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Current Organic Chemistry, 2013, 17, 125-131 125
1385-2728/13 $58.00+.00 2013 Bentham Science Publishers
Wound Dressing Based Collagen Biomaterials Containing Usnic Acid
as Quorum
Sensing Inhibitor Agent: Synthesis, Characterization and
Bioevaluation
Alexandru Mihai Grumezescu1, Ecaterina Andronescu1*, Madalina
Georgiana Albu2, Anton Ficai1, Coralia Bleotu3,4, Denisa Dragu3 and
Veronica Lazar4
1Department of Science and Engineering of Oxidic Materials and
Nanomaterials, Faculty of Applied Chemistry and Materials
Science, University Politehnica of Bucharest, Polizu Street no
1-7, 011061, Bucharest, Romania 2INCDTP Leather & Footwear Res
Inst, Collagen Dept, Bucharest 031215, Romania
3Stefan Nicolau Institute of Virology, 285 Mihai Bravu Avenue,
030304, Bucharest, Romania
4Department of Microbiology, Faculty of Biology, Universtity of
Bucharest, Aleea Portocalelor no. 1-3, 060101, Bucharest,
Romania
Abstract: The aims of this research were to obtain improved
wound dressings based on collagen (COLL), polysaccharides (dextran=
DEX, diethylaminoethyl-cellulose= DEAEC), silica network and usnic
acid, as quorum sensing inhibitor. FT-IR, SEM, interaction with
eukaryotic cells and a novel protocol to evaluate the antimicrobial
activity of the new wound dressing, firstly reported in literature
were used for the characterization of fabricated wound dressings.
The obtained wound dressings are not cytotoxic, do not influences
the mes-enchymal stem and exhibit good anti-biofilm properties.
Taken together, these results are suggesting that the new systems
can be safely used for local applications on the lesional
tissues.
Keywords: Collagen biomaterial, Wound dressing, Usnic acid,
Anti-biofilm.
INTRODUCTION
The major causes of skin loss are burn injuries, long term
chronic wounds (e.g. venous, diabetic and pressure ulcers) trauma,
excisions of skin tumours or other dermatological conditions
(dis-eases). Rapid re-epithelialisation of a wound is essential to
confer protection on the underlying tissues and prevent uid loss
[1,2], infection development and homeostasis [3]. Tissue
engineering has been used to generate bioengineered substitutes for
skin which pro-duce greater expansion of surface area from donor
skin than con-ventional methods [4]. In both clinical and
preclinical models of skin substitutes, collagen is the most
commonly used scaffold mate-rial [5-7]. Collagen membrane has
excellent cell affinity and biocompatibility to regenerate tissues
[8]. However, membrane made from non-mineralized collagen is
normally weak in strength and is therefore difficult to manipulate.
Furthermore, the resorption rate is difficult to match with normal
tissue-healing process [9].
Human skin represents the largest barrier to outside
environ-mental pathogens in the body, however, its protective
mechanisms become compromised on creation of a wound, allowing for
expo-sure to a variety of bacterial microbiota. The moist,
nutritionally supportive microenvironment of the wound bed matrix
becomes an ideal setting for formation of bacterial bio lm,
creating a destruc-tive and sustainable interaction that impairs
host wound healing [10, 11].
Bacterial bio lms are a key factor whose importance to wound
chronicity and persistence has only recently become widely
appre-ciated [12, 13]. Within the bio lm, the microorganisms are
sur-
*Address correspondence to this author at the Department of
Science and Engineering of Oxidic Materials and Nanomaterials,
Faculty of Applied Chemistry and Materials Science, University
Politehnica of Bucharest, Polizu Street no 1-7, 011061, Bucharest,
Romania; Tel: +4021 402 38 30; Fax: + 4021 318 10 10; E-mail:
[email protected]
rounded by a glycocalyx composed of a combination of an
extracel-lular matrix that is produced by the microorganisms and
the host surrounding tissues [14]. The glycocalix contributes to
the en-hanced resistance of microorganisms to the host response as
well as to various antibiotic treatments [15, 16].
Polysaccharide-based hydrogels are non-cytotoxic and
biode-gradable [17]. From a structural point of view,
polysaccharides have reactive functional groups that can be modi ed
to form hydro-gels with speci c characteristics of interest [18].
Dextran is a hy-drophilic natural polysaccharide and has attracted
much attention for use in controlled drug-delivery system because
of its excellent hydrophilic nature and biocompatibility [19, 20].
Cellulose, is a highly interesting material due to its
renewability, low price, high availability, good mechanical
properties and has safe characters such as no taste and odorless,
biodegradability, insolubility in water and most organic solvents
[21-23]. Cellulose and its derivatives are regarded as one of the
most popular polymeric materials to prepare nanoparticles for drug
delivery systems [24, 25].
Silica has increasingly attracted interest due to their unique
properties and potential applications in biotechnology and
materials science [26]. Due to its excellent biocompatibility,
silica is also an ideal candidate for biomedical applications such
is the targeted drug release [27, 28]. The porosity of silica,
which efficiently encapsu-lates drugs at high concentrations
assures the afore mentioned prop-erties [29]. A surface enriched in
silica in the presence of surface Si-OH groups provides intrinsic
hydrophilicity, thus allowing sur-face attachment of specific
biomolecules and increasing target specificity [30-32].
Usnic acid, a yellowgreen cortical pigment, is a derivative of
dibenzofuran produced by several lichen species, as a product of
fungal secondary metabolism [33]. The antibacterial activity of
usnic acid was recognized early against a number of planktonic
Gram-positive bacteria, to which ndings of anticancer,
antiviral,
126 Current Organic Chemistry, 2013, Vol. 17, No. 2 Grumezescu
et al.
antioxidant, anti-in ammatory, and analgesic properties have
been more recently added [34]. Also, recent studies report
successful fabrication of nanofluid based magnetite and usnic acid
highlight-ing the potential use as controlled release vehicle of
the anti-biofilm agents, opening a new perspective for obtaining
new antimicrobial and anti-biofilm surfaces, based on hybrid
functionalized nanos-tructured biomaterials [35].
In this context, the aim of this paper is to fabricate a new
bio-material based on collagen, polysaccharides, silica and usnic
acid to be used for local applications on lesional tissues in order
both to assure the tissue healing and to prevent the bacterial
colonization and the occurrence of would infections.
MATERIALS AND METHODS
Materials
All chemicals used for the preparation of the compounds were of
reagent grade quality and were purchased from Sigma- Aldrich.
Collagen (300.000 Da; COLL) gel was obtained in the Leather and
Footwear Research Institute- Collagen Department starting from calf
hides by chemical and enzymatic extraction. The collagen gel
concentration was 2.54 % and pH=7 [36].
Fabrication of Wound Dressing Based Collagen, Polysaccha-rides
and Silica
Wound dressing based collagen, polysaccharides and silica was
prepared as follow: 50 mL of polymeric suspension (1,27% dextran
(DEX); 1,27 % diethylaminoethylcellulose (DEAEC)) is added onto the
collagen gel (50 mL 2,54 %) and let to interact for 30 min-utes.
Polysaccharides (DEX and DEAEC) and collagen were mixed in 0.5 M
acetic acid by stirring and homogenizing several times. Silica
network was obtained from Na2SiO3 solution (50 mL; 1.27 %) dropped
into COLL-DEX/DEAEC solutions until the gel pH=7. COLL/DEX/SiO2 and
COLL/DEAEC/SiO2 were divided in two halfs, one being cross-linked
(CL) with 0,5 % (w/v) glutaraldehyde solution [37] and the other
one being not cross-linked (NCL). Ob-tained CL and NCL gels were
casted into glass Petri dishes (12.5 cm in diameter; 20 mL) to be
lyophilized.
Characterization of Wound Dressing Based Collagen,
Polysac-charides and Silica
FT-IR. A Nicolet 6700 FT-IR spectrometer (Thermo Nicolet,
Madison, WI) connected to software of the OMNIC operating sys-tem
(Version 7.0 Thermo Nicolet) was used to obtain FT-IR spectra of
hybrid materials. The samples were placed in contact with
at-tenuated total reflectance (ATR) on a multibounce plate of ZnSe
crystal at controlled ambient temperature (25oC). FT-IR spectra
were collected in the frequency range of 4,000650 cm-1 by co-adding
32 scans and at a resolution of 4 cm-1 with strong apodiza-tion.
All spectra were ratioed against a background of an air
spec-trum.
SEM. SEM analysis was performed on a HITACHI S2600N electron
microscope, at 15 and 25 keV, in primary electrons fasci-cle, on
samples covered with a thin silver layer.
Isolation, Culture and Characterization of Human Bone Mar-row
Mesenchymal Stem Cells (MSCs)
Mesenchymal stem cells were isolated using Sirbu-Boeti method
[38] slightly modified. Briefly, MCSs were obtained by
centrifugation of bone marrow aspirate in Biocoll (Biochrom,
den-sity 1.077 g/mL). The cells from inner (containing
mononuclear
cells and mesenchymal stem cells) were cultivated in Alpha MEM
(Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal
calf serum (Sigma-Aldrich Corp., St. Louis, MO, USA) and bFGF 10
ng/mL (Sigma-Aldrich Corp). The peripheral blood mononuclear cells
were removed by changing the media after the first 24 hours. MSCs
were selected by adherence and purified after 3 successive
passages. The characterization of MSCs based on posi-tive/negative
stain of monoclonal antibodies specific for CD105, CD90, CD34, CD45
(BD Pharmingen, San Diego, CA, USA) was performed on an Beckman
Coulter Epics XL flow cytometer (Beckman Coulter Inc, CA, USA).
Assessment of the Obtained Materials Biocompatibility
Materials were placed in six-well plates and injected with 3 x
105 mesenchymal stem cells. Thereafter, 1 mL of alpha-DMEM
supplemented with 10% bovine calf serum has been added. At 24 hours
the effect of the tested materials has been evaluated after
staining with propidium iodide (10 g/mL) and fluorescein diace-tate
(10 g/mL). The stained specimens have been examined in fluo-rescent
microscopy and photographed both in visible and ultraviolet fields.
At least three separated fields have been photographed with a
magnification of 100x and 200x. Viable cells occurred in green,
while the dead cells were stained in red.
The in vitro Assessment of the Anti-biofilm Activity of the
Obtained Materials
S. aureus ATCC 25923 reference strain was used to create an
artificial biofilm [39]. In order to assesss the antibiofilm
activity of the usnic acid adsorbed on the bandages with collagen
biopolimers and amorphous mineral phase, three experimental
versions, noted T0, T1 and T2 have been tested to simulate
different microbial load-ings of the infection site. At T0 the
bandage is combined with usnic acid and placed on the solid culture
medium, immediately after seeding it with a microbial suspension of
1-3 x 108 CFU/mL density (corresponding to the 0.5 Mac Farland
standard) [40, 41]. At T1 the seeded plates were incubated for 6
hours at 37oC, to allow the bac-terial growth and multiplication,
simulating the multiplication in the conditions of the host body,
prior to the placement of the bandages specimens, then continuing
the incubation for 24 hours. At T2- the seeded plates were
incubated for 12 hours, the bacterial cultures reaching high
densities and developing a confluent culture on the culture medium
surface, the materials specimens being placed over the bacterial
culture and the incubation being continued for another 24
hours.
RESULTS AND DISCUSSIONS
Figure 1 presents the IR spectra of lyophilized cross-linked
col-lagen (COLL(CL)), COLL/polysaccharides/SiO2 cross-linked (CL)
and not cross-linked (NCL). The broad band at 3283 cm-1, amide A,
is due to the NH stretching vibration. It is also due to the OH
com-ponent, con rming the active participation of water in the
collagen molecule. The amide B band is observed at around 30503180
cm-1, with a maximum at 3063 cm1. This band also shifts to a lower
wave number and becomes less in intensity [42]. The amide I band
appears in the range 16001700 cm-1 with a maximum near 1631 cm-1.
It is produced mainly by the peptide bond C=O stretching vibration.
The amide II band with a maximum at 1542 cm-1 is con-nected with
CNH groups [43]. By analyzing the FT-IR spectrum of the
COLL/DEX/SiO2 and COLL/DEAEC/SiO2, the same stretching bands
characteristic for the silica pattern are observed. The peak at
1059 cm-1 signify the bending vibration of the SiO functional
Wound Dressing Based Collagen Biomaterials Containing Usnic Acid
Current Organic Chemistry, 2013, Vol. 17, No. 2 131
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Received: July 12, 2012 Revised: October 14, 2012 Accepted:
October 20, 2012
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