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International Journal of Pharmaceutics 436 (2012) 141– 153

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

jo ur nal homep a ge: www.elsev ier .com/ locate / i jpharm

n innovative bi-layered wound dressing made of silk and gelatin for acceleratedound healing

orada Kanokpanonta, Siriporn Damrongsakkula, Juthamas Ratanavaraporna, Pornanong Aramwitb,∗

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

r t i c l e i n f o

rticle history:eceived 17 April 2012eceived in revised form 18 June 2012ccepted 21 June 2012vailable online 4 July 2012

eywords:ilk fibroinelatinilk sericini-layered wound dressing

a b s t r a c t

In this study, the novel silk fibroin-based bi-layered wound dressing was developed. Wax-coated silkfibroin woven fabric was introduced as a non-adhesive layer while the sponge made of sericin andglutaraldehyde-crosslinked silk fibroin/gelatin was fabricated as a bioactive layer. Wax-coated silk fibroinfabrics showed improved mechanical properties compared with the non-coated fabrics, but less adhesivethan the commercial wound dressing mesh. This confirmed by results of peel test on both the partial-and full-thickness wounds. The sericin-silk fibroin/gelatin spongy bioactive layers showed homogeneousporous structure and controllable biodegradation depending on the degree of crosslinking. The bi-layeredwound dressings supported the attachment and proliferation of L929 mouse fibroblasts, particularlyfor the silk fibroin/gelatin ratio of 20/80 and 0.02% GA crosslinked. Furthermore, we proved that thebi-layered wound dressings promoted wound healing in full-thickness wounds, comparing with the

ound healing clinically used wound dressing. The wounds treated with the bi-layered wound dressings showed thegreater extent of wound size reduction, epithelialization, and collagen formation. The superior propertiesof the silk fibroin-based bi-layered wound dressings compared with those of the clinically used wounddressings were less adhesive and had improved biological functions to promote cell activities and woundhealing. This novel bi-layered wound dressing should be a good candidate for the healing of full-thicknesswounds.

. Introduction

A large number of wound dressings are currently used in thereatment of burns, chronic ulcers, decubitus ulcers, etc. (Suzukit al., 1997; Tanihara et al., 1998). An ideal wound dressing shouldrevent dehydration of the wound and retain a favorable moistnvironment at the wound interface, allow gas permeability, andct as a barrier against dust and microorganisms. Also, it shoulde non-adherent and easily removed without trauma. Woundressings are generally made of readily available biomaterialshat require minimal processing, possess nontoxic, non-allergenic,nd antimicrobial properties, as well as promote wound heal-ng (Jayakumar et al., 2011). Recently, many research groups areocusing in the production of the novel wound dressings by synthe-izing or modifying biocompatible materials (Shibata et al., 1997;

lubayram et al., 2001). Current strategies also point out the accel-ration of the wound repair by systematically designed dressingaterials. By this direction, most efforts have experimentally and

∗ Corresponding author. Tel.: +66 89 921 7255; fax: +66 2 218 8403.E-mail address: aramwit@gmail.com (P. Aramwit).

378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2012.06.046

© 2012 Elsevier B.V. All rights reserved.

clinically utilized the biologically derived materials such as col-lagen, chitin, chitosan, etc., which are capable of accelerating thehealing processes at molecular, cellular, and systemic levels, asmaterials 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 compo-nents, i.e. glycine and alanine, are specific sequence of non-polaramino acids. It has been widely used as biomaterials by humansfor centuries, such as suture materials from silk fibers of silk-worm (Bombyx mori). Silk fibroin has also been received even moreinterests on the broad biomedical applications due to its uniquephysical, mechanical and biological properties, including strength,toughness, elasticity, lightweight, biocompatibility, biodegradabil-ity, minimal inflammatory reaction, capability to promote woundhealing, and easy chemical modification to suit the applications(Moy et al., 1991). Then, it has recently become a new familyof advanced tissue engineered biomaterials. Due to the abilityto promote adhesion and proliferation of various cells includ-

ing keratinocytes and fibroblasts, silk fibroin has been a potentialbiomaterial 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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uccess of wound dressings made of silk fibroin for the recoveryf skin wounds by its favorable physical, mechanical and bio-ogical properties (Baoyong et al., 2010; Sugihara et al., 2000b;eo et al., 2000). However, a few researches focus on the non-dherent property of the silk fibroin dressing. In a part of thistudy, we have established the silk fibroin woven fabric coatedith carnauba wax as a non-adhesive layer of a bi-layered woundressing.

Carnauba wax is a natural edible coating material, whichs recovered from the underside of the leaves of a Brazilianalm tree (Copernica cerifera). It is mainly used to retard mois-ure loss and impart glossiness (Embuscado and Huber, 2009).he beneficial role of carnauba wax is well known for enhanc-ng shelf life and maintaining post-harvest quality of fruitsKhuyen et al., 2008). Our wax-coated silk fibroin woven fabricould be advantageous in terms of the prevention of dehy-ration for the wound and easily removal to reduce trauma.echanical properties of the wax-coated silk fibroin woven

abric were characterized. Peel test of this fabric layer was per-ormed in the partial- and full-thickness wounds of porcine skin,omparing with the commercial wound dressing mesh “Sofra-ulle®”.

Apart from the non-adhesive layer, bioactive layer is introducedo accelerate wound healing and function as a three-dimensional

atrix to support cell activities and tissue regeneration. In thistudy, a composite of silk fibroin/gelatin (SF/G) was selected toroduce the spongy bioactive layer that attached to the wax-oated silk fibroin woven fabric. Silk fibroin/gelatin compositesave been investigated in terms of their superior properties overhe single or other combinations (Jetbumpenkul et al., 2012;khawilai et al., 2010; Shubhra et al., 2011). The outstandingoints of the silk fibroin/gelatin composites are the unique mechan-

cal properties derived from �-sheet structure of silk fibroinnd the improved specific biological characteristics derived fromrg-Gly-Asp (RGD) sequence of gelatin. In addition, sericin of

silk gum was introduced into the silk fibroin/gelatin com-osites of this study. Sericin is highly hydrophilic with strongolar side chains such as hydroxyl, carboxyl and amino groups.hus, it is easy for the cross-linking, copolymerization and blend-ng with other polymers to produce materials with improvedroperties (Ahn et al., 2001; Nagura et al., 2001). It has beenemonstrated that after blending with gelatin or other poly-ers, silk sericin can form a scaffold and be a good candidate

or tissue engineering applications (Mandal et al., 2009; Aramwitt al., 2010). It was also reported that sericin enhanced mam-alian cell attachment and proliferation of human skin fibroblasts

Terada et al., 2002; Tsubouchi et al., 2005). Our previous studyhowed that sericin enhances wound healing by promoting col-agen production (Aramwit and Sangcakul, 2007; Aramwit et al.,009).

In this study, silk fibroin/gelatin at different mixing ratios waslended with sericin solution to prepare the spongy bioactive lay-rs that attached to the wax-coated silk fibroin fabrics by thereeze-drying and glutaraldehyde (GA) crosslinking techniques,efined as the bi-layered wound dressings. Different crosslinkingegrees were varied by adjusting the GA concentration. Mor-hology, crosslinking extent, and in vitro degradation rate of theilk fibroin/gelatin bioactive layers were investigated. L929 mousebroblast cells were cultured on the bi-layered wound dress-

ngs developed to evaluate cell attachment and proliferation. Inddition, our bi-layered wound dressings were tested with the full-hickness wounds of rat model to evaluate the efficiency of wound

ealing characterized by the reduction of wound area, epithelial-

zation, and the production of collagen tissue, comparing with thelinically used wound dressing “3 MTM Tegaderm high performanceoam adhesive dressing”.

f Pharmaceutics 436 (2012) 141– 153

2. Materials and methods

2.1. Materials

Silk fibroin (SF) woven fabric was purchased from Chul ThaiSilk Co., Ltd., Phetchabul province, Thailand. Thai silk strain, B.mori (Nangnoi Srisaket 1) was supplied by Queen Sirikit SericultureCenter, Nakornratchasima province, Thailand. Carnauba wax (No.1, yellow) was purchased from Sigma–Aldrich Laborchemikelien,Germany. A gelatin (G) sample prepared by an acidic treatment ofporcine skin collagen (isoelectric point (IEP) = 9.0) was kindly sup-plied by Nitta Gelatin Inc., Osaka, Japan. Glutaraldehyde (GA) andother chemicals were analytical grade and used without furtherpurification.

2.2. Preparation of wax-coated silk fibroin woven fabrics

Carnauba wax was dissolved in morpholine solution at differ-ent concentrations (0.025, 0.050, and 0.100% w/v). The silk fibroinwoven fabric (5 in. × 5 in.) was stretched and immersed in the waxsolution at room temperature for 20 min, and then dried overnightto obtain the wax-coated silk fibroin woven fabric, defined as wSFfabric.

2.3. Characterization of wax-coated silk fibroin woven fabrics

2.3.1. Mechanical characterizationThe tensile test was performed on the wSF fabrics (150 mm in

length and 25 mm in width) at room temperature using a uni-versal testing machine (Instron, No. 5567) at a constant rate of30 mm/min. The curves of force as a function of deformation (mm)were automatically recorded by the software. The tensile modu-lus (MPa) and elongation at break were calculated according to theASTM D638-01 method (n = 6).

2.3.2. Weight increased after wax coatingThe fabrics before and after coating with different concen-

trations of carnauba wax were weighed. Percentage of weightincreased was calculated (n = 3).

2.3.3. Peel test with porcine skinPorcine skin was used within 2 h after sacrifice. Partial- and

full-thickness (1 cm in depth) wounds were prepared by scrap-ing the outer skin layer and by cutting the skin at 1 cm in depth,respectively. The wSF fabrics and the commercial wound dressingmesh “Sofra-tulle®” (Patheon UK Limited, Swindon, UK) immersedin phosphate-buffered saline solution (PBS, pH 7.4) were attachedon the wounds. After 12 h, the dressings were removed and fixedin 2.5% (v/v) GA solution at 4 ◦C for 1 h. Then, the dressings weredehydrated in serial dilutions of ethanol (50, 70, 80, 90, 95, 99 and100%, v/v, respectively) for 5 min each and the hexamethyldisi-lazane solution was dropped on the dressings for the critical pointdrying. The attachment of cells on the dressings was observed ona scanning electron microscope (SEM, JSM 5400, JEOL) at an accel-erating voltage of 12–15 kV after sputter-coating with gold. Thenumber of cells attached on the dressings was analyzed by the flu-orometric quantification of cellular DNA according to the methodreported by Takahashi et al. (2005). The adhesive force applied topeel the dressings from wounds was also determined by a modi-fied fixed peeling angle peel test (Zhang et al., 2012). Briefly, theporcine skin attached with the dressings (150 mm in length and25 mm in width) was placed on a liner translation stage of a uni-

versal testing machine (Instron, No. 5567) at a fixed peeling angleof 135◦. The sample holder was fixed at the upper side of the dress-ings and the peeling force was applied at a constant tensile rate of5 mm/min. The adhesive force defined as the steady-state peeling

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orce used to peel the dressings from wounds was determined from load–displacement curve (n = 5).

.4. Fabrication of bi-layered wound dressings

.4.1. Preparation of Thai silk fibroin and sericin solutionsThai silk fibroin solution was prepared according to the method

reviously described by Kim et al. (2005). In brief, cocoons wereoiled in an aqueous solution of 0.02 M sodium carbonate (Na2CO3)nd then rinsed thoroughly with deionized (DI) water to removeericin. The degummed silk fibroin was mixed with 9.3 M lithiumromide (LiBr) solution at the ratio 1:3 (w/w). The mixture wastirred occasionally at 60 ◦C for 4 h until the silk fibroin was com-letely dissolved. The solution was dialyzed in DI water for 2 days.he dialyzed water was changed regularly until its conductivityas the same as that of the DI water. The final concentration of

ilk fibroin aqueous solution was about 6–6.5% (w/w). Silk sericinolution was extracted using a high temperature and pressureegumming technique (Lee et al., 2003). Briefly, the silkwormocoons were mixed with DI water (1 g of dry silk cocoon: 30 mLf water) and the samples were autoclaved at 120 ◦C for 60 min.fter filtration through a membrane to remove fibroin, the con-entration of sericin solution was measured by BCA Protein Assayeagent (Pierce, Rockford, IL, USA).

.4.2. Preparation of sericin-silk fibroin/gelatin spongy bioactiveayers attached to the wax-coated silk fibroin woven fabrics

Silk fibroin (SF) and gelatin (G) solutions at the SF/G mixingatios of 50/50 and 20/80 were mixed with 1% (w/w) sericin solutiono obtain a final solution concentration at 4% (w/w), defined as ser-F50/G50 and ser-SF20/G80, respectively. Then, GA was added tohe mixture at the final concentrations of 0.005, 0.010, 0.020, 0.050,nd 0.100% (v/v). After mixing, the mixture was cast onto the wSFabric stretched on the Teflon mold and placed at 4 ◦C for 24 h tollow for the crosslinking reaction. The crosslinked gels were thengitated in 100 mM aqueous glycine solution at room temperatureor 2 h to block the residual aldehyde groups of glutaraldehyde. Fol-owing washing three times with DI water, the gels were frozen at50 ◦C overnight prior to lyophilization for 48 h to obtain the bi-

ayered wound dressings of wax-coated silk fibroin woven fabricsith sericin-silk fibroin/gelatin spongy bioactive layers, defined asSF fabric + ser-SF50/G50 or ser-SF20/G80.

.5. Physico-chemical characterization of sericin-silkbroin/gelatin spongy bioactive layers

.5.1. Morphological observationCross-sectional structure of the ser-SF50/G50 and ser-SF20/G80

ponges was observed on a SEM as described previously. Pore sizeas determined using ImageJ software (the US National Institutes

f Health, USA). The porosity of the sponges was measured by a liq-id displacement (Nazarov et al., 2004). Absolute ethanol was useds the displacement liquid as it permeates through the spongesithout swelling or shrinking the matrix. A known weight of dried

ponge was immersed in a known volume (V1) of ethanol for 5 min.he total volume of ethanol and the ethanol-impregnated spongeas recorded as V2. The ethanol-impregnated sponge was then

emoved and the residual volume of ethanol was recorded as V3.he porosity of the sponge (ε) was obtained by:

V1 − V3

(%) =V2 − V3

× 100

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

f Pharmaceutics 436 (2012) 141– 153 143

2.5.2. Evaluation of crosslinking degreeColor intensity of the crosslinked ser-SF50/G50 and ser-

SF20/G80 sponges was determined using a Minolta colorimeter CR400 Series (Osaka, Japan) calibrated with a standard (Rivero et al.,2010). The CIELab scale was used; lightness (L) and chromatic-ity parameter b* (yellow–blue) were measured (n = 3). In term ofweight loss, a known weight sponge was placed in DI water at roomtemperature for 24 h. The remained sponge was then freeze-driedand weighed. Percentage of weight loss indicating the success ofcrosslinking was calculated (n = 3).

2.5.3. In vitro degradation testA 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, washedwith DI water, freeze dried, and weighed. Percentage of weightremaining was calculated (n = 3).

2.6. In vitro attachment and proliferation tests with L929 cells

L929 mouse fibroblast cells were seeded onto the sterilizedbi-layered wound dressings of wSF fabric + ser-SF50/G50 or ser-SF20/G80 (5 mm in diameter and 2 mm in height) at a densityof 5 × 105 cells/sample and cultured in Dulbecco’s Modified EagleMedium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum(FBS) and 100 U/ml penicillin/streptomycin at 37 ◦C, 5% CO2. Thenumber of cells attached after 6 h and cells proliferated after 5 daysof culture was quantified using the DNA assay (Takahashi et al.,2005). The morphology and density of L929 cells cultured in thesamples for 5 days were observed on a SEM after fixing in 2.5%(v/v) GA solution, dehydration in serial dilutions of ethanol, andcritical point drying as described previously.

2.7. In vivo study of full-thickness wound model

2.7.1. In vivo wound model in ratsAll animal experiments were performed in agreement with the

Chulalongkorn University Animal Care and Use Committee andwith ethics approval from the research ethical committee, Facultyof Medicine, Chulalongkorn University and the Mahidol Univer-sity Animal Care and Use Committee (MU-ACUC). Sprague-Dawleyrats (28-week-old, 250 ± 5 g, n = 24) were purchased from NationalLaboratory Animal Centre, Mahidol University, Nakhon Pathom,Thailand. After being anesthetized and injected with antibiotic, thefull-thickness wounds (1.5 cm × 1.5 cm) were prepared on both leftand right sides of the their back (2 wounds/rat). Wounds weredressed with the bi-layered wound dressing developed and thecommercial wound dressing “3 MTM Tegaderm high performancefoam adhesive dressing (3 M Corporate Headquarters, MN, USA)”.

2.7.2. Measurement of wound size reductionSize 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 cal-culated (n = 6).

2.7.3. Histological and immunohistochemical evaluationThe tissue regenerated at the wounds was collected at 3, 7,

and 14 days after operation, fixed in 10% (v/v) formalin, and thenembedded in a paraffin block. The paraffin-embedded tissues weresectioned (4 �m thickness) and stained with Hematoxylin andEosin (H&E) to evaluate the epithelialization, collagen formation,

and infiltration of cells. For the immunohistochemical staining, thesections were washed with PBS and blocked with a normal goatserum (Santa Cruz Biotechnology, Inc., CA, USA) at room temper-ature for 1 h. The sections were incubated with a mouse anti-rat

144 S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153

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- andf ® rics aff e partg

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All the results were statistically analyzed by the unpairedstudent’s t test and p < 0.05 was considered to be statistically sig-nificant. Data were expressed as mean ± standard deviation.

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

Concentrationof wax (%)

Tensile modulus(MPa)

Elongation (%) Weight increased(%)

0.000 211.2 ± 44.7 21.6 ± 0.1 –

ull-thickness wounds for 12 h. (C) Number of cells on the Sofra-tulle 0.1% wSF faborce applied to peel the Sofra-tulle® and 0.1% wSF fabrics off after attached to throups.

acrophage class HIS 36 (1:800, Santa Cruz Biotechnology, Inc.,A, USA) or a rabbit anti-rat collagen type III monoclonal antibody1:80, Santa Cruz Biotechnology, Inc., CA, USA) at 4 ◦C overnight forhe macrophages and type III collagen, respectively. Then, the sec-ions were washed with PBS and stained with a biotinylated goatnti-mouse antibody (Santa Cruz Biotechnology, Inc., CA, USA) or aoat anti-rabbit antibody (Dako, Denmark), respectively, for 30 mint room temperature. For bright-field microscopy, bound pri-ary antibodies were detected using DAKO EnVision-Horseradish

eroxidase and 3, 30-diaminobenzidine (DAB) substrate kit (Vec-or Laboratories, Burlingame, CA, USA), and counter-stained withematoxylin to visualize the cell nuclei. Then, the sections were

ounted with vectamount (Vector Laboratories, Inc., Burlingame,

A, USA) and the images were taken on a microscope. The num-er of macrophages was counted from the images taken at 100×agnification randomly selected.

ter attached to the partial- (�) and full-thickness wounds (�) for 12 h. (D) Adhesiveial- (�) and full-thickness wounds (�) for 12 h. * p < 0.05, significant between the

2.8. Statistical analysis

0.025 209.1 ± 23.9 28.7 ± 0.4* 1.1 ± 0.10.050 200.8 ± 43.6 29.3 ± 1.7* 6.8 ± 0.10.100 245.7 ± 10.0 * 30.7 ± 1.1* 10.2 ± 1.9

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

S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153 145

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 andser-SF20/G80 spongy bioactive layers crosslinked with different concentrations of GA (0.005, 0.010, 0.020, 0.050, and 0.100%).

146 S. Kanokpanont et al. / International Journal o

Fig. 3. In vitro degradation profiles of (A) ser-SF50/G50 and (B) ser-SF20/G80 spongybioactive layers crosslinked with different concentrations of GA in a PBS solutioncontaining collagenase (1 Unit/ml, pH 7.4) at 37 ◦C. Concentrations of GA crosslink-i

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. Results

.1. Properties of wax-coated silk fibroin woven fabrics

Table 1 presents the physical properties of silk fibroin wovenabrics coated with different concentrations of carnauba wax. Ten-ile modulus was improved for the fabrics coated with 0.1% wax

able 2orphological properties of silk fibroin/gelatin (SF/G) spongy bioactive layers crosslinked

Concentration of GA crosslinking (%) Pore size (�m)

SF50/G50 SF20/G80

0.005 240.9 ± 78.7 171.4 ± 54.9

0.010 209.7 ± 58.5 165.6 ± 61.1

0.020 230.9 ± 53.8 145.6 ± 43.1

0.050 205.1 ± 45.1 164.6 ± 67.2

0.100 166.9 ± 45.4 191.6 ± 43.9

able 3olorimetric properties and weight loss of silk fibroin/gelatin (SF/G) spongy bioactive lay

Concentration of GA crosslinking (%) Brightness (L)

SF50/G50 SF20/G80

0.005 79.02 79.27

0.010 79.67 79.02

0.020 80.42 81.15

0.050 83.97 86.94

0.100 77.76 89.97

f Pharmaceutics 436 (2012) 141– 153

(245.7 MPa) while the percentage of elongation was significantlyincreased for any concentration of wax coating. Percentage ofweight increased rose along the increasing concentration of waxcoated.

The macro- and microscopic images of wSF fabrics showed thesmooth surface (Fig. 1A). Fig. 1B shows the microscopic imagesof Sofra-tulle® and 0.1% wSF fabric after attached to the partial-and full-thickness wounds for 12 h. A large number of cells wereattached on the Sofra-tulle® when applied at both the partial- andfull-thickness wounds. On the other hand, less number of cells wasobserved on the 0.1% wSF fabric. The number of cells attached wasquantitatively confirmed by DNA assay (Fig. 1C). The number ofcells attached on the 0.1% wSF fabric was significantly lower thanthat on the Sofra-tulle® for both the partial- and full-thicknesswounds. The adhesive force of the 0.1% wSF fabric attached tothe wounds was significantly lower than that of the Sofra-tulle®

(Fig. 1D).

3.2. Morphology of sericin-silk fibroin/gelatin spongy bioactivelayers

The procedure of sericin-silk fibroin/gelatin solution castinginto Teflon mold to prepare the bi-layered wound dressings ofwSF fabric + ser-SF50/G50 or ser-SF20/G80 is shown in Fig. 2A.Fig. 2B shows the cross-sectioned images of ser-SF50/G50 andser-SF20/G80 sponges crosslinked with different concentrationsof GA. All formulations of sponges presented homogeneous porestructure; however, pores of the sponges crosslinked with lowestconcentration of GA (0.005%) seemed more irregular in shape. Thestructure 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 theporous sponges was around 0.09–0.17 mg/mm3.

3.3. Crosslinking properties of sericin-silk fibroin/gelatin spongybioactive layers

Brightness of all sponges was around 77.76–89.97. However, theintensity of yellowness index tended to increase along the increas-

ing concentration of GA, representing higher crosslinking degree ofthe sponges (Table 3). Correspondingly, lower percentage of weightloss was observed for the sponges crosslinked with higher concen-tration of GA (Table 3).

with different concentrations of GA.

Porosity (%) Density (mg/mm3)

SF50/G50 SF20/G80 SF50/G50 SF20/G80

80.6 ± 0.2 89.3 ± 1.7 0.09 ± 0.005 0.12 ± 0.00589.1 ± 0.6 92.4 ± 1.1 0.11 ± 0.004 0.14 ± 0.00881.7 ± 1.3 94.8 ± 1.4 0.12 ± 0.012 0.17 ± 0.00190.8 ± 0.5 92.7 ± 0.4 0.13 ± 0.007 0.15 ± 0.00796.3 ± 2.5 86.2 ± 2.1 0.14 ± 0.029 0.14 ± 0.009

ers crosslinked with different concentrations of GA.

Intensity of yellowness index (b*) Weight loss (%)

SF50/G50 SF20/G80 SF50/G50 SF20/G80

11.10 9.45 36.1 ± 0.4 41.0 ± 5.714.47 11.10 25.6 ± 10.9 35.7 ± 3.916.65 14.47 10.5 ± 2.1 10.1 ± 2.218.89 16.90 17.8 ± 3.7 11.5 ± 3.920.58 18.90 21.8 ± 3.0 19.4 ± 2.9

S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153 147

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 ser-SF20/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 cellsc 20/G8d

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ultured on the bi-layered wound dressings of wSF fabric + ser-SF50/G50 or ser-SFays.

.4. Degradation profiles of sericin-silk fibroin/gelatin spongyioactive layers

Fig. 3 shows the in vitro degradation profiles of ser-SF50/G50nd ser-SF20/G80 sponges crosslinked with different concentra-

ions of GA. The sponges crosslinked with higher GA concentrationegraded slower than those crosslinked with lower GA concentra-ion. The sponges crosslinked with 0.020–0.100% GA remained evenfter 14 days of the degradation period while those crosslinked with

0 crosslinked with different concentrations of GA (0.020, 0.050, and 0.100%) for 5

0.005 and 0.010% GA degraded completely within 14 days. The sim-ilar degradation profiles were observed for both the ser-SF50/G50and 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 thebi-layered wound dressings of wSF fabric + ser-SF50/G50 or

148 S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153

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-layeredw of fup ric + s

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ound dressing of wSF fabric + ser-SF20/G80 (right) for 3, 7, and 14 days. (B) Areaerformance foam adhesive dressing (�) and bi-layered wound dressing of wSF fab

er-SF20/G80 were shown in Fig. 4A. After 6 h of culture, the num-er of cells initially attached on the wSF fabric + ser-SF20/G80 wasigher than that of the wSF fabric + ser-SF50/G50, irrespective ofA concentration. Furthermore, after 5 days of culture, the numberf cells proliferated on the wSF fabric + ser-SF20/G80 crosslinkedt 0.020% GA was significantly higher than that of other groups.he cells showed spreading morphology and proliferated homoge-eously throughout the spongy structure (Fig. 4B). High density ofells proliferated on the wSF fabric + ser-SF20/G80 crosslinked at.020% GA was confirmed.

.6. Regeneration of full-thickness wounds

Fig. 5 shows gross images and the area of wounds afterressed with 3 MTM Tegaderm high performance foam adhe-ive dressing and the bi-layered wound dressings of the wSFabric + ser-SF20/G80 crosslinked at 0.020% GA for 3, 7, and 14ays. After dressed with both types of dressings, wound areaas reduced along the treatment period. However, the area ofounds dressed with the wSF fabric + ser-SF20/G80 seemed to be

maller than that of the 3 MTM Tegaderm high performance foamdhesive dressing. The wSF fabric + ser-SF20/G80 almost com-letely healed the wounds within 14 days of treatment.

H&E-staining images of the wounds after dressed with 3 MTM

egaderm high performance foam adhesive dressing and the bi-ayered wound dressings for 3, 7, and 14 days were presented in

ig. 6. At 3 days post-operation, a number of inflammatory cellsere observed for both samples. The wounds dressed with the bi-

ayered wound dressings showed higher extent of newly formedollagen tissue than those of 3 MTM Tegaderm high performance

ll-thickness wounds before (0 days) and after dressed with 3 MTM Tegaderm higher-SF20/G80 (�) for 3, 7, and 14 days.

foam adhesive dressing along treatment period. Matured collagenwas formed in the wounds dressed with the bi-layered wounddressings from 7 days, while that was observed at 14 days forthe wounds dressed with 3 MTM Tegaderm high performance foamadhesive dressing.

Fig. 7A shows the gap between epithelial tips and epithelialtongue of the wounds after dressed with 3 MTM Tegaderm highperformance foam adhesive dressing and the bi-layered wounddressings of the wSF fabric + ser-SF20/G80 crosslinked at 0.020%GA for 7 and 14 days. Epithelialization percentage of the woundsdressed with the wSF fabric + ser-SF20/G80 was increased withtime (Fig. 7B). The epithelialization was reached at 50% in thewounds dressed with the wSF fabric + ser-SF20/G80 for 14 dayswhile the treatment with 3 MTM Tegaderm high performance foamadhesive dressing did not promote any epithelialization.

More number of macrophages was found in the wounds dressedwith the wSF fabric + ser-SF20/G80 along 14-day period, comparingwith that of the 3 MTM Tegaderm high performance foam adhe-sive dressing treatment (Fig. 8A). Earlier and higher extent of typeIII collagen formation (stronger purple-blue) was also observedin the wounds dressed with the wSF fabric + ser-SF20/G80 thanthat of 3 MTM Tegaderm high performance foam adhesive dress-ing (Fig. 8B). The matured collagen with well-arranged structurewas observed, particularly at 14 days of treatment.

4. Discussion

In this study, we have developed the novel bi-layered wounddressings made of silk sericin, silk fibroin and gelatin which areboth non-toxic, biodegradable, and widely used clinically. The

S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153 149

F highf

sdasatsccetossSsab0fs

ig. 6. H&E-staining images of full-thickness wounds dressed with 3 MTM Tegadermabric + ser-SF20/G80 (right) for 3, 7, and 14 days.

ilk fibroin woven fabric was introduced as a mesh-based layerue to its suitable mechanical and biological properties, as wells slow degradation rate. The carnauba wax was coated on theurface of silk fibroin woven fabric in order to achieve the non-dhesive property and to prevent the dehydration when appliedo the wound. Herein, the wax was successfully coated on theilk fibroin woven fabric, as can be confirmed by the higher per-entage of weight increased of wSF fabrics along the increasingoncentration of wax (Table 1). Furthermore, mechanical prop-rties including tensile modulus and elongation percentage ofhe fabrics were improved after the wax coating (Table 1). Basedn the mechanical data, the fabrics coated with 0.1% wax wereelected for the peel test with porcine skin to evaluate its adhe-ive property, comparing with the clinically wound dressing mesh,ofra-tulle®. It was proved that our wSF fabrics were less adhe-ive than the Sofra-tulle®, as confirmed by the less number of cellsttached after peeling off and less adhesive force when applied to

oth the partial- and full-thickness wounds (Fig. 1). Therefore, the.1% wSF fabric was used as a slow-degraded non-adhesive layeror the further fabrication of bi-layered wound dressings of thistudy.

performance foam adhesive dressing (left) and bi-layered wound dressing of wSF

As a bioactive layer, the sponges made of silk fibroin/gelatincomposites at different mixing ratios were fabricated. The silkfibroin/gelatin composites have been recently investigated bysome researchers (Fan et al., 2008; Jetbumpenkul et al., 2012;Okhawilai et al., 2010; Shubhra et al., 2011). Our previous studiesalso reported that the chemical crosslinked Thai silk fibroin/gelatinscaffolds offered good mechanical strength from Thai silk fibroinand 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 previouspublished studies (Jetbumpenkul et al., 2012; Okhawilai et al.,2010). These ratios of SF/G provided good physical and biologicalproperties when fabricated into 3D porous scaffolds or electrospunfiber mats. Notably, in this study, the silk fibroin-based mixture(SF80G20) could not form gel during preparation which waspossibly due to the imbalanced electrostatic interaction of this

composition. Therefore, only the other two mixing ratios wereselected for the further investigation. In addition, sericin was addedinto the silk fibroin/gelatin composites due to its capability topromote wound healing (Aramwit and Sangcakul, 2007; Aramwit

150 S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153

F adermo izationa (�) for

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ig. 7. (A) H&E-staining images of full-thickness wounds dressed with 3 MTM Tegf wSF fabric + ser-SF20/G80 (right) for 7 and 14 days. (B) Percentage of epithelialdhesive dressing (�) and bi-layered wound dressing of wSF fabric + ser-SF20/G80

t al., 2009). The mixture of silk fibroin/gelatin and sericin wererosslinked with various concentrations of GA in order to alter thehysico-chemical properties as well as the degradation rate of theponges. By GA reaction, crosslinking bonds between amino groupsNH2) of protein and aldehyde groups of GA were formed (Kulkarnit al., 1999). Thus, in this study, the degree of crosslinking dependedn the contents of NH2 groups (composition of silk fibroin/gelatin)nd aldehyde groups (GA concentration). It was found that therosslinking degree was influenced mainly by the concentration ofA rather than the composition of silk fibroin/gelatin. The higherA concentration resulted in the higher crosslinking degree, asonfirmed by the increased intensity of yellowness index andecreased percentage of weight loss (Table 3).

In term of morphology, mixing ratio of silk fibroin/gelatin andoncentration of GA did not much affect on the porous struc-ure of the sponges. All formulations of sericin-silk fibroin/gelatinponges showed interconnected pore structure with similar poreize and porosity (Table 2). On the other hand, the degree ofrosslinking strongly altered the degradation rate of the sponges.he slow-degraded sponges crosslinked with 0.020–0.100% GAould remain even after 14 days, irrespective of the mixing ratiof silk fibroin/gelatin (Fig. 3). The controllable degradation rate ofhe sponges by adjusting the crosslinking degree was widely estab-ished (Kulkarni et al., 1999; Tanigo et al., 2010).

For the further investigation of in vitro cell culture and in vivoound model, the maintained three-dimensional matrix of

ponges was required to support cell activities and tissue regen-ration (Palsson et al., 2003). Then, the slow-degraded sericin-silkbroin/gelatin sponges crosslinked with 0.020–0.100% GA washosen to attach to the non-adhesive wSF fabrics in order to prepare

high performance foam adhesive dressing (left) and bi-layered wound dressing of full-thickness wounds dressed with 3 MTM Tegaderm high performance foam

3, 7, and 14 days.

the bi-layered wound dressings. The wSF fabric + ser-SF20/G80bi-layered wound dressings supported the initial attachment andproliferation of L929 cells at any crosslinking degree (Fig. 3A) possi-bly due to that the composition of SF20/G80 provided appropriatebiological functional groups and surface properties to allow the pre-adsorption of some proteins, such as fibronectin and vitronectinwhich would subsequently promoted cell activities (Basson et al.,1990; Kirkpatrick et al., 2007). Interestingly, the wSF fabric + ser-SF20/G80 crosslinked with lowest concentration of GA (0.02%) wasmore preferable for cell proliferation (Fig. 3). The reason for thiseffect was not clear at present. This might be explained that the lesscrosslinked bi-layered wound dressings degraded faster and wouldrelease more amount of sericin to promote the proliferation offibroblasts (Terada et al., 2002; Tsubouchi et al., 2005). Moreover,it is possible that more content of free amino groups would beremained in the less crosslinked structure. It was reported thatthe interaction of amino groups with the cell surface receptorscould promote cell activities (Belmonte et al., 2005; Curran et al.,2005, 2006; Keselowsky et al., 2004). Based on the results, the wSFfabric + ser-SF20/G80 crosslinked with 0.02% GA was selected as aformulation of bi-layered wound dressing for further in vivo study.

Naturally, wound healing process involves coordinated infil-tration of dermal cells together with ECM deposition, collagenformation, and re-epithelialization (Reynolds et al., 2005). Epithe-lialization or epidermal recover is the migration and growthof keratinocytes on neodermis followed by the formation of a

complete basement membrane that ensures the structural andmechanical stability of the dermo-epidermal junction (Briggamanand Wheeler, 1975). The epithelialization process depends on self-renewal, proliferation, and migration of keratinocytes residing at

S. Kanokpanont et al. / International Journal of Pharmaceutics 436 (2012) 141– 153 151

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 wounddressing 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 thefull-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) for3

twwpwtoI

, 7, and 14 days.

he basal cell layer. In this study, the full-thickness wounds dressedith the wSF fabric + ser-SF20/G80 bi-layered wound dressingsere healed faster than those dressed with 3 MTM Tegaderm higherformance foam adhesive dressing, as confirmed by the reduced

ound area, the formation and maturation of collagen tissue, and

he increased percentage of epithelialization (Figs. 5–7). Moreover,ur bi-layered wound dressings induced the production of typeII collagen (Fig. 8B) which is the second most abundant collagen

found in extensible connective tissues such as skin, lung, and thevascular system, in association with type I collagen.

To explain the accelerated wound healing, it is supposed thatour bi-layered wound dressings were produced from the natural

proteins (silk sericin, silk fibroin and gelatin) which are both goodbiomaterial candidates to promote tissue regeneration (Baoyonget 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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52 S. Kanokpanont et al. / International Jou

dhesion and proliferation of keratinocytes and fibroblasts andromote wound healing. It has been used as material for woundressings in various formulations, such as films, sponges, hydro-els and non-woven mats (Pra et al., 2005; Schneider et al., 2009;iri and Maensiri, 2010). Baoyong et al. (2010) reported that theecombinant spider silk protein membrane promotes the recoveryf wound skin by increasing the expression and secretion of therowth factor bFGF and hydroxyproline. Silk fibroin films employedor the treatment of full-thickness skin wounds in rats healed theounds faster with a lower inflammatory response than traditionalorcine-based wound dressings (Sugihara et al., 2000a). Spongesabricated from a blend of poly(vinyl alcohol) (PVA), chitosan andilk fibroin potentially healed the epidermal and dermal woundsf rats (Yeo et al., 2000). Sugihara et al. (2000b) reported thatounds dressed with sterilized silk film healed faster than those

overed with traditional dressing by promoting the epithelializa-ion. Gelatin is a denatured collagen which is the main componentf skin and connective tissue. Gelatin is practically more convenienthan commercially used collagen because it is easier to prepare aolution in mild condition (neutral pH) and more economical (Hongt al., 2011). Gelatin contains a number of biological functionalroups like amino acids that promote cell activities. Gelatin is alsonown to exhibit the activation of macrophages and a high haemo-tatic effect (Tabata and Ikada, 1987). Gelatin-based scaffolds haveeen experimentally and clinically used as wound dressings to pro-ote wound healing (Marois et al., 1995; Ulubayram et al., 2001;ang et al., 2006).In addition, sericin was added into the silk fibroin/gelatin com-

osites. Sericin has been proved in term of promoting proliferationf human skin fibroblasts, collagen production and wound heal-ng (Akturk et al., 2011; Aramwit and Sangcakul, 2007; Aramwitt al., 2009; Terada et al., 2002; Tsubouchi et al., 2005). Herein, its supposed that the degradable silk fibroin/gelatin sponges wouldontrol release the sericin to accelerate the wound healing.

Furthermore, we found the more number of macrophages inhe wounds dressed with the bi-layered wound dressings (Fig. 8A).lthough the number of inflammatory cells indicates the extentf inflammation, macrophages play a functional role to trigger theound regeneration (Davis and Lennon, 2005; Leibovich and Ross,

975). Macrophages are important in recruiting and activatingbroblasts and other inflammatory cells and producing numer-us soluble factors that stimulate fibroblast proliferation (Changt al., 2000). Moreover, corneal wound healing studies have shownhat macrophages are potent stimulators of angiogenesis and col-agen synthesis in a cell number dependent fashion (Hunt et al.,984). Davis and Lennon (2005) found that the increased num-er of macrophage progenitor cells contributes to the acceleratednd scarless tissue regenerative repair response. The induction ofacrophages would be another possible reason for the accelerated

ealing of the wounds dressed with the bi-layered wound dress-ngs.

Taken together, the wSF fabric + ser-SF20/G80 bi-layered woundressings showed the promising results of wound healing, com-aring with the clinically used wound dressing “3 MTM Tegadermigh performance foam adhesive dressing”. The superior proper-ies of our bi-layered wound dressings were less adhesive and itsiological functions to promote cell activities and wound healing.

. Conclusion

The bi-layered wound dressings of wax-coated silk fibroin

oven fabrics with sericin-silk fibroin/gelatin spongy bioactive lay-

rs were developed. The layer of wax-coated silk fibroin wovenabrics showed improved mechanical but less adhesive proper-ies than the commercial wound dressing mesh “Sofra-tulle®”. On

f Pharmaceutics 436 (2012) 141– 153

the other hand, the sericin-silk fibroin/gelatin spongy bioactivelayers were biodegradable with controlled rate depending on thedegree of crosslinking. The bi-layered wound dressings supportedthe attachment and proliferation of L929 mouse fibroblasts andpromoted the healing in full-thickness wounds, comparing with theclinically used wound dressing “3 MTM Tegaderm high performancefoam adhesive dressing”.

Acknowledgment

This research was supported by Agricultural Research Develop-ment Agency.

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