Research Article Preliminary Characterization of Genipin...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 904314 13 pageshttpdxdoiorg1011552013904314

Research ArticlePreliminary Characterization of Genipin-Cross-Linked SilkSericinPoly(vinyl alcohol) Films as Two-Dimensional WoundDressings for the Healing of Superficial Wounds

Tippawan Siritientong12 Juthamas Ratanavaraporn3

Teerapol Srichana4 and Pornanong Aramwit12

1 Bioactive Resources for Innovative Clinical Applications Research Unit Chulalongkorn University Phayathai RoadPathumwan Bangkok 10330 Thailand

2Department of Pharmacy Practice Faculty of Pharmaceutical Sciences Chulalongkorn University Phayathai RoadPathumwan Bangkok 10330 Thailand

3Department of Chemical Engineering Faculty of Engineering Chulalongkorn University Phayathai Road PathumwanBangkok 10330 Thailand

4Department of Pharmaceutical Technology and Drug Delivery System Excellence Center Faculty of Pharmaceutical SciencesPrince of Songkla University Hat Yai Songkhla 90110 Thailand

Correspondence should be addressed to Pornanong Aramwit aramwitgmailcom

Received 9 May 2013 Accepted 7 August 2013

Academic Editor Joshua R Mauney

Copyright copy 2013 Tippawan Siritientong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The genipin-cross-linked silk sericinpoly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensionalwound dressings for the treatment of superficial wounds The effects of genipin cross-linking concentration on the physical andbiological properties of the films were investigated The genipin-cross-linked silk sericinPVA films showed the increased surfacedensity tensile strength and percentage of elongation but decreased percentage of light transmission water vapor transmissionrate and water swelling compared to the non-cross-linked films This explained that the cross-linking bonds between genipinand silk sericin would reduce the mobility of molecular chains within the films resulting in the more rigid molecular structureSilk sericin was released from the genipin-cross-linked films in a sustained manner In addition either L929 mouse fibroblast orHaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericinPVA films cross-linked with 0075and 01wv genipin The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silksericinPVA films were safe for the medical usages The efficacy of the films for the treatment of superficial skin wounds will befurther investigated in vivo and clinically The genipin-cross-linked silk sericinPVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds

1 Introduction

Epidermal damage as a result of ulcer burn or other trau-matic incidents requires the treatment with wound dressingfor the accelerated regeneration with functional satisfactionRoles of wounddressing are to protect thewound and providethe optimal microenvironment as well as activate cellularresponses [1] Despite the availability of numerous typesof wound dressings there is still a high demand for the

development of more effective and affordable ones Wounddressings made of natural proteins or polysaccharides (egcollagen calcium alginate chitin and chitosan) and syntheticmaterials (eg silicone gel poly(ethylene glycol) and poly(l-leucine)) have been widely studied in research area and alsoused clinically [2 3] Silk protein sericin derived from thesilkworm cocoon has been recently investigated for possiblenew applications in the biomedical field [4ndash9]We previouslyfound that silk sericin could activate collagen production in

2 BioMed Research International

wounds which subsequently induced epithelialization [4ndash6]It was also reported that silk sericin-treated wounds showedmuch lesser inflammatory reactions than the wounds treatedwith cream base (formula control) which showed acuteinflammation [4] Furthermore silk sericin could promotethe attachment and proliferation of human skin fibroblastsand keratinocytes [10ndash14] These properties contributed tothe excellent suitability of silk sericin as a wound dressingmaterial

In order to fabricate wound dressing silk sericin itselfforms fragile materials that are not suitable for the med-ical usages Our previous study reported that silk sericincould form strong and stable scaffolds by combining it withpoly(vinyl alcohol) (PVA) glycerin (as a plasticizer) andgenipin (as a cross-linking agent) [15] Genipin is a naturalcross-linking agent which abundantly found in gardenia fruitextract It can cross-link proteins including silk sericin by aspontaneous reaction [16] The three-dimensional genipin-cross-linked silk sericinPVA scaffolds exhibited good phys-ical and mechanical properties and potentially healed thepartial- or full-thickness skin wounds [15 17] Howeverfor the healing of epidermal or superficial wounds two-dimensional wound dressings are rather suitable than thethree-dimensional ones As a novelty of this study thetwo-dimensional genipin-cross-linked silk sericinPVA filmsfabricated were aimed to be applied for the treatment ofsuperficial wounds The effects of genipin concentration onproperties of the films were investigated Physical and chem-ical properties of the genipin-cross-linked silk sericinPVAfilms including surface density cross-linking degree watercontact angle light and water vapor transmission humidityabsorption water swelling ability and chemical structureas well as mechanical properties were characterized Thein vitro release test of silk sericin from the films and thedegradation test of the films were performed In addition theviability nitric oxide and collagen production of L929mousefibroblast and HaCat keratinocyte cells cultured on the filmswere assessed Finally the in vivo test on safety of the filmswasperformed according to ISO 10993-6 Biological evaluation ofmedical devices

This preliminary study would be beneficial for furtherin vivo and clinical investigations on efficacy of the genipin-cross-linked silk sericinPVAfilms as two-dimensional dress-ings for the treatment of superficial wounds

2 Materials and Methods

21 Materials The fresh bivoltine white-shell cocoons ofBombyx mori produced in a controlled environment werekindly supplied by Chul Thai Silk Co Ltd (Petchabunprovince Thailand) Poly(vinyl alcohol) (PVA MW 77000ndash82000) was purchased from Ajax Finechem (New SouthWales Australia) Genipin was obtained from Wako PureChemical Industries Ltd Tokyo Japan while glycerin waspurchased from Sigma-Aldrich St Louis MO USA Otherchemicals were analytical grade and used without furtherpurification

22 Preparation of Silk Sericin The silkworm cocoons werecut into small pieces and the silk sericin was extracted usinga high temperature and pressure degumming technique [18]Briefly silkworm cocoons were put into deionized (DI) waterand then autoclaved at 120∘C for 60min After filtrationthrough a filter paper to remove fibroin fibers silk sericinsolution was concentrated until the desired concentrationwas achieved (approximately 7 wv measured by the BCAProtein Assay Reagent Pierce Rockford IL USA) Themolecular weight of silk sericin obtained was ranging from25 to 150 kDa

23 Fabrication of Genipin-Cross-Linked Silk SericinPVAFilms PVA was dissolved at 80∘C with a constant stir-ring for 4 h Genipin was dissolved in ethyl alcohol at aconcentration of 20 wv Silk sericin solution (3ww)PVA solution (2ww) and glycerin solution (1 ww)were mixed Genipin solution was added into the mixtureto the final concentrations of 001 0025 005 0075 and01wv and thenmixed at room temperature for 30minThemixture was cast onto a petri dish and air-dried for 24 h toobtain the genipin-cross-linked silk sericinPVA films Thefilm prepared by the same procedures but without genipinaddition was used as a control

24 Morphological Observation Surface morphology of thefilms was observed on a scanning electronmicroscope (SEMJSM 5410LV JEOL Tokyo Japan) at 15 keV after sputtercoated with gold Surface density of the films was determinedby dividing the weight with the dimension (119899 = 6)

25 Determination of Cross-Linking Degree The amountof free NH

2groups in the silk sericinPVA films after

cross-linking with genipin was evaluated by 246-trinitrobenzenesulfonic acid (TNBS) method [19] Brieflya known weight of film was treated with 1mL of 4 wvsodium hydrogen carbonate (NaHCO

3 pH 85) and 1mL

of 05 wv TNBS at 40∘C for 2 h Then 2mL of 6Nhydrochloric acid (HCl) was added to the reacting solutionand further incubated at 60∘C for 15 h The absorbanceof the solution was spectrophotometrically determined at415 nm after suitable dilution and the amount of free NH

2

groups was determined from a standard curve preparedfrom different concentrations of 120573-alanine The amount offree NH

2groups remained in the cross-linked films was

calculated in relative to the non-cross-linked film as a control(119899 = 3)

26 Determination of Water Contact Angle Water contactangle of the genipin-cross-linked silk sericinPVA films wasevaluated using a sessile drop method [20] Thin films ofgenipin-cross-linked silk sericinPVA were prepared on glassslides Deionizedwater (DI) was dropped onto the films usinga syringe and the water contact angle was measured at 30 safter dropping using a computerized video water contactangle system (CAM RE0110 UK) The baseline and thetangent were drawn using software and the contact angles

BioMed Research International 3

were measured from three different points on the films (119899 =3)

27 Evaluation of Light Transmission and Water Vapor Trans-mission Each film was cut into a rectangle (1 cm times 45 cm)and carefully placed on the internal side of a spectroscopy cellPercentage of light transmission of the films was measuredbetween the wavelength of 400 to 800 nm at 40 nm intervalsusing a UVV spectrophotometer (PerkinElmer Ltd Bun-desverband Solarwirtschaft Germany)

Water vapor transmission rate (WVTR) was determinedaccording to ASTM E96-80 with a slight modification(ASTM 1989) A container with DI water was closed firmlywith a film Then the container was placed in a desiccatorwith silica gel at 37∘C The film was weighed on an analyticalbalance model AB204-S (Mettler Toledo Inc Ohio USA)at the predetermining times The WVTR was calculatedaccording to (1)

WVTR = 119908 times 119909119905 times 119860

(1)

where 119908 represents weight of the film 119860 represents thepermeation area and119909119905was calculated by a linear regressionfrom the points of weight gain and time during constant rateperiod (119899 = 6)

28 Moisture Absorption andWater Swelling Tests Themois-ture absorption capability of the films was evaluated byplacing the dried films in the desiccators in which the relativehumidity was controlled by salt solution Potassium chloridewas used to obtain a relative humidity of 8147plusmn 148 at 25∘C[21] The films were removed from the desiccators at the pre-determining times and carefully weighed The percentage ofweight increase of the films was calculated relatively to theirinitial weights (119899 = 6) Swelling test was carried out accordingto the method of Mandal et al with a slight modification [8]Briefly a known weight of dried film was immersed in 10mLof DI water After 24 h the films were carefully taken out andweighed in the swollen state The equilibrium swelling of thefilms was calculated according to

Degree of water swelling () =119908119905minus 1199080

1199080

times 100 (2)

where 1199080and 119908

119905represent weights of the dried and swollen

films respectively (119899 = 6)

29 Mechanical Test The tensile test was performed on thefilms at room temperature using a universal testing machine(Hounsfield H10KM UK) equipped with a 10 kN load cellat a constant rate of 30mmmin The curves of force as afunction of deformation (mm) were automatically recordedby the softwareThe tensile strength (Nmm2) and percentageof elongation at breakwere calculated according to the ASTMD638-01 method (119899 = 6)

210 FT-IR Measurement The functional groups presentedin the films were examined using fourier transform infrared

(FT-IR) spectroscopy (PerkinElmer USA) of dried samplesThe information on structural contribution was collected inthe FT-IR analysis using PerkinElmer Spectrum GX (FT-IRsystem) All spectra were recorded in the wave number rangefrom 4000 to 650 cmminus1 at the resolution of 4 cmminus1The FT-IRanalysis was based on the identification of absorption bandsconcerned with the vibrations of functional groups presentedin the samples

211 In Vitro Release Test of Silk Sericin from Films Thefilms were placed into phosphate-buffered saline solution(PBS pH 74) at 37∘C with a continuous stirring in a closedcontainer The PBS solutions (15mL) were collected at thepredetermined times and the amount of silk sericin releasedinto the solutionswasmeasured using a BCAprotein assay kit(Pierce Rockford IL USA) The absorbance of the solutionwas measured at 562 nm and the amount of silk sericin wasdetermined from a standard curve prepared from differentconcentrations of bovine serum albumin (119899 = 3)

212 In Vitro Enzymatic Biodegradation Test The films wereincubated in 16120583gmL lysozyme solution (hen egg white92717Unitmg lot no 1316456 BioChemika Fluka Sigma-Aldrich Switzerland) at pH 74 37∘C The enzyme solutionwas changed every 2 days to ensure continuous enzymeactivity At each interval of time the remained films weretaken out of the solution rinsed repeatedly with deionizedwater and freeze dried The dried films were weighed andthe percentage of weight loss was calculated as follows

Percentage of weight loss () = (1198820minus119882119905

1198820

) times 100 (3)

where 1198820and 119882

119905represent the initial weight of the film

before degradation and the weight of the film after degrada-tion at different time intervals respectively (119899 = 3)

213 Cell Viability Test The films were sterilized by ethyleneoxide gas at 55∘C before cell culture Either L929 mousefibroblast or HaCat keratinocyte cells were seeded onto thesterilized films at a density of 5 times 105 cellsfilm and culturedin Dulbeccorsquos Modified Eagle Medium (DMEM) supple-mented with 10 vv fetal bovine serum (FBS) and 100UmLpenicillinstreptomycin at 37∘C 5 CO

2 After 24 and 72 h

of culture the number of cells was quantified using theconventional 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay (119899 = 3) [22] Morphology ofcells was observed under optical microscope

214 Nitric Oxide Assay L929 cells were cultured on the filmsat the same conditions as those of viability test After 24 and72 h of culture nitric oxide (NO) levels were determined bythe Griess reaction In brief 100 120583L Griess reagent (1 vvN-(1-naphthyl)-ethylenediamine dihydrochloride and 1 vvsulfanilamide in 25 vv phosphoric acid)wasmixedwith anequal volume of medium supernatant The resulting solutionyields a pink solution for a positive result and a yellowsolution for a negative result The absorbance of reacting


solution was measured at 570 nm based on a standard curveof sodium nitrate (NaNO

3) (119899 = 3)

215 Determination of Soluble Collagen Production L929cells were cultured on the films at the same conditions asthose of viability test After 24 and 72 h of culture themediumsupernatants were collected The total amount of solubletype I collagen was assayed using the Sircol collagen assaykit (Biocolor UK) The absorbance was determined by amicroplate reader (Biohit 830 Biohit) at a wavelength of500 nm The amount of collagen was calculated based on astandard curve of soluble collagen (type I collagen standardfrom American disease-free animals) (119899 = 3)

216 In Vivo Test on Safety of the Films (ISO 10993-6 Stan-dard) In vivo test was approved by the Ethics Committeeof the Faculty of Medicine Chulalongkorn University Theanimal experiments were performed according to Chula-longkorn University Animal Care and Use Committee (CU-ACUC) under standard sterile conditions The implantationof non-cross-linked and 01 wv genipin-cross-linked silksericinPVA films (2 times 2 cm2) into the subcutaneous tissueof female Wistar rats (8-week-old 200ndash300 g) was carriedout Sofra tulle a clinically available dressing consists of acotton leno weave fabric impregnated with a base composedof white soft paraffin anhydrous lanolin and 10wwframycetin sulphate was used as a control Briefly the ratswere anesthetized shaved the hair and disinfected with70 vol ethyl alcohol A 1 cm skin incision was made to formpockets in the subcutaneous tissue then the sample wasinserted into each pocket The wound was closed with 6ndash0 prolene suture and disinfected with Betadine (povidone-iodine topical antiseptics) solution After 3 7 14 and 28 daysof implantation the rats were sacrificed with an overdoseof thiopental sodium The samples and surrounding tissuewere retrieved fixed with 10 vol formalin solution andembedded in paraffin The paraffin-embedded samples weresectioned and stained with Hematoxylin and Eosin (HampE)For histological assessment the HampE slides were semiquan-titatively scored following ISO 10993-6 Inflammatory celltypes neovascularization fibrosis and fatty infiltrate werescored by one pathologist at two different times Intensityof inflammatory cells neovascularization fibrosis and fattyinfiltrate was recorded using 0ndash4 scales (0 = not observed1 = rare 2 = minimal 3 = heavily infiltrate and 4 = packedinfiltrate)The final score was calculated according to (3) andclassified as follows 00ndash29 (sample is nonirritant) 30ndash89(sample is slight irritant) 90ndash15 (sample ismoderate irritant)andgt15 (sample is severely irritant)The level of irritationwascompared to that of the control (Sofra tulle)

Final score = [2119868119905+ 119873119905] minus [2119868

119888+ 119873119888] (4)

where 119868119894is the total number of polymorphonuclear cell

lymphocytes plasma cells macrophages giant cells andnecrosis of sample 119894 (119894= test sample (119905) and control (119888)) and119873

119894

is the total number of neovascularization fibrosis and fattyinfiltrate of sample 119894 (119894 = test sample (119905) and control (119888))

217 Statistical Analysis All quantitative data were shownas mean plusmn SD For physical characterization the statisticalsignificancewas determined by paired and unpaired Studentrsquos119905-tests along with ANOVA For in vivo study all treatmentgroups were compared by ANOVA and the differencesbetween groups at different time points were compared bypost hoc 119905-test A value of 119875 lt 005 was considered to besignificant

3 Results

31 Surface Morphology and Density of the Films Surfacemorphologies for example surface roughness of non-cross-linked and genipin-cross-linked silk sericinPVA films weresimilar (unpublished data) Surface density of the films ispresented in Table 1 The surface densities of all genipin-cross-linked silk sericinPVA films (133ndash135mgmm3) weresignificantly higher than that of the non-cross-linked films(128mgmm3) By comparing the genipin-cross-linkedfilmstheir surface densities were not significantly different

32 Cross-Linking Degree of the Films Cross-linking degreeof the films was increased with the increasing concentrationof genipin as shown in Table 1 The films cross-linked with005 0075 and 01wv genipin showed significantly highercross-linking degree (22ndash38) comparing with those cross-linked with lower concentration of genipin (13ndash18)

33 Water Contact Angle of the Films Water contactangles of the non-cross-linked and genipin-cross-linked silksericinPVA films were around 50ndash55 degrees as presentedin Table 1 The significant difference in water contact angleamong the films was not observed

34 Light and Water Vapor Transmission of the Films Fig-ure 1(a) shows the percentage of light transmission throughthe films For the non-cross-linked films the percentageof light transmission was gradually increased from 20up to 60 along the increasing wavelength from 400 to800 nm The increasing percentage of light transmissionalong the increasing wavelength was also observed for thegenipin-cross-linked films however their light transmissionpercentages tended to drop at the wavelength around 520ndash680 nm Every genipin-cross-linked film showed the mini-mum percentage of light transmission at 600 nm The silksericinPVA films cross-linked with a higher concentrationof genipin showed less percentage of light transmission thanthose cross-linked with lower concentration of genipin orwithout cross-linking

Figure 1(b) shows the water vapor transmission rate(WVTR) through the filmsWVTR through all filmswas dra-matically increased initially and became stable thereafter Allgenipin-cross-linked silk sericinPVA films showed slowerWVTR than the non-cross-linked films along the periodThefilms cross-linked with 001 and 0025wv genipin tendedto have faster WVTR than those cross-linked with higherconcentration of genipin particularly at the initial period


Table 1 Surface density degree of cross-link and water contact angle of silk sericinPVA films cross-linked with genipin at differentconcentrations (0 001 0025 005 0075 and 01 wv)

Genipin concentration ( wv) Surface density (mgmm3) Degree of cross-link () Contact angle (∘)0 128 plusmn 004 mdash 5491 plusmn 429

0010 133 plusmn 006lowast

1384 plusmn 259 5471 plusmn 974


1882 plusmn 400 5206 plusmn 766


2264 plusmn 189dagger

5385 plusmn 329



5263 plusmn 357



5404 plusmn 1010

lowastP lt 005 significant against the value of non-cross-linked filmdaggerP lt 005 significant against the value of films cross-linked with 001 wv

0

10

20

30

40

50

60

70

400 440 480 520 560 600 640 680 720 760 800

Ligh

t tra

nsm

issio

n (

)

Wavelength (nm)

(a)

000

040

080

120

160

200

0 200 400 600 800 1000Time (min)

Wat

er v

apor

tran

smiss

ion

rate

(g m

inminus1

mminus2)

(b)

Figure 1 (a) Percentage of light transmission and (b) water vapor transmission rate of silk sericinPVA films cross-linked with genipin atdifferent concentrations (I) 0 (e) 001 (998771) 0025 (X) 005 (◼) 0075 and (lowast) 01 wv lowast119875 lt 005 significant against the values of non-cross-linked films at corresponding time

Table 2 Tensile strength and percentage of elongation of silksericinPVA films cross-linked with genipin at different concentra-tions (0 001 0025 005 0075 and 01 wv)

Genipin concentration( wv)

Tensile strength(Nmm2) Elongation ()

Sofra tulle 2641 plusmn 259 1107 plusmn 171

0 497 plusmn 059 18854 plusmn 5522

0010 466 plusmn 072 15650 plusmn 3193

0025 582 plusmn 091 19868 plusmn 5564


30960plusmn6336lowast





lowastP lt 005 significant against the values of non-cross-linked film

35 Moisture Absorption and Water Swelling Ability of theFilms Moisture absorption ability of the films is shown inFigure 2(a) The percentage of weight increased of all filmsrose from 10 to around 50 along the increasing time Thedifference in moisture absorption among the films was notobserved Figure 2(b) shows the percentage of water swellingof the films All genipin-cross-linked silk sericinPVA films

showed slightly lower percentage of water swelling com-paring with the non-cross-linked films but the significantdifference was not observed

36 Tensile Strength and Percentage of Elongation of theFilms Table 2 presents the tensile strength and percentageof elongation of the films and Sofra tulle Increasing concen-tration of genipin resulted in the higher tensile strength andpercentage of elongation of the films The silk sericinPVAfilms cross-linked with 005 0075 and 01wv genipinshowed significantly higher tensile strength and percentage ofelongation than those cross-linked with 001 and 0025wvgenipin or without cross-linking (119875 lt 005) The cross-linking with 001 and 0025 wv genipin seemed not toimprove both tensile strength and elongation of the filmscomparing to the non-cross-linked films On the otherhand the Sofra tulle a cotton leno-weave fabric showeda significantly higher tensile strength (sim2641Nmm2) butlower elongation percentage (sim11) than the films

37 FT-IR Spectra of Films Figure 3 shows FT-IR spectra ofthe filmsThe spectra of all films showed the obvious presenceof amide bands of silk sericin protein [23]The peak positionsat 1600 cmminus1 indicated random coil structure of amide I


0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)

Genipin concentration (wv)

(b)

Figure 2 (a) Moisture absorption rate and (b) percentage of water swelling of silk sericinPVA films cross-linked with genipin at differentconcentrations (I) 0 (e) 001 (998771) 0025 (X) 005 (◼) 0075 and (lowast) 01 wv

Table 3 Average intensity of inflammatory cells necrosis fibrosis neovascularization and fatty infiltrate in non-cross-linked silk sericinPVAfilms 01 wv genipin-cross-linked silk sericinPVA films and Sofra tulle after subcutaneous implantation for 3 7 14 and 28 days

3 d 7 d 14 d 28 dAlowast Blowastlowast Clowastlowastlowast A B C A B C A B C

PMN 4dagger 4 3 28 33 08 08 3 0 0 38 0Lymphocytes 28 28 15 3 3 2 35 3 23 3 3 1Plasma cells 0 0 0 0 0 0 0 0 0 0 08 0Macrophages 23 28 3 3 3 2 33 3 3 3 3 23Giant cells 0 0 0 0 0 23 33 13 38 23 1 4Necrosis 1 0 0 1 0 0 15 15 0 15 0 0Fibrosis 18 08 18 33 4 28 35 4 3 3 35 25Neovascularization 3 3 3 3 28 23 3 3 28 1 3 13Fatty infiltrate 0 0 0 0 1 23 0 0 23 05 0 3lowastA non-cross-linked silk sericinPVA filmslowastlowastB 01 wv genipin-cross-linked silk sericinPVA filmslowastlowastlowastC Sofra tulle (control)daggerIntensity 0ndash4 0 = not observed 1 = rare 2 = minimal 3 = heavily infiltrate and 4 = packed infiltrate

Table 4 Level of tissue irritation after subcutaneous implantationwith non-cross-linked silk sericinPVA films and 01 wv genipin-cross-linked silk sericinPVA films for 3 7 14 and 28 days relativeto Sofra tulle as a control

Level of irritation

Non-cross-linked silksericinPVA films

01 wvgenipin-cross-linked silk

sericinPVA films3 d Slightly irritant Slightly irritant7 d Slightly irritant Slightly irritant14 d Slightly irritant Slightly irritant28 d Nonirritant Slightly irritant

(C=O stretching)The peaks at around 1530 cmminus1 were amideII (NndashH deformation and CndashN stretching) and those at1250 cmminus1 indicated random coil structure of amide III (CndashN stretching and NndashH deformation) The peak positions at

800 cmminus1 indicated amide V (out-of-plane NH bending)Thecharacteristic absorption peaks of PVA at about 3000 cmminus1(ndashOH stretching) and at about 1082 and 1400 cmminus1 (ndashCndashO)were observed [24] The peak positions at around 1150 cmminus1(CndashO stretching) and 1400 cmminus1 (CndashOndashHbending) indicatedthe characteristics of glycerin [25] It was observed that theheight of peaks at 900 and 1000 cmminus1 of all genpin-cross-linked films was increased comparing to those of non-cross-linked filmThese peaks may indicate the intramolecular andintermolecular cross-linking bonds in which genipin had aheterocyclic structure with primary amine groups [26]

38 In Vitro Release Profiles of Silk Sericin from Films In vitrorelease profiles of silk sericin from the films are shown inFigure 4 At initial time a burst release of silk sericin wasobserved for all films particularly for the films without cross-linking or those cross-linked with lower concentration ofgenipin (001 0025 and 005wv) However the amount of


60012001800240030003600Wavenumber (cmminus1)

00010025

005007501wv

Figure 3 FT-IR spectra of silk sericinPVA films cross-linked withgenipin at different concentrations (black line) 0 (blue line) 001(green line) 0025 (yellow line) 005 (purple line) 0075 and (redline) 01 wv

lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0

Figure 4 In vitro release profiles of silk sericin from the filmscross-linked with genipin at different concentrations (I) 0 (e) 001(998771) 0025 (X) 005 (◼) 0075 and (lowast) 01 wv after incubatedin a phosphate-buffered saline solution (PBS pH 74) at 37∘C andassessed by a BCA protein assay kit lowast119875 lt 005 significant againstthe values of non-cross-linked films at corresponding time

silk sericin releasedwas reduced and became stable thereafterThe films cross-linked with 0075 and 01wv genipinshowed less amount of silk sericin release than those cross-linked with lower concentration of genipin

39 In Vitro Degradation of the Films Figure 5 shows thedegradation profiles of the films Percentage of weight lossof all films gradually increased along the incubation periodThe non-cross-linked films and the films cross-linked with001 and 0025wv genipin showed higher percentage of

0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)

Figure 5 In vitro degradation profiles of silk sericinPVA filmscross-linked with genipin at different concentrations (I) 0 (e) 001(998771) 0025 (X) 005 (◼) 0075 and (lowast) 01 wv after incubated in16120583gmL lysozyme solution (pH 74) at 37∘C lowast119875 lt 005 significantagainst the values of non-cross-linked films at corresponding time

weight loss than the films cross-linked with 005 0075 and01wv genipin After 168 h of incubation the weight lossesof non-cross-linked films and the films cross-linked with 001and 0025wv genipin were upto 60 while those of thefilms cross-linkedwith 005 0075 and 01wv genipinwerearound 50

310 Viability of Cells Cultured on the Films Figure 6(a)shows the viability of L929 mouse fibroblast cells cultured onthe films After 24 and 72 h of culture the higher percentageof cell viability was found on the films cross-linked with0075 and 01wv genipin comparing to those of non-cross-linked film and the films cross-linked with lowerconcentration of genipin (119875 lt 005) Morphology of cellswas round when cultured on the non-cross-linked filmswhile those cultured on the films cross-linked with 01wvgenipin showed spindle shape (Figure 6(b)) The numbers ofHaCat keratinocyte cells cultured on the films for 24 and 72 hare shown in Figure 7(a)The trendwas similar to that of L929mouse fibroblast cells culture The HaCat keratinocyte cellscultured on the films cross-linked with 0075 and 01wvgenipin showed higher number than those cultured on thefilms cross-linked with 001 and 0025wv genipin Whencultured for 72 h the number of cells cultured on the filmscross-linked with 01wv genipin was comparable to that ofSofra tulle Figure 7(b) presents the images of MTT-stainedHaCat cells cultured on the films for 24 h It can be seen thatthe extent of MTT-stained cells was consistent with the cellnumber reported in Figure 7(a)

311 Production of NO and Soluble Collagen Production ofNO and soluble collagen by cells cultured on the films wasshown in Figures 7(a) and 7(b) respectively After 24 h ofculture cells cultured on the films cross-linked with 01wvgenipin produced the highest concentration of NO whilethose cultured on other films seemed to produce very lowconcentration of NO After 72 h of culture the production


0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)

0 wv genipin 01 wv genipin

L929 cells cultured on genipin-cross-linked silk sericinPVA films for 24 h

(b)

Figure 6 (a) Viability of L929 mouse fibroblast cells cultured on silk sericinPVA films cross-linked with genipin at different concentrations(0 001 0025 005 0075 and 01wv) for (◻) 24 h and (◼) 72 h (b)Morphology of L929 cells cultured on non-cross-linked silk sericinPVAfilms and silk sericinPVAfilms cross-linkedwith 01wv genipin for 24 h lowast119875 lt 005 significant against the values of non-cross-linked films

of NO of cells cultured on the genipin-cross-linked filmswas significantly higher than that of non-cross-linked film(119875 lt 005) The production of soluble collagen was seen onlyfor cells cultured on the genipin-cross-linked films and thattended to be higher for the films cross-linked with genipin athigher concentrationThe soluble collagenwas not detectablefor cells cultured on non-cross-linked films at both 24 and72 h of culture

312 In Vivo Safety of the Films The rats which receivedthe implantation of all samples were healthy throughoutthe implantation period and no signs of inflammation (ieredness swelling pain and heat) were observed Figure 8showed the images of HampE-stained sections of non-cross-linked 01wv genipin-cross-linked silk sericinPVA filmsand Sofra tulle The arrows indicated the interface betweensample implanted and surrounding tissue No excessiveinflammatory reaction was detected around the implanta-tion sites The infiltration of inflammatory cells into thesamples implanted was shown in Figure 9 The numberof inflammatory cells infiltrated into the non-cross-linkedand 01wv genipin-cross-linked silk sericinPVA films wasslightly higher than that of Sofra tulle along the implanta-tion period The intensity of inflammatory cells necrosis

fibrosis neovascularization and fatty infiltrate was graded aspresented in Table 3 After 3 days of implantation polymor-phonuclear cells were packed infiltrated into the non-cross-linked and 01wv genipin-cross-linked silk sericinPVAfilms and heavily infiltrated into the Sofra tulle However theintensity of polymorphonuclear cells was gradually reducedthereafter Lymphocytes macrophages and neovasculariza-tion were found along the implantation period Heavy infil-tration of giant cells and fibrosis was observed particularlyafter 14 and 28 days of implantation (see Figure 10) Sofratulle implantation showed more intensity of fatty infiltratethan both films implantation On the other hand plasmacells were not observed for all samples along the implantationperiod It was evaluated that the implantation of both filmswas non- to slightly irritant relative to the Sofra tulle as acontrol (Table 4)

4 Discussion

Wound dressing made of silk sericin would accelerate woundhealing because silk sericin could promote the attachmentand proliferation of fibroblasts and keratinocytes [10ndash14]and induce the collagen production and epithelialization[4ndash6] We previously reported that the three-dimensional


0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)

HaCat cells cultured on genipin-cross-linked silk sericinPVA films for 24 hSofra tulle 0 wv genipin 001 wv genipin

005 wv genipin 0075 wv genipin 01 wv genipin

0025 wv genipin

(b)

Figure 7 (a) Number of HaCat keratinocyte cells cultured on silk sericinPVA films cross-linked with genipin at different concentrations(0 001 0025 005 0075 and 01 wv) for (◻) 24 h and (◼) 72 h (b) MTT-stained HaCat keratinocyte cells cultured on the films for 24 hlowast

119875 lt 005 significant against the values of non-cross-linked films

0

05

1

15

2

25

3

35

4

45

0 001 0025 005 0075 01Genipin concentration (wv)

NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)

Figure 8 (a) NO and (b) collagen production of L929 cells cultured on silk sericinPVA films cross-linked with genipin at differentconcentrations (0 001 0025 005 0075 and 01 wv) for (◻) 24 h and (◼) 72 h lowast119875 lt 005 significant against the values of non-cross-linked films

genipin-cross-linked silk sericinPVA scaffolds exhibitedgood physical properties and potentially healed the partial-or full-thickness skin wounds [15 16] In this study thetwo-dimensional genipin-cross-linked silk sericinPVA filmsdeveloped were aimed to be applied for the treatment ofsuperficial wounds The effects of genipin cross-linking onthe physical and biological properties of silk sericinPVAfilms were reported Herein it was shown that the increasingconcentration of genipin resulted in the higher cross-linkingdegree of the films (Table 1) This was correlated with theincreased surface density (Table 1) andmechanical properties(Table 2) but decreased percentage of light transmissionand WVTR (Figure 1) and poorer water swelling ability(Figure 2(b)) of the genipin-cross-linked silk sericinPVAfilms

The cross-linking mechanism of genipin with a methy-lamine of proteins or other molecules containing primary

amines was proposed [26] The reaction occurred through anucleophilic attack of the primary amine on the C3 carbonof genipin This caused an opening of the dihydropyran ringfollowed by an attack on the resulting aldehyde group bythe secondary amine group The final step of the forma-tion of cross-linking bonds was the dimerization producedby radical reactions The intra- and intermolecular cross-linking bonds in which genipin had a heterocyclic structurewith the molecules containing primary amine groups wereformed The increased height of peaks of FT-IR spectraat 900 and 1000 cmminus1 may confirm these intra- and inter-molecular cross-linking bonds of the genipin-cross-linkedsilk sericinPVA films (Figure 3)

The increased density and improved mechanical prop-erties of the films as a result of cross-linking were reportedelsewhere [27 28] Huang et al have studied the effects ofcalcium chloride cross-linking on structure and properties


3 d

7 d

14 d

28 d

Non-cross-linkedsilk sericinPVA film

01 genipin-cross-linkedsilk sericinPVA film Sofra tulle

Figure 9 Microscopic images of HampE-stained sections of non-cross-linked silk sericinPVA films 01 wv genipin-cross-linked silksericinPVA films and Sofra tulle after subcutaneous implantation for 3 7 14 and 28 days Scale bar = 300120583m arrow interface betweensample implanted and surrounding tissue

of waterborne polyurethane-carboxymethylated guar gumfilms [28] The cross-linked films formed a relatively densearchitecture which led to better miscibility and higher tensilestrength On the other hand the genipin-cross-linked filmsshowed lower percentage of light and water vapor transmis-sion because the cross-linking bonds reduced the mobility ofmolecular chains within the films [29] The rigid structureof the cross-linked films would obstruct the transmission oflight and water vapor This behavior has also been reportedin alginate films with higher concentrations of calcium [30]We here also elucidated the effect of genipin cross-linkingon the degradation behavior of the silk sericinPVA filmsIt was found that the genipin-cross-linking particularly athigh concentration could prolong the degradation of the silksericinPVA films (Figure 5)

The films which are capable of swelling would supportthe adsorption of proteins or bioactive molecules fromculture medium or body fluid and subsequently promotebioactivity of cells [31] In this study the genipin-cross-linking slightly reduced the water swelling ability of the filmsIt is possible that the rigid molecular chains of the cross-linked films impeded the penetration of water moleculesthrough the films The effects of chemical cross-linking onthe molecular crystallinity and diffusion coefficient of waterwere also reported by Hasimi et al [32] However from ourresults the significant difference in water swelling ability

between the genipin-cross-linked and non-cross-linked filmswas not observed Thus the genipin-cross-linking would notdiminish the capability of the silk sericinPVA films to adsorbproteins for biological signaling

The release of silk sericin from the films was possiblygoverned by two main mechanisms which are diffusion andmaterial degradation [33] The former mechanism was seenfrom the burst release of non-cross-linked silk sericin whilethe latter mechanism explained the release of the genipin-cross-linked silk sericin Initially the small amount of silksericin was diffusionally released from the films cross-linkedwith 0075 and 01 genipin (Figure 4) because most of thesilk sericin was cross-linked with genipin On the other handhigh amount of non-cross-linked silk sericin found in thefilms cross-linked with lower concentration of genipin (001ndash005) was diffusionally released at initiation Howeverthereafter the genipin-cross-linked silk sericin was sustainedand released from all films along the degrading of the films

For the biological activities it was found that L929mousefibroblast cells cultured on the silk sericinPVA films cross-linked with high concentration of genipin (0075 and 01wv) showed higher percentage of viability (Figure 6(a)) andproduced higher concentrations of NO and soluble collagen(Figure 8) Nitric oxide is usually produced during inflam-matory conditions by the inducible isoform of the enzymeNO synthase [34] It was reported that some concentrations




3 d

7 d

14 d

28 d

Figure 10 Microscopic images of HampE-stained sections indicating inflammatory cells in non-cross-linked silk sericinPVA films 01 wvgenipin-cross-linked silk sericinPVA films and Sofra tulle after subcutaneous implantation for 3 7 14 and 28 days Scale bar = 30120583m

of NO can be potentially beneficial for cell activities [35ndash38] Witte et al reported that the synthesis of fibroblasticcollagen together with total protein was enhanced in thepresence of low concentrations of NO [34 36] Howeverit should be noted that there are adverse effects of NO onwound healingThe excessive generation of intracellular ROSand NOS in the skin may result in subsequent oxidativestress damage and apoptosis NO overexpression may beinvolved directly or indirectly through production of perox-ynitrite in the pathogenesis and delayed healing due to theimpaired vasculature inflammation and toxicity In additionto the L929 mouse fibroblast cells the viability of HaCatkeratinocyte cells when cultured on the films was shown(Figure 7) The results further confirmed that the films cross-linked with high concentration of genipin promoted cellproliferation It might be that the films cross-linked with0075 and 01wv genipin could entrap high amount ofsilk sericin and sustained its release which probably improvebiological properties of the films It has been reported that silksericin could activate collagen production [4ndash6] and promoteattachment and proliferation of human skin fibroblasts andkeratinocytes [10ndash14]

The in vivo evaluation on safety of the films was per-formed according to ISO 10993-6 comparingwith Sofra tulle

to confirm their suitability for medical applications It wasshown that the implantation of the 01wv genipin-cross-linked silk sericinPVA films was evaluated as slightly irritantwhich is clinically acceptable for topical applications relativeto the Sofra tulle implantation (Table 4) Therefore the01wv genipin-cross-linked silk sericinPVA films wouldbe safe for the further in vivo and clinical efficacy tests astwo-dimensional dressings for the treatment of superficialwounds

It was concluded from this study that the silk sericinPVAfilms cross-linked with genipin particularly at high con-centration showed good physical and biological propertiesas well as the sustained release of sericin which would beadvantageous for the healing of superficial wounds

5 Conclusion

The silk sericinPVA films cross-linked with genipin showedincreased surface density and mechanical properties butdecreased percentage of light transmission and WVTR andpoorer water swelling ability comparing with the non-cross-linked films Silk sericin was released from the genipin-cross-linked films in a sustained manner Either L929 mouse


fibroblast or HaCat keratinocyte cells cultured on the silksericinPVA films cross-linked with high concentration ofgenipin (0075 and 01wv) showed higher percentage ofviability and produced higher concentrations of NO andsoluble collagen The in vivo safety test performed accord-ing to ISO 10993-6 confirmed that the 01wv genipin-cross-linked silk sericinPVA films would be safe for themedical usages Therefore the 01wv genipin-cross-linkedsilk sericinPVA films would be promising choices of two-dimensional wound dressings for the treatment of superficialwounds

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgment

This research was supported by theThailand Research Fund

References

[1] R Okabayashi M Nakamura T Okabayashi Y Tanaka ANagai and K Yamashita ldquoEfficacy of polarized hydroxyapatiteand silk fibroin composite dressing gel on epidermal recoveryfrom full-thickness skin woundsrdquo Journal of Biomedical Mate-rials Research vol 90 no 2 pp 641ndash646 2009

[2] L Cuttle M Kempf G E Phillips et al ldquoA porcine deep dermalpartial thickness burn model with hypertrophic scarringrdquoBurns vol 32 no 7 pp 806ndash820 2006

[3] N S Gibran S Boyce and D G Greenhalgh ldquoCutaneouswound healingrdquo Journal of Burn Care and Research vol 28 no4 pp 577ndash579 2007

[4] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[5] P Aramwit S Kanokpanont T Nakpheng and T SrichanaldquoThe effect of sericin from various extraction methods oncell viability and collagen productionrdquo International Journal ofMolecular Sciences vol 11 no 5 pp 2200ndash2211 2010

[6] P Aramwit S Kanokpanont W De-Eknamkul K Kamei andT Srichana ldquoThe effect of sericin with variable amino-acidcontent from different silk strains on the production of collagenand nitric oxiderdquo Journal of Biomaterials Science PolymerEdition vol 20 no 9 pp 1295ndash1306 2009

[7] S C Kundu B C Dash R Dash and D L Kaplan ldquoNaturalprotective glue protein sericin bioengineered by silkwormspotential for biomedical and biotechnological applicationsrdquoProgress in Polymer Science vol 33 no 10 pp 998ndash1012 2008

[8] B B Mandal A S Priya and S C Kundu ldquoNovel silksericingelatin 3-D scaffolds and 2-D films fabrication andcharacterization for potential tissue engineering applicationsrdquoActa Biomaterialia vol 5 no 8 pp 3007ndash3020 2009

[9] B C Dash B B Mandal and S C Kundu ldquoSilk gland sericinproteinmembranes fabrication and characterization for poten-tial biotechnological applicationsrdquo Journal of Biotechnology vol144 no 4 pp 321ndash329 2009

[10] M Sasaki Y Kato H Yamada and S Terada ldquoDevelopmentof a novel serum-free freezing medium for mammalian cellsusing the silk protein sericinrdquo Biotechnology and Applied Bio-chemistry vol 42 no 2 pp 183ndash188 2005

[11] K Tsubouchi Y Igarashi Y Takasu and H Yamada ldquoSericinenhances attachment of cultured human skin fibroblastsrdquo Bio-science Biotechnology and Biochemistry vol 69 no 2 pp 403ndash405 2005

[12] S Terada T Nishimura M Sasaki H Yamada and MMiki ldquoSericin a protein derived from silkworms acceleratesthe proliferation of several mammalian cell lines including ahybridomardquo Cytotechnology vol 40 no 1ndash3 pp 3ndash12 2003

[13] F Zhang Z Zhang X Zhu E T Kang and K G NeohldquoSilk-functionalized titanium surfaces for enhancing osteoblastfunctions and reducing bacterial adhesionrdquo Biomaterials vol29 no 36 pp 4751ndash4759 2008

[14] R Dash M Mandal S K Ghosh and S C Kundu ldquoSilksericin protein of tropical tasar silkworm inhibits UVB-inducedapoptosis in human skin keratinocytesrdquoMolecular and CellularBiochemistry vol 311 no 1-2 pp 111ndash119 2008

[15] P Aramwit T Siritientong S Kanokpanont and T SrichanaldquoFormulation and characterization of silk sericin-PVA scaffoldcrosslinked with genipinrdquo International Journal of BiologicalMacromolecules vol 47 no 5 pp 668ndash675 2010

[16] J S Yoo Y J Kim S H Kim and S H Choi ldquoStudy ongenipin a new alternative natural crosslinking agent for fixingheterograft tissuerdquoKorean Journal ofThoracic and Cardiovascu-lar Surgery vol 44 no 3 pp 197ndash207 2011

[17] T Siritientong T Srichana and P Aramwit ldquoThe effect ofsterilization methods on the physical properties of silk sericinscaffoldsrdquo AAPS PharmSciTech vol 12 no 2 pp 771ndash781 2011

[18] K LeeHKweon JH Yeo et al ldquoEffect ofmethyl alcohol on themorphology and conformational characteristics of silk sericinrdquoInternational Journal of Biological Macromolecules vol 33 no1ndash3 pp 75ndash80 2003

[19] W A Bubnis and C M Ofner III ldquoThe determination of120576-amino groups in soluble and poorly soluble proteinaceousmaterials by a spectrophotometric method using trinitroben-zenesulfonic acidrdquo Analytical Biochemistry vol 207 no 1 pp129ndash133 1992

[20] J Zhou J Zhang Y Ma and J Tong ldquoSurface photo-crosslinking of corn starch sheetsrdquo Carbohydrate Polymers vol74 no 3 pp 405ndash410 2008

[21] L Greenspan ldquoHumidity fixed points of binary saturatedaqueous solutionsrdquo Journal of Research of the National Bureauof Standards A vol 81 no 1 pp 89ndash96 1977

[22] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[23] J Kong and S Yu ldquoFourier transform infrared spectroscopicanalysis of protein secondary structuresrdquo Acta Biochimica etBiophysica Sinica vol 39 no 8 pp 549ndash559 2007

[24] I R Rodrigues M M de Camargo Forte D S Azambujaand K R L Castagno ldquoSynthesis and characterization ofhybrid polymeric networks (HPN) based on polyvinyl alco-holchitosanrdquo Reactive and Functional Polymers vol 67 no 8pp 708ndash715 2007

[25] E N Hidawati and A M Mimi Sakinah ldquoTreatment ofglycerin pitch from biodiesel productionrdquo International Journalof Chemical and Environmental Engineering vol 2 pp 309ndash3132011

[26] W H Chang Y Chang P H Lai and H W Sung ldquoA genipin-crosslinked gelatin membrane as wound-dressing material invitro and in vivo studiesrdquo Journal of Biomaterials SciencePolymer Edition vol 14 no 5 pp 481ndash495 2003


[27] D I Lee ldquoThe effects of latex coalescence and interfacialcrosslinking on the mechanical properties of latex filmsrdquoPolymer vol 46 no 4 pp 1287ndash1293 2005

[28] Y Huang H Yu and C Xiao ldquoEffects of Ca2+ crosslink-ing on structure and properties of waterborne polyurethane-carboxymethylated guar gum filmsrdquo Carbohydrate Polymersvol 66 no 4 pp 500ndash513 2006

[29] S Benavides R Villalobos-Carvajal and J E Reyes ldquoPhysicalmechanical and antibacterial properties of alginate film effectof the crosslinking degree and oregano essential oil concentra-tionrdquo Journal of Food Engineering vol 110 no 2 pp 232ndash2392011

[30] J W Rhim ldquoPhysical and mechanical properties of waterresistant sodium alginate filmsrdquo Food Science and Technologyvol 37 no 3 pp 323ndash330 2004

[31] DW Hutmacher ldquoScaffold design and fabrication technologiesfor engineering tissuesmdashstate of the art and future perspectivesrdquoJournal of Biomaterials Science Polymer Edition vol 12 no 1pp 107ndash124 2001

[32] A Hasimi A Stavropoulou K G Papadokostaki and MSanopoulou ldquoTransport of water in polyvinyl alcohol filmseffect of thermal treatment and chemical crosslinkingrdquo Euro-pean Polymer Journal vol 44 no 12 pp 4098ndash4107 2008

[33] J Ratanavaraporn H Furuya H Kohara and Y TabataldquoSynergistic effects of the dual release of stromal cell-derivedfactor-1 and bone morphogenetic protein-2 from hydrogels onbone regenerationrdquo Biomaterials vol 32 no 11 pp 2797ndash28112011

[34] C Nathan ldquoInducible nitric oxide synthase what differencedoes it makerdquo Journal of Clinical Investigation vol 100 no 10pp 2417ndash2423 1997

[35] M B Witte F J Thornton D T Efron and A BarbulldquoEnhancement of fibroblast collagen synthesis by nitric oxiderdquoNitric Oxide vol 4 no 6 pp 572ndash582 2000

[36] D Miljkovic I Cvetkovic S Stosic-Grujicic and V TrajkovicldquoMycophenolic acid inhibits activation of inducible nitricoxide synthase in rodent fibroblastsrdquo Clinical and ExperimentalImmunology vol 132 no 2 pp 239ndash246 2003

[37] M Du M M Islam L Lin Y Ohmura Y Moriyama and SFujimura ldquoPromotion of proliferation of murine BALBc 3T3fibroblasts mediated by nitric oxide at lower concentrationsrdquoBiochemistry and Molecular Biology International vol 41 no 3pp 625ndash631 1997

[38] D T Efron S J Kirk M C Regan H L Wasserkrug and ABarbul ldquoNitric oxide generation from L-arginine is required foroptimal human peripheral blood lymphocyte DNA synthesisrdquoSurgery vol 110 no 2 pp 327ndash334 1991

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


wounds which subsequently induced epithelialization [4ndash6]It was also reported that silk sericin-treated wounds showedmuch lesser inflammatory reactions than the wounds treatedwith cream base (formula control) which showed acuteinflammation [4] Furthermore silk sericin could promotethe attachment and proliferation of human skin fibroblastsand keratinocytes [10ndash14] These properties contributed tothe excellent suitability of silk sericin as a wound dressingmaterial

In order to fabricate wound dressing silk sericin itselfforms fragile materials that are not suitable for the med-ical usages Our previous study reported that silk sericincould form strong and stable scaffolds by combining it withpoly(vinyl alcohol) (PVA) glycerin (as a plasticizer) andgenipin (as a cross-linking agent) [15] Genipin is a naturalcross-linking agent which abundantly found in gardenia fruitextract It can cross-link proteins including silk sericin by aspontaneous reaction [16] The three-dimensional genipin-cross-linked silk sericinPVA scaffolds exhibited good phys-ical and mechanical properties and potentially healed thepartial- or full-thickness skin wounds [15 17] Howeverfor the healing of epidermal or superficial wounds two-dimensional wound dressings are rather suitable than thethree-dimensional ones As a novelty of this study thetwo-dimensional genipin-cross-linked silk sericinPVA filmsfabricated were aimed to be applied for the treatment ofsuperficial wounds The effects of genipin concentration onproperties of the films were investigated Physical and chem-ical properties of the genipin-cross-linked silk sericinPVAfilms including surface density cross-linking degree watercontact angle light and water vapor transmission humidityabsorption water swelling ability and chemical structureas well as mechanical properties were characterized Thein vitro release test of silk sericin from the films and thedegradation test of the films were performed In addition theviability nitric oxide and collagen production of L929mousefibroblast and HaCat keratinocyte cells cultured on the filmswere assessed Finally the in vivo test on safety of the filmswasperformed according to ISO 10993-6 Biological evaluation ofmedical devices

This preliminary study would be beneficial for furtherin vivo and clinical investigations on efficacy of the genipin-cross-linked silk sericinPVAfilms as two-dimensional dress-ings for the treatment of superficial wounds

2 Materials and Methods

21 Materials The fresh bivoltine white-shell cocoons ofBombyx mori produced in a controlled environment werekindly supplied by Chul Thai Silk Co Ltd (Petchabunprovince Thailand) Poly(vinyl alcohol) (PVA MW 77000ndash82000) was purchased from Ajax Finechem (New SouthWales Australia) Genipin was obtained from Wako PureChemical Industries Ltd Tokyo Japan while glycerin waspurchased from Sigma-Aldrich St Louis MO USA Otherchemicals were analytical grade and used without furtherpurification

22 Preparation of Silk Sericin The silkworm cocoons werecut into small pieces and the silk sericin was extracted usinga high temperature and pressure degumming technique [18]Briefly silkworm cocoons were put into deionized (DI) waterand then autoclaved at 120∘C for 60min After filtrationthrough a filter paper to remove fibroin fibers silk sericinsolution was concentrated until the desired concentrationwas achieved (approximately 7 wv measured by the BCAProtein Assay Reagent Pierce Rockford IL USA) Themolecular weight of silk sericin obtained was ranging from25 to 150 kDa

23 Fabrication of Genipin-Cross-Linked Silk SericinPVAFilms PVA was dissolved at 80∘C with a constant stir-ring for 4 h Genipin was dissolved in ethyl alcohol at aconcentration of 20 wv Silk sericin solution (3ww)PVA solution (2ww) and glycerin solution (1 ww)were mixed Genipin solution was added into the mixtureto the final concentrations of 001 0025 005 0075 and01wv and thenmixed at room temperature for 30minThemixture was cast onto a petri dish and air-dried for 24 h toobtain the genipin-cross-linked silk sericinPVA films Thefilm prepared by the same procedures but without genipinaddition was used as a control

24 Morphological Observation Surface morphology of thefilms was observed on a scanning electronmicroscope (SEMJSM 5410LV JEOL Tokyo Japan) at 15 keV after sputtercoated with gold Surface density of the films was determinedby dividing the weight with the dimension (119899 = 6)

25 Determination of Cross-Linking Degree The amountof free NH

2groups in the silk sericinPVA films after

cross-linking with genipin was evaluated by 246-trinitrobenzenesulfonic acid (TNBS) method [19] Brieflya known weight of film was treated with 1mL of 4 wvsodium hydrogen carbonate (NaHCO

3 pH 85) and 1mL

of 05 wv TNBS at 40∘C for 2 h Then 2mL of 6Nhydrochloric acid (HCl) was added to the reacting solutionand further incubated at 60∘C for 15 h The absorbanceof the solution was spectrophotometrically determined at415 nm after suitable dilution and the amount of free NH

2

groups was determined from a standard curve preparedfrom different concentrations of 120573-alanine The amount offree NH

2groups remained in the cross-linked films was

calculated in relative to the non-cross-linked film as a control(119899 = 3)

26 Determination of Water Contact Angle Water contactangle of the genipin-cross-linked silk sericinPVA films wasevaluated using a sessile drop method [20] Thin films ofgenipin-cross-linked silk sericinPVA were prepared on glassslides Deionizedwater (DI) was dropped onto the films usinga syringe and the water contact angle was measured at 30 safter dropping using a computerized video water contactangle system (CAM RE0110 UK) The baseline and thetangent were drawn using software and the contact angles





WVTR = 119908 times 119909119905 times 119860

(1)




1199080

times 100 (2)

where 1199080and 119908









1198820

) times 100 (3)

where 1198820and 119882




2 After 24 and 72 h





3) (119899 = 3)




119888+ 119873119888] (4)



119894



3 Results










1384 plusmn 259 5471 plusmn 974


1882 plusmn 400 5206 plusmn 766



5385 plusmn 329



5263 plusmn 357



5404 plusmn 1010


0

10

20

30

40

50

60

70

400 440 480 520 560 600 640 680 720 760 800

Ligh

t tra

nsm

issio

n (

)

Wavelength (nm)

(a)

000

040

080

120

160

200

0 200 400 600 800 1000Time (min)

Wat

er v

apor

tran

smiss

ion

rate

(g m

inminus1

mminus2)

(b)






0 497 plusmn 059 18854 plusmn 5522

0010 466 plusmn 072 15650 plusmn 3193

0025 582 plusmn 091 19868 plusmn 5564













0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)


(b)






Level of irritation









00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







WVTR = 119908 times 119909119905 times 119860

(1)




1199080

times 100 (2)

where 1199080and 119908









1198820

) times 100 (3)

where 1198820and 119882




2 After 24 and 72 h





3) (119899 = 3)




119888+ 119873119888] (4)



119894



3 Results










1384 plusmn 259 5471 plusmn 974


1882 plusmn 400 5206 plusmn 766



5385 plusmn 329



5263 plusmn 357



5404 plusmn 1010


0

10

20

30

40

50

60

70

400 440 480 520 560 600 640 680 720 760 800

Ligh

t tra

nsm

issio

n (

)

Wavelength (nm)

(a)

000

040

080

120

160

200

0 200 400 600 800 1000Time (min)

Wat

er v

apor

tran

smiss

ion

rate

(g m

inminus1

mminus2)

(b)






0 497 plusmn 059 18854 plusmn 5522

0010 466 plusmn 072 15650 plusmn 3193

0025 582 plusmn 091 19868 plusmn 5564













0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)


(b)






Level of irritation









00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





3) (119899 = 3)




119888+ 119873119888] (4)



119894



3 Results










1384 plusmn 259 5471 plusmn 974


1882 plusmn 400 5206 plusmn 766



5385 plusmn 329



5263 plusmn 357



5404 plusmn 1010


0

10

20

30

40

50

60

70

400 440 480 520 560 600 640 680 720 760 800

Ligh

t tra

nsm

issio

n (

)

Wavelength (nm)

(a)

000

040

080

120

160

200

0 200 400 600 800 1000Time (min)

Wat

er v

apor

tran

smiss

ion

rate

(g m

inminus1

mminus2)

(b)






0 497 plusmn 059 18854 plusmn 5522

0010 466 plusmn 072 15650 plusmn 3193

0025 582 plusmn 091 19868 plusmn 5564













0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)


(b)






Level of irritation









00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







1384 plusmn 259 5471 plusmn 974


1882 plusmn 400 5206 plusmn 766



5385 plusmn 329



5263 plusmn 357



5404 plusmn 1010


0

10

20

30

40

50

60

70

400 440 480 520 560 600 640 680 720 760 800

Ligh

t tra

nsm

issio

n (

)

Wavelength (nm)

(a)

000

040

080

120

160

200

0 200 400 600 800 1000Time (min)

Wat

er v

apor

tran

smiss

ion

rate

(g m

inminus1

mminus2)

(b)






0 497 plusmn 059 18854 plusmn 5522

0010 466 plusmn 072 15650 plusmn 3193

0025 582 plusmn 091 19868 plusmn 5564













0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)


(b)






Level of irritation









00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

10

20

30

40

50

60

0 20 40 60 80 100 120Time (h)

Wei

ght i

ncre

ased

()

(a)

0

100

200

300

400

0 001 0025 005 0075 01

Deg

ree o

f sw

ellin

g (

)


(b)






Level of irritation









00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





00010025

005007501wv


lowastlowast

lowast

lowast

lowast

lowast

0

5

10

15

20

25

30

24 48 72 96

Cum

ulat

ive p

rote

in re

leas

e (

)

Time (h)0




0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Time (h)

Wei

ght l

oss (

)






0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

20

40

60

80

100

120

0 001 0025 005 0075 01

Cel

l via

bilit

y (

)


lowast

lowastlowast

lowast

(a)



(b)





4 Discussion



0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

2

4

6

8

10

12

14

0 001 0025 005 0075 01


Num

ber o

f cel

ls (times104)

Sulfratulle

lowast

lowast

lowastlowast

(a)



0025 wv genipin

(b)



0

05

1

15

2

25

3

35

4

45


NO

pro

duct

ion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

(a)

0

20

40

60

80

100


Col

lage

n pr

oduc

tion

(120583g

mL)

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)







3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




3 d

7 d

14 d

28 d












3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






3 d

7 d

14 d

28 d






5 Conclusion






Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







Acknowledgment


References















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Preliminary Characterization of Genipin...

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