The Impact of Using Laser and Milling Techniques to Create Zirconia Patterns on Streptococcus oralis Biofilm Formation

Abstract

This study aimed to evaluate zirconia dental implant surfaces patterned using Nd:YAG laser or conventional milling techniques against Streptococcus oralis adhesion and biofilm formation. Zirconia dental implant discs were subjected to surface patterning treatments and categorized into four groups: groove texturing by conventional milling (GM), pore texturing by conventional milling (PM), groove texturing by Nd:YAG laser (GL), and pore texturing by Nd: YAG laser (PL). Streptococcus oralis CECT 907T was cultivated on enriched blood agar plates and then transferred to a brain–heart infusion modified medium and incubated at 37 °C under anaerobic conditions until reaching the exponential growth phase. The bacterial suspension was then seeded on 24-well plates containing the treated discs. The viability of bacteria within the biofilm was determined based on colony-forming unit (CFU) counts, while the total biofilm was quantified by measuring its biomass. A qualitative analysis was conducted using scanning electron microscopy (SEM) images to evaluate the bacterial morphology. The statistical analysis of multigroup comparisons was performed using Kruskal–Wallis test with post hoc pairwise comparison, as well as Mann Whiney U test, with significance set at p < 0.05. After both 1 h and 24 h of incubation of Streptococcus oralis on the discs, all groups showed similar results, with no statistically significant differences (p > 0.05). A comparison of the Nd: YAG laser-treated surfaces with conventionally milled surfaces, as well as grooves versus pores for CFU counts, also revealed no statistically significant differences (p > 0.05) for both 1 h and 24 h of culture. Biomass quantification at both the 1 h and 24-h time points showed similar results across the groups, without statistical differences. When comparing the conventionally machined surfaces to Nd: YAG laser-treated surfaces in terms of biomass, no significant differences were observed (p > 0.05). Similarly, the comparison between groove-patterned surfaces and pore-patterned surfaces showed no statistically significant difference. The groove and pore patterns on zirconia surfaces with Nd: YAG laser or conventional milling did not change the Streptococcus oralis adhesion and biofilm formation behavior. Additional studies are recommended to expand our knowledge in this area.

1. Introduction

The use of dental implants to replace missing teeth in edentulous patients is a well-established practice in dentistry [1]. The number of implant placements has increased significantly, with over 800,000 implants being placed annually in the United States and more than 1.8 in the European Union [2].

Titanium (Ti) has traditionally been the material of choice for dental implants due to its proven success. However, its drawbacks such as soft tissue discoloration, hypersensibility, and its role in the progression of peri-implantites, have driven interest in alternative materials [3].

Zirconia (ZrO₂) is a non-resorbable, bioinert metal oxide that is known for its strong mechanical properties [4]. It has emerged as a promising alternative, with over 1 million hip joint replacements using zirconia, demonstrating its superior corrosion resistance, mechanical strength, and flexibility. Its natural tooth-like color and translucency also make it esthetically favorable in dental applications. Zirconia’s biocompatibility and potential for strong osseointegration further enhance its appeal for use in dental implants [5,6].

Among the types of zirconia_, 3 mol % yttrium partially stabilized tetragonal polycrystalline zirconia (3Y-TZP) is widely used in dental implants [2,7,8] due to its high flexural strength (900–1200 MPa) and fracture toughness (8–10 MPa m^½). This material also exhibits low microbial adhesion, favorable osseointegration, and a color close to that of natural teeth, providing esthetic benefits over titanium implants [5,9,10]. Zirconia applications include crowns, full-arch fixed implant prostheses, implant abutments, and dental implants [11].

The success of an implant rehabilitation depends heavily on osseointegration, which can be hindered by microbial biofilm formation on implant surfaces, leading to peri-implantites. Studies have shown a strong link between biofilm presence and implant failure, with periodontopathic bacteria such as Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans increasing the risk of peri-implantites. Many researchers have evaluated a variety of microorganisms using different culture models, including monoculture [2,4,12,13,14,15,16,17,18], multispecies biofilm [16,19,20,21], and co-culture [2,22], because it is essential to understand these dynamics, as the quality and quantity of bacteria that are present on the implant surface are critical factors in determining whether an implant succeeds or fails.

Compared to titanium, zirconia implants have demonstrated potential to reduce microbial adhesion. Al-Radha and coworkers studied the bacterial adhesion to zirconia, titanium, titanium blasted with zirconia, and titanium blasted with zirconia/acid and etched and discovered that zirconia and titanium blasted with zirconia showed superior effects compared to titanium in reducing the adhesion of bacteria [4].

To enhance the surface properties of zirconia and reduce biofilm formation, various surface modification techniques have been investigated, including the incorporation of antibacterial agents, antimicrobial coatings, and grafted antibacterial compounds. However, many of these approaches may compromise the implant’s mechanical strength. Thus, the development of antibacterial zirconia implants that retain both their mechanical integrity and biocompatibility is highly desirable [23].

While a significant amount of research has addressed biofilm formation on patterned titanium dental implants [24,25,26,27,28,29,30,31], there are comparatively limited data on biofilm formation on patterned zirconia dental implants [16,18,32,33,34,35,36]. In a recent study, Ghalandarzadeh and colleagues (2024) introduced a femtosecond laser-modified zirconia surface and evaluated its effects on periodontal cells, as well as on periodontopathic bacteria. Their findings indicated that textured zirconia surfaces significantly reduce bacterial adhesion, with cell viability assays, cell morphology observations, high levels of collagen I, and interleukin−1β further supporting the biocompatibility of these surfaces [36]. Additionally, our previous work has shown that groove and pore patterns on zirconia surfaces, created via Nd: YAG laser or conventional milling, support the biocompatibility of these surfaces [37,38]. These findings suggest that surface modification is a promising approach to achieve both antibacterial and biocompatible properties on zirconia surfaces. Consequently, we conducted this study to investigate Streptococcus oralis biofilm formation on these modified surfaces.

Therefore, this in vitro study aims to evaluate the impact of Nd: YAG lasers and conventional milling techniques on Streptococcus oralis biofilm formation on zirconia, comparing the impacts of different patterns, including grooves and pores, created by each technique.

2. Materials and Methods

2.1. Specimen Preparation

All discs used in this study were manufactured using commercial yttria-stabilized zirconia powder (YTZP), uniformly dispersed with 3 mol % yttria (Basic Grades, Tosoh Corporation©, Amsterdam, The Netherlands). The discs were precisely engineered to a diameter of 8 mm and a thickness of 2 mm through cold-pressing techniques, and then, the discs were textured and patterned via conventional milling and Nd: YAG laser (OEM Plus, Sisma, Vicenza, Italy), as extensively documented in previous publications [37,38,39,40,41,42].

Following manufacture, the zirconia discs were subjected to a sintering process in an oven (Zirkonofen 700, Gais BZ, Italy) and subsequently cleaned with isopropyl alcohol. The discs were then blasted with alumina particles (Al₂O₃) for 30 s at a pressure of 6 bar to standardize their surface roughness, followed by a washing procedure. Each disc was immersed in 48% hydrofluoric acid (HF) for 30 min at room temperature, after which it underwent a 5 min immersion in isopropyl alcohol. The samples were then subjected to ultrasonic cleaning in absolute ethanol (100%) (Laborspirit, Lisboa, Portugal) and sterilized via autoclaving to ensure their suitability for subsequent biological assays.

The physical and mechanical properties of the zirconia samples, including their surface roughness, wettability, hardness, and cutting resistance, were evaluated and compared according to the methodologies described in our previous publications [37,38,39,40,41,42].

Before the biological test, the surface morphologies and topographical features of the zirconia samples were meticulously examined using scanning electron microscopy (SEM) (JSM-6010 LV, JEOL Ltd., Tokyo, Japan). Images were acquired at a magnification of 100× with an acceleration voltage of 10 kV. Additionally, atomic contrast images were obtained utilizing a Backscattering Electron Detector (BSED) under an acceleration voltage of 15 kV, as per the protocols established in our previous studies [37,38,39,40,41,42].

The discs were randomly allocated into four experimental groups (n = 21 per group) based on the specific surface patterning method: groove patterns by conventional milling (GM), pore patterns by conventional milling (PM), groove patterns by Nd: YAG laser (GL), and pore patterns by Nd: YAG laser (PL), as shown Table 1 and Figure 1.

2.2. Bacterial Strain and Growth Conditions

The Streptococcus oralis CECT 907T strain (CECT, Valencia, Spain) was cultured on enriched blood agar (Frilabo, Portugal) plates at 37 °C for 72 h under anaerobic conditions (10% CO₂, 10% H₂, and balance N₂) (Acail Gás, Soure, Portugal). A single colony was subsequently inoculated into 15 mL of brain–heart infusion modified medium (BHI-2) (Fisher Scientific, Porto Salvo, Portugal) and incubated overnight at 37 °C under anaerobic conditions. A bacterial suspension with an initial optical density (OD) of 0.05 was prepared and incubated under anaerobic conditions at 37 °C until reaching the exponential growth phase. The exponential phase was confirmed spectrophotometrically at 550 nm using a Camspec M50 spectrophotometer (Spectronic CamSpec, West Yorkshire, UK) with the OD of 0.4. Zirconia discs from the four groups (GM, PM, GL, and PL) were randomly selected and placed in 24-well plates (Frilabo, Maia, Portugal). Streptococcus oralis in the exponential growth phase was added to each well, followed by incubation under anaerobic conditions at 37 °C.

2.2.1. Colony-Forming Unit (CFU)

The viable bacteria on the zirconia discs were assessed by quantifying the CFU after 1 h and 24 h of culture.

To determine the number of viable bacteria that adhered to the discs, each disc was washed with filtered phosphate-buffered saline (PBS) (VWR, Carnaxide, Portugal) and placed in a Falcon tube containing 3 mL of PBS. The tubes were vortexed at 1600 rpm for 1 min, followed by ultrasonication for 4 min and an additional 2 min of vortexing at 1600 rpm. Serial ten-fold dilutions were prepared up to 10⁻⁶, and 20 μL of each dilution was plated in triplicate on brain–heart infusion agar (BHIA) (Frilabo, Maia, Portugal). BHIA plates were incubated at 37 °C under anaerobic conditions, and the CFU was counted after 24 h incubation periods.

2.2.2. Biomass—Crystal Violet (CV)

The biomass on the zirconia discs was assessed after 1 h and 24 h of Streptococcus oralis culture. Following incubation, the discs were washed with sterile distilled water (dH₂O) and air-dried in a laminar flow chamber cabinet for 2 h. Subsequently, 500 μL of 1% crystal violet (certified by the Biological Stain Commission, Merck, Algés, Portugal) solution was added to each well and incubated for 20 min at room temperature. The dye was removed, and the samples were washed twice with 400 μL of sterile dH₂O to remove any non-adherent dye. The remaining biomass-bound dye was solubilized with 500 μL of 33% glacial acetic acid, and 100 μL of the resulting solution was transferred to a 96-well plate (Corning^®, Corning, NY, USA). The optical density was measured at 595 nm using a multimode reader (VICTOR NivoTM HH3500, PerkinElmer^®, Pontyclun, UK).

2.2.3. Scanning Electron Microscopy (SEM)

To examine the bacterial cell morphology, Streptococcus oralis was cultured on zirconia discs for 1 h and 24 h. The discs were washed with filtered PBS, fixed with 2.5% glutaraldehyde (VWR, Carnaxide, Portugal), and dehydrated in a graded ethanol (Laborspirit, Lisboa, Portugal) series (20 to 100%), with each concentration applied for 15 min. The samples were then coated with a 15 nm ultrathin gold–palladium film (80–20% in weight). SEM was conducted using JEOL JSM5200-LV microscopy (JEOL JSM5200-LV, Jeol Ltd., Tokyo, Japan) at 25 Kv with ×5000 magnification, using a 5 μm scale bar.

2.3. Statistical Analysis

All experiments were performed in triplicate, with three independent repetitions to ensure the reliability of the results. Statistical analyses were conducted using the IBM^® SPSS^® Statistics for Mac (version 28.0 SPSS, Armonk, NY, USA). Data normality was evaluated using the Shapiro–Wilk test. Comparisons between groups for CFU counts and biomass were conducted using the Kruskal–Wallis test with post hoc pairwise comparison to identify groups with significant differences, as well as Mann Whiney U test. A significance of p < 0.05 was applied. Data are presented as mean ± standard deviation (SD).

3. Results

3.1. Colony-Forming Unit

The CFU counts of Streptococcus oralis on the zirconia discs patterned using conventional milling or Nd: YAG laser techniques were assessed after 1 h and 24 h incubation periods, as shown in Figure 2, Figure 3 and Figure 4. Overall, the CFU count per milliliter increased from 1 h to the 24 h incubation period of Streptococcus oralis. After both 1 h and 24 h of incubation, the CFU counts across all groups were similar, with no statistically significant difference (p > 0.05).

3.1.1. Conventional Milling Versus Nd: YAG Laser

When comparing the CFU counts per milliliter on zirconia discs patterned using conventional milling versus Nd:YAG laser techniques, no statistically significant differences were observed between these groups after both 1 h and 24 h incubation periods (p > 0.05).

3.1.2. Groove Versus Pore

The CFU analysis indicated similar results for both groove- and pore-patterned zirconia discs, with no statistically significant differences observed after 1 h and 24 h incubation periods (p > 0.05).

3.2. Biomass (CV)

The biomass was quantified after 1 h and 24 h of incubation of Streptococcus oralis on the zirconia discs (Figure 5, Figure 6 and Figure 7). Across all groups, the biomass increased from the 1 h to the 24 hours’ time point. For both the 1 h and 24 h incubation periods, the biomass was similar across all groups, with no statistically significant differences (p > 0.05).

3.2.1. Conventional Milling Versus Nd: YAG Laser

After 1 h of incubation, the biomasses on the zirconia discs patterned via conventional milling and Nd: YAG laser showed no statistically significant differences (p < 0.05). Similarly, no statistically significant differences were observed after 24 h of incubation (p > 0.05).

3.2.2. Groove Versus Pore

When comparing the groove- and pore-patterned zirconia surfaces, the biomass results were similar across all groups, without statistically significant differences (p > 0.05).

3.3. SEM

The SEM images of Streptococcus oralis on the zirconia discs patterned using conventional milling and Nd:YAG laser techniques are presented in Figure 8. The images revealed a typical coccoid morphology of planktonic bacteria. Bacterial growth was evident in all groups, with more pronounced growth observed after 24 h of incubation, indicating increased adhesion and biofilm formation compared to the 1 h time point.

4. Discussion

This study evaluated zirconia surfaces patterned with grooves and pores, using conventional milling and Nd: YAG laser techniques, against Streptococcus oralis biofilm formation.

In our study, we tried to understand the real effect of surface patterning on zirconia, so all surfaces were initially sandblasted and acid-etched. This standardized pretreatment ensured that the bacterial adhesion and biofilm formation observed were primarily due to the surface patterns and the methods used to create them, providing a controlled comparison between different patterned zirconia surfaces [38,43].

An overall increase in CFU counts and biomass was observed from 1 h to 24 h incubation periods across all groups, which is consistent with the expected progression of bacterial biofilm development and the technique used. These results were corroborated by the biomass quantification and the SEM analyses [17,18].

The CFU, as well as the biomass, show similar results across the groups from 1 h to 24 h, without statistically significant differences, revealing that none of these tested groups had better results in terms of Streptococcus oralis adhesion and biofilm formation. Probably, the bacterial behavior is more influenced by other surfaces parameters, such as surface free energy, wettability, and bacterial proliferation dynamics, rather than roughness, grooves, or pores created by conventional milling or Nd:YAG laser [35,44].

In this context, we compare conventional milling with Nd:YAG laser patterning surfaces, but our study showed there were no statistical differences observed between groups. However, previous studies by Bihn et al. (2023) [34] have indicated that surfaces treated with nanosecond laser exhibit hydrophobic properties, which tend to reduce the bacterial adhesion and biofilm formation, but they also found that the inhibitory effect may vary depending on the type of bacteria. As they evaluated three periodontopathic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, they discovered that Porphyromonas gingivalis have less bacteria adhesion to hydrophobic surfaces, which was due to the hydrophobic surface topography achieved using a nanosecond laser [34]. Parmar and coworkers, in 2018 [45], reported that laser surfaces had lower bacterial adhesion than untreated samples. Ghalandarzadeh et al. (2024) studied the zirconia surface topography via femtosecond laser and found a substantial reduction in bacterial adhesion on textured zirconia surfaces. However, it is worth mentioning that they incorporated ciprofloxacin onto zirconia, which is an antibiotic in the fluoroquinolone class that is used to treat bacterial infections. Probably, these contradictory results with our study can be correlated with differences in the wavelength and energy of the used lasers, the roughness of the surface materials, and the incorporated compounds or antibacterial agents.

Quantifying the bacterial biomass is essential for understanding biofilm maturation, as the biomass contributes to biofilm stability through the production of extracellular polymeric substances (EPSs) [46], which facilitate initial bacterial attachment. However, it is important to know that the biomass measures all the bacteria that are present in a sample—either live or dead—which differs from the CFU, which only evaluates live bacteria that are present in the sample. Different results were found by Guastaldi et al. (2019) [47], who observed that laser irradiation increased the bacterial adhesion to titanium surfaces. Drago et al. (2016) [48] also reported different results from ours. They found significantly lower biofilm biomass and bacterial volume on laser-treated titanium discs compared to sandblasted discs. An interesting result was found by Wassmann and colleagues in 2017, when they were evaluating the effects of the surface texture and wettability on the initial bacterial adhesion on titanium and zirconia, suggesting that the influence of the surface modification on bacterial adhesion does not follow universal rules and differs between species. In this context, the comparison of textured zirconia surfaces with other works may be limited, since most of them used different bacteria, which can lead to different results. Furthermore, different implant materials may provide different results, which also limits the comparison between these studies.

Comparing groove versus pore patterning, there were no differences in the bacterial behavior in terms of the CFU and biomass quantification. It is difficult to compare our results with the recent literature, since there is no standardization of the types and techniques of patterning or types of bacteria used in different studies. For example, Uhlmann et al. (2018) [49] stated that microcavities supported less bacterial adhesion than microchannels when tested on laser-textured titanium implants. Additional studies with silver coatings combining different microgrooves showed an effective inhibition of bacterial adhesion and biofilm formation by Porphyromonas gingivalis [50]. The standardization of the types of patterns or texturing and techniques used to make them is essential, as it allows us to make a proper comparison between studies.

This study relates to our group’s research on zirconia surface modifications. These surfaces were previously improved and tested with periodontal cells, specifically fibroblasts and osteoblasts, which indicated that different types and techniques used on zirconia surfaces do not seems to influence the cellular behavior [37,38,39,40,41,42]. We therefore conducted this study to focus on bacterial interactions. The results reveal that bacterial adhesion and biofilm formation also do not seem to be improved by these surface modifications.

This study provides valuable insights into the influence of surface patterning on bacterial adhesion and biofilm formation on zirconia surfaces. However, some limitations should be noted, including the small sample size, the use of a single bacterial species, and the examination of a specific dental material. Future studies are needed to expand on these findings and further optimize zirconia surface modifications for enhanced antibacterial properties.

5. Conclusions

The use of dental implants has increased significantly over the years, yet complications, particularly peri-implantitis, remain common. To address this, it is essential to explore surface modification strategies, such as patterned surfaces, to enhance both the biocompatible and antibacterial properties of implants. Zirconia surfaces patterned with grooves and pores using conventional milling and Nd:YAG laser techniques do not seem to have significant effects against Streptococcus oralis adhesion and biofilm formation. Further studies are recommended to expand knowledge related to the interactions between surface patterns and bacterial behavior, which could inform future strategies for implants’ surfaces that work against bacterial adhesion and biofilm formation.

Author Contributions

Conceptualization, A.M., J.C., F.S. and M.B.d.C.; methodology, M.B.d.C., N.S. and J.M.; validation, A.M., J.C., F.S. and M.B.d.C.; formal analysis, N.S. and M.B.d.C.; investigation, N.S., M.B.d.C. and J.M.; resources, A.M., J.C. and F.S.; data curation, N.S. and M.B.d.C.; writing—original draft preparation, N.S.; writing—review and editing, M.B.d.C. and N.S.; visualization, M.B.d.C., N.S. and J.M.; supervision, M.B.d.C. and J.M.; project administration, A.M., J.C., F.S. and M.B.d.C.; funding acquisition, A.M., J.C. and F.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data will be available upon request.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1. Schematic representation of zirconia disc production followed by randomization, texturing and patterning, sandblasting, and acid-etching. YTZP—yttria-stabilized zirconia powder; GM—groove patterns by conventional milling; PM—pore patterns by conventional milling; GL—groove patterns by laser Nd: YAG; PL—pore patterns by laser Nd: YAG; SBAE—sandblasted and acid-etched.

Figure 2. Bar charts representing the mean CFU count per milliliter of Streptococcus oralis, presented as mean values, with error bars representing the standard deviation (n = 9). Group comparisons were performed using the Kruskal–Wallis test with post hoc pairwise comparisons. Statistical significance was set at p [less than] 0.05. GM—groove patterns by conventional milling; PM—pore patterns by conventional milling; GL—groove patterns by laser Nd: YAG; PL—pore patterns by laser Nd: YAG.

Figure 3. Bar charts representing the CFU count per milliliter of Streptococcus oralis on conventional milling versus Nd:YAG laser zirconia-patterned surfaces, presented as mean values, with error bars indicating standard deviation (n = 18). Group comparisons were conducted using the Mann–Whitney U Test. Statistical significance was set at p [less than] 0.05.

Figure 4. Bar charts representing the CFU count per milliliter of Streptococcus oralis groove-patterned zirconia discs versus pore-patterned zirconia discs, presented as mean values, with error bars representing the standard deviation (n = 18). Group comparisons were conducted using the Mann–Whitney U Test. Statistical significance was set at p [less than] 0.05.

Figure 5. Bar charts illustrating the biomass quantification of Streptococcus oralis, presented as mean values, with error bars indicating standard deviation (n = 9). Group comparisons were conducted using the Kruskal–Wallis test with post hoc pairwise comparisons. Statistical significance was set at p [less than] 0.05. GM—groove patterns by conventional milling; PM—pore patterns by conventional milling; GL—groove patterns by laser Nd: YAG; PL—pore patterns by laser Nd: YAG.

Figure 6. Bar charts depicting the biomass quantification of Streptococcus oralis on conventional milling versus Nd:YAG laser-patterned zirconia surfaces, presented as mean values, with error bars representing the standard deviation (n = 18). Group comparisons were conducted using the Mann–Whitney U Test. Statistical significance was set at p <0.05.

Figure 7. Bar charts depicting the biomass quantification of Streptococcus oralis on groove- versus pore-patterned zirconia surfaces, presented as mean values, with error bars representing the standard deviation (n = 18). Group comparisons were conducted using the Mann–Whitney U Test. Statistical significance was set at p [less than] 0.05.

Figure 8. SEM images of Streptococcus oralis cultured on patterned zirconia after 1 h and 24 h incubations periods (n = 3). Images were acquired at 25 kV, ×5000 magnification, with scale bars representing 5 μm. GM—groove patterns by conventional milling; PM—pore patterns by conventional milling; GL—groove patterns by laser Nd: YAG; PL—pore patterns by laser Nd: YAG.

Table 1

Description of the experimental groups and corresponding surface patterning methods.

Name	Abbreviation	n	Type of Surface Treatment
Zirconia groove patterns by conventional milling	GM	21	Groove patterns by conventional milling
Zirconia pore patterns by conventional milling	PM	21	Pore patterns by conventional milling
Zirconia groove patterns by laser Nd: YAG	GL	21	Groove patterns by laser Nd: YAG
Zirconia pore patterns by laser Nd: YAG	PL	21	Pore patterns by laser Nd: YAG

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