characterization of a novel active release coating to prevent biofilm implant-related infections

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Characterization of a novel active release coating to prevent biofilm implant-related infections Dustin L. Williams, 1,2,3 Kristofer D. Sinclair, 1,3 Sujee Jeyapalina, 1,3 Roy D. Bloebaum 1,2,3 1 George E. Wahlen Department of Veterans Affairs Medical Center, Salt Lake City, Utah 2 Department of Bioengineering, University of Utah, Salt Lake City, Utah 3 Department of Orthopaedics, University of Utah, Salt Lake City, Utah Received 9 October 2012; revised 24 January 2013; accepted 30 January 2013 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32918 Abstract: Biofilm implant-related infections cost the US healthcare system billions of dollars each year. For several decades, device coatings have been developed that actively release antimicrobial compounds in an attempt to prevent these infections from developing. To date, few coatings have been put into clinical use. These have shown limited to no efficacy in clinical trials. Recent data have shown the in vitro and in vivo efficacy of a novel active release coating that may address the limitations of coatings that are used clinically. In this study, the novel active release coating was characterized to gain an understanding of the effects of combining an anti- microbial additive, cationic steroid antimicrobial-13 (CSA-13), to a medical grade polydimethylsiloxane (PDMS) material. Results indicated that the addition of CSA-13 did influence the physical properties of the PDMS, but not with adverse effects to the desired material properties. Furthermore, there was no indication of chemical reactivity. It was shown that CSA-13 was uniformly dispersed as small particles throughout the PDMS matrix. These particles were able to dissolve and elute out of the PDMS within a 30-day period. The results of this work suggested that the PDMS with CSA-13 was thermally, chemically and physically stable when used as a device coat- ing to treat local infection and/or prevent biofilm implant- related infections from developing. V C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2013. Key Words: characterization, active release, antimicrobial, coating, PDMS, CSA-13 How to cite this article: Williams DL, Sinclair KD, Jeyapalina S, Bloebaum RD. 2013. Characterization of a novel active release coating to prevent biofilm implant-related infections. J Biomed Mater Res Part B 2013:00B:000–000. INTRODUCTION Biofilm implant-related infections have been shown to cost the current US healthcare system more than $3 billion dol- lars each year. 1 One of the most practical strategies that has been undertaken in recent years to prevent these infections has been the development of antimicrobial active release coatings. 2–4 These coatings are designed to release antimi- crobial compounds in a controlled fashion away from the surface of an implanted device, and into the surrounding periprosthetic tissues and fluids of a patient to deliver a local antimicrobial treatment. The intent of this treatment method is ultimately to prevent biofilm growth and subse- quent infection from developing on or near the surface of an implanted device. The potential benefits of active release coatings are twofold: first, high doses of antimicrobial can be delivered locally, which is an important requirement for effectively eradicating bacteria that reside in the biofilm phenotype. Second, local delivery of antimicrobial products may reduce the risk of adverse side effects that can accom- pany systemic and/or chronic antimicrobial therapy. 4 Importantly, active release coatings also have two possi- ble limitations. First, because bacteria are intentionally killed by active release agents, bacteria may in turn release endotoxins or other byproducts en masse, which may elicit an inflammatory or other adverse response. Second, an anti- microbial agent may adversely affect host eukaryotic cells either directly or indirectly (cell injury or toxicity) when it is delivered in high doses. A range of toxic reactions have been documented following administration of antimicrobial agents. 5 Thus, there is an important balance between Correspondence to: D. L. Williams; e-mail: [email protected] The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Arthritis and Musculoskeletal and skin Diseases or the National Institutes of Health. Contract grant sponsor: Office of Research and Development, Rehabilitation R and D Service, George E. Wahlen Department of Veterans Affairs, Salt Lake City, UT. Contract grant sponsor: National Institute of Arthritis and Musculoskeletal and Skin Diseases; contract grant number: R01AR057185 Contract grant sponsors: Albert and Margaret Hofmann Chair and the Department of Orthopaedics, University of Utah School of Medicine, Salt Lake City, UT V C 2013 WILEY PERIODICALS, INC. 1

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Page 1: Characterization of a novel active release coating to prevent biofilm implant-related infections

Characterization of a novel active release coating to prevent biofilmimplant-related infections

Dustin L. Williams,1,2,3 Kristofer D. Sinclair,1,3 Sujee Jeyapalina,1,3 Roy D. Bloebaum1,2,3

1George E. Wahlen Department of Veterans Affairs Medical Center, Salt Lake City, Utah2Department of Bioengineering, University of Utah, Salt Lake City, Utah3Department of Orthopaedics, University of Utah, Salt Lake City, Utah

Received 9 October 2012; revised 24 January 2013; accepted 30 January 2013

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.32918

Abstract: Biofilm implant-related infections cost the US

healthcare system billions of dollars each year. For several

decades, device coatings have been developed that actively

release antimicrobial compounds in an attempt to prevent

these infections from developing. To date, few coatings have

been put into clinical use. These have shown limited to no

efficacy in clinical trials. Recent data have shown the in vitro

and in vivo efficacy of a novel active release coating that may

address the limitations of coatings that are used clinically. In

this study, the novel active release coating was characterized

to gain an understanding of the effects of combining an anti-

microbial additive, cationic steroid antimicrobial-13 (CSA-13),

to a medical grade polydimethylsiloxane (PDMS) material.

Results indicated that the addition of CSA-13 did influence the

physical properties of the PDMS, but not with adverse effects

to the desired material properties. Furthermore, there was no

indication of chemical reactivity. It was shown that CSA-13

was uniformly dispersed as small particles throughout the

PDMS matrix. These particles were able to dissolve and elute

out of the PDMS within a 30-day period. The results of this

work suggested that the PDMS with CSA-13 was thermally,

chemically and physically stable when used as a device coat-

ing to treat local infection and/or prevent biofilm implant-

related infections from developing. VC 2013 Wiley Periodicals, Inc.

J Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2013.

Key Words: characterization, active release, antimicrobial,

coating, PDMS, CSA-13

How to cite this article: Williams DL, Sinclair KD, Jeyapalina S, Bloebaum RD. 2013. Characterization of a novel active releasecoating to prevent biofilm implant-related infections. J Biomed Mater Res Part B 2013:00B:000–000.

INTRODUCTION

Biofilm implant-related infections have been shown to costthe current US healthcare system more than $3 billion dol-lars each year.1 One of the most practical strategies that hasbeen undertaken in recent years to prevent these infectionshas been the development of antimicrobial active releasecoatings.2–4 These coatings are designed to release antimi-crobial compounds in a controlled fashion away from thesurface of an implanted device, and into the surroundingperiprosthetic tissues and fluids of a patient to deliver alocal antimicrobial treatment. The intent of this treatmentmethod is ultimately to prevent biofilm growth and subse-quent infection from developing on or near the surface ofan implanted device. The potential benefits of active releasecoatings are twofold: first, high doses of antimicrobial can

be delivered locally, which is an important requirement foreffectively eradicating bacteria that reside in the biofilmphenotype. Second, local delivery of antimicrobial productsmay reduce the risk of adverse side effects that can accom-pany systemic and/or chronic antimicrobial therapy.4

Importantly, active release coatings also have two possi-ble limitations. First, because bacteria are intentionallykilled by active release agents, bacteria may in turn releaseendotoxins or other byproducts en masse, which may elicitan inflammatory or other adverse response. Second, an anti-microbial agent may adversely affect host eukaryotic cellseither directly or indirectly (cell injury or toxicity) when itis delivered in high doses. A range of toxic reactions havebeen documented following administration of antimicrobialagents.5 Thus, there is an important balance between

Correspondence to: D. L. Williams; e-mail: [email protected]

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Arthritis

and Musculoskeletal and skin Diseases or the National Institutes of Health.

Contract grant sponsor: Office of Research and Development, Rehabilitation R and D Service, George E. Wahlen Department of Veterans Affairs,

Salt Lake City, UT.

Contract grant sponsor: National Institute of Arthritis and Musculoskeletal and Skin Diseases; contract grant number: R01AR057185

Contract grant sponsors: Albert and Margaret Hofmann Chair and the Department of Orthopaedics, University of Utah School of Medicine, Salt

Lake City, UT

VC 2013 WILEY PERIODICALS, INC. 1

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toxicity and antimicrobial efficacy, which must be identifiedand maintained in a coating during the developmentalphase.

To date, few active release coatings on medical deviceshave been approved for use in patients.6–17 Clinical studiesthat have been performed with these approved coatingshave had variable success. Some have indicated that coatedmaterials may be marginally effective,6–9 and others haveshown minimal to no difference in infection or healing out-comes.10–17 There may be at least three reasons for thislack of success. First, researchers have at times tested theperformance of an antimicrobial coating in vivo without firstoptimizing its efficacy in vitro.18,19 In cases where in vitrowork has been performed prior to in vivo testing, stagnantbroth solutions or hard agar surfaces have been used, andoften fail, to accurately model physiological environmentswherein fluid flow may be present.20–23 As such, there maybe limited understanding of the dosage required to effec-tively eradicate biofilm-related bacterial infections. Second,investigators have at times performed in vivo testing whilerelying on concentrations of antimicrobial that are based onminimum inhibitory concentration (MIC) data.18,19 However,MIC values are wholly based on data that is obtained fromplanktonic bacteria. MIC values are not likely to translate toconcentrations required to eradicate biofilms.24 Third, in allanimal models that have been tested to date, planktonicbacterial cells have been used as initial inocula.19,20,25–34

However, data have overwhelmingly indicated that bacteriafrom natural ecosystems, including areas of the humanbody, predominantly reside in the biofilm phenotype.35–42

Based on this information one could argue that wound sites,surgical sites and implanted devices are at risk of beingcontaminated with bacteria that reside in the biofilm pheno-type.24 Moreover, active release agents that are optimizedagainst planktonic bacteria may have reduced efficacy whenthey are exposed to biofilm residing bacteria, both in invitro and in vivo systems.

In order to address these limitations, a novel activerelease coating has recently been developed.43,44 This coat-ing consisted of a polydimethylsiloxane (PDMS) carrier poly-mer and a novel antimicrobial compound, cationic steroidantimicrobial-13 (CSA-13) that functioned as the activerelease agent. CSA-13 is a derivative of cholic acid with thechemical formula C41H84Cl4N4O3 (MW 822.9427). It is asynthetic analog of naturally occurring antimicrobial pep-tides, and has similar structural and antimicrobial character-istics to, for example, squalamine.45,46 Its structure is basedon the attachment of aminoalkyl chains to a cholic acidbackbone. These chains extend in the same plane resultingin an amphipathic molecule, which consists of a hydropho-bic backbone and a cationic face.45 Because it is not a pep-tide, it is not a target for proteases, which contributes to itsstability in vivo. It has a shelf life of several years, can besterilized by autoclaving, ethylene oxide (ETO) treatment orgamma radiation, and it displays broad spectrum activityagainst Gram negative and Gram positive bacteria in theplanktonic and biofilm phenotypes.43,44 Due to its rapid andglobal method of action on bacterial cell membranes, it has

reduced risk of engendering bacterial resistance.47,48 All ofthese characteristics make CSA-13 a promising compoundfor clinical use, in particular as the active release agent ofdevice coatings.

When tested in vitro using a flow cell system, the CSA-13-based active release coating had the ability to eradicatebiofilms of methicillin-resistant Staphylococcus aureus(MRSA) by greater than 108 colony forming units (CFU) in a24-h period.43 When tested in vivo, this coating was able toprevent infection from developing in 100% (9/9) of sheepwith minimal to no toxic effects compared to an untreatedgroup (n ¼ 9) of sheep.43 This work was performed bygrowing well-established biofilms containing approximately109 CFU of MRSA on the surface of polyetheretherketone(PEEK) membranes in a biofilm reactor.49,50 Biofilms wereplaced in apposition to the proximal medial aspect of sheeptibiae, then covered by simulated fracture fixation platesthat contained the novel coating. Each sheep received aninoculum of �7 � 109 CFU/PEEK membrane (1 cm2 each)of MRSA in well-established biofilms.43,51 In this previousstudy, sheep were monitored for signs of infection over a12-week period.43

To better understand the in vitro and in vivo efficacy ofthis coating, the goal of this study was to characterize thephysical and chemical properties of the PDMS/CSA-13 com-bination material for continuing the advancement of thisnovel coating for eventual Food and Drug Administration(FDA) clinical trials.

MATERIALS AND METHODS

ReagentsCSA-13 was manufactured by Dr. Paul Savage’s researchgroup at Brigham Young University, Provo, UT (although notcommercially available, CSA-13 may be received uponrequest for independent analysis). Analytical grade Naphthawas purchased from Fisher Scientific. A one-part room tem-perature vulcanizing PDMS and primer (catalog #s MED-6607 and MED-160, respectively) were purchased fromNuSil Technologies LLC (CA). This particular silicone type ison master file with the FDA for use in approved medicaldevices.

Simulated fracture fixation plate design and fabricationIn this study, sheets of medical grade 316L stainless steel(SS) were used to machine 2 cm � 2 cm square plates with2.7 mm screw holes drilled in each corner (Figure 1). Theseholes were machined for secure bone fixation with bonescrews in the animal model that has been performed previ-ously.43,51 Each plate had a thickness of 1.85 6 0.01mm.

Prior to dip coating, each plate was grit blasted at 90psi using fine grit silica beads in a dry media blast cabinet(model 36bp2; Trinity Tool Company, Fraser, MI) in order toenhance the roughness of the surface. After grit blasting, theplates were cleaned and passivated following the AmericanSociety for Testing and Materials (ASTM) standard F86-04.Briefly, this passivation process involved cleaning and soni-cating the plates in detergent, exposing them to a 35% ni-tric acid solution for 30–60 min and rinsing and sonicating

2 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING

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them in copious amount of water for 20 additional minutes.Each plate was then allowed to air dry prior to dip coating.

Preparation of CSA-13/PDMS dispersionMED-6607 PDMS was chosen for this application because itcan cure at room temperature with a tin catalyst, which wascompatible with CSA-13, and can be dispersed in naphtha, ahydrophobic organic solvent in which CSA-13 remained in-soluble (data not shown). As such, CSA-13 dispersed as par-ticles or particle aggregates that caused pores to form inthe final, cured PDMS product.

In order to optimize the coating composition, varyingratios of PDMS to naphtha (e.g., 0.5:1, 1:1, and 2:1) as wellas varying amounts of CSA-13 (from 2% to 20%, w/w)were tested to develop an optimized PDMS/CSA-13 disper-sion system for dip coating.43 Based on a series of in vitrotests that were performed in a flow cell system to model aphysiologically relevant environment, the final compositionwas formulated and was comprised of an 18% (w/w) con-centration of CSA-13.43

To produce an 18% (w/w) CSA-13/PDMS dispersion, jetmill (00 Jet-O-Mizer jet mill, Fluid Energy, Telford, PA)micronized CSA-13 (50–200 nm particle size) was added to10 mL of naphtha solvent and stirred in a flask with a stirbar for a minimum of 45 min. PDMS of 10 mL were thenadded to obtain a homogeneous suspension with 1:1PDMS:naphtha ratio and 18% (w/w) of CSA-13. The follow-ing equation shows how an 18% (w/w) concentration wascalculated:

x ¼ 0:18ðy þ xÞ0:825

(1)

where x equals the amount of CSA-13 in mg/mL and yequals the solids content of PDMS in mg/mL. The sum of yþ x represented the total solids content of the mixture. Thenumber 0.825 was used to take into account the salt con-tent of the CSA-13 powder (82.5% of CSA-13 was the free

base and 17.5% was comprised of a chloride anion). Equa-tion (1) was based on an assumption of 100% purity. Theactual purity was closer to 95% as determined by inde-pendent liquid chromatography/mass spectrometry (LC/MS) analysis at Southwest Research Institute, San Antonio,TX (data not shown). A purity of 95% resulted in an overallerror of �3% in the amount of CSA-13 that was in the coat-ing of each plate. This error was considered to beacceptable.

After PDMS was added to the dispersion, it was stirredfor a minimum of 3 h. For the control group, that is, thoseplates that were coated with PDMS only, the PDMS andnaphtha were stirred at a 1:1 ratio for a minimum of 3 h.After being stirred, all dispersions were degassed undervacuum (�25 in Hg) to remove air bubbles.

Hardness (durometer) testingTo determine the hardness of the cured PDMS and PDMSwith CSA-13, thin films were made from each formulationby pouring the dispersion into multiple 2 cm � 2 cm chasecontainers that had a depth of 2 mm. The dispersions wereallowed to cure for a 7-day period (as per the manufac-turer’s recommendation), and then removed from the con-tainers. Following ASTM standard D2240, multiple filmswere stacked one on top of the other to a height greaterthan 6 mm. An Asker XP-A type A durometer (www.Gauge-city.com, Northbrook, IL) was then used to determine thehardness of both material types.

Dip coating procedureFollowing passivation, SS plates were initially dipped intoMED-160 primer and allowed to dry for 45 min in ambientconditions on a rotating wheel set at a speed of 5 rpm. Thisrotating wheel was custom designed to hold each plate ontwo prongs that were perpendicular to the face of thewheel. It was experimentally determined that by rotatingthe wheel at 5 rpm, the primer spread and coated the plateuniformly.

After priming, SS plates were hand dipped either withthe dispersion of PDMS only or PDMS with CSA-13. Plateswere again placed onto the rotating wheel set at 5 rpm.This process was repeated two more times to obtain a uni-form coating with the desired thickness. There was a 10-min interval between each of the dip coatings, which waswithin the recommendations of the manufacturer if multiplelayers of dip coatings were to be performed. Importantly,the same person performed the dip coating procedureunder similar ambient conditions in an attempt to reducelot-to-lot variability. After the final dip coat, each SS platewas left on the rotating wheel at 5 rpm for 7 days underambient and aseptic conditions in a laminar flow cabinet toprevent dust particles from accumulating on the plates.

Uniformity of the coating was determined by measuringthe thickness of the coating in three different areas of theplate using standard thickness calipers with a resolution of10 lm. The thickness was further verified by cutting thecoating of several plates with a razor blade perpendicular tothe plate surface, then measuring the thickness using a

FIGURE 1. Image of a SS plate that was designed to simulate a frac-

ture fixation plate.

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JEOL-6100 (Joel, Peabody, MA) scanning electron micro-scope. The amount of CSA-13 present per plate was calcu-lated by weighing each plate before and after the dip coat-ing procedure, subtracting the weight of the plate, thenmultiplying the final coating weight by 0.18 to account forthe 18% (w/w) concentration of CSA-13.

To perform multiple tests, a large cohort of plates weredip coated with PDMS only or in PDMS that was mixed withCSA-13. Once the amount of CSA-13 was determined andthe thickness measured, each plate was sterilized with ETOat the University of Utah Hospital ETO facility. Followingsterilization, each plate was used in one of the characteriza-tion tests described below.

Scanning electron microscopyScanning electron microscopy (SEM) analysis was used forthree purposes. First, the surface of n ¼ 5 plates with PDMSonly and n ¼ 5 plates with PDMS and CSA-13 from threedifferent lots of the dip coating procedure were imagedunder secondary electron (SE) mode in an FEI NOVANanohigh resolution SEM (FEI, Hillsboro, OR). A total of 25images per coating type were collected from five differentareas of each plate to determine the surface morphology ofthe coating. These images were collected with a landingenergy of 7–10 keV. None of the plates were sputter coatedwith gold or carbon using the FEI NOVANano SEM.

Second, backscatter electron (BSE) images (n ¼ 5 plates;five images per plate) were collected using a JEOL-6100SEM at 20 keV. To collect these images, sample chargingwas limited by coating each plate with carbon using a 208cHigh Vacuum Turbo Carbon Coater (Ted Pella, Redding, CA).These images were subsequently used to measure the po-rosity and pore size distribution of each coating type. Tomeasure the distribution of pore sizes throughout the bulkof the polymer, a razor blade was used to cut through thepolymer to the surface of the metal.

Third, using the JEOL-6100 SEM, energy dispersive X-ray(EDX) analysis was performed, again in five areas of n ¼ 5plates coated with PDMS with and without CSA-13, to deter-mine the distribution of CSA-13 throughout the coating.Each plate was coated with carbon, as described above,prior to collecting images and as before, a razor blade wasused to cut the polymer and analyze the bulk of thematerial.

Importantly, an additional n ¼ 5 plates per coating typewere placed in a solution of 10% brain heart infusion (BHI)broth that flowed at a rate of 4.5 mL/h to model the flow offluid in the anatomical location of the animal model inwhich these plates were tested.43 This flow was continuedfor a 30-day period. This procedure was carried out toallow the CSA-13 to elute out of the coating. An n ¼ 5plates coated with PDMS only were also tested as negativecontrols. After the 30-day elution period, the coating oneach plate was cut at a 45� angle with a razor blade toallow the polymer to be analyzed from the top of its surfacedown to the metal plate. In doing so, SE images were col-lected to determine the morphology of the coating followingelution and EDX analysis was performed to confirm that

there were no detectable quantities of CSA-13 in the top orbottom portions of the coating.

Attenuated Total Reflectance Fourier TransformedInfrared SpectroscopyAttenuated total reflectance Fourier transformed infraredspectroscopy (ATR-FTIR) spectra were collected using aThermo Scientific (Waltham, MA) Nicolet 6700 FTIR spec-trometer. The purpose for performing ATR-FTIR was toobtain information with regard to the chemical interac-tion(s), if any, that were present between the PDMS andCSA-13. Spectra were acquired using a single reflection ATRSmartOrbit accessory equipped with a single-bounce dia-mond crystal. The data were then analyzed using OmnicTM

software (Thermo Scientific, Waltham, MA). FTIR spectrawere obtained first by collecting a baseline reading withoutany sample on the diamond crystal, followed by analyzingfive areas per plate on a total of n ¼ 5 plates per coatingtype. Soxhlet extraction was performed with isopropanol(70%) for a 48-h period after plates were analyzed initially.In doing so, the CSA-13 was eluted from the coating. Plateswere once again analyzed by ATR-FTIR to determine if thespectrum would be similar to the PDMS only profile.

Contact angleBy following a similar protocol as Hulterstrom et al.,52 con-tact angle measurements were obtained using a KSV Thetalite goniometer (KSV Instruments, Finland), wherein five dif-ferent areas of a single plate were analyzed by placing adrop of millipore water on the surface. Each drop wasallowed to sit for 10 s prior to reading a measurement. Intotal, there were n ¼ 5 plates analyzed from each of thetwo coating types for a total of 25 contact angle measure-ments of PDMS only and PDMS with CSA-13.

Tensile testingTensile testing was performed following ASTM standardD882-10 at Nelson Laboratories of Salt Lake City, UT. Usingthis standard procedure, the tensile strength, percent elon-gation and modulus of thin plastic materials that have lessthan a 1-mm thickness could be determined. For this work,thin films of PDMS only or PDMS with CSA-13 were madeby first mixing the dispersions as outlined above. Approxi-mately 1.5 mL of each dispersion was poured into a Teflonchase container that had a width of 1/2 in. (1.27 cm) and alength of 5 in. (12.7 cm) as per the ASTM standard. After a7-day curing period, films were conditioned as per theASTM standard, that is, at 23 6 2�C at 50 6 10% relativehumidity for greater than 40 h. After measuring the thick-ness, each film was uniaxially strained at a crosshead speedof 20 ft/min until break using an Instron tensometer (model5565, Instron Corp., Canton, MA). Ultimate force and elonga-tion at break were noted. The tensile strength was calcu-lated by dividing the ultimate force by the cross-sectionalarea of the thin film. Elastic modulus was calculated bydividing the stress by the strain using data points in the lin-ear elastic region. Although the ASTM standard suggested

4 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING

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that n ¼ 10 films be used for testing, in this study, n ¼ 15films were used per group.

Thermal gravimetric analysisThermal gravimetric analysis (TGA) was performed at Inter-tek Plastics Technology Laboratories (Pittsfield, MA) follow-ing ASTM standard E1131-08. In short, three samples (7–10mg) of each cured material type were subjected to TGAusing a Perkin Elmer Pyris 1 TGA (Perkin Elmer, Norwalk,CT). The samples were initially purged for 5 min with nitro-gen, then heated in air from 25�C to 850�C at a heating rateof 10�C/min. The flow of air through the furnace chamberwas controlled at 35 mL/min.

Optical profilometryIn order to determine the surface roughness and topogra-phy of the PDMS coating with CSA-13, an optical profilome-ter (Zygo NewViewTM 5032, Middlefield, CT) was used. Thecoating with PDMS only was unable to be analyzed byoptical profilometry because of its smooth translucent prop-erties. Quantification of the 3D surface roughness parame-ters (Ra) of the PDMS/CSA-13 coating was performed byMetroproTM metrology software (Zygo, Middlefiled, CT). Sim-ilar to the contact angle measurements, an n ¼ 5 plateswere used from three different lots of coated plates, andfive areas per plate were imaged at a 20� magnification toobtain a total of 25 measurements.

RESULTS

Hardness (durometer) testingDurometer testing indicated that the films of PDMS onlyand PDMS with CSA-13 compositions had durometer (ShoreA scale) scores of 35 and 45, respectively. Importantly,based on the quality assurance specifications of NuSilTechnologies, they recommend that the durometer scores oftheir cured material should be between 35 and 45. The datashowed that PDMS only and the PDMS with CSA-13 both

fell within this range, suggesting that the presence of 18%CSA-13 did not affect the manufacturer’s suggested durome-ter values for cured (vulcanized) PDMS.

Coating thicknessPlates that were coated with PDMS only [Figure 2(A)] had afinal coating weight of 75.2 6 11.8 mg and a thickness of83 6 12 lm. In contrast, plates that were coated withPDMS/CSA-13 [Figure 2(B)] had a coating thickness of 1126 17 lm and a final coating weight of 96.4 6 11.8 mg. Theamount of CSA-13 per plate was calculated to be 17.2 6

0.61 mg. Notably, the increase in coating thickness of platesthat were coated with the PDMS/CSA-13 dispersion com-pared to those that were coated with the PDMS only maybe attributed to the increase in the observed viscosity afterCSA-13 was added to PDMS dispersion. Although the viscos-ity was not measured, it was apparent that the dispersionshad distinctly different viscosities and it appeared that theincreased viscosity of PDMS with CSA-13 led to a slightincrease in the amount of solution adhering to the platesduring the dip coating process.

Surface analysis by SEMSE micrographs of both coating types prior to eluting theCSA-13 out of the coating are given in Figure 3. Theseimages indicated that those plates coated with the PDMSonly formulation had micron sized concave ‘‘dimple-like’’features on the outermost layer. The diameter of these dim-ples ranged from 1 lm to 2 lm. Overall, the PDMS onlycoating [Figure 3(A)] had a markedly smoother surface mor-phology compared to those that had CSA-13 in the coating.More specifically, it is believed that the presence of CSA-13created a more porous surface with small particles of CSA-13 entrapped in pores on the outer most surface of thecoating [Figure 3(B)]. These small particles were furtherconfirmed to be CSA-13 by EDX analysis (Figure 4).

FIGURE 2. (A) SS plate coated with PDMS only after being dip coated 3� in the 1:1 PDMS:naphtha dispersion, cured for 7 days and sterilized by

ETO. (B) SS plate coated with the 1:1 PDMS:naphtha þ 18% (w/w) CSA-13, cured for 7 days and sterilized by ETO. The slight jaggedness that

can be seen in the inner edges of the holes was due to the prongs that were used to hold the plates in place on the spinning wheel as the coat-

ing cured.

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The difference in the surface morphology of both coatingtypes became even more apparent after the CSA-13 hadbeen eluted out of the coating. The PDMS only coating hadno change in its surface morphology after being soakedin broth [Figure 3(C)], whereas the porosity of the PDMS/

CSA-13 coating type profoundly increased [Figure 3(D)]after soaking in broth. These results indicated that the pres-ence of CSA-13 in the dispersion caused various shapes andsizes of pores to form throughout the PDMS polymer coat-ing as it cured. It is believed that immiscible CSA-13

FIGURE 3. (A) Scanning electron micrograph of the PDMS only coating showing the surface morphology of the polymer without CSA-13. (B)

Micrograph of the PDMS coating that had CSA-13. (C) Micrograph of a PDMS only coating after being soaked in BHI broth for 30 days. Note the

lack of difference between this and the image in (A). (D) Micrograph of a coating after it was soaked in BHI broth for 30 days to allow CSA-13 to

elute out of the coating. Note the extensive amount of pores that were visible after the CSA-13 had eluted. (E) Micrograph of the bulk of the

PDMS only material after it had been cut with a razor blade. Note that the dimple-like appearance of the polymer can be seen. (F) Micrograph of

the bulk of the PDMS polymer with CSA-13 after it had been cut with a razor blade.

6 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING

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particles in the PDMS dispersion were entrapped within thepolymer matrix as it cured, and as a consequence, poreswere formed. It is likely that these pores allowed water topenetrate the coating and release CSA-13. Importantly,when multiple coatings were cut with a razor blade, perpen-dicular to the coating surface, to analyze the cross-sectionalmorphology of the coatings, it was confirmed that the smalldimple-like structures found on the surface of the PDMSonly coated plates were also present throughout the bulk ofthe coating [Figure 3(E)]. This was done by cutting the coat-ing with a razor blade and imaging the bulk of the polymer.The scanning electron micrographs of PDMS with CSA-13confirmed that pores were present from the outermost PDMS surface level down to the metal substrate[Figure 3(F)].

BSE images that were used to calculate the porosity ofeach the coating types indicated that the PDMS only coatinghad no detectable porosity (Figure 4, top row), whereas thecoating with CSA-13 had 17 6 3% porosity (Figure 4, middlerow). The pore sizes ranged from �0.5 lm to �20 lm withan average pore size of 5 6 8 lm. The porosity of the bulkmaterial, measured after samples were cut with a razor blade,was 10 6 2.3% with an average pore size of 6 6 5 lm.

Because the PDMS only coating had very similar chemi-cal constitution to CSA-13, the only defining element thatwas different and detectable by EDX in the CSA-13 coatingcompared to the PDMS coating was chloride (Cl�). The chlo-ride is the counter anion of the cationic CSA-13 compound.Using EDX mapping with silicone and chlorine channels, thelocation and distribution of CSA-13 throughout the coatingwas determined. The EDX map for chlorine (Figure 4)showed that CSA-13 particles and/or particle aggregates

were predominantly present within the pores of the PDMSfilm thereby supporting that CSA-13 particles were whatcaused the formation of pores in the PDMS film. These dataalso pointed out that the distribution of CSA-13 was uni-form throughout the coating.

The fact that there was no detectable Cl� in the surfaceportions of the PDMS/CSA-13 polymer or throughout thebulk of the polymer after soaking for 30 days in BHI brothindicated that all detectable amounts of CSA-13 had elutedout of the PDMS polymer matrix by this period (Figure 4,bottom row). Importantly, EDX analysis was performed onone plate that had been soaked in broth for just 24 h todetermine whether Cl� was still present. The data from thistest indicated that Cl� was present, albeit at reduced levels(data not shown). Moreover, microbiological data that hasbeen collected previously showed that there were sufficientamounts of CSA-13 eluted from the coating to kill MRSAbacteria for 10–15 days.43 After 10–15 days, MRSA began togrow at increased levels and CSA-13 was no longer detecta-ble by high-pressure LC/MS, which indicated that the CSA-13 had dropped below bactericidal concentrations.

Attenuated total reflectance Fourier transformedinfrared SpectroscopyThe rationale for performing ATR-FTIR was twofold: first, togain an understanding of infrared (IR) active chemicalbonds (functional groups) within the PDMS only and PDMSwith CSA-13 coatings and second, to determine if any cova-lent linkage may have formed between CSA-13 and thePDMS polymer. The ATR-FTIR spectra of PDMS with andwithout CSA-13 are shown in Figure 5. The data showedthat there were specific differences between the two coating

FIGURE 4. Top row—BSE images (grayscale channel) and EDX elemental maps for Si channel and Si þ Cl channel of a PDMS only coating.

Green indicates Si and magenta Cl. Middle row—BSE images of a PDMS coating with CSA-13. In the grayscale channel, pores can be seen.

These pores were believed to have formed due to the presence of CSA-13 particles and/or particle aggregates as indicated by the presence of

the conjugate anion, Cl�, within the pores. Bottom row—BSE images of a PDMS/CSA-13 coating that had been soaked for a period of 30 days in

BHI broth. No Cl was detected in any area (surface or bulk) of the eluted samples. [Color figure can be viewed in the online issue, which is

available at wileyonlinelibrary.com.]

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types. The spectrum of PDMS/CSA-13 samples showedcharacteristic IR scissoring absorptions of NH2

þ and NH3þ

at �1450–1600 cm�1 as well as asymmetrical and symmet-rical stretching of –CH2 at �2940 cm�1 and �2850 cm�1,respectively.53 These particular characteristic absorptionswere not seen in samples that had PDMS only. Importantly,the fingerprint region (between �500 cm�1 and �1200cm�1) of both coating types was very consistent with whatis found in the literature involving FTIR spectra of PDMSpolymers.53–57

If any potential chemical reaction between PDMS andCSA-13 molecules had occurred, based on the known rela-tivities of the functional groups of CSA-13 and PDMS, onemay have seen the formation of Si–N and/or N–O bonds inthe resultant products. N–O bonds would have hadcharacteristic IR absorption bands centered at �1300 cm�1.If Si–N bonds were present, they may have shown in the700–900 cm�1 range of the fingerprint region. As such, theSi–N bonds may have potentially been masked in thefingerprint region where Si–C absorption bands were pre-dominant. For this reason Soxhlet extraction was performedin isopropanol (70%) for a 48-h period to elute CSA-13 outof the coating. After extraction of CSA-13, ATR-FTIR wasperformed once again and the spectrum was found to beidentical to the PDMS only IR spectrum, thus indicating thatCSA-13 had not covalently bound to the PDMS material. TheEDX analysis data corroborated with the ATR-FTIR resultsindicating that after CSA-13 was extracted from the PDMSmaterial, it was no longer detectable. Taken together, thesedata suggested that CSA-13 particles and/or particle aggre-gates were physically suspended/present within the PDMSmatrix as opposed to being chemically cross-linked.

Contact angleContact angle data indicated that the coating with PDMS onlyhad a hydrophobic surface compared to the coating that hadCSA-13. Specifically, the PDMS only coating had a contact angleof 113.74 6 4.14�. These data were consistent with what hasbeen reported in the literature previously with respect to PDMSpolymers.52 The coating with PDMS/CSA-13 resulted in areduced contact angle of 84.18 6 6.33�. When compared usingan independent samples t test, these differences were signifi-cant (p < 0.05). This difference may be attributed to the pres-ence of the positively charged CSA-13 that decreased the surfacetension/increased surface energy or it may have also been par-tially attributed to the increased roughness of the surface.58,59

These data indicated that the coating with CSA-13 was morewettable, but still had a predominantly hydrophobic surface.

Tensile testingTable I provides the results from tensile testing. Resultsindicated that the coating with CSA-13 had a statisticallysignificant reduction in tensile strength (p < 0.05) as wellas percent elongation (p < 0.05; statistical analysis per-formed using an independent samples t test). Furthermore,there was an increase in the initial elastic modulus of thePDMS material with CSA-13, which was also significantlydifferent (p < 0.05). This increase suggested that the PDMSwith CSA-13 was stiffer compared to the PDMS only films.This may be attributed to the filler effect of CSA-13 withinthe PDMS matrix of the polymer.

Thermal gravimetric analysisThe rationale for performing TGA was to determine thethermal stability of the PDMS only and PDMS with CSA-13,

FIGURE 5. FTIR spectra of PDMS with CSA-13 (blue/top line) and PDMS only (red/bottom line). In the top left hand corner of the image,

chemical structures of CSA-13 and the PDMS backbone are provided for the reader to see what bonds were present and how they correspond

to the spectra. A detailed description of the bending/scissoring that occurred, as well as the major differences that were seen in absorption

patterns in the spectra are provided. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

8 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING

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as well as determine if there were any volatile componentsthat were released from each coating type. Each experimentwas run from room temperature to 850�C, with closeattention given to the temperature range of 35–41�C. Thistemperature range was chosen because it was within thebody temperatures of interest for sheep (in vivo model) andhumans (potential coating for clinical applications). Resultsfrom TGA testing indicated that both of the material typeswere heat stable up to greater than 100�C (Figure 6).

In both polymers, with and without CSA-13, the firstnotable onset of weight loss was seen at approximately150�C. From this point, weight loss occurred dramaticallybetween 300�C [Figure 6(A)] and 350�C [Figure 6(B)], thenagain between 450�C [Figure 6(A)] and 500�C [Figure 6(B)].In both cases, this weight loss was well above the tempera-ture range of interest, suggesting that the material wasthermally stable in the intended use range of 35–41�C.

Optical profilometryRoughness data measured using optical profilometry indi-cated that the average surface roughness of the PDMS withCSA-13 was 1.30 6 0.87 lm. In Figure 7, a representativesurface topography is shown that outlines the undulating,porous surface of the PDMS with CSA-13.

DISCUSSION

The goal of this work was to characterize the physical andchemical properties, as well as determine the reproducibil-ity of a novel active release coating, which carries significantpotential to prevent and/or treat local biofilm implant-related infections. Results showed that the incorporation ofCSA-13 into the PDMS polymer did influence the physicaland mechanical properties of the PDMS, but it did notappear that that influence was adverse to the desired mate-rial properties. No chemical cross-linking was observedbetween CSA-13 and the PDMS material that was used. Itwas found that the coating thickness and amount of CSA-13in the device coating was consistent over multiple lots ofdip coated plates. SEM data also showed that the coatingmorphology was consistent from lot-to-lot and that CSA-13was distributed uniformly throughout the PDMS polymer.

The SEM data demonstrated that CSA-13 particlesresided in the pores of the PDMS polymer and that the CSA-13 eluted out of the surface and bulk of the polymer afterbeing soaked in broth for a 30-day period. Furthermore,SEM data indicated that the PDMS with CSA-13 was highlyporous. The porosity of the material may have contributedto a reduction in tensile strength of the material. Morespecifically, the pores may have served as points of stressthat led to reduced elongation at break compared to the

PDMS only films. In addition, the presence of the particlesand particle aggregates seemed to influence the stiffness ofthe material, perhaps by acting as boundaries that inter-acted with polymer movement as the material was pulledunder tension.60,61 These particles and particle aggregatesalso appeared to influence the hardness of the material, asindicated by the durometer measurements.

Data from ATR-FTIR analysis strongly suggested thatthere were no other detectable byproducts in the CSA-13coating other than the CSA-13 antimicrobial when comparedto the PDMS only coating. In all of the ATR-FTIR data, theabsorption peaks that were present in the spectra corre-sponded to the bonds that were present in both coatingtypes with important differences distinguishing the presenceof CSA-13 from the PDMS only coating.

The results of this work indicated that the active releaseagent, CSA-13, was able to be incorporated homogeneouslyinto a PDMS dispersion. After plates were dipped into the

TABLE I. Tensile Testing Data (Mean 6 Standard Deviation) Indicating the Strength, Elongation, and Modulus of Films

of PDMS Only and PDMS With CSA-13

Material TypeTensile Strengthat Break (MPa)

Elongation(%) Strain

Elastic Modulus of theInitial Linear Region (MPa)

PDMS only 2.06 6 0.62 327.72 6 75.68 6.53 6 1.49 1.24 6 0.69PDMS with CSA-13 0.79 6 0.27 215.78 6 39.01 4.47 6 0.80 3.20 6 1.39

FIGURE 6. (A) A representative thermogravimetric curve of PDMS

with CSA-13. Note that the material had no significant weight loss

until approximately 300�C, suggesting that the material was stable in

the 35–41�C temperature range. (B) A representative thermogravimet-

ric curve of PDMS only material. This material showed no significant

signs of thermal degradation until approximately 380�C, also suggest-

ing that it was stable in the 35–41�C range. The initial slope seen in

both compound (1–2% weight loss) may be attributed to the evapora-

tion of absorbed water from the polymer matrix.

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dispersion, CSA-13 particles caused the PDMS to becomeporous upon curing. The particle and/or particle aggregatesizes in the final cured product corresponded to the size ofthe pores that they formed in the PDMS polymer. Becausethe PDMS polymer was made porous, water was able topenetrate the pores and facilitate the dissolution and diffu-sion of CSA-13 from the surface and bulk of the coating.Importantly, although the presence of CSA-13 did appear toaffect the physical properties of the polymer, the effect wasnot determined significant for this particular application.The desired mechanical properties of the material weremaintained. Finally, the thermal stability of both materialsas demonstrated by TGA suggested that the materials wouldremain stable in the range of body temperatures of sheepand humans.

In conclusion, several key characteristics of this novelactive coating were identified in this study. These include(1) the manner by which this coating functioned is basedon the fact that particles of CSA-13 are uniformly distrib-uted into a medical grade PDMS, and as the PDMS cures,these particles create pores in the polymer network. (2) Aswater penetrated the porous network, it caused the disso-lution and diffusion of CSA-13 out of the matrix. (3)Although not presented in detail here, this coating has dis-played promising bactericidal activity both in vitro and invivo.43,44 (4) Results showed that differences were seenbetween the PDMS only polymer and PDMS with CSA-13,but none of these differences suggested that they wouldhave deleterious effects on the function or stability of thePDMS with CSA-13 in vivo. If this coating were to be usedin applications wherein shear forces might be present,such as on intramedullary nails used in fracture fixationprocedures, further data would be needed to show itsstability under those forces. In conclusion, the thermal andmechanical stabilities, and antimicrobial activity of thisnovel active release coating, provide significant promise forit to translate to clinical use wherein it has the potential totreat and/or prevent local biofilm implant-related infec-tions from developing in orthopedic and other biomaterialapplications.

ACKNOWLEDGMENTS

The authors acknowledge the help of Bryan Haymond, JuliaLerdahl, and Andrew Grange for their help in preparingsamples for analysis as well as Brian van Devener for his helpwith the Optical Profilometry analysis. The authors also thankRandy Poulson, PhD, for his help in collecting SEM images,Dennis Romney for machining and grit blasting the SS plates,and Nitesh Madaan and Matt Linford, PhD, for their insightsand collection of ATR-FTIR data.

REFERENCES1. Darouiche RO. Treatment of infections associated with surgical

implants. N Engl J Med 2004;350:1422–1429.

2. Costerton JW. Biofilm theory can guide the treatment of device-

related orthopaedic infections. Clin Orthop Relat Res 2005;437:

7–11.

3. Zilberman M, Elsner JJ. Antibiotic-eluting medical devices for

various applications. J Control Release 2008;130:202–215.

4. Hetrick EM, Schoenfisch MH. Reducing implant-related infections:

Active release strategies. Chem Soc Rev 2006;35(9):780–790.

5. Sanders WE, Sanders CC. Toxicity of antibacterial agents: Mecha-

nism of action on mammalian cells. Ann Rev Pharmacol Toxicol

1979;19:53–83.

6. Rasic Z, Schwarz D, Adam VN, Sever M, Lojo N, Rasic D, Matejic

T. Efficacy of antimicrobial triclosan-coated polyglactin 910

(Vicryl* Plus) suture for closure of the abdominal wall after colo-

rectal surgery. Coll Antropol 2011;35(2):439–443.

7. Galal I, el-Hindawy K. Impact of using triclosan-antibacterial

sutures on incidence of surgical site infection. Am J Surg 2011;

202(2):133–138.

8. Ranucci M, Isgro G, Giomarelli PP, Pavesi M, Luzzani A, Cattab-

riga I, Carli M, Giomi P, Compostella A, Digito A, Mangani V,

Silvestri V, Mondelli E. Impact of oligon central venous catheters

on catheter colonization and catheter-related bloodstream

infection. Crit Care Med 2003;31(1):52–59.

9. Bologna RA, Tu LM, Polansky M, Fraimow HD, Gordon DA, Whit-

more KE. Hydrogel/silver ion-coated urinary catheter reduces

nosocomial urinary tract infection rates in intensive care unit

patients: A multicenter study. Urology 1999;54(6):982–987.

10. Moretti EW, Ofstead CL, Kristy RM, Wetzler HP. Impact of central

venous catheter type and methods on catheter-related coloniza-

tion and bacteraemia. J Hosp Infect 2005;61(2):139–145.

11. Liu XS, Zola JC, McGinnis DE, Squadrito JF, Zeltser IS. Do silver

alloy-coated catheters increase risk of urethral strictures after

robotic-assisted laparoscopic radical prostatectomy? Urology

2011;78(2):365–367.

12. Corral L, Nolla-Salas M, Ibanez-Nolla J, Leon MA, Diaz RM, Martin

MC, Iglesia R, Catalan R. A prospective, randomized study in

FIGURE 7. (A) A representative two-dimensional profilometer image of the surface of a PDMS coating that had 18% CSA-13 added to it. The

blue regions indicated lower areas whereas the red/pink areas indicated high points of the surface. These surface characteristics were present in

all samples analyzed. The black areas that are present are those areas that had vertical surfaces within the polymer, which cannot be analyzed

by optical profilometry because interference patterns do not develop. (B) Three-dimensional rendition of the same area of a surface as in (A).

The black areas were more prominent, yet the model outlined the undulating, porous nature of the coating. [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

10 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING

Page 11: Characterization of a novel active release coating to prevent biofilm implant-related infections

critically ill patients using the oligon vantex catheter. J Hosp

Infect 2003;55(3):212–219.

13. Justinger C, Slotta JE, Schilling MK. Incisional hernia after

abdominal closure with slowly absorbable versus fast absorbable,

antibacterial-coated sutures. Surgery 2012;151(3):398–403.

14. Chen SY, Chen TM, Dai NT, Fu JP, Chang SC, Deng SC, Chen SG.

Do antibacterial-coated sutures reduce wound infection in head

and neck cancer reconstruction? Eur J Surg Oncol 2011;37(4):

300–304.

15. Fraenkel D, Rickard C, Thomas P, Faoagali J, George N, Ware R.

A prospective, randomized trial of rifampicin-minocycline-coated

and silver–platinum–carbon-impregnated central venous cathe-

ters. Crit Care Med 2006;34(3):668–675.

16. Arvaniti K, Lathyris D, Clouva-Molyvdas P, Haidich AB, Mouloudi

E, Synnefaki E, Koulourida V, Georgopoulos D, Gerogianni N,

Nakos G, Matamis D. Comparison of oligon catheters and chlo-

rhexidine-impregnated sponges with standard multilumen central

venous catheters for prevention of associated colonization and

infections in intensive care unit patients: A multicenter, random-

ized, controlled study. Crit Care Med 2012;40(2):420–429.

17. Storch M, Perry LC, Davidson JM, Ward JJ. A 28-day study of the

effect of coated Vicryl* Plus antibacterial suture (coated polyglac-

tin 910 suture with triclosan) on wound healing in guinea pig lin-

ear incisional skin wounds. Surg Infect 2002;3:S89–S98.

18. Fei J, Yu H-j, Pan C-j, Zhao C-h, Zhou Y-g, Wang Y. Efficacy of a

norvancomycin-loaded, PDLLA-coated plate in preventing early

infection of rabbit tibia fracture. Orthopedics 2010;33(5):310.

19. Darouiche RO, Mansouri MD. Dalbavancin compared with vanco-

mycin for prevention of Staphylococcus aureus colonization of

devices in vivo. J Infect 2005;50(3):206–209.

20. Darouiche RO, Mansouri MD, Zakarevicz D, AlSharif A, Landon

GC. In vivo efficacy of antimicrobial-coated devices. J Bone Joint

Surg 2007;89(4):792–797.

21. Price JS, Tencer AF, Arm DM, Bohach GA. Controlled release of

antibiotics from coated orthopedic implants. J Biomed Mater Res

1996;30:281–286.

22. Schierholz JM, Steinhauser H, Rump AFE, Berkels R, Pulverer G.

Controlled release of antibiotics from biomedical polyurethanes:

Morphological and structural features. Biomaterials 1997;18:

839–844.

23. Kalicke T, Schierholz J, Schlegel U, Frangen TM, Koller M, Print-

zen G, Seybold D, Klockner S, Muhr G, Arenas S. Effect on infec-

tion resistance of a local antiseptic and antibiotic coating on

osteosynthesis implants: An in vitro and in vivo study. J Orthop

Res 2006;24:1622–1640.

24. Williams DL, Costerton JW. Using biofilms as initial inocula in

animal models of biofilm-related infections. J Biomed Mater Res

B 2011;100(4):1163–1169.

25. Buret A, Ward KH, Olson ME, Costerton JW. An in vivo model to

study the pathobiology of infectious biofilms on biomaterial

surfaces. J Biomed Mater Res 1991;25(7):865–874.

26. Cirioni O, Mocchegiani F, Ghiselli R, Silvestri C, Gabrielli E,

Marchionni E, Orlando F, Nicolini D, Risaliti A, Giacometti A. Dap-

tomycin and rifampin alone and in combination prevent vascular

graft biofilm formation and emergence of antibiotic resistance in

a subcutaneous rat pouch model of staphylococcal infection. Euro

J Vasc Endovasc Surg 2010;40(6):817–822.

27. Lambe DW Jr, Ferguson KP, Mayberry-Carson KJ, Tober-Meyer B,

Costerton JW. Foreign-body-associated experimental osteomyeli-

tis induced with Bacteroides fragilis and Staphylococcus epider-

midis in rabbits. Clin Orthop Relat Res 1991;266:285–294.

28. Darouiche RO, Farmer J, Chaput C, Mansouri M, Saleh G, Landon

GC. Anti-infective efficacy of antiseptic-coated intramedullary

nails. J Bone Joint Surg 1998;80(9):1336–1340.

29. Darouiche RO, Mansouri MD, Gawande PV, Madhyastha S. Anti-

microbial and antibiofilm efficacy of triclosan and dispersin B

combination. J Antimicrob Chemother 2009;64(1):88–93.

30. Davis SC, Ricotti C, Cazzaniga A, Welsh E, Eaglstein WH, Mertz PM.

Microscopic and physiologic evidence for biofilm-associated wound

colonization in vivo. Wound Repair Regen 2008;16(1):23–29.

31. Hansen LK, Berg K, Johnson D, Sanders M, Citron M. Efficacy of

local rifampin/minocycline delivery (AIGISRxVR

) to eliminate

biofilm formation on implanted pacing devices in a rabbit model.

Int J Artif Organs 2010;33(9):627–635.

32. Lucke M, Schmidmaier G, Sadoni S, Wildemann B, Schiller R,

Haas NP, Raschke M. Gentamicin coating of metallic implants

reduces implant-related osteomyelitis in rats. Bone 2003;32(5):

521–531.

33. Mayberry-Carson KJ, Tober-Meyer B, Smith JK, D.W. Lambe J,

Costerton JW. Bacterial adherence and glycocalyx formation in

osteomyelitis experimentally induced with Staphylococus aureus.

Infect Immun 1984;43(3):825–833.

34. Williams D, Bloebaum R, Petti CA. Characterization of Staphylo-

coccus aureus strains in a rabbit model of osseointegrated pin

infections. J Biomed Mater Res A 2008;85(2):366–370.

35. Wimpenny J, Manz W, Szewzyk U. Heterogeneity in biofilms.

FEMS Microbiol Rev 2000;24(5):661–671.

36. Costerton JW, Geesey GG, Cheng KJ. How bacteria stick. Sci Am

1978;238(1):86–95.

37. Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE.

Optical sectioning of microbial biofilms. J Bacteriol 1991;173:

6558–6567.

38. Geesey GG, Richardson WT, Yeomans HG, Irvin RT, Costerton

JW. Microscopic examination of natural sessile bacterial popula-

tions from an alpine stream. Can J Microbiol 1977;23(12):

1733–1736.

39. James GA, Swogger E, Wolcott R, Pulcini Ed, Secor P, Sestrich J,

Costerton JW, Stewart PS. Biofilms in chronic wounds. Wound

Repair Regen 2008;16:37–44.

40. Feazel LM, Baumgartner LK, Peterson KL, Frank DN, Harris JK,

Pace NR. Opportunistic pathogens enriched in showerhead bio-

films. Proc Natl Acad Sci USA 2009;106(38):16393–16399.

41. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA,

Wolcott RD. Survey of bacterial diversity in chronic wounds using

pyrosequencing, DGGE, and full ribosome shotgun sequencing.

BMC Microbiol 2008;6(8):43.

42. Williams DL, Costerton JW. Using biofilms as initial inocula in

animal models of biofilm-related infections. J Biomed Mater Res

Part B 2011;00B.

43. Williams DL, Haymond BS, Beck JP, Savage PB, Chaudhary V,

Epperson RT, Kawaguchi B, Bloebaum RD. In Vivo efficacy of a

silicone—Cationic steroid antimicrobial coating to prevent

implant-related infection. Biomaterials 2012;33(33):8641–8656.

44. Sinclair KD, Pham TX, Farnsworth RW, Williams DL, Loc-Carrillo

C, Horne LA, Ingebretsen SH, Bloebaum RD. Development of a

broad spectrum polymer-released antimicrobial coating for the

prevention of resistant strain bacterial infections. J Biomed Mater

Res A 2012;100(10):2732–2738.

45. Savage PB. Design, Synthesis and characterization of cationic

peptide and steroid antibiotics. Eur J Org Chem 2002;2002(5):

759–768.

46. Moore KS, Wehrli S, Roder H, Rogers M, Forrest JN, McCrimmon

D, Zasloff M. Squalamine: An aminosterol antibiotic from the

shark. PNAS 1993;90(4):1354–1358.

47. Bucki R, Sostarecz AG, Byfield FJ, Savage PB, Janmey PA. Resist-

ance of the antibacterial agent ceragenin CSA-13 to inactivation

by DNA or F-actin and its activity in cystic fibrosis sputum. J Anti-

microb Chemother 2007;60:535–545.

48. Epand RF, Pollard JE, Wright JO, Savage PB, Epand RM. Depolari-

zation, bacterial membrane composition, and the antimicrobial

action of ceragenins. Antimicrob Agents Chemother 2010;54(9):

3708–3713.

49. Williams DL, Haymond BS, Bloebaum RD. Use of delrin plastic in

a modified CDC biofilm reactor. Res J Microbiol 2011;6:425–429.

50. Williams DL, Woodbury KL, Haymond BS, Parker AE, Bloebaum

RD. A modified CDC biofilm reactor to produce mature biofilms

on the surface of PEEK membranes for an in vivo animal model

application. Curr Microbiol 2011;62(6):1657–1663.

51. Williams DL, Haymond BS, Woodbury KL, Beck JP, Moore DE,

Epperson RT, Bloebaum RD. Experimental model of biofilm

implant-related osteomyelitis to test combination biomaterials

using biofilms as initial inocula. J Biomed Mater Res A 2012;

100(7):1888–1900.

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2013 VOL 00B, ISSUE 00 11

Page 12: Characterization of a novel active release coating to prevent biofilm implant-related infections

52. Hulterstrom AK, Berglund A, Ruyter IE. Wettability, water sorption

and water solubility of seven silicone elastomers used for

maxillofacial prostheses. J Mater Sci 2008;19:225–231.

53. Cross AD. Inroduction to Practical Infra-Red Spectroscopy.

London, UK: Butterworths Scientific Publications; 1960. 80 pp.

54. Abbasi F, Mirzadeh H, Katbab A-A. Bulk and surface modification

of silicone rubber for biomedical applications. Polym Int 2002;

51(10):882–888.

55. Abbasi F, Mirzadeh H. Adhesion between modified and unmodi-

fied poly(dimethylsiloxane) layers for a biomedical application. Int

J Adhes Adhes 2004;24(3):247–257.

56. Orhan JB, Parashar VK, Flueckiger J, Gijs MA. Internal modifica-

tion of poly(dimethylsiloxane) microchannels with a borosilicate

glass coating. Langmuir 2008;24(16):9154–9161.

57. Khorasani MT, Mirzadeh H, Kermani Z. Wettability of porous poly-

dimethylsiloxane surface: Morphology study. Appl Surf Sci 2004;

242(3-4):339–345.

58. McHale G, Newton MI, Shirtcliffe NJ. Dynamic wetting and

spreading and the role of topography. J Phys: Condens Matter

2009;21(46):464122.

59. Quere D. Rough ideas on wetting. Physica A 2002;313(1-2):

32–46.

60. McCrum NG, Buckley CP, Bucknall CB. Principles of Polymer

Engineering. New York: Oxford University Press; 1997. pp 242–

245.

61. Bergstrom JS, Boyce MC. Mechanical behavior of particle filled

elastomers. Rubber Chem Technol 1999;72:633–656.

12 WILLIAMS ET AL. CHARACTERIZATION OF A NOVEL ACTIVE RELEASE COATING