isocyanate-terminated urethane prepolymer as bioadhesive material: evaluation of bioadhesion and...

15
This article was downloaded by: [Carnegie Mellon University] On: 17 October 2014, At: 04:54 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20 Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays N. Sheikh a , H. Mirzadeh b , A. A. Katbab c , P. Salehian d , M. Daliri e & S. Amanpour f a Gamma Irradiation Center, Atomic Energy Organization of Iran, P.O. Box 11365-8486, Tehran, Iran b Polymer Engineering Department, Amirkabir University, Tehran, Iran c Polymer Engineering Department, Amirkabir University, Tehran, Iran d Iran Medical Sciences University, Rassoud-Akram Hospital, Tehran, Iran e National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran f Razi Vaccine and Serum Research Institute, Karaj, Iran Published online: 02 Apr 2012. To cite this article: N. Sheikh , H. Mirzadeh , A. A. Katbab , P. Salehian , M. Daliri & S. Amanpour (2001) Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays , Journal of Biomaterials Science, Polymer Edition, 12:7, 707-719, DOI: 10.1163/156856201750411611

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Page 1: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

This article was downloaded by [Carnegie Mellon University]On 17 October 2014 At 0454Publisher Taylor amp FrancisInforma Ltd Registered in England and Wales Registered Number 1072954Registered office Mortimer House 37-41 Mortimer Street London W1T3JH UK

Journal of BiomaterialsScience Polymer EditionPublication details including instructions forauthors and subscription informationhttpwwwtandfonlinecomloitbsp20

Isocyanate-terminatedurethane prepolymer asbioadhesive materialEvaluation of bioadhesion andbiocompatibility in vitro andin vivo assaysN Sheikh a H Mirzadeh b A A Katbab c PSalehian d M Daliri e amp S Amanpour fa Gamma Irradiation Center Atomic EnergyOrganization of Iran PO Box 11365-8486 TehranIranb Polymer Engineering Department AmirkabirUniversity Tehran Iranc Polymer Engineering Department AmirkabirUniversity Tehran Irand Iran Medical Sciences University Rassoud-AkramHospital Tehran Irane National Research Center for Genetic Engineeringand Biotechnology Tehran Iranf Razi Vaccine and Serum Research Institute KarajIranPublished online 02 Apr 2012

To cite this article N Sheikh H Mirzadeh A A Katbab P Salehian M Daliriamp S Amanpour (2001) Isocyanate-terminated urethane prepolymer as bioadhesivematerial Evaluation of bioadhesion and biocompatibility in vitro and in vivoassays Journal of Biomaterials Science Polymer Edition 127 707-719 DOI101163156856201750411611

To link to this article httpdxdoiorg101163156856201750411611

PLEASE SCROLL DOWN FOR ARTICLE

Taylor amp Francis makes every effort to ensure the accuracy of allthe information (the ldquoContentrdquo) contained in the publications on ourplatform However Taylor amp Francis our agents and our licensorsmake no representations or warranties whatsoever as to the accuracycompleteness or suitability for any purpose of the Content Any opinionsand views expressed in this publication are the opinions and views ofthe authors and are not the views of or endorsed by Taylor amp FrancisThe accuracy of the Content should not be relied upon and should beindependently verified with primary sources of information Taylor andFrancis shall not be liable for any losses actions claims proceedingsdemands costs expenses damages and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with inrelation to or arising out of the use of the Content

This article may be used for research teaching and private studypurposes Any substantial or systematic reproduction redistributionreselling loan sub-licensing systematic supply or distribution in any formto anyone is expressly forbidden Terms amp Conditions of access and usecan be found at httpwwwtandfonlinecompageterms-and-conditions

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J Biomater Sci Polymer Edn Vol 12 No 7 pp 707ndash719 (2001)Oacute VSP 2001

Isocyanate-terminated urethane prepolymer as bioadhesivematerial Evaluation of bioadhesion and biocompatibilityin vitro and in vivo assays

N SHEIKH 1 H MIRZADEH 2 A A KATBAB 2curren P SALEHIAN3M DALIRI 4 and S AMANPOUR5

1 Gamma Irradiation Center Atomic Energy Organization of Iran PO Box 11365-8486Tehran Iran

2 Polymer Engineering Department Amirkabir University Tehran Iran3 Iran Medical Sciences University Rassoud-Akram Hospital Tehran Iran4 National Research Center for Genetic Engineering and BiotechnologyTehran Iran5 Razi Vaccine and Serum Research Institute Karaj Iran

Received 14 June 2000 revised 20 November 2000 accepted 4 January 2001

AbstractmdashAttempts have been made to evaluate the degree of bioadhesion and biocompatibilityof asynthesized urethane prepolymer with specially tailored microstructure Wetting behaviour and extentof interfacial adhesion of the prepared prepolymer towards biological substrates were examined byin vitro methods The former was carried out by measuring the contact angle between drops of theprepolymer liquid and a biological surface while the latter was determined from the force betweenthe prepolymer and tissue model or mucus The obtained results exhibited good tissue wettabilityand bioadhesion by the prepolymer Preliminary evaluation of biocompatibilityfor the uncatalyticallycured prepolymer lms was performed by cytotoxicity and histotoxicity experiments Results showeda signi cant growth for the adhered L929 broblast cells within a period of 5 days incubation Also nosevere in ammatory tissue response towards the samples implanted in rabbit for 16 weeks was seenThese observations can support the potentiality of the designed urethane prepolymer to be applied ashemostatic agent

Key words Bioadhesion urethane prepolymer sterilization biocompatibility cell culture implanta-tion

INTRODUCTION

A bioadhesive is de ned as a material with a substantial ability to interact withbiological substances and also capable to remain on the surface of biological

currenTo whom correspondence should be addressed E-mail katbabcicakuacir

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708 N Sheikh et al

substrates for a predetermined length of time and then be degraded [1] Asa bioadhesive the material needs to exhibit good bioadhesion through physical orchemical interaction with biological substrates No standard test methods have beenreported in the literature to measure and evaluate the speci c desired propertiesof bioadhesives However there exist a number of different test methods whichpredict the extent of the bioadhesion of a material [2 3] In order to improveour understanding of the bioadhesion phenomena there is no alternative other thanemploying various test methods to accumulate experimental data

The static contact angle between a drop of the liquid adhesive on a biologicalmodel can provide a reasonable estimation for its wetting characteristics in orderto be considered as a candidate for bioadhesion applications Also the adhesionstrength can be qualitatively and quantitatively assessed by detaching a spot or smallmass of the cured adhesive from the substrate This can provide information aboutthe adhesive force and also mode of failure The ability of the mucus to adhere toa bioadhesive can also be assessed by some techniques designed on the basis of theWilhelmy plate method [2]

Any material which is to be used in the human body has to satisfy biocompatibilityevaluation tests These include in vitro and in vivo assays to determine the potentialtoxicity of the biomaterial before any clinical trials [4] The preliminary in vitrostudy is designed to evaluate the cytotoxicity of the material by cell culture test [5]Moreover in vivo implantation is employed as a complementary test to predict thehost response by histologic examination of the tissue at the implant site [6]

The objective of the present study is to investigate the bioadhesion characteristicsand biocompatibility of a synthesized isocyanate terminated urethane prepolymerto be used for hemostasis applications

MATERIALS AND METHODS

Materials

Polyethylene glycol (PEG 1000 Sigma) and castor oil (Aldrich) were used aspolyether polyol and polyester polyol respectively and dried at 80plusmnC undervacuum 24 Toluene diisocyanate (TDI) supplied by Riedel-de Haen co was usedas-received

Synthesis and sterilization of the prepolymer

The urethane prepolymer which was studied as a bioadhesive was obtained bythe reaction between a mixture of PEG and castor oil (5050 ww) with TDIThe amount of isocyanate- and hydroxyl-containing material was adjusted so thata ratio of 2 was obtained for NCO to OH groups To accelerate the curingof the prepolymer Ovalbumin was used with a concentration of 10 (wwcatalyst prepolymer) The synthesized prepolymer was characterized according

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Reactive urethane prepolymer as bioadhesive 709

to the procedures detailed in a previously-published work [7] The stability ofthe puri ed urethane prepolymer towards gamma radiation sterilization was alsoevaluated For this purpose the glass vial containing the prepolymer was sealedunder an atmosphere of dry nitrogen and then irradiated by gamma-ray at asterilizing dose (25 kGy) The infrared spectrum of the irradiated prepolymer wasstudied and compared with that previous to sterilization

Bioadhesion tests

To evaluate the wetting behavior of the synthesized urethane prepolymer on thebiological model a piece of frozen beef tissue was freshly thawed prior to use andkept damp by moistening with phosphate buffered saline (PBS) The thin fatty layerwas then separated from the beef tissue surface and covered on a glass plate andstored in a desiccator to be used in the contact angle experiment The static contactangle formed between a small drop of the urethane prepolymer and the surface ofthis thin fatty layer was then measured using a Kruss G10 goniometer For thispurpose the contact angle measurement was carried out for at least three points onthe surface and the reading of the angle was done 1 min after dropping

In order to assess qualitatively the mode of bonding of the synthesized urethaneprepolymer to the tissue model the standard test method ASTM-D3808 lsquoQualitativedetermination of adhesion of adhesives to substrates by spot adhesionrsquo wasemployed According to this method the degree of bondability of the test adhesiveto the substrate and any visual interaction between the adhesive and substrate arequalitatively evaluated and reported For this assessment the spots of urethaneprepolymer were placed onto the surface of the beef tissue and left to be curedBonding strength was then determined by the mode that the cured prepolymer spotscould be pried from the substrate

Moreover another test was designed for the preliminary evaluation of the adhe-sion strength between the synthesized prepolymer and beef tissue In this experi-ment the prepolymer was applied as a thin layer with a spatula on the surface ofa piece of wood of 23 cm diameter and 2 cm height and then this surface waspressed by a weight of 210 g onto the surface of the beef tissue After curing ofthe prepolymer the adhesion strength was immediately determined by detaching thebonded surfaces using a handy scale which had been vertically connected to thewood

The interfacial bonding of the uncatalyzed and catalyzed urethane prepolymer tomucus was assessed using the method reported by Kellaway and his co-workers [2]which is based on the Wilhelmy plate method for surface tension measurement Inthis technique a microscope glass plate coated with the urethane prepolymer wassuspended from a microbalance and then immersed into a beaker which containednatural mucus (saliva) with a pH of 65 and kept for 10 min After this periodthe beaker is slowly lowered so that the plate can be exposed to the air and themicroforce is displayed by the microbalance The microforce is correlated to theinterfacial adhesion strength between the urethane prepolymer layer and natural

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710 N Sheikh et al

mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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712 N Sheikh et al

(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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718 N Sheikh et al

(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 2: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

To link to this article httpdxdoiorg101163156856201750411611

PLEASE SCROLL DOWN FOR ARTICLE

Taylor amp Francis makes every effort to ensure the accuracy of allthe information (the ldquoContentrdquo) contained in the publications on ourplatform However Taylor amp Francis our agents and our licensorsmake no representations or warranties whatsoever as to the accuracycompleteness or suitability for any purpose of the Content Any opinionsand views expressed in this publication are the opinions and views ofthe authors and are not the views of or endorsed by Taylor amp FrancisThe accuracy of the Content should not be relied upon and should beindependently verified with primary sources of information Taylor andFrancis shall not be liable for any losses actions claims proceedingsdemands costs expenses damages and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with inrelation to or arising out of the use of the Content

This article may be used for research teaching and private studypurposes Any substantial or systematic reproduction redistributionreselling loan sub-licensing systematic supply or distribution in any formto anyone is expressly forbidden Terms amp Conditions of access and usecan be found at httpwwwtandfonlinecompageterms-and-conditions

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J Biomater Sci Polymer Edn Vol 12 No 7 pp 707ndash719 (2001)Oacute VSP 2001

Isocyanate-terminated urethane prepolymer as bioadhesivematerial Evaluation of bioadhesion and biocompatibilityin vitro and in vivo assays

N SHEIKH 1 H MIRZADEH 2 A A KATBAB 2curren P SALEHIAN3M DALIRI 4 and S AMANPOUR5

1 Gamma Irradiation Center Atomic Energy Organization of Iran PO Box 11365-8486Tehran Iran

2 Polymer Engineering Department Amirkabir University Tehran Iran3 Iran Medical Sciences University Rassoud-Akram Hospital Tehran Iran4 National Research Center for Genetic Engineering and BiotechnologyTehran Iran5 Razi Vaccine and Serum Research Institute Karaj Iran

Received 14 June 2000 revised 20 November 2000 accepted 4 January 2001

AbstractmdashAttempts have been made to evaluate the degree of bioadhesion and biocompatibilityof asynthesized urethane prepolymer with specially tailored microstructure Wetting behaviour and extentof interfacial adhesion of the prepared prepolymer towards biological substrates were examined byin vitro methods The former was carried out by measuring the contact angle between drops of theprepolymer liquid and a biological surface while the latter was determined from the force betweenthe prepolymer and tissue model or mucus The obtained results exhibited good tissue wettabilityand bioadhesion by the prepolymer Preliminary evaluation of biocompatibilityfor the uncatalyticallycured prepolymer lms was performed by cytotoxicity and histotoxicity experiments Results showeda signi cant growth for the adhered L929 broblast cells within a period of 5 days incubation Also nosevere in ammatory tissue response towards the samples implanted in rabbit for 16 weeks was seenThese observations can support the potentiality of the designed urethane prepolymer to be applied ashemostatic agent

Key words Bioadhesion urethane prepolymer sterilization biocompatibility cell culture implanta-tion

INTRODUCTION

A bioadhesive is de ned as a material with a substantial ability to interact withbiological substances and also capable to remain on the surface of biological

currenTo whom correspondence should be addressed E-mail katbabcicakuacir

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substrates for a predetermined length of time and then be degraded [1] Asa bioadhesive the material needs to exhibit good bioadhesion through physical orchemical interaction with biological substrates No standard test methods have beenreported in the literature to measure and evaluate the speci c desired propertiesof bioadhesives However there exist a number of different test methods whichpredict the extent of the bioadhesion of a material [2 3] In order to improveour understanding of the bioadhesion phenomena there is no alternative other thanemploying various test methods to accumulate experimental data

The static contact angle between a drop of the liquid adhesive on a biologicalmodel can provide a reasonable estimation for its wetting characteristics in orderto be considered as a candidate for bioadhesion applications Also the adhesionstrength can be qualitatively and quantitatively assessed by detaching a spot or smallmass of the cured adhesive from the substrate This can provide information aboutthe adhesive force and also mode of failure The ability of the mucus to adhere toa bioadhesive can also be assessed by some techniques designed on the basis of theWilhelmy plate method [2]

Any material which is to be used in the human body has to satisfy biocompatibilityevaluation tests These include in vitro and in vivo assays to determine the potentialtoxicity of the biomaterial before any clinical trials [4] The preliminary in vitrostudy is designed to evaluate the cytotoxicity of the material by cell culture test [5]Moreover in vivo implantation is employed as a complementary test to predict thehost response by histologic examination of the tissue at the implant site [6]

The objective of the present study is to investigate the bioadhesion characteristicsand biocompatibility of a synthesized isocyanate terminated urethane prepolymerto be used for hemostasis applications

MATERIALS AND METHODS

Materials

Polyethylene glycol (PEG 1000 Sigma) and castor oil (Aldrich) were used aspolyether polyol and polyester polyol respectively and dried at 80plusmnC undervacuum 24 Toluene diisocyanate (TDI) supplied by Riedel-de Haen co was usedas-received

Synthesis and sterilization of the prepolymer

The urethane prepolymer which was studied as a bioadhesive was obtained bythe reaction between a mixture of PEG and castor oil (5050 ww) with TDIThe amount of isocyanate- and hydroxyl-containing material was adjusted so thata ratio of 2 was obtained for NCO to OH groups To accelerate the curingof the prepolymer Ovalbumin was used with a concentration of 10 (wwcatalyst prepolymer) The synthesized prepolymer was characterized according

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Reactive urethane prepolymer as bioadhesive 709

to the procedures detailed in a previously-published work [7] The stability ofthe puri ed urethane prepolymer towards gamma radiation sterilization was alsoevaluated For this purpose the glass vial containing the prepolymer was sealedunder an atmosphere of dry nitrogen and then irradiated by gamma-ray at asterilizing dose (25 kGy) The infrared spectrum of the irradiated prepolymer wasstudied and compared with that previous to sterilization

Bioadhesion tests

To evaluate the wetting behavior of the synthesized urethane prepolymer on thebiological model a piece of frozen beef tissue was freshly thawed prior to use andkept damp by moistening with phosphate buffered saline (PBS) The thin fatty layerwas then separated from the beef tissue surface and covered on a glass plate andstored in a desiccator to be used in the contact angle experiment The static contactangle formed between a small drop of the urethane prepolymer and the surface ofthis thin fatty layer was then measured using a Kruss G10 goniometer For thispurpose the contact angle measurement was carried out for at least three points onthe surface and the reading of the angle was done 1 min after dropping

In order to assess qualitatively the mode of bonding of the synthesized urethaneprepolymer to the tissue model the standard test method ASTM-D3808 lsquoQualitativedetermination of adhesion of adhesives to substrates by spot adhesionrsquo wasemployed According to this method the degree of bondability of the test adhesiveto the substrate and any visual interaction between the adhesive and substrate arequalitatively evaluated and reported For this assessment the spots of urethaneprepolymer were placed onto the surface of the beef tissue and left to be curedBonding strength was then determined by the mode that the cured prepolymer spotscould be pried from the substrate

Moreover another test was designed for the preliminary evaluation of the adhe-sion strength between the synthesized prepolymer and beef tissue In this experi-ment the prepolymer was applied as a thin layer with a spatula on the surface ofa piece of wood of 23 cm diameter and 2 cm height and then this surface waspressed by a weight of 210 g onto the surface of the beef tissue After curing ofthe prepolymer the adhesion strength was immediately determined by detaching thebonded surfaces using a handy scale which had been vertically connected to thewood

The interfacial bonding of the uncatalyzed and catalyzed urethane prepolymer tomucus was assessed using the method reported by Kellaway and his co-workers [2]which is based on the Wilhelmy plate method for surface tension measurement Inthis technique a microscope glass plate coated with the urethane prepolymer wassuspended from a microbalance and then immersed into a beaker which containednatural mucus (saliva) with a pH of 65 and kept for 10 min After this periodthe beaker is slowly lowered so that the plate can be exposed to the air and themicroforce is displayed by the microbalance The microforce is correlated to theinterfacial adhesion strength between the urethane prepolymer layer and natural

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mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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718 N Sheikh et al

(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 3: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

J Biomater Sci Polymer Edn Vol 12 No 7 pp 707ndash719 (2001)Oacute VSP 2001

Isocyanate-terminated urethane prepolymer as bioadhesivematerial Evaluation of bioadhesion and biocompatibilityin vitro and in vivo assays

N SHEIKH 1 H MIRZADEH 2 A A KATBAB 2curren P SALEHIAN3M DALIRI 4 and S AMANPOUR5

1 Gamma Irradiation Center Atomic Energy Organization of Iran PO Box 11365-8486Tehran Iran

2 Polymer Engineering Department Amirkabir University Tehran Iran3 Iran Medical Sciences University Rassoud-Akram Hospital Tehran Iran4 National Research Center for Genetic Engineering and BiotechnologyTehran Iran5 Razi Vaccine and Serum Research Institute Karaj Iran

Received 14 June 2000 revised 20 November 2000 accepted 4 January 2001

AbstractmdashAttempts have been made to evaluate the degree of bioadhesion and biocompatibilityof asynthesized urethane prepolymer with specially tailored microstructure Wetting behaviour and extentof interfacial adhesion of the prepared prepolymer towards biological substrates were examined byin vitro methods The former was carried out by measuring the contact angle between drops of theprepolymer liquid and a biological surface while the latter was determined from the force betweenthe prepolymer and tissue model or mucus The obtained results exhibited good tissue wettabilityand bioadhesion by the prepolymer Preliminary evaluation of biocompatibilityfor the uncatalyticallycured prepolymer lms was performed by cytotoxicity and histotoxicity experiments Results showeda signi cant growth for the adhered L929 broblast cells within a period of 5 days incubation Also nosevere in ammatory tissue response towards the samples implanted in rabbit for 16 weeks was seenThese observations can support the potentiality of the designed urethane prepolymer to be applied ashemostatic agent

Key words Bioadhesion urethane prepolymer sterilization biocompatibility cell culture implanta-tion

INTRODUCTION

A bioadhesive is de ned as a material with a substantial ability to interact withbiological substances and also capable to remain on the surface of biological

currenTo whom correspondence should be addressed E-mail katbabcicakuacir

Dow

nloa

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by [

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708 N Sheikh et al

substrates for a predetermined length of time and then be degraded [1] Asa bioadhesive the material needs to exhibit good bioadhesion through physical orchemical interaction with biological substrates No standard test methods have beenreported in the literature to measure and evaluate the speci c desired propertiesof bioadhesives However there exist a number of different test methods whichpredict the extent of the bioadhesion of a material [2 3] In order to improveour understanding of the bioadhesion phenomena there is no alternative other thanemploying various test methods to accumulate experimental data

The static contact angle between a drop of the liquid adhesive on a biologicalmodel can provide a reasonable estimation for its wetting characteristics in orderto be considered as a candidate for bioadhesion applications Also the adhesionstrength can be qualitatively and quantitatively assessed by detaching a spot or smallmass of the cured adhesive from the substrate This can provide information aboutthe adhesive force and also mode of failure The ability of the mucus to adhere toa bioadhesive can also be assessed by some techniques designed on the basis of theWilhelmy plate method [2]

Any material which is to be used in the human body has to satisfy biocompatibilityevaluation tests These include in vitro and in vivo assays to determine the potentialtoxicity of the biomaterial before any clinical trials [4] The preliminary in vitrostudy is designed to evaluate the cytotoxicity of the material by cell culture test [5]Moreover in vivo implantation is employed as a complementary test to predict thehost response by histologic examination of the tissue at the implant site [6]

The objective of the present study is to investigate the bioadhesion characteristicsand biocompatibility of a synthesized isocyanate terminated urethane prepolymerto be used for hemostasis applications

MATERIALS AND METHODS

Materials

Polyethylene glycol (PEG 1000 Sigma) and castor oil (Aldrich) were used aspolyether polyol and polyester polyol respectively and dried at 80plusmnC undervacuum 24 Toluene diisocyanate (TDI) supplied by Riedel-de Haen co was usedas-received

Synthesis and sterilization of the prepolymer

The urethane prepolymer which was studied as a bioadhesive was obtained bythe reaction between a mixture of PEG and castor oil (5050 ww) with TDIThe amount of isocyanate- and hydroxyl-containing material was adjusted so thata ratio of 2 was obtained for NCO to OH groups To accelerate the curingof the prepolymer Ovalbumin was used with a concentration of 10 (wwcatalyst prepolymer) The synthesized prepolymer was characterized according

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Reactive urethane prepolymer as bioadhesive 709

to the procedures detailed in a previously-published work [7] The stability ofthe puri ed urethane prepolymer towards gamma radiation sterilization was alsoevaluated For this purpose the glass vial containing the prepolymer was sealedunder an atmosphere of dry nitrogen and then irradiated by gamma-ray at asterilizing dose (25 kGy) The infrared spectrum of the irradiated prepolymer wasstudied and compared with that previous to sterilization

Bioadhesion tests

To evaluate the wetting behavior of the synthesized urethane prepolymer on thebiological model a piece of frozen beef tissue was freshly thawed prior to use andkept damp by moistening with phosphate buffered saline (PBS) The thin fatty layerwas then separated from the beef tissue surface and covered on a glass plate andstored in a desiccator to be used in the contact angle experiment The static contactangle formed between a small drop of the urethane prepolymer and the surface ofthis thin fatty layer was then measured using a Kruss G10 goniometer For thispurpose the contact angle measurement was carried out for at least three points onthe surface and the reading of the angle was done 1 min after dropping

In order to assess qualitatively the mode of bonding of the synthesized urethaneprepolymer to the tissue model the standard test method ASTM-D3808 lsquoQualitativedetermination of adhesion of adhesives to substrates by spot adhesionrsquo wasemployed According to this method the degree of bondability of the test adhesiveto the substrate and any visual interaction between the adhesive and substrate arequalitatively evaluated and reported For this assessment the spots of urethaneprepolymer were placed onto the surface of the beef tissue and left to be curedBonding strength was then determined by the mode that the cured prepolymer spotscould be pried from the substrate

Moreover another test was designed for the preliminary evaluation of the adhe-sion strength between the synthesized prepolymer and beef tissue In this experi-ment the prepolymer was applied as a thin layer with a spatula on the surface ofa piece of wood of 23 cm diameter and 2 cm height and then this surface waspressed by a weight of 210 g onto the surface of the beef tissue After curing ofthe prepolymer the adhesion strength was immediately determined by detaching thebonded surfaces using a handy scale which had been vertically connected to thewood

The interfacial bonding of the uncatalyzed and catalyzed urethane prepolymer tomucus was assessed using the method reported by Kellaway and his co-workers [2]which is based on the Wilhelmy plate method for surface tension measurement Inthis technique a microscope glass plate coated with the urethane prepolymer wassuspended from a microbalance and then immersed into a beaker which containednatural mucus (saliva) with a pH of 65 and kept for 10 min After this periodthe beaker is slowly lowered so that the plate can be exposed to the air and themicroforce is displayed by the microbalance The microforce is correlated to theinterfacial adhesion strength between the urethane prepolymer layer and natural

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mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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716 N Sheikh et al

(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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718 N Sheikh et al

(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 4: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

708 N Sheikh et al

substrates for a predetermined length of time and then be degraded [1] Asa bioadhesive the material needs to exhibit good bioadhesion through physical orchemical interaction with biological substrates No standard test methods have beenreported in the literature to measure and evaluate the speci c desired propertiesof bioadhesives However there exist a number of different test methods whichpredict the extent of the bioadhesion of a material [2 3] In order to improveour understanding of the bioadhesion phenomena there is no alternative other thanemploying various test methods to accumulate experimental data

The static contact angle between a drop of the liquid adhesive on a biologicalmodel can provide a reasonable estimation for its wetting characteristics in orderto be considered as a candidate for bioadhesion applications Also the adhesionstrength can be qualitatively and quantitatively assessed by detaching a spot or smallmass of the cured adhesive from the substrate This can provide information aboutthe adhesive force and also mode of failure The ability of the mucus to adhere toa bioadhesive can also be assessed by some techniques designed on the basis of theWilhelmy plate method [2]

Any material which is to be used in the human body has to satisfy biocompatibilityevaluation tests These include in vitro and in vivo assays to determine the potentialtoxicity of the biomaterial before any clinical trials [4] The preliminary in vitrostudy is designed to evaluate the cytotoxicity of the material by cell culture test [5]Moreover in vivo implantation is employed as a complementary test to predict thehost response by histologic examination of the tissue at the implant site [6]

The objective of the present study is to investigate the bioadhesion characteristicsand biocompatibility of a synthesized isocyanate terminated urethane prepolymerto be used for hemostasis applications

MATERIALS AND METHODS

Materials

Polyethylene glycol (PEG 1000 Sigma) and castor oil (Aldrich) were used aspolyether polyol and polyester polyol respectively and dried at 80plusmnC undervacuum 24 Toluene diisocyanate (TDI) supplied by Riedel-de Haen co was usedas-received

Synthesis and sterilization of the prepolymer

The urethane prepolymer which was studied as a bioadhesive was obtained bythe reaction between a mixture of PEG and castor oil (5050 ww) with TDIThe amount of isocyanate- and hydroxyl-containing material was adjusted so thata ratio of 2 was obtained for NCO to OH groups To accelerate the curingof the prepolymer Ovalbumin was used with a concentration of 10 (wwcatalyst prepolymer) The synthesized prepolymer was characterized according

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Reactive urethane prepolymer as bioadhesive 709

to the procedures detailed in a previously-published work [7] The stability ofthe puri ed urethane prepolymer towards gamma radiation sterilization was alsoevaluated For this purpose the glass vial containing the prepolymer was sealedunder an atmosphere of dry nitrogen and then irradiated by gamma-ray at asterilizing dose (25 kGy) The infrared spectrum of the irradiated prepolymer wasstudied and compared with that previous to sterilization

Bioadhesion tests

To evaluate the wetting behavior of the synthesized urethane prepolymer on thebiological model a piece of frozen beef tissue was freshly thawed prior to use andkept damp by moistening with phosphate buffered saline (PBS) The thin fatty layerwas then separated from the beef tissue surface and covered on a glass plate andstored in a desiccator to be used in the contact angle experiment The static contactangle formed between a small drop of the urethane prepolymer and the surface ofthis thin fatty layer was then measured using a Kruss G10 goniometer For thispurpose the contact angle measurement was carried out for at least three points onthe surface and the reading of the angle was done 1 min after dropping

In order to assess qualitatively the mode of bonding of the synthesized urethaneprepolymer to the tissue model the standard test method ASTM-D3808 lsquoQualitativedetermination of adhesion of adhesives to substrates by spot adhesionrsquo wasemployed According to this method the degree of bondability of the test adhesiveto the substrate and any visual interaction between the adhesive and substrate arequalitatively evaluated and reported For this assessment the spots of urethaneprepolymer were placed onto the surface of the beef tissue and left to be curedBonding strength was then determined by the mode that the cured prepolymer spotscould be pried from the substrate

Moreover another test was designed for the preliminary evaluation of the adhe-sion strength between the synthesized prepolymer and beef tissue In this experi-ment the prepolymer was applied as a thin layer with a spatula on the surface ofa piece of wood of 23 cm diameter and 2 cm height and then this surface waspressed by a weight of 210 g onto the surface of the beef tissue After curing ofthe prepolymer the adhesion strength was immediately determined by detaching thebonded surfaces using a handy scale which had been vertically connected to thewood

The interfacial bonding of the uncatalyzed and catalyzed urethane prepolymer tomucus was assessed using the method reported by Kellaway and his co-workers [2]which is based on the Wilhelmy plate method for surface tension measurement Inthis technique a microscope glass plate coated with the urethane prepolymer wassuspended from a microbalance and then immersed into a beaker which containednatural mucus (saliva) with a pH of 65 and kept for 10 min After this periodthe beaker is slowly lowered so that the plate can be exposed to the air and themicroforce is displayed by the microbalance The microforce is correlated to theinterfacial adhesion strength between the urethane prepolymer layer and natural

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710 N Sheikh et al

mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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712 N Sheikh et al

(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 5: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

Reactive urethane prepolymer as bioadhesive 709

to the procedures detailed in a previously-published work [7] The stability ofthe puri ed urethane prepolymer towards gamma radiation sterilization was alsoevaluated For this purpose the glass vial containing the prepolymer was sealedunder an atmosphere of dry nitrogen and then irradiated by gamma-ray at asterilizing dose (25 kGy) The infrared spectrum of the irradiated prepolymer wasstudied and compared with that previous to sterilization

Bioadhesion tests

To evaluate the wetting behavior of the synthesized urethane prepolymer on thebiological model a piece of frozen beef tissue was freshly thawed prior to use andkept damp by moistening with phosphate buffered saline (PBS) The thin fatty layerwas then separated from the beef tissue surface and covered on a glass plate andstored in a desiccator to be used in the contact angle experiment The static contactangle formed between a small drop of the urethane prepolymer and the surface ofthis thin fatty layer was then measured using a Kruss G10 goniometer For thispurpose the contact angle measurement was carried out for at least three points onthe surface and the reading of the angle was done 1 min after dropping

In order to assess qualitatively the mode of bonding of the synthesized urethaneprepolymer to the tissue model the standard test method ASTM-D3808 lsquoQualitativedetermination of adhesion of adhesives to substrates by spot adhesionrsquo wasemployed According to this method the degree of bondability of the test adhesiveto the substrate and any visual interaction between the adhesive and substrate arequalitatively evaluated and reported For this assessment the spots of urethaneprepolymer were placed onto the surface of the beef tissue and left to be curedBonding strength was then determined by the mode that the cured prepolymer spotscould be pried from the substrate

Moreover another test was designed for the preliminary evaluation of the adhe-sion strength between the synthesized prepolymer and beef tissue In this experi-ment the prepolymer was applied as a thin layer with a spatula on the surface ofa piece of wood of 23 cm diameter and 2 cm height and then this surface waspressed by a weight of 210 g onto the surface of the beef tissue After curing ofthe prepolymer the adhesion strength was immediately determined by detaching thebonded surfaces using a handy scale which had been vertically connected to thewood

The interfacial bonding of the uncatalyzed and catalyzed urethane prepolymer tomucus was assessed using the method reported by Kellaway and his co-workers [2]which is based on the Wilhelmy plate method for surface tension measurement Inthis technique a microscope glass plate coated with the urethane prepolymer wassuspended from a microbalance and then immersed into a beaker which containednatural mucus (saliva) with a pH of 65 and kept for 10 min After this periodthe beaker is slowly lowered so that the plate can be exposed to the air and themicroforce is displayed by the microbalance The microforce is correlated to theinterfacial adhesion strength between the urethane prepolymer layer and natural

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mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 6: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

710 N Sheikh et al

mucus A similar test was performed on each clean microscope glass plate beforebeing coated with the test material The coated plate force was then expressed as apercentage of the clean plate force

Film preparation and surface characterization

For the preparation of the polyurethane lms the liquid prepolymer was cast ontothe surface of a glass plate and then left to be cured under ambient conditions Thecured prepolymer lms were then ultrasonically rinsed in distilled water acetonand stored in a desiccator prior to use The prepared lms were handled onlywith forceps to eliminate contamination with oily materials of the skin Thesurface tension of the cured prepolymer lm was measured by a Kruss K12tesiometer on the basis of the OwensWendt method using water formamideethylene glycol benzyl alcohol and 15-pentandiol as the test liquids Also theWilhelmy plate technique was employed for the determination of the dynamiccontact angle between water and the surface of the cured prepolymer lm usingthe same instrument

In vitro cell system

Mouse C34 connective tissue (L929) was obtained from the National Cell BankPasteur Institute of Iran A modi ed technique of Wan et al (1997) was usedas the test cell model to follow the substrate effects on cell attachment andmorphological cell change The cells were maintained in growth media RPMI-1640 and supplemented with 10 fetal calf serum (PCS) 100 U mliexcl1 penicillinand 100 sup1g mliexcl1 streptomycin (Gibco BRL Laboratories) A routine subculturemethod was used to maintain the cell line The cells were incubated in a humidi edatmosphere with 5 CO2 at a temperature of 37plusmnC After incubation of 1 weekthe monolayer was then harvested by trypsinization A cell suspension of 4 pound105 cells mliexcl1 was prepared before seeding

Cell attachment and growth assay

This experiment was carried out using a surgical glove grade of natural rubber latexand tissue culture polystyrene (TCPS) as positive and negative control respectivelyThe cured urethane prepolymer (PU) and control lms were punched into discsof 15 mm diameter and then sterilized by autoclaving (121plusmnC 20 min) or gammaradiation (25 kGy) The polymeric discs were individually placed into a multiwellplate (Nunc Denmark) Five ml cell suspension was seeded into each well Thesamples were maintained separately in the incubator for 1 and 5 days The test wascarried out in duplicate for both the prepared prepolymer lms and control samplesThe samples were removed from the wells and washed with PBS twice Theywere then placed on glass slides and their attached cells were xed in a series ofaqueous ethanol solutions of decreasing concentration a total of 20 min and nally

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Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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712 N Sheikh et al

(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 7: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

Reactive urethane prepolymer as bioadhesive 711

stained with crystal violet or giemsa solution for 10 min All air-dried sampleswere covered with coverslips and then examined by a Hund light microscope modelH500 Adherent cells were counted and their areas were determined after anincubation period of 5 days using Image Pro Plus software in seven areas randomlychosen in the central and peripheral regions of the polymer substrates

Statistical analysis

The data were analyzed using Studentrsquos t-test for unpaired samples

In vivo biocompatibility assay

The biocompatibility of the prepolymer lms was also evaluated in vivo using rabbit(Duch-2 kg) as the test animal The back areas of the rabbits were shaven anddisinfected by betadine General anaesthesia was given to the test rabbits usingIM injection of a mixture of ketamine hydrochloride (50 mg kgiexcl1 body weight)and xylazine hydrochloride (5 mg kgiexcl1 body weight) Six sterilized urethaneprepolymer lms (2 cm pound 1 cm pound 003 cm) were implanted subcutaneously in theparavertebral areas of three rabbits In each case the implanted and control (sham)operation sites were arranged opposite to each other After a period of 16 weeks theimplanted specimens together with their connected tissues were removed from theimplant sites and xed in a 10 solution of formalin Paraf n sections were thenprepared These sections were stained routinely by hematoxylin and eosin (HampE)and in ammatory reaction of the implants were assessed

RESULTS AND DISCUSSION

Structural properties of the prepared prepolymer

The reaction between the PEGcastor oil and TDI with an NCOOH ratio of 2 ledto the formation of a clear yellowish viscous liquid which could change intoa white solid with contact with water and other materials with active hydrogenatoms It was found that the use of a mixture of polyether polyol and polyesterpolyol gives the possibility of tailoring and optimizing the required properties ofthe urethane prepolymer such as uidability and adhesiveness in the liquid stateand also exibility and mechanical strength in a solid lm The addition of 10(ww) Ovalbumin into the prepolymer was found to have a signi cant effect uponits curing time [7] The infrared spectra of the unsterilized and gamma-sterilizedurethane prepolymer are given in Fig 1a and b respectively It is obvious that thechemical structure of the prepolymer has remained intact after irradiation Also theviscosity of the irradiated prepolymer was found to be the same as the unsterilizedprepolymer This indicates the capability of the synthesized prepolymer to beradiation-sterilized

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(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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712 N Sheikh et al

(a)

(b)

Figure 1 (a) Infrared spectrum of the unsterilized urethane prepolymer and (b) infrared spectrum ofthe sterilized urethane prepolymer

Bioadhesion evaluation

The results obtained from the evaluation of wetting behavior of the uncatalyzedurethane prepolymer onto the surface of the the thin fatty beef tissue showed acontact angle of 347 sect 2 deg The low value of the contact angle indicates goodwetting and low interfacial tension between the synthesized prepolymer and thebeef tissue substrate This can be attributed to the high polarity of the prepolymerdue to isocyanate groups which leads to a higher spreadability of the prepolymerdrop The contact angle between the catalyzed liquid prepolymer and biologicalmodel could not be easily determined because of its quick solidi cation and hightime-dependency

From the standard spot adhesion test it was observed that the spot of urethaneprepolymer placed on the tissue model changed into a white elastic mass after beingcured The elastic mass could not be separated easily from the surface of the beeftissue but was nally detached from the substrate adhesively The high elasticity ofthe cured urethane prepolymers have been attributed to the presence of hard and softsegments in their microstructure [8] This can considered a superior characteristicof urethane-based bioadhesives for soft tissue applications

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Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Reactive urethane prepolymer as bioadhesive 713

Table 1Degree of adhesion between the urethane prepolymer layer and natural mucus (saliva)

Max clean plate force (mN) Max coated plate force (mN) relative force

uncatalyzed layer catalyzed layer uncatalyzed layer catalyzed layer

172 sect 001 29 sect 009 255 sect 004 169 sect 65 148 sect 22

The results are presented as mean sect SD n D 3

The adhesion strength test of the prepolymer on the tissue model showed an initialadhering strength of 200 sect 22 gf cmiexcl2 The extend of interaction and adhesionbetween the uncatalyzed and catalyzed prepolymer layers with saliva have beendemonstrated and compared in Table 1 It is obvious that the surface coated withuncatalyzed prepolymer has resulted in an increase of 169 adhesion towards salivacompared with the uncoated clean plate surface This may be explained due tothe greater interaction between the chemical groups of the prepolymer and salivacomponents The surface coated with the catalyzed prepolymer has presentedless adhesion towards saliva which could be due to a faster rate of curing andtherefore less surface concentration of the isocyanate groups for interacting withsaliva

Relationship between surface properties and cellular responses

The extent of broblast cell adhesion and its relationship with surface properties ofthe prepared polyurethane and also tissue culture polystyrene and natural rubberlatex lms are presented in Table 2 It can be observed that the adhesion of broblast cells to the surface of the latex lm is inhibited compared with theprepared polyurethane and also tissue culture polystyrene which have been able tosupport cell adhesion The af nity of the broblast cells towards the polyurethanesurface is related to its high surface energy resulting from the presence of thepolar chemical groups on the surface of the polyurethane lm Also the existenceand ratio of the hard and soft segments in the structure of the polyurethanesubstrates have been reported to be important in controlling the amount of surfaceenergy and therefore the degree of wettability [9] Polystyrene in the pure andnonmodi ed state shows high surface hydrophobicity [10] while the tissue culturepolystyrene posses some surface polarity which causes cell attachment and morewater compatibility These results are in agreement with the high cell adhesionreported in the literature for a range of biomaterials possessing low to moderatehydrophilicity [11] Moreover from the results presented in this table a signi cantdifference in the number of attached cells can be observed between the TCPS andPU surfaces p lt 001 Also the PU surface supports a signi cantly higherdegree of cell spreading than that of TCPS p lt 001 This can be attributedto the rubbery elastic behavior of the polyurethane substrate which can toleratethe forces imposed by the cells for being stretched However the natural rubber

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Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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714 N Sheikh et al

Table 2Relation between broblast cell adhesion and surface properties of the polyurethane and control lms

Polymer Dynamic contact angle Surface tension degs No of attached cells Area of cellssurface (deg) (mN miexcl1) (5 day) (sup1m)2

microa micror

PU 81 321 50 124 sect 22a 447 sect 117b

TCPS 1017 58 466 225 sect 67 244 sect 85

Latexc mdash mdash mdash 0 0

a p lt 001 when compared with the corresponding TCPS valueb p lt 001 when compared with the corresponding TCPS valuec Surface properties of latex could not be measured due to the quick folding of the latex lm in

contact with test liquidThe results are expressed as the average of three measurements or mean sect SD n D 7Mean standard deviation advancing contact angle D microa sect 3 deg receding contact angle D

micror sect 2 deg surface tension D degs sect 5 mN miexcl1

(a)

Figure 2 Light photomicrographswith the magni cation of pound200 illustrating the attached broblastcells on the surface of the lms (a) latex 5 day incubation (b) TCPS 5 day incubation(c) PU 1 day incubation and (d) PU 5 day incubation (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

latex compounded with vulcanizing ingredients has been reported to have cytotoxicbehavior [12] which leads to the inhibition of cell adhesion and cell proliferation

Figure 2andashd exhibits the number and morphological changes of the broblastcells attached to the surface of the polyurethane and control lms It is obvious

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(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Reactive urethane prepolymer as bioadhesive 715

(b)

(c)

Figure 2 (Continued)

from these photomicrographs that only a few detached cells are on the latex surfacewithout any growth after an incubation period of 5 days (Fig 2a) However asigni cant number of cells attached to the surface of tissue culture polystyrenewith considerable growth can be observed under the same incubation conditions

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Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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716 N Sheikh et al

(d)

Figure 2 (Continued)

(Fig 2b) In the case of the prepared polyurethane lms (Fig 2d) similar behaviorhas been shown to the tissue culture polystyrene substrate Comparison betweenthe photomicrographs demonstrated in Fig 2c and d shows that the number ofthe attached broblast cells increases with the incubation time with more cells attening

Two mechanisms have been proposed to explain the cell attachment and growthIn one mechanism the deposition of the proteins from the serum component of theculture media onto the substrate surface has been thought to be the main drivingforce for further cell attachment [13] Another mechanism suggests that the proteinmolecules synthesized by the rst attached cell layer are adsorbed on the substratesurface which leads to the enhancement of cell attachment [14] However itseems that the extent of protein deposition depends on the surface properties ofthe substrate and is more probable for the moderately wettable surfaces such aspolyurethane substrate

In vivo implantation and histological observations

The operation sites and implanted areas for the rabbit model are shown in Fig 3During post-operative care no abnormal changes were found on the implantationsites by direct visual observation The tissue reaction for the test sample (PU lm)at 16 weeks of implantation revealed the following changes in the implanted zoneencapsulation of the prosthesis by the collagen ber with a thickness of 2ndash3 sup1m

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Reactive urethane prepolymer as bioadhesive 717

Figure 3 Schematic of the operation sites in the rabbit model

(a)

Figure 4 Optical micrographs of implanted PU lms cross-sectioned after 16 weeks implantationin rabbit (HampE stain magni cation pound200) (a) the implant is surrounded by a thin brouscapsule no acute or chronic in ammatory reaction is observed and (b) encapsulation with atsquamous cells with stromal edema and mild vascularization no in ammation is found (PU Dprosthesis T D tissue (iexcl) indicates the brous capsule) (This gure is published in colour onhttpwwwvsppubcomjcontsJBS)

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718 N Sheikh et al

(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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718 N Sheikh et al

(b)

Figure 4 (Continued)

could be observed Neither acute or chronic in ammatory reaction nor foreign bodytype granulomatous in ammation were evidenced (Fig 4a and b) The formationof thin brous capsules around the implanted specimens is reported to be a sign ofimproved tissue compatibility [15] However to judge the suitability of the preparedurethane prepolymer adhesive for clinical applications needs more complementarystudies

CONCLUSIONS

The synthesized prepolymer indicated good wettability and spreadability to thethin fatty beef tissue layer Curing of the prepolymer on the tissue model surfacepresented a exible interface with moderate adhesion strength From the in vitro cellculture test it was found that the cured urethane prepolymer lms with moderatehydrophilicity can present good cytocompatibility responses Implantation of thecured prepolymer lms in the rabbit model showed no evidence of acute or chronicin ammatory reaction which indicates the suitability of the prepared urethaneprepolymer as a biomaterial Also the obtained results showed that gammairradiation can be considered as a desirable method for the sterilization of thesynthesized urethane prepolymer

Dow

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by [

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rsity

] at

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Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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Page 15: Isocyanate-terminated urethane prepolymer as bioadhesive material: Evaluation of bioadhesion and biocompatibility, in vitro and in vivo assays

Reactive urethane prepolymer as bioadhesive 719

Acknowledgements

The authors would like to acknowledge the nancial support of the GammaIrradiation Centre AEOI given to this work We also appreciate the Laser Polymergroup of AEOI for providing us the cell culture facilities

REFERENCES

1 L Gross and R Hoffman in Handbook of Adhesives I Skeisk (Ed) p 818 Van NostrandReinhold New York (1977)

2 N A Peppas and P A Buri J Control Rel 2 257 (1985)3 K Park and H Park in Bioadhesive Drug Delivery Systems V Lenaerts and R Gurny (Eds)

p 45 CRC Press Boca Raton FL (1990)4 A Pizzoferrato C R Arciola E Cenni G Ciapetti D Granchi L Savarino and S Stea

in EncyclopediaHandbook of Biomaterialsand Bioengineering D L Wise (Ed) Part A Vol 1p 329 Marcel Dekker New York (1995)

5 M Cannas A Pigatto and A Masse in Encyclopedia Handbook of Biomaterials and Bioengi-neering D L Wise (Ed) Part A Vol 1 p 433 Marcel Dekker New York (1995)

6 R Marchant A Hiltner C Hamlin A Rabinovitch R Slobodkin and J M AndersonJ Biomed Mater Res 17 301 (1983)

7 N Sheikh A A Katbab and H Mirzadeh Int J Adhesion Adhesives 20 299 (2000)8 M D Lelah and S L Cooper in Polyurethanes in Medicine p 49 CRC Press Boca Raton FL

(1986)9 F Garbassi M Morra and E Occhiello in Polymer Surfaces from Physics to Technology

p 177 John Wiley New York (1995)10 P B Van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G Van Aken

Biomaterials 6 403 (1985)11 M J Lydon T W Minett and B J Tighe Biomaterials 6 396 (1985)12 Y Ikarashi K Toyoda N Ohsava T Uchima T Tsuchiya M Kaniwa M Sato M Takahashi

and A Nakamura J Biomed Mater Res 26 339 (1992)13 H Lin C Echeverria S Asakura W Sun D F Mosher and S L Cooper Biomaterials 13 905

(1992)14 P B Van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G Van

Aken Biomaterials 8 323 (1987)15 J B Park and R S Lakes in Biomaterials An Introduction p 223 Plenum Press New York

(1992)

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by [

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ello

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] at

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