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Combining xanthan and chitosan membranes to multipotent mesenchymal

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Article  in  Journal of Biomaterials Applications · October 2014

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Soft Tissues and Materials
Journal of Biomaterials Applications
2015, Vol. 29(8) 1155–1166
! The Author(s) 2014
Combining xanthan and chitosan Reprints and permissions:
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membranes to multipotent DOI: 10.1177/0885328214553959
jba.sagepub.com
mesenchymal stromal cells as bioactive
dressings for dermo-epidermal wounds

Márcia Z Bellini1,2, Carolina Caliari-Oliveira3, Amanda Mizukami3, Kamilla Swiech4,

Dimas T Covas3, Eduardo A Donadi3, Pedro Oliva-Neto5 and Ângela M Moraes1

Abstract
The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining
viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to
regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds
is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture,
potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others.
Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal
stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional struc-
ture for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the
applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesen-
chymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed
efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal
stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus
indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Keywords
Scaffolds, chitosan, xanthan gum, stem cells, dressings, regenerative medicine, skin lesions

Introduction
to provide faster and more effective healing of the
The skin, the largest organ of the human body, consti- damaged skin are of great relevance.
tutes the first barrier of organism defense, playing
important roles in homeostasis, such as body tempera-
ture regulation and protection against dehydration, as
well as providing support to blood vessels and nerves.1,2 1
School of Chemical Engineering, University of Campinas (UNICAMP),
In severe burns and chronic wounds of various etiolo- Campinas, SP, Brazil
2
gies, the spontaneous regeneration process is severely Integrated Adamantinenses Colleges (FAI), Adamantina, SP, Brazil
3
School of Medicine of Ribeirão Preto, University of São Paulo (USP),
impaired, raising the need for therapeutic intervention. Ribeirão Preto, SP, Brazil
The rapid healing of the skin area damaged or lost 4
School of Pharmaceutical Sciences of Ribeirão Preto, University of
may represent an improvement of prognosis of the São Paulo (USP), Ribeirão Preto, SP, Brazil
5
patient, and for this purpose, conventional dressings School of Sciences and Languages of Assis, São Paulo State University
are extensively used. However, ideally, a dressing (UNESP), Assis, SP, Brazil
should not only protect the wound but also promote These first two authors contributed equally to this work.
the healing process by providing a suitable microenvir- Corresponding author:
Ângela M Moraes, Department of Engineering of Materials and
onment, hydrated and with thermal insulation, allow- Bioprocesses, University of Campinas (UNICAMP), Campinas, SP,
ing the elimination of the excess of exudate and CEP 13083-852, Brazil.
promoting gas exchange.3 Thus, approaches intended Email: ammoraes@feq.unicamp.br

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1156
Human skin may be successfully reconstituted
in vitro through the culture of skin cells on biological
scaffolds such as de-epidermized human dermis4 and
bovine collagen,5 being morphologically and function-
ally compatible with in vivo human skin. Examples
of products for skin tissue engineering already
approved by the Food and Drug Administration
(FDA, USA) are ApligrafÕ (produced by
Organogenesis, Massachusetts, USA) and OrCelÕ
(Forticell Bioscience, New York, USA), both consti-
tuted of human keratinocytes and fibroblasts cultivated
in scaffolds of bovine collagen type I.
Approaches involving the use of scaffolds obtained
from animal origin, human or not, as supports for cell
growth may raise concerns due to shortage of donors
and to the possibility of transmission of pathogens.
However, the use of certain bioactive polymers as
scaffolds for tissue engineering is attractive due to
the fact that it is possible, through their use, to repro-
duce relevant characteristics of the native extracellular
matrix and also to obtain materials with rates of deg-
radation in vivo compatible to those of new tissue
development.6
Several studies have demonstrated the potential of
natural-origin bioabsorbable polymers such as chitosan
(Ch) combined or not with alginate,7,8 gelatin,9 cellu-
lose and its derivatives10 in tissue engineering. Lamellar
dense (compacted) and porous scaffolds of Ch com-
plexed with xanthan (Xn) seems to be particularly pro-
mising for this purpose, given that Xn is a
polysaccharide obtained by fermentation, with more
controlled quality in contrast to the natural counter-
parts processed from animal or vegetable sources.
Dense and porous Ch-Xn scaffolds were analyzed in
detail and extensively characterized by Veiga and
Moraes11 and Bellini et al.12 The dense membranes
are thin, transparent in both wet and dry states, and
present no apparent pores on their surfaces, having a
highly compacted structure when dry. The porous
Ch-Xn membranes, on the other hand, are opaque
and have a large number of uniformly distributed
pores. Both membrane types show high capacity of
absorption of physiological solutions and body fluids,
excellent stability in cell culture medium, mechanical
properties compatible with those of the skin and negli-
gible cytotoxicity to L929 cells, comprising a set of rele-
vant characteristics for application as wound dressings
and scaffolds for cell growth.
Artificial autologous epidermis can be successfully
obtained by inoculation of epidermal and dermal cells
derived from the patient himself on polymeric scaffolds
based on Ch.13,14 However, this approach is frequently
hampered by the long time required for obtaining it,
about 4 to 5 weeks.5 This waiting period may result in
dehydration of the patient, increasing his or her
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Journal of Biomaterials Applications 29(8)
predisposition to infections. In this sense, artificial epi-
dermis produced from multipotent cells, even those of
allogenous origin, could function as a temporary sub-
stitute while the patient waits for an autologous graft
obtained in vitro.
Several factors are favorable to the use of multipo-
tent mesenchymal stromal cells (MSCs) in the treat-
ment of skin wounds, such as their regenerating and
immunoregulation properties, their easy isolation and
in vitro expansion, added to satisfactory results in
recent preclinical and clinical studies.15 Moreover,
MSCs are poorly immunogenic,16 allowing therapies
based on previously established cultures of healthy
donors, what represents a considerable benefit for
patients with severe wounds requiring immediate
treatment.
Thus, in the search for alternatives for more effective
treatments for dermo-epidermal lesions, this study
aimed to demonstrate the feasibility of the association
of Xn and Ch membranes with MSCs and the develop-
ment of a biodressing for the therapy of this type of
wounds.
Materials and methods
Animals and cells
Wistar rats weighting 200 to 300 g with 4 to 6 weeks in
age were kept in boxes lined with sawdust, in a room
with controlled temperature and light (22 C, 12 h light
and 12 h dark). Throughout the procedure, the animals
received food and filtered water ad libtum.
Multipotent mesenchymal stromal cells were isolated
from the bone marrow of at least three animals per
batch, through flushing with PBS/EDTA of their
femurs and tibias after the removal of bone epiphysis.17
The animals were sacrificed prior to bone extraction
and the collected cells were cultivated in Alpha
Minimum Essential Medium with 0.58 g/L L-gluta-
mine, 4.50 g/L D-glucose (Gibco, Grand Island, NY),
supplemented with 3.70 g/L NaHCO3 (Merck,
Germany), antibiotic solution containing 10,000 UI/L
penicillin and 10,000 mg/L streptomycin (Gibco,
Grand Island, NY), 5.96 g/L Hepes (Gibco, Grand
Island, NY), and 15% (v/v) fetal bovine serum (SFB,
Thermo Scientific, Rockford, IL, EUA) and main-
tained at 37 C and 5% of CO2 in an incubator
(Tecnal, Brazil, model TE-399). Cells at the third or
fourth passage were then tested regarding adhesion
and proliferation on the Ch:Xn membranes as well as
for substrate consumption and metabolite production
during cultivation and for cell therapy assays in an
in vivo model.
The homogeneity of the MSCs population after suc-
cessive subculture procedures was assessed by flow
Bellini et al.
cytometry at the Flow Cytometry Laboratory of the
Regional Blood Center (Ribeirão Preto/FMRP-USP,
São Paulo, Brazil). Monoclonal antibodies against the
markers CD45, CD11b, CD31, CD29, CD90, CD105,
and CD44 (eBioscience, USA) were used.
The procedures in vitro and in vivo are consistent
with the standards of the Brazilian College of Animal
Experimentation (COBEA) and were reviewed and
approved by the Ethics Committee on Animal
Experimentation (CETEA) of the School of Medicine
of Ribeirão Preto, University of São Paulo, where the
experiments were conducted under the registration
number 158/2010.
Membrane preparation and characterization
The dense and porous membranes of Ch complexed
with Xn were prepared according to the procedures
described by Bellini et al.,12 at a mass ratio of Xn to
Ch equal to 1:1, using 96% deacetylated Ch, xanthan
gum, and Pluronic F68Õ from Sigma-Aldrich
(St. Louis, MO, USA) and glacial acetic acid from
Merck (São Paulo, Brazil). The water used was distilled
and deionized in a MilliQ system (Millipore, São Paulo,
Brazil).
After sterilization with ethylene oxide, the mem-
branes showed the characteristics summarized in
Table 1. Prior to in vitro and in vivo tests with MSCs,
the mutagenic potential of the membranes was evalu-
ated by the AMES test, which is based on the induction
of reverse mutation in histidine auxotrophic Salmonella
typhimurium bacteria18 at the Tecam Laboratórios (São
Paulo, Brazil), following the procedures described in
the OECD Guidelines for Testing of Chemicals
(1997),19 ISO10993-3 (1992),20 ISO10993-1 (2003),21
and ISO10993-12 (2007).22
Table 1. Properties of the dense and porous Ch-Xn membranes used (summarized from Bellini et al.12).
Property
Thickness of dry membranes (mm)
Uptake of water after 24 h at 37 C (g/g)
Uptake of simulated body fluid after 24 h at 37 C (g/g)
Membrane mass loss after exposure to water for 24 h at 37 C (%)
Membrane mass loss after exposure to simulated body fluid for 24 h at 37 C (%)
Tensile strength of dry membranes (MPa)
Elongation at break of dry membranes (%)
Cytotoxicity of membrane extracts to L929 cells (%)
Ch: chitosan; Xn: xanthan.
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1157
Analysis of MSCs behavior after inoculation on the
Ch:Xn membranes
Membrane samples (1.5 cm in diameter) were individu-
ally arranged in 24-well flat-bottom plates (TPP, Brazil)
and washed three times with PBS/EDTA buffer. After
washing, 700 mL of a-MEM supplemented medium
were added to each well and the plates were incubated
at 37 C and 5% CO2 overnight to swell. Afterwards,
the excess of culture medium was removed and 100 mL
of a suspension of 3.0  105 cells/mL were inoculated
into each well. After 2 to 3 h in the incubator, 900 mL
of a-MEM supplemented medium were added and the
samples were maintained in the incubator for up to
168 h, with exchange of the culture medium every
48 h. As a control, the adhesion of cells to the 24-well
plate without the membranes was also monitored.
During this period, samples were collected to determine
cell adhesion kinetics and the concentrations of glucose,
lactate, glutamine, and ammonium in the culture
medium and of cells grown in the biomaterials, as
well as MSCs morphology on the scaffolds as described
below. All experiments were performed in at least
triplicate.
Kinetics of cell adhesion. Cell adhesion was determined by
monitoring the decrease in the number of cells sus-
pended in the culture medium after inoculation.
Samples from the supernatant were collected at every
hour during 6 h and analyzed for cell concentration and
viability using the Vi-Cell XR analyzer Beckman
Coulter (Brea, CA, USA).
Concentration of glucose, lactate, glutamine, and
ammonium. Samples of culture medium supernatant
were collected every 24 h to determine the basic
Values
Dense Ch-Xn Porous Ch-Xn
membrane membrane
0.10  0.04 1.84  0.23
85.6  3.7 29.8  1.8
15.8  0.1 18.0  0.3
14.0  0.9 16.9  0.4
10.3  1.4 34.7  7.5
25.1  7.8 1.25  0.2
2.0  0.7 2.1  0.6
1.6  0.7 2.6  0.4
1158
metabolic activities of MSCs in the biomaterial.
Glucose, lactate, and glutamine were determined
using an automatic analyzer (YSI 2700, Yellow
Springs Instruments, OH) and ammonium was deter-
mined using an ion-selective electrode (Thermo
Scientific, ORION 720A).
Concentration of cells adhered to the scaffolds. The concen-
tration of viable cells was determined indirectly by the
MTT method (3-(4,5-dimethylthiazol-2-yil)-2,5-diphe-
nyltetrazolium bromide) (Sigma-Aldrich, St. Louis,
USA). The membrane samples were removed from
the plate, washed once with PBS/EDTA buffer, and
incubated with 200 mL of a-MEM without phenol red
and 20 mL of MTT reagent (5 mg/mL in PBS) at 37 C
for 4 h. The formazan crystals formed were dissolved
with 200 mL of 0.1 N HCl in anhydrous isopropanol
and the absorbance of the supernatant was measured
at 570 nm in a plate reader (Multiskan FC Thermo
Scientific) and compared to a calibration curve previ-
ously established. Three independent samples were ana-
lyzed daily and subsequently discarded.
Morphology of cells adhered to the scaffolds. The morph-
ology of cells attached to the biomaterial was analyzed
by scanning electron microscopy (SEM, model JSM
5800 LV, JEOL) by immersing the samples into different
fixative solutions, one consisting of 4% paraformalde-
hyde (v/v) and glutaraldehyde 2% (m/v) in 0.2 M caco-
dylate buffer and the other of osmium tetroxide (1% m/
v) in cacodylate buffer at 0.2 M, all from Sigma
Chemical Co, Brazil. Afterwards, the samples were
sequentially dehydrated in alcoholic solutions of differ-
ent concentrations and dried through the critical point
drying procedure with CO2. The samples were then
metal-coated through depositing a thin gold layer (92 Å).
In vivo wound healing assay
Wistar rats were anaesthetized intraperitoneally with 1 mg
of anesthetic solution per gram of animal. The anesthetic
solution consisted of a 1:1 (v/v) mixture of ketamine solu-
tion at 10% (Agener Union, Brazil) and xylazine at
20 mg/mL (Dopaser-Calier, Brazil). After the anesthesia,
the back of the animals was shaved and two circular
wounds of 1.5 cm in diameter were performed with sur-
gical scissors in previously marked areas. All layers of the
skin, including the panniculus carnosus, were removed
with the aid of the scissors, ensuring the production of
lesions of about the same thickness in all animals. After
15 min of completion, the animals still anesthetized were
divided into four groups of at least three rats each and
subjected to the different treatments tested.
In the control group (Ctrl), the wounds were not
covered by any dressing. In the second group (Memb),
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Journal of Biomaterials Applications 29(8)
the wounds were treated with dense membranes of
1.5 cm in diameter previously hydrated with a-MEM
culture medium supplemented with 15% fetal bovine
serum, but with no cells inoculated on the surface. In
the third group (MSCs), 1  105 MSCs were directly
applied at the edges of the wound, and no coverage
with Ch:Xn membrane was provided. Depositing the
cells in the incision edges facilitated their attachment
to the lesion and prevented their displacement to
regions away from the lesion during the early stages
of cell adhesion. In the last group (Memb þ MSCs),
the wounds were dressed with the dense scaffold
inoculated with the MSCs 96 h before the in vivo test
as previously described. The blood exuded from the
lesions aided in the adhesion of the membrane to the
wound bed, avoiding its displacement thereafter. No
secondary dressings were used as additional protection
of the tested treatments and as a preventive measure
against the occurrence of trauma injuries, individual
cages relatively large in size (49  34  16 cm3) were
used to accommodate the animals during the healing
period.
Photos were taken (Fuji S2000HD FilmFinepix digi-
tal camera, resolution of 10.0 megapixels) and wound
healing percentage was analyzed immediately after
the start of the experiment (day 0) and 20 days after-
wards. The wound healing percentage was calculated
by the ratio between the final and the initial area of
the wound through the Image JÕ software. Statistical
analyses were based on one-way ANOVA and the
Tukey’s test to determine the differences among the
experimental groups. Differences were considered
statistically significant when the p value was lower
than 5% (p < 0.05). These tests were performed using
the software PRISM 5.0.
Results
Mutagenicity of the Ch-Xn membranes
The dense and porous Ch-Xn membranes tested
herein were extensively characterized referring to sev-
eral relevant physico-chemical properties in a previous
work,12 showing a set of features to a large extent
adequate for their potential application in the tissue
engineering area (Table 1), however, their mutageni-
city potential was not assessed at that time. Hence,
their potential to cause mutations was analyzed in
the present work. It was observed (Table 2) that
after 72 h of incubation of the membranes with
TA98, TA100, TA102, TA1535, and TA1537 strains
of Salmonella typhimurium in the absence and presence
of metabolic activation, none of the samples showed
mutagenic characteristics.
Bellini et al. 1159

Table 2. Results of the AMES mutagenicity test performed on the dense and porous Ch-Xn membranes.

Salmonella typhimurium

Sample description TA98 TA100 TA102 TA1535 TA1537

In the absence of metabolic activation

Negative controla () () () () ()
Positive controlb (þ) (þ) (þ) (þ) (þ)
Dense Ch-Xn membrane () () () () ()
Porous Ch-Xn membrane () () () () ()
In the presence of metabolic activation
Negative controla () () () () ()
Positive controlc (þ) (þ) (þ) (þ) (þ)
Dense Ch-Xn membrane () () () () ()
Porous Ch-Xn membrane () () () () ()
Ch: chitosan; Xn: xanthan; (þ): mutagenic; (): not mutagenic.
a
Deionized water.
b
5.0 mg per dish of sodium azide.
c
2.5 mg/dish of 2-aminoantracene.

Behavior of MSCs inoculated on the Ch:Xn
membranes
All experiments involving MSCs were performed with
cultures established from the third or fourth passage,
with CD45, CD11b, and CD31 expression levels lower
than 5.5% and high expression of CD29 and CD44
surface markers, as characterized by flow cytometry
(Table 3).
The adhesion of MSCs to the materials tested, in
terms of the gradual reduction in the number of cells
in suspension, is shown in Figure 1. More than 50% of
the total initial population had already adhered to the
materials 2 h after MSCs inoculation in all tested sys-
tems, however, the cells adhered more rapidly to the
surface of the control culture plate than to the mem-
branes (Figure 1a). Three hours after the inoculation,
the number of cells in the supernatant of the control
culture plate and of the vessels containing dense mem-
brane samples was similar and more than 98% of the
cells inoculated was capable of adhering to the
membranes.
As shown in Figure 1(b), the viability of the MSCs
in the supernatant of all systems decreased with
time, indicating that cells that were unable to adhere
to the materials tested were not able to survive in
suspension.
Growth and metabolism of MSCs in the membranes. MSCs
were inoculated at an initial concentration of 3.0  104
cells per well on the surface of each membrane sample.
Intense cell growth was noticed for up to 96 h of culti-
vation and cell concentration reached maximum values
of 1.1  105 and 1.27  105 cells/per membrane for
Table 3. Expression of surface antigen markers by mesenchy-
mal stromal cells (MSCs) on 3rd passage.
Surface antigen Mean expression value (%)
CD45 5.5  4.7
CD11b 4.8  1.8
CD31 2.5  1.8
CD29 92.4  1.2
CD44 85.8  8.6
dense and porous Ch:Xn membrane samples, respect-
ively (Figure 2). Afterwards, cell death occurred.
The concentrations of glucose, lactate, glutamine,
and ammonium in the culture media were monitored
over 168 h (Figure 3). Due to the frequent exchange of
culture medium, depletion of glucose and glutamine
was not observed. Lactic acid was produced as a bypro-
duct of the glucose metabolism, reaching maximum
values of 3.86 and 7.78 mM in porous and dense mem-
branes, respectively. The maximum accumulation of
ammonium attained was 0.64 mM in the supernatant
of the porous membranes and equal to 0.51 mM for
the dense Ch-Xn membranes.
Morphology of MSCs. The morphological aspect of the
cells cultured on the surface of the tested materials
was examined by SEM and it is shown in Figure 4.
MSCs were distributed over the surface of both formu-
lations studied (Figure 4e and f). Cells cultured in the
porous sample proliferated intensely within the pores
(Figure 4f), in which it is possible to observe the pres-
ence of cytoplasmic extensions as well as the production
of extracellular matrix. The dense membranes Ch-Xn

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1160 Journal of Biomaterials Applications 29(8)

(a) (b)
1.2x10 5 100
Cell population in the supernatant
Porous Ch-Xn Membrane Porous Ch-Xn Membrane
Dense Ch-Xn Membrane Dense Ch-Xn Membrane
5
1.0x10 Control - plate Control - plate
80
4
8.0x10

Viability (%)
60
6.0x10 4

40
4.0x10 4

2.0x10 4 20

0.0
0
0 1 2 3 4 5 0 1 2 3 4 5
Time (hours) Time (hours)

Figure 1. Adhesion of MSCs to control polystyrene culture plates and to dense and porous Ch:Xn membranes: (a) cell population
remaining in the supernatant; (b) percentage of viable cells during culture. Data are reported as means  SD for experiments in
triplicate.

marrow cells were extracted because those animals

2.0x105 were sacrificed prior to extraction.
Dense Ch-Xn Membrane
Viable cell density (cells/ membrane)

Porous Ch-Xn Membrane
1.5x105
1.0x105
5.0x104
0.0
0 24
Figure 2. MSCs proliferation on dense and porous Ch-Xn
membranes during cultivation for 7 days in supplemented a-MEM
medium, assessed through the use of the MTT assay. Data are
reported as means  SD for experiments in triplicate.
remained transparent even after inoculation and culti-
vation of cells and this is a characteristic of great
importance, since it would allow the effective observa-
tion of the wound bed during the lesion recovery
process.
In vivo healing assays
The in vivo wound healing assays were performed using
Wistar rats subjected to surgical wounds aiming to
assess the applicability of the tested approach upon
the principles of translational medicine using allogeneic
MSCs, given that it was not possible to use in this part
of the study the same animals from which the bone
The dense Ch-Xn membrane was selected over the
porous biomaterial due to the lower thickness and the
higher transparency of the former. These characteristics
might contribute positively to aspects related to
improved comfort for the patient with the thinner dres-
sing and also of better visual monitoring of lesion
healing.
The usual aspect of the lesions before treatment, the
percentage of wound healing 20 days after the begin-
ning of the treatment, and the typical aspect of the
48 72 96 120 144 168 192 wounds of animals representative from each group
Time (hours)
are respectively shown in Figure 5(a–c).
It was observed that healing progressed well in all
animals and that on day 20 after the beginning of the
treatment all animals were already in the final process
of reepithelialization, even those which did not receive
membranes or MSCs.
The group treated with the membrane containing
adhered MSCs (Memb þ MSCs) showed healing of
wounds significantly higher (96.49  1.62%) when com-
pared to the Ctrl group (90.01  2.38%, p ¼ 0.03). The
difference was also significant when compared to the
group treated with the membrane without cells
(Memb), which reached 87.24  3.10% (p ¼ 0.02).
Statistically significant difference was not noticed
when comparing the percentage of healing in groups
Memb þ MSCs and MSCs (animals with lesions that
received only MSCs and reached 99.27  0.73% of
healing). Consequently, the percentage of wound heal-
ing in group MSCs was significantly higher than that of
the Ctrl group and of the Memb group that received the
acellular biomembrane (p ¼ 0.003 and p ¼ 0.001,
respectively).

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Bellini et al. 1161

(a) 10 (b) 5
Glucose Glutamine
Lactate Ammonium
8 4
Concentration (mM)

Concentration (mM)
6 3

4 2

2 1

0 0
0 24 48 72 96 120 144 168 192 0 24 48 72 96 120 144 168 192
Time (hours) Time (hours)

(c) 10 (d) 5
Glucose Glutamine
Lactate Ammonium

8 4

Concentration (mM)
Concentration (mM)

6 3

4 2

2 1

0 0
0 24 48 72 96 120 144 168 192 0 24 48 72 96 120 144 168 192
Time (hours) Time (hours)

Figure 3. Variation in the concentrations of glucose, glutamine, lactate, and ammonium during MSCs cell cultivation on: (a and b)
dense Ch-Xn membranes; (a, c and d) porous Ch-Xn membranes. Dashed lines refer to the time points of culture medium changes.

agents in vivo could lead to consequences as serious as

Discussion
Over the last years tissue engineering has advanced sig-
nificantly, especially in the dermatology field. One of
the trends in this field is the use of biomaterials pro-
duced by the combination of inert and low-complexity
materials with cells that satisfactorily meet require-
ments of biosafety and are active on the healing of
the type of wound focused. In this sense, the develop-
ment of a dermal biodressing through the association of
Ch-Xn membranes which were already characterized in
a previous study12 with multipotent MSCs, a widely
studied cell type especially interesting for regenerative
medicine and tissue engineering, was investigated
in vitro and in vivo on Wistar rats. The results obtained
indicate that the formulations tested present suitable
properties aiming application as scaffolds for cell
growth.
None of the formulations tested were mutagenic.
This property is of particular importance in the devel-
opment of biomaterials, since the release of genotoxic
cancer induction, even when negative results are
obtained in in vitro cytocompatibility analysis.23 The
negative mutagenic activity results attained evidenced
then that compounds with potential to alter cell growth
were not released.
Once the safety of use of the biomaterials has been
established, assays involving the association of the
membranes with MSCs were performed. Despite sup-
posedly higher area for cell accommodation would be
available in the porous biomaterial than in the dense
membranes, the cells cultivated in the biomaterials pre-
sented similar behavior in both membrane formulations
studied. High population increase was noticed in the
first 96 h of culture, followed by marked decline in
viable cells in the subsequent hours. The decrease in
cell viability observed after 96 h cannot be attributed
to the exhaustion of the substrates glucose and glutam-
ine, since they were replenished during culture medium
exchange, or to the accumulation of toxic byproducts
such as lactic acid and ammonium, which reached

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1162 Journal of Biomaterials Applications 29(8)

Figure 4. Scanning electron micrographs of scaffolds. Dense Ch-Xn membranes: (a) before MSCs inoculation; (b) 24 h after cell
inoculation; (c) 96 h after cell inoculation. Porous Ch-Xn membranes: (d) before MSCs inoculation; (e) 24 h after cell inoculation;
(f) 96 h after cell inoculation.

concentrations of only 3.86 and 0.64 mM, respectively,
for the Ch-Xn porous membranes, and 7.78 and
0.51 mM, respectively, for the Ch-Xn dense mem-
branes. Since lactic acid concentrations over 28.0 mM
and ammonium concentrations over 3.0 mM are con-
sidered inhibitory for MSCs,24 and those high concen-
trations were not observed in the supernatant culture
medium, the decrease in the number of viable cells in
the biomaterials evaluated might be attributed to mass
transfer issues related both to limited diffusion of nutri-
ents (even of oxygen) to the membranes and of toxic
metabolites to the medium outside the scaffolds
(including carbon dioxide produced during cell respir-
ation), causing then fast depletion and accumulation
respectively of nutrients and metabolites in the inner
parts of the scaffolds. Given that the thickness of the
porous membranes is significantly higher than that of
the dense biomaterials, more severe limitation to cell
growth in the inner parts of the scaffolds would be
expected for the porous scaffolds. However, the
growth curves for both types of membranes are very
similar regarding cell population in both systems in
all periods, then the hypotheses pointing to more
severe problems in the porous systems alone cannot
fully satisfactorily explain the results obtained.
Another possible explanation for the decrease in cell
population in the dense and porous scaffolds could be
the degradation of the polymer matrix during cell
growth. According to Bellini et al.,12 Ch-Xn dense
and porous membranes exhibit weight loss between 17
and 34% after 144 h of exposure to a sterile culture
medium traditionally used for mammalian cell in vitro
(RPMI supplemented with 10% fetal bovine serum).
The presence of cells in the scaffold could have further

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Bellini et al. 1163

(a) #
#
*
(b) 100 *

Wound healing (%)

75

Ctrl Memb MSCs Memb+MSCs

(c)

0 days 7 days 15 days 20 days

Ctrl
Memb
MSCs
Memb+
MSCs

Figure 5. Wound healing progression in animals of the different groups: control group (Ctrl); animals with lesions treated with only
the dense membrane (Memb); animals with wounds treated only with the mesenchymal stromal cells (MSCs); and rats treated with the
dense membrane inoculated with cells (Memb þ MSCs). (a) Usual aspect of the wound in day 0; (b) Percentage of wound healing in
animals of each study group 20 days after the beginning of the treatment (values determined by photographic analysis with the
software Image JÕ and statistical analysis performed using One-Way ANOVA, PRISMA 5.0: *groups with statistical difference from the
Ctrl group; #groups with statistical difference from the Memb group); and (c) Typical aspect of the lesions of animals representative of
each study group 20 days after the beginning of the treatment.

accelerated the degradation process. Nevertheless, bio-
degradation may be seen as a positive factor for a bio-
material intended to the application as a bioactive
dermal healing device, since in vivo it would facilitate
the delivery of bioactive agents incorporated therein, in
the present case, the cells cultivated on it, to the deepest
layers of the damaged tissue.
The morphological analysis of the cells grown in the
membranes indicated that the MSCs adhered
satisfactorily to both membrane formulations.
Evidence of extracellular matrix formation was also
noticed. Similar successful results in the culture of
MSCs in scaffolds based on Ch have also been reported
by Ragetly et al.25,26 The production of extracellular
matrix is important to provide additional physical sup-
port for growing cells and is also relevant in processes
of tissue morphogenesis, differentiation, and homeosta-
sis.27 According to Yang,28 scaffolds containing Ch

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1164
may stimulate the production of extracellular matrix.
Due to its charge distribution at physiological condi-
tions, Ch can increase, by adsorption, the local concen-
tration of important negatively charged molecules such
as glycosaminoglycans (GAG) and proteoglycans,
which are essential components of the extracellular
matrix.
Morphological differences were observed when com-
paring MSCs cultured on the Ch-Xn scaffolds to the
cells cultivated on the polysterene plate (data not
shown). This result was expected, given that MSCs
behave differently when grown on scaffolds with differ-
ent stiffness.29 As observed by Engler et al.,29 soft
matrices mimicking the brain are neurogenic, while stif-
fer matrices mimicking muscles are myogenic, and
matrices with higher rigidity may be osteogenic
and there is evidence that Ch scaffolds have physical
and mechanical characteristics effectively useful for the
cultivation of cells such as human dermal fibroblasts
for dermal regeneration.30
The results obtained when assessing the adhesion
and proliferation of MSCs on both dense and porous
formulations showed that the two membrane types are
suitable for application in tissue engineering as sup-
ports for cell culture. Lower thickness and higher trans-
parency before and after cell inoculation though
pointed that the dense formulation might be more
appropriate for application in vivo as a bioactive
dermal dressing. In addition, the processing time to
obtain porous membranes is significantly higher when
compared to that of dense films.
Highly satisfactory results were observed in the
Memb þ MSCs group treated with the biodressing
combining the dense membrane with the MSCs. The
cells were able to remain viable and active in the mem-
brane, showing promising therapeutic perspectives.
Despite the cells in suspension also showing very effect-
ive healing performance (MSCs group), their applica-
tion to the wound site is not as straightforward as that
of the biodressing. The transference of the cells already
adhered to the membrane surface is much simpler and,
in addition, the membrane provides greater mechanical
protection to the wound and to the MSCs themselves.
Another advantage is that the membrane can function
as a physical barrier, preventing microbial contamin-
ation from the outside environment.
Local application of Ch:Xn acellular biomembrane
on the bed ulcers apparently did not interfere in the
healing process in comparison to the untreated animals
(Ctrl group), suggesting that the improved therapeutic
potential in groups MSCs and Memb þ MSCs may be
attributed to the MSCs themselves. Despite the fact
that the use of the membrane to cover the wound
implies in an additional barrier to gas permeation
from and to the wound and that membranes composed
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Journal of Biomaterials Applications 29(8)
solely of Ch or of Ch complexed with alginate have low
permeability to oxygen (unpublished results), that
might not be the case for the Ch:Xn membrane, since
no significant difference was observed when comparing
Ctrl and Memb groups. These results confirm that the
membrane is a biocompatible biomaterial and appar-
ently inert in this microenvironment, representing a
good alternative for the administration of these cells
and consisting in a non-invasive and faster alternative
for the treatment of skin wounds. Furthermore, the fact
that the membranes need to be applied to the surface of
the lesions only in the beginning of the treatment is
quite attractive in the clinical point of view, and given
that the produced dressings are very transparent, their
periodic removal to inspect the progress of the lesion is
not required.
Several works demonstrate the therapeutic potential
of local application of MSCs in the healing of skin
wounds. Most of them use topical or intravenous
application of MSCs,31–34 whereas others use fibrin
sealant for the fixation of the cells to the
wound bed.35,36 Effective results were also reported
by Liu et al.37 when using allogeneic MSCs in a
collagen scaffold for the reepithelialization of superfi-
cial dermal burns in mini pigs. Despite successful out-
come is attained with fibrin or collagen-based
biomaterials, less limitations might be involved with
the use of Ch and xanthan membranes as MSCs sup-
ports for the treatment of skin wounds, since these
polysaccharides have lower probability of transference
of adventitious agents than proteins from mammalian
sources. Also, less potential to cause immunogenic
problems might be associated to Ch:Xn scaffolds
than with supports obtained from proteins of heterol-
ogous sources.
Keratinocytes may be also used for skin lesion regen-
eration, but MSCs cells have a significant advantage
over these cells. MSCs may be used in allogeneic sys-
tems, given that they are not too immunogenic, present-
ing low MHC-I expression and absence of expression of
MHC-II and of costimulatory molecules.38,39 Since
relatively long periods of culture are required for both
keratinocytes and MSCs, the former type of cells could
be successfully used for the treatment of chronic ulcers,
being isolated from the patient himself, while MSCs
previously cultured and stored in banks might be
more useful in the therapy of acute injuries, such as
severe burns, that require immediate treatment and
for which limited availability of autologous cells
might be a concern.
Conclusions
In this work, the association of dense and porous mem-
branes of Ch-Xn with mesenchymal stromal cells was
Bellini et al.
tested to verify their applicability as bioactive dermal
healing dressings. The results showed that the mem-
branes are not mutagenic and present favorable archi-
tecture for adhesion, maintenance, and proliferation of
MSCs. In vivo assays performed with the non-porous
membranes containing MSCs on the surface showed sig-
nificant acceleration of dermo-epidermal wound heal-
ing, leading to the conclusion that this membrane-cells
association might be considered viable in tissue engin-
eering and should be considered for further studies.
Acknowledgements
The authors are grateful to the São Paulo Research
Foundation (FAPESP, Brazil) for the financial support to
this research. MZ Bellini would also like to acknowledge
the Coordination for the Improvement of Higher Education
Personnel (CAPES, Brazil) for a PhD fellowship and ÂM
Moraes acknowledges the National Council for Scientific
and Technological Development (CNPq, Brazil) for a fellow-
ship. Thanks are also due to the assistance of Alessandra
Almeida for the English review and of Sandra Navarro
Bresciani for the graphic design.
Declaration of conflicting interests
None declared.
Funding
This research received grants from São Paulo Research
Foundation (FAPESP, Brazil), the Coordination for the
Improvement of Higher Education Personnel (CAPES,
Brazil) and from the National Council for Scientificand
Technological Development (CNPq, Brazil).
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