mitigation of staphylococcus aureus‐mediated surgical site

7
www.advhealthmat.de www.MaterialsViews.com FULL PAPER © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 wileyonlinelibrary.com Adv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201100032 Mitigation of Staphylococcus aureus-Mediated Surgical Site Infections with IR Photoactivated TiO 2 coatings on Ti Implants Asem Aboelzahab, Abdul-Majeed Azad,* Shawn Dolan, and Vijay Goel DOI: 10.1002/adhm.201100032 A. Aboelzahab, V. Goel Department of Bioengineering The University of Toledo Toledo, OH 43606-3390, USA A.-M. Azad Chemical Engineering The University of Toledo Toledo, OH 43606-3390, USA E-mail: [email protected] S. Dolan Henkel Corporation Madison Heights, MI, 48071, USA V. Goel Department of Orthopedic Surgery Health Science Campus The University of Toledo Toledo, OH 43606-3390, USA Surgical site infections caused by methicillin-resistant and methicillin-susceptible Staphylococcus aureus (MRSA, MSSA) lead to patient hospitalization for an extended period coupled with concomitant hospitalization resources and cost. The detrimental effect resulting from the onset of these infections poses great health risks, leading to death in some instances. Titanium dioxide (TiO 2 ) is endowed with the unique capability of photoactivity which has been extensively exploited in antibacterial activities. It has been shown to be very effective in its bactericidal efficacy against infection-causing bacterial strains, namely, E. coli and S. aureus. In this study, the use of IR-photoactivated TiO 2 nanocoatings on titanium implants to mitigate the onset of surgical site infections is described. TiO 2 coatings were created on implantable materials by way of an aqueous plasma electrodeposition technique and were used to mitigate the harmful bacte- rial growth upon brief activation by an infrared (IR) laser source. The necrosis of S. aureus cells was found to exceed 90% within 30 min. following a 30s exposure of the titania-coated model implants (Ti mesh and plate). Promising potential of antibacterial coatings in mitigating surgical site infections has been shown. such as spinal tumors. [3] One study per- taining to the assessment of the cost and length of patient hospitalization found that SSI onset after surgery resulted in an average increase in cost up to $26,000 per patient, in addition to longer hospi- talization for 8 days. [4] These outstanding outcomes of SSIs make it increasingly important to focus attention on ways to mitigate the very onset of infection, fol- lowing the surgery. It is therefore, relevant and particularly important to explore new and radical strategies in lieu of traditional treatments via antibiotics that have been shown to fail in many cases to eradicate the infection buildup fully. [5–9] In this connection, systematic research was launched where the unique photoac- tive attributes of nanostructured titanium dioxide (titania, TiO 2 ) were examined. The bactericidal efficacy of infrared (IR) photo- activated TiO 2 -coated Ti plates and wires against E. coli has been reported previously, which demon- strated their effectiveness in causing quantitative cell necrosis in record time. [10] In another application, the antibacterial effect of IR photoactivated electrospun nanofibrillar titania has been demonstrated with bacterial necrosis exceeding 90%. [11,12] Other researchers have also reported the efficacy of titania powder and thin films in their cell necrosis potency against S. aureus and other infection-causing bacterial species upon activation by ultraviolet radiation (UV). [13–15] The photocatalytic properties of titanium dioxide were dis- covered by Akira Fujishima, [16] who showed the photo-induced cleavage of water molecules, known as the Honda-Fujishima effect, [17] on TiO 2 electrodes. Since then, photocatalysts based on titania have been extensively studied for the past 40 years, mainly for the destructions of organic compounds in water and in the environment. Titania is a semiconducting oxide with a bandgap of 3.2 eV. Upon illumination by light, the photoenergy generates an electron-hole pair which comes to the titania sur- face, as shown below: TiO 2 hν −→ e cb (TiO 2 ) + h + vb (TiO 2 ) The electron in the conduction band can reduce O 2 molecule and produce superoxide ions ( O 2 ), and the hole in the valence band can react with H 2 O and produce hydroxyl radical ( OH). 1. Introduction Surgical site infections (SSI) are the leading cause of increased patient hospitalization, and in some cases patient mortality. According to some studies, approximately 500,000 cases of patient infection occur per year in the United States alone. [1,2] These are exacerbated with the presence of other complications

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Mitigation of Staphylococcus aureus -Mediated Surgical Site Infections with IR Photoactivated TiO 2 coatings on Ti Implants

Asem Aboelzahab, Abdul-Majeed Azad,* Shawn Dolan, and Vijay Goel

Surgical site infections caused by methicillin-resistant and methicillin-susceptible Staphylococcus aureus (MRSA, MSSA) lead to patient hospitalization for an extended period coupled with concomitant hospitalization resources and cost. The detrimental effect resulting from the onset of these infections poses great health risks, leading to death in some instances. Titanium dioxide (TiO 2 ) is endowed with the unique capability of photoactivity which has been extensively exploited in antibacterial activities. It has been shown to be very effective in its bactericidal effi cacy against infection-causing bacterial strains, namely, E. coli and S. aureus . In this study, the use of IR-photoactivated TiO 2 nanocoatings on titanium implants to mitigate the onset of surgical site infections is described. TiO 2 coatings were created on implantable materials by way of an aqueous plasma electrodeposition technique and were used to mitigate the harmful bacte-rial growth upon brief activation by an infrared (IR) laser source. The necrosis of S. aureus cells was found to exceed 90% within 30 min. following a 30s exposure of the titania-coated model implants (Ti mesh and plate). Promising potential of antibacterial coatings in mitigating surgical site infections has been shown.

1. Introduction

Surgical site infections (SSI) are the leading cause of increased patient hospitalization, and in some cases patient mortality. According to some studies, approximately 500,000 cases of patient infection occur per year in the United States alone. [ 1 , 2 ] These are exacerbated with the presence of other complications

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheiAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201100032

DOI: 10.1002/adhm.201100032

A. Aboelzahab , V. Goel Department of BioengineeringThe University of ToledoToledo, OH 43606-3390, USA A.-M. Azad Chemical EngineeringThe University of ToledoToledo, OH 43606-3390, USAE-mail: [email protected] S. Dolan Henkel CorporationMadison Heights, MI, 48071, USA V. Goel Department of Orthopedic SurgeryHealth Science CampusThe University of ToledoToledo, OH 43606-3390, USA

such as spinal tumors. [ 3 ] One study per-taining to the assessment of the cost and length of patient hospitalization found that SSI onset after surgery resulted in an average increase in cost up to $26,000 per patient, in addition to longer hospi-talization for 8 days. [ 4 ] These outstanding outcomes of SSIs make it increasingly important to focus attention on ways to mitigate the very onset of infection, fol-lowing the surgery. It is therefore, relevant and particularly important to explore new and radical strategies in lieu of traditional treatments via antibiotics that have been shown to fail in many cases to eradicate the infection buildup fully. [ 5–9 ]

In this connection, systematic research was launched where the unique photoac-tive attributes of nanostructured titanium dioxide (titania, TiO 2 ) were examined. The bactericidal effi cacy of infrared (IR) photo-activated TiO 2 -coated Ti plates and wires

against E. coli has been reported previously, which demon-strated their effectiveness in causing quantitative cell necrosis in record time. [ 10 ] In another application, the antibacterial effect of IR photoactivated electrospun nanofi brillar titania has been demonstrated with bacterial necrosis exceeding 90%. [ 11 , 12 ] Other researchers have also reported the effi cacy of titania powder and thin fi lms in their cell necrosis potency against S. aureus and other infection-causing bacterial species upon activation by ultraviolet radiation (UV). [ 13–15 ]

The photocatalytic properties of titanium dioxide were dis-covered by Akira Fujishima, [ 16 ] who showed the photo-induced cleavage of water molecules, known as the Honda-Fujishima effect, [ 17 ] on TiO 2 electrodes. Since then, photocatalysts based on titania have been extensively studied for the past 40 years, mainly for the destructions of organic compounds in water and in the environment. Titania is a semiconducting oxide with a bandgap of 3.2 eV. Upon illumination by light, the photoenergy generates an electron-hole pair which comes to the titania sur-face, as shown below:

TiO2hν−→ e−

cb (Ti O2) + h+vb (Ti O2)

The electron in the conduction band can reduce O 2 molecule and produce superoxide ions ( O 2 − ), and the hole in the valence band can react with H 2 O and produce hydroxyl radical ( OH ).

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Both these reactive oxygen species (ROS) are short-lived but extremely potent. In addition, other ROS such as hydrogen peroxide and singlet oxygen radicals have also been detected. The reaction of ROS with organic compounds is extremely agile leading to their complete oxidation to carbon dioxide. The process of cell destruction in the case of living micro-organisms also involves this oxidation mechanism, where the photo-induced generation of these radical oxygen spe-cies (ROS) creates a hostile environment that mitigates bac-terial proliferation and survival, thereby inducing bacterial cell necrosis. [ 18 , 19 ] Killing of microbial cells in contact with a Pt-TiO 2 catalyst in water, upon illumination with near-UV light for 1 to 2 h was fi rst reported by Matsunaga et al. [ 20 ] It should be pointed out that in the absence of O 2 or other suitable elec-tron acceptor, no photocatalytic reaction is likely to occur, due to the equally rapid and deleterious process of electron-hole recombination. [ 21 ]

This paper describes the development of titania coating on Ti plates and mesh samples by using a patented aqueous plasma electro-deposition process. [22] Photoactivation using a hand-held IR laser was used to illustrate the advantage of shorter exposure time while obtaining comparable and in some case, increased cell necrosis as compared to far longer illumination by UV radiation. [ 10–15 ]

Although plates and other net-shaped confi gurations are more robust mechanically in cases where geometrically standard implants are preferred, wires and mesh give more fl exibility in those instances where reaching the intricate areas of injury poses diffi culty to the fi rm plates and rods. This fl ex-ible behavior is also of relevance in the use of shape memory alloys in conjunction with the bactericidal coatings being devel-oped in this research program.

2. Experimental Section

Titanium plates (of commercial purity 99.99%, Alfa-Aesar, MA) and titanium mesh (99% purity, Cleveland Wire Cloth & Manu-facturing Company, OH) were used as implant surrogates. The use of diverse materials also permitted a direct comparison between the nanostructured morphologies formed on each, as well as their corresponding bactericidal potency.

2.1. TiO 2 Coating on cp Ti Plates and Mesh

The two titanium implant surrogates were coated by Henkel Corporation (Madison Heights, MI, USA) using a patented aqueous plasma electrodeposition (PED) process. The method employs a pulsed DC voltage of 240V for 10 ms on and 30 ms off with a current density of 1500 A/m 2 , which creates a very adherent TiO 2 coating from the precursor solution on the sub-strate. In this case, the Ti substrates (plate or mesh) were used as the anode, onto which titanium dioxide deposits are created from solution; the coatings are cured in situ by the plasma glow on the surface. [ 22 ] By increasing the time of PED process, sequentially thicker titania coatings were obtained on the Ti substrates. The TiO 2 -coated samples were subsequently heated

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to 800 ° C for 4 h in static air at a ramp rate of 10 ° /min in both heating and cooling cycles.

Systematic structural and microstructural analysis of the samples was carried out using X-ray diffraction (XRD-PAN-alytical X’Pert Pro MPD) and scanning electron microscopy (Hitachi S-4800 UHR SEM). The quantitative elemental anal-ysis was accomplished by using the energy dispersive spectros-copy (EDS) attached to the SEM unit. Prior to SEM imaging, the sample surfaces were sputter-coated with a gold-palladium target for approximately 40s. While the coated plates were ana-lyzed with both XRD and SEM/EDS, the analysis on mesh sample was done by SEM/EDS alone because of the diffi culty of doing XRD on mesh confi gurations.

2.2. Bacterial Growth

For bacterial growth, two sub-culture fl asks with 150 mL of TSB superbroth (32g of tryptone, 20g of yeast extract and 5g of sodium chloride per 1L DI water, Fisher Scientifi c, Waltham, MA) each were inoculated with 1mL of S. aureus (NRS72, Sanger476; MSSA), which was taken from a previously purifi ed batch culture growth. A standard calibration curve was created with the aid of streaking of cell suspensions on TSB/agar to enu-merate cell concentration. The S. aureus cells were grown to sta-tionary phase, diluted to a concentration of 9.52 × 10 8 cells mL − 1 , and then divided into 1mL aliquots in 3mL capacity eppen-dorf tubes for individual experiments. Stock solutions were also stored in 1mL aliquots with the addition of 30% glycerol for future growth of more bacterial samples. All samples were stored at −80 ° C until use.

2.3. In-Situ Image Analysis

For analysis and imaging of cell necrosis of the bacterial sus-pension during experimentation, an Invitrogen Baclight Live/Dead Assay kit (Invitrogen, Carlsbad, CA) was used in addition to confocal microscopy to obtain real-time imaging of the bac-terial cells while in suspension with the titania-coated Ti sub-strates, upon being activated by the IR laser. This assay uses SYTO9 and propidium iodide stains, which were stored in 1 μ l aliquots for individual experimental use.

100 μ L of S. aureus suspension were diluted in ultra-pure water (with a conductivity of 5.5 × 10 − 8 S cm − 1 ) to a 1:3 v/v ratio to a volume of 300 μ L and cell concentration of 3.17 × 10 8 cells/ml, with SYTO9 and PI stains added in 1 μ L quantities, respec-tively. The suspension was set in the dark for 20 min. to com-plete staining. 10 μ L of the suspension was then mixed with 30 uL of ultra-pure water (to cause 4-fold dilution) and added to a 35 mm × 14 mm glass bottom microwell dish (No. 15 cover glass). This gave a fi nal concentration of 7.93 × 10 7 cells/ml for imaging.

A 1 cm × 1 cm TiO 2 -coated Ti specimen was fi rst activated by a handheld IR laser ( λ = 808 nm; power = 1 W) from www.freaklasers.com for 30s, then introduced to the bacterial sus-pension. This laser introduces much greater power than the IR fl ashlight used in previous studies. [ 10 , 12 ] The time-lapse imaging was recorded for 40 min. using a confocal multiphoton

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Figure 1 . SEM images of TiO 2 -coatings of increasing thickness (a = 1.3 μ m, b = 2.4 μ m, c = 3.7 μ m d = 5.0 μ m) on Ti plates; the samples were fi red at 800 ° C for 4 h after coating by PED process.

microscope system (Leica TCS SP5 MP, Leica Microsystems, Bannockburn, IL). Upon photoexcitation, the S. aureus cells stained with SYTO9 fl uoresce green and those stained with pro-pidium iodide fl uoresce red. The excitation/emission of SYTO9 and propidium iodide occur at 480/500 nm and 490/645 nm, respectively. The survival/necrosis rate of the microorganism was determined by using the imaging software called ImageJ. By post-confocal imaging, this software allows one to count the bacteria in order to determine the amount of live cells com-pared to total number of cells.

Figure 2 . XRD signatures of the coating on Ti plates after fi ring at 800 ° C for 4 h. The diffraction peaks predominantly belong to rutile titania with minor peaks conforming to the anatase (A) modifi cation.

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3. Results and Discussion

3.1. Surface Analysis of Henkel Coated Samples

The Henkel coatings of titania on the plates and mesh were created with different thicknesses. They were imaged by SEM and analyzed for their gross chemical composition by EDS; the TiO 2 -coated plates were also subjected to x-ray diffraction for phase identifi cation. Figure 1 shows the systematic evolution of the surface morphological features, with increasing thickness of titania coating on the plate samples.

As can be seen, with increasing fi lm thickness, the structure goes gradually from all high aspect (L/D) ratio 1-D fi bers, to a mixture of 1-D fi bers and 2-D platelets, to 2-D platelets and 3-D rods. Moreover, the surface densifi cation also increases; a thinner titania fi lm is endowed with more nanofeatures and more void space which may allow for higher and direct exposure of the bacterial species with the

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, WeinhAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201100032

photoactive surface. On the other hand, the microscale platelets and rods offer a larger surface area, which also could be benefi cial in the bacterial necrosis per unit area.

The XRD analysis showed the presence of TiO 2 in rutile phase in all the four samples, as can be seen from Figure 2 . The identical XRD signatures confi rm the reproducibility of the PED coating technique developed by Henkel and the uniformity in fi lm growth.

The morphological features of the titania fi lms deposited on Ti mesh are distinctly different from those on Ti plates, as seen in Figure 3 .

Figure 4 shows the systematic variation in the thickness of TiO 2 fi lm coated by PED process on the mesh samples. The fi lm thick-ness increases monotonically and almost lin-early, which is also corroborated by the SEM images of the successive fi lms, taken at same magnifi cation (inset).

A similar trend was observed in the case of plates as well, but is not shown here to avoid redundancy.

3.2. Evaluation of Bactericidal Effect of Photoactivated Titania-Coated Plates and Mesh

We have previously reported the fabrication of titania coatings by anodization, hydrothermal processing and the PED tech-nique. [ 10 ] Their bactericidal effi cacy against E. coli was also suc-cessfully demonstrated. Activation by IR laser ( λ = 808 nm) for 30 s proved effective in the necrosis of E. coli cells up to 40%. The photocatalytic response of free-standing titania in other for-mats (powder, nanofi bers, etc.) in the presence of UV radiations has also been reported. [ 10 , 12–15 ]

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Figure 3 . Morphology of a) pure Ti-mesh and b) Henkel TiO 2 -coated Ti mesh fi red at 800 ° C for 4 h; c) a higher magnifi cation image of coating (b).

Figure 4 . Variation in the thickness of TiO 2 coating on Ti mesh. Inset: SEM images of TiO 2 –Ti cross-sectional interface for sample with varying titania fi lm thickness (red arrow is a measure of the thickness of titania fi lm on the Ti substrate).

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The UV radiations ( λ = 365 nm) employed hitherto are of lower intensity though of larger photon energy (de Broglie’s equation of wave length-energy equivalence: E = h c/ λ ; where h is Planck’s constant, c the speed of light and λ the radiation wave length). Consequently, longer exposure time (reaching 60 min. to 2h in some cases) is needed to obtain signifi cant results. On the other hand, the IR laser source used by the present authors in this as well as previous work, is of larger wave lengths, and hence of smaller photon energy. However,

Figure 5 . Confocal images of the bacterial colonies at different times in S. aureus suspension exposed to a handheld IR laser ( λ = 808 nm) for 30 s (green/SYTO9 = live cells; red/PI = dead cells).

these laser beams are of much higher source intensity (5 W/cm 2 ). This effectively creates a larger population of incident photons per unit area and it is this parameter that functions as a deciding factor in the observed photoacti-vation-mediated effect in the cell necrosis. A larger photon fl ux (number per unit incident area) in the case of IR source as opposed to UV source is likely the reason for observed agile and more quantitative bactericidal activ-ities in relatively shorter span of time on the titania surfaces. A shorter exposure would limit the time the patient is exposed to radia-tions, thereby making the effectiveness of the technique more attractive and applicable for clinical applications. Furthermore, in the

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light of the fact that longer exposure to UV radiation (up to 60 min. or more) does not induce quantitative necrosis, repeated exposures would be needed. Under these conditions, one might reach dose levels that are harmful to human tissues.

This explanation corroborates the brief but effective use of an IR source and strengthens the following results obtained on the titania-coated plate and mesh samples; to the best of our knowledge no such data exists with a parallel study using UV activation by others.

Figure 5 shows the images of an S. aureus suspension not subjected to any TiO 2 or IR activation over an interval of 20 min.; with no cell necrosis occurring, this was used to serve as a control.

The titania-coated plate and mesh samples were evaluated for their antibacterial effi cacy against S. aureus (MSSA). Confocal imaging using SYTO9 and PI stains was employed to monitor the bacterial cells as a function of time-lapse subsequent to irra-diation of the sample and its immersion in the microbial bath. A Ti mesh coupon with a 1.5 μ m thick TiO 2 coating (sample #2) was subjected to IR activation for 30 s followed by immer-sion in a bacterial suspension of stained S. aureus cells. The specimen was placed so as to expose the bacteria to the photo-activated region of the mesh. Confocal imaging was recorded in real-time as the bactericidal effects of the titania coating took effect. Figure 6 illustrates the quick effect of the Henkel-coated mesh in causing S. aureus cell necrosis.

Within approximately 5 min. of exposure, only 23.71% of the S. aureus cells survived; the concentration of the surviving cells

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Figure 6 . Confocal images of the bacterial colonies at different times in S. aureus suspension con-taining Henkel TiO 2 -coated mesh samples (top to bottom) 2, 4, and 5 (fi red at 800 ° C/4 h) and exposed to a handheld IR laser ( λ = 808 nm) for 30 (green/SYTO9 = live cells; red/PI = dead cells).

Figure 7 . Confocal images of the bacterial colonies at different times in S. aureus suspension containing Henkel TiO 2 -coated plate sample (fi red at 800 ° C/4 h) and exposed to a handheld IR laser ( λ = 808 nm) for 30 s (green/SYTO9 = live cells; red/PI = dead cells).

was 9% after 30 min. The green pixels indi-cate live cells while the red pixels denote dead ones.

A note of clarifi cation with regard to the time stamp of 0 min. on the confocal images is warranted. The images marked as ‘0 min.’ were realistically taken a few moments after actual exposure to the titania-coated mesh due to the time elapsed between activation and placement in the suspension, before being placed inside the slide holder of the apparatus. An addi-tional amount of time also elapsed in posi-tioning the center of the microscopic lens on the activated area prior to imaging. This would explain the observed cell necrosis

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, WeinhAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201100032

already taken place at 0 min. in some of the images.

Figure 6 also shows the bactericidal effi -cacy of the mesh samples with titania coating thicknesses of 2.6 μ m (#4) and 3.2 μ m (#5), respectively. Although these mesh samples did cause evident cell necrosis, they were not found to be as effective as the sample with a smaller coating thickness.

The survival count after 5 min. of exposure was 50.7% for sample 4, which dropped down to 21.5% after a time-lapse of 30 min. In comparison to sample (#2) with a thinner titania coating, the cell sur-vival concentration was slightly more than twice on sample #4.

Increasing the coating thickness of the Ti mesh to 3.2 μ m (sample 5) did increase the bactericidal effi cacy as compared to Sample 4 by 10% after only 5 min., but the survival after 30 min. for this sample was approxi-mately 22.3%. This is slightly less effective than sample 4, but portrays relatively equiva-lent results.

The bactericidal effi cacy of Ti plates also was tested against S. aureus . It was found that coated mesh samples were more effec-tive because of their more immediate access to the bacterial suspension as is apparent by the structural difference between plates and mesh. Therefore, cell necrosis was apparently detected in one Henkel-coated plate sample of thickness 5 μ m, the results of which are seen in Figure 7 .

It should be noted here that placement of the plates among the bacterial suspension incorporated a further degree of diffi culty because of the dense and bulky nature of the material. It was necessary for the plate to be fully exposed to the S. aureus cells while not “crushing” the cells or distorting bacterial movement, which therefore leads to distortion of the fl uorescent imaging by

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Table 1. Summary of the effi cacy of the TiO 2 -coated mesh and plate samples activated by IR-laser source ( λ = 808 nm; power = 5 W cm − 2 ) for 30s against S. aureus . The data represent an average of 10 measure-ments with each of the mesh and plate samples and has an uncertainty of ± 2%

Material Titania fi lm thickness

Post-exposure cell survival [%] after a time-lapse of

5 min. 30 min.

Control (no exposure to TiO 2 /IR) - 100 100

TiO 2 -coated Ti mesh (2) 1.5 μ m 23.7 9.0

TiO 2 -coated Ti mesh (4) 2.6 μ m 50.7 21.5

TiO 2 -coated Ti mesh (5) 3.2 μ m 42.6 22.3

TiO 2 -coated Ti plate (4) 5.0 μ m 55.7 12.9

confocal microscopy. For this reason, bactericidal effi cacy of the Henkel-coated plates was not consistent throughout. Still, the results for Henkel-coated plate showed its successful anti-bacterial effect against S. aureus, indicating that only 55.7% of cells survived after 5 min.; the survival diminished to about 12.9% after 30 min. The data is summarized in Table 1 .

From the data sown in Table 1 , it can be concluded that titania-coated Ti mesh samples had an increased effi cacy towards cell necrosis as compared to Ti plates. This increased effect can be attributed to the ability of mesh to be more acces-sible to the bacterial suspension because of its porous struc-ture. In addition, thinner coatings were more effective in causing necrosis of the bacterial cells. Ti plates and mesh with increasing coating thicknesses caused slightly lower cell death after activation, though still comparable with the highest sur-vival count being around 20%.

At fi rst sight, these results might appear counterintuitive, but the increased effi cacy observed in this study can be explained as a combination of a number of factors related mainly to the structural artifacts of the coating. For example, the particle den-sity of TiO 2 is much greater in thinner coatings, thus allowing for more titania particles packing within a unit space. Second, there is a gradual but defi nite change in shape and particle dis-tribution between thin and thick coatings. This results in a cor-responding change in the aspect ratio (length to diameter; l/d ) as seen clearly from the SEM images in Figure 1 . The effec-tiveness of the thinner fi lm (with larger aspect ratio) is there-fore due to the availability of larger surface area on the thinner coating within the contour of limited activation area of the laser beam.

This behavior of titania coatings could be used for the implant activation in both pre and post-surgery situations. In post-surgery cases, this may simplify the reactivation step if the infection persists after surgery. Furthermore, IR radiations are capable of penetrating skin and other soft tissue, which might necessitate little or no surgical incision. Currently, the persistence of surgical site infection often requires re-opening and removal of implants for ex-vivo sterilization and even replacement in some cases. With a titania pre-coated implant in place, that could be activated and re-activated as required, the necessity of additional surgical procedures would poten-tially diminish.

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4. Conclusion

Ti mesh and plate coupons were coated with uniform TiO 2 fi lm using a patented aqueous plasma electrodeposition (PED) process. The bactericidal effi cacy of the coated samples was tested against S. aureus (MSSA) with cell necrosis of greater than 90% achieved after a 30 min. time-lapse following 30 s of IR activation.

In comparison to the bactericidal effi cacy of titania with UV illumination, activation by IR laser adopted in this study illus-trated the superiority of the method used to create the photoac-tive fi lms. The advantage of IR activation in terms of approxi-mately 90% cell death after only 30 s of exposure is outstanding. Cell necrosis rates less than 10% have been reported by other groups, and comparable results were obtained only after very long UV activation (cf. 60 min.).

Acknowledgements Image processing and analysis was carried out in the in Java–National Institute of Health (http.://rsbweb.nih.gov/ij/download.html). The authors wish to express their gratitude to Ms. Tamara Phares of the Bioengineering Department, Dr. Andrea Kalinoski of the Advanced Microscopy and Imaging Center (Department of Surgery, Health Science Campus), Mr. Robert Kinner of the Chemical Engineering Department and Dr. Kristen Williams of the Microbiology Department (Health Science Campus) for their assistance in various stages of this work. Financial support from DePuy Spine (Johnson & Johnson, Raynham, MA) is also gratefully acknowledged.

Received: February 13, 2012Published online:

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