Materials and methods

Ethics committee approval

As stated in the letter dated September 19, 2025, issued by the Department of Oral Implantology, Faculty of Dentistry, Saint-Joseph University of Beirut, this study (file no. Tfemd-2026-18) was reviewed at the meeting of the Saint-Joseph University of Beirut, Faculty of Dentistry Clinical Research Ethics Committee and was found ethically appropriate.

Determination of sample size

This was an in vitro controlled comparative study evaluating four implant surface conditions: untreated control, 0.12% CHX decontamination, GalvoSurge® (Straumann, Basel, Switzerland), and Er,Cr:YSGG laser solid-state.

A total of 48 SLA tissue-level titanium dental implants (Straumann® SP WN SLA, 4.8 mm × 10 mm) were included (n = 12/group). The sample size of 12 implants per group was selected based on the expected large effects reported in previous in vitro implant decontamination studies using CFU outcomes [11,16,17]. In controlled microbiological models, variability is generally limited, and treatment effects are typically large, allowing detection of biologically relevant differences with moderate group sizes. After data collection, effect-size estimation for the overall treatment effect showed η² = 0.883, indicating that 88.3% of the variance in bacterial load was attributable to treatment at α = 0.05. This value is an effect-size estimate and was not interpreted as statistical power. The primary outcome was viable bacterial load expressed as CFU per implant. All procedures were performed by a single operator in Wakim Laboratory (Jounieh, Lebanon).

Experimental model, biofilm formation, and decontamination procedures

Each implant was mounted in a custom three-dimensional-printed model designed to simulate a standardized peri-implant defect while ensuring mechanical stability during contamination, decontamination, and sampling procedures. The model was designed using computer-aided design (CAD) software and fabricated using additive manufacturing technology. The defect geometry was standardized to expose the coronal implant threads while maintaining complete embedding of the apical portion, thereby simulating the implant surface exposure encountered in peri-implant bone loss. All implants were positioned at the same depth and orientation within the model to ensure identical exposure of the contaminated surface area among specimens. The model covered the apical portion of the implant while leaving the predetermined coronal threads exposed, restricting contact exclusively to the contamination medium, the assigned decontamination protocol, and the sampling instrument.

To standardize the simulated peri-implant defect, the level of implant exposure was predetermined before specimen preparation, with the same number of implant threads exposed in all samples. This approach ensured comparable biofilm accumulation and equivalent accessibility of the contaminated implant surface during decontamination procedures.

Biofilm formation was achieved by placing each implant individually in a sterile container containing 40 mL of brain-heart infusion broth supplemented with 4 mL of _Staphylococcus aureus _inoculum adjusted to 3 McFarland in sterile saline. Samples were incubated at 37°C for 72 hours under controlled conditions to allow biofilm development. All decontamination procedures were performed at room temperature under standardized laboratory conditions. The 72-hour incubation period was selected because in vitro biofilm susceptibility studies have used 72-hour Staphylococcus aureus biofilms as older or mature biofilm models for antimicrobial testing, reporting increased cellular density and/or greater antimicrobial tolerance compared with 24-hour biofilms [18,19]. Individual contamination prevented cross-interference and preserved each implant as an independent experimental unit.

Following contamination, implants were randomly assigned to four groups (n = 12 per group): Group 1, Control (no decontamination); Group 2, 0.12% (CHX); Group 3, GalvoSurge; Group 4, Er,Cr:YSGG laser. Decontamination procedures were performed immediately after removal from the contamination medium under standardized environmental conditions to minimize variability.

Implants in the control group did not undergo any decontamination procedure but were handled identically and exposed to ambient conditions for a duration equivalent to the longest treatment time prior to sampling. Chemical decontamination consisted of immersion in 0.12% CHX for 60 seconds, followed by rinsing with sterile saline (0.9% NaCl) for 20 seconds to neutralize residual antimicrobial activity and prevent carryover effects during microbiological analysis. Electrolytic cleaning was carried out using the GalvoSurge system (Straumann AG, Basel, Switzerland) according to the manufacturer's clinical workflow. The system generates electrochemical reactions at the implant surface through the controlled application of an electrolyte solution and an electric current, resulting in the disruption and removal of biofilm deposits. The applicator was positioned to ensure complete electrolyte contact and coverage of all exposed implant threads. The device was activated for 120 seconds, corresponding to the recommended clinical application time, while maintaining continuous electrolyte flow throughout the procedure. Laser decontamination was performed using a Waterlase MD Er,Cr:YSGG system (BIOLASE, Irvine, CA, USA; 2780 nm) operated in H mode with an MZ6 tip at 2.5 W and 30 Hz, with 70% water and 60% air in non-contact mode. The tip was oriented at 90 degrees relative to the implant surface and applied along the exposed threads for a standardized duration of 30 seconds to ensure complete coverage of the contaminated area. A cohort flowchart illustrating the implant grouping was presented in Table 1.

Group | n | Protocol | Key parameters | Sampling after treatment
Control | 12 | No decontamination | Handled identically and exposed to ambient conditions for equivalent time | Microbrush recovery into 1 mL sterile saline
CHX | 12 | Chemical decontamination | 0.12% CHX immersion for 60 s; sterile saline rinse 20 s | Microbrush recovery into 1 mL sterile saline
GalvoSurge | 12 | Electrolytic cleaning | Manufacturer workflow; standardized 120 s activation; complete electrolyte contact with exposed threads | Microbrush recovery into 1 mL sterile saline
Laser | 12 | Er,Cr:YSGG laser | Waterlase MD (BIOLASE, Irvine, CA, USA); 2780 nm; H mode; MZ6 tip; 2.5 W; 30 Hz; 70% water; 60% air; 30 s | Microbrush recovery into 1 mL sterile saline

Following biofilm formation, implants were randomly allocated to the experimental groups using a computer-generated randomization sequence to ensure balanced distribution among treatment conditions. The operator responsible for biofilm recovery and CFU quantification was blinded to the treatment allocation.

Biofilm recovery and CFU quantification

After treatment, residual biofilm was collected using a sterile microbrush passed circumferentially along all exposed implant threads for approximately 15 seconds per implant. The microbrush tip was transferred into 1 mL of sterile saline, vortexed, and subjected to serial dilution. Aliquots of 0.1 mL were plated onto agar media for colony enumeration. CFU per implant was calculated using the standard plate count method, with preference given to plates containing 30 to 300 colonies. CFU values were log10-transformed prior to analysis to account for the skewed distribution typical of microbiological data [14,15]. Colony counting was performed by a calibrated examiner using standardized plate-counting criteria. Plates containing 30-300 colonies were considered valid for enumeration, and duplicate readings were performed to ensure counting consistency. Representative images of the experimental setup have been added to enhance methodological transparency and facilitate replication of the experimental protocol (Figure 1).

Figure 1: Experimental setup and decontamination protocol(A) Close-up view of the implant specimen during electrolytic cleaning treatment, showing the working tip positioned adjacent to the implant surface. (B) Standardized implant specimens mounted in individual experimental blocks before allocation to the study groups. (C) Er,Cr:YSGG laser unit used for implant surface decontamination, showing the selected operating parameters. (D) Implant specimen immersed in storage medium, showing a macroscopically visible structure suggestive of biofilm maturation

Statistical analysis

Statistical analyses were conducted using RStudio (version 2025.09.0, “Cucumberleaf Sunflower” Release; R Foundation for Statistical Computing, Vienna, Austria). One-way analysis of variance (ANOVA) was used for group comparison, with Tukey post hoc testing for pairwise contrasts. When homogeneity of variance was not fully satisfied, Welch robust testing and complementary nonparametric analysis were used to assess the stability of the findings. Statistical significance was set at p < 0.05. Several steps were incorporated to improve reproducibility: the experimental unit was the implant, all groups had equal sample size, decontamination times were standardized, and the same recovery, dilution, plating, and counting workflow was applied across all samples. These details are reported to facilitate replication and comparison with future implant-biofilm studies. A schematic flowchart illustrating the experimental workflow is presented in Figure 2.

Figure 2: The experimental workflowImage credit: created by the author (RB) using Microsoft PowerPoint (Microsoft Corporation, Redmond, WA, USA)