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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: ec_andronescu@yahoo.com

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. 2 127

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 com-posite matrix. The pores formed possibly due to sublimation of ice

inside the composite, and they could help to form a high-water-content wound dressing. On the other hand, the COLL/DEX/SiO2 (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 colla-gen 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.

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

Fig. (2). SEM micrographs of COLL/DEX/SiO2 (CL): a,b; and (NCL): c,d;

128 Current Organic Chemistry, 2013, Vol. 17, No. 2 Grumezescu et al.

Fig. (3). SEM micrographs of COLL/DEAC/SiO2 (CL): a,b; and (NCL): c,d.

Fig. (4). Biocompatibility evaluation of COLL/DEAEC/SiO2 (a,b) and COLL/DEX/SiO2 (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 struc-ture was apparent in the 10-20 m size range. Interconnected po-rous 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 fabri-cated biocomposites showed that cell morphology and also retain their viability (Fig. 4).

(a) (b)

(c) (d)

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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/SiO2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO2/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 dis-tributed 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 po-lymeric 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

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/SiO2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO2/UA (NCL); (e) Coll/DEAEC/SiO2/UA (NCL).

130 Current Organic Chemistry, 2013, Vol. 17, No. 2 Grumezescu et al.

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 expres-sion 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 improve-ment 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 struc-tural and functional properties (bacterial killing, biofilm inhibition and disruption, lack of cytotoxicity) that recommend them for fur-ther 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 Hu-man Resources 135/2010 grant (Contract no. 76/29.07.2010).

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/SiO2/UA (NCL); (c) UA; (d) Coll/DEAEC/SiO2/UA (NCL); (e) Coll/DEAEC/SiO2/UA (NCL).

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Received: July 12, 2012 Revised: October 14, 2012 Accepted: October 20, 2012

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