silk fibroin scaffolds in infected full-thickness burns.

Acta Biomaterialia xxx (2014) xxx–xxx

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Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns Yong Lan a,b, Weichang Li a,c, Yanpeng Jiao a,b, Rui Guo a,d,⇑, Yi Zhang a,d, Wei Xue a,d, Yuanming Zhang a,c,⇑ a

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, People’s Republic of China Department of Materials Science and Engineering, Jinan University, Guangzhou 510632, People’s Republic of China c Department of Chemistry, Jinan University, Guangzhou 510632, People’s Republic of China d Department of Biomedical Engineering, Jinan University, Guangzhou 510632, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 25 November 2013 Received in revised form 24 March 2014 Accepted 26 March 2014 Available online xxxx Keywords: Silk fibroin Gentamycin sulfate Gelatin microspheres Full-thickness burn infection Wound healing

a b s t r a c t Despite advances in burn treatment, burn infection remains a major cause of morbidity and mortality. In this study, an antibacterial silk fibroin (SF) scaffold for burn treatment was designed; gelatin microspheres (GMs) were impregnated with the antibiotic gentamycin sulfate (GS), and the GS-impregnated GMs were then embedded in a SF matrix to fabricate GS/GM/SF scaffolds. The developed GS/GM/SF scaffolds could serve as a dermal regeneration template in full-thickness burns. The average pore size and porosity of the GS/GM/SF scaffolds were 40–80 lm and 85%, respectively. Furthermore, the drug release rate of the scaffolds was significantly slower than that of either GS/GM or GS/SF scaffolds. And the composite scaffold exhibited stronger antimicrobial activities against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Hence, we evaluated the wound-healing effects and antibacterial properties of the GS/GM/SF scaffolds in a rat full-thickness burn infection model. Over 21 days, the GS/GM/SF scaffolds not only significantly reduced burn infection by P. aeruginosa but also accelerated the regeneration of the dermis and exhibited higher epithelialization rates than did GS/SF and SF scaffolds. Thus, GS/GM/SF scaffolds are potentially effective for treatment of full-thickness infected burns, and GS/GM/SF scaffolds are a promising therapeutic tool for severely burned patients. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Burn infection has been a constant threat to human health throughout history, because infection prevents and delays wound healing and can lead to death [1,2]. It is reported that 50–75% of the morbidity in burn patients is related to infection [3]. The most common pathogens responsible for serious infections in burn patients include Staphylococcus aureus, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) [4]. Complications associated with infected burns constitute serious problems in the care of severely burned patients [5]. Treatment of these infections is frequently accomplished by debridement to remove as much of the source of infection as possible [6]. However, full-thickness burns are particularly difficult to heal and pose a high risk of bacterial infection. Therefore, development of a dermal

⇑ Corresponding authors at: Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, People’s Republic of China. Tel./fax: +86 20 85222756. E-mail addresses: [email protected] (R. Guo), [email protected] (Y. Zhang).

regeneration template to reduce the incidence of burn infection and promote full-thickness burn healing is desired. Dermal regeneration templates for treatment of full-thickness burns should exhibit several characteristics, including excellent biocompatibility in terms of a lack of toxicity and immunogenicity, and a microstructure that promotes burn healing. Various materials have been used to fabricate dermal regeneration templates, such as collagen, chitosan, chondroitin sulfate and silk fibroin (SF) [7–10]. These scaffolds significantly improve skin recovery from full-thickness defects. In particular, SF, which is derived from the silkworm Bombyx mori, is a structural polymer possessing unique physical properties, including good biocompatibility, low immunity, non-toxicity and biodegradability [10,11]. Therefore, SF is currently being investigated for a number of biomedical applications [12]. For example, SF has been exploited as a biomaterial for in vitro cell culture and in vivo tissue engineering [13]. Furthermore, due to its peptide constituents, SF promotes the proliferation of human skin fibroblasts [14] and can be used as a wound dressing. Sugihara et al. examined the influence of silk film on full-thickness skin wounds; the silk film promoted greater skin regeneration than Alloask D, which is a commonly used dressing for burns and ulcers [15]. However, SF itself

http://dx.doi.org/10.1016/j.actbio.2014.03.029 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lan Y et al. Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.03.029

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Y. Lan et al. / Acta Biomaterialia xxx (2014) xxx–xxx

does not exhibit antimicrobial properties, which are crucial for the prevention of bacterial infection of burn wounds. To introduce antibacterial activity in a SF scaffold, we developed a composite SF scaffold using gelatin microspheres (GMs) loaded with vancomycin (Vm) as the antimicrobial agent [16]. The use of the GMs for Vm encapsulation improved the drug-release properties; GMs are widely accepted as efficient drug carriers for various administration routes, including nasal, gastrointestinal and rectal delivery [17]. The Vm-impregnated GM/SF (Vm/GM/SF) scaffold exhibited strong antimicrobial activity against S. aureus in vitro. However, Vm is active only against Gram-positive bacteria, such as S. aureus [18], which restricts the application of composite Vm/GM/SF scaffolds in burn treatment. Furthermore, the therapeutic efficacy of antibiotic-loaded GM/SF scaffolds in infected fullthickness burns has not been evaluated. Because of its broad activity against both Gram-negative and -positive bacteria, gentamycin sulfate (GS) has been used as a topical antibiotic in the treatment of superficial skin infections and sepsis [19]. In this study, we explored the applications of GS-impregnated GM/SF (GS/GM/SF) scaffolds for treatment of infected full-thickness burns. The morphology and properties of GMs, SF and GS/ GM/SF scaffolds were investigated, and the release behaviour of GS from GS/GMs, GS/SF scaffolds and GS/GM/SF scaffolds were evaluated. The antimicrobial activities of the SF scaffolds, GS/SF scaffolds and GS/GM/SF scaffolds were investigated using the Kirby–Bauer (KB) test. Next, we prepared a wound-infection model based on full-thickness burns in male Sprague–Dawley (SD) rats, in which the dorsal skin was artificially burned and infected with P. aeruginosa. Thereafter, the burns were treated with the different SF scaffolds. The dynamic wound healing of the full-thickness skin defects was then investigated. 2. Materials and methods

ing for 24 h to remove any remaining solvent. The GS/GM/SF scaffold was produced by mixing 1 ml of 4% (w/v) SF solution and 10 mg of GS/GMs, and then pouring the solution into a 24-well plate and freeze-drying for 24 h. The GS/SF scaffold was fabricated by mixing 1 ml of 4% (w/v) SF solution and 4 ll of GS solution (25 mg ml1) and then pouring the mixture into a 24-well plate and freeze-drying for 24 h. Pure SF scaffolds were also prepared. All SF-based scaffolds were immersed in 90% (v/v) methanol aqueous solution for 30 min to induce a structural transition that generated water-insoluble SF scaffolds [17,24]. This methanol treatment has little effect on GS, due to GS being insoluble in most organic liquids, including acetone and methanol [25]. 2.4. Swelling of the GMs Dried and wet (saturated with deionized (DI) water for 4 h at room temperature) microspheres were observed under a microscope (Axio Scope A1 FL; Carl Zeiss, Wetzlar, Germany). At least 100 of both dried and wet microspheres were viewed. Their diameter was measured, from which the volume of respective microspheres was calculated [21]. The swelling ratios were then calculated using the following formula:

Swelling ratio ¼

Volume of wet microsphere Volume of dried microsphere

2.5. Fourier transform infrared spectroscopy The infrared (IR) spectra of GMs, GS and GS/GMs were obtained using a Fourier transform infrared (FTIR) spectrometer (Vertex 70; Bruker, Billerica, MA). The IR spectra in the absorbance mode were recorded using a diamond crystal plate and obtained in the spectral region 400–4000 cm1 with a resolution of 4 cm1 and 20 scans per sample.

2.1. Materials 2.6. Scanning electron microscopy Bovine gelatin (isoelectric point of 5.0) was purchased from Acros Organics (Geel, Belgium). B. mori silkworm cocoons used in the experiment were kindly donated by Sijia Min from Zhejiang University. GS and pentobarbital sodium were obtained from Sigma– Aldrich (St. Louis, MO, USA). E. coli (ATCC8099), S. aureus (ATCC6538) and P. aeruginosa (ATCC9027) were obtained from the Department of Biomedical Engineering of Jinan University and were maintained on solid agar medium at 4 °C. All other reagents were of analytical grade and used without further processing.

The morphologies of GMs, SF scaffolds and GS/GM/SF scaffolds were characterized by scanning electron microscopy (SEM; LEO1530 VP, Philips, Amsterdam, the Netherlands). The pore sizes of the scaffolds were evaluated by measurement of 25 random pores in SEM images of the same sample using ImageJ software (NIH, Bethesda, MD, USA). The porosity of the SF scaffolds was measured according to a method published previously [26]. 2.7. In vitro release of GS

2.2. Preparation of the GS-impregnated GMs (GS/GMs) An emulsion solvent evaporation method was used to prepare the GMs [20]. GS was incorporated into the GMs according to a method reported previously [16,21], with slight modification. Briefly, 4 ll of GS solution (25 mg ml1) was added dropwise to 1 mg of freeze-dried GMs that were cross-linked with glutaraldehyde, and then the GMs were maintained at 4 °C overnight. Because the volume of GS solution that was added to the GMs was considerably less than the theoretical swelling saturation volume of the GMs, the GMs absorbed all of the GS solution [22]. Therefore, the incorporation efficiency of GS into GMs was 100%. The composite microspheres were then freeze-dried for 24 h to remove any remaining solvent.

The release profiles of the GS/GMs, GS/SF scaffolds and GS/GM/ SF scaffolds were determined by immersion in 10 ml of phosphatebuffered saline (PBS) and incubation at 37 °C [27]. At pre-set time intervals, 1 ml of supernatant was collected and replaced with an equal volume of fresh PBS. The amount of GS in the supernatant was determined spectrophotometrically at 248 nm using an ultraviolet/visible (UV/Vis) spectrophotometer (UV-2550; Shimadzu, Otsu, Japan) and calculated using a standard curve, which was obtained in previous experiments. Quintuplicate experiments were carried out. The percentage of drug released was calculated using the following formula:

Release ð%Þ ¼

Released Gentamycin sulfate Total Gentamycin sulfate

2.3. Preparation of the composite SF scaffolds 2.8. Antibacterial activity SF solution was prepared according to a method established previously [23]. The final concentration of the SF solution was 4% (w/v), which was determined gravimetrically after freeze-dry-

The antimicrobial activity of the free GS and GS-containing SF scaffolds were investigated using the Kirby–Bauer (KB) test against



E. coli as a model Gram-negative bacterium and S. aureus as a model Gram-positive bacterium [28]. The SF, GS/SF and GS/GM/ SF scaffolds, which were sterilized under UV light on a laminar flow bench for 24 h, were placed in a Petri dish containing agar upon which E. coli and S. aureus had been cultured. After incubation for 24 h (37 °C, 5% CO2), the semi-diameter between the inhibition zone and SF scaffold was measured to assess the antimicrobial efficacy of the SF scaffolds. 2.9. Induction of burn infection in rats and treatment with composite SF scaffolds

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2.12. Statistical analysis Data are expressed as the means of at least three replicates ± standard deviations (SD). Statistical comparisons were performed using ANOVA (t-test). All statistical computations were performed using the SPSS software (v. 16.0; SPSS, Chicago, IL). Differences with a value of P < 0.05 were considered statistically significant. 3. Results 3.1. Characterization of GMs

All procedures and handling of the animals were carried out in accordance with the guidelines of Jinan University and the US National Institutes of Health. Male SD rats weighing 200–250 g were used in the study, for which approval by the Institute’s Animal Ethics Committee of Jinan University was obtained. Food and water were supplied ad libitum. The night before the experiments took place, the backs of the rats were denuded with 8% Na2S aqueous solution [29]. The rats were anaesthetized by intraperitoneal injection of pentobarbital sodium (Sigma Aldrich) at 30 mg kg1, in accordance with the instructions. After anaesthetization, the dorsal hair of the rats was removed. The dorsal skin area was then burned with a hot circular copper billet (15 mm diameter, 90 °C, 20 s) to induce a full-thickness burn. To mimic the clinical treatment of burns, the burns were excised to the level of the panniculus carnosus 24 h later. A total of four full-thickness burns (20 mm diameter) were created on the dorsum of each rat (Fig. S2A) [30]. Each wound was 30 mm apart. To prevent dehydration, the rats were resuscitated with 0.9% saline (1 ml) subcutaneously. After producing the full-thickness burn, a 100 ll aliquot of P. aeruginosa suspension (1 108 CFU) was applied topically to the wound area (Fig. S2B and C) [5]. After 2 h of bacterial challenge to the full-thickness burns [31,32], the wounds were covered with GS/GM/SF, GS/SF or SF scaffolds (Fig. S1D). The entire wound area was then wrapped with sterile gauze and fixed with an elastic bandage. As a control group, one of the full-thickness burns was covered with (Fig. 2D) sterile gauze only. Samples were harvested at days 3, 7, 10, 14 and 21 postoperatively. The five rats in each group were killed so that skin repair at each time point could be evaluated. No any other medicine was administered during the surgery.

GMs were prepared using a water-in-oil method and were cross-linked with glutaraldehyde. The surface morphology of GMs was examined by SEM (Fig. 1A). As shown in Fig. 1A and Table 1, all of the GMs were spherical, uniformly sized (average diameter 18.8 lm), and smooth. Fig. S1 shows optical images of the GMs before and after swelling in DI water for 4 h at room temperature. After immersion in DI water, the GMs swelled and became transparent. The average diameter of the wet GMs was 30.6 ± 5.4 lm, with a range of 21–48 lm. The swelling ratio of the GMs based on volume was 4.3 ± 0.6. The FTIR analyses of GMs, GS and GS/GMs composites, as shown in Fig. 2, indicated the presence of GS inside the GMs; characteristic GS peaks at 619, 1120 and 1242 cm1 were observed in all spectra [34]. 3.2. Characterization of SF scaffolds As shown in the SEM image of Fig. 1B and C, typical SF scaffolds exhibited uniform pore distribution with an average pore size of 90–120 lm. The porosity of the SF scaffold was 92 ± 0.5% with marked interconnectivity. In GS/GM/SF scaffolds (Fig. 4B), the embedded GS/GMs were well distributed and integrated throughout the SF structure and were fixed firmly to the walls or intersections of the scaffolds. Throughout, all embedded GS/GMs appeared to be wrapped in a thin layer of SF. The porosity (85%) and interconnectivity of the pores were well maintained and not affected by the embedded GS/GMs. However, the average pore size of the GS/ GM/SF scaffolds decreased to 40–80 lm. 3.3. In vitro drug release studies

2.10. Histology evaluation For histological analyses, the harvested samples were fixed in 4% formaldehyde in PBS at 4 °C, dehydrated in a graded series of ethanol, and then embedded in paraffin for routine haematoxylin–eosin (H&E) staining and Masson’s trichrome staining for collagen fibres [33]. H&E- and Masson’s-trichrome-stained sections were observed with a light microscope (Axio Scope A1 FL; Carl Zeiss, Wetzlar, Germany).

GS release studies were performed to evaluate the ability of GMs to control the release of the loaded drug. The GS release profile is shown in Fig. 3. In GS/SF scaffolds, >60% of the GS was rapidly released from the GS/SF scaffold within 4 h. After 48 h, all GS loaded in the GS/SF scaffold was released. In GS/GMs, 35% of the GS was released in the first 4 h. After 72 h, 100% of the GS was released. The drug release rate was the slowest in the GS/ GM/SF scaffolds; GS was released gradually from the GS/GM/SF scaffolds over 144 h, with only 25% of the GS released after 4 h.

2.11. Immunohistochemistry 3.4. Antimicrobial activity For the immunohistochemical staining, the paraffin sections (5 lm) were de-paraffinized, washed three times in PBS for 5 min, and then blocked with 5% serum for 30 min. The slides were subsequently incubated with primary antibodies against interleukin-1b (IL-1b) (SANTA CRUZ), interleukin-6 (IL-6) (Abcam, Cambridge, UK) or tumour necrosis factor-a (TNF-a) (Abcam) at 4 °C overnight. After rinsing three times with PBS, the slides were incubated with secondary antibodies at 37 °C for 20–30 min, further developed with 3,30 -diaminobenzidine tetrahydrochloride (DAB) solution, and then finally counterstained with haematoxylin.

The antimicrobial activities of three types of scaffolds were tested using the KB method against E. coli, and S. aureus; the results are shown in Fig. 4. No inhibition of E. coli, and S. aureus was observed with pure SF. The semi-diameters of the inhibition zones of GS/SF scaffolds against E. coli, and S. aureus were 8.5 and 9.5 mm, respectively. In contrast, the semi-diameters of the inhibition zones (7.5 and 8.8 mm) of GS/GM/SF scaffolds against E. coli and S. aureus were slightly smaller than those of the GS/SF scaffolds. The results were in accordance with the release profile, because


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Fig. 1. Scanning electron micrographs of (A) GMs, (B) SF scaffolds and (C) GS/GMs/SF scaffolds.

Table 1 Particle sizes and swelling ratio of GMs. Wet diameter ± SD (lm)

Swelling ratio ± SD (volume)

20.8 ± 6.5

30.6 ± 5.4

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the GS/SF scaffolds exhibited more rapid release over 24 h, and the amount of GS was greater than in the GS/GM/SF scaffolds. It has been reported that GS is effective against many Gram-negative bacteria, especially Pseudomonas species [35]. Furthermore, the minimum inhibitory concentration of GS for P. aeruginosa is 4 lg ml1 [36]. 3.5. In vivo wound healing The burn lesions were covered with the various SF scaffolds after inoculation with P. aeruginosa. Representative macroscopic images of full-thickness burns treated with a sterile gauze, GS/ GM/SF scaffolds, GS/SF scaffolds and SF scaffolds at various postoperative days are shown in Fig. 5. Throughout this experiment all rats survived until they were killed, and no obvious necrosis of the epidermal tissue was observed. The wound surfaces were reduced markedly in all groups over 21 days. At day 3, whitecoloured SF scaffolds were evident on the burns, and inflammation

Fig. 3. GS release profiles from (A) GS/SF scaffolds, (B) GS/GMs and (C) GS/GM/SF scaffolds.

was detected in all rats. However, haemorrhage was observed in wounds treated with sterile gauze only. Furthermore, the surfaces of scaffold-treated wounds were moist, whereas wounds in the control group exhibited a dry surface and a scab. At day 7, the group treated with gauze only exhibited surface scabs, while no blood clotting was detected in groups treated with GS/GM/SF scaffolds, GS/SF scaffolds or SF scaffolds. At day 10, all SF-based scaffolds integrated well with the wounds, and all wounds exhibited a ruddy surface. At day 14, wounds treated with GS/GM/SF, GS/ SF or SF scaffolds were mostly healed and nearly sealed. However, pustules were observed in wounds treated with sterile gauze only. At day 21, all wounds were sealed; however, scabs were observed in wounds treated with sterile gauze only. 3.6. Histological observation Histological analyses of dorsal skin lesions stained with H&E are shown in Fig. 6 and a higher-magnification image is shown in Fig. S4.


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Fig. 4. Antimicrobial activities of SF scaffolds, GS/SF scaffolds and GS/GM/SF scaffolds. (A) E. coli and (B) S. aureus.

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Fig. 5. Change in appearance of wounds dressed with SF scaffolds after full-thickness burn infection.

At day 3, infected burns treated with GS/GM/SF scaffolds (Fig. 6A) or GS/SF scaffolds (Fig. 6B) did not exhibit severe inflammatory tissue response. On the other hand, infected burns treated with SF scaffolds (Fig. 6C) or sterile gauze (Fig. 6D) exhibited oedema and severe inflammation. At day 7, severe inflammation and oedema remained evident in wounds treated with sterile gauze only. Typical infiltration of granulocytes was observed in all SF-based groups, and macrophages were also observed at this stage. Furthermore, the occurrence of infiltrated inflammatory cells in granulation tissues was less in wounds treated with GS/GM/SF scaffolds than in the other groups. At day 10, a large number of blood vessels were present in both the GS/GM/SF group and the GS/SF group. At day 10, epithelialization began to occur in the rim of the wound treated with GS/ GM/SF scaffolds (Fig. 6I). However, neutrophils and lymphocytes were still present in the control group (Fig. 6L). At day 14, epithelialization was observed in all groups. However, moderate inflammation and oedema remained in wounds treated with sterile gauze only. Furthermore, the epidermis and the newly formed dermis were connected tightly in the GS/GM/SF group, suggesting more effective regeneration in infected full-thickness wounds in the presence of GS/GM/SF scaffolds than in the presence of other scaffolds or gauze only. At day 21, all burns were mostly covered with epidermis. A papillary structure was observed in the GS/GM/SF scaffold group, and the structure of regenerated skin was similar to that of normal skin

(Fig. S3A). The GS/SF group also exhibited more re-epithelialization and reconstruction of skin tissues than the SF scaffold and gauzeonly control groups. Fig. 7 depicts the deposition of collagen using Masson’s trichrome stain in the various scaffolds at the indicated time intervals. At day 7, numerous collagen fibres and proliferation of a few blood vessels was observed in the regenerated skin of the GS/GM/SF group. At day 10, collagen bundles were observed in both the GS/GM/SF group and the GS/SF group. Moreover, at day 10, re-epithelialization also occurred in the GS/GM/SF group. At day 14, more neotissue formation accompanied by more collagen deposition in the defect sites (Fig. 7M, N and P) was observed. In the GS/GM/SF group, the deposition of collagen increased and its distribution was uniform. At day 21, deposition of collagen in the GS/GM/SF group was greater and more regular than in the other groups, which demonstrated that neotissue formation was enhanced by the GS/GM/SF scaffold. Furthermore, the alignment of the collagen was regular and similar to that of normal dermal tissue (Fig. S3B). 3.7. Immunohistochemistry IL-1b, which induces a variety of acute-phase reactions, is an endogenous pyrogen [37]. IL-6 is an indicator of severity of the infectious phase [38], while TNF-a is a primary mediator of the


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Fig. 6. Histological analyses of dorsal skin stained with H&E after full-thickness burn infection. The bar corresponds to 200 lm.

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Fig. 7. Histological analyses of dorsal skin stained with Masson’s trichrome after full-thickness burn infection. The block diagram indicates new collagen deposition and the bar corresponds to 200 lm.

host inflammatory response [39]. Thus, the roles of IL-1b, IL-6 and TNF-a have been investigated in a number of inflammatory responses to burns [40]. Expression levels of IL-1b, IL-6 and TNFa in dorsal skin after full-thickness burn infection of the various groups at the indicated time points are illustrated in Figs. 8–10 and higher-magnification images are shown in Figs. S5–S7. At day 3, severe inflammatory tissue response was observed in all groups. However, the group treated with GS/GM/SF scaffolds exhibited lower cytokine expression levels. At day 7, the GS/GM/ SF group exhibited significantly lower expression of IL-1b, IL-6 and TNF-A than all other groups. Expression of IL-1b, IL-6 and TNF-a was persistent and widespread over the burns in the gauze-only group until day 14. In contrast, the GS/GM/SF group exhibited lower-level expression of proinflammatory cytokines, and epithelialization was observed at day 10. At day 14, wounds treated with GS/GM/SF scaffolds exhibited almost no inflammatory response, and the tissue was similar to that of normal skin (Fig. S3C–E). At day 21, expression levels of all proinflammatory cytokines were significantly lower in all groups.

4. Discussion Infection caused by bacteria is a major problem in treating fullthickness burns, because infection can impair the proliferation of dermal cells and thereby delay the wound-healing process [2]. Therefore, dermal regeneration templates must both have good antibacterial properties and promote wound healing. Currently, various antibiotic drug delivery systems are available, including hydrogels, scaffolds and microspheres [41–44]. Microspheres are widely used to control drug release, because they not only have potential for drug delivery, but also provide a diffusion barrier to retard rapid drug release [45,46]. Due to their good biocompatibility and degradation to nontoxic and readily excreted products, GMs have attracted much attention in the drug delivery field [47]. The SEM analyses of GMs indicate a spherical morphology with an average diameter of 18.8 lm and a smooth surface (Fig. 1). Moreover, the water absorption profile of GMs can be used to evaluate the merits of specific GMs as drug carriers. As shown in Fig. 2 and Table 1, the swelling ratio of the GMs used in this study


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Fig. 8. Expression of IL-1b in dorsal skin after full-thickness burn infection. The arrows indicate the neovasculatures and the bar corresponds to 200 lm.

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Fig. 9. Expression of IL-6 in dorsal skin after full-thickness burn infection. The arrows indicate the neovasculatures and the bar corresponds to 200 lm.

was 4.3 ± 0.6, which was similar to values reported previously [47,48]. Hence, the morphology and high water absorption capability of GMs suggest that they can be easily loaded with drug. The signal of GS was weak in the FTIR spectra of GS/GMs, which maybe attributed to the small quantity of GS in the composite GMs [49]. However, the FTIR spectra of GS/GMs indicated that GMs can be successfully impregnated with GS by means of a simple soaking procedure, and that no chemical linkage between GS and GMs is necessary. The drug was incorporated into the GMs via hydration [50], and most of the absorbed drug was located in the interior of the particles. Furthermore, the drug release behaviours of GS/ SF and GS/GM/SF scaffolds (Fig. 3) indicated that GMs can be used to control drug release. The GS/GM/SF scaffolds were composed of the following three types of compound: GS as the antibiotic agent, GMs as the carrier for the antibiotic, and SF as the scaffold material. The use of SF as a scaffold material not only improved cell proliferation and differentiation but also adherence and migration [10,51,52]. The GS/GM/SF scaffolds exhibited a uniform pore distribution with an average size of 40–80 lm and a porosity of 85%. Scaffolds with an opti-

mum pore size ranging from 20 to 125 lm support regeneration of adult mammalian skin [53]. As shown in Fig. 4, GS/GM/SF scaffolds exhibited the lowest GS release rate, which was attributed to the distribution and integration of GS/GMs throughout the SF scaffold structure. As the release profiles show, the release rate of GS/ GM was slower than the GS/SF scaffolds. In GS/SF scaffolds, most of the GS was located around the scaffold; thus, GS was released rapidly from the GS/SF scaffolds by diffusion. These revealed that most of the drug absorbed by GMs was presented in the particle interior. Furthermore, these results also demonstrated that the GS/GM/SF scaffold had the best sustained-release function and that the GMs were the key determining factor in the controlled release of GS. Both GS/GM/SF scaffolds and GS/SF scaffolds exhibited antimicrobial activity against Gram-negative (E. coli) and Gram–positive (S. aureus) bacteria. However, the antimicrobial activity of GS/ GM/SF scaffolds was slightly weaker than that of GS/SF scaffolds, which was attributed to a higher GS release rate from the GS/SF scaffolds over 24 h. These results were in accordance with the release profile. Previous reports showed that the dorsal skin of rats


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Control

Fig. 10. Expression of TNF-a in dorsal skin after full-thickness burn infection. The arrows indicate the neovasculatures and the bar corresponds to 200 lm.

is fragile after burn injury, thereby making the damaged skin particularly susceptible to infection by P. aeruginosa, which causes systemic inflammation. Therefore, a rat full-thickness burn infection model was used to evaluate the in vivo antimicrobial properties of the GS/GM/SF scaffolds. Wound healing is a complex process involving interactions among various cells, cytokines and extracellular matrices (ECMs). Wound healing processes and neotissue formation were evaluated in vivo by macroscopic observations, H&E staining, Masson’s trichrome staining and proinflammatory cytokine (IL-1b, IL-6 and TNF-a) immunohistochemistry at the indicated time points. At all time points, the GS/GM/SF group exhibited a lower inflammatory response, which would be favourable to skin repair. The GS/ SF group also exhibited a lower inflammatory response at day 3, which was attributed to the greater GS release rate, and consequent higher antibacterial activity, of GS/SF scaffolds compared to GS/GM/SF scaffolds. Compared to the other groups, wound healing was accelerated in the GS/SF group due to deactivation of bacteria in the infected wounds [54]. IL-1b, IL-6, and TNF-a are major cytokine mediators of the acute inflammatory response that occurs following skin injury [55]. There is evidence that IL-1b, IL-6 and TNF-a stimulate the release of somatostatin, which inhibits the release of growth hormone [56–58]. Growth hormone increases protein synthesis and improves wound healing [59,60]; thus, the inhibited release of growth hormone will delay the wound healing process. In the GS/GM/SF group, expression of the three proinflammatory cytokines was lower than in all other groups, which may be favourable to wound healing. Moreover, GS/GM/SF scaffolds have the appropriate pore size and a highly porous structure, which should promote matrix swelling and absorption of wound exudates. These features may activate the generation of vascular endothelial cells and fibroblasts, enhancing neovascularization and neotissue formation. The use of GS/GM/SF scaffolds reduced the inflammatory response (Figs. 8– 10) and accelerated regeneration of dermal tissue in comparison to the other groups (Figs. 6 and 7). In general, the scaffold degradation ratio should match the neotissue formation ratio [61]; in this study, the tissue regeneration ratio matched well the scaffold degradation ratio reported in our previous study [16]. Conventional wound dressings are changed daily to monitor infection of the wound site. However, this procedure increases the risk of bacterial infection. GS/GMs/SF scaffolds can be used to

achieve longer-term release of effective concentrations of antibiotics, thereby reducing the risk of infection and the pain experienced by patients.

5. Conclusions We prepared GS/GM/SF scaffolds for use as dermal regeneration templates and investigated their antimicrobial activities in a fullthickness burn infection treatment in a SD rat model. The GS/ GM/SF scaffolds exhibited long-duration drug release and potent antibacterial activities. This treatment effectively inhibited local infection in vivo, thereby accelerating dermal regeneration and re-epithelialization. Hence, GS/GM/SF scaffolds are applicable as a novel, antimicrobial dermal regeneration template for severely burned patients.

Acknowledgments This study was supported financially by the Natural Science Foundation of China (51303064), the Natural Science Foundation of Guangdong (S2012040008003), Guangzhou Science and Technology Plan Project (No. 11C32070752), the Key Project of DEGP (cxzd1109), the Ph.D. Programs Foundation of the Ministry of Education of China, and the Fundamental Research Funds of the Central Universities (21612327).

Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–10 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio. 2014.03.029.

Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio. 2014.03.029.



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