Surface Hydrophilicity and Antifungal Properties of Ti[O.sub.2] Films Coated on a Co-Cr Substrate.
The demand for prosthetic treatment is of increasing tendency as a result of the aging issue globally [1, 2]. Among all available techniques, removable partial denture (RPD) is regarded as a simple and economic option to repair the missing teeth . Therefore, RPDs have been the commonly used treatment for edentulism to date. Co-Cr alloys have a wide range of applications as a material for metallic framework owing to their good mechanical properties as well as superior biocompatibility. However, it has been reported that yeasts and bacteria are prone to colonize on the fitting surface of the dentures forming biofilms [4,5]. These biofilms consequently cause damage to the oral mucosabelow, commonly associated with denture stomatitis (DS). DS affects up to two-thirds or even more of denture wearers, the majority of whom are asymptomatic and unaware of the problem [6-8].
The etiology of DS is multifactorial, among which the candida primarily C. albicans infection and poor denture hygiene have been widely accepted as critical risk factors [9-11]. Yoshijima et al.  have reported an association of hydrophobic surface with the colonization of C. albicans. It is therefore believed that a proper deactivation of the metallic fitting surface may alleviate the problem associated with biofilms. This is attributed to the increase of hydrophilicity and the antimicrobial properties relying on surface coating techniques. Recently, titanium dioxide (Ti[O.sub.2]) coating has drawn great attentions due to its photocatalytic, antimicrobial, and self-cleaning properties [13-17]. In addition, Ti[O.sub.2] films have shown enhanced biocompatibilities and good corrosion resistance of the substrate [18,19]. In the published studies, Ti[O.sub.2] thin film has been applied to denture base acrylic resin, resulting in increased hydrophilic properties and decreased attachment of food bolus accumulation, as well as an inhibitory effect on adhesion of microorganisms [20, 21]. However, to the best of our knowledge, the study of Ti[O.sub.2] film on Co-Cr alloy frameworks is still not available, since Ti[O.sub.2] films growing on a Co-Cr alloy surface with good bonding strength are challenging, restricting their applications so far.
A large magnitude of techniques, including sol-gel processes, chemical vapor deposition, electrophoretic deposition, plasma immersion ion implantation, plasma spray, and atomic layer deposition (ALD), have been made available for depositing Ti[O.sub.2] films on a variety of surfaces [22-25]. Among the aforementioned techniques, ALD has been widely applied due to its figure of merits including high-quality and largearea flat coating, perfect structure, and process controllability. Although significant efforts have been devoted to depositing Ti[O.sub.2] films on various substrates, such as silicon and metallic and polymer materials [23, 26, 27], there is little information in the literature with regard to the application of ALD in dentistry. Therefore, this study was innovatively designed to deposit a thin layer of Ti[O.sub.2] on a Co-Cr substrate, with the help of ALD. Concerning potential dental applications, our hypothesis was that the hydrophilic and antimicrobial capacity of Ti[O.sub.2] films might have the potential to inhibit the growth of C. albicans adhered to the metallic frameworks and thus lower the risk of associated denture stomatitis. In particular, an optimized ALD process was adopted via a homemade ALD system. The crystalline structure, microstructure, and adhesion strength of deposited Ti[O.sub.2] film were examined. Moreover, the water contact angles and antifungal properties of the Ti[O.sub.2]-deposited Co-Cr samples were investigated and further compared with those of uncoated Co-Cr substrates.
2. Materials and Methods
2.1. Preparation of Ti[O.sub.2] Film. The experiments were carried out in a hot-wall ALD system (home-made system, School of Chemistry and Chemical Engineering, Nanjing University, China). The Co-Cr alloy (Bego, Bremen, Germany) was used as substrate material, with the alloy composition given in Table 1. Small disc samples (diameter = 15 mm; thickness = 2.5 mm) were casted from the Co-Cr alloy. Prior to deposition, the substrate specimens were mechanically grounded by water-proof SiC abrasive papers with various sizes of up to 2000 grit and then polished with 30-50 nm alumina powder. The alloy discs were further ultrasonically cleaned in diluted sodium hydroxide solution, acetone, ethanol, and deionized water sequentially to remove residual surface contamination completely and finally dried [N.sub.2].
Ti[Cl.sub.4] and water, held in separate external reservoirs at room temperature, were alternatively introduced into the reactor chamber. The sketch is shown in Figure 1(a). One ALD deposition cycle consists of 3.0 s Ti[Cl.sub.4] pulse time, 250 s pump-down, 2.0 s water pulse time, and 300 s pumpdown, respectively. Herein, a longer pump-down period was employed instead of using high purity [N.sub.2]/Ar gas as purging gas to remove any residual reactants and by-products. The deposition temperature was held at 300[degrees]C constantly and 1000 ALD cycles were deposited for each sample.
2.2. Film Characterization Measurements. The crystalline structure of films was identified by X-ray diffraction (XRD) (D8, Bruker AXS GmbH, Karlsruhe, Germany). CoK[[alpha].sub.1] radiation ([lambda] = 0.178897 nm) was over a range from 20[degrees] to 90[degrees] (2[theta]). The surface morphology of the samples was examined by scanning electron microscopy (SEM) (S-3400N II, Hitachi, Tokyo, Japan). The cross-sectional microstructure and thickness of deposited films were determined by field emission scanning electron microscopy (Quanta FEG 250, FEI, Hillsboro, America).
A scratch tester was used to examine the adhesion strength between the Ti[O.sub.2] film and the Co-Cr substrate (UMT Multi-Specimen Test System, CETR, CA, USA). The coated surface was scratched using a conical Rockwell C tip with diameter of 5 [micro]m. Scratch (3 mm in length) was made with the applied loads ranged from 0.15 to 5.0 Kg at a linear speed of 0.01 mm/s, where the acoustic emission (AE) signal intensity (due to interface exfoliating) was monitored at the same time. The critical load was identified with the continuous increase in the AE signal during scratching. After testing, the scratch on the surface of Ti[O.sub.2] film was evaluated by optical microscope (Gx41, Olympus, Tokyo, Japan) at 16x magnification.
The static contact angles of coated and uncoated samples were measured using the sessile drop method at room temperature (OCA30 video contact angle system, Dataphysics, Filderstadt, Germany). A 2.0 [micro]L droplet of deionized water was dropped on the testing surface using a computer-controlled microsyringe. The contact angle was then determined from the magnified image collected with a camera in the system. Six samples from each group were selected randomly and three replicate measurements were performed at different positions. The averaged value was presented as the contact angle for each sample. The data is presented as the mean [+ or -] standard deviation. A t test was applied for statistical analysis, with P < 0.05 being considered significant.
2.3. Antifungal Property Tests. C. albicans strain (ATCC 10231) was used as the pathogen for the microbiological tests. Cultures of microorganisms were grown in Martin Broth, modified (liquid) at 37[degrees]C with 150 rpm shaking for 12 h. 10-fold serial dilutions of the incubation fluid were applied to reach the concentration of [10.sup.7] colony-forming units per milliliter (CFU/ml). The tested samples were sterilized under ultraviolet light (245 nm) for 2 h, prior to the investigations. A total of 100 [micro]L C. albicans diluted suspension was added onto the Ti[O.sub.2]-coated and uncoated surface, respectively. After illumination under an 8W UV lamp (365 nm) for 1h, the droplets were washed from the sample surface using 0.9 ml saline solution repeatedly. Afterwards 100 [micro]L of each serially diluted washing suspension was dispersed on the modified Martin Agar Medium and incubated for 24 h at 37[degrees]C. The number of colonies on the mediums was quantified via the direct counting method after incubation.
3.1. Structural Characterization of Ti[O.sub.2] Film. Figure 1(b) displayed a typical XRD spectrum of the Ti[O.sub.2]-deposited Co-Cr sample. Besides the typical peaks corresponding to elements of Co-Cr substrate, the dominating (101), (112), (200), and (211) crystalline peaks corresponded to the anatase Ti[O.sub.2] phase. This suggested a highly crystalline Ti[O.sub.2] film formed on the Co-Cr alloy. Anatase Ti[O.sub.2] was the dominating crystalline form of the surface layer under current experimental conditions.
The microstructures of the sample with and without Ti[O.sub.2] film were shown in SEM images (Figures 2(a)-2(c)). The surface of the Ti[O.sub.2] film was relatively dense, uniform, and smooth without noticeable pinholes or cracks (Figure 2(b)). It was noted that the Ti[O.sub.2] film has entirely covered the polishing scratches on the Co-Cr surface (Figure 2(a)). In addition, a large number of rounded grains with the size of about 100 nm were observed in a high-magnification SEM image of Ti[O.sub.2] film (Figure 2(c)). It could be seen from the cross-sectional scanning of the Ti[O.sub.2]-coated sample that the Ti[O.sub.2] layer was continuous, and its thickness was estimated to be 1.2-1.4 [micro]m (Figure 2(d)).
3.2. Adhesion Strength. The adhesion strength between the coating layer and substrate is listed as one of the most important factors determining the lifetime and performance of the coated component. The association curve between AE signal and the load on C tip showed that the Ti[O.sub.2] coating starts to peel off when the load reached approximately 4.0 Kg with continuously peaks (Figure 3(a)). The scratch morphology image (Figure 3(b)) revealed neither observed neighboring cracks nor coating detachment, besides the scratch corresponding to the applied force. All aforementioned observations suggested a good adhesion between the Ti[O.sub.2] film and Co-Cr substrate.
3.3. Water Contact Angle. The optical images of water drops sprayed onto the tested samples demonstrated that the average surface contact angle of deionized water was 103.0[degrees] [+ or -] 1.2[degrees] on the Co-Cr alloy and 37.3[degrees] [+ or -] 3.8[degrees] on the Ti[O.sub.2] -coated samples, respectively (Figure 4), indicating that the water contact angle of Co-Cr alloy showed a significant decrease (P < 0.05) after the Ti[O.sub.2] film deposition.
3.4. Ti[O.sub.2]-Coated Film Exhibited a Powerful Antifungal Property. In order to clarify the relative number of survival C. albicans on tested surface after 1 h UV illumination, the diluted suspensions washed from the uncoated and Ti[O.sub.2]-coated sample surface were incubated, respectively. Figures 5(a) and 5(b) showed photo images of colonies on the media after incubation for 24 h. The number of survival C. albicans on Ti[O.sub.2]-coated sample was much lesser than that on uncoated one. As shown in Figure 5(c), the quantified number of colonies from Ti[O.sub.2]-coated group was significantly less than uncoated group (P < 0.05). These results demonstrated that the Ti[O.sub.2] film presented a powerful antifungal effect under UV illumination.
The majority of ALD reactions rely on two gaseous precursors introduced into the chamber in a sequential, self-limiting manner. An inert gas, such as Ar and nitrogen, is infused between the precursor pulses to remove extra reactants and by-products. For example, Cheng et al. obtained Ti[O.sub.2] films using Ti[Cl.sub.4] and [H.sub.2]O, employing Ar as the purging gas, on a magnitude of substrate materials . In the current study, the application of ALD has been successfully extended to coat Ti[O.sub.2] films on a Co-Cr alloy. Moreover, the method optimization includes longer pump-down instead of purging gas. This modification greatly simplified the experimental procedure by obviating additional purging gases. On the other hand, the experimental period was slightly extended. Further optimization in terms of pumping capacity and tubing design will help to improve the working efficiency of current setup.
It is well-known that the phase composition and photocatalytic properties of the ALD Ti[O.sub.2] films are dependent on many parameters such as the chamber pressure, the deposition temperature, and the substrate material. On the basis of XRD pattern, it was concluded that the film grown under the current experimental conditions at 300[degrees]C was pure anatase structure. Earlier studies have also shown that the anatase structure with lower conduction bandedge exhibits a stronger photocatalytic and bactericidal ability [22, 28-30].
The surface smoothness is considered as an important factor influencing the adherence of oral pathogenic microorganisms [31-33]. Surface scratches and cracks can enhance the attachment of microorganisms and the growth of biofilms [8,34]. Furthermore, wettability is also an important property of biomaterials because hydrophilic surfaces are more resistant to microbial adhesion than hydrophobic surfaces [12, 35, 36]. Usually, surface with a contact angle more than 70[degrees] is identified as hydrophobic, while a hydrophilic surface has a contact angle below 70[degrees] . Results from the water contact angle measurements suggested that the Co-Cr alloy was hydrophobic and then became hydrophilic when coated with the Ti[O.sub.2] film. Yoshijima et al.  have reported that the decreasing surface hydrophobicity can diminish the ability of C. albicans to attach and colonize the denture surface. Our results revealed that coating Ti[O.sub.2] film can cover the polished scratches and increase the surface hydrophilicity of Co-Cr alloy at the same time. This may prevent the microbial attachment to certain extent and thus resist the development of biofilms leading to denture stomatitis.
More importantly, some studies have identified Ti[O.sub.2] as a surface coating to enhance the antibacterial capability of substrate materials [13,26,27]. This observation is most likely due to its photo-induced superhydrophilicity and photocatalytic property, though the exact bactericidal mechanisms of Ti[O.sub.2] under UV-irradiation are still under debate . In general, the cell wall and membrane damage by reactive oxygen species generated from the photocatalytic activity of Ti[O.sub.2] is the mostly accepted killing action . Furthermore, not only bacteria but also viruses, fungi, and other microorganisms can also be killed by UV-irradiated Ti[O.sub.2] . Evaluation of microbial survivability is one of in vitro techniques to assess antimicrobial properties . Our study complemented the previous studies by evaluating growth inhibition of C. albicans on Ti[O.sub.2]-coated and uncoated samples. As can be observed from the antifungal experiment, the Ti[O.sub.2] coating exhibited a great inhibitory effect against C. albicans, with a statistically significant reduction in the number of colony-forming units (CFU) compared to the control group (P < 0.05). Thus, it is fair to assume that the powerful oxidative ability of Ti[O.sub.2] can induce the apoptosis and necrosis in C. albicans cells, although the multilayer composition of C. albicans cell wall is more complicated and resistant to antimicrobial activity than that of bacteria . To better understand the underlying mechanism and mimic the real scenario, it is beneficial to perform a time-resolved antifungal experiment lasting a longer period of days or even weeks. Moreover, the illuminating UV conditions in terms of strength and duration can be further studied to match more practical values.
In conclusion, Ti[O.sub.2] thin coatings have been successfully deposited on Co-Cr substrate via an optimized ALD process. The figure of merits included pure anatase structure, dense and smooth surface, strong bonding, and full coverage to the substrate surface. More importantly, the Ti[O.sub.2] coatings possessed a high antifungal activity that eliminates most of the C. albicans after the UV-irradiation. Therefore, there is a great potential of Ti[O.sub.2] coating on future RPD items against possible denture stomatitis.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Lijuan Huang and Shuanglin Jing contributed equally to this work.
This work was supported by Translational Medicine Center of Nanjing.
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Lijuan Huang, (1) Shuanglin Jing, (2) Ou Zhuo, (3) Xiangfeng Meng, (1) and Xizhang Wang (3)
(1) Department of Prosthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, 30 Zhongyang Road, Nanjing, Jiangsu 210008, China
(2) Jiangsu Key Laboratory of Oral Diseases and Department of Endodontics, Affiliated Hospital of Stomatology, Nanjing Medical University, 136 Hanzhong Road, Nanjing, Jiangsu 210029, China
(3) Key Laboratory of Mesoscopic Chemistry ofMOE, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
Correspondence should be addressed to Xiangfeng Meng; email@example.com and Xizhang Wang; firstname.lastname@example.org
Received 26 April 2017; Revised 5 June 2017; Accepted 15 June 2017; Published 7 August 2017
Academic Editor: Nick Silikas
Caption: Figure 1: (a) Sketch of ALD technique (Pump 1: turbomolecular pump. Pump 2: sliding-vane rotary vacuum pump) and (b) XRD pattern of the surface of the treated sample.
Caption: Figure 2: SEM images of (a) Co-Cr substrate in low magnification. (b) Ti[O.sub.2] film in low magnification. (c) Ti[O.sub.2] film in high magnification. (d) Cross-sectional view of Ti[O.sub.2] film on Co-Cr substrate. Caption: Figure 3: Scratch test (a) applied load and AE signal intensity. (b) Optical image of the scratch test specimen.
Caption: Figure 4: Optical images of a water droplet in contact with Co-Cr (a) and Ti[O.sub.2] (b) surface.
Caption: Figure 5: Photo images of incubated C. albicans colonies washed from surface of uncoated (a) and Ti[O.sub.2] - coated sample (b). (c) The numbers of C. albicans colonies.
Table 1: Chemical composition of the Co-Cr alloy. Element Co Cr Mo Ni Others (wt.%) 62.3% 29.3% 6.2% 1.0% 1.2%
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|Title Annotation:||Research Article|
|Author:||Huang, Lijuan; Jing, Shuanglin; Zhuo, Ou; Meng, Xiangfeng; Wang, Xizhang|
|Publication:||BioMed Research International|
|Date:||Jan 1, 2017|
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