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Keywords:TitaniumNanotopographyBacteriaAdhesionFibronectin

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and, thus, a high need for revision surgery [3]. Joint prostheticinfection costs about $50,000 U.S. dollars per episode while theassociated mortality rate may be as high as 2.5% [3]. In addition, ifthe infection persists into the deep tissue, amputation may also berequired.

around the biomaterial exit site due to bacteria invasion [9].As bacteria colonize either the implant surface or adjacentdamaged tissue sites, biomaterial exit sites become the gateway toinfection [1012], possibly leading to bacteria spreading internallyand causing osteomyelitis [12,13]. According to previous studies,the occurrence of osteomyelitis after insertion of an external xatorcan be up to 4% [1012]. In addition to osteomyelitis, infection leadsto bone implant loosening [11] and fracture malunion or nonunion,leading to early failure of the device.

* Corresponding author. Tel.: 1 401 523 3802; fax: 1 401 523 9107.E-mail address: thomas_webster@brown.edu (T.J. Webster).

1

Contents lists availab

Biomat

ev

Biomaterials 31 (2010) 706713Contributed equally.including central venous catheters and needleless connectors,endotracheal tubes, intrauterine devices, mechanical heart valves,pacemakers, peritoneal dialysis catheters, tympanostomy tubes,voice prostheses, orthopedic joint prosthetics, and percutaneousorthopedic devices (including external xators and bone-anchoredamputee prosthetics) [1,2]. Prosthetic joint replacements are beingused with increasing frequency to alleviate pain, to promotemobility, and to improve the quality of life. Yet, such implantationsuffers from the added risk of infection occurring in about 1.52.5%of all hip and knee arthroplasties resulting in failure of the device

Of these, an estimated 3352 THA (1.23%) and 5838 TKA (1.21%) weretreated for infection [4]. It was further revealed that of these totalarthroplasties performed in 2004, 38,629 THA and 36,425 TKAwere due to revision surgeries [4]. Revision surgeries are a result ofimplant failure that can be caused from stressstrain imbalances,implant migration, wear debris, lack of integration, and infection[1,58]. Of these implant failure modes, about 8% of THA and 15% ofTKA revision surgeries were a direct result of infection [4].

In addition to orthopedic joint prosthetics, percutaneousimplant devices suffer from a lack of successful skin integration1. Introduction

Infection has been reported on an0142-9612/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.09.081shown to enhance select protein adsorption and subsequent osteoblast (bone-forming cell) functions,were investigated as a means to also reduce bacteria adhesion. This study examined the adhesion ofStaphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa on conventional Ti,nanorough Ti produced by electron beam evaporation, and nanotubular and nanotextured Ti producedby two different anodization processes. This study found that compared to conventional (nano-smooth)Ti, the nanorough Ti surfaces produced by electron beam evaporation decreased the adherence of all ofthe aforementioned bacteria the most. The conventional and nanorough Ti surfaces were found to havecrystalline TiO2 while the nanotubular and nanotextured Ti surfaces were found to be amorphous. Thesurface chemistries were similar for the conventional and nanorough Ti while the anodized Ti surfacescontained uorine. Therefore, the results of this study in vitro study demonstrated that certain nano-meter sized Ti topographies may be useful for reducing bacteria adhesion while promoting bone tissueformation and, thus, should be further studied for improving the efcacy of Ti-based orthopedicimplants.

2009 Elsevier Ltd. All rights reserved.

of implantable devices

To put these percentages into perspective, it is important to notethat in 2004, 265,441 total hip arthroplasties (THA) and 496,018total knee arthroplasties (TKA) were performed in the U.S. alone [4].Available online 30 October 2009

infection and allow for subsequent appropriate tissue integration with the biomaterial surface. In this invitro study, nanometer sized topographical features of titanium (Ti) surfaces, which have been previouslyReceived 10 August 2009Accepted 21 September 2009

implant. Reducing the adhesion of a broad range of bacteria could be an attractive means to decreaseThe relationship between the nanostrucbacterial attachment

Sabrina D. Puckett 1, Erik Taylor 1, Theresa RaimondDivision of Engineering and Department of Orthopaedics, Brown University, Providence

a r t i c l e i n f o

Article history:

a b s t r a c t

Infection of an orthopedic

journal homepage: www.elsAll rights reserved.re of titanium surfaces and

Thomas J. Webster*

02917, USA

osthesis is undesirable and causes a decrease in the success rate of an

le at ScienceDirect

erials

ier .com/locate/biomater ia ls

2.3. Anodization

analysis, X-ray photoelectron spectroscopy (XPS) was performed using a PerkinElmer 5500 Multitechnique Surface Analyzer System (Waltham, MA, USA). XPS was

2.5. Surface energy and contact angles

aterReduction of microbial adhesion to an implant (without the useof drugs) could be an attractive method for reducing infection.Planktonic (suspended) bacteria present in the body can be clearedby host defense mechanisms and are more susceptible to antibiotictreatment [14]. However, once bacteria bind to the biomaterialsurface, changes in their functions occur. More specically, geneexpression changes, growth rates are altered, host defense mech-anisms are no longer able to remove them from the body, andformation of an antibiotic resistant biolm occurs [1,1416].Development of this biolm is responsible for many chronicinfections [1,1416] and prevents proper integration of the implantto the surrounding tissue. In addition, antibiotic resistant strainscannot be treated by antibiotic therapy after adhesion to theprosthetic surface. Multiple antibiotic resistant strains includingStaphylococcus aureus (S. aureus) and Staphylococcus epidermidis(S. epidermidis) are well documented in the clinical orthopedicsetting [17,18]. Thus, it can be argued that the prevention of bacteriaadhesion without drugs may be one of the best ways to reduceorthopedic implant infection [18].

Along these lines, altering surface roughness of an implantmaterial from one that possesses conventional, micron size featuresto one that possesses nanometer size features has been shown toenhance certain cellular, such as osteoblasts (bone-forming cells)[1924], adhesion and subsequent cellular functions (such ascalcium deposition) while simultaneously decreasing competitivecell, such as broblast (cells that create the brous tissue around animplanted material preventing proper bone integration) function[25,26]. Research has specically demonstrated that nanorough Ti(created through electron beam evaporation) [27] and nanotubularand nanotextured Ti (created through anodization) can enhanceosteoblast adhesion and other functions (such as alkaline phos-phatase synthesis, calcium deposition, and collagen secretion)compared to their micron nano-smooth counterparts [28].Increased select protein adsorption on such Ti surfaces containingnanofeatures has been correlated to the improved functions ofosteoblasts [2931]. Previous studies have also shown that byvarying the surface roughness of a biomaterial, bacteria adhesiondecreases [32]. However, more research is required to understandthe underlying factors for such a phenomenon and translating suchresults to metals commonly used in orthopedics.

Based on the evidence from these previous studies, this studyexplored the adhesion of multiple bacteria species well-known tolead to orthopedic implant infection on nanotubular, nanotextured,nanorough, and conventional Ti. Moreover, initial protein adsorp-tion events were explored that may explain such bacteria adhesiontrends. Specically, gram positive S. aureus and S. epidermidis alongwith gram negative Pseudomonas aeruginosa (P. aeruginosa) wereexamined since these strains have been shown to be clinicallyprevalent in orthopedic prosthetic infections [18]. With selectivelyimproved bone cell responses, as already demonstrated, anddecreased bacteria adhesion, integration between bone and theimplant surface would be promoted, thus, improving the successrate of orthopedic prosthetics.

2. Materials and methods

2.1. 2.1. Titanium substrates

Titanium(Ti) foils (1001001 mm;99.2%pure;AlfaAesar,WardHill,MA,USA)were cut into 1010 mm squares using a shear cutter. All substrates were ultra-sonically cleaned with a diluted cleaning solution (Branson, Dabury, CT, USA) for20 min followed by sonication in acetone, 70% ethanol, and deionized water (DI) for10 min. Substrates were then dried in an oven (VWR) at 40 C for 15 min. Some ofthese substrates served as the conventional, unmodied Ti substrates usedthroughout thepresentwork,whileotherswereusedbelowinSection2.2 forelectron

S.D. Puckett et al. / Biombeam evaporation or Section 2.3 for anodization. Prior to cell culture experiments,these substrates were sterilized in a steam autoclave at 120 C and 17 psi for 30 min.Material surface energy and wettability were investigated with a drop shapeanalysis system (EasyDrop, Kruss, Hamburg, Germany). The contact angle of 3 mLsessile droplets was measured at two locations on each of the four samples (thenanotubular, nanotextured, nanorough, and unmodied Ti). To determine surfaceenergy, three different liquid solvents, distilled water, glycerol, and polyethyleneglycol, were used. Measurements were taken 5 sec after placing the droplet on thesample surface under ambient conditions. Drop shape analysis software (DSA1,Kruss, Hamburg, Germany) was used to calculate surface energy by entering surfacetension and contact angle data into the OwensWendt equation:

1 g1 cos q 2

gds gd1

q gpcg

p1

q (1)

Here, gds and gps are the respective dispersion and polar terms of the solid surface

tension, gs; gd1 and gp1 are the respective dispersion and polar terms of the liquid

surface tension, gl. Other theories were investigated (Fowkes and Zisman) butspecically used to determine the composition of the surface oxide formed on the Tisubstrates. An aluminum K-alpha monochromatized X-ray source was used tostimulate photoemission of the inner shell electrons on the Ti surface. The energyfrom these electrons was then recorded and analyzed for identication purposedconcerning chemical composition of the nanotubular, nanorough, and unmodied Tisubstrates.

For qualitative surface roughness analysis, scanning electron microscopy (SEM)was performed on the conventional, nanorough, nanotubular, and nanotextured Tisubstrates. Images were taken using a LEO 1530VP SEM at varying magnications.Digital images were created using secondary electrons collected with an in-lensdetector at an accelerating voltage of 3 kV for nanorough and conventional Tisubstrates and 5 kV for nanotubular and nanotextured Ti substrates.

Phase analysis was carried out by X-ray diffraction (XRD) analysis using a D500Siemens Diffractometer (Bruker AXS, WI). Spectra were taken using a power supplyof 30.0 mA and 40.0 kV.Prior to anodization, Ti substrates were immersed in a dilute acidic mixture ofnitric acid (HNO3) and hydrouoric acid (HF) for 5 min to remove the thin oxidelayer that spontaneously forms on Ti while in the presence of air. Titania nanotubearrays were then formed into the Ti surface by anodization. Anodization is anelectrolytic passivation process used to increase the thickness of the natural oxidelayer on metal surfaces, in this case Ti. This process was conducted with a DCpowered electrochemical cell which consisted of a two electrode conguration:a platinummeshwhich served as the cathode and Ti foils which served as the anode.To fabricate nanotubular Ti surfaces, the anodization process took place in 1.5 wt%HF for 10 min at a constant voltage of 20 V [28]. To fabricate nanotextured Tisurfaces, the anodization process took place in 0.5 wt% HF for one minute ata constant voltage of 20 V [28]. The nanotubular and nanotextured Ti substrateswere rinsed with large amounts of DI immediately after anodization, air dried, andsterilized under ultraviolet light for 3 h per substrate side.

2.4. Surface characterization

The conventional, nanorough, nanotubular, and nanotextured Ti substrates werecharacterized for chemistry, surface roughness, and crystallinity. For chemical2.2. Electron beam evaporation

A Temescal Electron Beam Evaporator (Reston, VA, USA) was used to createnanorough Ti substrates. Electron beam evaporation concentrates a large amount ofheat produced by high energy electron beam bombardment on the source materialto be deposited, in this case 99.995% pure Ti pellets (Kamis, Mahopac Falls, NY, USA).The electron beam is generated by an electron gun that uses the thermoionicemission of electrons produced by an incandescent lament. A magnet focuses andbends the electron trajectory so that the beam is accelerated towards a graphitecrucible (Lesker, Clairton, PA, USA) containing the source material. As the beamrotates and hits the surface of the source material, heating and vaporization occur.The vapor ow then condenses onto the substrate surface located at the top of thevacuum chamber. In this study, Ti was deposited onto the Ti substrates at a rate of3.5 /s and at a thickness of 500 nm. Following deposition, the nanorough Tisamples were rinsed thoroughly with DI, air dried, and sterilized in a steam auto-clave at 120 C and 17 psi for 30 min.

ials 31 (2010) 706713 707results showed the same trends of surface energy as those obtained with theOwensWendt model.

2.6. Bacteria culture

Bacteria cell lines used in this study were S. epidermidis, P. aeruginosa, andS. aureus obtained in freeze-dried form from the American Type Culture Collection(35984, 25668, and 25923 respectively). The dry pellet was rehydrated in 6 mL ofLuria broth (LB) consisting of 10 g tryptone, 5 g yeast extract, and 5 g NaCl per literdouble distilled water with the pH adjusted to 7.4 (all chemicals were obtained fromSigma Aldrich, St. Louis, MO, USA). The bacteria solutionwas agitated under standardcell conditions (5% CO2/95% humidied air at 37 C) for 24 h until the stationaryphase was reached. The second passage of bacteria was diluted at a ratio of 1:200into fresh LB and incubated until it reached stationary phase. The second passagewas then frozen in one part LB and one part glycerol (Sigma Aldrich) and stored at18 C. All experiments were conducted from this frozen stock. One day beforebacterial seeding for experiments, a sterile 10 ml loop was used to withdraw bacteriafrom the frozen stock and to inoculate a centrifuge tube with 3 mL of fresh LB.

2.7. Bacteria adhesion

Prior to seeding, sterilized substrates were placed into a standard 24-wellculture plate and were washed twice with phosphate buffer saline (PBS). Bacteriawere seeded on the substrates at a density of 1107 bacteria/mL (as estimated bythe McFarland scale) by diluting the LB bacteria cultures to an optical density of0.52 at 562 nm and then further diluted at a ratio of 1:90 in Dulbeccos ModiedEagles Medium (DMEM, Hyclone; Logan, UT/USA) supplemented with 10% fetalbovine serum (FBS, Hyclone), 1% penicillinstreptomycin (P/S, Hyclone), 50 mg/mLL-ascorbate acid (Sigma Aldrich), and 10 mM b-glycerophosphate (Sigma Aldrich).The bacteria were allowed to adhere for one hour under standard cell conditions(5% CO2/95% humidied air at 37 C) with constant shaking at 200 rpm to preventsettling of the cell solution. At the end of the prescribed time period, the substrateswere rinsed twice with Tris-buffered saline (TBS) comprised of 42 mM TrisHCl,8 mM Tris Base, and 0.15 M NaCl (Sigma Aldrich) and then incubated for 15 min withthe BacLight Live/Dead solution (Life Technologies Corporation, Carlsbad, CA)dissolved in TBS at the concentration recommended by themanufacturer. Substrateswere then rinsed twice with TBS and placed into a 50% glycerol solution in TBS priorto imaging. Bacteria were then visualized and counted in situ using a LeicaDM5500 B uorescence microscope with image analysis software captured usinga Retiga 4000R camera. Adhesion experiments were run in duplicate and repeated

The data was represented by the mean value with the standard error of the mean(SEM) noted. A students t-test was used to check statistical signicance betweenmeans and p< 0.1 was considered statistically signicant.

2.8. Fibronectin adsorption (ELISA)

The enzyme-linked immunosorbent assay (ELISA) is a well established proce-dure for measuring the amount of protein, in this study bronectin, adsorbed to theconventional, nanorough, nanotextured, and nanotubular Ti substrates. Substrateswere placed in a standard 24-well culture plate and immersed in 1 mL of DulbeccosModied Eagles Medium (DMEM, Hyclone; Logan, UT, USA) supplemented with andwithout 10% FBS and 1% P/S for 24 h at 37 C in 5% CO2/95% humidied air. Afterrinsing in PBS, areas that did not adsorb proteinswere blocked and incubated for onehour in bovine serum albumin, BSA (2 wt% in PBS, Sigma Aldrich, MO, USA).Substrates were again rinsed twice with PBS before bronectin was directly linkedwith primary rabbit anti-bovine bronectin (AB2047, Millipore, CA, USA) ata concentration of 6 mg/mL in 1% BSA for 1 h at 37 C in 5% CO2/95% humidied air.After rinsing 3 times with 0.05% Tween 20 for 5 min with each rinse, the sampleswere further incubated for another 1 h with a secondary goat anti-rabbit conjugatedwith horseradish peroxidase (HRP, Bio-Rad, MD, USA) at a concentration of 10 mg/mLin 1% BSA. Followed by another 3 rinses with 0.05% Tween 20 for 5 min with eachrinse, the amount of bronectin adsorbed to the surfaces was measured with anABTS substrate kit (Vector Laboratories, CA, USA) that reacted only with the HRP.Light absorbance was measured at 405 nm on a spectrophotometer and analyzedwith computer software. The average adsorbance was subtracted by the averageadsorbance obtained from the negative controls soaked in DMEMwith no FBS or P/S.ELISA was performed in duplicate and repeated three different times per substrate.

3. Results

3.1. Surface characterization

The unmodied titanium (Ti) as purchased from the vendorpossessed micron rough surface features as displayed under SEM(Fig. 1(a)). After electron beam evaporation, the Ti substrates

S.D. Puckett et al. / Biomaterials 31 (2010) 706713708three different times per substrate type. Total bacteria colonies were determined bysumming the number of live and dead bacteria colonies found using Image J.Fig. 1. SEM micrographs of Ti before and after electron beam evaporation and anodization: (evaporation; (c) nanotextured Ti after anodization for 1 min in 0.5% HF at 20 V; (d) nanotupossessed a high degree of nanometer surface features, thus,creating a more nanometer rough surface topography (Fig. 1(b)).a) conventional Ti as purchased from the vendor; (b) nanorough Ti after electron beambular Ti after anodization for 10 min in 1.5% HF at 20 V. Scale bars 200 nm.

Completion of anodization for 1 min in 0.5% hydrouoric acid (HF)at 20 V resulted in a Ti substrate containing nanotextured surfacefeatures (Fig. 1(c)). Increasing the anodization time (10 min) andconcentration of HF (1.5%) resulted in a Ti surface that containednanotubular like structures with an inner diameter from 60 to70 nm, as estimated from the SEM images (Fig. 1(d)).

One high resolution XPS spot was taken on each sample toexamine the Ti 2 p binding energy. Data indicated that other thanTiO2, nootherTi species, suchasTiOandTi2O3,werepresent (Table1).XPS results also showed that for all sample types the outermost layerof oxide contained O and Ti (Table 2). The nanotubular and nano-textured Ti substrates also contained a small amount of F in the

compared to the conventional, nanotubular, and nanotextured Tisubstrates (Fig. 3). In addition, when further examining bacteriabehavior on the anodized Ti surfaces, results indicated that bacteriasignicantly adhered more to the nanotubular Ti compared to thenanotextured Ti (Fig. 3). Fig. 4 qualitatively highlights the decreasedattachment of S. aureus, S. epidermidis, and P. aeruginosa on thenanorough Ti substrates. Interestingly, data also indicated that thenanotubular and nanotextured Ti substrates had the highestnumber of bacterial colonies for all cell lines compared toconventional and nanorough Ti substrates (Fig. 3). Fig. 4 alsovisually highlights the signicantly greater number of S. aureus,S. epidermidis, and P. aeruginosa present on the nanotextured andnanotubular Ti substrates compared to conventional and nano-

Table 2Atomic percentage of selective elements in the outermost layers of Ti before andafter electron beam evaporation (nanorough Ti) and anodization (nanotubular andnanotextured Ti) as examined by XPS.

Substrates O Ti F

Conventional Ti 51.23 48.77 0Nanorough Ti 49.34 50.66 0Nanotubular Ti 57.00 33.62 9.38Nanotextured Ti 56.27 34.95 6.03

Fig. 2. Total surface energy of the Ti before and after electron beam evaporation(nanorough Ti) and anodization (nanotubular and nanotextured Ti). Surface energywas calculated for each sample by measuring the contact angle of three liquids at thesample surface and entering values into the OwensWendt equation. Values are

S.D. Puckett et al. / Biomaterials 31 (2010) 706713 709outermost level, due to the anodization process involving the use ofHF (Table 2). In particular, nanotubular Ti contained a higherpercentage of uorine compared to the nanotextured Ti surfaces(Table 2).

XRD spectra (data not shown) conrmed the presence ofamorphous titania (no anatase or rutile phasewas observed) for thenanotextured and nanotubular Ti substrates. XRD spectra alsoconrmed the presence of crystalline titania for the nanorough andconventional Ti substrates. More specically, the conventional Ticontained rutile TiO2 and no anatase TiO2, while the nanorough Ticontained anatase TiO2 and no rutile TiO2.

3.2. Surface energy and contact angles

Surface energy calculations from contact angle data indicatedthat increasing surface roughness increased surface energy.All nanofabricated surfaces (nanorough, nanotextured, and nano-tubular Ti) had a surface energy signicantly higher than that ofunmodied, conventional Ti surfaces (Fig. 2). In addition, thenanofabricated surfaces had a lower contact angle for each liquidused to determine surface energy (Table 3), indicating increasedsurface energy for such samples compared to the unmodied Ti.Interestingly, among the nanofabricated surfaces, nanotexturedand nanotubular Ti had much lower contact angles for each liquidused to determine surface energy compared to the nanorough Ti(Table 3), indicating that nanotextured and nanotubular Ti surfaceshad the greatest surface energy of the substrates created in thisstudy.

3.3. Bacterial adhesion

Fig. 3 shows the total bacteria colonies, including both live anddead bacteria, attached after 1 h on the substrates of interest to thisstudy. The results of this study revealed that bacteria adhered theleast to the nanorough Ti substrates. More specically, whennormalized to the projected surface area, there was a signicantlylower attachment of colonies for all bacteria lines (S. aureus,S. epidermidis, and P. aeruginosa) on the nanorough Ti substrates

Table 1Binding energy of the high resolution Ti 2p peaks for Ti before and after electronbeam evaporation (nanorough Ti) and anodization (nanotubular and nanotexturedTi) as examined by XPS.

Substrates Peak BindingEnergy (ev)

Conventional Ti Ti 2p3/2 458.8Ti 2p1/2 464.5

Nanorough Ti Ti 2p3/2 458.8Ti 2p1/2 464.5

Nanotubular Ti Ti 2p3/2 458.8Ti 2p1/2 464.5

Nanotextured Ti Ti 2p3/2 458.8

Ti 2p1/2 464.5rough Ti.Fig. 5(a) displays the number of live bacteria colonies present on

the surfaces after 1 h while Fig. 5(b) displays the number of deadbacteria colonies present on the surfaces after 1 h. Results indicatedthat nanorough Ti substrates had the least amount of living bacteriaafter 1 h. In other words, when normalized to the projected surfacearea, there was a signicantly lower attachment of live bacteriacolonies for all strains (S. aureus, S. epidermidis, and P. aeruginosa)on the nanorough Ti substrates compared to conventional, nano-tubular, and nanotextured Ti substrates (Fig. 5(a)). In addition,results showed that the nanotubular and nanotextured Tisubstrates hadmore live colonies for each bacteria line compared toconventional Ti substrates (Fig. 5(a)). Furthermore, upon examiningthe amount of dead bacteria present on the surfaces, nanotubularand nanotextured Ti substrates contained the greatest number ofdead bacteria colonies for all bacteria lines, while the nanorough Timean SEM; n 4; *p< 0.01 compared to unmodied Ti; **p< 0.01 compared tonanorough Ti; ***p< 0.05 compared to nanotextured Ti.

substrates contained the least amount of dead bacteria colonies(Fig. 5(b)).

Fig. 6 shows the percentage of live bacteria, as calculated fromthe data provided in Fig. 5. The results indicated that the nanoroughTi substrates contained the signicantly highest percentage of livebacteria for all strains attached to the surface after 1 h (Fig. 6).When examining this data as well as the total number of bacterialcolonies (Fig. 3), it can be concluded that the nanorough Tisubstrates appeared to be the best surface for inhibiting bacteria

Table 3Contact angle measurements (deg) of three liquids on Ti before and after electronbeam evaporation (nanorough Ti) and anodization (nanotubular and nanotexturedTi). Contact angle data was used to determine surface energy via the OwensWendtequation. Values are mean SEM, n 4.

Substrates Contact angle ofDI water

Contact angleof glycerol

Contact angleof PEG

Conventional Ti 70.6 1.58 69.3 0.84 41.18 1.20Nanorough Ti 59.3 1.13 57.6 0.89 28.3 1.74Nanotubular Ti 26.5 2.40 21.9 1.32 11.1 0.80Nanotextured Ti 29.5 1.13 25.4 1.35 17.2 1.43

S.D. Puckett et al. / Biomater710leading to early failure of the device as well as preventing properintegration between the tissue and implant. Bacteria that arenanotextured, and nanotubular Ti) increased the adsorption ofbronectin compared to the conventional Ti surfaces (Fig. 7).Among the nanofabricated surfaces, nanotextured and nanotubularTi signicantly increased bronectin adsorption compared to thenanorough Ti (Fig. 7). Interestingly, bronectin adsorption corre-lated to the surface energy data with the greatest surface energysamples adsorbing the most bronectin (Fig. 2).

4. Discussion

Infection carries a signicant burden for orthopedic devices bycompared to the conventional, nanotubular, and nanotextured Tisubstrates.

3.4. Protein adsorption

Fig. 7 shows that all nanofabricated surfaces (nanorough,Fig. 3. Decreased S. aureus, S. epidermidis, and P. aeruginosa colonies on nanorough andconventional Ti compared to nanotubular and nanotextured Ti after 1 h. Data aremean SEM; n 3; *p< 0.01 compared to nanorough Ti; **p< 0.01 compared toconventional Ti; ***p< 0.01 compared to nanotextured Ti; #p< 0.1 compared tonanotextured Ti; ##p< 0.05 compared to nanotextured Ti for respective bacteria lines.surface bound versus planktonic are clinically more relevant tobiomaterial infection. Reducing bacteria adhesion has been previ-ously explored on implant surfaces, but needs to be furtheraddressed. Nanotechnology has specically been investigated forimproving the success of orthopedic implants. Surfaces containingfeatures in the nanometer regime have been shown to increase cellbehavior, such as osteoblasts, while simultaneously reducing theadhesion of bacteria. Yet, the role of nanotechnology as a potentialsolution for decreasing bacteria adhesion warrants further expla-nation. The purpose of this study was to explore a possible meansfor the reduction of bacteria attachment to the implant surfaces.

Results from this study indicated that the presently preparednanorough Ti surfaces are the best surfaces for inhibiting bacterialadhesion. Compared to conventional surfaces, nanostructuredmaterials have excellent biocompatibility properties due toenhanced protein interaction (including adsorption and confor-mation) resulting in improved cellular adhesion and tissue growth[2931]. It has been demonstrated here that there is a linear rela-tionship between nano-roughness, surface energy, and proteinadsorption. More specically, a surface that has more nanoroughfeatures possesses increased surface energy which leads to greaterprotein adsorption [2931]. This study also conrmed the samecorrelation as it revealed that nanorough, nanotubular, and nano-textured Ti possessed higher degrees of nanometer features, highersurface energy, and increased bronectin adsorption compared toconventional Ti. Furthermore, research has also shown thatincreased protein adsorption, such as bronectin, results indecreased bacteria attachment [33,34]. In the present study, thistrend was observed between the nanorough Ti, which promotedthe least amount of bacteria attachment, and conventional Ti.Compared to conventional Ti, nanorough Ti possessed no chemicaldifference, and, thus, the presence of nanometer features alone(higher surface energy) increased bronectin adsorption whichdecreased bacterial attachment.

Based on this thinking, decreased bacterial attachment wouldalso be expected for the nanotubular and nanotextured Tisubstrates since these surfaces had greater nanometer surfaceroughness, surface energy, and bronectin adsorption. However,increased bacteria attachment was observed on both the nano-tubular and nanotextured Ti compared to the nanorough andconventional Ti. It is possible that uorine present on the nano-tubular and nanotextured on Ti surfaces (Table 2) increasedbacterial adhesion compared to conventional and nanorough Tisurfaces. Further examining bacteria attachment on the anodizednanotubular surfaces revealed the highest bacteria attachmentcompared to the anodized nanotextured surfaces (Fig. 3), corre-lating well to the possible role of greater bacteria attachment withuorine concentration (Table 2). Other studies have conrmed thistrend that uorine present on a material surface increases bacterialadhesion [3538]. Specically, Katsikogianni and colleaguesexamined bacteria (S. epidermidis) function on polymers with andwithout uorine [38]. They showed that the polymers containinguorine increased bacteria attachment [38]. Li and colleaguesshowed that increasing the concentration of surface ions encour-aged the binding for both gram positive (Bacillus subtilis) and gramnegative (two P. aeruginosa strains, three Escherichia coli strains,and two Burkholderia cepacia strains) bacteria to glass or metaloxide surfaces [37]. This observationwas not affected by the surfacecharge or hydrophobicity/hydrophilicity of the bacteria surface[37]. These ndings may explain the results of this current studywhich demonstrated that nanotubular and nanotextured Tisurfaces containing uorine ions increased bacteria attachmentdespite observed increases in bronectin adsorption. It is alsointeresting to note that a previous study by Popat and colleagues

ials 31 (2010) 706713were able to decrease the adhesion of bacteria (S. epidermidis) on

aterS.D. Puckett et al. / Biomnanotubular Ti (prepared by an anodization similar to the processused in this study) compared to conventional counterparts, onlyafter loading antibiotics into the Ti nanotubes [39].

Although total bacteria adhered the most to the anodizednanotubular surfaces, this study also revealed that the anodizedsurfaces (nanotubular and nanotextured Ti) decreased thepercentage of living cells compared to the non-uorinated surfaces(nanorough and conventional Ti). This could be a result of theantibacterial effects caused by the presence of uorine, as shown byother studies [4042]. For example, Raulio and colleagues foundthat by coating stainless steel with uoropolymers, it was possibleto reduce biolm formation of several bacteria strains, includingS. epidermidis [41]. In addition, Yoshinari and colleagues founduorine ion-implanted Ti surfaces contained fewer viable bacteriacolony forming units further suggesting antibacterial properties ofuorine. This study demonstrated that uorine ions were notreleased suggesting that the formation of metal uoride bondswere sufcient for producing antibacterial effects.

Another reason for the observation of increased total bacteriacolonies on nanotubular and nanotextured Ti surfaces compared tonanorough and conventional Ti surfaces could be explained by the

Fig. 4. Fluorescent micrographs of decreased S. aureus colonies on (b) nanorough Ti compa(d) nanotubular Ti compared to (a) conventional Ti after 1 h. These micrographs were reprials 31 (2010) 706713 711large number of adherent dead bacteria (Fig. 5 (b)). Dead bacteriabound to a biomaterial surface can aid in the adhesion of subse-quent live bacteria [43,44]. Specically, dead or dying P. aeruginosacan release intracellular lectins to promote the adhesion of livingbacteria [43]. In a similar manner, S. aureus can release an inter-cellular protein upon death to enhance the adhesion to othermicroorganisms [44]. Clearly, the release of such compounds fromadherent dead bacteria may have promoted subsequent livebacterial adhesion in this study (Fig. 5(a)).

Furthermore, there was also a difference in the crystallinitybetween the anodized Ti surfaces, nanorough, and conventional Tithat can be linked to bacteria adherence. Nanotextured and nano-tubular Ti contained amorphous TiO2 while the nanorough andconventional surfaces contained crystalline TiO2 (anatase and rutilephase). Research has shown that amorphous TiO2 promotedbacteria attachment compared to anatase TiO2 (which is known topossess antibacterial properties [45,46]). Thus, despite the fact thatnanotubular and nanotextured Ti surfaces increased nanometersurface roughness, surface energy, and bronectin adsorption overconventional Ti, the presence of amorphous TiO2 may have alsoincreased bacterial attachment.

red to all other substrates and increased bacteria colonies on the (c) nanotextured andesentative of S. epidermidis and P. aeruginosa.

Fig. 5. Increased S. aureus, S. epidermidis, and P. aeruginosa live (a) and dead (b)colonies on nanotubular and nanotextured Ti compared to nanorough and conven-tional Ti after 1 h. Data are mean SEM; n 3; *p< 0.05 compared to nanorough Ti;**p< 0.01 compared to nanorough Ti; ***p< 0.01 compared to conventional Ti;#p< 0.05 compared to conventional Ti; ##p< 0.01 compared to nanotextured Ti;###p< 0.05 compared to nanotextured Ti for respective bacteria lines.

Fig. 6. The highest percentage of live bacteria colonies for S. aureus, S. epidermidis, andP. aeruginosa attached to the nanorough Ti surfaces after 1 h compared to theconventional, nanotextured, and nanotubular Ti surfaces. Data are mean SEM; n 3;*p< 0.1 compared to nanotextured Ti; **p< 0.01 compared to nanotextured Ti;***p< 0.05 compared to nanotubular Ti; #p< 0.05 compared to conventional Ti;##p< 0.01 compared to nanotubular Ti; ###p< 0.1 compared to nanotubular Ti;p< 0.1 compared to conventional Ti; p< 0.1 compared to nanotubular Ti forrespective bacteria lines.

S.D. Puckett et al. / Biomater7125. Conclusions

A simple means for the reduction of bacteria on and subsequentinfection of titanium using nanometer sized Ti surface features wasexplored here for orthopedic applications. In summary, results ofthis in vitro study demonstrated the decreased adhesion ofS. aureus, S. epidermidis, and P. aeruginosa (bacteria that limitorthopedic implant function and efcacy) on nanorough Ti surfacescreated through electron beam evaporation while nanotubular andnanorough Ti created through anodization resulted in an increaseof bacteria attachment. This research demonstrated that throughcareful selection of nanometer surface properties to increasebronectin adsorption, while maintaining favorable chemistry andcrystallinity (specically, anatase TiO2) it was possible to decreasebacteria adhesion. This study, thus, provided further knowledge tothe orthopedic eld on ways to reduce bacteria colonization,a prerequisite for infection, which should be investigated asa means to improve the longevity of orthopedic implants.

Fig. 7. Increased bronectin adsorption on nanorough, nanotubular, and nanotexturedTi compared to conventional Ti. Data are mean SEM; n 3; *p< 0.01 comparedto conventional Ti; **p< 0.1 compared to conventional Ti; ***p< 0.05 compared tonanorough Ti; #p< 0.01 compared to nanorough Ti; ##p< 0.1 compared tonanotextured Ti.

ials 31 (2010) 706713Acknowledgements

The authors wish to acknowledge the VA Pre-Doctoral Associ-ated Health Rehabilitation Research Fellowship Program and theDepartment of Veterans Affairs, RR & D, A3772C for funding alongwith the National Science Foundation Graduate K-12 TeachingFellowship. The authors also wish to acknowledge the Microelec-tronics Facility and the Leduc Bioimaging Facility at BrownUniversity as well as the University of Rhode Island Surface Char-acterization Laboratory. Lastly, the authors would like to thankMr. Anthony McCormick for his help with SEM.

Appendix

The full color images can be found in the online version, at doi:10.1016/j.biomaterials.2009.09.081.

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The relationship between the nanostructure of titanium surfaces and bacterial attachmentIntroductionMaterials and methods2.1. Titanium substratesElectron beam evaporationAnodizationSurface characterizationSurface energy and contact anglesBacteria cultureBacteria adhesionFibronectin adsorption (ELISA)

ResultsSurface characterizationSurface energy and contact anglesBacterial adhesionProtein adsorption

DiscussionConclusionsAcknowledgementsAppendixReferences