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Current Organic Chemistry, 2013, 17, 125-131

125

Wound Dressing Based Collagen Biomaterials Containing Usnic Acid as Quorum


Sensing Inhibitor Agent: Synthesis, Characterization and Bioevaluation
Alexandru Mihai Grumezescu1, Ecaterina Andronescu 1*, Madalina Georgiana Albu2, Anton Ficai1, Coralia
Bleotu3,4, Denisa Dragu3 and Veronica Lazar4
1

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

INCDTP Leather & Footwear Res Inst, Collagen Dept, Bucharest 031215, Romania

Stefan Nicolau Institute of Virology, 285 Mihai Bravu Avenue, 030304, Bucharest, Romania

Department 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 mesenchymal 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 (diseases). 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 produce greater expansion of surface area from donor skin than conventional methods [4]. In both clinical and preclinical models of
skin substitutes, collagen is the most commonly used scaffold material [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 environmental pathogens in the body, however, its protective mechanisms
become compromised on creation of a wound, allowing for exposure 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 destructive 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 appreciated [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: ec_andronescu@yahoo.com

1385-2728/13 $58.00+.00

rounded by a glycocalyx composed of a combination of an extracellular matrix that is produced by the microorganisms and the host
surrounding tissues [14]. The glycocalix contributes to the enhanced resistance of microorganisms to the host response as well as
to various antibiotic treatments [15, 16].
Polysaccharide-based hydrogels are non-cytotoxic and biodegradable [17]. From a structural point of view, polysaccharides
have reactive functional groups that can be modi ed to form hydrogels with speci c characteristics of interest [18]. Dextran is a hydrophilic 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 encapsulates drugs at high concentrations assures the afore mentioned properties [29]. A surface enriched in silica in the presence of surface
Si-OH groups provides intrinsic hydrophilicity, thus allowing surface 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,
2013 Bentham Science Publishers

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 highlighting 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 nanostructured biomaterials [35].
In this context, the aim of this paper is to fabricate a new biomaterial 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.

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 positive/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).

MATERIALS AND METHODS

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 diacetate (10 g/mL). The stained specimens have been examined in fluorescent 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.

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, Polysaccharides 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 minutes. 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). Obtained 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, Polysaccharides and Silica
FT-IR. A Nicolet 6700 FT-IR spectrometer (Thermo Nicolet,
Madison, WI) connected to software of the OMNIC operating system (Version 7.0 Thermo Nicolet) was used to obtain FT-IR spectra
of hybrid materials. The samples were placed in contact with attenuated 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 coadding 32 scans and at a resolution of 4 cm-1 with strong apodization. All spectra were ratioed against a background of an air spectrum.
SEM. SEM analysis was performed on a HITACHI S2600N
electron microscope, at 15 and 25 keV, in primary electrons fascicle, on samples covered with a thin silver layer.
Isolation, Culture and Characterization of Human Bone Marrow 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, density 1.077 g/mL). The cells from inner (containing mononuclear

Assessment of the Obtained Materials Biocompatibility

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 loadings 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 bacterial 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 collagen (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 component, con rming the active participation of water in the collagen
molecule. The amide B band is observed at around 30503180 cm1
, 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 connected 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

127

Fig. (1). FT-IR spectra of fabricated wound dressings.

Fig. (2). SEM micrographs of COLL/DEX/SiO 2 (CL): a,b; and (NCL): c,d;

group and the peak at 961 cm -1 is assigned to the SiOH functional


group [44].
The inner micro-structure of the lyophilized COLL/DEX/SiO2
wound dressing was analyzed by scanning electron microscopy
(SEM), and the micrographs were shown in Fig. (2). The wound
dressing present an interconnected porous structure, many irregular
pores with size from of several micros could be found in the composite matrix. The pores formed possibly due to sublimation of ice

inside the composite, and they could help to form a high-watercontent wound dressing. On the other hand, the COLL/DEX/SiO 2
(CL) showed morphology of continuous polymer matrix compared
to COLL/DEX/SiO2 (NCL) composite which displayed an
unconsolidated and fragile pattern. The reason might be that collagen fibers could enhance the formability of the composite. Some
lamentous fibers even could be seen in the SEM micrographs.
There were large numbers of SiO2 microcrystals on the wound
dressing surface.

128 Current Organic Chemistry, 2013, Vol. 17, No. 2

Grumezescu et al.

Fig. (3). SEM micrographs of COLL/DEAC/SiO 2 (CL): a,b; and (NCL): c,d.

(a)

(c)

(b)

(d)

Fig. (4). Biocompatibility evaluation of COLL/DEAEC/SiO 2 (a,b) and COLL/DEX/SiO 2 (c,d) with mesenchymal stem cell (100X: a,c; 200X: b,d)

The morphology of COLL/DEAC/SiO2 wound dressing, CL


and NCL, was investigated using SEM (Fig. 3). The COLL/DEAC/
SiO2 (NCL) exhibited a rough, brous surface due to the underlying
collagen structure together with DEAEC. An irregular pore structure was apparent in the 10-20 m size range. Interconnected porous network structures were found within the macropores of the
wound dressing, while very few micropores were seen on the wall

of the wound dressing. In comparison, the COLL/DEAC/SiO2 (CL)


displayed a homogenous microstructure, with bers organized in
layers, with a moderate orientation.
Analysis of mesenchymal stem cells grown 24 hours in fabricated biocomposites showed that cell morphology and also retain
their viability (Fig. 4).

Wound Dressing Based Collagen Biomaterials Containing Usnic Acid

Current Organic Chemistry, 2013, Vol. 17, No. 2

129

Fig. (5). The aspect of bacterial growth inhibition zones after the removal of the obtained specimens (T0): (a) Coll/DEX/SiO2 /UA (CL); (b)
Coll/DEX/SiO 2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO2 /UA (NCL); (e) Coll/DEAEC/SiO2 /UA (NCL).

Fig. (6). The aspect of bacterial growth inhibition zones after the removal of
the obtained specimens (T1): (a) Coll/DEX/SiO2/UA (CL); (b)
Coll/DEX/SiO 2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO 2/UA (NCL); (e)
Coll/DEAEC/SiO2 /UA (NCL);

Concerning the influence of the tested materials on the viability


of S. aureus cultures, a strong microbicidal effect was noticed for
all working variants. At T0, the number of the bacterial cells distributed on the surface of the culture medium was reduced, this
density being comparable with the minimal infectious dose required
for an opportunistic microorganism to initiate an infectious process.
Our results are showing that the UA (usnic acid), responsible for
the antimicrobial activity was released in active form from the polymeric matrix exhibiting a microbicidal effect comparable to that
observed for the UA control solution (Fig. 5).
At T1, after six hours of incubation at 37 oC, taking into account
that the average time of a multiplication cycle in case of S. aureus
is about 40 minutes, the microbial density is reaching about 109
CFU/mL. Although the bacterial density is significantly higher, the
same microbicidal effect as for the T0 was noticed (Fig. 6).
An interesting result has been obtained at T2. In this case, the
bacterial density is very high, the bacterial culture forming a
confluent layer covering the surface of the culture medium. In this
case, the UA solution exhibited no visible inhibition of the
microbial growth, in exchange, in case of the tested systems
containing UA, the bacterial culture has been practically disolved,
probably due to the dispersing effect of UA on the bacterial dense
culture resembling a biofilm (Fig. 7).
This intense bactericidal activity was also noticed by other
authors. Francollini et al. (2004) showed that the relative proportion
of S. aureus live cells attached to polyurethane charged with UA

130 Current Organic Chemistry, 2013, Vol. 17, No. 2

Grumezescu et al.

Fig. (7). The aspect of bacterial growth inhibition zones after the removal of the obtained specimens (T2): (a) Coll/DEX/SiO2 /UA (CL); (b)
Coll/DEX/SiO 2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO2 /UA (NCL); (e) Coll/DEAEC/SiO2 /UA (NCL).

decreased from approximately 80% after 30 minutes to less than


1% after 24 hours [45]. The selective killing activity of UA on
Gram-positive microorganisms has been previously demonstrated
using dental plaque specimens, in which UA selectively inhibited
the biofilm development by Gram positive bacteria and the expression of haemolytic properties of strains isolated from the dental
plaque [46].
The absence of this effect in case of the UA solution could
suggest the efficiency of the polymeric mathrix in the controlled
release of UA in active forms. The proposed solution could also
provide a moisturized wound healing environment, with efficient
bactericidal action determining practically the lysis of bacterial
culture even at high density as in the case of biofilms developed on
tissues or medical devices and removing drainage liquid and debris.
Thus, an efficient bactericidal activity was noticed, irrespective
to the bacterial density and no significant differences for the
crosslinked versus not crosslinked wound dressings have been
noticed. The newly fabricated wound dressing offers the improvement of the UA release in active forms. The results show that the
fabricated wound dressing (Coll/DEX/SiO2/UA and Coll/DEAEC/
SiO2/UA) can be safely used as efficient wound dressing systems,
either for preventing of the microbial contamination of a wound or
for the local treatment of an infected wound.

CONCLUSION
Preparation and characterization of COLL/DEX/SiO2/UA and
COLL/DEAEC/SiO2/UA wound dressing including the morphology
and their in vitro biological efficacy are reported. The FT-IR, SEM,
interaction with mesenchymal stem cell and a novel protocol to
evaluate wound dressing, firstly reported in literature were used for
the characterization of fabricated wound dressings. The results
demonstrated that the newly obtained materials are exhibiting structural and functional properties (bacterial killing, biofilm inhibition
and disruption, lack of cytotoxicity) that recommend them for further and safe applications in the biomedical field, as efficient
wound dressing systems.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts
of interest.
ACKNOWLEDGEMENT
The results presented in this work were supported by the Human Resources 135/2010 grant (Contract no. 76/29.07.2010).

Wound Dressing Based Collagen Biomaterials Containing Usnic Acid


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Accepted: October 20, 2012