Lin-Shu Liu, Richard A. Berg

FzioMed, Inc., 170-A Granada Drive, San Luis Obispo, California 93401

Received 28 September 2001; revised 11 December 2001; accepted 17 December 2001Published online 3 April 2002 in Wiley 1nterScience (www.interscience.wiley.com). DOl: 10.1002/jbm.10211

Abstract: Composite gels and films of CMC and PEO have been used to separate healingtissues and have been demonstrated to reduce postsurgical adhesions in animal models ofadhesion formation. Gels of CMC/PEO were studied here to elucidate the mechanism bywhich the combination of PEO with CMC is effective in reducing adhesions between tissues.CMC and PEO were demonstrated to undergo micro phase separation to form a two-phasesystem. Protein partitioning was measured in this system for albumin, fibrinogen, and gammaglobulin. All of these proteins were found to partition preferentially into the CMC phase.When gels of CMC and PEO were examined for tissue adherence, the addition of PEOreduced the adherence of CMC to tissues. To further investigate the effects of PEO on tissueadherence of the gel, the extent of thrombus formation of citrated blood initiated by calciumchloride in CMC/PEO gels was measured in vitro. The extent of thrombus formation by CMCwas reduced proportionally to the content of PEO in gels of CMC/PEO. A model wasdeveloped to explain how CMC and PEO contribute to the effectiveness of CMC/PEO gels thatform a barrier between healing tissues to reduce postsurgical adhesions. In an open systemPEO is released from the gel faster than CMC is dissolved, resulting in a shell structure withCMC coated by PEO. The PEO-rich outer layer functions to inhibit protein deposition andthrombus formation. The CMC-rich layer functions to anchor the gel to the tissue surface.@ 2002 Wiley Periodicals, Inc, J Biomed Mater Res (Appl Biomater) 63: 326-332, 2002

Keywords: carboxymethylcellulose; polyethylene oxide; protein partitioning; adhesions

INTRODUCTION polyethylene oxide that has a large unfavorable free energy ofinteraction with proteins. 13 It is a biocompatible polymer thathas been used as a component of a tissue sealantl4, 15 and as

a coating for,medical devicesl6 and it has recently been usedfor conjugation to therapeutic proteins to extend their circu-lation time for drug delivery.17

Solutions of CMC have been tested to inhibit adhesions inseveral animal models. I 1 To achieve sufficient coverage, the

solutions were applied both intraoperatively and postopera-tively.11 PEa has also been tested in animals as an antiadhe-sive solution. 18,19 However, because of the difficulty of keep-

ing PEa in contact with tissues, large volumes must beused. 19

More recently, a composite material composed of bothCMC and PEa in the form of a film2o or a gefl has beendemonstrated to reduce adhesion formation in several animalstudies of peritoneal or peridural adhesions. Composites ofCMC and PEa in the form of both films and gels are tissueadherent and are effective in reducing adhesions!O, 21 Be-cause neither polymer was as effective when used alone7, 18as when used in the composire,2O, 21 the contribution of each

component was evaluated here. It was shown that when CMCand PEa are formed into a composite gel, the unique prop-

Postsurgical adhesions are a serious complication in manysurgeries, as they result from a normal wound-healing pro-cess that is triggered by inflammation or tissue trauma. 1

Peritoneal adhesions and adnexal adhesions develop in ,pa-tients undergoing abdominal or gynecological surgeries.2Peridural adhesions develop in patients undergoing spinesurgery3 and perineural adhesions develop in patients under-going peripheral nerve surgery.4 An important consequenceof adhesion formation is the development of fibrosis that cancause a serious compromise of organ function in terms ofbowel obstruction,5 infertility,6 or peridural fibrosis in thespine7, 8 resulting in pain or neurological dysfunction.

CMC is a water-soluble anionic linear polymer that hasbeen used in several medical applications9 and more recentlyas a component of an antiadhesion gelID. 11 or membrane.I2

PEa is a nonpolar, high-molecular-weight linear polymer of

Correspondence to: Richard Berg, FzioMed, Inc., 170-A Granada Dr., San LuisObispo, CA 93401 (E-mail: raberg@fziomed.com)

@ 2002 Wiley Periodicals, Inc.

326

TABLE I. Characteristics of PEO/CMC Gelsa solved in PBS separately. The polymers were mixed togetheras described in detail below. To study the kinetics of PEDreleased from the gel, gels were prepared with the use offluorescein-PEG (mol.wt. 5000). For evaluation of the gelstructure, gels were also formulated at a concentration of6.7% w/v CMC in PBS containing methylene blue (50 ppm)and 0.764% w/v PED in PBS and mixed as described above.The samples were centrifuged at 3500 rpm for 20 min.Photographs were taken after mixing and centrifugation.

Gel Viscosity

The viscosity of synthesized gels was measured at roomtemperature with the use of a digital viscometer (Brookfieldmodel DV-ll, Brookfield Engineering Laboratory, Inc.,Stoughton, MA) at shear rate 0.5 rpm and spindle No. 29.

erties of each provide the basis for a mechanism to explainthe composite's antiadhesive function.

Gel TurbidityMATERIALS AND METHODS

The turbidity of gels was measured at 580 nm with the use ofa UV spectrophotometer (Model 160U, Shimadzu, Japan)after any air bubbles entrapped in the gels were carefullyremoved by brief centrifugation.

Materials

Sodium carboxymethylcellulose (CMC, mol.wt. 7 X 105Daltons) was purchased from Hercules, Inc. (Wilmington,DE). Polyethylene oxide (PEO; mol.wt. 4.4 X 106 Daltons)was obtained from Rita, Inc. (Woodstock, IL). Fluorescein-PEG (mol.wt. 5 X 103 Daltons) was obtained from Shearwa-ter (Huntsville, AL). PEG (mol.wt. 5000 Daltons.), sodiumcitrate solution (4%, aseptically filled), as well as bovinefibrinogen and 'Y globulin were from Sigma Chemical Co. (St.Louis, MO). Bovine plasma albumin was from CalbiochemInc. (La Jolla, CA). Methylene blue (0.05%, w/v, ACSgrade), sulfuric acid (98%), phenol (double distilled) andother chemicals were from VWR (San Francisco, CA). Por-cine intestine was from a local market. Citrated bovine bloodwas prepared by mixing one part of sodium citrate solutionwith nine parts of whole blood from a healthy adult bull(courtesy of Dr. William Plummer of the Animal ScienceDepartment, California Polytechnic State University, SanLuis Obispo, CA).

Release of CMC and PEa from Gels

Studies on the release of CMC and PEa from gels wereperformed with PBS used as the release medium. Membranesof porcine intestine were mounted on the bottoms of petridishes (diam= 50 mm) with double-sided adhesive tape. Gelswere prepared with fluore~cein-labeled PEa. An aliquot of5.0 rnl of each gel was evenly spread over the surface of themembrane. PBS (10 rnl) was carefully loaded on the top ofthe gel layer, followed by incubation at room temperatureunder gentle shaking. At the time periods of 3 min, 10 min,20 min, 1 h, and 2 h the dish was tipped to one side and 1.0rnl of PBS was pipetted from the solution above the gel andanalyzed for the amount of PEa and CMC released.

The released PEa was quantified by measuring the absor-bance of the fluorescein moiety attached at the PEG chain at500 nm. The CMC released into the incubation medium wasdetermined by measuring hexose content, by the method ofphenol-sulfuric acid reaction, where the absorbance at 480nm is measured after the CMC is incubated with phenol andsulfuric acid at 30 °C for 20 min.22

Protein Partitioning

Protein partitioning in an aqueous CMC/PEO system wasperformed as described by Johansson.23 Proteins tested in thecurrent study were bovine plasma albumin, bovine fibrino-gen, and bovine 'Y globulin. Solutions of 6.7 % (w/v) CMCand 0.74 % (w/v) PEO (mol.wt. 8000) were prepared sepa-rately as described in the paragraph on gel preparation, exceptthat PBS was used as solvent instead of deionized H2O.Proteins were added into the polymer solutions at the end ofthe blending step at a concentration of 0.01 % (w/v). A vol-ume of 45 rnl of CMC/protein in PBS was placed in a 100-ml

Gel Preparation

Gels of various compositions (Table I) were prepared byblending CMC and PEG powders in deionized water contain-ing 0.43% (w/v) NaCl and 0.47% (w/v) CaCl2 . 2H2G atroom temperature with the use of a heavy-duty blender (Ar-row 2000, Arrow Engineering Co., Inc., Hillside, NJ). Thepolymers are blended to a smooth gel.

Gels thus formed were filtered through a stainless-steelmembrane with the pore size of 53 JLm (Millipore Corp.,Bedford, MA), and loaded into 5-rnl polypropylene syringes(Becton Dickinson & Co., Franklin Lakes, NJ). All gels werestored at 4 DC.

For a blood coagulation test, gels were cast on polystyrenepetri dishes (100 X 15 mm, Fisher Scientific, Santa Clara,CA). Each dish contained 30 ml of the gel. For the protein-partitioning study, CMC and PEG (mol.wt. 8000) were dis-

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was then placed on the gel surface, followed by the additionof 0.4 m1 of 0.1 M CaCl2 solution to initiate clotting. A largevolume of deionized H2O was added to the gel to stop the clotformation in 20 min. The thrombus was carefully removedfrom the gel with a clean, flat-end spatula, and fixed in PBScontaining 2% glutaraldehyde for at least 4 h. The weights ofclots were recorded after removing them from the fixingsolution and removing the surface water by tapping withKimwipes.

Empty polystyrene petri dishes were used as controls.Each sample was tested four times and the mean weights andstandard deviations were calculated.

Figure 1. Transparency of CMC/PEO gels with various ratios be.tween the two components. Statistical Analysis

All data are expressed as the mean ::t standard deviation.Significance was determined with the use of a student's t test.volumetric column and 45 ml of PED in PBS containing

related protein was added on the top of CMC solution. Thecolumn was capped, and the contents were mixed by gentlyturning the column up and down 15 times to complete sur-face-to-surface contact. The column was allowed to stand atroom temperature for 48 h. Fractions of the top layer and thebottom layer were then pipetted and the protein concentrationwas measured by measuring the absorbance at 280 nm againsta blank.

RESULTS

Gels were prepared by blending CMC with PEa at variousratios in the presence of CaClz in an aqueous solution to forma composite gel. Gels were first characterized for viscosity bydetermining the resistance to shear force at 0.5 rpm and forturbidity by measuring the optical density at 580 nm. Asshown in Table I, gel composed of CMC alone has thehighest viscosity; gel prepared from PEa alone has a lowerviscosity than that of CMC at the same concentration (%solids, w/v). After mixing CMC with PEa, the viscosities ofcomposite gels varied as the function of PEa content. At theweight ratio of 50/50 of CMC and PEa, the composite gelshowed the lowest viscosity.

The turbidity results of CMC/PEO gels as demonstratedby transparency are shown in Figure 1. At a concentration of3.7% (w/v) , gels prepared either from CMC alone or fromPEa alone were transparent. However, for CMC/PEO com-

Tissue Adhesion

The tissue-adhesive property of the gels was determined bymeasuring the force needed to detach the gels from themembrane using a modified Tape Loop Tack Tester (ModelLT-IOO; Chern. Instruments, Fairfield, OR) equipped with adigital force meter (Chatillon Model DFM; Greensboro, NJ).Membranes of porcine intestine were used as the receiver.The membranes were mounted onto each surface of both thetest panel and specimen jaw, which was attached to thetension head by means of a yoke and release pin.

The gap between the two membranes was adjusted to 2 :t1 mm by releasing and tightening the release pin. An aliquotof 5.0 :t 0.1 ml of the gel was applied on the membranebound to the test panel. All measurements were performedwith settings as follows: specimen jaw lowering speed: 9rnrn/s, contact time: 3 min, contact area: 5.31 cm2, specimenjaw withdrawal rate: 9 mm/s, withdrawal height: 4.5 cm.

Each experiment was carried out five times. The force (N)needed to detach the membranes was recorded and repre-sented as the mean value with standard deviation.

1 ~2 4

Thrombogenic Property

The thrombogenic property of gels was determined by mea-suring the extent of clot formation after contact with citratedwhole bovine blood at 37 DC for 20 min.24 Bovine blood waschosen because the protein-partitioning studies describedabove were performed with bovine proteins. Polystyrene petridishes with precast gels were incubated at 37 DC for 30 minprior to each experiment: 4.0 ml citrated whole bovine blood

Figure 2. Phase separation of CMC/PEO. Gels were prepared from6.7% (w/v) CMC, mol.wt. 7 x 105 in PBS and 0..74% (w/v) PEG,mol.wt. 8 x 103 in PBS. (a) CMC solution; (b) PEG solution; (c)mechanically mixed solutions of CMC and PEG; (d) CMC/PEO mix-ture containing methylene blue, 50 ppm. The mixture was centrifugedat 3500 rnm Rt rnnm tAmnArRnJrA fnr ?n min

weight, proteins can diffuse freely from one phase to another,driven by the affinity of proteins toward each polymer phase.As indicated by the results summarized in Table II, bloodplasma proteins were preferentially partitioned into CMC-rich phase. There was an insignificant difference in the pro-tein concentration observed in the CMC-rich phase amongthe three proteins, indicating that the CMC/PEG system iscomparatively insensitive in the nature of proteins (Table II).

The stability of a composite gel in an open system, such asin contacting with body fluid or incubating with media, wasinvestigated in vitro. An experiment was performed by coat-ing the gel onto a piece of porcine intestine membrane, whichwas then incubated with the gel in PBS at room temperature.As shown in Figure 3, fluorescein-labeled PEO of molecularweight 5 X 103 was released from the gel at a much higherrate than that of CMC. The amount of PEO released wasmeasurable after 3 min and increased over 60 min. Within1 h, 25% of the fluorescein-labeled PEO was released fromthe gel coated onto the porcine intestine membrane into thePBS (tl/2 = 108 min). In contrast, there was no CMC in therelease medium detected in the first 20 min period of incu-bation, and more than 97% of CMC was adhered to the tissuesurface at the end of experiment (Table III, Figure 3). After1 h of incubation, the gel was swollen enough to begin todissolve and release CMC.

posite gels, the transparency of gels changed as the ratio ofthe two components changed. The gel composed of equalamounts of CMC and PED had the highest turbidity. Athigher concentrations of polymers, 7.5% (w/v), the transpar-ency was easily seen as shown by photographs in Figure 2.Gels of CMC or PED were transparent (Figure 2, Tubes I and2); the mixture of CMC and PED at high concentration wascloudy (Figure 2, Tube 3). Data from above viscosity andturbidity measurement demonstrated the existence of microphase separation in the CMC/PED gel system. This phaseseparation could be further demonstrated by centrifugation ofthe gel at 3500 rpm for 20 min to separate the system as aCMC phase in the bottom and a PED phase in the top (Figure2, Tube 4). In this study, a specific binder ofCMC, methyleneblue, was added into the system prior to mixing to visualizethe CMC. As shown in the photo, there was no color thatcould be observed in the top phase, indicating that a completephase separation occurred. Without centrifugation, the CMC/PED composite gel retained the heterogeneous structure (Fig-ure 2, Tube 3) at the temperature ranging from 4 to 25 DC,demonstrating the physical stability of gel when stored innormal conditions.

Because the turbidity experiments indicated a phase sep-aration of CMC and PED, serum proteins were used tomeasure their partitioning between the phases with the use ofa modified protein partitioning method. Because both phasesof CMC and PED contain more than 90% of buffer by

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Figure 3. Release of PEG and CMC from CMC/PEG gels. The gelwas composed of 3.3% (w/v) CMC, mol.wt. 7 x 105 and 0.34% (w/v)fluorescein-PEG, mol.wt. 5 x 103. The gel was incubated with pas inpetri dishes covered with membranes of porcine intestine at ambienttemperature. The amount of released CMC and PEG was detected byUV spectrometer as described in the Methods section. Data is ex-

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CMC Content (%)

Figure 4. Tissue adherence of CMC/ PEG gels as a function of theCMC content. Peak force for detachment was measured on a tapeloop tester at ambient temperature. The gap between two testedtissues were adjusted to 2 :!: 1 mm. Data expressed as mean:!: SD(n=5).

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CompositionFigure 5. Thrombogenesis of CMC/PEO gels as a function of com-position. Thrombus formation was initiated by the addition 0.4 mlCaCI2 into 4.0 ml citrated whole blood followed by incubation at 37 °Cfor 20 min. Data expressed as mean:!: SD (n=5) p<.01 compared togel composed of 50% PEO and the gel prepared from PEO alone.

The tissue adhesiveness of CMC/PEa gels was deter-mined by measuring the peak detachment force required toseparate two pieces of porcine intestine, between which issandwiched a thin layer of the gel. As shown in Figure 4, theforce recorded for tissue adhesiveness decreased with in-creasing fraction of PEa in the composition. For the PEa-alone gel, the value of peak detachment force was only about20% of that contributed by the CMC gel with same density.This indicated that CMC possesses a better tissue-adhesiveproperty than PEa.

The thrombogenicity of gels was investigated by deter-mining whole blood clot formation on the gel surface. Asshown in Figure 5, the extent of thrombus formation on CMCwas similar to plastic in that it provided a surface for theformation of a thrombus initiated by Ca + + in citrated blood,

whereas PEa itself was inhibitory to thrombus formationcompared with polystyrene and CMC. In addition, a largerthrombus formed on the gels having lower PEa content,indicating that the presence of PEa in the composition in-hibited the extent of thrombus formation.

DISCUSSION

PEG forms aqueous two-phase systems with several poly-mers including polysaccharides, such as dextran, methylcel-lulose, and hyaluronate.2S, 26 Phase separation of the two

polymers depends on the composition of the polymers and thesolvent. Aqueous two-phase systems have been extensivelystudied and have been developed for protein purification,26macromolecular partitioning,27 and protein delivery?8 PEGhas unique properties in aqueous solution owning to the factthat it is nonionic, yet highly soluble in water .29 This is due

to its unique ethylene oxide monomeric unit, which displaysa high degree of polymer solvent interaction in aqueoussolutions. Because of the biocompatibility of PEa and itssolubility in aqueous solution, polyethylene oxide has beenused recently to coat a variety of materials to limit theirinteraction with proteins.3o One hypothesis to explain thisproperty of PEa is that at room temperature, the dipolemoment of ethylene oxide is maximal and PEa undergoesextensive hydration in water.31 This gives rise to a stericrepulsion force between PEa and proteins that increases withthe molecular weight of the polymer and the extent of surfacecoverage with PEO.3O,31

Adhesion formation requires the apposition of traumatizedtissue with the suppression of fibrinolysis.2,32 Typically, ad-hesion formation is initiated by the deposition of a fibrinnetwork that bridges the apposed, traumatized tissues.2 Alocal temporary ischemia may contribute to adhesion forma-tion by reducing the availability of fibrinolytic factors.32Because tissue apposition is critical, adhesion prevention hasbeen addressed primarily by developing materials that can beplaced between healing tissues to provide a physical barrier tothe transfer of proteins or cells between tissues. The proper-ties of antiadhesive materials include their ability to providea temporary barrier to reduce thrombogenicity and fibrinattachments between apposed tissues, which is recognized asa first step in adhesion formation.2 To be effective suchbarriers need to be present for a short time to prevent theapposed tissues from forming attachments that develop intoadhesions; attract macrophages, fibroblasts; and blood ves-sels; and slowly become organized into scars.2 The presenceof PEa with the CMC in the composites studied here wouldbe hypothesized to reduce the partitioning of plasma proteinsonto the coated tissue and reduce the extent of thrombusformation of whole blood, if present, thereby providing atemporary barrier to the development of adhesions.

PEa and CMC have each individually been used to inhibitadhesion formation. PEa itself has been tested in animals foradhesion prevention. IS One difficulty has been how to keep

PEa in contact with tissues long enough for it to be effective.One novel approach has been to covalently cross-link thePEa to tissue in situ.33 In this case two different, reactive,PEa derivatives are directly administrated on the tissue site,where they react with one another to form a hydrogel thatcovers the tissue surface.15, 33

Polycarbohydrates have been tested as antiadhesion mate-rials. Hyaluronic acid in the form of a gel has been tested34and a polymer of glucose (icodextran) has been tested inanimal studies.35 CMC has also been used to reduce postsur-gical adhesions in several animal models 11 and as a compo-

nent in an anti-adhesive membrane consisting of HA andCMC.12

In order to take advantage of the unique properties of bothCMC and PEa, composites of these polymers have beendeveloped to reduce postsurgical adhesions. The CMC/PEOcomposite materials described here have been shown to beeffective antiadhesion barriers in several animal studies.2o, 21

CMClPEQ Gel

Figure 6. Schematic structure ofbarrier. PEa is released from theform a CMC-rich layer covered byan open system.

Several properties of the individual polymers and propertiesof the composite may be responsible for the effectiveness ofsuch composite gels. As demonstrated in this study, CMC istissue adhesive and provides a barrier function. PEa inhibitsthe interaction of proteins with the CMC and functions tolimit tissue interaction.

A scheme to explain these effects is presented in Figure 6.In a closed system, PEa and the calcium-stabilized CMCform a network of interpenetrating polymers that undergo amicrophase separation. In an open system, PEa is releasedfrom the gel at a rate faster than that of CMC, resulting in ashell structure of CMC surrounded by PEa. The PEa-richoutside layer plays a role in inhibition of protein depositionand thrombus formation on the surface of tissues. The CMC-rich layer anchors the gel to the tissue surface, functioning intissue adhesion. Together CMC and PEa are an effectiveantiadhesive material because CMC coats and separates trau-matized tissue, and PEa inhibits protein-protein interactionand is known to exclude proteins.

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