of KSFM and alpha-MEM 10% FBS
(KSFM/MEM) in a 1:1 proportion (Figure 1B).
2.8. Analysis by Flow Cytometry
The phenotype of hKC before the homo- and heterotypic cultures
was screened by flow cytometry. Cells were detached from the
culture flask with 0.5% trypsin-EDTA and permeabilized using
permeabilization buffer 1 (eBioscience, USA) for 10 min at RT.
Cells were then incubated with Cytokeratin 10 (K10, Abcam, UK),
Cytokeratin 14 (K14, FITC-conjugated, AbDserotec, UK) and
Involucrin (Imgenex, USA) primary antibodies for 1 h at RT and
at the concentration advised by the manufacturer. Cells were then
washed with a 2% bovine serum albumin (BSA, Sigma, USA) PBS
solution and incubated with the secondary antibody Alexa fluor
488 (Molecular Probes, USA) for 1 h at 4 8C, except when
fluorochrome-conjugated primary antibodies were used. Cells
were washed with PBS/BSA, re-suspended in PBS with 1%
formaldehyde (VWR, UK) and analyzed on a BD FACScalibur flow
cytometer (BD Biosciences, USA). For each sample, 15 000 events
were acquired and analyzed using CellQuest software.
2.9. Scanning Electron Microscopy (SEM)
Samples from cell culture were washed in PBS, fixed in 2.5%
glutaraldehyde (VWR UK) for two hours, and then dehydrated in
increasing concentrations of ethanol (30, 50, 70, 90, and 100%)
and critical point-dried (Autosamdri-815, Tousimis, USA).
Dehydrated cell culture samples, as well as samples before and
after degradation studies were mounted on aluminum stumps,
coated with 20 nm Au-Pd (80/20 wt%) using a high resolution
sputter coater (208HR, Cressington Company, UK) coupled to a
MTM-20 High Resolution Thickness Controller (Cressington
Company, UK), and analyzed on a Nova NanoSEM 200 (FEI, The
Netherlands) at 5.00 kV.
SEM images of three different samples of solvent cast
membranes were analyzed using ImageJ software to determine
the thickness and porosity.
2.10. Calcein AM Staining
Cell viability along the cultures was assessed after Calcein AM
staining. After each cell culture time point, samples were washed
with PBS and incubated for 20 min with a 0.002% (v/v) calcein AM
(Molecular Probes, USA) solution prepared in alpha-MEM without
phenol red and FBS. Samples were then fixed with 10% formalin for
30 min and the nuclei were counterstained with 4’,6-diamidino-2-
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Poly(hydroxybutyrate-co-hydroxyvalerate) Bilayer Skin Tissue Engineering Contructs
phenylindole (DAPI; Sigma, USA) for 5 min. Samples were observed
with an Axioplan Imager Z1 fluorescence microscope (Zeiss,
Germany) and images were acquired and processed with the
AxioVision V.4.8 software (Zeiss, Germany).
2.11. Double-stranded DNA Quantification
Proliferation of hKC and hDFb respectively on and in the PHBV
bilayer structures was assessed by the quantification of the total
amount of double-stranded DNA along the culture time. Briefly, at
each time point, constructs were immersed in 500 mL of ultrapure
water at 37 8C and frozen at –80 8C, respectively for osmotic and
thermal shocks. Double-stranded DNA was quantified in the lysed
cell suspension using the Quant-iT PicoGreen dsDNA assay kit
(Molecular Probes, USA), according to the manufacturer’s instruc-
tions. Fluorescence was measured at an excitation wavelength of
485/20 nm and at an emission wavelength of 528/20 nm in a
Synergie HT microplate reader (Bio-Tek, USA).
2.12. Immunofluorescence
After 3 d of co-culture, hKC were analyzed for the expression of
keratinocytes markers associated to different maturation/differ-
entiation degrees. The cells were fixed with 10% formalin for
30 min at RT and then incubated with 0.2% triton X-100 (Sigma,
USA) for 10 min, again at RT. After washing with PBS, the samples
were incubated for 1 h at RT with the primary antibodies Ki-67
(Abcam, UK), K10, K14 (Covance, USA) and involucrin, at the
concentration advised by the manufacturers. Following a PBS
washing, samples were incubated with the secondary antibody
Alexa fluor 488. F-actin cytoskeleton fibers were stained with
rhodamine-conjugated phalloidin (Sigma, USA) and cell nuclei
with DAPI. Samples were observed in a FluoView 1000 confocal
microscope (Olympus, Japan). ImageJ software was used to
quantify the number of cells positive for Ki-67. Cells were counted
in three different 20 magnification fields in each sample (n ¼ 3)
and plotted in relation to the total number of cells, determined by
the DAPI stained nuclei.
2.13. Statistical Analysis
All quantitative data is presented as an average of the number of
samples indicated for each methodology and standard deviation is
reported as a measure of sample deviation. In vitro biological data
was obtained from three independent experiments with at least
three replicates for each condition.
Statistical analysis was performed using GraphPad Prism 5.00
software for Windows (GraphPad Software, San Diego, USA). Firstly,
a Shapiro-Wilk test was used to ascertain about the data normality.
All results showed a normal distribution and were analyzed by one-
way ANOVA (hDFb DNA quantification), two-way ANOVA (water
uptake, hKC DNA quantification, co-culture DNA quantification
and Ki-67 expression) or unpaired two-tailed t-Student (tensile
properties) tests. For one-way ANOVA and two-way ANOVA,
Bonferroni’s was used as a post-hoc pairwise comparison test.
Values were considered statistically significant for p < 0.05.
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3. Results
3.1. Characterization of PHBV Structures
3.1.1. Microarchitecture
A bilayer scaffold combining a PHBV membrane, obtained
by solvent casting, and a 3D freeze dried porous structure,
was designed having in consideration the main skin strata,
epidermis and dermis. SEM analysis revealed that the
solvent cast layer is composed of a thin dense surface
with a thickness of 28.48 mm  7.13 mm and nanopores of
75 nm  19 nm (Figure 2A,C). In opposition, the freeze dried
layer was formed by a highly porous structure (Figure 2B
and C), with a porosity of 82.2%  0.5% and an average pore
size of 122.4 mm  58.1 mm (Figure 2D).
3.1.2. Water Retention Capacity
The water uptake capacity of the PHBV bilayer structures, as
well as of the independent solvent cast membranes and
freeze-dried scaffolds, was assessed in order to determine
their water retention capacity (Figure 3). The solvent cast
membranes presented the lowest capability to absorb
water represented by an increase in weight of about 45%
within the first hour of immersion in PBS, which remained
unchanged up to 48 h. Contrarily, the freeze-dried scaffolds
and the bilayer structures showed an increase in weight
of respectively 870% and 1050% within the first 15 min of
immersion. Moreover, the significantly higher (p < 0.05)
water uptake capacity observed for the bilayer structures in
comparison to the freeze-dried scaffolds within the first
15 min of immersion was not detected for longer times of
immersion. In fact, while the bilayer structures reached
the water uptake equilibrium (around 1370% increase
in weight) between 1 and 2 h, the freeze-dried scaffolds
uptake water up to 3 h reached significantly higher
(p < 0.001) weight gain (about 1670%) at the equilibrium
state.
3.1.3. Mechanical Performance
The tensile properties of the PHBV solvent cast membranes,
freeze-dried scaffolds and bilayer structures were measured
in dry and wet conditions as summarized in Table 1. Overall,
the combination of the solvent cast membranes with the
freeze-dried scaffolds lead to a bilayer structure with
increased (p < 0.01, in the dry and wet state) tensile
modulus and maximum stress, but diminished (p < 0.05,
in the wet state) strain at break in comparison to the freeze-
dried scaffolds alone. The immersion in PBS did not
significantly affect the tensile properties of the solvent
cast membranes and the strain at break of freeze-dried
scaffolds and bilayer structures. However, wet freeze-
dried scaffolds depicted reduced tensile modulus (p < 0.01)
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Figure 2. Morphological characterization of PHBV structures. Scanning electron micrograph of A) solvent cast membrane, B) freeze-dried
scaffold, and C) cross-section of PHBV bilayer structure, combining I) the membrane and II) the porous scaffold. D) 3D image obtained by
micro-CT reconstruction model. Upper right corner: micrograph in (A) represent a magnified area, highlighting the topography and the
nanoporosity of the membrane.

and maximum stress (p < 0.001), while wet bilayer
structures only showed reduced tensile modulus (p < 0.05)
in relation to the respective dry structures.
3.1.4. In Vitro Sensitivity to Enzymatic Degradation
The susceptibility of the PHBV freeze-dried scaffolds to
enzymatic degradation was assessed in the presence of
Figure 3. Water uptake of the PHBV solvent cast membranes, freeze-
dried scaffolds and bilayer structures. # indicates significant
difference between the PHBV solvent cast membranes and the
freeze-dried scaffolds and the PHBV bilayer structures within
the same time point. Data was obtained from 5 different
measurements,  p < 0.05,  , # p < 0.001, two-way ANOVA and
Bonferroni’s post-hoc test.
lipase and/or lysozyme at concentrations similar to the
ones found in human serum. The percentage of weight
loss in the different degradative solutions along the time
is shown in Figure 4A. Independent of the conditions, no
variations of weight were detected within the first 2 weeks.
Weight loss was noticed from that timepoint on but only
in the presence of lipase and lipase plus lysozyme. After
4 weeks, the freeze-dried scaffolds lost about 30% of their
initial weight in the presence of lipase and around 50% in
the concomitant presence of lipase and lysozyme. At the
end of the experiment, 8 weeks, the samples immersed
in the solution containing lipase plus lysozyme were
completely degraded and the samples incubated only with
lipase had lost about 87% of their initial weight. Contrarily,
samples immersed in the solution containing only lyso-
zyme or in PBS (control) did not show any weight variation
during the time of the experiment. The alterations
caused in the freeze-dried scaffolds structure along the
degradation process were investigated by SEM (Figure 4B).
Topographic changes in the pores walls surface were
observed in the presence of lipase and lipase plus lysozyme,
the conditions where weight loss was detected. Up to
2 weeks no significant changes were observed in all
conditions (data not shown) but reduced size and rougher
pore walls surfaces were detected for the lipase conditions

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Table 1. Tensile properties of PHBV solvent cast membrane, freeze-dried scaffold and bilayer structure in the dry and wet states.

Tensile Modulusa) Maximum Stressa) Strain at Breaka)

[MPa] [MPa] [%]

Solvent cast membranes Dry 1188  160 22.33  1.23 6.17  1.58
Wet 1159  217 21.73  5.76 4.79  0.93
 f  f
Freeze-dried scaffolds Dry 0.69  0.27 , 0.017  0.003 , 7.93  2.48
Wet 0.19  0.09### 0.007  0.001## 10.70  2.20#
Bilayer structures Dry 10.26  3.00 0.23  0.05 4.93  1.84
Wet 6.01  1.34 0.17  0.08 7.37  1.72

Data was obtained from 5 different measurements;  Indicates significant difference between dry and wet condition for the same structure;
a)

f
indicates significant difference between dry freeze-dried scaffolds and dry bilayer structures; #indicates significant difference between
wet freeze-dried scaffolds and wet bilayer structures.  , # p < 0.05,  ,## p < 0.01 and  ,f, ### p < 0.001, Student’s t-test.

after 4 and 8 weeks. Similar observations were made in the
lipase plus lysozyme conditions but only for 4 weeks, as
after 8 weeks the structure was completely degraded and
only small fragments of material were collected.
3.2. Optimal hKC Density
Two different hKC concentrations were used to determine
the optimal density to reach a confluent cell layer on the
PHBV solvent casting membranes within 3 d of culture.
Independently of the initial cell number, hKC were able to
adhere on the solvent cast membranes of the PHBV bilayer
structures exhibiting the typical polygonal shape. How-
ever, while a cohesive cell layer was formed after 3 d of
culture when 3.5  105 cells cm
2 were initially seeded on
the membranes (Figure 5A), a similar behavior was only
detected for the lower seeding density after 5 d of culture
(data not shown). Cell proliferation data confirmed that
when seeded at higher density cells were able to proliferate
up to 3 d of culture (Figure 5B) occupying the whole
membrane surface and remaining viable (Figure 5C).
Moreover, when a lower seeding density was used, an
increased number of hKC was still detected from day 3 to
day 5 of culture (Figure 5B).
3.3. Behavior of hDFb in the Freeze-dried Scaffolds
The capacity of the freeze-dried scaffolds of the PHBV
bilayer structures to support hDFb adhesion and prolifera-
tion was assessed. SEM analysis revealed that hDFb
attached to the scaffolds within 1 d of culture exhibiting
the typical fibroblastic morphology (Figure 6A.1). Moreover,
hDFb were able to proliferate along the culture period,
(Figure 6A.1-3,B) colonizing the interior of the scaffolds
(Figure 6A.4) and remaining viable (Figure 6C).
3.4. Heterotypic Communication between hKC and
hDFb as Part of the PHBV Bilayer Construct
The previous knowledge acquired with the homotypic
cultures was used to create heterotypic PHBV bilayer
constructs. hKC and hDFb were respectively cultured on the
solvent cast membranes and on the freeze-dried scaffolds,
at the previously defined cell densities and following
subsequent cell seeding as illustrated in Figure 1B. The
initial hKC culture was performed in KSFM, the optimal
hKC culture medium, but deprived of serum. Considering
that the absence of serum could be detrimental for hDFb
adhesion in the co-culture, after the initial 1 d culture of hKC
on the solvent cast membranes, co-culture was performed
in KSFM and in KSFM/alpha-MEM in an equal proportion.
Independently of the cell culture medium, hKC and hDFb in
co-culture were able to adhere to the respective PHBV
structure layer and remained viable for the time period
tested (Figure 7A). Nonetheless, DNA quantification data
showed that total DNA content did not vary from day 1
to day 3 when cells were cultured in KSFM but cells were
able to proliferate when cultured in KSFM/MEM medium
as confirmed by the significantly higher (p < 0.05) DNA
amount at day 3 (Figure 7B).
The analysis of the hKC behavior in the homotypic and
co-cultures performed in the different medium showed
that, although forming a cohesive cell layer in both media,
the total number of cells and the number of proliferative
cells, Ki-67 positive hKC, varied. In the KSFM the hKC had
grown as a monolayer in both homotypic and heterotypic
cultures (Figure 8A), but a significantly higher (p < 0.05)
percentage of proliferative cells was found in the co-culture,
32.76  4.17% compared to 22.18  3.61% in the homotypic
culture (Figure 8C). Contrarily, and despite the apparent
similarities in terms of growth, the percentage of Ki-67
positive-cells cultured in KSFM/MEM was significantly

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Figure 4. Degradation of PHBV freeze-dried scaffolds. A) Percentage of weight loss along the immersion time in PBS, Lipase, Lysozyme, and
Lipase/Lysozyme, at pH 7.4 and 37 8C. Macroscopic images represent the appearance of the freeze-dried scaffolds in the different conditions
at specific time points. B) SEM images of the PHBV scaffolds before (day 0) and after 4 and 8 weeks of immersion in PBS, Lipase, Lysozyme
and Lipase/Lysozyme. Images on upper right corners represent magnified areas highlighting the topography of the pore walls surfaces.
Samples immersed in Lipase/Lysozyme solution for 8 weeks were completely degraded and therefore no image corresponding to that
condition is shown.

higher (p < 0.05) than in KSFM and was not affected by the
presence of hDFb. Moreover, in the co-culture established
with KSFM/MEM, the hKC organized in multiple layers
(basal and suprabasal). A higher number of cells presenting
a smaller size and a cuboidal shape (Figure 8B.1) was
observed in the basal layer, whereas in the upper layers,
cells became larger and flattened (Figure 8B.2,3). Moreover,
almost all the Ki-67 positive cells, 49.12  3.48% of the total
number of hKC (Figure 8C) were confined to the basal layer
(Figure 8B.1). Few Ki-67 positive-cells were found in the
suprabasal layer (Figure 8B.2) and no expression of Ki-67
was detected in the upper layer (Figure 8B.3).
In addition to the proliferative character of the hKC
adhered to the solvent cast membranes along the co-

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Poly(hydroxybutyrate-co-hydroxyvalerate) Bilayer Skin Tissue Engineering Contructs

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Figure 5. hKC behavior on the solvent cast membranes of the PHBV bilayer structures. A) SEM images of hKC, seeded at an initial density of
3.5  105 cells  cm
2, adhered on the solvent cast membranes of the PHBV bilayer structures after (A.1) 1 and (A.2) 3 d of culture. B) hKC
proliferation on the solvent cast membranes of the PHBV bilayer structures indicated by the amount of double-strand DNA along the
culture. C) Viable hKC (green) stained with Calcein AM after 3 ds of culture and seeded at an initial density of 3.5  105 cells cm
2. Cell nuclei
were stained with DAPI (blue). Data was obtained from three independent experiments with three replicates for each condition,

p < 0.001, two-way ANOVA and Bonferroni’s post-hoc test.

culture, the degree of differentiation of the hKC forming
the different layers was assessed. Flow cytometry analysis
of the hKC prior to seeding revealed that the cell
populations were composed mostly by K14 positive-cells
(54.59  14.65%) but few K10- (1.03  0.59%) and involu-
crin-positive (7.62  5.75%) cells. After the co-culture,
immunostaining of the cells forming the constructs
revealed that when using KSFM medium, the majority of
the cells continued expressing K14, no cells were expressing
K10 and few cells were positive for involucrin (Figure 9A–C).
When the co-culture was established in KSFM/MEM
medium, the expression of K14 and involucrin was
restricted to the upper layer and only few K10 positive-
cells were found in the suprabasal layer (Figure 9D–L).
4. Discussion
Engineering skin substitutes represents a prospective
approach to combat acute and chronic skin wounds as
for instance large burn injuries or ulcers.[1] Bilayer skin
substitutes exploring the interaction between epidermal
and dermal components, as regulators of wound healing,
represent a valuable approach towards improved out-
comes. Taking this into consideration, a bilayer structure of
PHBV composed of a thin nanoporous upper layer and a
porous sub-layer was developed combining solvent casting
and freeze-drying. This structure was designed to attain
specific properties that were respectively expected not
only to positively modulate hKC and hDFb behavior,
allowing biochemical crosstalk between them, but to
particularly contribute to retain wound moisture and to
withstand the mechanical stresses diminishing wound
contraction. The solvent cast membrane where hKC were
seeded, presented a rough surface with small pores in
the range of 75  19 nm. These porous dimensions are
important to physically separate cells within the different
layers, but allowing the diffusion of nutrients and the
communication between hKC and hDFb. In fact, the
exchange of cytokines and growth factors released by
hDFb and hKC are crucial for keratinocyte proliferation and
differentiation and for the deposition of various basement
membrane proteins and hemi-desmosome components
controlling epidermal homeostasis.[43,44]
Concerning the freeze-drying layer, it was designed to
depict a highly porous structure intended to favor hDFb
motility within the structure and subsequent ECM deposi-
tion and organization. Moreover, this morphology is
expected to favor water uptake, thus significantly contrib-
uting to maintain the wound moist environment. The

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Figure 6. Behavior of hDFb cultured in the freeze-dried scaffolds of the PHBV bilayer structures. A) SEM images of hDFb adhered to the
freeze-dried scaffolds of the PHBV bi-layer structures after (A.1) 1 d, (A.2) 3 d, and (A.3) 7 d of culture. (A.4) Constructs cross-section showing
cells in the interior of the scaffolds after 7 d of culture. Images on upper right corners represent magnified areas. B) hDFb proliferation in the
freeze-dried scaffolds of the PHBV bilayer structures indicated by the amount of double-strand DNA along the culture. C) Viable hDFb
(green), stained with calcein AM, after 7 d of culture in the interior of the freeze-dried scaffolds of the PHBV bilayer structures. Cell nuclei
were stained with DAPI (blue). Data was obtained from three independent experiments with three replicates for each condition,  p < 0.01,
one-way ANOVA and Bonferroni’s post-hoc test.

Figure 7. Viability and proliferation of hKC and hDFb in co-culture. A) hKC and hDFb viability after different times of co-culture in KSFM/
MEM medium (1 and 3 d). Green: Calcein AM, blue: DAPI. Scale bar: 50 mm. B) DNA content of co-culture samples in different medium as a
function of time. Data was obtained from three independent experiments with three replicates for each condition,  p < 0.05, two way
ANOVA and Bonferroni’s post-hoc test.

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Figure 8. Ki-67 expression (green) of hKC adhered on the solvent cast membranes and in co-culture with hDFb in A) KSFM and B) KSFM/MEM
medium after 3 d of co-culture. Z-scan images showing the Ki-67 expression on the basal (B.1), suprabasal (B.2) and upper (B.3) layers. Nuclei
were stained with DAPI (blue) and F-actin cytoskeleton fibers with rhodamine-conjugated phalloidin (red). C) Percentage of Ki-67 positive
cells in the different culture conditions. Data was obtained from three independent experiments with three replicates for each condition,

p < 0.05,  p < 0.01,  p < 0.001, two way ANOVA and Bonferroni’s post-hoc test.

freeze-dried scaffold demonstrated a high water retention
capacity being able to uptake around 900% of water, in
relation to its initial weight, within the first 15 min and
a maximum of 1600% within 2 h of immersion in PBS.
Despite the low water uptake capability of the solvent
cast membrane, and its impact over the overall total
percentage of water uptake by the bilayer structure upon
combination with the freeze-dried scaffold, we consider
that the proposed bilayer structure still depicts a water
retention degree that complies with the requirement of
maintaining the injury environment moist enough in order
to enhance wound healing.[45] The combination of the
stiffer PHBV solvent cast membrane with the freeze-
dried scaffold also impacted the mechanical properties
of the bilayer structure. However, the stiffer and less
ductile character of the combined structure, in relation to
the freeze-dried scaffold, is considered to be positive in the
sense that the combined structure is expected to better
withstand the mechanical stresses that occur during the
deposition of fibrotic tissue, thus diminishing wound
contraction.[17]
One of the major limitations, particularly for biomedical
applications, that have been pointed out to PHBV is its
slow hydrolytic degradation.[46–48] Accordingly, no evi-
dence of weight loss was detected in PHBV scaffold after
eight weeks of immersion in PBS and in the presence of
lysozyme. Nevertheless, lipase leads to the degradation
of the structures detectable after two weeks resulting in
a reduction of 87% of the initial weight after eight weeks.
The degradation caused by lipase probably exposed
binding sites for lysozyme thus, when both enzymes
were used, PHBV scaffold was completely degraded after
the same period of time, eight weeks. Lipase and lysozyme
are enzymes present both in healthy skin but also at
the inflammatory stage of wound healing.[49,50] In deep
injuries, such as burns, that cause fat inflammation or fat
necrosis, lipase can be secreted by mononuclear phag-
ocytes to facilitate the digestion and clearance of the lipid
material released by the damaged adipocytes.[50] Upon
skin injury polymorphonuclear leukocytes are also re-
sponsible for the release of lysozyme, which has an
important antimicrobial action in infected wounds.[51,52]
The in vitro degradation tests were carried out in the
presence of those enzymes at blood serum concentrations.
Thus, considering that due to the inflammatory process
resulting from skin tissue injury the local tissue concen-
tration of both lipase and lysozyme will be higher, the
degradation of the PHBV structure in vivo is expected to
start earlier.
The ultimate goal of this work was to obtain a skin
analogue to be used as an autologous substitute. From a
clinical perspective, the time needed to expand patient’s
own cells is nowadays considered a limitation of cellular
skin substitutes[53] which is even worsened by the
additional time required to obtain living-like tissues that,
histologically, closely resemble native dermis and epider-
mis. Bearing this in mind, we propose a co-culture strategy
that allows the complete coverage of the PHBV solvent cast
membrane by hKC, and the partial colonization of the
freeze-dried porous layer by hDFb, after 4 d of culture. The

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Figure 9. Expression patterns of keratinocyte differentiation markers in hKC adhered on the solvent cast membranes and in co-culture with
hDFb in A–C) KSFM and D–L) KSFM/MEM medium. When cultured with KSFM medium hKC grown as a monolayer with the majority of
the cells expressing K14 (A, green), no cells expressing K10 (B, green) and some cells expressing Involucrin (C, green). When cultured
with KSFM/MEM medium, hKC organized in multiple cell layers. Z-scan images showing the localization of hKC expressing K14 (D,G,J),
K10 (E,H,K) and Involucrin (F,I,L). (D-L) depicts x-y digital sectioning images of the upper (D,E,F), suprabasal (GH,I) and basal (J,K,L) layers.
Nuclei were stained with DAPI (blue) and cells cytoskeleton with rhodamine-conjugated phalloidin (red).

membrane revealed adequate surface properties to support
the attachment and growth of hKC, confirming previously
reported results,[32] and allows to define an optimal initial
cell density of 350 000 hKC per cm2 to achieve a cohesive
layer of cells within 3 d in homotypic cultures and 4 d in the
co-cultured construct. One million of hKC can be harvest
from 1 cm2 of skin sample, and after 1 week of expansion
almost 20 million cells can be obtained.[54] Several studies
using bilayer approaches, have reported the need of
1 million of hKC per cm2 to obtain a stratified epidermis-
like structure, throughout a 14–21 d period.[55–57] Therefore,
we tested lower cell concentrations and demonstrated
that while using PHBV bilayer structure we were able
to diminish the number of cells, consequently optimizing
the usage of the biopsy samples.
The hDFb within the freeze-dried porous layer and under
the defined co-culture timeframe were able to support the
hKC on the upper layer of the bilayer structure providing, in
addition to the culture media, the necessary biochemical
signals to retain cells in a high proliferative state. This effect
is mainly due to paracrine signaling, and it is well reported
that cells secretome results from the direct interaction with
either extracellular matrix or biomaterials surface.[19] Thus,
the bilayer structure was very important to provide the
physical structure with the adequate properties to support
the growth of hKC and hDFb in separate layers which

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Poly(hydroxybutyrate-co-hydroxyvalerate) Bilayer Skin Tissue Engineering Contructs
resulted in specific signaling that allowed a particular hKC
rearrangement. In fact, the presence of hDFb in the co-
culture system lead to hKC differentiation and organization
in multiple layers that contrasted with the monolayer of
cells observed in the homotypic cultures. It is known that
fibroblasts play key roles promoting keratinocyte growth
and differentiation.[15,58] Additionally, the presence of
calcium, serum, or both, can induce keratinocyte prolifera-
tion and differentiation.[59,60] These observations are in
agreement with the obtained results that clearly showed
the rearrangement and phenotypic variations of hKC to be
dependent on the presence of hDFb and serum in the
heterotypic culture established in KSFM/MEM. In fact when
culturing with KSFM/MEM, proliferative Ki-67 positive hKC
were detected in the basal layer. The expression of K14, a
marker of early differentiation that is usually confined to
the basal layer, was detected on the cells of the upper layer.
The same expression pattern was observed for K5 (data
not shown). Although without achieving a fully stratified
epidermis-like structure, the presence of a hKC multilayer
with cells expressing the terminal marker involucrin, a
cytoplasmic protein precursor of the cornified envelope
that forms a barrier to protect underlying tissue from
infection, dehydration, chemicals and mechanical
stress,[61,62] was observed as the outer layer. This might
be beneficial upon implantation by more rapidly providing
a barrier against the external environment. Our approach
provides a one-step system that has already fibroblasts and
keratinocytes organized in the different tissue-like layers,
using lower cell concentration within 4 d, a significantly
less time than the one required for the TissueTech Autograft
System.[13,14] Moreover, this pre-rearrangement associated
to the PHBV degradation at early time points is likely to
promote in a faster way epidermal-dermal junction and
consequently improved healing.
5. Conclusion
PHBV bilayer structures were successfully designed and
produced by assembling a solvent cast membrane and a 3D
freeze-dried scaffold. The combination of the distinct
qualities of the two systems such as susceptibility to
inflammatory enzymes degradative action, high water
retention capability and stiff character, allowed to achieve
an improved system expected to contribute to a better
wound healing. The bilayer structures design and associat-
ed properties favored hDFb and hKC performance under
defined heterotypic culture conditions that lead to the
particular rearrangement of hKC with more differentiated
cells in the outermost side. These results demonstrate that
the proposed PHBV-based bilayer skin tissue engineered
construct has potential to be used as an improved
autologous graft.
Macromol. Biosci. 2014, 14, 977–990
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www.mbs-journal.de
Acknowledgements: A.Z. acknowledges the Brazilian National
Council for Scientific and Technological Development (CNPq) for a
PhD Grant (141775/2010-6/141787/2012) and financial support of
the projects 564779/2010-5 and 490414/2010-9, also the Brazilian
Coordination of Improvement of Higher Education Personnel
(CAPES) for providing the PhD scholarship abroad: PDEE0046/11-6.
This work was also financed by the project RL1 – ABMR – NORTE-
01-0124-FEDER-000016 co-financed by North Portugal Regional
Operational Programme (ON.2 – O Novo Norte), under the
National Strategic Reference Framework (NSRF), through the
European Regional Development Fund (ERDF). Authors would also
like to acknowledge Lucılia Goreti Ribeiro Pinto for the help with
confocal images and Edith Ariza Avila for the help with SEM
images.
Received: January 6, 2014; Revised: February 4, 2014; Published
online: March 4, 2014; DOI: 10.1002/mabi.201400005
Keywords: biomimetic; bioengineering; in vitro epidermal
rearrangement; polyesters; wound healing
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