-
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Keywords:TitaniumNanotopographyBacteriaAdhesionFibronectin
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array
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: [email protected] (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