An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing

International Journal of Pharmaceutics 436 (2012) 141–153

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

An innovative bi-layered wound dressing made of silk and gelatin for accelerated wound healing Sorada Kanokpanont a , Siriporn Damrongsakkul a , Juthamas Ratanavaraporn a , Pornanong Aramwit b,∗ a

Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, PhayaThai Road, Phatumwan, Bangkok 10330, Thailand Bioactive Resources for Innovative Clinical Applications Research Unit and Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, PhayaThai Road, Phatumwan, Bangkok 10330, Thailand b

a r t i c l e

i n f o

Article history: Received 17 April 2012 Received in revised form 18 June 2012 Accepted 21 June 2012 Available online 4 July 2012 Keywords: Silk fibroin Gelatin Silk sericin Bi-layered wound dressing Wound healing

a b s t r a c t In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silk fibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin and glutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroin fabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesive than the commercial wound dressing mesh. This confirmed by results of peel test on both the partialand full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneous porous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularly for the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that the bi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed the greater extent of wound size reduction, epithelialization, and collagen formation. The superior properties of the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wound dressings were less adhesive and had improved biological functions to promote cell activities and wound healing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thickness wounds. © 2012 Elsevier B.V. All rights reserved.

1. Introduction A large number of wound dressings are currently used in the treatment of burns, chronic ulcers, decubitus ulcers, etc. (Suzuki et al., 1997; Tanihara et al., 1998). An ideal wound dressing should prevent dehydration of the wound and retain a favorable moist environment at the wound interface, allow gas permeability, and act as a barrier against dust and microorganisms. Also, it should be non-adherent and easily removed without trauma. Wound dressings are generally made of readily available biomaterials that require minimal processing, possess nontoxic, non-allergenic, and antimicrobial properties, as well as promote wound healing (Jayakumar et al., 2011). Recently, many research groups are focusing in the production of the novel wound dressings by synthesizing or modifying biocompatible materials (Shibata et al., 1997; Ulubayram et al., 2001). Current strategies also point out the acceleration of the wound repair by systematically designed dressing materials. By this direction, most efforts have experimentally and

∗ Corresponding author. Tel.: +66 89 921 7255; fax: +66 2 218 8403. E-mail address: [email protected] (P. Aramwit). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.06.046

clinically utilized the biologically derived materials such as collagen, chitin, chitosan, etc., which are capable of accelerating the healing processes at molecular, cellular, and systemic levels, as materials to produce wound dressings (Balasubamani et al., 2001; Howling et al., 2001; Jayakumar et al., 2011). Silk fibroin (SF) is a fibrous protein in which the main components, i.e. glycine and alanine, are specific sequence of non-polar amino acids. It has been widely used as biomaterials by humans for centuries, such as suture materials from silk fibers of silkworm (Bombyx mori). Silk fibroin has also been received even more interests on the broad biomedical applications due to its unique physical, mechanical and biological properties, including strength, toughness, elasticity, lightweight, biocompatibility, biodegradability, minimal inflammatory reaction, capability to promote wound healing, and easy chemical modification to suit the applications (Moy et al., 1991). Then, it has recently become a new family of advanced tissue engineered biomaterials. Due to the ability to promote adhesion and proliferation of various cells including keratinocytes and fibroblasts, silk fibroin has been a potential biomaterial to fabricate wound dressings in various formulations (Baoyong et al., 2010; Chiarini et al., 2003; Minoura et al., 1995; Sugihara et al., 2000a; Yeo et al., 2000). Most of them reported the

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success of wound dressings made of silk fibroin for the recovery of skin wounds by its favorable physical, mechanical and biological properties (Baoyong et al., 2010; Sugihara et al., 2000b; Yeo et al., 2000). However, a few researches focus on the nonadherent property of the silk fibroin dressing. In a part of this study, we have established the silk fibroin woven fabric coated with carnauba wax as a non-adhesive layer of a bi-layered wound dressing. Carnauba wax is a natural edible coating material, which is recovered from the underside of the leaves of a Brazilian palm tree (Copernica cerifera). It is mainly used to retard moisture loss and impart glossiness (Embuscado and Huber, 2009). The beneficial role of carnauba wax is well known for enhancing shelf life and maintaining post-harvest quality of fruits (Khuyen et al., 2008). Our wax-coated silk fibroin woven fabric would be advantageous in terms of the prevention of dehydration for the wound and easily removal to reduce trauma. Mechanical properties of the wax-coated silk fibroin woven fabric were characterized. Peel test of this fabric layer was performed in the partial- and full-thickness wounds of porcine skin, comparing with the commercial wound dressing mesh “Sofratulle® ”. Apart from the non-adhesive layer, bioactive layer is introduced to accelerate wound healing and function as a three-dimensional matrix to support cell activities and tissue regeneration. In this study, a composite of silk fibroin/gelatin (SF/G) was selected to produce the spongy bioactive layer that attached to the waxcoated silk fibroin woven fabric. Silk fibroin/gelatin composites have been investigated in terms of their superior properties over the single or other combinations (Jetbumpenkul et al., 2012; Okhawilai et al., 2010; Shubhra et al., 2011). The outstanding points of the silk fibroin/gelatin composites are the unique mechanical properties derived from ␤-sheet structure of silk fibroin and the improved specific biological characteristics derived from Arg-Gly-Asp (RGD) sequence of gelatin. In addition, sericin of a silk gum was introduced into the silk fibroin/gelatin composites of this study. Sericin is highly hydrophilic with strong polar side chains such as hydroxyl, carboxyl and amino groups. Thus, it is easy for the cross-linking, copolymerization and blending with other polymers to produce materials with improved properties (Ahn et al., 2001; Nagura et al., 2001). It has been demonstrated that after blending with gelatin or other polymers, silk sericin can form a scaffold and be a good candidate for tissue engineering applications (Mandal et al., 2009; Aramwit et al., 2010). It was also reported that sericin enhanced mammalian cell attachment and proliferation of human skin fibroblasts (Terada et al., 2002; Tsubouchi et al., 2005). Our previous study showed that sericin enhances wound healing by promoting collagen production (Aramwit and Sangcakul, 2007; Aramwit et al., 2009). In this study, silk fibroin/gelatin at different mixing ratios was blended with sericin solution to prepare the spongy bioactive layers that attached to the wax-coated silk fibroin fabrics by the freeze-drying and glutaraldehyde (GA) crosslinking techniques, defined as the bi-layered wound dressings. Different crosslinking degrees were varied by adjusting the GA concentration. Morphology, crosslinking extent, and in vitro degradation rate of the silk fibroin/gelatin bioactive layers were investigated. L929 mouse fibroblast cells were cultured on the bi-layered wound dressings developed to evaluate cell attachment and proliferation. In addition, our bi-layered wound dressings were tested with the fullthickness wounds of rat model to evaluate the efficiency of wound healing characterized by the reduction of wound area, epithelialization, and the production of collagen tissue, comparing with the clinically used wound dressing “3 MTM Tegaderm high performance foam adhesive dressing”.

2. Materials and methods 2.1. Materials Silk fibroin (SF) woven fabric was purchased from Chul Thai Silk Co., Ltd., Phetchabul province, Thailand. Thai silk strain, B. mori (Nangnoi Srisaket 1) was supplied by Queen Sirikit Sericulture Center, Nakornratchasima province, Thailand. Carnauba wax (No. 1, yellow) was purchased from Sigma–Aldrich Laborchemikelien, Germany. A gelatin (G) sample prepared by an acidic treatment of porcine skin collagen (isoelectric point (IEP) = 9.0) was kindly supplied by Nitta Gelatin Inc., Osaka, Japan. Glutaraldehyde (GA) and other chemicals were analytical grade and used without further purification. 2.2. Preparation of wax-coated silk fibroin woven fabrics Carnauba wax was dissolved in morpholine solution at different concentrations (0.025, 0.050, and 0.100% w/v). The silk fibroin woven fabric (5 in. × 5 in.) was stretched and immersed in the wax solution at room temperature for 20 min, and then dried overnight to obtain the wax-coated silk fibroin woven fabric, defined as wSF fabric. 2.3. Characterization of wax-coated silk fibroin woven fabrics 2.3.1. Mechanical characterization The tensile test was performed on the wSF fabrics (150 mm in length and 25 mm in width) at room temperature using a universal testing machine (Instron, No. 5567) at a constant rate of 30 mm/min. The curves of force as a function of deformation (mm) were automatically recorded by the software. The tensile modulus (MPa) and elongation at break were calculated according to the ASTM D638-01 method (n = 6). 2.3.2. Weight increased after wax coating The fabrics before and after coating with different concentrations of carnauba wax were weighed. Percentage of weight increased was calculated (n = 3). 2.3.3. Peel test with porcine skin Porcine skin was used within 2 h after sacrifice. Partial- and full-thickness (1 cm in depth) wounds were prepared by scraping the outer skin layer and by cutting the skin at 1 cm in depth, respectively. The wSF fabrics and the commercial wound dressing mesh “Sofra-tulle® ” (Patheon UK Limited, Swindon, UK) immersed in phosphate-buffered saline solution (PBS, pH 7.4) were attached on the wounds. After 12 h, the dressings were removed and fixed in 2.5% (v/v) GA solution at 4 ◦ C for 1 h. Then, the dressings were dehydrated in serial dilutions of ethanol (50, 70, 80, 90, 95, 99 and 100%, v/v, respectively) for 5 min each and the hexamethyldisilazane solution was dropped on the dressings for the critical point drying. The attachment of cells on the dressings was observed on a scanning electron microscope (SEM, JSM 5400, JEOL) at an accelerating voltage of 12–15 kV after sputter-coating with gold. The number of cells attached on the dressings was analyzed by the fluorometric quantification of cellular DNA according to the method reported by Takahashi et al. (2005). The adhesive force applied to peel the dressings from wounds was also determined by a modified fixed peeling angle peel test (Zhang et al., 2012). Briefly, the porcine skin attached with the dressings (150 mm in length and 25 mm in width) was placed on a liner translation stage of a universal testing machine (Instron, No. 5567) at a fixed peeling angle of 135◦ . The sample holder was fixed at the upper side of the dressings and the peeling force was applied at a constant tensile rate of 5 mm/min. The adhesive force defined as the steady-state peeling

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force used to peel the dressings from wounds was determined from a load–displacement curve (n = 5). 2.4. Fabrication of bi-layered wound dressings 2.4.1. Preparation of Thai silk fibroin and sericin solutions Thai silk fibroin solution was prepared according to the method previously described by Kim et al. (2005). In brief, cocoons were boiled in an aqueous solution of 0.02 M sodium carbonate (Na2 CO3 ) and then rinsed thoroughly with deionized (DI) water to remove sericin. The degummed silk fibroin was mixed with 9.3 M lithium bromide (LiBr) solution at the ratio 1:3 (w/w). The mixture was stirred occasionally at 60 ◦ C for 4 h until the silk fibroin was completely dissolved. The solution was dialyzed in DI water for 2 days. The dialyzed water was changed regularly until its conductivity was the same as that of the DI water. The final concentration of silk fibroin aqueous solution was about 6–6.5% (w/w). Silk sericin solution was extracted using a high temperature and pressure degumming technique (Lee et al., 2003). Briefly, the silkworm cocoons were mixed with DI water (1 g of dry silk cocoon: 30 mL of water) and the samples were autoclaved at 120 ◦ C for 60 min. After filtration through a membrane to remove fibroin, the concentration of sericin solution was measured by BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). 2.4.2. Preparation of sericin-silk fibroin/gelatin spongy bioactive layers attached to the wax-coated silk fibroin woven fabrics Silk fibroin (SF) and gelatin (G) solutions at the SF/G mixing ratios of 50/50 and 20/80 were mixed with 1% (w/w) sericin solution to obtain a final solution concentration at 4% (w/w), defined as serSF50/G50 and ser-SF20/G80, respectively. Then, GA was added to the mixture at the final concentrations of 0.005, 0.010, 0.020, 0.050, and 0.100% (v/v). After mixing, the mixture was cast onto the wSF fabric stretched on the Teflon mold and placed at 4 ◦ C for 24 h to allow for the crosslinking reaction. The crosslinked gels were then agitated in 100 mM aqueous glycine solution at room temperature for 2 h to block the residual aldehyde groups of glutaraldehyde. Following washing three times with DI water, the gels were frozen at −50 ◦ C overnight prior to lyophilization for 48 h to obtain the bilayered wound dressings of wax-coated silk fibroin woven fabrics with sericin-silk fibroin/gelatin spongy bioactive layers, defined as wSF fabric + ser-SF50/G50 or ser-SF20/G80. 2.5. Physico-chemical characterization of sericin-silk fibroin/gelatin spongy bioactive layers 2.5.1. Morphological observation Cross-sectional structure of the ser-SF50/G50 and ser-SF20/G80 sponges was observed on a SEM as described previously. Pore size was determined using ImageJ software (the US National Institutes of Health, USA). The porosity of the sponges was measured by a liquid displacement (Nazarov et al., 2004). Absolute ethanol was used as the displacement liquid as it permeates through the sponges without swelling or shrinking the matrix. A known weight of dried sponge was immersed in a known volume (V1 ) of ethanol for 5 min. The total volume of ethanol and the ethanol-impregnated sponge was recorded as V2 . The ethanol-impregnated sponge was then removed and the residual volume of ethanol was recorded as V3 . The porosity of the sponge (ε) was obtained by: ε(%) =

V1 − V3 × 100 V2 − V3

Density of the sponges was calculated by the weight per volume of the dried sponges (mg/mm3 ).

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2.5.2. Evaluation of crosslinking degree Color intensity of the crosslinked ser-SF50/G50 and serSF20/G80 sponges was determined using a Minolta colorimeter CR 400 Series (Osaka, Japan) calibrated with a standard (Rivero et al., 2010). The CIELab scale was used; lightness (L) and chromaticity parameter b* (yellow–blue) were measured (n = 3). In term of weight loss, a known weight sponge was placed in DI water at room temperature for 24 h. The remained sponge was then freeze-dried and weighed. Percentage of weight loss indicating the success of crosslinking was calculated (n = 3). 2.5.3. In vitro degradation test A known weight sponge was subjected to the degradation in PBS (pH 7.4) containing collagenase (1 Unit/ml) at 37 ◦ C for 14 days. At each time point, the remained sponge was collected, washed with DI water, freeze dried, and weighed. Percentage of weight remaining was calculated (n = 3). 2.6. In vitro attachment and proliferation tests with L929 cells L929 mouse fibroblast cells were seeded onto the sterilized bi-layered wound dressings of wSF fabric + ser-SF50/G50 or serSF20/G80 (5 mm in diameter and 2 mm in height) at a density of 5 × 105 cells/sample and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 100 U/ml penicillin/streptomycin at 37 ◦ C, 5% CO2 . The number of cells attached after 6 h and cells proliferated after 5 days of culture was quantified using the DNA assay (Takahashi et al., 2005). The morphology and density of L929 cells cultured in the samples for 5 days were observed on a SEM after fixing in 2.5% (v/v) GA solution, dehydration in serial dilutions of ethanol, and critical point drying as described previously. 2.7. In vivo study of full-thickness wound model 2.7.1. In vivo wound model in rats All animal experiments were performed in agreement with the Chulalongkorn University Animal Care and Use Committee and with ethics approval from the research ethical committee, Faculty of Medicine, Chulalongkorn University and the Mahidol University Animal Care and Use Committee (MU-ACUC). Sprague-Dawley rats (28-week-old, 250 ± 5 g, n = 24) were purchased from National Laboratory Animal Centre, Mahidol University, Nakhon Pathom, Thailand. After being anesthetized and injected with antibiotic, the full-thickness wounds (1.5 cm × 1.5 cm) were prepared on both left and right sides of the their back (2 wounds/rat). Wounds were dressed with the bi-layered wound dressing developed and the commercial wound dressing “3 MTM Tegaderm high performance foam adhesive dressing (3 M Corporate Headquarters, MN, USA)”. 2.7.2. Measurement of wound size reduction Size of wounds was measured immediately after operation and at 3, 7, and 14 days after operation using a stereomicroscope (1024 × 768 pixels). Percentage of wound size reduction was calculated (n = 6). 2.7.3. Histological and immunohistochemical evaluation The tissue regenerated at the wounds was collected at 3, 7, and 14 days after operation, fixed in 10% (v/v) formalin, and then embedded in a paraffin block. The paraffin-embedded tissues were sectioned (4 ␮m thickness) and stained with Hematoxylin and Eosin (H&E) to evaluate the epithelialization, collagen formation, and infiltration of cells. For the immunohistochemical staining, the sections were washed with PBS and blocked with a normal goat serum (Santa Cruz Biotechnology, Inc., CA, USA) at room temperature for 1 h. The sections were incubated with a mouse anti-rat

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Fig. 1. (A) Macroscopic (left) and microscopic images (right) of 0.1% wSF fabrics. (B) Microscopic images of Sofra-tulle® and 0.1% wSF fabrics after attached to the partial- and full-thickness wounds for 12 h. (C) Number of cells on the Sofra-tulle® 0.1% wSF fabrics after attached to the partial- () and full-thickness wounds () for 12 h. (D) Adhesive force applied to peel the Sofra-tulle® and 0.1% wSF fabrics off after attached to the partial- () and full-thickness wounds () for 12 h. * p < 0.05, significant between the groups.

macrophage class HIS 36 (1:800, Santa Cruz Biotechnology, Inc., CA, USA) or a rabbit anti-rat collagen type III monoclonal antibody (1:80, Santa Cruz Biotechnology, Inc., CA, USA) at 4 ◦ C overnight for the macrophages and type III collagen, respectively. Then, the sections were washed with PBS and stained with a biotinylated goat anti-mouse antibody (Santa Cruz Biotechnology, Inc., CA, USA) or a goat anti-rabbit antibody (Dako, Denmark), respectively, for 30 min at room temperature. For bright-field microscopy, bound primary antibodies were detected using DAKO EnVision-Horseradish Peroxidase and 3, 30-diaminobenzidine (DAB) substrate kit (Vector Laboratories, Burlingame, CA, USA), and counter-stained with Hematoxylin to visualize the cell nuclei. Then, the sections were mounted with vectamount (Vector Laboratories, Inc., Burlingame, CA, USA) and the images were taken on a microscope. The number of macrophages was counted from the images taken at 100× magnification randomly selected.

2.8. Statistical analysis All the results were statistically analyzed by the unpaired student’s t test and p < 0.05 was considered to be statistically significant. Data were expressed as mean ± standard deviation.

Table 1 Physical properties of wax-coated silk fibroin (wSF) woven fabrics. Concentration of wax (%)

Tensile modulus (MPa)

0.000 0.025 0.050 0.100

211.2 209.1 200.8 245.7

*

± ± ± ±

44.7 23.9 43.6 10.0 *

Elongation (%) 21.6 28.7 29.3 30.7

± ± ± ±

0.1 0.4* 1.7* 1.1*

Weight increased (%) – 1.1 ± 0.1 6.8 ± 0.1 10.2 ± 1.9

p < 0.05, significant against the value of non-coated fibroin woven fabric.

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Fig. 2. (A) Image of sericin-silk fibroin/gelatin solution casting into Teflon mold to prepare the bi-layered wound dressings. (B) Cross-sectioned images of ser-SF50/G50 and ser-SF20/G80 spongy bioactive layers crosslinked with different concentrations of GA (0.005, 0.010, 0.020, 0.050, and 0.100%).

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(245.7 MPa) while the percentage of elongation was significantly increased for any concentration of wax coating. Percentage of weight increased rose along the increasing concentration of wax coated. The macro- and microscopic images of wSF fabrics showed the smooth surface (Fig. 1A). Fig. 1B shows the microscopic images of Sofra-tulle® and 0.1% wSF fabric after attached to the partialand full-thickness wounds for 12 h. A large number of cells were attached on the Sofra-tulle® when applied at both the partial- and full-thickness wounds. On the other hand, less number of cells was observed on the 0.1% wSF fabric. The number of cells attached was quantitatively confirmed by DNA assay (Fig. 1C). The number of cells attached on the 0.1% wSF fabric was significantly lower than that on the Sofra-tulle® for both the partial- and full-thickness wounds. The adhesive force of the 0.1% wSF fabric attached to the wounds was significantly lower than that of the Sofra-tulle® (Fig. 1D).

3.2. Morphology of sericin-silk fibroin/gelatin spongy bioactive layers

Fig. 3. In vitro degradation profiles of (A) ser-SF50/G50 and (B) ser-SF20/G80 spongy bioactive layers crosslinked with different concentrations of GA in a PBS solution containing collagenase (1 Unit/ml, pH 7.4) at 37 ◦ C. Concentrations of GA crosslinking: 0.005 (), 0.010 (䊉), 0.020 (), 0.050 (), and 0.100% ().

The procedure of sericin-silk fibroin/gelatin solution casting into Teflon mold to prepare the bi-layered wound dressings of wSF fabric + ser-SF50/G50 or ser-SF20/G80 is shown in Fig. 2A. Fig. 2B shows the cross-sectioned images of ser-SF50/G50 and ser-SF20/G80 sponges crosslinked with different concentrations of GA. All formulations of sponges presented homogeneous pore structure; however, pores of the sponges crosslinked with lowest concentration of GA (0.005%) seemed more irregular in shape. The structure of all formulations showed high porosity (80.6–96.3%) with the pore sizes of 145.6–240.9 ␮m (Table 2). The density of the porous sponges was around 0.09–0.17 mg/mm3 .

3.3. Crosslinking properties of sericin-silk fibroin/gelatin spongy bioactive layers 3. Results 3.1. Properties of wax-coated silk fibroin woven fabrics Table 1 presents the physical properties of silk fibroin woven fabrics coated with different concentrations of carnauba wax. Tensile modulus was improved for the fabrics coated with 0.1% wax

Brightness of all sponges was around 77.76–89.97. However, the intensity of yellowness index tended to increase along the increasing concentration of GA, representing higher crosslinking degree of the sponges (Table 3). Correspondingly, lower percentage of weight loss was observed for the sponges crosslinked with higher concentration of GA (Table 3).

Table 2 Morphological properties of silk fibroin/gelatin (SF/G) spongy bioactive layers crosslinked with different concentrations of GA. Concentration of GA crosslinking (%)

Pore size (␮m) SF50/G50

0.005 0.010 0.020 0.050 0.100

240.9 209.7 230.9 205.1 166.9

± ± ± ± ±

78.7 58.5 53.8 45.1 45.4

Density (mg/mm3 )

Porosity (%) SF20/G80 171.4 165.6 145.6 164.6 191.6

± ± ± ± ±

54.9 61.1 43.1 67.2 43.9

SF50/G50 80.6 89.1 81.7 90.8 96.3

± ± ± ± ±

0.2 0.6 1.3 0.5 2.5

SF20/G80 89.3 92.4 94.8 92.7 86.2

± ± ± ± ±

1.7 1.1 1.4 0.4 2.1

SF50/G50 0.09 0.11 0.12 0.13 0.14

± ± ± ± ±

0.005 0.004 0.012 0.007 0.029

SF20/G80 0.12 0.14 0.17 0.15 0.14

± ± ± ± ±

0.005 0.008 0.001 0.007 0.009

Table 3 Colorimetric properties and weight loss of silk fibroin/gelatin (SF/G) spongy bioactive layers crosslinked with different concentrations of GA. Concentration of GA crosslinking (%)

0.005 0.010 0.020 0.050 0.100

Brightness (L)

Intensity of yellowness index (b*)

Weight loss (%)

SF50/G50

SF20/G80

SF50/G50

SF20/G80

SF50/G50

79.02 79.67 80.42 83.97 77.76

79.27 79.02 81.15 86.94 89.97

11.10 14.47 16.65 18.89 20.58

9.45 11.10 14.47 16.90 18.90

36.1 25.6 10.5 17.8 21.8

± ± ± ± ±

0.4 10.9 2.1 3.7 3.0

SF20/G80 41.0 35.7 10.1 11.5 19.4

± ± ± ± ±

5.7 3.9 2.2 3.9 2.9

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Fig. 4. (A) Attachment (6 h) and proliferation (5 days) of L929 mouse fibroblast cells cultured on the bi-layered wound dressings of wSF fabric + ser-SF50/G50 () or serSF20/G80 () crosslinked with different concentrations of GA (0.020, 0.050, and 0.100%). * p < 0.05, significant between the groups. (B) Microscopic images of L929 cells cultured on the bi-layered wound dressings of wSF fabric + ser-SF50/G50 or ser-SF20/G80 crosslinked with different concentrations of GA (0.020, 0.050, and 0.100%) for 5 days.

3.4. Degradation profiles of sericin-silk fibroin/gelatin spongy bioactive layers Fig. 3 shows the in vitro degradation profiles of ser-SF50/G50 and ser-SF20/G80 sponges crosslinked with different concentrations of GA. The sponges crosslinked with higher GA concentration degraded slower than those crosslinked with lower GA concentration. The sponges crosslinked with 0.020–0.100% GA remained even after 14 days of the degradation period while those crosslinked with

0.005 and 0.010% GA degraded completely within 14 days. The similar degradation profiles were observed for both the ser-SF50/G50 and ser-SF20/G80 sponges, irrespective of the GA concentration. 3.5. Attachment and proliferation of L929 cells on bi-layered wound dressings Attachment and proliferation of L929 cells cultured on the bi-layered wound dressings of wSF fabric + ser-SF50/G50 or

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Fig. 5. (A) Gross images of full-thickness wounds before (0 days) and after dressed with 3 MTM Tegaderm high performance foam adhesive dressing (left) and bi-layered wound dressing of wSF fabric + ser-SF20/G80 (right) for 3, 7, and 14 days. (B) Area of full-thickness wounds before (0 days) and after dressed with 3 MTM Tegaderm high performance foam adhesive dressing () and bi-layered wound dressing of wSF fabric + ser-SF20/G80 () for 3, 7, and 14 days.

ser-SF20/G80 were shown in Fig. 4A. After 6 h of culture, the number of cells initially attached on the wSF fabric + ser-SF20/G80 was higher than that of the wSF fabric + ser-SF50/G50, irrespective of GA concentration. Furthermore, after 5 days of culture, the number of cells proliferated on the wSF fabric + ser-SF20/G80 crosslinked at 0.020% GA was significantly higher than that of other groups. The cells showed spreading morphology and proliferated homogeneously throughout the spongy structure (Fig. 4B). High density of cells proliferated on the wSF fabric + ser-SF20/G80 crosslinked at 0.020% GA was confirmed. 3.6. Regeneration of full-thickness wounds Fig. 5 shows gross images and the area of wounds after dressed with 3 MTM Tegaderm high performance foam adhesive dressing and the bi-layered wound dressings of the wSF fabric + ser-SF20/G80 crosslinked at 0.020% GA for 3, 7, and 14 days. After dressed with both types of dressings, wound area was reduced along the treatment period. However, the area of wounds dressed with the wSF fabric + ser-SF20/G80 seemed to be smaller than that of the 3 MTM Tegaderm high performance foam adhesive dressing. The wSF fabric + ser-SF20/G80 almost completely healed the wounds within 14 days of treatment. H&E-staining images of the wounds after dressed with 3 MTM Tegaderm high performance foam adhesive dressing and the bilayered wound dressings for 3, 7, and 14 days were presented in Fig. 6. At 3 days post-operation, a number of inflammatory cells were observed for both samples. The wounds dressed with the bilayered wound dressings showed higher extent of newly formed collagen tissue than those of 3 MTM Tegaderm high performance

foam adhesive dressing along treatment period. Matured collagen was formed in the wounds dressed with the bi-layered wound dressings from 7 days, while that was observed at 14 days for the wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing. Fig. 7A shows the gap between epithelial tips and epithelial tongue of the wounds after dressed with 3 MTM Tegaderm high performance foam adhesive dressing and the bi-layered wound dressings of the wSF fabric + ser-SF20/G80 crosslinked at 0.020% GA for 7 and 14 days. Epithelialization percentage of the wounds dressed with the wSF fabric + ser-SF20/G80 was increased with time (Fig. 7B). The epithelialization was reached at 50% in the wounds dressed with the wSF fabric + ser-SF20/G80 for 14 days while the treatment with 3 MTM Tegaderm high performance foam adhesive dressing did not promote any epithelialization. More number of macrophages was found in the wounds dressed with the wSF fabric + ser-SF20/G80 along 14-day period, comparing with that of the 3 MTM Tegaderm high performance foam adhesive dressing treatment (Fig. 8A). Earlier and higher extent of type III collagen formation (stronger purple-blue) was also observed in the wounds dressed with the wSF fabric + ser-SF20/G80 than that of 3 MTM Tegaderm high performance foam adhesive dressing (Fig. 8B). The matured collagen with well-arranged structure was observed, particularly at 14 days of treatment. 4. Discussion In this study, we have developed the novel bi-layered wound dressings made of silk sericin, silk fibroin and gelatin which are both non-toxic, biodegradable, and widely used clinically. The

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Fig. 6. H&E-staining images of full-thickness wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing (left) and bi-layered wound dressing of wSF fabric + ser-SF20/G80 (right) for 3, 7, and 14 days.

silk fibroin woven fabric was introduced as a mesh-based layer due to its suitable mechanical and biological properties, as well as slow degradation rate. The carnauba wax was coated on the surface of silk fibroin woven fabric in order to achieve the nonadhesive property and to prevent the dehydration when applied to the wound. Herein, the wax was successfully coated on the silk fibroin woven fabric, as can be confirmed by the higher percentage of weight increased of wSF fabrics along the increasing concentration of wax (Table 1). Furthermore, mechanical properties including tensile modulus and elongation percentage of the fabrics were improved after the wax coating (Table 1). Based on the mechanical data, the fabrics coated with 0.1% wax were selected for the peel test with porcine skin to evaluate its adhesive property, comparing with the clinically wound dressing mesh, Sofra-tulle® . It was proved that our wSF fabrics were less adhesive than the Sofra-tulle® , as confirmed by the less number of cells attached after peeling off and less adhesive force when applied to both the partial- and full-thickness wounds (Fig. 1). Therefore, the 0.1% wSF fabric was used as a slow-degraded non-adhesive layer for the further fabrication of bi-layered wound dressings of this study.

As a bioactive layer, the sponges made of silk fibroin/gelatin composites at different mixing ratios were fabricated. The silk fibroin/gelatin composites have been recently investigated by some researchers (Fan et al., 2008; Jetbumpenkul et al., 2012; Okhawilai et al., 2010; Shubhra et al., 2011). Our previous studies also reported that the chemical crosslinked Thai silk fibroin/gelatin scaffolds offered good mechanical strength from Thai silk fibroin and favored cell attraction from gelatin (Jetbumpenkul et al., 2012; Okhawilai et al., 2010). In this study, various mixing ratios, from silk fibroin-based to gelatin-based (SF80/G20, SF50/G50, and SF20/G80) were studied based on the results of our previous published studies (Jetbumpenkul et al., 2012; Okhawilai et al., 2010). These ratios of SF/G provided good physical and biological properties when fabricated into 3D porous scaffolds or electrospun fiber mats. Notably, in this study, the silk fibroin-based mixture (SF80G20) could not form gel during preparation which was possibly due to the imbalanced electrostatic interaction of this composition. Therefore, only the other two mixing ratios were selected for the further investigation. In addition, sericin was added into the silk fibroin/gelatin composites due to its capability to promote wound healing (Aramwit and Sangcakul, 2007; Aramwit

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Fig. 7. (A) H&E-staining images of full-thickness wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing (left) and bi-layered wound dressing of wSF fabric + ser-SF20/G80 (right) for 7 and 14 days. (B) Percentage of epithelialization of full-thickness wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing () and bi-layered wound dressing of wSF fabric + ser-SF20/G80 () for 3, 7, and 14 days.

et al., 2009). The mixture of silk fibroin/gelatin and sericin were crosslinked with various concentrations of GA in order to alter the physico-chemical properties as well as the degradation rate of the sponges. By GA reaction, crosslinking bonds between amino groups (NH2 ) of protein and aldehyde groups of GA were formed (Kulkarni et al., 1999). Thus, in this study, the degree of crosslinking depended on the contents of NH2 groups (composition of silk fibroin/gelatin) and aldehyde groups (GA concentration). It was found that the crosslinking degree was influenced mainly by the concentration of GA rather than the composition of silk fibroin/gelatin. The higher GA concentration resulted in the higher crosslinking degree, as confirmed by the increased intensity of yellowness index and decreased percentage of weight loss (Table 3). In term of morphology, mixing ratio of silk fibroin/gelatin and concentration of GA did not much affect on the porous structure of the sponges. All formulations of sericin-silk fibroin/gelatin sponges showed interconnected pore structure with similar pore size and porosity (Table 2). On the other hand, the degree of crosslinking strongly altered the degradation rate of the sponges. The slow-degraded sponges crosslinked with 0.020–0.100% GA could remain even after 14 days, irrespective of the mixing ratio of silk fibroin/gelatin (Fig. 3). The controllable degradation rate of the sponges by adjusting the crosslinking degree was widely established (Kulkarni et al., 1999; Tanigo et al., 2010). For the further investigation of in vitro cell culture and in vivo wound model, the maintained three-dimensional matrix of sponges was required to support cell activities and tissue regeneration (Palsson et al., 2003). Then, the slow-degraded sericin-silk fibroin/gelatin sponges crosslinked with 0.020–0.100% GA was chosen to attach to the non-adhesive wSF fabrics in order to prepare

the bi-layered wound dressings. The wSF fabric + ser-SF20/G80 bi-layered wound dressings supported the initial attachment and proliferation of L929 cells at any crosslinking degree (Fig. 3A) possibly due to that the composition of SF20/G80 provided appropriate biological functional groups and surface properties to allow the preadsorption of some proteins, such as fibronectin and vitronectin which would subsequently promoted cell activities (Basson et al., 1990; Kirkpatrick et al., 2007). Interestingly, the wSF fabric + serSF20/G80 crosslinked with lowest concentration of GA (0.02%) was more preferable for cell proliferation (Fig. 3). The reason for this effect was not clear at present. This might be explained that the less crosslinked bi-layered wound dressings degraded faster and would release more amount of sericin to promote the proliferation of fibroblasts (Terada et al., 2002; Tsubouchi et al., 2005). Moreover, it is possible that more content of free amino groups would be remained in the less crosslinked structure. It was reported that the interaction of amino groups with the cell surface receptors could promote cell activities (Belmonte et al., 2005; Curran et al., 2005, 2006; Keselowsky et al., 2004). Based on the results, the wSF fabric + ser-SF20/G80 crosslinked with 0.02% GA was selected as a formulation of bi-layered wound dressing for further in vivo study. Naturally, wound healing process involves coordinated infiltration of dermal cells together with ECM deposition, collagen formation, and re-epithelialization (Reynolds et al., 2005). Epithelialization or epidermal recover is the migration and growth of keratinocytes on neodermis followed by the formation of a complete basement membrane that ensures the structural and mechanical stability of the dermo-epidermal junction (Briggaman and Wheeler, 1975). The epithelialization process depends on selfrenewal, proliferation, and migration of keratinocytes residing at

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Fig. 8. (A) Number of macrophages per field (100×) in the wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing () and bi-layered wound dressing of wSF fabric + ser-SF20/G80 () for 3, 7, and 14 days. *p < 0.05, significant between the groups. (B) Immunohistochemical-staining images of collagen formed in the full-thickness wounds dressed with 3 MTM Tegaderm high performance foam adhesive dressing (left) and bi-layered wound dressing of wSF fabric + ser-SF20/G80 (right) for 3, 7, and 14 days.

the basal cell layer. In this study, the full-thickness wounds dressed with the wSF fabric + ser-SF20/G80 bi-layered wound dressings were healed faster than those dressed with 3 MTM Tegaderm high performance foam adhesive dressing, as confirmed by the reduced wound area, the formation and maturation of collagen tissue, and the increased percentage of epithelialization (Figs. 5–7). Moreover, our bi-layered wound dressings induced the production of type III collagen (Fig. 8B) which is the second most abundant collagen

found in extensible connective tissues such as skin, lung, and the vascular system, in association with type I collagen. To explain the accelerated wound healing, it is supposed that our bi-layered wound dressings were produced from the natural proteins (silk sericin, silk fibroin and gelatin) which are both good biomaterial candidates to promote tissue regeneration (Baoyong et al., 2010; Pra et al., 2005; Schneider et al., 2009; Siri and Maensiri, 2010). Silk fibroin has been reported for its capability to promote

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adhesion and proliferation of keratinocytes and fibroblasts and promote wound healing. It has been used as material for wound dressings in various formulations, such as films, sponges, hydrogels and non-woven mats (Pra et al., 2005; Schneider et al., 2009; Siri and Maensiri, 2010). Baoyong et al. (2010) reported that the recombinant spider silk protein membrane promotes the recovery of wound skin by increasing the expression and secretion of the growth factor bFGF and hydroxyproline. Silk fibroin films employed for the treatment of full-thickness skin wounds in rats healed the wounds faster with a lower inflammatory response than traditional porcine-based wound dressings (Sugihara et al., 2000a). Sponges fabricated from a blend of poly(vinyl alcohol) (PVA), chitosan and silk fibroin potentially healed the epidermal and dermal wounds of rats (Yeo et al., 2000). Sugihara et al. (2000b) reported that wounds dressed with sterilized silk film healed faster than those covered with traditional dressing by promoting the epithelialization. Gelatin is a denatured collagen which is the main component of skin and connective tissue. Gelatin is practically more convenient than commercially used collagen because it is easier to prepare a solution in mild condition (neutral pH) and more economical (Hong et al., 2011). Gelatin contains a number of biological functional groups like amino acids that promote cell activities. Gelatin is also known to exhibit the activation of macrophages and a high haemostatic effect (Tabata and Ikada, 1987). Gelatin-based scaffolds have been experimentally and clinically used as wound dressings to promote wound healing (Marois et al., 1995; Ulubayram et al., 2001; Wang et al., 2006). In addition, sericin was added into the silk fibroin/gelatin composites. Sericin has been proved in term of promoting proliferation of human skin fibroblasts, collagen production and wound healing (Akturk et al., 2011; Aramwit and Sangcakul, 2007; Aramwit et al., 2009; Terada et al., 2002; Tsubouchi et al., 2005). Herein, it is supposed that the degradable silk fibroin/gelatin sponges would control release the sericin to accelerate the wound healing. Furthermore, we found the more number of macrophages in the wounds dressed with the bi-layered wound dressings (Fig. 8A). Although the number of inflammatory cells indicates the extent of inflammation, macrophages play a functional role to trigger the wound regeneration (Davis and Lennon, 2005; Leibovich and Ross, 1975). Macrophages are important in recruiting and activating fibroblasts and other inflammatory cells and producing numerous soluble factors that stimulate fibroblast proliferation (Chang et al., 2000). Moreover, corneal wound healing studies have shown that macrophages are potent stimulators of angiogenesis and collagen synthesis in a cell number dependent fashion (Hunt et al., 1984). Davis and Lennon (2005) found that the increased number of macrophage progenitor cells contributes to the accelerated and scarless tissue regenerative repair response. The induction of macrophages would be another possible reason for the accelerated healing of the wounds dressed with the bi-layered wound dressings. Taken together, the wSF fabric + ser-SF20/G80 bi-layered wound dressings showed the promising results of wound healing, comparing with the clinically used wound dressing “3 MTM Tegaderm high performance foam adhesive dressing”. The superior properties of our bi-layered wound dressings were less adhesive and its biological functions to promote cell activities and wound healing.

5. Conclusion The bi-layered wound dressings of wax-coated silk fibroin woven fabrics with sericin-silk fibroin/gelatin spongy bioactive layers were developed. The layer of wax-coated silk fibroin woven fabrics showed improved mechanical but less adhesive properties than the commercial wound dressing mesh “Sofra-tulle® ”. On

the other hand, the sericin-silk fibroin/gelatin spongy bioactive layers were biodegradable with controlled rate depending on the degree of crosslinking. The bi-layered wound dressings supported the attachment and proliferation of L929 mouse fibroblasts and promoted the healing in full-thickness wounds, comparing with the clinically used wound dressing “3 MTM Tegaderm high performance foam adhesive dressing”.

Acknowledgment This research was supported by Agricultural Research Development Agency.

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