Materials Science and Engineering C 49 (2015) 201–209
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Silver doped titanium oxide–PDMS hybrid coating inhibits Staphylococcus aureus and Staphylococcus epidermidis growth on PEEK Nhiem Tran a,b, Michael N. Kelley a,b, Phong A. Tran a,b, Dioscaris R. Garcia a,b, John D. Jarrell a,c, Roman A. Hayda a,b, Christopher T. Born a,b,c,⁎ a b c
Department of Orthopaedic Surgery, Alpert Medical School, Brown University, Providence, RI, USA Weiss Center for Orthopaedic Trauma Research, Rhode Island Hospital, Providence, RI, USA BioIntraface Inc., North Kingstown, RI, USA
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 18 November 2014 Accepted 20 December 2014 Available online 23 December 2014 Keywords: Coating Antibacterial Silver PEEK Biofilm
a b s t r a c t Bacterial infection remains one of the most serious issues affecting the successful installation and retention of orthopedic implants. Many bacteria develop resistance to current antibiotics, which complicates or prevents traditional antibiotic-dependent eradication therapy. In this study, a hybrid coating of titanium dioxide and polydimethylsiloxane (PDMS) was synthesized to regulate the release of silver. The coatings were benefited from the antimicrobial activity of silver ion, the biocompatibility of titanium dioxide, and the flexibility of the polymer. Three studied silver doped coatings with different titanium dioxide–PDMS ratios effectively inhibited the attachment and growth of Staphylococcus aureus and Staphylococcus epidermidis in a dose-dependent manner. The coatings were successfully applied on the discs of polyether ether ketone (PEEK), a common spinal implant material and antibacterial property of these coatings was assessed via Kirby Bauer assay. More importantly, these selected coatings completely inhibited biofilm formation. The release study demonstrated that the release rate of silver from the coating depended on doping levels and also the ratios of titanium dioxide and PDMS. This result is crucial for designing coatings with desired silver release rate on PEEK materials for antimicrobial applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bacterial infection during and after implantation of a medical device is a common complication in reparative orthopedic surgeries. Nosocomial infections have been calculated to cost $11 billion in the US per year, with 45% of these infections developing from device related implants [1]. Specifically, the gram positive bacteria Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are the leading causes of infection associated with implanted medical devices (IMDs) [2]. These infections are difficult to treat because of the formation of a biofilm, a highly organized multilayered structure of bacteria and their secreted polysaccharide material, which protects the bacteria from antibiotics and foreign body responses [3]. The presence of a biofilm requires removal of the implant to eliminate the infection, thereby prolonging the recovering period of the patient. Current therapeutic approaches aim to inhibit initial bacterial adherence and proliferation, reducing the capacity of the bacteria to form biofilms in the first place. One potential approach is loading antibiotics
⁎ Corresponding author at: Department of Orthopaedic Surgery, Brown University, Suite 200, 2 Dudley Street, Providence, RI 02905, USA. E-mail address: [email protected] (C.T. Born).
http://dx.doi.org/10.1016/j.msec.2014.12.072 0928-4931/© 2014 Elsevier B.V. All rights reserved.
into biodegradable polymers and inorganic compositions that leach out of the coating as the materials degrade. Examples of this approach include the loading of gentamycin into titanium nanotubes [4], vancomycin doped poly(glycolic-lactic) acid [5], and calcium phosphate/hydroxyapatite scaffolds [6]. Similarly, sol–gel films can be loaded to display zero order release for up to two weeks [7]. However, the excessive use of traditional antibiotic drugs can lead to bacterial resistance, a consequence that has greatly reduced the widespread adoption of antibiotic drug based coatings on orthopedic medical implants. Some polymers have also been developed to decrease bacterial adherence. Coatings of silk sericin immobilized poly(methylacrylic) acid, a hydrophilic polymer, has been shown to greatly reduce the adhesion of S. aureus and S. epidermidis on titanium surfaces [8]. Another agent that has been shown to reduce the adhesion of bacteria is nitric oxide (NO), which has also been incorporated to be released from films created by sol–gel methods [9,10]. NO is a molecule that is commonly found in many bodily responses, including phagocytosis and wound healing which is the posited reason for its efficacy as a bactericidal agent [11]. Recently, there has been an increasing interest in using inorganic metallic agents, such as copper, zinc, and silver ions to kill bacteria [12,13]. These inorganic metallic agents (silver in particular), show great promise for bactericidal use because they have been shown to be effective against both gram positive and negative bacteria [14].
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There are many suggested mechanisms for the bactericidal activity of silver [15–18]. Silver ions bind to thiol groups [19] and can also catalyze the formation of a disulfide bond [20]. These linkages can change the shape of the protein and render it inactive. This is particularly important when proteins involved in chemiosmotic energy generation are affected because the cell is deprived of energy. Additionally, silver has been hypothesized to disrupt the base pairing within a DNA molecule, thereby denaturing it [19]. Yamanaka et al. have suggested that the bactericidal activity of silver actually stems from its association with bacterial ribosomes and synthesis inhibition of energy generating proteins [21]. In this study, a hybrid coating of titanium oxide and polydimethylsiloxane (PDMS) doped with metal-organic derived silver is investigated. The hybrid coating combines the advantages of polymer (e.g., flexibility), metal oxide (e.g., bioactivity, biocompatibility), and silver ion (antimicrobial activity). The coating is applied on polyether ether ketone (PEEK), a polymer which has gained popularity as a spinal and trauma implant material [22]. PEEK exhibits many desirable properties such as formability, mechanical strength, and biocompatibility but lacks the bioactivity found in titanium based implants [23]. Therefore, a bioactive coating which is conducive to implant integration and prevents infection of PEEK is highly desirable.
2. Materials and methods 2.1. Silver doped titanium oxide/PDMS hybrid coating synthesis Titanium oxide/PDMS hybrids doped with silver were prepared using a patented metal-organic synthesis method (BioIntraface®, North Kingstown, RI, USA). Briefly, titanium precursor and PDMS precursor were prepared by diluting titanium isopropoxide (Sigma-Aldrich, St. Louis, MO, USA) in isopropanol (10% v/v) and PDMS (Nusil Technology, Carpinteria, CA, USA) in a hexane/isopropanol (70/30) mixture (10% v/v). The titanium precursor and PDMS precursor were combined at three different volume ratios, 50:50, 75:25 and 95:5. These ratios were chosen according to our previous studies showing that the hybrid coating improved osteoblast and fibroblast cell growth at this range of concentrations [24,25]. In a glass vial, 1.4 mL of the combined solutions was diluted by 9.8 mL of isopropanol. Silver neodecanoate (25% in xylene, Gelest, Morrisville, PA, USA) was then added to the mixture with increasing doping levels of silver to evaluate its antibacterial properties against S. aureus and S. epidermidis (Table 1). A coating solution of only silver neodecanoate was used as control and noted as Ag 100%. 20 μL of the hybrid mixtures were dispensed into each well of 96-well plates. The plates were dried overnight and heat treated on a hot plate at 95 °C for 1 h. Polystyrene (PS) wells without any coating were also used as controls.
2.2. S. aureus and S. epidermidis culture Cultures of S. aureus and S. epidermidis were obtained frozen from the American Type Culture Collection (ATCC 25923 and ATCC 35894, respectively, ATCC, Manassas, VA, USA). The bacteria were thawed on ice for 20 min before being placed on agar plates. The plates were incubated at 37 °C for 24 h. A single colony of bacteria was selected using a 10 μL loop (Sigma, St. Louis, MO) and inoculated into centrifuge tubes containing 5 mL of tryptic soy broth (TSB). Bacteria in centrifuge tubes were then incubated at 37 °C under agitation at 200 rpm for another 16 h. At that point, the bacteria solution was diluted in TSB to an optical density (OD) of 0.50 at 562 nm using a microplate reader (SPECTRAmax® PLUS 384, Molecular Devices Corporation, Sunnyvale, CA, USA). The amount of bacteria was 5 × 108 cfu/mL according to a pre-established correlation between OD562 and bacteria colony forming unit. Bacteria solutions were diluted another 100 folds with TSB before being used for further experiments.
2.3. S. aureus and S. epidermidis adhesion and growth on silver doped hybrid coated surfaces To investigate the levels of silver needed to inhibit S. aureus and S. epidermidis growth, silver doped hybrids coated 96-well plates were prepared as described above. 200 μL of bacteria solution were put into each well at a density of 5 × 106 cfu/mL. The bacteria were allowed to adhere to the surface for 4 h before being rinsed with PBS and replaced with 250 μL of new media. After 24 h of incubation at 37 °C, 200 μL of the bacteria solution was transferred to a new 96-well plate to avoid the influence of the coatings on measured optical density. Bacterial solutions were then analyzed using a spectrophotometer (SPECTRAmax® PLUS 384, Molecular Devices Corporation, Sunnyvale, CA, USA) at OD 562. Bacteria densities were normalized to OD of non-coated polystyrene control substrates. Wells coated with 100% Ag solutions were used as positive control.
2.4. Hybrid coated PEEK preparation and characterization Six coating solutions were chosen and applied to PEEK discs (Table 1). Specifically, coating solutions with three titanium dioxide: PDMS ratios with two silver doping levels (38.4 μL and 384 μL) were prepared and named H50-38.4, H50-384, H75-38.4, H75-384, H9538.4, and H95-384. PEEK discs with 1 mm thick and 9 mm diameter were cleaned with ethanol and autoclaved. The discs were dip coated, dried overnight, and heat treated on a hot plate at 95 °C for 1 h. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle goniometry were used to examine the surface characteristics of the hybrid coated PEEK samples.
Table 1 The compositions of hybrid coatings doped with silver neodecanoate in the final 11.2 mL solution. Titanium precursor: PDMS precursor ratios (referred to as “H50”)
Titanium precursor: PDMS precursor ratios (referred to as “H75”)
Titanium precursor: PDMS precursor ratios (referred to as “H95”)
Volume of silver neodecanoate (μL)
Calculated silver concentration in coating solution (mM)
50:50 50:50 50:50 50:50 50:50 50:50 (H50-38.4) ⁎ 50:50 50:50 50:50 50:50 (H50-384) ⁎
75:25 75:25 75:25 75:25 75:25 75:25 (H75-38.4) ⁎ 75:25 75: 25 75:25 75:25 (H75-384)
95:5 95:5 95:5 95:5 95:5 95:5 (H95-38.4) ⁎ 95:5 95:5 95:5 95:5 (H95-384) ⁎
0 2 4 8 16 38.4 ⁎ 64 128 256 384 ⁎
0 0.15 0.3 0.6 1.2 2.88 4.8 9.6 19.2 28.8
⁎ Samples that were chosen for PEEK coating and release study in the later phases.
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of silver in each coating, and plotted against time. The rate of silver release was also calculated from the release amount and time.
2.8. Statistical analysis All statistical analysis was performed using Student's t-test to determine significance (p b .05) with at least 3 samples per test (N = 3).
3. Results 3.1. Dose response
Fig. 1. Dose response of S. aureus growth after 24 h with respect to amount of silver doping in the hybrid coatings. Data = Mean ± SD. N = 3.
2.5. Kirby–Bauer tests To study antibacterial property of coated PEEK discs, S. aureus and S. epidermidis at 5 × 106 cfu/mL was streaked onto tryptic soy agar plates. Nine mm diameter hybrid coated PEEK discs were placed onto the seeded agar plates and incubated for 24 h at 37 °C. The “kill-zone” around the samples where the bacteria do not grow depends on the amount of silver diffused out from the coating. The plates were photographed and analyzed using ImageJ software (NIH). For each kill zone, three difference measurements of diameter were recorded and averaged to account for any irregularities in the kill zone shape. 2.6. Biofilm growth on silver doped hybrid coatings To understand whether the silver doped coatings stops bacterial biofilm formation or not, S. aureus was seeded on PEEK samples at 5 × 106 cfu/mL and incubated for 4 h. The samples were then rinsed with PBS and placed into another well plate with fresh TSB and incubated for an additional 48 h. TSB for biofilm growth was pre-diluted three fold from original stock. After the incubation period, PEEK discs were rinsed with 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) buffer and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) buffer for 30 min. The samples were then dehydrated with 30%, 50%, 70%, 80% and 95% ethanol for 10 min each and 100% ethanol for 10 min (three times) and finally 100% ethanol for 40 min. The samples were then critically point dried in CO2 and sputter coated with Au/Pt before examination by SEM (LEO 1530, LEO Electron Microscopy Ltd, England). Uncoated PEEK discs were used as control.
S. aureus and S. epidermidis are two pathogenic bacteria commonly found in infected implanted medical devices. Therefore, in this study, the coatings were tested against these two bacteria. Bacteria were inoculated and allowed to adhere to the coatings before rinsing away of the non-adherent bacteria and replenished with fresh culture medium. The attached bacteria were then cultured for 24 h during which adherent bacteria continued to colonize the coating surface. Some bacteria are also released into the culture media where they propagate. Therefore, the bacteria in solution (planktonic bacteria) are in a pseudo equilibrium state with those adhered on the coatings and monitoring their concentration can provide a measurement of the ability of the coating to resist bacterial attachment and prevent bacteria growth. Fig. 1 shows clearly that increasing the amount of Ag in the coatings resulted in decreased optical density of S. aureus solution which indicated lower bacteria concentrations. All the coatings H50, H75, and H95 show very similar dose-dependent responses which identify the doping volume of 38.4 μL of Ag that completely inhibited bacteria growth. It can also be seen from Fig. 1 that different titanium dioxide to PDMS ratios have negligible effects on the dependence of growth inhibition of S. aureus on Ag content. The coatings without any silver still resulted in approximately 10–20% reduction in S. aureus growth compared to the polystyrene control (PS). A similar dose-dependent inhibition trend was observed in the results for S. epidermidis (Fig. 2). However, the results also indicated that S. epidermidis were more sensitive to the coating than S. aureus. The concentration at which growth was completely inhibited is 38.4 μL which is the same with S. aureus (Fig. 1). The TiO2 and PDMS compositions in the coatings did not significantly influence the inhibition of bacterial growth. The coatings without any silver still resulted in approximately 20% reduction in S. epidermidis growth compared to the polystyrene control (PS).
2.7. Release of silver from hybrid coatings To quantify the release of silver from the coatings, 100 μL of the hybrid solutions (H50-38.4, H50-384, H75-38.4, H75-384, H95-38.4, and H95-384) were applied to the bottom of glass scintillation vials. From silver concentration data presented in Table 1, the amount of silver in the coatings was calculated to be 0.03 mg and 0.3 mg for doping levels 38.4 and 384, respectively. Once the coatings were dried and heat treated, 10 mL of deionized water was added to each vial. At time points 2, 24, 48, 168, 336, 672, and 1008 h all of the solution in the vials were collected and replaced with fresh water. The silver concentration in the collected solutions was analyzed using a graphite furnace atomic absorption spectrometer (GFAAS). Accumulated silver released from the coatings were calculated from GFAAS data, normalized to total amount
Fig. 2. Dose response of S. epidermidis growth after 24 h with respect to amount of silver doping in the hybrid coatings. Data = Mean ± SD. N = 3.
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Fig. 3. XPS data of PEEK discs coated with H95-38.4 (a, b, c) and H95-384 (d, e, f). Full spectrum survey data (a, d) confirmed the presence of Ti, Ag, O, and C on coated surface. Multiplex scans (b, e) further identified phase of titanium as TiO2. Multiplex scans (c, f) also showed that on sample with lower Ag doping (H95-38.4), silver was in oxide form while on coating with higher Ag content (H95-384), the presence of elemental silver was more significant.
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The result indicated that highest loading of Ag neodecanoate was reduced to elemental Ag in our system (Fig. 3f). 3.3. Wetting properties of coated surfaces The hydrophobicity of coated surfaces was studied using contact angle measurement (Fig. 4). The original PEEK disc (control) was hydrophobic with water contact angle of 90°. The contact angles on coated PEEKs were all higher than that of the control sample, meaning coated surfaces were even more hydrophobic. Hydrophobicity also increased with higher level of silver doping. 3.4. Kirby Bauer test
Fig. 4. Water contact angle data of silver doped hybrid coated PEEK discs. Non-coated PEEK discs were used as control. *p b 0.05 compared to control, **p b 0.05 compared to sample with the same TiO2 to PDMS ratio but lower level of Ag doping. Data = Mean ± SD. N = 3.
3.2. PEEK coating characterization To investigate the surface chemistry of the coatings, X-ray photoelectron spectroscopy (XPS) was used. Representative data of H95 samples with two concentrations of Ag loading (38.4 μL and 384 μL) are shown in Fig. 3. A survey scans of the coating loaded with 38.4 μL silver solution showed peaks due to C, O, Ag and Ti (Fig. 3a). Peaks of C at 285 nm and O at 532 nm are expected from the PDMS in the coating. However, peaks of Si were not detected most likely due to the low concentration of PDMS in this coating. High resolution multiplex scans were used to identify positions of Ti 2p and Ag 3d peaks in the H95-38.4 coating and provided information about oxidation state of Ti and Ag (Fig. 3b, c). The positions of Ti 2p1/2 and 2p3/2 peaks at 464.45 eV and 458.60 eV, respectively, suggest that Ti is in the form of TiO2 [26]. The formation of TiO2 was expected as a result of the hydrolysis followed by condensation of titanium isopropoxide in the synthesis mixture when contacting with moisture in the air during air-drying. The position of Ag3d5/2 at binding energy of 367.6 eV indicated the presence of silver oxide [26] which is expected from the reduction of silver neodecanoate in the synthesis solution by agents such as isopropanol and hexanes. Fig. 3d, e and f show XPS spectrum of the coating with the same titanium precursor to PDMS ratio but with higher doping level of Ag (H95384). It can be clearly seen from these graphs that the intensity of Ag peaks had increased confirming the higher Ag loading. The silver peak position also shows the presence of elemental silver on coated surface.
The Kirby Bauer test is a standard method in microbiology to evaluate antimicrobial activity of a compound. This method relies on measuring the diameter of the “inhibition zones” surrounding discs coated with the compound where there is no growth of bacteria due to the diffusion of the compound from the discs. In our study, the diameter was measured at three different locations and averaged to account for the non-uniformity of the inhibition zones. The results show that higher Ag loading (H50-384, H75-384, and H95-384) resulted in a significant increase in inhibition zone diameter, indicating higher antimicrobial activity against both types of bacteria tested (Fig. 5). For coating loaded with lower amount of silver (H50-38.4, H75-38.4, and H95-38.4), minimal inhibition zones were observed indicating reduced antimicrobial activities on these coatings. These results suggest that higher Ag loading had led to increased amounts of Ag diffused out from the coatings. Therefore, the dose-dependent growth inhibition observed in Figs. 1 and 2 are likely the result of a combination of reduced attachment on the coating and bactericidal activity from the increased release of Ag. Inhibition zones were similar for both S. aureus and S. epidermidis. 3.5. Biofilm growth on silver doped coated PEEKs One of the reasons why infections are difficult to treat is because of the ability of bacteria to form dense biofilms which prevent the penetration of antibiotics. To test the effectiveness of the hybrid coatings against the formation of such biofilm, the coatings were applied on PEEK discs. S. aureus were allowed to adhere to surface and biofilm formation was evaluated using SEM. Biofilm formations on these substrates are presented in Fig. 6. As expected, on non-coated control PEEK, a thick and dense biofilm was formed (Fig. 6d). On surfaces coated with the low level of Ag (H50-38.4, H75 38.4 and H95-38.4), smaller colonies of S. aureus were found, indicating biofilm formation was delayed on these samples (Fig. 6a, b, and c). However, on surfaces coated with the higher level of Ag (H50-384, H75 384 and H95-384), no colonies of bacteria were found (Fig. 6e, f, and g). The results suggested that higher
Fig. 5. Kirby–Bauer test results of hybrid coated PEEK discs. Inhibition zone diameter measurements for S. aureus and S. epidermidis show significantly larger inhibition zones for samples with higher doping of Ag. Non-coated PEEK discs with diameter of 9 mm (dotted line) were used as control. *p b 0.05 compared to sample with lower doping of Ag. Data = Mean ± SD. N = 3.
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Fig. 6. SEM images of S. aureus biofilm grown on silver doped hybrid coated PEEK discs. High resolution (scale bar 5 μm) and low resolution (scale bar 50 μm) images of tested samples H5038.4 (a), H75-38.4 (b), H95-38.4 (c), control (d), H50-384 (e), H75-384 (f), and H95-384 (g) are presented.
silver doped coatings completely prevent the formation of S. aureus biofilm on PEEK. 3.6. Release of silver from the hybrid coatings Release experiments were conducted to investigate the elution of Ag from the coatings to the surrounding aqueous environment. Coatings were immersed in de-ionized water which was replaced by fresh deionized water after the indicated time periods and analyzed using GFAAS. The results show that when the Ag loading was 384 μL in the coatings, high initial release of Ag was observed in all samples (H50-384,
H75-384 and H95-384) during the 150 h (Fig. 7a). 50% of loaded Ag or 150 μg Ag in the coating was quickly released during the first 200 h but then slowed down for the remainder of the test period. The longterm release for the coating with 50% titanium precursor (H50-384) was lower than that of the other two coatings (H75-384 and H95384). At the end of the experiment, about 65% of Ag was released from the H75-384 and H95-384 while 58% of Ag was released from the H50-384 coating. When the Ag loading was reduced to 38.4 μL, the release of Ag from all coatings was in a relatively linear fashion during the tested time periods (Fig. 7b). Release from the H95-38.4 coatings was lower than that
Fig. 7. Accumulated release of silver from hybrid coatings. Released amount was normalized to total amount of silver in the coating. Data = Mean ± SD. N = 3.
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Fig. 8. Averaged release rate of Ag from hybrid coatings. Data = Mean ± SD. N = 3.
from the other two coatings which have identical release profiles. At the end of the experiment, 10% of Ag (or 3 μg Ag) was released from H5038.4 and H75-38.4 coatings while 7% of Ag was released from the H95-38.4 coating. However, as seen from Figs. 1, 2 and 5, this difference in release did not cause observable differences in the results from bacteria tests. Compared with the coatings with lower Ag loadings, the amount of Ag released from the higher loading coatings is much larger (~2 orders of magnitude when taking into account the ~ 1 order of magnitude higher in the loading concentration). This significant increase in release is strongly correlated with the increase in kill zone diameter (Fig. 5). The release rates of Ag from coatings were calculated based on the release amount and release time periods (Fig. 8a). In samples loaded with higher amount of Ag (H50-384, H75-384, and H95-384), the difference in release rate after 24 h was minimal. The rate quickly dropped to around 2000 ng/h after 24 h and continued to decrease to about 25 ng/h after 1000 h. However, the rate of release during the first 2 h in sample with 50% titanium (H50) (10,000 ng/h) was significantly higher than that of the other two samples (H75-384, and H95-384) (5,500 ng/h). There were no statistical difference between release rate of H75-384 and H95-384 (~5,500 ng/h) during this first 2 h. For samples with lower amount of Ag (H50-38.4, H75-38.4, and H95-38.4), the release rates also dropped quickly after the first 2 h. The release rates stayed relatively stable at 2 ng/h until the end of the experiment. In contrast to samples with higher level of Ag, highest rate of release during the first 2 h was observed in sample with 75% TiO2 (HD75-38.4) at 85 ng/h. 4. Discussions Bacterial infections to implants have been one of the most serious issues for our orthopedic community. Common bacteria such as S. aureus and S. epidermidis can form biofilms on implant surface and avoid eradication by antibiotic treatments. Applying antimicrobial compound on an implant as a coating is a novel strategy to prevent bacterial adhesion and subsequent biofilm formation [27]. Many studies have shown the potential of antibiotic release coatings in combating bacterial infection [5,28,29]. For example, Neut et al. investigated the effectiveness of gentamycin-released coating on titanium [29]. Their study showed that the coatings inhibited the growth of both gentamycin-sensitive and gentamycin-resistant S. aureus strains and was effective for 4 days. Another recent study by Stewart et al. showed that vancomycin-modified plates inhibited biofilm formation by S. aureus but did not prevent healing of fractures in a sheep model [28]. However, with the increasing number of antibiotic resistant bacteria, alternative treatments using compound such as silver in our study is necessary [13]. The current study focused on hybrid silver-released coatings and the applicability of the coatings on a popular orthopedic implant material, PEEK. The coating combined the bioactivity of titanium oxide [30], the flexibility of polymer PDMS, and the antibacterial property of silver.
The coatings were formulated and characterized using XPS and water contact angle. However, it was challenging to measure the coating thickness. Previously, we have applied the coating on smooth stainless steel and measured the thickness of the coating using a laser scanning microscope. The result revealed that the coating was about 103 nm thick. On a rougher surface such as that of the PEEK discs, a reliable thickness measurement could not be obtained, even though it is expected that the average coating thickness would increase. Coatings with different metal oxide to polymer ratios showed similar antibacterial effects against both S. aureus and S. epidermidis. These coatings when applied on PEEK also showed inhibition of biofilm formation after 24 h of incubation. There was no significant difference in Ag content-dependent inhibition for the three different concentrations of TiO2 in the coatings. Therefore, we postulate two possibilities to account for this finding: (i) the release of Ag from the coatings was not significantly different for the three TiO2 concentrations tested here; or (ii) the difference in the release of Ag from the coatings with the three TiO2 concentrations did not cause significant difference in growth inhibition of bacteria. The results from silver release study showed that there are differences in Ag release profile of each coating in both short-term (under 24 h) and long-term. When considering short-term release (under 24 h), the coating with more polymer (i.e., H50-384) showed higher burst release compared to coatings with less polymer (H75-384 and H95-384). The initial burst release may be responsible for lower long-term accumulated release of H50-384 with only 58% of silver released after 1008 h compared to 65% total release from H75384 and H95-384. With the lower doping of Ag, the release profile changed with lowest long-term accumulated release from coating with the least polymer (H95-38.4). The release profile also indicated a more sustained-release with only 10% of Ag released after 1008 h. Additionally, the sustained release of silver suggested that the coatings were robust and adhered to PEEK discs after at least 1008 h. Our preliminary tape tests also confirmed this assumption. The results in this study suggest that accumulated release and release rate of silver from the coatings depend on the titanium and PDMS content of the coatings. Adjustment of these parameters can lead to a prolonged release of Ag at desired rate. It is important to note that the mechanical property and bioactivity of the coating is largely influenced by the metal oxide to PDMS ratios. Hybrid coatings of ceramic metal oxides such as TiO2, Nb2O5, or ZrO2 and polymers such as PDMS in this case can inherit the advantages of both materials, the bioactivity of ceramics and the flexibility of the polymer. Our previous studies also demonstrated that hybrid coatings enhanced cell growth and provided a more sustained release profile compared to pure metal oxide films or pure PDMS. [24,25,30–32] Via thorough optimization of metal oxide to PDMS ratios as well as level of silver doping, a bioactive orthopedic coating which inhibits bacterial biofilm growth and at the same time promotes osseointegration can be achieved. There is evidence in other studies which have shown the effects of surface hydrophobicity to bacterial adhesion and subsequent biofilm
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formation on surfaces. [33,34] It is generally accepted that bacteria adhere more extensively on hydrophilic surface [33,35]. However, different species with different surface tensions show variable levels of adhesion to surfaces. In this current study, all coated surfaces were much more hydrophobic than bare PEEK. Increases in water contact angles from the coatings were expected due to the presence of PDMS, which was more hydrophobic than PEEK, on the coated surfaces. Coatings with the same Ag doping levels have similar contact angles for all TiO2 concentrations tested. This result suggests that differences in TiO2: PDMS ratios do not influence initial wettability of coated surface. Higher Ag (i.e., H50-384, H75-384, and H95-384) loading resulted in a significant increase in contact angles. This is an interesting finding as Ag is a hydrophilic material with a water contact angle of ~ 60° on pure Ag surface [36]. However, it has been also recognized that surface roughness can increase water contact angles on a hydrophobic surface according to the Wenzel's theory [37]. It is possible that the higher Ag loading in these coatings resulted in rougher coating surface leading to more hydrophobic coatings. These hydrophobic surfaces might be one of the factors contributed to antibacterial effects of the coated samples. It is worth mention that the hydrophobicity of the coated surfaces did not prevent osteoblast and fibroblast cell growth. Previous results on hybrid coatings of niobium and zirconium with PDMS showed the formation of hydrophobic surfaces. [25,30,32] At certain compositions, cell proliferation on these hydrophobic surfaces was higher than that on hydrophilic pure metal oxides. Furthermore, with trauma application in mind, the coated implant will be removed eventually. Therefore, it is preferable to have a coating that does not prevent cell growth but also not overly encourage cell attachment. When a compound is released into the body, there have always been concerns with the potential for in vivo toxicity. Silver compounds, especially silver nanoparticles, have been identified to have inflammatory, oxidative, genotoxic, and possible cytotoxic effects [38–40]. Therefore, it is critical to establish a balance between antimicrobial property and potential toxicity. According to United States Environmental Protection Agency, a normal adult can take 5 mg/kg/day orally of silver daily for a prolonged time without apprehensive risk [41]. In our release study, the coated surface area was approximately 500 mm2 and the highest amount of silver in the coating was 0.3 mg (doped volume 384 μL). Assuming this coating is applied on a common orthopedic intramedullary implant with surface area of 5000 mm2, the total amount of silver on the nail is only 3 mg. Furthermore, this amount of silver is released gradually from the coating over a period of weeks. Therefore, even at the highest doping level used in this study, the amount of silver released would still be considered safe. 5. Conclusion In this study, novel titanium dioxide–PDMS hybrid coatings were formulated and doped with silver. The coatings showed the ability to inhibit the growth of both S. aureus and S. epidermidis in a dose-dependent manner with high potency. Moreover, the coatings were successfully applied on a commonly used orthopedic material, PEEK. Coated PEEK doped with the higher level of silver (H50-384, H75-384, and H95-384) showed large inhibition zones and completely prevented the formation of S. aureus biofilms indicating the successful release of ionic silver at bactericidal concentrations. The release study demonstrated that Ag was effectively eluted from the coatings. The amount and rate of release depended on the titanium to PDMS ratio and the doping level of Ag, which allows for customization of the pharmacokinetics of silver release. Since an antibacterial, anti-biofilm coating is highly desirable; these novel coatings will likely improve the treatments using implanted biomaterials. Disclosure John D. Jarrell is President of BioIntraface, Inc. and has stock ownership. Christopher T. Born is the head of the Clinical Advisory Board and
Chief Technical Officer (CTO) for BioIntraface, Inc. and has stock ownership. Roman A. Hayda is an unpaid consultant on the Advisory Board for BioIntraface, Inc. The other authors have no conflict of interests to declare.
Acknowledgments NT, PAT and DG have been supported by Stein/Bellet Postdoctoral Fellowship. The authors thank Mike Platek for XPS and Joseph Orchado for GFAAS technical supports.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Silver doped titanium oxide–PDMS hybrid coating inhibits Staphylococcus aureus and Staphylococcus epidermidis growth on PEEK Nhiem Tran a,b, Michael N. Kelley a,b, Phong A. Tran a,b, Dioscaris R. Garcia a,b, John D. Jarrell a,c, Roman A. Hayda a,b, Christopher T. Born a,b,c,⁎ a b c
Department of Orthopaedic Surgery, Alpert Medical School, Brown University, Providence, RI, USA Weiss Center for Orthopaedic Trauma Research, Rhode Island Hospital, Providence, RI, USA BioIntraface Inc., North Kingstown, RI, USA
a r t i c l e
i n f o
Article history: Received 16 July 2014 Received in revised form 18 November 2014 Accepted 20 December 2014 Available online 23 December 2014 Keywords: Coating Antibacterial Silver PEEK Biofilm
a b s t r a c t Bacterial infection remains one of the most serious issues affecting the successful installation and retention of orthopedic implants. Many bacteria develop resistance to current antibiotics, which complicates or prevents traditional antibiotic-dependent eradication therapy. In this study, a hybrid coating of titanium dioxide and polydimethylsiloxane (PDMS) was synthesized to regulate the release of silver. The coatings were benefited from the antimicrobial activity of silver ion, the biocompatibility of titanium dioxide, and the flexibility of the polymer. Three studied silver doped coatings with different titanium dioxide–PDMS ratios effectively inhibited the attachment and growth of Staphylococcus aureus and Staphylococcus epidermidis in a dose-dependent manner. The coatings were successfully applied on the discs of polyether ether ketone (PEEK), a common spinal implant material and antibacterial property of these coatings was assessed via Kirby Bauer assay. More importantly, these selected coatings completely inhibited biofilm formation. The release study demonstrated that the release rate of silver from the coating depended on doping levels and also the ratios of titanium dioxide and PDMS. This result is crucial for designing coatings with desired silver release rate on PEEK materials for antimicrobial applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bacterial infection during and after implantation of a medical device is a common complication in reparative orthopedic surgeries. Nosocomial infections have been calculated to cost $11 billion in the US per year, with 45% of these infections developing from device related implants [1]. Specifically, the gram positive bacteria Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are the leading causes of infection associated with implanted medical devices (IMDs) [2]. These infections are difficult to treat because of the formation of a biofilm, a highly organized multilayered structure of bacteria and their secreted polysaccharide material, which protects the bacteria from antibiotics and foreign body responses [3]. The presence of a biofilm requires removal of the implant to eliminate the infection, thereby prolonging the recovering period of the patient. Current therapeutic approaches aim to inhibit initial bacterial adherence and proliferation, reducing the capacity of the bacteria to form biofilms in the first place. One potential approach is loading antibiotics
⁎ Corresponding author at: Department of Orthopaedic Surgery, Brown University, Suite 200, 2 Dudley Street, Providence, RI 02905, USA. E-mail address: [email protected] (C.T. Born).
http://dx.doi.org/10.1016/j.msec.2014.12.072 0928-4931/© 2014 Elsevier B.V. All rights reserved.
into biodegradable polymers and inorganic compositions that leach out of the coating as the materials degrade. Examples of this approach include the loading of gentamycin into titanium nanotubes [4], vancomycin doped poly(glycolic-lactic) acid [5], and calcium phosphate/hydroxyapatite scaffolds [6]. Similarly, sol–gel films can be loaded to display zero order release for up to two weeks [7]. However, the excessive use of traditional antibiotic drugs can lead to bacterial resistance, a consequence that has greatly reduced the widespread adoption of antibiotic drug based coatings on orthopedic medical implants. Some polymers have also been developed to decrease bacterial adherence. Coatings of silk sericin immobilized poly(methylacrylic) acid, a hydrophilic polymer, has been shown to greatly reduce the adhesion of S. aureus and S. epidermidis on titanium surfaces [8]. Another agent that has been shown to reduce the adhesion of bacteria is nitric oxide (NO), which has also been incorporated to be released from films created by sol–gel methods [9,10]. NO is a molecule that is commonly found in many bodily responses, including phagocytosis and wound healing which is the posited reason for its efficacy as a bactericidal agent [11]. Recently, there has been an increasing interest in using inorganic metallic agents, such as copper, zinc, and silver ions to kill bacteria [12,13]. These inorganic metallic agents (silver in particular), show great promise for bactericidal use because they have been shown to be effective against both gram positive and negative bacteria [14].
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There are many suggested mechanisms for the bactericidal activity of silver [15–18]. Silver ions bind to thiol groups [19] and can also catalyze the formation of a disulfide bond [20]. These linkages can change the shape of the protein and render it inactive. This is particularly important when proteins involved in chemiosmotic energy generation are affected because the cell is deprived of energy. Additionally, silver has been hypothesized to disrupt the base pairing within a DNA molecule, thereby denaturing it [19]. Yamanaka et al. have suggested that the bactericidal activity of silver actually stems from its association with bacterial ribosomes and synthesis inhibition of energy generating proteins [21]. In this study, a hybrid coating of titanium oxide and polydimethylsiloxane (PDMS) doped with metal-organic derived silver is investigated. The hybrid coating combines the advantages of polymer (e.g., flexibility), metal oxide (e.g., bioactivity, biocompatibility), and silver ion (antimicrobial activity). The coating is applied on polyether ether ketone (PEEK), a polymer which has gained popularity as a spinal and trauma implant material [22]. PEEK exhibits many desirable properties such as formability, mechanical strength, and biocompatibility but lacks the bioactivity found in titanium based implants [23]. Therefore, a bioactive coating which is conducive to implant integration and prevents infection of PEEK is highly desirable.
2. Materials and methods 2.1. Silver doped titanium oxide/PDMS hybrid coating synthesis Titanium oxide/PDMS hybrids doped with silver were prepared using a patented metal-organic synthesis method (BioIntraface®, North Kingstown, RI, USA). Briefly, titanium precursor and PDMS precursor were prepared by diluting titanium isopropoxide (Sigma-Aldrich, St. Louis, MO, USA) in isopropanol (10% v/v) and PDMS (Nusil Technology, Carpinteria, CA, USA) in a hexane/isopropanol (70/30) mixture (10% v/v). The titanium precursor and PDMS precursor were combined at three different volume ratios, 50:50, 75:25 and 95:5. These ratios were chosen according to our previous studies showing that the hybrid coating improved osteoblast and fibroblast cell growth at this range of concentrations [24,25]. In a glass vial, 1.4 mL of the combined solutions was diluted by 9.8 mL of isopropanol. Silver neodecanoate (25% in xylene, Gelest, Morrisville, PA, USA) was then added to the mixture with increasing doping levels of silver to evaluate its antibacterial properties against S. aureus and S. epidermidis (Table 1). A coating solution of only silver neodecanoate was used as control and noted as Ag 100%. 20 μL of the hybrid mixtures were dispensed into each well of 96-well plates. The plates were dried overnight and heat treated on a hot plate at 95 °C for 1 h. Polystyrene (PS) wells without any coating were also used as controls.
2.2. S. aureus and S. epidermidis culture Cultures of S. aureus and S. epidermidis were obtained frozen from the American Type Culture Collection (ATCC 25923 and ATCC 35894, respectively, ATCC, Manassas, VA, USA). The bacteria were thawed on ice for 20 min before being placed on agar plates. The plates were incubated at 37 °C for 24 h. A single colony of bacteria was selected using a 10 μL loop (Sigma, St. Louis, MO) and inoculated into centrifuge tubes containing 5 mL of tryptic soy broth (TSB). Bacteria in centrifuge tubes were then incubated at 37 °C under agitation at 200 rpm for another 16 h. At that point, the bacteria solution was diluted in TSB to an optical density (OD) of 0.50 at 562 nm using a microplate reader (SPECTRAmax® PLUS 384, Molecular Devices Corporation, Sunnyvale, CA, USA). The amount of bacteria was 5 × 108 cfu/mL according to a pre-established correlation between OD562 and bacteria colony forming unit. Bacteria solutions were diluted another 100 folds with TSB before being used for further experiments.
2.3. S. aureus and S. epidermidis adhesion and growth on silver doped hybrid coated surfaces To investigate the levels of silver needed to inhibit S. aureus and S. epidermidis growth, silver doped hybrids coated 96-well plates were prepared as described above. 200 μL of bacteria solution were put into each well at a density of 5 × 106 cfu/mL. The bacteria were allowed to adhere to the surface for 4 h before being rinsed with PBS and replaced with 250 μL of new media. After 24 h of incubation at 37 °C, 200 μL of the bacteria solution was transferred to a new 96-well plate to avoid the influence of the coatings on measured optical density. Bacterial solutions were then analyzed using a spectrophotometer (SPECTRAmax® PLUS 384, Molecular Devices Corporation, Sunnyvale, CA, USA) at OD 562. Bacteria densities were normalized to OD of non-coated polystyrene control substrates. Wells coated with 100% Ag solutions were used as positive control.
2.4. Hybrid coated PEEK preparation and characterization Six coating solutions were chosen and applied to PEEK discs (Table 1). Specifically, coating solutions with three titanium dioxide: PDMS ratios with two silver doping levels (38.4 μL and 384 μL) were prepared and named H50-38.4, H50-384, H75-38.4, H75-384, H9538.4, and H95-384. PEEK discs with 1 mm thick and 9 mm diameter were cleaned with ethanol and autoclaved. The discs were dip coated, dried overnight, and heat treated on a hot plate at 95 °C for 1 h. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and contact angle goniometry were used to examine the surface characteristics of the hybrid coated PEEK samples.
Table 1 The compositions of hybrid coatings doped with silver neodecanoate in the final 11.2 mL solution. Titanium precursor: PDMS precursor ratios (referred to as “H50”)
Titanium precursor: PDMS precursor ratios (referred to as “H75”)
Titanium precursor: PDMS precursor ratios (referred to as “H95”)
Volume of silver neodecanoate (μL)
Calculated silver concentration in coating solution (mM)
50:50 50:50 50:50 50:50 50:50 50:50 (H50-38.4) ⁎ 50:50 50:50 50:50 50:50 (H50-384) ⁎
75:25 75:25 75:25 75:25 75:25 75:25 (H75-38.4) ⁎ 75:25 75: 25 75:25 75:25 (H75-384)
95:5 95:5 95:5 95:5 95:5 95:5 (H95-38.4) ⁎ 95:5 95:5 95:5 95:5 (H95-384) ⁎
0 2 4 8 16 38.4 ⁎ 64 128 256 384 ⁎
0 0.15 0.3 0.6 1.2 2.88 4.8 9.6 19.2 28.8
⁎ Samples that were chosen for PEEK coating and release study in the later phases.
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of silver in each coating, and plotted against time. The rate of silver release was also calculated from the release amount and time.
2.8. Statistical analysis All statistical analysis was performed using Student's t-test to determine significance (p b .05) with at least 3 samples per test (N = 3).
3. Results 3.1. Dose response
Fig. 1. Dose response of S. aureus growth after 24 h with respect to amount of silver doping in the hybrid coatings. Data = Mean ± SD. N = 3.
2.5. Kirby–Bauer tests To study antibacterial property of coated PEEK discs, S. aureus and S. epidermidis at 5 × 106 cfu/mL was streaked onto tryptic soy agar plates. Nine mm diameter hybrid coated PEEK discs were placed onto the seeded agar plates and incubated for 24 h at 37 °C. The “kill-zone” around the samples where the bacteria do not grow depends on the amount of silver diffused out from the coating. The plates were photographed and analyzed using ImageJ software (NIH). For each kill zone, three difference measurements of diameter were recorded and averaged to account for any irregularities in the kill zone shape. 2.6. Biofilm growth on silver doped hybrid coatings To understand whether the silver doped coatings stops bacterial biofilm formation or not, S. aureus was seeded on PEEK samples at 5 × 106 cfu/mL and incubated for 4 h. The samples were then rinsed with PBS and placed into another well plate with fresh TSB and incubated for an additional 48 h. TSB for biofilm growth was pre-diluted three fold from original stock. After the incubation period, PEEK discs were rinsed with 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) buffer and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate and 0.1 M sucrose (pH 7.4) buffer for 30 min. The samples were then dehydrated with 30%, 50%, 70%, 80% and 95% ethanol for 10 min each and 100% ethanol for 10 min (three times) and finally 100% ethanol for 40 min. The samples were then critically point dried in CO2 and sputter coated with Au/Pt before examination by SEM (LEO 1530, LEO Electron Microscopy Ltd, England). Uncoated PEEK discs were used as control.
S. aureus and S. epidermidis are two pathogenic bacteria commonly found in infected implanted medical devices. Therefore, in this study, the coatings were tested against these two bacteria. Bacteria were inoculated and allowed to adhere to the coatings before rinsing away of the non-adherent bacteria and replenished with fresh culture medium. The attached bacteria were then cultured for 24 h during which adherent bacteria continued to colonize the coating surface. Some bacteria are also released into the culture media where they propagate. Therefore, the bacteria in solution (planktonic bacteria) are in a pseudo equilibrium state with those adhered on the coatings and monitoring their concentration can provide a measurement of the ability of the coating to resist bacterial attachment and prevent bacteria growth. Fig. 1 shows clearly that increasing the amount of Ag in the coatings resulted in decreased optical density of S. aureus solution which indicated lower bacteria concentrations. All the coatings H50, H75, and H95 show very similar dose-dependent responses which identify the doping volume of 38.4 μL of Ag that completely inhibited bacteria growth. It can also be seen from Fig. 1 that different titanium dioxide to PDMS ratios have negligible effects on the dependence of growth inhibition of S. aureus on Ag content. The coatings without any silver still resulted in approximately 10–20% reduction in S. aureus growth compared to the polystyrene control (PS). A similar dose-dependent inhibition trend was observed in the results for S. epidermidis (Fig. 2). However, the results also indicated that S. epidermidis were more sensitive to the coating than S. aureus. The concentration at which growth was completely inhibited is 38.4 μL which is the same with S. aureus (Fig. 1). The TiO2 and PDMS compositions in the coatings did not significantly influence the inhibition of bacterial growth. The coatings without any silver still resulted in approximately 20% reduction in S. epidermidis growth compared to the polystyrene control (PS).
2.7. Release of silver from hybrid coatings To quantify the release of silver from the coatings, 100 μL of the hybrid solutions (H50-38.4, H50-384, H75-38.4, H75-384, H95-38.4, and H95-384) were applied to the bottom of glass scintillation vials. From silver concentration data presented in Table 1, the amount of silver in the coatings was calculated to be 0.03 mg and 0.3 mg for doping levels 38.4 and 384, respectively. Once the coatings were dried and heat treated, 10 mL of deionized water was added to each vial. At time points 2, 24, 48, 168, 336, 672, and 1008 h all of the solution in the vials were collected and replaced with fresh water. The silver concentration in the collected solutions was analyzed using a graphite furnace atomic absorption spectrometer (GFAAS). Accumulated silver released from the coatings were calculated from GFAAS data, normalized to total amount
Fig. 2. Dose response of S. epidermidis growth after 24 h with respect to amount of silver doping in the hybrid coatings. Data = Mean ± SD. N = 3.
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Fig. 3. XPS data of PEEK discs coated with H95-38.4 (a, b, c) and H95-384 (d, e, f). Full spectrum survey data (a, d) confirmed the presence of Ti, Ag, O, and C on coated surface. Multiplex scans (b, e) further identified phase of titanium as TiO2. Multiplex scans (c, f) also showed that on sample with lower Ag doping (H95-38.4), silver was in oxide form while on coating with higher Ag content (H95-384), the presence of elemental silver was more significant.
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The result indicated that highest loading of Ag neodecanoate was reduced to elemental Ag in our system (Fig. 3f). 3.3. Wetting properties of coated surfaces The hydrophobicity of coated surfaces was studied using contact angle measurement (Fig. 4). The original PEEK disc (control) was hydrophobic with water contact angle of 90°. The contact angles on coated PEEKs were all higher than that of the control sample, meaning coated surfaces were even more hydrophobic. Hydrophobicity also increased with higher level of silver doping. 3.4. Kirby Bauer test
Fig. 4. Water contact angle data of silver doped hybrid coated PEEK discs. Non-coated PEEK discs were used as control. *p b 0.05 compared to control, **p b 0.05 compared to sample with the same TiO2 to PDMS ratio but lower level of Ag doping. Data = Mean ± SD. N = 3.
3.2. PEEK coating characterization To investigate the surface chemistry of the coatings, X-ray photoelectron spectroscopy (XPS) was used. Representative data of H95 samples with two concentrations of Ag loading (38.4 μL and 384 μL) are shown in Fig. 3. A survey scans of the coating loaded with 38.4 μL silver solution showed peaks due to C, O, Ag and Ti (Fig. 3a). Peaks of C at 285 nm and O at 532 nm are expected from the PDMS in the coating. However, peaks of Si were not detected most likely due to the low concentration of PDMS in this coating. High resolution multiplex scans were used to identify positions of Ti 2p and Ag 3d peaks in the H95-38.4 coating and provided information about oxidation state of Ti and Ag (Fig. 3b, c). The positions of Ti 2p1/2 and 2p3/2 peaks at 464.45 eV and 458.60 eV, respectively, suggest that Ti is in the form of TiO2 [26]. The formation of TiO2 was expected as a result of the hydrolysis followed by condensation of titanium isopropoxide in the synthesis mixture when contacting with moisture in the air during air-drying. The position of Ag3d5/2 at binding energy of 367.6 eV indicated the presence of silver oxide [26] which is expected from the reduction of silver neodecanoate in the synthesis solution by agents such as isopropanol and hexanes. Fig. 3d, e and f show XPS spectrum of the coating with the same titanium precursor to PDMS ratio but with higher doping level of Ag (H95384). It can be clearly seen from these graphs that the intensity of Ag peaks had increased confirming the higher Ag loading. The silver peak position also shows the presence of elemental silver on coated surface.
The Kirby Bauer test is a standard method in microbiology to evaluate antimicrobial activity of a compound. This method relies on measuring the diameter of the “inhibition zones” surrounding discs coated with the compound where there is no growth of bacteria due to the diffusion of the compound from the discs. In our study, the diameter was measured at three different locations and averaged to account for the non-uniformity of the inhibition zones. The results show that higher Ag loading (H50-384, H75-384, and H95-384) resulted in a significant increase in inhibition zone diameter, indicating higher antimicrobial activity against both types of bacteria tested (Fig. 5). For coating loaded with lower amount of silver (H50-38.4, H75-38.4, and H95-38.4), minimal inhibition zones were observed indicating reduced antimicrobial activities on these coatings. These results suggest that higher Ag loading had led to increased amounts of Ag diffused out from the coatings. Therefore, the dose-dependent growth inhibition observed in Figs. 1 and 2 are likely the result of a combination of reduced attachment on the coating and bactericidal activity from the increased release of Ag. Inhibition zones were similar for both S. aureus and S. epidermidis. 3.5. Biofilm growth on silver doped coated PEEKs One of the reasons why infections are difficult to treat is because of the ability of bacteria to form dense biofilms which prevent the penetration of antibiotics. To test the effectiveness of the hybrid coatings against the formation of such biofilm, the coatings were applied on PEEK discs. S. aureus were allowed to adhere to surface and biofilm formation was evaluated using SEM. Biofilm formations on these substrates are presented in Fig. 6. As expected, on non-coated control PEEK, a thick and dense biofilm was formed (Fig. 6d). On surfaces coated with the low level of Ag (H50-38.4, H75 38.4 and H95-38.4), smaller colonies of S. aureus were found, indicating biofilm formation was delayed on these samples (Fig. 6a, b, and c). However, on surfaces coated with the higher level of Ag (H50-384, H75 384 and H95-384), no colonies of bacteria were found (Fig. 6e, f, and g). The results suggested that higher
Fig. 5. Kirby–Bauer test results of hybrid coated PEEK discs. Inhibition zone diameter measurements for S. aureus and S. epidermidis show significantly larger inhibition zones for samples with higher doping of Ag. Non-coated PEEK discs with diameter of 9 mm (dotted line) were used as control. *p b 0.05 compared to sample with lower doping of Ag. Data = Mean ± SD. N = 3.
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Fig. 6. SEM images of S. aureus biofilm grown on silver doped hybrid coated PEEK discs. High resolution (scale bar 5 μm) and low resolution (scale bar 50 μm) images of tested samples H5038.4 (a), H75-38.4 (b), H95-38.4 (c), control (d), H50-384 (e), H75-384 (f), and H95-384 (g) are presented.
silver doped coatings completely prevent the formation of S. aureus biofilm on PEEK. 3.6. Release of silver from the hybrid coatings Release experiments were conducted to investigate the elution of Ag from the coatings to the surrounding aqueous environment. Coatings were immersed in de-ionized water which was replaced by fresh deionized water after the indicated time periods and analyzed using GFAAS. The results show that when the Ag loading was 384 μL in the coatings, high initial release of Ag was observed in all samples (H50-384,
H75-384 and H95-384) during the 150 h (Fig. 7a). 50% of loaded Ag or 150 μg Ag in the coating was quickly released during the first 200 h but then slowed down for the remainder of the test period. The longterm release for the coating with 50% titanium precursor (H50-384) was lower than that of the other two coatings (H75-384 and H95384). At the end of the experiment, about 65% of Ag was released from the H75-384 and H95-384 while 58% of Ag was released from the H50-384 coating. When the Ag loading was reduced to 38.4 μL, the release of Ag from all coatings was in a relatively linear fashion during the tested time periods (Fig. 7b). Release from the H95-38.4 coatings was lower than that
Fig. 7. Accumulated release of silver from hybrid coatings. Released amount was normalized to total amount of silver in the coating. Data = Mean ± SD. N = 3.
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Fig. 8. Averaged release rate of Ag from hybrid coatings. Data = Mean ± SD. N = 3.
from the other two coatings which have identical release profiles. At the end of the experiment, 10% of Ag (or 3 μg Ag) was released from H5038.4 and H75-38.4 coatings while 7% of Ag was released from the H95-38.4 coating. However, as seen from Figs. 1, 2 and 5, this difference in release did not cause observable differences in the results from bacteria tests. Compared with the coatings with lower Ag loadings, the amount of Ag released from the higher loading coatings is much larger (~2 orders of magnitude when taking into account the ~ 1 order of magnitude higher in the loading concentration). This significant increase in release is strongly correlated with the increase in kill zone diameter (Fig. 5). The release rates of Ag from coatings were calculated based on the release amount and release time periods (Fig. 8a). In samples loaded with higher amount of Ag (H50-384, H75-384, and H95-384), the difference in release rate after 24 h was minimal. The rate quickly dropped to around 2000 ng/h after 24 h and continued to decrease to about 25 ng/h after 1000 h. However, the rate of release during the first 2 h in sample with 50% titanium (H50) (10,000 ng/h) was significantly higher than that of the other two samples (H75-384, and H95-384) (5,500 ng/h). There were no statistical difference between release rate of H75-384 and H95-384 (~5,500 ng/h) during this first 2 h. For samples with lower amount of Ag (H50-38.4, H75-38.4, and H95-38.4), the release rates also dropped quickly after the first 2 h. The release rates stayed relatively stable at 2 ng/h until the end of the experiment. In contrast to samples with higher level of Ag, highest rate of release during the first 2 h was observed in sample with 75% TiO2 (HD75-38.4) at 85 ng/h. 4. Discussions Bacterial infections to implants have been one of the most serious issues for our orthopedic community. Common bacteria such as S. aureus and S. epidermidis can form biofilms on implant surface and avoid eradication by antibiotic treatments. Applying antimicrobial compound on an implant as a coating is a novel strategy to prevent bacterial adhesion and subsequent biofilm formation [27]. Many studies have shown the potential of antibiotic release coatings in combating bacterial infection [5,28,29]. For example, Neut et al. investigated the effectiveness of gentamycin-released coating on titanium [29]. Their study showed that the coatings inhibited the growth of both gentamycin-sensitive and gentamycin-resistant S. aureus strains and was effective for 4 days. Another recent study by Stewart et al. showed that vancomycin-modified plates inhibited biofilm formation by S. aureus but did not prevent healing of fractures in a sheep model [28]. However, with the increasing number of antibiotic resistant bacteria, alternative treatments using compound such as silver in our study is necessary [13]. The current study focused on hybrid silver-released coatings and the applicability of the coatings on a popular orthopedic implant material, PEEK. The coating combined the bioactivity of titanium oxide [30], the flexibility of polymer PDMS, and the antibacterial property of silver.
The coatings were formulated and characterized using XPS and water contact angle. However, it was challenging to measure the coating thickness. Previously, we have applied the coating on smooth stainless steel and measured the thickness of the coating using a laser scanning microscope. The result revealed that the coating was about 103 nm thick. On a rougher surface such as that of the PEEK discs, a reliable thickness measurement could not be obtained, even though it is expected that the average coating thickness would increase. Coatings with different metal oxide to polymer ratios showed similar antibacterial effects against both S. aureus and S. epidermidis. These coatings when applied on PEEK also showed inhibition of biofilm formation after 24 h of incubation. There was no significant difference in Ag content-dependent inhibition for the three different concentrations of TiO2 in the coatings. Therefore, we postulate two possibilities to account for this finding: (i) the release of Ag from the coatings was not significantly different for the three TiO2 concentrations tested here; or (ii) the difference in the release of Ag from the coatings with the three TiO2 concentrations did not cause significant difference in growth inhibition of bacteria. The results from silver release study showed that there are differences in Ag release profile of each coating in both short-term (under 24 h) and long-term. When considering short-term release (under 24 h), the coating with more polymer (i.e., H50-384) showed higher burst release compared to coatings with less polymer (H75-384 and H95-384). The initial burst release may be responsible for lower long-term accumulated release of H50-384 with only 58% of silver released after 1008 h compared to 65% total release from H75384 and H95-384. With the lower doping of Ag, the release profile changed with lowest long-term accumulated release from coating with the least polymer (H95-38.4). The release profile also indicated a more sustained-release with only 10% of Ag released after 1008 h. Additionally, the sustained release of silver suggested that the coatings were robust and adhered to PEEK discs after at least 1008 h. Our preliminary tape tests also confirmed this assumption. The results in this study suggest that accumulated release and release rate of silver from the coatings depend on the titanium and PDMS content of the coatings. Adjustment of these parameters can lead to a prolonged release of Ag at desired rate. It is important to note that the mechanical property and bioactivity of the coating is largely influenced by the metal oxide to PDMS ratios. Hybrid coatings of ceramic metal oxides such as TiO2, Nb2O5, or ZrO2 and polymers such as PDMS in this case can inherit the advantages of both materials, the bioactivity of ceramics and the flexibility of the polymer. Our previous studies also demonstrated that hybrid coatings enhanced cell growth and provided a more sustained release profile compared to pure metal oxide films or pure PDMS. [24,25,30–32] Via thorough optimization of metal oxide to PDMS ratios as well as level of silver doping, a bioactive orthopedic coating which inhibits bacterial biofilm growth and at the same time promotes osseointegration can be achieved. There is evidence in other studies which have shown the effects of surface hydrophobicity to bacterial adhesion and subsequent biofilm
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formation on surfaces. [33,34] It is generally accepted that bacteria adhere more extensively on hydrophilic surface [33,35]. However, different species with different surface tensions show variable levels of adhesion to surfaces. In this current study, all coated surfaces were much more hydrophobic than bare PEEK. Increases in water contact angles from the coatings were expected due to the presence of PDMS, which was more hydrophobic than PEEK, on the coated surfaces. Coatings with the same Ag doping levels have similar contact angles for all TiO2 concentrations tested. This result suggests that differences in TiO2: PDMS ratios do not influence initial wettability of coated surface. Higher Ag (i.e., H50-384, H75-384, and H95-384) loading resulted in a significant increase in contact angles. This is an interesting finding as Ag is a hydrophilic material with a water contact angle of ~ 60° on pure Ag surface [36]. However, it has been also recognized that surface roughness can increase water contact angles on a hydrophobic surface according to the Wenzel's theory [37]. It is possible that the higher Ag loading in these coatings resulted in rougher coating surface leading to more hydrophobic coatings. These hydrophobic surfaces might be one of the factors contributed to antibacterial effects of the coated samples. It is worth mention that the hydrophobicity of the coated surfaces did not prevent osteoblast and fibroblast cell growth. Previous results on hybrid coatings of niobium and zirconium with PDMS showed the formation of hydrophobic surfaces. [25,30,32] At certain compositions, cell proliferation on these hydrophobic surfaces was higher than that on hydrophilic pure metal oxides. Furthermore, with trauma application in mind, the coated implant will be removed eventually. Therefore, it is preferable to have a coating that does not prevent cell growth but also not overly encourage cell attachment. When a compound is released into the body, there have always been concerns with the potential for in vivo toxicity. Silver compounds, especially silver nanoparticles, have been identified to have inflammatory, oxidative, genotoxic, and possible cytotoxic effects [38–40]. Therefore, it is critical to establish a balance between antimicrobial property and potential toxicity. According to United States Environmental Protection Agency, a normal adult can take 5 mg/kg/day orally of silver daily for a prolonged time without apprehensive risk [41]. In our release study, the coated surface area was approximately 500 mm2 and the highest amount of silver in the coating was 0.3 mg (doped volume 384 μL). Assuming this coating is applied on a common orthopedic intramedullary implant with surface area of 5000 mm2, the total amount of silver on the nail is only 3 mg. Furthermore, this amount of silver is released gradually from the coating over a period of weeks. Therefore, even at the highest doping level used in this study, the amount of silver released would still be considered safe. 5. Conclusion In this study, novel titanium dioxide–PDMS hybrid coatings were formulated and doped with silver. The coatings showed the ability to inhibit the growth of both S. aureus and S. epidermidis in a dose-dependent manner with high potency. Moreover, the coatings were successfully applied on a commonly used orthopedic material, PEEK. Coated PEEK doped with the higher level of silver (H50-384, H75-384, and H95-384) showed large inhibition zones and completely prevented the formation of S. aureus biofilms indicating the successful release of ionic silver at bactericidal concentrations. The release study demonstrated that Ag was effectively eluted from the coatings. The amount and rate of release depended on the titanium to PDMS ratio and the doping level of Ag, which allows for customization of the pharmacokinetics of silver release. Since an antibacterial, anti-biofilm coating is highly desirable; these novel coatings will likely improve the treatments using implanted biomaterials. Disclosure John D. Jarrell is President of BioIntraface, Inc. and has stock ownership. Christopher T. Born is the head of the Clinical Advisory Board and
Chief Technical Officer (CTO) for BioIntraface, Inc. and has stock ownership. Roman A. Hayda is an unpaid consultant on the Advisory Board for BioIntraface, Inc. The other authors have no conflict of interests to declare.
Acknowledgments NT, PAT and DG have been supported by Stein/Bellet Postdoctoral Fellowship. The authors thank Mike Platek for XPS and Joseph Orchado for GFAAS technical supports.
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