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Silver nanoparticle/bacterial cellulose gel membranes for antibacterial wound dressing: investigation in vitro and in vivo

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Biomedical Materials Biomed. Mater. 9 (2014) 035005 (12pp)

doi:10.1088/1748-6041/9/3/035005

Silver nanoparticle/bacterial cellulose gel membranes for antibacterial wound dressing: investigation in vitro and in vivo Jian Wu 1 , Yudong Zheng 1,4 , Xiaoxiao Wen 1 , Qinghua Lin 1 , Xiaohua Chen 2 and Zhigu Wu 3 1 School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, People’s Republic of China 2 State Key Laboratory of Advanced Metals and Materials, University of Science and Technology Beijing, Beijing 100083, People’s Republic of China 3 Hospital affiliated to Chinese PLA General Hospital, Beijing 100853, People’s Republic of China

E-mail: [email protected] Received 9 September 2013, revised 2 March 2013 Accepted for publication 12 March 2014 Published 16 April 2014 Abstract

Bacterial cellulose (BC) has attracted increasing attention as a novel wound dressing material, but its antimicrobial activity, which is one of the critical skin-barrier functions in wound healing, is not sufficient for use in practical applications. To overcome such a deficiency, silver nanoparticles were generated and self-assembled on the surface of BC nanofibers, forming a stable and evenly distributed Ag nanoparticle coated BC nanofiber (AgNP-BC). The performance of AgNP-BC was systematically studied in terms of antibacterial activities, cytocompatibility and effects on wound healing. The results showed that AgNP-BC exhibited significant antibacterial activity against Staphylococcus aureus. Moreover, AgNP-BC allowed attachment, and growth of rat fibroblasts with low cytotoxicity emerged. Based on these advantages, AgNP-BC samples were applied in a second-degree rat wound model. Wound flora showed a significant reduction during the healing. The fresh epidermal and dermis thicknesses with AgNP-BC samples were 111 and 855 μm respectively, higher than 74 and 619 μm for BC groups and 57 and 473 μm for untreated control wounds. The results demonstrated that AgNP-BC could reduce inflammation and promote scald wound healing. Keywords: bacterial cellulose, silver nanoparticles, antimicrobial activities, biocompatibility, second-degree wound (Some figures may appear in colour only in the online journal)

wounds [4] and burns [5]. In the processing of skin tissue repair, the moisture environment provided by the dressing has been shown to promote ulcer healing and to reduce pain of patients [6]. The intrinsic properties of BC make it an attractive, novel wound dressing material. However, the lack of antimicrobial activity of BC is the main issue to be tackled, which is critical to provide a bacteria-proof barrier to the outside world in wound healing [7]. To solve this problem, studies have introduced different materials such as chitosan [9], benzalkonium chloride [8], silver sulfadiazine [10] and metallic nanoparticles into BC.

1. Introduction Bacterial cellulose (BC), a natural biopolymer synthesized in abundance by different strains of bacteria, namely, Acetobacter strains [1], displays high water content, high wet strength and chemical purity. Due to its special properties, there is increasing research interest in developing a wide range of biomedical applications, such as artificial blood vessels [2], tissue-engineering scaffolds [3] and wound dressings for 4

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Biomed. Mater. 9 (2014) 035005

Among them, metallic nanoparticles have been recently reported as excellent antimicrobial agents [11–13], owing to their nanoscale structure and large surface area to volume ratio compared to their bulk form. In particular, silver nanoparticles (AgNPs) have been intensively studied due to their strong antimicrobial activities toward many different bacteria, fungi and viruses [14–17]. However, the aggregation tendency of AgNPs can undermine their unique properties at nanoscales. One strategy to prevent aggregation is the controlled deposition of metal particles through hybridization by using a nanoporous material as a template to ensure a well-defined spatial distribution [18]. Sheet-shaped BC supports can make it easy to incorporate nanosized metallic particles [19]. Researchers have proposed several methods to obtain the AgNP impregnated BC composites through triethanolamine (TEA) [20]; NaBH4 and UV radiation [21]; hydrazine, hydroxylamine or ascorbic acid [22]; self-polymerized polydopamine [23]; and TEMPOmediated oxidized BC followed by thermal reduction [19]. For impregnation, most of these studies utilized the electrostatic interactions between metallic ions and dipole moments of cellulose molecules [21, 23–26]. However, the anchor metallic ions on cellulose fibers generated through these methods are normally weak [22], resulting in a low yield and sparse distribution of metallic nanoparticles [19]. It has been reported that silver is a recognized cause of argyrosis and argyria [27, 28] and a high concentration of silver nanoparticles is toxic to mammalian cells [29]. Thus, the weak interaction between AgNPs and BC may cause cell toxicity due to the instability and uncontrollable release of AgNPs, which has not been tackled up to now. Besides, there are no systematic studies on the effects of these compounds on antiinfection and concrescence in vitro and in vivo wound healing tests. In this work, we put forward a new method of producing silver nanoparticle/bacterial cellulose (AgNP-BC) hybrid gel membranes, aiming to improve the antimicrobial activity of bacterial cellulose. Silver nanoparticles were synthesized in situ through the reaction of Tollens’ reagent with bacterial cellulose. Such a reaction formed a stable and strong interaction between silver nanoparticles and BC fibers. The structure of AgNP-BC was investigated by XRD and SEM. The antibacterial activities of AgNP-BC against Staphylococcus aureus were demonstrated using the disc diffusion test method. Effects of AgNP-BC on cell growth were investigated through cultivation of fibroblasts in vitro. Finally, effects of AgNP-BC on anti-infection and concrescence in vivo were investigated in a second-degree (deep partial thickness) rat wound model.

ethylene tetrazolium bromide (3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H, MTT), and trypsin were offered by Merck. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibcobrl. All other reagents and solvents were purchased from domestic suppliers and used as received. 2.2. In situ formation of silver nanoparticles into BC

The bacterial cellulose membranes were washed with deionized water and then immersed in 0.1 N NaOH solution at 100 ◦ C for 1 h to remove impurities such as medium, endotoxin, etc on the membranes. Finally, samples were rinsed with deionized water to pH = 7 and stored in deionized water at room temperature prior to further experimentation. Silver ammonia solution (Tollens’ reagent, 0.01 mol L−1) was prepared. The purified BC membranes were soaked in silver ammonia solution for 24 h and then thoroughly washed to remove residual chemicals on the surface. Subsequently, BC membranes were kept in a warm water bath at 80 ◦ C for 10 min, and after that the resulting membranes were washed with running deionized water to remove the residual chemicals. 2.3. Characterization 2.3.1. Contents of Ag. AgNP-BC hybrid gel membranes were dissolved in 1 mL concentrated sulfuric acid. The amount of Ag in the acquired solution was determined by flame atomic absorption spectrophotometry (FAAS; AA300, Perkin Elmer, USA).

X-ray diffraction (XRD) tests of pure BC and AgNP-BC (all samples were freeze-dried under −40 to −50 ◦ C) were performed using a Rigaku D/max-RB x-ray diffractometer (Japan) with a thin pellicle attachment. The samples were placed in the sample holder and scanned at a rate of 9◦ /min from 10◦ to 85◦ (2θ ).

2.3.2. X-ray diffraction.

Samples of BC and AgNP-BC were freeze-dried (−40 to −50 ◦ C) to constant weight before being examined using scanning electron microscopy (SEM). For improved contrast, the specimens were spattered with a thin layer of evaporated gold. The morphology of BC and AgNP-BC was observed by an Apollo 300 scanning electron microscope (USA) equipped with an electron optical system with a 10 keV capacity electron gun and an electron detector.

2.3.3. Scanning electron microscopy.

2.4. Release of silver in vitro

The kinetics of silver release was studied from the prepared BC samples. AgNP-BC membranes were cut into round pieces with a diameter of 10 mm. These samples were then immersed in 10 mL phosphate buffered saline (PBS, pH = 6.0) at 37 ◦ C. A 0.2 mL solution was taken at regular time intervals (0.5, 1, 2, 3, 6, 12, 24, 48 and 72 h) and analyzed for the amount of Ag released using FAAS. After removing the aliquots of 0.2 mL for each sample for analysis, the same volume of fresh phosphate buffered saline solution was added.

2. Materials and methods 2.1. Materials

Bacterial cellulose was offered by Hainan Yida Food Co. Ltd (China). Silver nitrate (AgNO3) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. Diamine tetraacetic acid (EDTA), 2

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Figure 1. The process of experiment in vivo: (A) anesthetization; (B) shaving and disinfection; (C) preparing the deep partial thickness skin wounds; (D) photographing wound; (E) covering with AgNP-BC; (F) fixing with elastic adhesive bandage; (G) histology HE of deep partial thickness skin wounds.

added to dissolve the formazan formed previously. Solubilized formazan products were quantified by spectrophotometry at 570 nm using an ELx800 Universal Microplate Reader (BioTEK instruments, Inc., USA). Cells cultured with medium alone were used as control and the cell viability was considered to be 100%.

2.5. Antimicrobial activity studies

The antimicrobial activity of the composite membranes was investigated against S. aureus (ATCC26085, purchased from Beijing Land Bridge Technology CO., Ltd). In the disc diffusion method (ISO 20645:2004), the test specimen and pure BC (control samples) were cut into a disc shape with 6.5 mm diameter, sterilized by autoclaving for 20 min at 121 ◦ C, and in the process, all samples were put in vacuumsealed bags to avoid oxidization. Before culturing in an incubator at 37 ◦ C for 24 h, samples were pressed into intimate contact with an agar culture medium inoculated with the S. aureus solution. Subsequently, the zone of bacterial inhibition was monitored.

2.7. In vivo wound healing

The wound healing efficacy of the in situ synthetic bacterial cellulose containing silver nanoparticles was evaluated using a rat model, and we have complied with institutional ethical use protocols. The process of the experiment is shown in figure 1. Wistar rats (half male and half female) weighing approximately 250 g were anesthetized by intraperitoneal injection of pentobarbital sodium, at a dose of 40 and 5 mg per kilogram body weight (figure 1(A)). The skin of the animal was shaved and disinfected using 70% ethanol (figure 1(B)). After disinfection, the surgical site was prepared for aseptic surgery. Two second-degree (deep partial thickness) skin wounds (figure 1(G)) of 4 cm2 area (20 mm × 20 mm) were prepared by burning the dorsum of each animal at 80 ◦ C with a self-made scald apparatus (figure 1(C)). A photograph of the wound was taken with a sterile ruler along wound to measure the wound area (figure 1(D)). The test wounds (n > 8) were then covered with AgNP-BC (S1) gel membranes (figure 1(E)), fixed with an elastic adhesive bandage (figure 1(F)). Another test group of wounds (n > 8) was covered with pure BC membranes and elastic adhesive bandages in the same way. Control wounds (n > 8) were just covered with gauze without any other treatment, as an untreated control. After the experiment, animals were kept in separate cages and fed with commercial rat feed and water ad libitum until the sacrifice, which was performed using an excess dose of sodium pentobarbital on day 7, 10, 21 and 28 after surgery. The wounds were grossly examined and photographed to measure the wound size reduction. For histology, the skin, including the entire wound with adjacent normal skin, was excised and fixed in 10% buffered formalin. The specimen included the dermis and subcutaneous tissue.

2.6. Assay of biocompatibility

Westar fetal rat epidermal cells (Experimental Animal Center of Military Medical Sciences, China) were incubated in 2 mL of DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO). Samples were cut into 15 mm diameter to fit 24 well cell culture polystyrene plates. Before usage, the membranes were sterilized by γ -irradiation. The samples were placed in 24 well cell culture polystyrene plates, seeded with 1 mL Westar fetal rat epidermal cells (1 × 105 per mL) per disk and cultured in 5% CO2 at 37 ◦ C for 1, 4 and 7 days. The culture medium was changed on the fourth day. To observe cell morphology and distribution, rat fibroblasts cultured for 1, 4 and 7 days were observed respectively using a Leica DMI 4000 semi-automatic inverted biological microscope (Germany). Culture medium was replaced with a 0.5 mL blended solution of 0.25% trypsin and 0.02% EDTA. Ten minutes later, 0.5 mL fetal calf serumDMEM was added to terminate the digestion. Cell viability was tested by MTT assays on 1, 4 and 7 days, respectively. Briefly, 100 μL of MTT (3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H tetrazolium bromide, 5 mg mL−1) was added into each well followed by 4 h incubation at 37 ◦ C in 5% CO2 incubator. Subsequently, the medium was removed carefully and 500 μL of DMSO (dimethyl sulfoxide) was 3

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Figure 2. Scanning electron microscopic morphology of (A) BC, (B) AgNP-BC (5000X), and (C) AgNP-BC (20000X); the insert shows the size distribution of silver particles.

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Figure 3. XRD patterns of (A) bacterial cellulose and (B) AgNP-BC

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composite materials.

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Time (h) The wound size measurements taken at the time of surgery and biopsy were used to calculate the reduction percentage in wound size using the equation

Figure 4. Release quantity changes of silver ion in PBS solution; the

inside curve is log(release Ag+) versus log t curve.

Wound Size Reduction (%) = (A0 − At )/A0 × 100%,

Masson’s trichrome to visualize collagen fibers in the wound bed.

where A0 and At are the initial wound area and wound area after time interval ‘t’. Area was measured from the photographs of the wounds using Image-Pro Plus 6.0 after calibration. Samples for culture were obtained from the skin surfaces (0.25 cm2) of rats by applying a sterile filter paper and swabbing for 30 s, rotating clockwise three times in the wound bed, at 4, 7, 10, 14 and 21 days after the procedure. Swab samples were placed in a tube containing 1 mL of sterile saline, vibrated thoroughly, serially diluted, and plated onto agar plates. The number of index microorganisms per swab was estimated. Excised wound sites fixed in formalin were processed and embedded in paraffin with 3–5 mm thick sections stained with hematoxylin and eosin. The percentage of wound reepithelialization was determined by using image analysis software (Image-Pro Plus 6.0). The distance from the right wound margin to the left wound margin was measured. The length of newly generated epithelium across the surface of the wound was determined as the sum of the new epidermis growing from right and left margins of the wound. This length was expressed as a percentage of the entire wound length. Representative sections of each wound were also stained with

2.8. Statistical analysis

Statistical analysis of the data was performed using one-way analysis of variance (ANOVA), assuming a confidence level of 95% (p < 0.05) for statistical significance. All the data were expressed as means ± standard deviation (SD). 3. Results and discussion 3.1. Synthesis of AgNP-BC composite membrane

The guest molecules of Tollens’ reagent diffused into the inner spaces of BC nanopores and reacted with cellulose fibers, and BC played the role of reactive template at nanoscale during the formation of silver nanoparticles. Purified BC membranes were initially soaked in Tollens’ reagent for 24 h, resulting in the attachment of Ag(NH3)2+ to BC fibers by electrostatic adsorption or hydrogen bond. Subsequently, BC membranes were kept in a warm water bath at 80 ◦ C for 10 min. BC could react with Ag(NH3)2OH to form more Ag (as shown 4

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Diamter of inhibition ring / mm

Figure 5. The growth inhibition ring of bacterial cellulose, gauze, positive control and AgNP-BC against S. aureus.

particle diameter of more than 90% of silver nanoparticles generated in BC nanofibers was in the range of 10 to 100 nm (figure 2(C)).

15 14 13 12 11 10 9 8 7 6 5

3.3. Crystal structure of the AgNP-BC composite membrane

Gauze

Positive control

BC

An x-ray diffractogram of BC showed three characteristic peaks (figure 3(A)) located at 14.60◦ , 16.82◦ and 22.78◦ , corresponding to (1 1 0), (1 1¯ 0) and (2 0 0) crystal planes. According to Bragg’s law, the identity distances of each crystal plane are 0.606, 0.527 and 0.390 nm, demonstrating the existence of a typical cellulose I crystal [30]. The degree of crystallinity of BC membranes calculated by jade6.0 software is 85.77%. An x-ray diffractogram of AgNP-BC also shows the same characteristic peaks of cellulose I crystal (figure 3(B)). Crystallinity of BC in AgNP-BC is about 83.68%, and thus there is no obvious difference in crystallinity of pure BC and AgNP-BC membranes. Five sharper characteristic peaks could be seen in figure 3(B), at wider Bragg angles of 38.1◦ , 44.2◦ , 64.2◦ , 77.5◦ and 81.5◦ , which respectively corresponded to (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) crystal planes based on the face-centered cubic (fcc) structure of silver. These results further proved that metallic crystalline silver was produced in the BC network.

AgNP-BC

Figure 6. Diameter of inhibition ring. significance (p < 0.05): •,

greater than gauze; , greater than BC; , greater than positive control.

in formula (1)). Silver nanoparticles (AgNPs) were gradually generated and closely deposited on BC nanofibers, forming a stable and uniform Ag nanoparticle/BC hybrid, which is proven by the following observation using SEM (figure 2): OH

BC

CH 2

+ 4Ag(NH 3)2OH

BC

O C OH

3.4. Ag+ releasing properties of the AgNP-BC composite membrane

+ 4Ag + 8NH 3 + 3H 2O

The silver content of AgNP-BC was about 2.62 wt%, which was determined by FAAS; Ag+ releasing profiles of AgNP-BC in PBS solution are presented in figure 4. The released Ag+(t) ∝ tn (as shown in the inside curve of figure 4), the power law exponent, n, the slope of a linear fitting between log (Ag+) and log t are indicative of different transport mechanisms. The power law exponent for the Ag+ release of AgNP-BCs is 0.703, with an R2 fitting coefficient of 99.6%, indicating the high static correlation between the exponential power law and experimental measurements. Such release power laws partly follow a t1/2 time dependence of physical diffusion-controlled drug transport of a polymer thin film based on the Higuchi

3.2. Morphology of BC and the AgNP-BC composite membrane

Figure 2 shows SEM micrographs of the morphology of BC and AgNP-BC. BC nanofibers form an interconnective porous structure (figure 2(A)); its three-dimensional (3D) network allowed guest molecules to diffuse throughout its inner space easily. As shown in figure 2(B), the hybrid nanostructure of the resulting AgNP-BC consisted of stable, silver nanoparticles evenly distributed and anchored on a BC nanofiber 3D network. The granularity analysis results showed that the 5

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Figure 7. Epidermal cell morphologies of the BC group on days 1 (A), 4 (B) and 7 (C), and epidermal cell morphologies of AgNP-BC group on days 1 (D), 4 (E) and 7 (F).

equation or pure Fick’s second diffusion law in a thin polymer film [31, 32]. The Ag+ release of AgNP-BC in PBS solution was slowed down significantly with a gradual increase of release concentration of Ag+ over time, which means Ag+ was released in a controllable way.

market products as a positive control group; it developed an inhibition ring zone against S. aureus with about 2.03 mm in diameter (figure 6). The inhibition zone of AgNP-BC was greater than that of the positive control, which demonstrated that the antibacterial activity of AgNP-BC is not less than the wound dressing in the current market products.

3.5. Antimicrobial effects of AgNP-BC

The antibacterial activities of AgNP-BC membranes against S. aureus, which is a common bacteria of wound infections, were measured using the disc diffusion method after a 24 h incubation at 37 ◦ C (figure 5). It was found that Ag nanoparticle impregnated BC composites developed an inhibition ring zone against S. aureus of about 3.46 mm in diameter (figure 6). No inhibition zone was observed for the pure BC membrane and gauze as control (figure 5(A)). This clearly demonstrates that the antimicrobial activity of AgNP-BC is only attributed to the presence of Ag nanoparticles impregnated into BC rather than BC itself. We chose a wound dressing from the current

3.6. Cytotoxicity of AgNP-BC for rat fibroblasts

A number of nanomaterials are reported to be potentially harmful due to their influence on organisms at the level of cells, subcellular fractions and proteins [33]. The cell toxicity is of crucial concern due to the large surface areas of nanomaterials [34]. The strong adhesion between Ag and BC nanofibers observed above is promising for preventing Ag nanoparticles from leaching from the BC network and entering organisms, which is imperative for the application of AgNPBC in a biomedical field. The biocompatibility of AgNP-BC 6

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Absorbency MTT Colorimetry

Biomed. Mater. 9 (2014) 035005

3.7. In vivo wound healing

0.6

Control BC AgNP-BC

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The wound healing effects of BC and AgNP-BC used as wound dressing for experimental rats with the second-degree (deep partial thickness) wound model were investigated and compared.

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3.7.1. Gross examination.

0.5

Generally, each wound (both test and control) was photographically observed for time periods of 4, 7, 10, 14 and 21 days post-treatment (figure 9). On the fourth day, there was no evidence of infection or contraction of the wound, while skin was hemorrhagic for some control samples and a scab was present on the wound bed. During the whole wound healing, the high-water-containing hydrogel structure of BC could form an occlusive and a semi-occlusive moist environment on the wound bed, and AgNP-BC could provide both a moist and an extra-aseptic environment. It has been reported that the epithelialization was retarded by the dry scab. Winter showed that epithelialization was accelerated if the wound was kept moist [35]. One explanation for this was that keratinocytes migrated more easily over a moist wound surface than underneath a scab [36]. Rat fibroblasts can migrate at a speed of about 0.5 mm/day over a moist wound surface, which is twice as fast as under a scab in dry wounds. After seven days, there was severe inflammation in the wounds of the untreated group, and a large amount of wound fluid was observed in figure 9(B1), while wounds of the AgNP-BC groups converted to local crusta, and the margins of the skin defects contracted. Meanwhile, there are no obvious scabs on the wounds of the BC groups. Subcutaneous aspects appeared grossly normal for AgNP-BC and BC group wounds on the tenth day. Scabs grew in the wound of the untreated group, and AgNP-BC groups began to scab and a small area of scabs fell off from the wounds. After 14 days, most of the scabs fell off from the wounds, and AgNP-BC groups even started to grow hair on the wounds. At the same time, BC groups were fully covered with scabs, and the wound of the untreated group did not scab completely. On the 21st day, a majority of the AgNPBC groups appeared to be healed, but a small area of the scabs did not fall off from the wounds of BC groups, and the wound of the untreated group still had mild swelling and redness.

0.2 0.1 0.0

4day 1day Time of the Co-culture

7day

Figure 8. MTT results of rat fibroblasts co-cultivated with different samples for 1, 4 and 7 days. Significance (p < 0.05): ∗ greater than C0.

was evaluated by co-culturing with rat fibroblasts of neonatal rats on days 1, 4 and 7. Cell proliferations and morphologic changes are shown in figure 7, where the orbicular transparent small cells observed under an optical microscope are rat fibroblasts. After culturing for one day, rat fibroblasts were sparsely spread more or less everywhere with some noticeable dense cell domains. Most rat fibroblasts appeared in spindle shape. There were no significant differences between groups. On the fourth day, the number of cells increased obviously with more spindle and larger flat epidermal cells produced, demonstrating that epidermal cell proliferation had begun. Cells continued the growth in well-spread ways on the seventh day, and the cells were intensive. A confluence of epidermal cells occurred on the seventh day, and there were no significant differences among the groups. The observation above showed that there were no significant differences between pure BC and AgNP-BC, indicating that AgNP-BC possessed good biocompatibility, similar to BC. The MTT data describe the relative viability of rat fibroblasts growing on different samples. The results analyzed in figure 8 are comparable since the same number of cells was added to all samples. The formazan absorbance indicates that the rat fibroblasts seeded onto the control sample (the polystyrene cell culture plate) and different membranes were able to convert MTT into a blue formazan product. Though the BC sample exhibited slightly higher MTT absorbance results than the AgNP-BC samples, there were no obvious differences between BC and AgNP-BC. Furthermore, with increasing culture time, the viability of cells on each sample increased. Therefore, it can be concluded that the addition of the AgNPs to BC did not greatly affect the cytocompatibility of the membrane. The in vitro experimental results demonstrated that AgNPBC samples had relatively low cytotoxicity, which is an essential characteristic for biomaterials as wound dressing. The positive effects of AgNP-BC on cells in vitro encouraged us to carry on the following investigation in vivo.

The reduction in wound defect area was estimated by measuring the wound area before and after definite intervals of time (figure 9(F)); the data show the proportion of the healing rate. After four days, there was no significant reduction in the wound defect area for both tests and control. About 36.28% of the wound defect area of the AgNP-BC groups healed on the seventh day, whereas for BC groups this was only 21.67%, and 11.53% for the untreated group. However, statistical analysis revealed that this difference was not significant (p > 0.05). On the tenth day, the wound defect filled up to about 62.13% for the AgNPBC groups, but only 42.82% for the BC groups and 27.94% for the untreated group. Statistical analysis revealed that this difference became significant (p < 0.05). After 14 days, the wound defect filled up to about 85.92% for the AgNP-BC groups, whereas for the BC groups this was only 60.77%, and

3.7.2. Wound size reduction.

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Figure 9. Representative photographs of the macroscopic appearance of a 20 mm × 20 mm wound excised on a rat; control wounds of the untreated group on the 4th (A1), 7th (B1), 10th (C1), 14th (D1) and 21st (E1) day; test wounds of BC group on the 4th (A2), 7th (B2), 10th (C2), 14th (D2) and 21st (E2) day; and test wounds of AgNP-BC group on the 4th (A3), 7th (B3), 10th (C3), 14th (D3) and 21st (E3) day. The wound healing rate (F) and bacteria of wound surface (G) post-wounding are also shown.

43.39% for the untreated group. The healing area continued to grow with 99.3% recovered in the test wounds of the AgNPBC groups on the 21st day, whereas for the BC groups this was about 82.37%, and 68.36% for the untreated group, which was statistically significant (p < 0.05). After 28 days, healing was complete for wounds that received AgNP-BC, leading to about 100% fill in of the wound defect; for the BC groups this was about 96.42%, and only 81.58% for the untreated group. These results further quantitatively demonstrate that AgNPBC membranes could accelerate the healing process in the second-degree rat wound model.

A bacterial infection can retard the healing process of the wound. To study AgNP-BC’s inhibition on bacteria proliferation, the amount of bacteria on the wound surface was examined by the method described in section 2.5. Results showed that AgNP-BC could effectively control the infection of common bacteria and drugresistance bacteria on the wound surface (figure 9(G)). The bacteria of the wound surface were observed for periods of 1, 4, 7, 10, 14 and 21 days post-treatment. Results show that in all untreated, AgNP-BC and BC groups, the bacteria of the wound surface grew obviously over 4–14 days after surgery.

3.7.3. Bacteria reduction on the wound surface.

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Figure 10. Histology of control wound sections (untreated) stained with hematoxylin and eosin at 7 days (A1), 14 days (B1), 21 days (C1)

and 28 days (D1). Histology of test wound sections (covered with BC) stained with hematoxylin and eosin at 7 days (A2), 14 days (B2), 21 days (C2) and 28 days (D2). Histology of test wound sections (covered with AgNP-BC) stained with hematoxylin and eosin at 7 days (A3), 14 days (B3), 21 days (C3) and 28 days (D3).

only 40.00 × 103 CFU cm−2. After that, we saw a gradual decline in the number of bacteria on the wound surface. On the seventh day, the bacteria reduced to 85.00 × 103 CFU cm−2 on the wound surface of the BC groups, 132.49 × 103 CFU cm−2 on the wound surface of the untreated groups, and only 37.75 × 103 CFU cm−2 for the AgNP-BC groups. From 10 to 14 days, the decline of bacteria on the wound surface of the BC groups slowed down and the number of bacteria leveled off with small variation from 73.88 × 103 to 75.88 × 103 CFU cm−2. The number of bacteria on the wound surface of the untreated groups showed a small reduction from 86.17 × 103 to 82.60 × 103 CFU cm−2. In contrast, the

Notably, the bacteria of the wound surface of the BC groups are two to four times higher than those of the AgNP-BC groups, and there was more bacteria on the wound surface of untreated groups than that of the BC groups. Statistical analysis revealed that this difference was significant (p < 0.05). On the first day there was no significant difference of bacteria on the wound surface for both the AgNP-BC and BC groups, while there was 37.24 × 103 CFU cm−2 on the wound surface of the untreated groups. After four days, the presence of bacteria achieved the highest level with 128.13 × 103 CFU cm−2 on the wound surface of the BC groups, and 161.48 × 103 CFU cm−2 on the wound surface of the untreated groups, which is more than 3–4 times higher than the AgNP-BC groups with 9

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(D3)

Figure 11. Collagen content was visualized using Masson’s trichrome staining. Masson’s trichrome staining of control wound sections

(untreated) shown at 7 days (A1), 14 days (B1), 21 days (C1) and 28 days (D1). Masson’s trichrome staining of test wound sections (covered with BC) shown at 7 days (A2), 14 days (B2), 21 days (C2) and 28 days (D2). Masson’s trichrome staining of test wound sections (covered with AgNP-BC) shown at 7 days (A3), 14 days (B3), 21 days (C3) and 28 days (D3).

bacteria from the AgNP-BC groups continued to slide down with a reduction from 20.88 × 103 to 17.63 × 103 CFU cm−2. Bacteria of the wound surface were restored to the level of pre-operation 21–28 days later. These data indicate that AgNP-BC could effectively decrease the bacteria proliferation on the wound surface and provide a better local environment for scald wound healing, contributing to the acceleration of the wound healing process.

tests and untreated control samples at 7, 14, 21 and 28 days after wounding. On the seventh day after wounding, histology analysis revealed that the wounds of the untreated groups had moderate necrosis with inflammatory infiltration (figure 10(A1)), the wounds of the BC groups had moderate necrosis with slight inflammatory infiltration (figure 10(A2)), and the wounds of the AgNP-BC groups had slight necrosis with no inflammatory infiltration (figure 10(A3)). Fragments of dressing began a foreign body response. An area of coagulative necrosis and integration appeared in the epidermis and some parts of dermal tissues. Meanwhile, the hair follicle structure was destroyed,

The healing pattern of wounds was studied by examining the histology of the two

3.7.4. Histological examination.

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Table 1. The fresh epidermal and dermis thickness (x¯ ± s, μm).

Healthy rats [37] Control BC AgNP-BC

Fresh epidermal thickness

Fresh dermis thickness

Whole epiderm: 31.2–61.4 μm 57 ± 21 μm 74 ± 30 μm 111 ± 43 μm

Whole skin: 2.04–2.80 mm 473 ± 284 μm 619 ± 306 μm 855 ± 103 μm

and hair follicles twisted out of shape; nuclei of epithelial cells from hair follicles were cracked or dissolution disappeared; sebaceous glands disappeared; nuclei of fibroblasts were in a bent position; collagen fibers solidified into conglomerate; and its eosinophilic stain declined. In addition, considerable infiltration of neutrophil cells was seen in the BC groups. The main features of the wounds after 14 days were the formation of granulation tissue with a unique loose structure, which consisted of a large number of collagen tissues; neovascularization; and fibroblasts (figures 10(B1)–(B3)). Deposition of collagen could be seen in the granulation tissue, and the collagen was irregular and in disorder (figures 11(B1)– (B3)). Infiltration of neutrophil cells showed up in the untreated and BC groups (figures 10(B1) and (B2)). The epidermal tissue appeared to extend into the wound in the AgNP-BC groups (figure 10(B3)), while no epidermal tissue was seen in the untreated and BC groups. On the 21st day, collagen in granulation tissue became shorter and thinner, containing neovascularization and a large number of fibroblasts (figures 11(C1)–(C3)). The epidermal tissue slowly extended into the wounds of the BC groups (figure 10(C2)), and still no epidermal tissue was observed in the wounds of the untreated groups (figure 10(C1)). The wound skin of the AgNP-BC groups was already fully covered with epidermal tissue, and some parts in the wound healed (figure 10(C3)). After 28 days, the wound of the AgNP-BC groups was completely healed with a few shallow wrinkles of the new epidermis and no epidermal keratinization found in the epidermal tissue (figure 9(D3)). In comparison, the wound skin of the BC groups was covered with a thin layer of epidermal tissue and some parts of the wound had also healed (figure 10(D2)); moreover, the epidermal tissue had started to slowly extend into the wound of the untreated groups (figure 10(C1)). Collagen in new dermis tissues of both test and control wounds was dense, and there were no appendages of skin such as hair follicles or glandula subarea in the dermis tissues. By measuring the wound area before and after definite intervals of time, the thickness of fresh epidermal and dermis was calculated (table 1). The fresh epidermal average thickness for the AgNP-BC group was 111 μm, while only about 74 μm for the BC group and 57 μm for untreated group. The fresh dermis average thickness for the AgNP-BC group was 855 μm, while for control wound the value was only about 619 μm and for untreated group was 284 μm. Robert et al [37] measured the thickness of healthy rat skin (as shown in table 1); the whole skin of healthy rats is about 2.04–2.80 mm, and the whole epiderm of healthy rats is about 31.2–61.4 μm. It was clear that fresh epidermal of all three groups was thicker than that

of healthy rats, and potentially, parts of the fresh epidermal would transform into cuticle. Furthermore, the fresh dermis of three experimental groups was thinner than that of healthy rats, and the dermis will continue growing with the collagen deposition until it recovers completely. In summary, the in vivo results of gross examination, wound size reduction, bacteria reduction and histological examination show that AgNP-BC gel membranes can promote wound healing and reduce bacteria proliferation in the wounds of rats. 4. Conclusions To conclude, in situ synthesis and deposition of silver nanoparticles into BC fibrous membrane have been successfully developed through the reaction of Tollens’ reagent under ambient conditions. BC nanofibers and their interconnective nanoporous structure played the roles of reactive template and confined nanoreactor during the reaction. The resulting silver nanoparticles (AgNP) were evenly distributed in the network of BC gel membranes, forming a robust hybrid nanostructure with tailorable Ag loading. AgNP-BC exhibited significant antibacterial activity against S. aureus. AgNP-BC allows attachment and growth of rat fibroblasts with low cytotoxicity to emerge in culture with rat fibroblasts. The in vivo study demonstrated excellent healing effects of AgNP-BC in a second-degree rat wound model. The regenerated fresh epidermal and dermis thickness under the AgNP-BC gel membrane were 111 and 855 μm, respectively, while for BC wounds the values were only about 74 and 619 μm, and for the untreated group were 57 and 473 μm. In addition, AgNP-BC could effectively decrease the bacteria on the wound surface and provide a better local environment for scald wound healing. In short, systematic in vitro and in vivo results manifest that the nanostructural AgNP-BC gel membrane is promising for antimicrobial wound dressing with good biocompatibility to promote scald wound healing. Acknowledgments This study is financially supported by the National Natural Science Foundation of China Project (grant no. 51073024 and grant no. 51273021) and the National Science and Technology Support Project of China (grant no. 2011BAK15B04). References [1] Brown A J 1886 XIX.—The chemical action of pure cultivations of bacterium aceti J. Chem. Soc. Trans. 49 172–87 11

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