Journal of Applied Polymer Science Journal...Journal of Applied Polymer Science Journal Copy of...

Journal of Applied Polymer Science Journal Copy of e-mail Notification

Journal of Applied Polymer Science Published by John Wiley & Sons, Inc.

Dear Author,

YOUR PAGE PROOFS ARE AVAILABLE IN PDF FORMAT; please refer to this URL address:

http://121.240.155.8/jw/retrieval.aspx?pwd=31d070ccab19

Login: your e-mail address

Password: 31d070ccab19

The site contains 1 file. You will need to have Adobe Acrobat Reader software to read these files. This is free

software and is available for user downloading at http://www.adobe.com/products/acrobat/readstep.html.

Alternatively, if you would prefer to receive a paper proof by regular mail, please contact Prashant/Sankar/Balaji (e-

mail: [email protected], phone: +91 (44) 4205-8888 (ext.217)). Be sure to include your article number.

This file contains:

Author Instructions Checklist

Adobe Acrobat Users - NOTES tool sheet

Reprint Order Information

A copy of your page proofs for your article

Please read the page proofs carefully and:

1) indicate changes or corrections in the margin of the page proofs;

2) answer all queries (footnotes AQ1, AQ2, etc.) on the last page of the PDF proof;

3) proofread any tables and equations carefully;

4) check that any Greek symbols, especially "mu", have translated correctly.

Special Notes:

Please return corrections to Wiley via REPLY E-MAIL as soon as possible.

Your article will be published online via our EarlyView service after correction receipt. Your prompt attention to and

return of page proofs is crucial to faster publication of your work. Thank you for your cooperation.

NEW! Return corrections via reply e-mail to:

APP Journal Team

[email protected]

Journal of Applied Polymer Science Journal Copy of e-mail Notification

If you experience technical problems, please contact Prashant/Sankar/Balaji (e-mail: [email protected],

phone: +91 (44) 4205-8888 (ext.217)).

PLEASE NOTE: This e-proof is to be used ONLY for the purpose of returning corrections to the publisher. The

[email protected] address is to be used ONLY for the submission of proof corrections, the reprint order

form, and the copyright transfer agreement (if you have not already signed one). PLEASE ALWAYS INCLUDE

YOUR FIVE-DIGIT ARTICLE NO. (32813) WITH ALL CORRESPONDENCE.

Thank you.

Sincerely,

APP Journal Team

E-mail: [email protected]

Softproofing for advanced Adobe Acrobat Users - NOTES tool NOTE: ACROBAT READER FROM THE INTERNET DOES NOT CONTAIN THE NOTES TOOL USED IN THIS PROCEDURE.

Acrobat annotation tools can be very useful for indicating changes to the PDF proof of your article. By using Acrobat annotation tools, a full digital pathway can be maintained for your page proofs. The NOTES annotation tool can be used with either Adobe Acrobat 6.0 or Adobe Acrobat 7.0. Other annotation tools are also available in Acrobat 6.0, but this instruction sheet will concentrate on how to use the NOTES tool. Acrobat Reader, the free Internet download software from Adobe, DOES NOT contain the NOTES tool. In order to softproof using the NOTES tool you must have the full software suite Adobe Acrobat Exchange 6.0 or Adobe Acrobat 7.0 installed on your computer. Steps for Softproofing using Adobe Acrobat NOTES tool: 1. Open the PDF page proof of your article using either Adobe Acrobat Exchange 6.0 or Adobe Acrobat 7.0. Proof your article on-screen or print a copy for markup of changes. 2. Go to Edit/Preferences/Commenting (in Acrobat 6.0) or Edit/Preferences/Commenting (in Acrobat 7.0) check “Always use login name for author name” option. Also, set the font size at 9 or 10 point. 3. When you have decided on the corrections to your article, select the NOTES tool from the Acrobat toolbox (Acrobat 6.0) and click to display note text to be changed, or Comments/Add Note (in Acrobat 7.0). 4. Enter your corrections into the NOTES text box window. Be sure to clearly indicate where the correction is to be placed and what text it will effect. If necessary to avoid confusion, you can use your TEXT SELECTION tool to copy the text to be corrected and paste it into the NOTES text box window. At this point, you can type the corrections directly into the NOTES text box window. DO NOT correct the text by typing directly on the PDF page. 5. Go through your entire article using the NOTES tool as described in Step 4. 6. When you have completed the corrections to your article, go to Document/Export Comments (in Acrobat 6.0) or Comments/Export Comments (in Acrobat 7.0). Save your NOTES file to a place on your harddrive where you can easily locate it. Name your NOTES file with the article number assigned to your article in the original softproofing e-mail message.

7. When closing your article PDF be sure NOT to save changes to original file. 8. To make changes to a NOTES file you have exported, simply re-open the original PDF proof file, go to Document/Import Comments and import the NOTES file you saved. Make changes and reexport NOTES file keeping the same file name. 9. When complete, attach your NOTES file to a reply e-mail message. Be sure to include your name, the date, and the title of the journal your article will be printed in.

Additional reprint purchases

Should you wish to purchase additional copies of your article, please click on the link and follow the instructions provided:

https://caesar.sheridan.com/reprints/redir.php?pub=10089&acro=APP

Corresponding authors are invited to inform their co-authors of the reprint options available.

Please note that regardless of the form in which they are acquired, reprints should not be resold, nor further disseminated in electronic form, nor deployed in part or in whole in any marketing, promotional or educational contexts without authorization from Wiley. Permissions requests should be directed to mail to: [email protected]

For information about ‘Pay-Per-View and Article Select’ click on the following link: http://www3.interscience.wiley.com/aboutus/ppv-articleselect.html

mailto:[email protected]

http://www3.interscience.wiley.com/aboutus/ppv-articleselect.html

http://www3.interscience.wiley.com/aboutus/ppv-articleselect.html

Asymmetric Polyurethane Membrane with In Situ-Generated Nano-TiO2 as Wound Dressing

Yi Chen,1 Lidan Yan,2 Tun Yuan,3 Qiyi Zhang,2 Haojun Fan1

1National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University,Chengdu 610065, People’s Republic of ChinaAQ12School of Chemical Engineering, Sichuan University, Chengdu 610065, People’s Republic of China3Engineering Research Center in Biomaterials, Sichuan University, Chengdu 610065, People’s Republic of China

Received 9 October 2009; accepted 13 May 2010DOI 10.1002/app.32813Published online 00 Month 2010 in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: For potential application as an ideal wounddressing, a novel asymmetric polyurethane membrane within situ-generated nano-TiO2 (PUNT) was successfully pre-pared via a combination of solvent evaporation, wet phaseinversion, and organic–inorganic hybridization. According tothis combination method, the PUNT membrane consisted ofan integral and dense skin layer supported by a porous sub-layer, with nano-TiO2 particles dispersed evenly throughoutthe sample. The skin layer was found to be impermeable tobacteria penetration, and the porous sublayer was designedfor absorbing high amounts of exudates. Nitrogen adsorp-tion/desorption experiment proved that extra mesoporeswere created in the PUNT membrane after organic–inorganichybridization, which resulted in promoted gas permeability,water vapor transmission rate, and exudate absorption capa-bility. The PUNT membrane, as a consequence, could acceler-

ate gas exchange and also provide an optimal level ofmoisture over the wound beds without risking dehydrationor exudate accumulation. Shake flask testing and cell culture(L929) assay indicated that the PUNT membrane exhib-ited antibacterial activity against Pseudomonas aeruginosa,Escherichia coli, and Staphylococcus aureus, whereas showedno cytotoxicity. From in vivo animal studies, the curativeeffect of PUNT membrane was found to be better thangauze and a commercial polyurethane membrane dress-ing. These results indicated that the PUNT membranewith multifunctions prepared in this study has potentialfor application as an ideal wound dressing. VC 2010 WileyPeriodicals, Inc. J Appl Polym Sci 000: 000–000, 2010

Key words: polyurethanes; nanocomposites; films;biomaterials

INTRODUCTION

In the treatment of burns, donor site wounds, skinulcers, and surgical wounds, immediate coverage ofwound beds with dressings is necessary for rapidand proper healing. According to modern medicalinsights,1,2 there are several functions that are desira-ble for an ideal wound dressing: (1) a suitable watervapor transmission rate (WVTR) that produces amoist environment on the wound beds, without risk-ing dehydration or exudate accumulation; (2) suffi-cient gas permeability for oxygen-requiring repara-tive processes; (3) a high level of fluid absorptioncapability to remove excessive exudates that containnutrients for bacteria from the wound beds; (4) agood barrier against the penetration of infection-causing microorganisms; (5) antibacterial activity to

suppress bacteria growth beneath the dressing; and(6) the absence of any cytotoxic effects in case of sec-ondary damage to the neonatal tissues.

Among various synthetic dressings in the market,polyurethane (PU) membrane dressings are increas-ingly popular considering their good physicalstrength, abrasion resistance, fatigue life, and tissuecompatibility.3 However, their totally dense structureresults in certain inconvenient limitations, includinglow water vapor/gas transmission rate and poorfluid absorption capability. These disadvantages fre-quently cause exudate accumulation and infection-related complications in clinic cases. Meanwhile, lowgas permeability inhibits oxygen exchange throughthe dressings, which is detrimental to cell migrationand mitosis. Another major problem with PU mem-brane dressings is the incorporation of aggressiveantimicrobial compounds. Despite their efficiencyagainst bacteria contamination, these compoundsmay exhibit cytotoxic effects to newly formed tissue.Thus, it seems that the PU membrane dressings cur-rently available are far from ideal, and modificationis necessary to overcome the above limitations.

Nowadays, asymmetric polymer membranes havefound numerous application fields, ranging from gas

Correspondence to: H. Fan ([email protected]).Contract grant sponsor: Hi-tech Research and

Development Program of China (863 Program); contractgrant number: 2007AA03Z341.

Journal of Applied Polymer Science, Vol. 000, 000–000 (2010)VC 2010 Wiley Periodicals, Inc.

J_ID: Z8E Customer A_ID: APP32813 Date: 15-June-10 Stage: Page: 1

ID: jaishankarn I Black Lining: [ON] I Time: 13:34 I Path: N:/3b2/APP#/Vol00000/100931/APPFile/C2APP#100931

separation, textile to biomedical industries.4,5 Amongthe methods proposed to prepare asymmetric mem-branes, wet phase inversion method is the mostwidely used technique. In this method, a castingpolymer solution containing solvent is immersedinto a nonsolvent coagulant that has a low mutualaffinity to the solvent. Because of delayed phase sep-aration at the outmost interface region of the castingsolution, a compact top layer with porous substruc-ture can be obtained. If this asymmetric membraneis used as a wound dressing, there seems to be twodisadvantages. First, the top layer formed by thismethod is quite thin (less than 1 lm),5–7 which can-not prevent excessive water vapor evaporation fromthe wound beds. Second, although the top layer hasa compact structure, it contains defects5–8 that signif-icantly compromise its barrier function against out-side contamination.

Polymeric nanocomposites have been established asan exciting new class of materials that are particle-filled, with at least one dimension of the dispersedparticles being in the nanometer range, which usuallyexhibit superior performances compared with conven-tional filler composites. Basically, the most commonlyused methods to prepare polymeric nanocompositesthus far are melt mixing and solution blending.9 Aspreformed nanoparticles are chemically fused together,it is hard for these two methods to achieve even distri-bution of nanoparticles in the polymer, which affectsthe final properties of the composites. Besides, oxida-tion and b-scission lead to degradation of polymerduring melt mixing, not to mention that this methodis energy intensive. In this study, solvent evaporation,wet phase inversion, and organic–inorganic hybridiza-tion were combined to design a novel asymmetric PUmembrane with in situ-generated nano-TiO2 (PUNT)as an ideal wound dressing. After a solvent evapora-tion process, organic–inorganic hybridization occurredsynchronously as PU solution coagulated by wetphase inversion. The PUNT membrane according tothis design consisted of an integral, dense skin layersupported by a porous sublayer, with well-separatednano-TiO2 particles dispersed evenly throughout thesample. Because of this special structure, the crucialperformances of the PUNT membrane fitted the basicrequirements of an ideal wound dressing. Finally, thecurative effect of the PUNT membrane was evaluatedby in vivo animal study using rat model.

EXPERIMENTAL

Materials

The commercial biomedical PU resin (BioSpan, 20 wt% solution in N,N-dimethylacetamide) used in thisstudy was supplied by Polymer Technology Group(United States). It was synthesized from methylene

diphenylene diisocyanate, polytetramethylene oxidewith molecular weight of 2000 g/mol, and mixed dia-mine chain extenders (ethylenediamine and 1,3-cyclo-hexanediamine). Tetrabutyl titanate and acetylacetonewere purchased from Aldrich Chemical (UnitedStates). Anhydrous n-butanol and acetic acid wereobtained from Junsei Chemical (Japan). Tegaderm(3M) brand commercial PU membrane dressing(9543HP) was used as a comparison material.

Membrane preparation

The modification of tetrabutyl titanate was performedunder dry argon with Schlenk-line technique to avoidany partial hydrolysis by atmospheric moisture. Apredetermined amount of tetrabutyl titanate (con-verted into TiO2 concentration, by weight of the solidcontent in BioSpan) was added dropwise under stir-ring into an optimized volume of acetylacetonediluted with n-butanol (the molar ratio of acac : Tiwas 4 : 1). This mixture was ultrasonicated for 1 h toobtain a transparent and yellow solution. The modi-fied nanoprecursor prepared above was then dis-solved in BioSpan under ultrasonic oscillation. Afterdegassed adequately, the mixture was cast on a poly-tetrafluoroethylene release paper to a thickness of 150lm using a Gardner knife. The casting solution waspreheated at 50�C for 5 min with a dry argon streamfor solvent evaporation before immersed into anaqueous bath at 25�C (pH 4.5–5.0, adjusted by aceticacid) for 30 min. During the wet phase inversion pro-cess, as the PU solution coagulated, the nanoprecur-sor hydrolyzed and condensated, in situ generatinginorganic nano-TiO2 particles in the organic PU mem-branes. Finally, the resultant membranes were rinsedin distilled water and dried at 50�C for 24 h. Themembrane samples in this study were referred to asPUNT-x, wherein PU and NT represented polyur-ethane and nano-TiO2, respectively, while x showedthe TiO2 concentration.

SEM/AFM analysis

Scanning electron microscope (SEM, Hitachi ModelS520, Japan) was used to observe the cross-sectionmorphologies of PUNT membranes. The cross sec-tions were prepared by cryogenically fracturing themembranes in liquid nitrogen and then coated withaurum before observation. The morphologies ofmembrane surfaces were observed by atomic forcemicroscope (AFM, SHIMADZU, SPM-9600, Japan) atambient temperature using a tapping mode.

Measurement of WVTRs

WVTRs of the PUNT membranes were determinedaccording to a published method.10 A specially



2 CHEN ET AL.

Journal of Applied Polymer Science DOI 10.1002/app

designed cup containing human plasma was sealedwith sample in such a manner that the cup mouthdefined the area of the sample exposed to the envi-ronment. Human plasma (supplied by the BloodBand at West China Hospital) was adopted becauseit was similar in composition to wound exudates.The edges of the sample were thoroughly sealed toprevent plasma leakage. Then, the assembly washanged inverted in a test chamber at 35�C 6 0.5�C,with a constant relative humidity of 35% 6 1%. Theselected temperature corresponded to the averagesurface temperature of injured skin reported byLamke et al.,11 and the relative humidity fell withinthe range of 22–49% observed by Wu for a hospitalburns ward.12 The weight change of permeation cupwas recorded after 24 h, and the WVTR was calcu-lated by the following equation.

WVTR ¼ a1 � a2

S;

where (a1 � a2) is the weight change of permeationcup (g) and S is the area of permeation (m2).

Measurement of gas permeability

Gas (H2, N2, and O2) permeation testing of PUNTmembranes was conducted by using Yanaco GTR-10gas permeability analyzer (Japan). The testing tem-perature was 35�C 6 0.5�C, and the pressure differ-ence over the membranes was 1 atm. When the plotof Q (cm3, the amount of gas that permeatedthrough the sample) versus t (s, testing time) becamelinear, a steady-state gas permeability coefficientP (cm3 cm/cm2 s atm) could be calculated as follows.

P ¼ dQ=dt

S� Dp=l;

where S and l stand for the permeation area (cm2)and the sample thickness (cm), respectively; Dp isthe pressure driving force (atm).

Nitrogen adsorption/desorption experiment

Low-temperature nitrogen adsorption/desorptionexperiments were conducted to determine the poreinformation and specific surface area of PUNT mem-branes at the boiling temperature of liquid nitrogen(�196�C) by using ASAP 2020 Accelerated SpecificSurface Area and Porosimetry Analyzer from Micro-meritics (United States). Samples were degassedovernight at 100�C under high vacuum before analy-sis. The obtained adsorption/desorption isothermsprovided information on total pore volume (Vtot)and size distribution in micro-(pore diameter d < 2nm), meso-(2 < d < 10 nm), and macropore range.

Vtot was obtained at the condition near to the satura-tion pressure p/p0¼ 0.99. The Horvath–Kawazoe(HK) model was used for micropore size calculation.In meso- and macrodomain, classical thermody-namic Barrett–Joyner–Halenda (BJH) theory wasused. Brunauer–Emmitt–Teller (BET) theory wasapplied for specific surface area calculation from theobtained adsorption isotherms. The range of fit forthe BET equation was restricted to nitrogen relativepressure (p/p0) from 0.05 up to 0.35.

PBS solution absorption

The water absorption and equilibrium water contentof PUNT membranes were determined by swellingthe samples in pH 7.4 of phosphate-buffered saline(PBS) at 35�C 6 0.5�C. A known weight of PUNTmembrane was placed in the PBS media for 7 daysto reach equilibrium. Then, the sample was weighedimmediately after it was blotted with filter paper toremove excessive surface water. The water absorp-tion and equilibrium water content were calculatedas follows.

Water absorption ð%Þ ¼Ws �W0

W0� 100%

Equilibrium water content ð%Þ ¼Ws �W0

W0� 100%;

where Ws is the weight of swollen sample and W0 isthe initial sample weight.

Bacteria experiments

Pseudomonas aeruginosa (P. aeruginosa, ATCC 27317,Gram-negative), Escherichia coli (E. coli, ATCC 25922,Gram-negative), and Staphylococcus aureus (S. aureus,ATCC 25923, Gram-positive) were selected as indica-tors of experimental bacteria, as they remain themost common and dangerous pathogen in burn inju-ries.13,14 Bacteria were cultivated in Luria Bertani(LB) broth at 37�C for 24 h and diluted to 1 � 105

CFM/mL with sterile distilled water.Bacteria penetration experiment was carried out

using an assembly composed of an automatic injec-tor and a filtration cell, which was drawn schemati-cally in Figure F11. Samples with diameter of 5 cmwere cut from the PUNT membranes. They weresterilized with ethylene oxide gas, sandwiched bytwo flat rubber gaskets to prevent fluid leakage, andmounted on the perforated stainless steel disk (di-ameter of 2.5 cm) in the filtration cell. Before inject-ing a suspension of P. aeruginosa, E. coli, or S. aureus(1 � 105CFM/mL) through the sample, precondi-tioning was conducted by injecting sterile distilledwater for five times. The injection pressure was kept



POLYURETHANE MEMBRANE WITH IN SITU-GENERATED NANO-TiO2AQ2 3


at 1 atm. The presence of bacteria in the outflowwas checked by agar plate culture method.

The antibacterial activity of PUNT membraneswas measured by shake flask testing according tothe national standard GB15979-2002 defined by thePublic Healthy Department of China: (1) Samplepieces (5 g) were sterilized with ethylene oxide gasbefore use and then put into a 250-mL flask with 5mL bacteria suspension (1 � 105 CFM/mL) and 70mL phosphate-buffered saline. (2) The mixed solu-tion was shook on an agitation shaker with thespeed of 300 rpm/min at 25�C for 2 h. This processwas exposed to UVA (315–400 nm) irradiation fromtwo 20 W black light lamps (Philips, Holland). TheUVA intensity was determined to be 1 mW/cm2 bya UVR-400 radiometer (S-365 UV-sensor, wavelengthsensitivity of 315–400 nm, Iuchi, Osaka, Japan). Thedistance between the light source and the mixedsolution was set at 20 cm. (3) The bacteria concentra-tions before and after shaking could be counted byagar plate counting method. The antibacterial activ-ity was calculated as follows.

Antibacterial activity ð%Þ ¼ A� B

A� 100%;

where A and B are the bacteria concentrations beforeand after shaking, respectively.

Cell adhesion and proliferation

Cell adhesion and proliferation on PUNT mem-branes were investigated to evaluate cytotoxicityaccording to ISO 10993-5. Test samples (1 cm in di-ameter) cut from PUNT membranes were washedwith 99% ethanol for three times and then immersedin 70% aqueous ethanol for 24 h for sterilization.After washed with PBS three times to remove resid-ual alcohol, the test samples were placed into thecenter of wells in a 24-well plate, respectively. Then,L929 fibroblasts (1 � 105 cells) in 2 mL Dulbecco’smodified Eagle medium with 10% fetal calf serumwere seeded onto the samples. Polystyrene well andlatex were used as negative and positive control,

respectively. After incubated at 37�C with 5% CO2

for 3 days, the attached cells on samples were fixedwith 0.5% glutaraldehyde, washed with PBS threetimes, and dehydrated in ethanol graded series.Finally, the samples were treated with tert-butanolfor ethanol replacement and dried by CO2 criticalpoint drying method. The attached cells were aurumdeposited in vacuum and observed by SEM.

The cell proliferation was measured after the cellswere seeded for 1, 3, and 7 days, respectively, usingthe procedure of MTT (methyl thiazolyl tetrazolium)assay.

In vivo wound healing test

The curative effect of PUNT-4 was evaluated byusing rat model. Male rats aged 3 months andweighing 300 6 50 g were anesthetized with anintramuscular injection of pentobarbital at a dose of50 mL/kg of body weight. Then, the dorsal hair ofeach animal was shaved. After the dehaired areawas disinfected with 70% ethanol, a full thicknesswound with a surface area of 2 cm � 2 cm wasexcised from the back of each rat. For comparison,the rats were divided randomly into three groups (n¼ 25/group). The wound surfaces in one groupwere dressed with an equal size of PUNT-4, and theother two groups were covered with sterile gauzeand Tegaderm (9543HP), respectively. The treatedanimals were kept in separate cages and fed withcommercial rat feed and water ad libitum during thehealing period. The size of the wound was measuredat 3rd, 6th, 12th, 15th, 18th, and 21st day, and thepercentage of wound healing was calculated by theratio of the observed size to the original wound size.The mean 6 standard deviation of samples in eachgroup was reported.

Statistics

The reported data were mean 6 standard deviationof quintuplicate samples for each measurement. Sta-tistical analysis was performed with Student’s t-test.P < 0.05 was accepted as statistically significant.

RESULTS AND DISCUSSION

Membrane morphologies

By combining solvent evaporation, wet phase inver-sion, and organic–inorganic hybridization, a novelasymmetric PU membrane with in situ-generatednano-TiO2 was prepared in this study, which fittedthe basic requirements of an ideal wound dressing.During the solvent evaporation process, the loss ofsolvent drove the outermost region of the originalpolymer solution to form an integral and dense skin

Figure 1 Schematic illustration of bacteria penetrationexperiment.



4 CHEN ET AL.


layer. This skin layer was expected to provide a bar-rier on wound beds against outside contamination.The underlying polymer solution with formed skinlayer was then immersed into a coagulation bath,in which wet phase inversion was induced by diffu-sion of nonsolvent into and solvent out of the poly-mer solution. Because of this solvent/nonsolventexchange, the underlying polymer solution sepa-rated into a polymer-rich and a polymer-lean phase.The former resulted in polymer skeleton, and the lat-ter formed the pores. This porous sublayer wasdesigned as a reservoir for excessive exudates on thewound beds. While the sublayer shaped during wetphase inversion, nanoprecursor hydrolyzed and con-densated, in situ generating inorganic nano-TiO2

throughout the organic PU membrane. Through thisorganic–inorganic hybridization, crucial performan-ces of the PUNT membrane as a wound dressing

were promoted, which were beneficial for rapid andproper wound healing.

Figure F22 illustrates SEM images concerning thecross-section morphologies of PUNT membranes. Itwas revealed that the PUNT membranes exhibiteddouble-layer structure as designed. The outmostlayer was integral and dense, � 10 lm, and the sub-layer exhibited a finger-like porous structure. De-spite the in situ generation of nano-TiO2, this asym-metric morphology did not change significantly.Higher magnification further showed that thein situ-generated TiO2 particles were well separatedand dispersed evenly in both skin layer and sub-layer. The size of these particles ranged from 70 to130 nm. As the sublayer surface would contact thewound beds directly, the morphologies of this sur-face were specially investigated by AFM, and theresults are illustrated in Figure F33. Similar to those inthe cross section, the in situ-generated TiO2 particleson the sublayer surface were also in nanoscale anddispersed evenly. As already known, tetrabutyl tita-nate is an extremely moisture-sensitive compound.Its rapid hydrolysis and condensation in BioSpanthat unavoidably contained a tiny amount of mois-ture yielded undesirable TiO2 agglomerates and pre-cipitates. Therefore, acetylacetone (acacH) wasadopted in this study as a chelating agent to reducethe chemical reactivity of the nanoprecursor. Accord-ing to prior report,15 the chelating product wasassumed to be Ti(OC4H9)3-xacacx based on the fol-lowing reaction.

TiðOC4H9Þ4 þ xacacH! TiðOC4H9Þ3�xacacx

þ xC4H9ðOHÞ:

By substituting the easily hydrolyzable alkoxygroups, the less reactive Ti-acac groups acted as poi-sons toward hydrolysis and condensation, restrain-ing the presence of TiO2 agglomerates and precipi-tates in the PU membrane. On the other hand, theformation of TiO2 could be hindered sterically bythe PU macromolecular matrix, which also helpedto keep their size in nanoscale and preventagglomeration.

Water vapor transmission rate andgas permeability

Exudate retention below a wound dressing, causedby poor WVTR, or the dehydration of a granulatingwound bed, caused by rapid water loss, can pose se-rious problems to the healings of wounds. An idealwound dressing therefore should control evapora-tive water loss from a wound at an optimal rate,preventing excessive dehydration and exudate accu-mulation. Lamke et al. reported that the WVTRs fornormal skin, first degree burns, and granulating

Figure 2 SEM images concerning the cross section ofPUNT membranes. (a1) whole image of PUNT-0; (a2)whole image of PUNT-5; (b1) skin layer of PUNT-0(3000�); (b2) skin layer of PUNT-5 (3000�); (c1) skin layerof PUNT-0 (10,000�); (c2) skin layer of PUNT-5 (10,000�);(d1) sublayer of PUNT-0 (10,000�); and (d2) sublayer ofPUNT-5 (10,000�).



POLYURETHANE MEMBRANE WITH IN SITU-GENERATED NANO-TiO2 5


wounds are 204 6 12, 279 6 26, and 5138 6 202g/m2 day, respectively.11 It was further recommendedby Wong that wound dressings with WVTRs in therange of 2000–2500 g/m2 day, half the loss of granu-lating wounds, would be sufficient to give adequatemoisture and prevent exudate accumulation.16 Typi-cal PU membrane dressings commercially availableinclude Tegaderm (3M), Bioclusive (Johnson-John-son), and Op Site (Smith & Nephew), which haveWVTRs of 491 6 44, 394 6 12, and 792 6 32 g/m2

day, respectively.17,18 Such low WVTRs lead to theexudate accumulation, decelerating the healing pro-cess, and risking of bacteria contamination. In thisstudy, special membrane-forming method and or-ganic–inorganic hybridization were combined to pre-pare PU membrane dressing with ideal WVTR.TableT1 I shows the WVTRs and gas (H2, N2, and O2)permeability coefficients of various PUNT mem-branes. It could be seen that the WVTRs were ashigh as 1258–2689 g/m2 day and increased withincreasing nano-TiO2 concentration. According toWong’s recommendation, it seemed that PUNT-3and PUNT-4 were quite suitable as dressing mem-branes in terms of WVTRs. Similarly, the gas perme-ability coefficients also increased with increasingnano-TiO2 concentration. Increased gas permeability

could improve gas exchange through the dressingmembranes, which was beneficial for oxygen-requir-ing reparative processes.

According to conventional principle, adding imper-meable filler particles to polymer will lead to a sys-tematic reduction in mass transport. Physically, suchbarrier phenomenon can be attributed to an increaseddiffusion path length, as penetrants are forced to takea tortuous course around filler particles to traverse amembrane.19 However, the establishment of suchprinciple is based on the assumption that the surfaceof the filler particles is completely wetted by sur-rounding polymer. It does not take account of theinterfacial effects between polymer and some specialfillers such as inorganic nanoparticles. As for the per-meability behaviors of PUNT membranes in thisstudy, the deviate from conventional prediction couldbe ascribed to the incompatibility between organic PUand inorganic nano-TiO2, which resulted in pores atthe interface. The effects of these interfacial poresbecame influential because of the tremendous surfacearea of nano-TiO2. In the process of mass transporta-tion, these pores helped mass diffusion and conse-quently resulted in an increase in permeability. SEMand AFM were useful for visualizing composite mor-phologies but did not readily reveal delicate

Figure 3 AFM images concerning the sublayer surface of PUNT membranes. (a) PUNT-0, (b) PUNT-1, (c) PUNT-3, and(d) PUNT-5. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]AQ3



6 CHEN ET AL.


interfacial structure between components. Therefore,nitrogen adsorption/desorption experiment was per-formed to confirm the existence of such interfacialpores.

Pore information and specific surfacearea of PUNT membranes

Nitrogen adsorption/desorption experiment providesa convenient way to characterize the pore informationand specific surface area of porous materials. Thetotal pore volumes of PUNT membranes are shownin Table I. The detected values increased as nano-TiO2 concentration increased. This indicated that thein situ generation of nano-TiO2 created extra pores inthe PU membranes. To provide detailed informationabout these extra pores, the pore size distribution ofPUNT membranes was calculated by using HKmodel for micropore, and BJH model for mesoporeand macropore, respectively. By comparing the poresize distribution of different PUNT membranethroughout the entire size range, the only differencewas found in the mesopore diameter range as dis-played in FigureF4 4. It was revealed that all the otherPUNT membranes contained extra mesopores com-

pared with PUNT-0, and the size of these extra meso-pores increased with increasing nano-TiO2 concentra-tion, varying from 2.6 nm for PUNT-1 to 3.3 nm forPUNT-5. These results indicated that the incompati-bility-induced interfacial pores mentioned previouslydid exist in the PUNT membranes, and it seemed asif the more nano-TiO2 in situ generated, the more sig-nificantly such incompatibility worked.

Another evidence for the presence of extra poresin PUNT membranes could be obtained from theanalysis of specific surface area of samples. As illus-trated in Figure F55, the BET specific surface area ofPUNT membranes was plotted against the nano-TiO2 concentration. If in situ-generated TiO2 particleswere completely wetted by PU, a linear decrease ofspecific surface with increasing TiO2 concentrationwas expected (represented by the dashed line). Asfor the measured values, however, sharp deviatewas recorded. The more nano-TiO2 in situ generated,the higher BET specific surface the sample exhibited.This distinct deviation could also be ascribed to theformation of pores between organic–inorganic

TABLE IWVTRs, Gas Permeability Coefficients (P), and Total Pore Volume (Vtot) of PUNT Membranes

Sample WVTR (g/m2 day)

P �10�7 (cm3 cm/cm2 satm)

Vtot � 10�3 (cm3/g)H2 N2 O2

PUNT-0 1258 6 29 451 6 7 26 6 1 134 6 4 4.41 6 0.05PUNT-1 1435 6 34 538 6 10 29 6 1 156 6 3 4.62 6 0.07PUNT-2 1871 6 20 605 6 8 32 6 1 189 6 3 4.77 6 0.05PUNT-3 2267 6 31 686 6 7 36 6 2 208 6 5 4.96 6 0.05PUNT-4 2412 6 26 739 6 8 39 6 2 219 6 3 5.12 6 0.04PUNT-5 2689 6 37 819 6 14 43 6 1 233 6 5 5.32 6 0.06

Figure 4 Mesopore size distribution of PUNT mem-branes. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

Figure 5 BET specific surface area of PUNT membranes.The dashed line represents the theoretical decrease thatcould be expected when the in situ-generated TiO2 wascompletely wetted by surrounding PU. [Color figure canbe viewed in the online issue, which is available atwww.interscience.wiley.com.]





phases, which created extra specific surface for thePU membranes.

Fluid absorption capability

The capability to absorb excessive exudates from thewound beds is another important characteristic for anideal dressing. In general, commercially available PUmembrane dressings exhibit low fluid absorptioncapability due to their totally dense structure. Forexample, the water absorption of Tegaderm, Bioclu-sive, and Op Site is only in the range of 31–46%.18 Asa consequence, their applications are limited to lowexudates cases, or exudates accumulation and infec-tion occur. TableT2 II shows the water absorption andequilibrium water content of various PUNT mem-branes. Because of the porous sublayer designed as areservoir for exudates, the water absorption of PUNTmembranes was in the range of 489.5–653.2%. Thesehigh values, according to previous literature,18,20 wereenough to prevent wound beds from exudate accu-mulation. On the other hand, the water absorptionincreased with increasing nano-TiO2 concentration.This was because the extra interfacial pores betweenPU and nano-TiO2 were beneficial for more capillaryadsorbed water. In addition, the equilibrium watercontent of PUNT membranes was also much higherthan those of Tegaderm, Bioclusive, and Op Site,which are as low as 21–36%.18 High equilibriumwater content guaranteed a moist environment overthe wound beds.18,20

Bacteria penetration and in vitroantibacterial activity

To protect the wound beds from outside microor-ganism, an ideal dressing should act as a barrieragainst microorganism penetration. In this study, nobacteria were detected in the outflow through PUNTmembranes, despite that the pore size at the sub-layer of the PUNT membranes was much largerthan the minimal size of the bacteria (0.5 lm). Thisimpermeability should be attributed to the integraland dense skin layer of samples formed in the sol-vent evaporation process.

Although the PUNT membranes could exclude out-side bacteria, bacteria survived in the depth of sweatglands and hair follicles might still colonize thewounds easily to cause infection after injury, unlesstopical antibacterial agents were used.21 Table T3IIIshows the antibacterial activity of PUNT membranesmeasured by shake flask testing. It was found thatPUNT-0 exhibited almost no antibacterial activityagainst P. aeruginosa, E. coli, or S. aureus, whereas theantibacterial activity of other PUNT membranesincreased with increasing nano-TiO2 concentration.Especially for PUNT-4 and PUNT-5, the percentagereduction ratios for three stains exceeded 68%. The ori-gin of such antibacterial activity could be ascribed tothe well-known photocatalytic activity of TiO2. In brief,when exposed to ultraviolet light (k < 400 nm), TiO2

will be activated to generate electron-hole pairs. Theelectrons reduce oxygen to produce superoxide anions.The holes oxidize water to produce hydroxyl radicals.These reactive species are powerful for degradingorganic pollutants, such as bacteria and viruses.22,23

The chemistry of tetrabutyl titanate to produceTiO2 has been established for decades. As the PUNTmembranes were prepared without calcination, the insitu-generated nano-TiO2 should be mostly present inan amorphous form with hydrated water molecules(TiO2.nH2O).24–26 Previous literature claimed thatamorphous TiO2 was inactive compared with crystal-lite forms because it contained high concentrations ofdefects that functioned as rapid electro-hole recombi-nation centers.27 However, the amorphous TiO2 usedin this literature was nonhydrated, because it wasprecalcined at around 300–400�C before use. Actually,the presence of hydrated water could substantiallysuppress the recombination of holes with electrons atdefects, making amorphous TiO2.nH2O active, or anti-bacterial herein, as TiO2 crystallites.26 Additionally,the extremely high surface area of nanoscaled TiO2

facilitated the adsorption of target bacteria, whichaccelerated the rate of antibacterial reaction.28 There-fore, although containing amorphous TiO2, the PUNTmembranes still exhibited obvious antibacterial activ-ity. In fact, the photocatalysis of hydrated form ofamorphous TiO2 had been reported in manyliteratures.25,26,29

TABLE IIWater Absorption and Equilibrium Water Content

of PUNT Membranes

SampleWater

adsorption (%)Equilibrium water

content (%)

PUNT-0 489.5 6 9.2 83.2 6 0.2PUNT-1 527.6 6 4.5 84.1 6 0.3PUNT-2 559.0 6 7.7 84.9 6 0.3PUNT-3 582.3 6 6.4 85.5 6 0.2PUNT-4 610.9 6 8.6 86.3 6 0.2PUNT-5 653.2 6 5.9 86.9 6 0.2

TABLE IIIAntibacterial Activity of PUNT Membranes

Sample

Antibacterial activity (%)

P. aeruginosa E. coli S. aureus

PUNT-0 2.6 6 0.6 3.2 6 0.9 2.2 6 1.1PUNT-1 32.3 6 2.1 22.0 6 1.8 28.1 6 2.5PUNT-2 47.2 6 1.9 35.4 6 2.2 40.5 6 2.4PUNT-3 58.3 6 2.7 49.3 6 2.0 54.8 6 1.9PUNT-4 74.0 6 1.9 68.6 6 2.8 70.3 6 2.6PUNT-5 86.9 6 2.3 78.5 6 1.7 81.5 6 2.2



8 CHEN ET AL.


Cytotoxicity

As specified by the supplier, BioSpan shows no cyto-toxicity. However, after any modification such as

incorporating aggressive antimicrobial agents, cyto-toxic effect may be induced. The morphologies ofL929 fibroblasts adhesive on PUNT membranes,polystyrene well (negative control), and latex (posi-tive control) after 3 days of seeding are shown inFigure F66. The cells on PUNT membranes showedsimilar morphologies to those on polystyrene well.They all exhibited typical fibroblast morphologies,an elongated and polygonal shape. However, fewcells on latex elongated as they were influenced bytoxic materials released from latex. Figure F77 showsthe MTT absorbance in cell proliferation experiment.No significant difference among PUNT membraneswas found after the same testing time. They were allsignificantly better at promoting cell proliferationthan negative control. The MTT absorbance of latexpositive control was significantly lower than nega-tive control throughout the testing period. This wasbecause the cells were damaged and finally died bytoxic materials released from latex. According tothese results and the evaluation criteria defined inISO 10993-5, it could be concluded that the in situ-generated nano-TiO2 in PUNT membranes woundnot induce any cytotoxicity to the seeded cells.

In vivo wound healing test

After comprehensive consideration, PUNT-4 waschosen in the animal test to compare its curativeeffect with some other dressings. Figure F88 illustratesthe reduction in wound areas at different postopera-tive time using PUNT-4, sterile gauze, and Tega-derm (9543HP), respectively. The wound areadecreased gradually and reached 54% after 21 daysfor sterile gauze. In the case of Tegaderm (9543HP)-treated wound beds, the healing rate was faster and

Figure 6 Morphologies of L929 fibroblasts on PUNTmembranes, polystyrene well (negative control), and latex(positive control) after incubating for 3 days. (a) PUNT-0,(b) PUNT-1, (c) PUNT-3, (d) PUNT-5, (e) polystyrene well,and (f) latex.

Figure 7 Cell proliferation on PUNT membranes, poly-styrene well (negative control), and latex (positive control).The absorbance was normalized with that of negative con-trol at each time interval. *p < 0.05 compared with nega-tive control.

Figure 8 Comparison of gauze, Tegaderm (9543HP), andPUNT-4 in animal test. *p < 0.05 compared with gauze.[Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]





about 93% wound closure was achieved at the endof the test. As for wounds covered with PUNT-4, thepercentage of wound healing almost equivalent tothose treated with Tegaderm (9543HP) was observedafter the same interval time. Except for the abovestatistical data, some other important facts observedduring the animal test were worth noting. After only6 days in the test, dried scabs began to appear onwound beds dressed with gauzes. The fibers of thegauzes become trapped in the nascent tissue, whichmade uncover of gauzes for measuring the woundarea difficult. This was because gauzes exhibited ex-cessive WVTR, and thus they could not maintain amoist environment at the wound/dressing interface.Obvious exudate leakage could be observed onwounds dressed with Tegaderm (9543HP). At the9th day in the test, infections were encountered onsix Tegaderm (9543HP)-treated rats, four of whomeven sacrificed at the 15th–17th day. We thoughtthat this phenomenon was due to the poor WVTRand fluid absorption capability of Tegaderm(9543HP), which hindered the removal of exudatesthat contained nutrients for the bacteria. Favorably,PUNT-4 was found to adhere uniformly on thewound surface without any exudate leakageobserved. It could also be peeled off smoothly fromthe wound beds. The healed area was healthy clean,pinkish red in color, and glistening in appearance.Considering the above facts and the statistical datapresent in Figure 8, it suggested that the curativeeffect of PUNT-4 was better than gauze and Tega-derm (9543HP).

CONCLUSIONS

By combining solvent evaporation, wet phase inver-sion, and organic–inorganic hybridization, a novelasymmetric PU membrane with in situ-generatednano-TiO2 (PUNT) was successfully prepared forapplication as an ideal wound dressing. An integraland dense skin layer was formed during the solventevaporation process, and the following wet phaseinversion and synchronously occurred organic–inor-ganic hybridization could produce a porous sublayerand evenly distributed nano-TiO2 throughout thesample. Each component part of this PUNT mem-brane could serve respective function on woundbeds to provide a suitable environment for properhealing. The skin layer prevented bacteria penetra-tion, and the sublayer could absorb high amounts ofexudates. In the presence of in situ-generated nano-TiO2, this PUNT membrane could control evapora-tive water loss from wound beds at an optimal level

and absorb more exudates, keeping the wound bedsmoist without risking dehydration or exudates accu-mulation. Nano-TiO2 also promoted the gas perme-ability of PUNT membrane, which was beneficial togas exchange between the wound and the environ-ment. After nanomodification, the PUNT membranebecame antibacterial against P. aeruginosa, E coli, andS. aureus, whereas still remained noncytotoxic. Ani-mal studies suggested that the curative effect of thisPUNT membrane was better than gauze and a com-mercial PU membrane dressing.

References

1. Livshits, V. S. Pharm Chem J 1988, 22, 515.2. Ovington, L. G. Clin Dermatol 2007, 25, 33.3. Yang, J. M.; Yang, S. J.; Lin, H. T.; Chen, J. K. J Biomed Mater

Res B Appl Biomater 2007, 80, 43.4. Hinrichs, W. L. J.; Lommen, E. J. C. M. P.; Wildevuur, Ch. R.

H.; Feijen, J. J Appl Biomater 2004, 3, 287.5. Kurdi, J.; Tremblay, A. Y. J Appl Polym Sci 1999, 73, 1471.6. Ismail, A. F.; Lai, P. Y. Sep Purif Technol 2004, 40, 191.7. Li, X.; Chen, C. X.; Li, J. D. J Membr Sci 2008, 314, 206.8. Lee, W. J.; Kim, D. S.; Kim, J. H. Korean J Chem Eng 2000, 17,

143.9. Jain, S.; Goossens, H.; Picchioni, F.; Magusin, P.; Mezari, B.;

Duin, M. V. Polymer 2005, 46, 6666.10. Queen, D.; Gaylor, J. D. S.; Evans, J. H.; Courtney, J. M. Bio-

materials 1987, 8, 367.11. Lamke, L. O.; Nilsson, G. E.; Reithner, H. L. Burns 1977, 3,

159.12. Wu, P. PhD Thesis, Bioengineering Unit, Strathclyde Univer-

sity, Glasgow, 1993.13. Agnihotri, N.; Gupta, V.; Joshi, R. M. Burns 2004, 30, 241.14. Fang, S. D.; Xia, Z. P. Clinical Infection in Surgery; Shenyang

Press: Shenyang, 1999.15. Puri, D. M.; Pande, K. C.; Mehrotra, R. C. J Less-Common Met

1962, 4, 393.16. Wong, P. MSc Thesis, University of Strathclyde, 1980.17. Wu, P.; Fisher, A. C.; Foo, P. P.; Queen, D.; Gaylor, J. D. S.

Biomaterials 1995, 16, 171.18. Yoo, H. J.; Kim, H. D. J Appl Polym Sci 2008, 107, 331.19. Merkel, T. C.; Freeman, B. D.; Spontak, R. J.; He, Z.; Pinnau, I.;

Meakin, P.; Hill, A. J. Science 2002, 296, 519.20. Yoo, H. J.; Kim, H. D. J Biomed Mater Res B Appl Biomater

2008, 85, 326.21. Vindenes, H.; Bjerknes, R. Burns 1995, 21, 575.22. Robertson, J. M. C.; Robertson, P. K. J.; Lawton, L. A. J Photo-

chem Photobiol Chem 2005, 175, 51.23. Acosta, D. R.; Martinez, A. I.; Lopez, A. A.; Magana, C. R.

J Mol Catal A: Chem 2005, 228, 183.24. Khalil, K. M. S.; Zaki, M. I. Powder Technol 1997, 92, 233.25. Randorn, C.; Wongnawa, S.; Boonsin, P. Sci Asia 2004, 30, 149.26. Zhang, Z.; Maggard, P. A. J Photochem Photobiol Chem 2007,

186, 8.27. Ohtani, B.; Ogawa, Y.; Nishimoto, S. J Phys Chem B 1997, 101,

3746.28. Kanna, M.; Wongnawa, S. Mater Chem Phys 2008, 110, 166.29. Ruan, J. F.; Li, C. X.; Wang, Z. H. J Beijing Univ Chem Tech-

nol 2008, 29, 1.



10 CHEN ET AL.


AQ1: Kindly provide department/division name (if available) for all the affiliations.

AQ2: Kindly check whether the short title is OK as given.

AQ3: Please note that it is the policy of this journal to charge authors for the additional cost of reproducingcolor figures in the printed version of the journal. Authors wishing to have figures printed in color in thejournal should contact the journal’s production office ([email protected]) for a price quote. Unless thepublisher of this journal receives other instructions from the author, this figure will be printed in black andwhite and will appear in color in the online version of the article only.



Journal of Applied Polymer Science Journal...Journal of Applied Polymer Science Journal Copy of...

Documents

Transcript of Journal of Applied Polymer Science Journal...Journal of Applied Polymer Science Journal Copy of...