Impact of physical decontamination methods on zirconia implant surface and subsequent bacterial adhesion: An in‐vitro study

Abstract Objective To evaluate the effect of routinely used physical decontamination methods on the surface characteristics of zirconia implants and subsequent ability of bacteria to adhere in vitro. Background Physical decontamination methods commonly used in peri‐implantitis therapy and routine implant maintenance can potentially alter zirconia implant surfaces. Methods Acid‐etched zirconia discs were instrumented with titanium curette (TC), plastic curette, air abrasive device, ultrasonic scaler (US) with stainless steel tip. Following instrumentation, surface topography, and surface elemental composition was analyzed using 3D‐laser scanning microscopy and energy‐dispersive X‐ray spectroscopy, respectively. Subsequently, plaque biofilm was cultured on zirconia discs for 48 h and bacterial adhesion assessed using a turbidity test and scanning electron microscopy. Results A significant difference in surface roughness was observed between the US and control group (p < 0.05). The US and TC caused gray surface discolouration on zirconia discs due to deposition of metallic residue as confirmed by X‐ray spectroscopy. No significant difference in bacterial adhesion was noted among all treatment groups (p > 0.05). Conclusion TC and US with stainless steel tips should be used with caution due to deposition of metallic residue on the surface. Air abrasive devices and plastic curettes caused minimal surface alterations and are, therefore, safer for zirconia implant decontamination.

. The detrimental effects of edentulism include, but are not limited to, difficulties in eating and speaking, concerns about appearance, lowered self-confidence, and feelings of bereavement (Dosumu et al., 2014).
Since the discovery of osseointegration in the late 1950s by Peri-Ingvar Branemark, titanium implants have remained the gold standard in dental implantology (Guglielmotti et al., 2019;Klinge et al., 2018).
Titanium implants are known for their high success rate owing to their excellent biocompatibility and favorable mechanical properties (Ozkurt & Kazazoglu, 2011). The main disadvantage of titanium as an implant material is its gray metallic appearance, which can be an aesthetic concern especially in the presence of thin gingival biotype or gingival recession (Apratim et al., 2015;Ozkurt & Kazazoglu, 2011;Sivaraman et al., 2018). It has also been reported that implant failure can occasionally occur due to the release of titanium ions into surrounding tissues, triggering a hypersensitivity reaction in susceptible patients (Kim et al., 2019). To overcome these drawbacks, zirconia implants have emerged as a viable alternative to titanium implants.
Zirconia is a chemically inert material with minimal local and systemic side effects and is already extensively used in clinical dentistry for the fabrication of crowns, bridges, and implant abutments (Grech & Antunes, 2019;Munro et al., 2020). Zirconia is also a highly biocompatible material with an aesthetically pleasing tooth-colored appearance, acceding to the increasing demand for metal-free dental implants (Grech & Antunes, 2020;Ozkurt & Kazazoglu, 2011).
Much like natural teeth, dental implants are susceptible to developing diseases and complications. According to the 2017 World Workshop Classification of Periodontal and Peri-implant Disease and Conditions (Caton et al., 2018), two types of peri-implant disease known as periimplant mucositis and peri-implantitis exist. Peri-implant mucositis is a reversible inflammatory condition affecting the soft tissues surrounding an implant and is characterized by redness, swelling and bleeding (Caton et al., 2018). If left untreated, peri-implant mucositis can progress to periimplantitis which involves the irreversible and progressive destruction of peri-implant bone (Caton et al., 2018). Peri-implantitis is one of the main causes of implant failure and is estimated to affect up to 18.8% of implant patients (Atieh et al., 2012). Routine supportive periodontal care is crucial in the prevention and management of peri-implant disease (Gulati et al., 2014;Renvert et al., 2019).

Various instruments have been proposed for implant maintenance
and peri-implantitis therapy, including the use of metal and plastic curettes, ultrasonic scalers, air abrasive devices, prophylaxis cups, and laser systems (Gulati et al., 2014;Louropoulou et al., 2012). However, some of the currently used decontamination methods can roughen implant surfaces, creating niche environments for bacterial colonization which in turn, increases the risk of peri-implant disease (Louropoulou et al., 2012;Yeo et al., 2012). As such, physical decontamination methods should not only be effective in removing plaque and calculus but also safe in terms of preventing surface alterations and biocompatibility issues (Louropoulou et al., 2015).
To date, studies have primarily focused on instruments for decontamination of titanium implants and little is known about their suitability for zirconia. Hence, the primary aim of this in-vitro study was to determine the effects of various physical decontamination methods on the surface characteristics of zirconia implant surface. The secondary aim was to assess changes in bacterial adhesion on treated zirconia surfaces following instrumentation. Our null hypothesis was that the physical decontamination methods tested would not alter the surface characteristics of the yttria-tetragonal zirconia discs and, therefore, there would be no change in bacterial adhesion after treatment.

| Sample preparation
Yttria-tetragonal zirconia polycrystal (Y-TZP) discs measuring 16 mm in diameter and 3 mm in thickness were fabricated by uniaxial pressing and sintering commercial 3 mol% yttria-partially stabilized zirconia powder (70% tetragonal, 30% monoclinic) using the protocol described in Munro et al. (2020). Y-TZP discs were then immersed in 40% hydrofluoric acid (Scharlab, Barcelona, Spain) for 30 min to create an acid-etched zirconia implant surface before being rinsed with purified water to remove any remaining acid or residue on the surface. Individual discs were oriented horizontally on a flat table and manually stabilized to prevent movement during treatment. All cleaning procedures were performed by an experienced dental clinician (N.T.).

| Titanium curette and plastic curette
Fifty overlapping strokes were performed along the entire surface of each sample using the cutting edge of the curette. Moderate finger pressure was applied with the aim of replicating the amount of force normally used in clinical practice to remove calculus from an implant surface. A new curette was used for each sample to ensure that instruments were sharp prior to use.

| Air abrasive device
The air abrasive device (AA) was loaded with glycine powder to the recommended level according to the manufacturer's instructions before being applied onto each sample. The AA was moved steadily over the entire surface for 1 min with the nozzle directed perpendicular to the sample at a distance 0.5 cm to 1 cm away.

| Ultrasonic Scaler
The water coolant supply on the ultrasonic scaler (US) (Suprasson P5 Satelec, Aceton, France) was adjusted to a level consistent with routine use in clinical practice and confirmed via visual inspection. The working lateral surface of the US tip was applied for 1 min at 70% power setting on each sample.
Following instrumentation, all samples were wiped with minimal pressure using a lint-free cloth soaked with 70% ethanol to remove debris and contaminants before being dried.

| Laser scanning microscopy
Surface characterization of three Y-TZP discs from each treatment group was performed using laser scanning microscopy (LEXT OLS4100, Olympus Corporation, Japan). Three scanned areas, each measuring 1.29 Â 1.28 mm in dimension, were randomly selected on each sample for surface measurements. These measurements were carried out using a Gaussian filter, a low-pass smoothing filter designed to reduce noise and separate roughness from waviness and form (Munro et al., 2020). The following parameters were chosen to provide information related to various facets of surface topography:

| Scanning electron microscopy/Energydispersive X-ray spectroscopy (SEM-EDS)
To determine the elemental composition of zirconia discs following treatment, one Y-TZP disc from each treatment group was coated with a thin layer of carbon. An SEM (JSM-5410LV, Jeol, Japan) equipped with an EDS detector (Oxford instruments, X-Max detector, Oxford, UK) was used for surface analysis. EDS analysis was performed in three randomly selected points on each sample to detect and quantify the elemental composition of the zirconia discs before and after each treatment.

| Bacterial adhesion assay
Following surface analysis, Y-TZP discs were wiped with 70% ethanol and autoclaved at 134 C for 3.5 min in a steam sterilizer. The discs were placed into individual wells of 12-well cell culture plates in preparation for bacterial adhesion assay using a protocol adapted from Park et al. (2015).
After ethical approval was obtained from the James Cook University Human Research Ethics Committee (#H8260), pooled saliva was collected from healthy participants with no active dental disease or known medical conditions (n = 5) and centrifuged at 1500g for 10 min to remove debris. The supernatant containing salivary bacteria was collected and diluted in a 1:2 ratio with Todd-Hewitt Broth growth medium. A 5 mL aliquot of undiluted supernatant was centrifuged further at 8000g for 10 min to retrieve salivary glycoproteins essential for bacterial adherence. The supernatant containing the glycoproteins was removed and a 250 μL aliquot carefully dispensed onto each disc. The glycoproteins were allowed to attach for 30 mins to form an acquired pellicle. Subsequently, 2 mL of saliva/growth medium was added to each well containing a Y-TZP disc before being incubated at 37 C for 48 h. Following incubation, the saliva/growth medium was removed and discs rinsed with phosphate-buffered saline (PBS; pH 7.4) to remove any unattached bacteria. 1 mL of PBS was added to each well and discs sonicated for 10 min to detach adhered bacteria into the solution. The solution from each well was then aliquoted in triplicate into a 96 well cell culture plate. The number of bacteria present in each sample was estimated by determining optical density (OD 600 ) in a microplate absorbance reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories Inc, CA, USA).

| Scanning electron microscopy
Qualitative analysis of bacterial adhesion on treated Y-TZP discs was conducted using scanning electron microscopy (SEM) (Phenom™ G2 pro, Phenom-World BV, Netherlands). Bacteria were grown on Y-TZP discs for 48 h using the protocol described above. After rinsing, attached bacteria were fixed by immersion in 3% glutaraldehyde for 15 min followed by dehydration in graded concentrations of ethanol (25%, 50%, 75%, 95% and 100% ethanol for 5 mins at each concentration). The discs were then immersed in a 1:1 solution containing ethanol and hexamethyldilazane (HMDS) for 15 min followed by 100% HMDS for 5 min before being left to dry inside a fume hood for 24 h. The samples were mounted onto aluminum stubs using conductive carbon tabs before being sputter-coated with gold (Spi-Module™ Sputter Coater, SPI Supplies, USA) prior to SEM evaluation. Three areas on each sample were randomly selected for bacterial adhesion evaluation at 10,000Â magnification.

| Statistical analysis
Statistical analysis of data was performed using GraphPad 8.4 (GraphPad Software, CA, USA). Data related to surface parameters (Sa, Sz, Sku, and Ssk) and optical density (OD) was expressed as mean ± standard error measurements and analyzed using one-way ANOVA. The post-hoc Tukey test was used for multiple comparisons between groups. A p-value <0.05 was considered statistically significant.

| Energy dispersive X-ray spectroscopy
The results of the Energy dispersive X-ray spectroscopy (EDS) are shown in Table 1. Analysis revealed that all zirconia discs had a relatively high proportion of zirconium (Zr) and oxygen which are constituents of zirconium dioxide (ZrO 2 ), small amounts of yttrium (Y) that is F I G U R E 1 Changes to the surface of zirconia discs are visible after instrumentation. Discs were treated with instruments before being visually inspected and analyzed by laser scanning microscopy. iron (Fe) which are metallic elements commonly found in stainless steel. The deposition of metallic remnants, titanium (Ti), and barium (Ba), was also observed on TC treated surfaces. In terms of AA, unusual traces of gallium (Ga) and osmium (Os) were found along with calcium (Ca) and potassium (K) which are likely due to residual glycine powder remaining on the surface. Low levels of potassium (K) and sodium (Na) were also detected on surfaces treated with PC.

| DISCUSSION
This in-vitro study was designed to explore the effects of various physical decontamination methods on the surface characteristics of zirconia implant surface and subsequent bacterial adhesion following instrumentation. The results showed that zirconia implant surfaces can be altered based on the type of decontamination method used, although no significant differences in bacterial adhesion was observed.
Four decontamination methods were examined including the use of an US with stainless steel tip, plastic curette, TC and an air abrasive device with glycine powder. The TC, plastic curette and air abrasive device selected for this study were specifically designed and deemed F I G U R E 2 Three-dimensional laser scanning microscopy reveals differences in surface morphology after treatment. Images were obtained at three randomly selected sites using digital laser scanning microscopy and representative wireframes were generated. Wireframes are shown in micrometers (μm.) A-E representative images (10X magnification) of acid-etched Y-TZP samples following (a) no treatment (b); titanium curette (TC); (c) air abrasive device (AA); (d); plastic curette (PC); (e) ultrasonic Scaler (US) 'implant safe' for maintenance procedures. In terms of the US, further investigations were needed to determine their effects on zirconia implants as US with metal tips have been found to damage titanium implants (Harrel et al., 2019;Kawashima et al., 2007). Acid-etched Y-TZP discs were used in this study as acid-etching is a common surface modification technique designed to enhance osseointegration and the effects of instrumentation on acid-etched zirconia implant surfaces had yet to be explored (Flamant et al., 2016;Hafezeqoran & Koodaryan, 2017). While the exact protocol used by manufacturers to fabricate acid-etched commercial dental implants is undisclosed, Y-TZP discs were etched with 40% hydrofluoric acid in accordance with recommendations provided by Flamant et al. (2016). The baseline surface roughness (Sa) value of control acid-etched Y-TZP discs in this study was found to be 1.6 μm, which is slightly higher than some commercially available acid-etched zirconia implants with Sa values ranging from 0.73 to 1.27 μm (Beger et al., 2018). However, according to Albrektsson and Wennerberg (2004), implant surfaces classified as "moderately rough" with a Sa value between 1.0 μm and 2.0 μm may have some clinical advantage over smoother and rougher surfaces due to a stronger bone response.
Previous studies (Checketts et al., 2014;Huang et al., 2019;Lang et al., 2016;Vigolo et al., 2017;Vigolo & Motterle, 2010) Results of the topographical analyses by laser scanning microscopy on acid-etched zirconia discs after treatment. Surface parameters indicative of changes in surface morphology were determined for treated Y-TZP samples using 3D laser scanning microscopy. (a) Sa, arithmetic mean height; (b) Sz, maximum surface height; (c) Sku, kurtosis; (d) Ssk, skewness. Data is presented as mean ± standard error (3 sites per disc). Titanium curette (TC); air abrasive device (AA); plastic curette (PC); ultrasonic Scaler (US). * indicates p < 0.05 between two treatment groups according to post hoc Tukey test; # indicates p < 0.05 with all treatment groups T A B L E 1 Elemental composition (mean Wt%) of treated Y-TZP surfaces analyzed (three sites per disc) using energy dispersive x-ray spectroscopy (EDS). Titanium curette (TC); air abrasive device (AA); plastic curette (PC); ultrasonic Scaler (US)  In separate experiments attached bacteria were dislodged and numbers estimated by measurement at OD600 (f). Results are presented as mean ± standard error, (three discs per group) than surface deterioration. Huang et al. (2019) and Lang et al. (2016) found no significant changes to the surface roughness of zirconia fol-  (Huang et al., 2019). Based on the present and past studies, a direct correlation between decontamination-induced surface roughness and bacterial adhesion on zirconia surfaces could not be established. This may be due to the influence of other surface factors such as wettability, surface-free energy and surface chemistry which also affect bacterial adhesion (Teughels et al., 2006).
One of the key limitations of the present study was that the effects of instrumentation were assessed on zirconia discs rather than root form implant fixtures consisting of numerous threads and valleys.
In addition, it is difficult to directly quantify bacterial numbers using OD measurements, especially in a salivary biofilm containing a diverse range of different bacteria species. To improve on this study, future studies investigating the effects of instrumentation on the surface topography of implant fixtures rather than the flat surface of zirconia discs are required. In addition, the effects of implant surface changes induced by instrumentation on cellular interactions needs to be explored. Saliva samples collected in peri-implant pockets would also provide a better representation of microbial species residing around dental implants due to variations in the oral microbiome within different areas of the oral cavity (Kilian et al., 2016). Finally, it remains to be determined how well these instruments perform in clinical practice.
The cleaning efficacy of these instruments should be assessed as their effectiveness depends upon their ability to access implant threads within the peri-implant region.

| CONCLUSION
Within the limitations of this study, air abrasive devices and plastic curettes may be a suitable option for zirconia implant decontamination as minimal surface changes were seen following their use. In contrast, US with stainless steel tips and TC should be used cautiously due to the deposition of metallic remnants on the surface that may present a biological and aesthetic concern. However, further studies are required to clarify the effects of these decontamination methods within the clinical setting.