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Effects of different sterilization methods on surface characteristics and biofilm formation on zirconia in vitro

Aifang Han a,b, James K.H. Tsoi a,∗, Jukka P. Matinlinna a, Yu Zhang c, Zhuofan Chen a,b,∗∗

a Dental Materials Science, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China b Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, PR China c Department of Biomaterials & Biomimetics, New York University College of Dentistry, New York, USA

a r t i c l e i n f o

Article history:

Received 2 May 2017

Received in revised form

1 September 2017

Accepted 9 November 2017

Keywords:

Zirconia

Sterilization methods

Bacteria

Colour

Gamma ray

UV

a b s t r a c t

Objective. The current laboratory study was to investigate the effect of different sterilization

treatments on surface characteristics of zirconia, and biofilm formation on zirconia surface

after exposure to these sterilization treatments.

Methods. Commercially available zirconia discs (Cerconbase, Degu-Dent, Hanau, Germany)

were prepared and polished to the same value of surface roughness. The discs were treated

with one of the following sterilization methods steam autoclave sterilization, dry heat ster-

ilization, ultraviolet C (UVC) irradiation, and gamma (�) ray irradiation. The characteristics

of zirconia surfaces were evaluated by scanning electron microscopy (SEM), surface rough-

ness, surface free energy (SFE), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction

(XRD) measurements. Then, Staphylococcus aureus (S.a.) and Porphyromonas gingivalis (P.g.) bac-

teria were used and cultured on the respective sterilized zirconia surfaces. The amount of

biofilm formation on zirconia surface was quantified by colony forming unit (CFU) counts.

Results. Significant modifications were detected on the colour and SFE of zirconia. The colour

of zirconia samples after UVC irradiation became light yellow whilst dark brown colour was

observed after gamma ray irradiation. Moreover, UVC and gamma ray irradiation increased

the hydrophilicity of zirconia surface. Overall, dry heat sterilized samples showed the signif-

icantly lowest amount of bacteria growth on zirconia, while UVC and gamma ray irradiation

resulted in the highest.

Significance. It is evident that various sterilization methods could change the surface which

contribute to different biofilm formation and colour on zirconia. emy

© 2017 The Acad

∗ Corresponding author at: Prince Philip Dental Hospital, 34 Hospital Ro ∗∗ Corresponding author at: Hospital of Stomatology, Sun Yat sen Univ

China. E-mail addresses: [email protected] (J.K.H. Tsoi), [email protected]

https://doi.org/10.1016/j.dental.2017.11.012 0109-5641/© 2017 The Academy of Dental Materials. Published by Elsev

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

ad, Sai Ying Pun, Hong Kong, China. ersity, No. 56 Lingyuan Xi Road, Yuexiu District, Guangzhou, PR

(Z. Chen).

ier Ltd. All rights reserved.

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d e n t a l m a t e r i a l s

. Introduction

irconia ceramic is an attractive material for dentistry and onsidered a material of choice. For example, metal implants ay reveal a bluish discoloration of the overlying gingiva hen apical bone loss and gingival recession occurred. How-

ver, a full zirconia implant avoids this aesthetic problem ecause of its more tooth-like colour [1]. Moreover, zirconia

s also popular due to its high flexural strength and toughness 2].

Conclusions from different studies about zirconia implants pplication and performance are controversial. A retrospec- ive clinical study by Roehling et al. investigated the clinical erformance of zirconia implants up to and after 7 years of

oading, and concluded that first-generation zirconia implants howed low overall survival (77.3%) and considered all surviv- ng implants, the success rate was 77.6% [3]. Another study by ashim et al. concluded that although the overall survival rate as 92% for zirconia implants after 1 year of function, further

linical studies are required to establish long-term results, and o determine the risk of technical and biological complica- ions [4]. However, some studies have reported that zirconia mplants have similar osseointegration capability compared o conventional titanium implants [5–7], and they could poten- ially be the alternative to titanium implants for a non-metallic mplant solution. Therefore, zirconia has been increasingly sed in implant dentistry due to its aesthetic performance [8], s well as good mechanical properties [9] and biocompatibility 10].

Implants, as surgical components that have intimate con- act with bone, need to be properly sterilized prior to the mplantation — or during the storage. Sterilization is consid- red as the final finishing procedure during the manufacturing rocess, because it may affect the implant surface and its odification, i.e., the physico-chemical properties of implant

urface might be changed and thus have an important clin- cal impact [11,12]. Moreover, sterilization is also applied as n essential step before in vitro biological tests, because it is a rocess enabling the device to be free from viable microor- anisms, preventing the proliferation and accumulation of nrelated microorganism that we do not want to focus on [13].

There are many issues to be considered when selecting sterilization method for a particular application condition, .g., cost and effectiveness. The ultimate goal is to sterilize mplant materials and devices properly without compromis- ng their key surface characteristics that may influence their nteraction with surrounding tissue [14]. Various steriliza- ion methods have been used in dentistry, depending on the esired application and material properties [15,16], such as team autoclaving and �-irradiation are the most commonly sed sterilization processes for implant materials storage ince they safe with respect to chemical contamination of the urface [15]. Dry heat and UV sterilization has been for dental mplements, e.g., reamer, drill [17–19]. In addition, enhanced steoblast function has been confirmed on ultraviolet light-

rradiated zirconia [20].

The sterilization techniques can be categorized by phys-

cal and chemical methods. Physical methods include dry eat sterilization, steam autoclave sterilization, UV radia-

( 2 0 1 8 ) 272–281 273

tion, and gamma ray irradiation. Treatments with chemicals such as ethylene oxide (EO), ozone, formaldehyde and phe- nols belong to chemical sterilization methods [21]. However, cold chemicals for routine sterilization of instruments are not recommend by the American Dental Association, since mon- itoring the solution can be difficult, and their efficaciousness can be limited by the inability to wrap the instruments in a sterile package [22]. Therefore, physical methods deemed to be the most suitable for sterilizing implant.

Steam autoclaving is a sterilization method commonly used in the dental field, due to its convenience, low cost and reliable sterilization effect [13]. Dry-heat sterilization can be used when the moisture in steam autoclaving would cause corrosion and deterioration of specific material [22]. Indeed, steam autoclave sterilization and dry heat sterilization offer both cost effectiveness and efficacy, but some materials can- not withstand invasive moisture or temperatures above 100 ◦C [23]. Therefore, other sterilization techniques, such as gamma (�) irradiation and ultraviolet (UV) irradiation, are also avail- able. Gamma irradiation from a cobalt-60 (Co-60) source is lethal to all forms of microorganism and it has the advantage of sterilizing without high temperature and pressure, chemi- cals or gases [13]. UV irradiation is divided into four distinct spectral areas according to wavelength, namely: vacuum- UV (100–200 nm), UVC (200–280 nm), UVB (280–315 nm), and UVA (315–400 nm), such that UVC is found to possess a high antimicrobial capability. UV sources, e.g., light-emitting diodes, lasers, and microwave-generated UV plasma, are avail- able for biomedical applications [24].

Cell and bacteria adhesion is sensitive to the surface prop- erties of implant materials [25,26] and different sterilization treatments may influence the surface chemistry and wetta- bility, consequently affecting cellular behavior [14]. A study by Vezeau et al. had explored the effects of sterilization on titanium surface characteristics and fibroblasts attachment in vitro. They reported that titanium surface characteristics can be altered by steam autoclave sterilization, and less murine fibroblasts attachment compared to that after UV irradiation [27]. However, there is still no published study investigating the effect of different sterilization treatments on zirconia sur- face characteristics, as well as the biological responses of biofilm formation.

The aims of this study are twofold: to examine the effects of sterilization methods, i.e., steam autoclave sterilization, dry heat sterilization, UVC irradiation, and gamma ray irradiation, on the surface characteristics of zirconia. Furthermore, after the four different sterilization treatments, the in vitro biofilm formation on zirconia surfaces was compared.

2. Materials and methods

2.1. Zirconia sample preparation

Commercially available cylindrical pre-sintered Y-TZP zirconia blocks (Cerconbase, DeguDent, Hanau, Germany) were used in

this study. The zirconia blocks were cut into quadrant-shaped specimen (12.5 mm in radius and 1 mm in thickness) using a diamond precision saw (IsoMetTM 5000, Buehler, USA) under cold running water. After being polished with 4000-grit SiC

274 d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 272–281

Table 1 – Sterilization treatment condition of different groups.

Group Treatment Equipment Treatment condition/protocol

Control Nil Nil No further treatment Group SA Steam autoclave sterilization Autoclave ASB300BT, ASTELL, UK 121 ◦C for 15 min [15] Group DH Dry heat sterilization Universal Oven U, MEMMERT,

Germany 160 ◦C for 2 h [30]

Group UVC UVC irradiation UV fluorescence cabinet CL-150, SPECTROLINE,USA

254 nm wavelength and 490 �W/cm2 at the distance of

gam Chin

Group � � ray irradiation Co-60 BINE,

abrasive paper, the samples were sintered according to the manufacturer instructions. Then, all the fully-sintered zirco- nia specimens were ultrasonically cleansed in 70% ethanol solution for 15 min, rinsed with de-ionized water, and allowed to dry in clean ambient air for 30 s. A polished only specimen was used as control.

2.2. Sterilization treatment

The zirconia samples were randomly divided into five study groups and treated with one of the following surface modifi- cation protocols (Table 1). The dose of � irradiation in Group � was set as 25 kGy, since it was recommended as standard dose for medical products in the European Union (EU) [28,29].

2.3. Surface characteristics of zirconia surface

2.3.1. Visual sample observation The colour of specimens before and after treatment was observed and assessed visually.

2.3.2. Scanning electron microscopy (SEM) A scanning electron microscope (SU-1510, HITACHI, Japan) was used to observe the surface morphology of different zirconia groups. The samples were gold sputtered and the analyzing procedures were carried out at 1000× magnification [32].

2.3.3. Surface roughness Three zirconia samples of each group were measured for Ra values using a profilometer (Surtronic3+, Taylor-Hobson, UK). The stylus tip radius of the diamond probe is 5 �m. The cut-off value was set at 0.8 mm. Each zirconia sample was tested for three times and the average value of each group was calcu- lated.

2.3.4. Surface free energy (SFE) Contact angle was determined using the sessile drop method (Drop shape analyzer DSA100, KRÜSS, Germany). Puri- fied water and diiodomethane were used as probe-liquids. Three zirconia samples of each group were tested. Surface free energy (SFE) was calculated in accordance with the Owens–Wendt–Rabel–Kaelble (OWRK) method [33].

2.3.5. X-ray photoelectron spectroscopy (XPS) The surface chemical composition of the samples was analyzed with the ultra-high vacuum chamber of an X-ray photoelectron spectrometer (X-ray Photoelectron Spec-

25 cm for 30 min on both sides [31] ma irradiator,BFT-3, a

25 kGy at room temperature [28,29]

troscopy/ESCA, Thermo Fisher Scientific, USA). The sample was irradiated with a monochromatic X-ray source Al K� (1486.6 eV) with an accelerating voltage of 15 kV. The working vacuum under X-ray irradiation was 2 × 10−9 mbar. Energy cal- ibration was performed based on the Ag 3d5/2 peak standard. Wide survey scans (with 100 eV pass energy) were recorded to identify the chemical elements of the surface. Then, high resolution narrow scans (20 eV pass energy) were applied on the main peaks to determine the elemental binding states. All spectra were aligned on the binding energy scale to the C 1s peak (284.8 eV). The quantitative evaluation of the chemical composition of the surface was performed with Al Thermo1 Library [34,35].

2.3.6. X-ray diffractionn analysis (XRD) X-ray diffraction (Empyrean, PANalytical, The Netherlands) examination was used to analyze the changes of surface crys- talline structure. The scanning was carried out within the 2� range between 5◦ and 80◦ at a speed of 10◦/min with a voltage of 40 kV and a current of 40 mA [36].

2.4. Bacteriological study

2.4.1. Biofilm formation To grow Staphylococcus aureus (S.a.), a solution with 107 bac- teria/ml in brain-heart infusion (BHI) medium was prepared and dispensed into a 24-well microtitre plates (Corning, USA) containing a zirconia disc in each well (1 ml/well). The growth medium was refreshed every 24 h. The biofilm formation was evaluated 2 days after inoculation.

For Porphyromonas gingivalis (P.g.), 108 bacteria/ml in P.g. broth (composed of 30 g TSB, 5 g yeast extract, 1 L distilled water, and 10 mL hemin/vitamin K stock solution) was pre- pared and dispensed into a 24-well microtitre plates (Corning, USA) containing a zirconia disc in each well (1 ml/well). The growth medium was refreshed every 3 days. The adhesion of bacterial cells or the biofilm formation was evaluated 7 days after inoculation.

In each test, all test groups contained three samples, and the experiment was repeated 3 times.

2.4.2. Quantification of biofilm formation At a designated biofilm collection time point, the zirconia discs with biofilms were rinsed once in phosphate buffered saline

(PBS) solution and transferred into 1 ml of growth medium (BHI for S.a. and P.g. broth for P.g.). The biofilms were removed from the discs and dispersed by vortexing for 30 s. Serially diluted samples were plated onto blood agar plates and incubated

d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 272–281 275

Fig. 1 – Zirconia samples sterilized with different methods including (left to right) Groups Control, SA, DH, UVC, and �. NB. UVC becomes yellow and � becomes brown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

F 1000 ( lizat

a w w s v f

ig. 2 – Results of SEM examination (original magnification a) control, (b) steam autoclave sterilization, (c) dry heat steri

naerobically at 37 ◦C to allow for colonies to grow. The plates ere taken out from the incubator and individual colonies

ere counted. This method for quantifying bacteria on sub-

trates was adapted from Seil et al. [37], which concluded that ortexing was an effective way to remove bacteria from sur- aces.

×) on zirconia surfaces sterilized with different methods: ion, (d) UVC irradiation, and (e) � ray irradiation.

2.5. Statistical analysis

The data were analyzed by Statistical Package for Social Sci- ence (SPSS

® , Version 23, IBM, USA). The statistical analysis was

performed using one-way analysis of variance (ANOVA) at a significance level of 5%.

276 d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 272–281

Fig. 3 – XPS spectra of Zr (3d) ionization for zirconia samples sterilized with different methods: (a) control, (b) steam autoclave sterilization, (c) dry heat sterilization, (d) UVC irradiation, and (e) � ray irradiation.

Table 2 – Surface roughness of different groups (Ra).

Group Mean (�m) ± standard deviation Control 0.24 ± 0.08 Group SA 0.22 ± 0.08

Fig. 4 – Results of XRD analysis on zirconia surface after different sterilization treatments (T represents tetragonal phase zirconia).

Group DH 0.22 ± 0.08 Group UVC 0.18 ± 0.03 Group � 0.22 ± 0.03

3. Results

3.1. Visual sample observation

The colour of the white zirconia samples did not have obvious change in Group SA and Group DH compared to the Control group. However, UVC and � ray irradiation caused discol- oration of zirconia samples. The colour of the zirconia changed to light yellow in Group UVC and dark brown in Group � (Fig. 1).

3.2. Scanning electron microscopy (SEM)

Fig. 2 shows the morphological appearance of zirconia sur- face before and after being sterilized, respectively. Zirconia appeared to have similar surface overall in all the groups after different sterilization treatments. Small pits and shal- low grooves generated in the polishing procedures could be observed. However, there seemed to be more debris on the zirconia surface after sterilization treatments compared to the zirconia in the control group.

3.3. Surface roughness

The average surface roughness (Ra) values in the five groups of zirconia after different sterilization treatments are shown in Table 2. There was no significant difference in Ra values between the five groups.

3.4. Surface free energy (SFE)

The changes in contact angles and SFE values in different groups after sterilization treatments are shown in Table 3. The hydrophilicity of zirconia surfaces increased after all the four types of sterilization treatments, compared to the control

group. The SFE values of different groups from the lowest to the highest were 30.41 (Control), 33.97 (Group SA), 34.81 (Group DH), 37.38 (Group UVC), and 43.28 (Group �).

3.5. X-ray photoelectron spectroscopy (XPS)

To investigate whether the chemical composition changes after sterilization, X-ray photoelectron spectroscopy (XPS) was used to analyze the atomic top layers of all five groups. The XPS analysis confirmed that surface chemistry was altered by sterilization treatments as shown in Table 4. It was shown that carbon decreased and oxygen increased obviously by atomic% in Group UVC and Group �. The Zr/O ratio in Group SA (0.3348) and Group DH (0.338) increased compared to the con- trol group (0.3259), whereas the Zr/O ratio decreased in Group UVC (0.3131) and Group � (0.2653) (Table 3). The lowest Zr/O ratio value was found in Group � (0.2653) while the highest value was found in Group DH (0.338). In addition, XPS spec-

tra showed Zr (3d) ionization for all the zirconia samples after different sterilization treatments, which is the same with the zirconia sample in the control group (Fig. 3).

d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 272–281 277

Table 3 – Surface free energy of different groups.

Group Water contact angle (◦) Diiodomethane contact angle (◦) Surface free energy (mN/m)

Control 100.24 ± 2.48 57.47 ± 5.25 30.41 Group SA 92.03 ± 4.52 53.03 ± 3.84 33.97 Group DH 96.14 ± 3.08 50.47 ± 1.90 34.81 Group UVC 81.58 ± 1.14 52.21 ± 4.00 37.38 Group � 69.89 ± 0.17 50.33 ± 1.43 43.28

Table 4 – Variation of surface composition of the carbon, oxygen, zirconium element and the ratio in different grous.

Group C1s Atomic % O1s Atomic % Zr3d Atomic % C1s/O1s ratio Zr3d/O1s ratio

Control 26.08 49.39 16.1 0.528 0.3259 Group SA 29.49 46.67 15.64 0.632 0.3348

1 1 1

3

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3

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Group DH 26.34 49.52 Group UVC 19.68 55.07 Group � 18.98 58.71

.6. Crystallographic structure analysis (XRD)

n Fig. 4, it is shown that only tetragonal (T) phase struc- ure could be detected on the zirconia surfaces of all the four roups. No monoclinic (M) phase was detected in zirconia amples after the four sterilization treatments.

.7. Biofilm formation as function of time

he amount of bacterial cells adhered to the zirconia surfaces as counted by the colony forming unit (CFU) method. Fig. 5 emonstrates the log transformed CFU counts of S. aureus on irconia surfaces sterilized with different methods after cul- ured for 2 days and P. gingivalis on zirconia surfaces sterilized ith different methods after cultured for 7 days. Significant ifference of biofilm formation of S. aureus was found on irconia surfaces between Group DH and Group � (p < 0.05). oreover, our study also found that there was significant dif-

erence of P. gingivalis biofilm growth between Group DH and roup UVC (p < 0.05). There was no significant difference of the acteria between Group SA and the other three groups both in he S. aureus and P. gingivalis biofilm formation.

. Discussion

his laboratory study investigated the effects of steriliza- ion treatments on the surface characteristics of zirconia and acteria adhesion on sterilized zirconia. Steam autoclave ster-

lization, dry heat sterilization, UVC irradiation and gamma ay irradiation were chosen as sterilization treatments in our tudy.

One of the clear differences after sterilization treatments hich was observed was the discoloration seen in Group UVC

nd Group �, based on visual observation and assessment. he colour of the zirconia samples changed to light yellow in roup UVC and brown in Group �, while there was no obvi- us discoloration in Group SA and Group DH compared to he white colour of zirconia in Group Control. The effect of igh-energy UVC or � radiation in zirconia may lead to the

eneration of free electrons with a range of energies through bsorption of the radiation energy and subsequent ionization 38]. Such a process can produce colour centers in zirconia,

hich may result in discoloration in surface and deeper layers

6.75 0.532 0.3380 8.36 0.357 0.3131 5.58 0.323 0.2653

of the material. This may also be the underlying reason for the discoloration phenomenon of zirconia-containing resin com- posites after UVC irradiation [39]. As a result, the discoloration phenomena indicated zirconia cannot be sterilized by � irra- diation if it is used as crown, since the discoloration will affect the aesthetic performance of zirconia, and additionally the aesthetic influence of UVC on the zirconia implant should be further investigated.

As determined by the SEM evaluations, the overall mor- phology of zirconia surface did not seem to be affected by sterilization treatments used in this study. This result was consistent with the finding by previous studies [13,27]. A study by Vezeau et al. investigated the effect of steriliza- tion treatments on pure titanium surfaces and concluded the sterilization treatments failed to alter the morphology of the samples [27]. Another previous study revealed the dentin sur- face morphology was not affected by gamma rays and no dentin structural change was detected [13].

For surface roughness, the zirconia samples did not show significant difference between different sterilization treat- ments in our study. A study by Park et al. also found that sterilization does not alter the surface roughness of pretreated titanium (PT, Ra = 0.4 �m) [40]. In fact, the limitation of our study is that we did not use the 3D-surface height, spatial, and hybrid roughness parameters (e.g., Sa, Sz, Ssk, Sku, Sal, Str and Sdr) to assess the surface characteristics, since certain studies suggested to choose a set of roughness parameters for the purpose of properly characterizing an implant surface [41]. However, it still remains to be explored, which rough- ness parameters are the most appropriate for characterizing an implant surface concerning with biological effect, such that Matinlinna et al. [42] has explicitly mentioned various study used different forms of roughness that is lack of unity. There- fore, while we might need to explore the effects of different sterilization methods on three-dimensional surface rough- ness of zirconia in further study, another focus could be the standardization of the roughness units or test methodology.

For surface free energy, the zirconia samples increased after sterilization treatments in all groups in our study. The result is consistent with a study by Wittenburg also revealed

that steam sterilized glass exhibited significantly lower con- tact angle values than untreated glass samples [43]. A study by Kummer et al. which found that UV irradiation on Ti could

278 d e n t a l m a t e r i a l s 3 4 ( 2 0 1 8 ) 272–281

Fig. 5 – Results of CFU counts of S. aureus on zirconia surfaces sterilized with different methods after cultured for 2 days and P. gingivalis on zirconia surfaces sterilized with different methods after cultured for 7 days. * denotes statistically significant

(p < 0.05) difference between different groups.

increase the formation of Ti-OH and result in the increase of surface wettability and surface energy [14]. Another reason illustrated by previous studies was that titanium and zirconia constantly adsorb organic impurities such as hydrocarbons from the environment, which could lead to an increase in hydrophobicity. UV irradiation could remarkably decrease car- bon content and increase the amount of hydroxyl groups on titanium and zirconia surface [20,44]. The underlying reason was that UV light irradiation could create surface O2 vacan- cies at bridging O2 sites [45]. This study was consistent with previous studies and we observed a decreased carbon contam- ination and increased oxide element on zirconia surface after UVC irradiation from XPS result. Our study demonstrated that gamma ray could also increase the hydrophilicity of zirconia surface as detected by the surface free energy test. Indeed, Ueno et al. found that, although gamma ray treatment on tita- nium did not change the surface topography, it could reduce contact angle. The mechanism possibly due to the gamma ray irradiation could decompose organic molecules adsorbed on titanium dioxide surfaces and generate activated oxygen species come from O2 in air, e.g., O2−, O−, and O (atom). These processes effectively contributed to the surface hydrophilicity [46]. Even so, exact mechanism on zirconia is still need to be further explored.

According to the XRD results, zirconia samples in our study only contained tetragonal (T) phase structure in all the groups. This result was consistent with a previous study which revealed that no evidence of zirconia phase trans- formation was observed in any zirconia/alumina composites after treated by 25 kGy � irradiation sterilization and 50 kGy � irradiation sterilization [47]. Although previous study found steam autoclave sterilization methods may cause a phase

change and surface roughening of yttria-stabilized zirconia femoral head components [48], no tetragonal (T) to mono- clinic (M) phase transformation could be observed in all the zirconia samples in our study. The underlying reason may

be the difference of sterilization condition between different studies. Y2O3-ZrO2 tetragonal polycrystal (Y-TZP) materials, containing 2–3 mol% Y2O3, are constituted predominantly of tetragonal zirconia grains. A previous study on hydrother- mal degradation behavior of a CAD/CAM-machined dental zirconia (3 mol% Y-TZP, IPS e.max ZirCAD, Ivoclar-Vivadent, Schaan, Liechtenstein) using identical steam autoclave tem- perature/cycles revealed that a much longer sterilization time and more sterilization cycles are required for the phase transformation of zirconia [49]. In addition, another study investigated the low temperature degradation behavior of a medical grade zirconia (3 mol% Y-TZP, Prozyr Y-TZP, Norton, East Granby, CT) in a universal oven at 200 ◦C in air [50]. It was found significant tetragonal to monoclinic phase transforma- tion occurred after a prolonged 12 h holding time.

The other aim of this study was to evaluate the effects of different sterilization treatments on biofilm formation on zirconia. Previous studies showed various factors may have influence on the bacteria adhesion on material surfaces, e.g., surface roughness [51,52], surface free energy [34,53], surface chemistry [54]. In this study, we choose the bacteria of S. aureus and P. gingivalis as the target bacteria. Recently, a clini- cal study was conducted to examine the prevalence and levels of six bacterial pathogens within the subgingival/submucosal microbiota at teeth versus implants with various clinical con- ditions A gram-positive cariogenic bacterial species, S. aureus was revealed to be the most commonly detected bacteria species in both periodontal and peri-implant sites, irrespec- tive of their health status [55]. Antimicrobial chemotherapy treatment which aimed to reduce the number of S. aureus enabled a better quality of bone repair of tibial surgical bone defects in rats [56]. P. gingivalis is considered to be one of the

most important microflora of dental peri-implantitis [57]. It has been a focus for peri-implantitis aetiology studies for a long time [58].

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The results showed S. aureus biofilm formation on zirconia urface treated by gamma irradiation sterilization was higher han that on zirconia surface treated by dry heat sterilization reatment after 2 days. The difference was significant (p < 0.05). owever, no significant difference of S. aureus biofilm forma-

ion on zirconia surfaces was found between the other groups. or P. gingivalis, significant difference of P.gingivalis biofilm rowth was found between zirconia surface after sterilized y dry heat and UVC irradiation (p < 0.05). More P. gingivalis acteria were found on zirconia surface after sterilized by VC irradiation compared to that by dry heat. Given this, it an be concluded that dry heat sterilization treatment may nduce less biofilm formation on zirconia compared to UVC nd gamma irradiation sterilization treatment.

Now, dry heat treatment of zirconia samples was shown o exhibit the lowest biofilm formation on zirconia samples etween different groups. The underlying reason is not quite lear. Dry heat sterilization simply means raising the temper- ture of an item to 160 ◦C under normal air pressure, during hich the sterilization process is conducted in dry condition ithout steam and high pressure compared to steam auto-

lave sterilization. Since water dissociation can produce OH−

nd is affected by pressure, we speculate that zirconia sur- ace of Group DH contained lower amount of OH− compared o Group SA, which requires further study to confirm. It was ound that OH− ions existing at the topmost surface layer are eneficial to the adsorption of cell adhesion proteins. This aid, the selective adsorption of cell adhesion proteins domi- ates cell adhesion onto a surface [59]. Therefore, the lowest mount of bacteria may adhere on the zirconia surface after ry heat sterilization treatment among all the sterilized study roups.

As for the UVC and � irradiation sterilized samples, they howed a significantly higher P. gingivalis bacteria amount on irconia surface after UVC irradiation and higher S. aureus bac- eria on zirconia surface after gamma irradiation respectively ompared to dry heat sterilized samples. This can be explained y the increased surface free energy of zirconia surface after VC and � irradiation. Our result is consistent with the pre- ious study by Zhao et al., who reported that UV sterilization ives rise to higher surface free energy of titania surfaces [31]. t has been reported that the hydrophilicity of the samples can ave drastic effects on cell adhesion [60]. Moreover, increased ydrophilicity can alter the adsorption of specific proteins nto a biomaterial surface which can further influence inter- ctions with cells [61]. In addition, some bacteria species were ore sensitive to surface chemistry whereas others to sur-

ace roughness [54]. Therefore, the underlying mechanism of . gingivalis and S. aureus adhesion on zirconia surfaces after ifferent sterilization treatments are complicated and need urther investigation.

. Conclusion

n conclusion, the surface free energy and surface chem-

stry of zirconia changed after sterilization treatments. The election of sterilization method used for zirconia plays an mportant role in the degree of biofilm formation onto the zir- onia surface. In addition, it is evident that the sterilization

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treatment, surface chemistry, and hydrophilicity are playing some role in the biofilm formation on zirconia material. More- over, the zirconia samples treated by dry heat sterilization showed decreased biofilm formation, while the zirconia sam- ples treated by UVC irradiation and � ray irradiation showed higher bacteria formation on zirconia surface compared to that treated by dry heat sterilization.

Acknowledgements

This work was done in partial fulfilment of the requirements of the degree of Doctor of Philosophy for AH at the Fac- ulty of Dentistry, The University of Hong Kong. Part of the results of this paper has been presented at the 2016 IADR Gen- eral Session, Seoul, South Korea. YZ would like to thank the United States National Institutes of Health, National Institute of Dental and Craniofacial Research (Grant Nos. R01DE017925 and R01DE026772) and the International Congress of Oral Implantologists (Implant Dentistry Research and Education Foundation Grant) for their support.

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