Advancing Biofilm Removal: Evaluating Electrolytic Methods for Decontaminating Dental Implants In Vitro

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B. Castro, Jill Hadisurya, Kenneth Simoens, Mrinal Gaurav Srivastava, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5402056/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study evaluated the decontaminating efficacy of two electrolytic cleaning systems on titanium implants contaminated with multispecies biofilm, compared to conventional treatments like 0.2% chlorhexidine (CHX) or local antibiotics (tetracycline), with phosphate-buffered saline (PBS) as a negative control. Materials and Methods A 14-species oral microbial community, developed using a bioreactor system, was used to grow biofilms on dental implant surfaces. Implants were then treated with two electrolytic systems, CHX, tetracycline, or phosphate-buffered saline (PBS). After cleaning, the implants were reincubated for 24 h. Biofilm viability was assessed through viability DNA extraction and quantitative PCR (v-qPCR). Additionally, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze biofilm structure and viability respectively. Results CHX and tetracycline treatments significantly reduced viable biofilm bacteria by 98.8% and 99.6%, respectively, compared to the negative control. The effect of the two electrolytic systems varied, with one reducing biofilm by 93.3%, similar to the positive controls, while the other showing only a 5.7% reduction in biofilm viability. SEM and CLSM imaging confirmed the distinct effects of the treatments on biofilm structure and viability. Conclusion The findings of this study highlight the potential of electrolytic cleaning as an effective minimally invasive approach for peri-implantitis management. However, this promising efficacy of the electrolytic cleaning systems alongside traditional antimicrobial agents in biofilm removal from dental implant surfaces greatly depend on the system. Further research is warranted to optimize different electrolytic cleaning protocols and validate their clinical efficacy in preventing and treating peri-implantitis. Dental Implants Peri-Implantitis Oral Biofilms Infection Decontamination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background According to the S3-level clinical practice guidelines by the European Federation of Periodontology (EFP), peri-implantitis is defined as an inflammatory condition triggered by the peri-implant accumulated biofilms ( 1 ). This condition manifests as either peri-implant mucositis or peri-implantitis. Peri-implant mucositis refers to inflammation confined to the peri-implant mucosa without marginal bone loss ( 2 ), while peri-implantitis involves both the soft tissues and the surrounding bone. The reported prevalence of peri-implantitis and peri-implant mucositis varies across studies. A systematic review commissioned at the XI European Workshop of Periodontology in 2014 reported a patient-level prevalence of 43% for peri-implant mucositis and 22% for peri-implantitis ( 3 ). The accumulation of peri-implant plaque biofilm is the primary etiological factor for the development and progression of peri-implantitis. In addition, a history of severe periodontitis, inadequate plaque control, and the lack of a rigorous maintenance regimen, are significant risk factors for peri-implantitis and peri-mucositis development. As to the growing numbers of dental implants, the prevalence of infected implants is also rising. Clinical manifestations include bleeding on probing (BoP), increased periodontal pocket depth (PPD), as well as inflammation signs such as erythema, swelling, and/or suppuration. In more advanced cases, marginal bone loss can take place, resulting in crater-like defects around the affected implants, which are often more severe than periodontitis bone loss ( 4 ). A radiographic examination is essential for confirming the diagnosis ( 5 , 6 ). The aim of peri-implantitis treatment is to alleviate inflammation via eliminating microbial biofilms and to prevent any further loss of the supporting bone. As outlined in the EFP S3 level clinical practice guidelines, managing peri-implantitis involves two distinct phases: an initial non-surgical treatment phase, which may be followed by surgical intervention depending on the observed outcomes ( 1 ). Even though non-surgical treatment, particularly sub-marginal instrumentation, plays a critical role in the initial phase of peri-implantitis management, its effectiveness remains a subject of ongoing research. Non-surgical debridement can help reduce inflammation and microbial load; however, its efficacy is often limited, particularly in advanced peri-implantitis cases where significant bone loss has already occurred ( 7 ). This limitation emphasize the necessity of subsequent surgical intervention, to allow direct access to the implant contaminated surface for thorough surface decontamination. Various techniques have been employed in peri-implantitis treatment, including mechanical subgingival debridement with hand instruments or ultrasound-driven curettes, as well as the use of brushes, lasers, pellets, cold plasma, and air-powder sprays, which may be used alone or combined with disinfectants or antibiotics. However, many current treatment modalities have shown limited efficacy in the majority of peri-implantitis cases ( 1 , 8 , 9 ). The ineffectiveness of these treatments can be attributed to several factors, including the complex morphology of bone defects, limited device access, suboptimal angles, and the implant surface design. Additionally, oversized cleaning particles often fail to reach and remove smaller bacteria from textured implant surfaces. Ultimately, the progression of the peri-implant bone loss may necessitate explantation and replacement of the implant, resulting in substantial costs and patient discomfort. This underscores the urgent need for more effective strategies to decontaminate infected implants and improve peri-implantitis outcomes. In 2008, the concept of electrochemical disinfection was introduced as a groundbreaking approach for sanitizing titanium (Ti) dental implant surfaces, providing a minimally invasive technique for biofilm eradication ( 10 , 11 ). A prominent system and an emerging one utilizing this technology for disinfecting Ti dental implants are GalvoSurge® and X-Implant, respectively. These systems are designed to tackle the limitations of traditional mechanical and chemical cleaning methods, offering a more comprehensive biofilm removal and microbial debris. They aim to enhance the long-term success rates of implants affected by peri-implantitis, providing a solution that is safe for the implant surface and efficient in biofilm eradication. This study aimed to evaluate the microbiological outcomes of the electrolytic cleaning of the GalvoSurge® system, developed by Straumann, that is specifically designed for screw-retained superstructures and the X-Implant system, developed by LED SpA, that also applies electrochemical principles to ensure effective biofilm eradication without damaging the implant surface. The study compared those two different electrolytic cleaning systems compared to treatments utilizing chlorhexidine (CHX) 0.2%, local antibiotics (Terramycin® gel), or phosphate-buffered saline (PBS), as a negative control, on implants contaminated with multispecies biofilms. Materials and Methods 1. Bacterial strains, media, and culture conditions Different oral bacterial strains representing various bacterial complexes (peri-implantitis associated, peri-implant health associated, and core microbiome bacteria) were included in the study (Table 1 ). They were maintained using blood agar supplemented with hemin (5.0 mg/L), menadione (1.0 mg/L), and 5% sterile horse blood. Brain Heart Infusion (BHI) broth supplemented with 0.04% L-cysteine HCl (BHIC), or only BHI were used to prepare broth cultures ( 12 ). Table 1 Bacterial strains included in the study Bacterial complex Bacterial species Peri-implantitis associated bacteria Prevotella intermedia ATCC † 25611 Porphyromonas gingivalis ATCC 33277 Fusobacterium nucleatum DSM † 20482 Aggregatibacter actinomycetemcomitans ATCC 43718 Peri-implant health associated bacteria Streptococcus sanguinis LM 14657 Actinomyces viscosus DSM 43327 Actinomyces naeslundii ATCC 51655 Core microbiome bacteria Streptococcus mutans ATCC 20523 Streptococcus sobrinus ATCC 20742 Veillonella parvula DSM 2008 Streptococcus gordonii ATCC 49818 Streptococcus oralis DSM 20627 Streptococcus mitis DSM 12643 Streptococcus salivarius TOVE-R † ATCC; American Type Culture Collection, DSM; Deutsche Sammlung von Mikroorganismen. 2. Multispecies oral microbial communities: A multispecies oral microbial community was established using a BIOSTAT® B TWIN bioreactor (Sartorius, Germany). Briefly, 750 mL of Brain Heart Infusion-2 (BHI-2) broth supplemented with 5.0 mg/L hemin, and 1.0 mg/L menadione was used to grow the microbial communities. The medium was pre-reduced over 24 hours at 37°C and pH 6.7 by bubbling 100% N 2 and 5% CO 2 in the medium under continuous stirring at 100 rpm. After 24 h, overnight cultures of the involved species (Table 1 ) were added at an equal optical density (OD) to the bioreactor medium. During the first 48 h, the medium was not replaced, and the different species were allowed to grow in harmony under controlled temperature and pH conditions. After that, the medium was refreshed at a rate of 200 mL/24 h. 3. Biofilm growth on the Ti implant surfaces: Bioreactor-derived biofilms were allowed to grow in BHI-2 medium with extra L-cysteine HCL (BHI-2C) on the surface of sterile Ti implants, 4 mm in diameter and 8 mm height (Ossean® surface implant, Intra-Lock), for 48 h in 96-well plates under microaerophilic conditions while shaking at 170 rpm according to Zayed et al. ( 12 ) (Fig. 1 ). The contaminated Ti implants were divided into five experimental groups. Five independent biological replicates were performed for each group. 4. Implants decontamination After 48 h of microaerophilic incubation for biofilm development, the following decontamination approaches were applied to the different experimental groups for biofilm removal: Electrolytic cleaning with Galvosurge® system utilizing the corresponding cleaning solution according to manufacturer instructions for 2 min (system provided by Straumann®) (Fig. 2 a). Electrolytic cleaning with X-Implant system® according to manufacturer instruction (system provided by LED SpA®), utilizing the appropriate size electrode to the tested implants (Fig. 2 b). Rinsing with chlorhexidine 0.2% for 2 min at room temperature (positive control group). Application of Terramycin® gel as a local antibiotic approach (positive control group) for 2 min. Terramycin® includes a combination of antibiotics: oxytetracycline and polymyxin B sulfate. Rinsing with sterile phosphate-buffered saline (PBS) for 2 min at room temperature (negative control group). After applying the different decontamination approaches, all the implants were PBS-washed to remove any detached biofilms or remaining cleaning materials, and re-incubated for 24 h in fresh sterile media to investigate the possible biofilm survival and recovery. 5. Biofilm collection, viability DNA extraction, and quantitative polymerase chain reaction (V-qPCR) After 24 h of incubation of the decontaminated implants in fresh sterile medium, biofilms were collected from the implants’ surfaces (n = 3 for each experimental group) using trypsin for 45 min at 37°C with shaking at 250 rpm. Viability DNA extraction and quantitative polymerase chain reaction (v-qPCR) were performed using propidium monoazide xx (PMAxx) and strain-specific primers and probes ( 13 ). 6. Scanning electron microscopy imaging of the decontaminated implant surfaces Following the 24-h recovery time, decontaminated implants (n = 1 for each condition) were collected for imaging using a scanning electron microscope (SEM) under high vacuum conditions and a 10-keV acceleration voltage (FEI XL30-FEG; FEI) ( 14 ). Prior to imaging, the implants were rinsed with PBS and the biofilms on their surfaces were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 30 min. Excess fixing solution was removed by washing with PBS. Dehydration was achieved by sequential incubation in ethanol solutions of increasing concentrations (30%, 50%, 70%, 90%, and 100%) for 20 min each. The implants were then air-dried and coated with a Pt/Pd (80/20) layer using a sputtering device (Quorum Technologies) before SEM imaging. 7. CLSM visualization A confocal laser scanning microscope (CLSM) was utilized to visualize the viability of any surviving biofilm bacteria following the 24-h recovery time from the applied decontamination approaches (n = 1 each). Biofilms were rinsed with Milli-Q® water, stained with LIVE/DEAD BacLight® fluorescent dye (Invitrogen Life Technologies) for 20 min at room temperature in darkness ( 12 ). The remaining biofilms were imaged fully hydrated using a Leica TCS SP8 inverted CLSM with a 63× glycerol immersion objective (Leica Lasertechnik). Z-stacks of 8-bit, 1,024 × 1,024-pixel resolution were acquired from at least three different locations for each decontamination condition, with a 1 µm z-step size. ImageJ software was employed to visualize the acquired image stacks and Imaris viewer was used for creating a 3D view of the stacks. 8. Statistical analysis Statistical analyses were conducted using R (v4.0.3). Residual normality was evaluated through the Shapiro–Wilk test and normal quantile plots, while Levene’s test was employed to assess homogeneity of variances across groups. For nonparametric data, the Kruskal–Wallis test, followed by Dunn multiple comparisons post hoc analysis, was applied to discern significant differences between treatment conditions and the control group. For parametric data, analysis of variance (ANOVA) test with Tukey honestly significant difference multiple comparisons analysis was employed. A confidence level of 95% was applied to all analyses. CLSM image processing was performed using ImageJ and Imaris viewer. Results Effects of various treatments on biofilm removal from implant surfaces: v-qPCR quantification The % of the 24-h biofilm recovery was calculated for the decontaminated implants (CHX, Terramycin®, Galvosurge®, or X-Implant groups) relative to the control PBS-treated biofilms (Fig. 3 ). A significant reduction ( p < 0.001) in the biofilm viable biomass on the implant surfaces was observed for the CHX- and the Terramycin®-treated implant group (98.8 ± 1% and 99.6 ± 0.2% respectively). On the other hand, the electrolytic cleaning results varied greatly depending on the employed system. For the Galvosurge®-decontaminated implants, a significant reduction (93.3 ± 6%) in the viable biofilm microbial load was detected ( p < 0.001), which did not significantly differ from the CHX- or the Terramycin®-treated groups. Contrastingly, the X-implant®-treated group did not significantly diverge from the control PBS-treated group ( p = 0.89), showing only 5.7 ± 16% reduction in the biofilm viable biomass on the treated implants surfaces compared to the control PBS-treated ones. The biofilm reduction due to the X-implant® treatment was significantly less ( p < 0.001) than the other employed cleaning techniques (CHX, Terramycin® or Galvosurge®). SEM visualization of recovered biofilms on implant surfaces Scanning electron micrographs were taken for the implant surfaces after 24-h recovery time to examine the microstructure of biofilms on their surfaces in all the control and experimental groups. As shown in Fig. 4 -a, the control group (PBS-rinsed) exhibited predominant biofilms, with abundant rod-shaped cells and filaments of elongated rods as well as cocci-shaped bacterial cells. Similarly, the surface of the X-implant®-treated group showed a comparable biofilm structure (Fig. 4 -e). On the other hand, both the Terramycin® and Galvosurge® treatments resulted in an obvious decreased bacterial count on the implant surfaces compared to the control group, indicating a diminished presence of the biofilms (Fig. 4 -c and 4 -d). Remarkably, the CHX decontaminated implant exhibited a higher cell density on the implant surface compared to the preceding experimental groups, with cocci- and rod-shaped cells and filaments. CLSM visualization of biofilms CLSM was employed to visualize the viability of bacterial cells within the remaining biofilms after 24 h of recovery. The 3D CLSM stacks of the CHX-, Galvosurge®- and Terramycin®- treated groups showed a remarkable reduction in the density of bacterial cells within the biofilm compared to the control group, with some differences (Fig. 5 ). The stacks of the CHX-treated implant surfaces showed dominance of red signals, indicating incidence of dead or compromised cells, while the stacks of the Terramycin®-treated implant surfaces showed more dominance of green signals, indicative for viable cells. On the other hand, the total observed signals after treatment with the Galvosurge® was less compared to the CHX- or the Terramycin®-treated groups. Discussion The investigations of this study compared the efficacy of different electrolytic decontamination systems to conventional approaches for biofilm removal from implant surfaces, revealing notable insights into the differential effects of different systems and methods. With the exception of the X-Implant® group, all the tested groups (CHX, Terramycin®, and Galvosurge®) demonstrated significant reductions in biofilm viability. The remarkable reduction in the viable biofilm bacteria on the implant surfaces treated with CHX highlights the efficacy of antiseptic treatments in controlling biofilms on implant surfaces, corroborating their established roles in periodontal and peri-implant disease management. Chlorhexidine (CHX) is widely regarded as the "gold standard" for oral antiseptics and is frequently included in studies as a positive control. In periodontitis, the adjunctive use of CHX formulations with subgingival debridement has been shown to result in a slightly greater reduction in probing pocket depth (PPD) compared to scaling and root planing (SRP) alone ( 15 , 16 ). However, despite these benefits, several studies found no improved outcomes in the (non-)surgical management of peri-implant mucositis and peri-implantitis with adjunctive CHX use ( 17 – 19 ). Consequently, the current EFP S3 level clinical guidelines do not recommend the adjunctive use of chlorhexidine for treating peri-implant diseases beyond (non-)surgical management ( 1 ). In this study, the v-qPCR analysis revealed a significant reduction in the viable bacterial population on implant surfaces following treatment with CHX. Nevertheless, SEM visualization of the implant surfaces showed a notable discrepancy compared to the aforementioned v-qPCR results. Additionally, the CLSM 3D stacks of the CHX-treated group displayed dominance of red signals, indicating the incidence of dead or compromised cells. This suggests that the bacteria visualized through SEM are presumably remnants of compromised or deceased cells. These findings imply that while CHX treatment effectively targets and compromises bacterial cells within the biofilm, reducing the overall biofilm viability on implant surfaces, it is not necessarily removing all the biofilm remnants from the surface. However, even if bacteria are no longer viable, their remnants can still trigger immune responses, hindering healing and promoting inflammation around the implant ( 20 , 21 ). Terramycin®, a well-known tetracycline antibiotic, demonstrated the highest reduction in viable bacterial population, achieving a 99.6% decrease in viable biofilm bacteria. This antibiotic functions by inhibiting bacterial protein synthesis, exhibiting bacteriostatic properties. The resurgence of localized antibiotic treatments, such as Terramycin®, stems from concerns over the side effects associated with systemic antibiotics ( 22 ). The Terramycin® group showed a significant reduction in the viable bacterial population, indicating effective biofilm control. SEM visualization revealed disruptions in the biofilm matrix and bacterial cell morphology following treatment. CLSM visualization was employed to assess the viability of bacterial cells within the remaining biofilm, showing a significant reduction in bacterial density in the Terramycin® group compared to the control. CLSM visualization in the Terramycin® group revealed a remarkable reduction in the density of bacterial cells within the biofilm compared to the control group. The predominance of green signals, suggesting viable cells, implies the bacteriostatic effect of the Terramycin® ( 23 ). According to Herrera et al., the adjunctive use of local antibiotics (e.g. doxycycline, tetracycline, and minocycline) with subgingival instrumentation in periodontitis has proven efficacy, yielding significant improvements in PPD ( 24 ). However, despite these positive findings, similar to CHX, the benefits of locally administered antibiotics in treating of peri-implant diseases remain limited, and current guidelines do not recommend their use ( 19 , 25 ). The lack of standardized and effective treatment highlights the significance of advancements in controlling or treating implant-related infections. One such advancement is electrochemical disinfection technology, which has recently shown promise in implant cleaning. A leading example is the GalvoSurge® cleaning system by Straumann®, designed specifically for implants with screwed superstructures. This system utilizes electrolytic cleaning by leveraging the electrical conductivity of the Ti implant surface to disinfect and eliminate adherent biofilms ( 26 ). The process involves applying controlled low voltage to the Ti implant while simultaneously irrigating it with an electrolyte solution. This generates small hydrogen bubbles at the implant surface, creating mechanical forces that aid in detaching biofilms and debris. The GalvoSurge® system demonstrated promising results in this study, resulting in a substantial reduction (93.3%) in viable biofilm microbial load, comparable to the reductions observed with CHX and Terramycin® treatments. SEM visualization provided further insights, revealing a marked decrease in biofilm cell counts on the implant surface, indicating effective biofilm removal. This, in turn, supports the potential for re-osseointegration of the affected implant, which is a key objective of such treatments. In an animal study by Schlee et al., complete re-osseointegration was observed histologically following electrolytic cleaning, further demonstrating the efficacy of GalvoSurge® system in treating peri-implantitis ( 27 ). Furthermore, CLSM provided additional perspectives on biofilm viability following Galvosurge® treatment in this study. The reduction in total observed signals compared to CHX and Terramycin® indicates greater biofilm removal effect, rather than just aiming to kill the biofilm bacteria. Consistent with our findings, Ratka et al. demonstrated a significant reduction in bacterial cell counts on contaminated implant surfaces following electrolytic cleaning. Further supporting evidence has been provided by Assunção et al., who conducted an in vitro study comparing electrolytic decontamination of dental implant surfaces to various mechanical approaches, revealing the effectiveness of the Galvosurge® treatment in removing Pseudomonas aeruginosa biofilms from implants ( 28 ). Recent clinical trials have provided valuable insights into the efficacy of electrolytic cleaning for managing peri-implantitis, confirming effective surface decontamination ( 29 , 30 ). In a randomized clinical trial conducted by Schlee et al., promising findings regarding the clinical application of Galvosurge® in patients with peri-implantitis were reported. Significant improvements in clinical parameters, such as bleeding on probing (BoP), suppuration, and probing pocket depth (PPD), were observed post-surgery, at 6 months following augmentation, and between 6 to 12 months after restoration replacement ( 29 ). Statistically significant radiographic bone fill was noted 18 months after therapy. The study also compared the effects of electrolytic cleaning alone to electrolytic cleaning combined with powder spray, finding no statistically significant difference between the two groups. This led to the conclusion that additional mechanical cleaning via powder spray following electrolytic cleaning is unnecessary. Further clinical evidence has been provided by Bosshardt et al., who observed similar improvements in BoP, PPD, and radiographic bone gain ( 31 ). Their study confirmed that re-osseointegration of contaminated implant surfaces following electrolytic cleaning was possible. Nevertheless, not all electrolytic cleaning approaches perform equally. Despite the promising outcomes seen with the Galvosurge® system, the efficacy of the X-Implant® system was notably limited in this study. The X-Implant® system, developed by LED SpA, employs a distinct method compared to the Galvosurge®. While the Galvosurge® utilizes an electrolyte solution in combination with a small electric current, the X-Implant® system operates without any solution, employing a dry technique. In this system, the active electrode is applied to the implant collar, while the patient holds the ground electrode. The X Implant® decontaminator is configured for treating peri-implantitis with an established specific program, delivering currents according to predefined durations and methods, with high-frequency electromagnetic waves facilitating the debonding and destruction of the bacterial biofilm. Despite its innovative approach, the X-Implant® system could not significantly reduce viable biofilm bacteria on implant surfaces compared to the control group, indicating potential challenges and limitations in its use for peri-implantitis management. SEM and the CLSM observations following treatment with the X-Implant® system revealed biofilm structure and bacterial viability similar to the control group. These findings underscores the need for further investigation into the optimization of the X-Implant® system's protocols or the exploration of complementary strategies to enhance its efficacy. This study emphasizes that not all electrolytic cleaning methods perform equally or effectively in decontaminating dental implant surfaces, highlighting the importance of ongoing research and refinement in this area. Even though the promising findings, several limitations of this study must be acknowledged. First, the body of evidence supporting the use of the GalvoSurge® device remains relatively limited compared to more established treatments like chlorhexidine (CHX) and Terramycin®. Although early studies and clinical trials have demonstrated promising results, the current scarcity of research specifically addressing the electrolytic cleaning approaches restricts the ability to draw comprehensive conclusions regarding its long-term efficacy. Future research should prioritize extensive, long-term clinical studies to validate these preliminary findings and fully explore the potential of electrolytic cleaning in implant dentistry. Second, this study was conducted in vitro using a semi-complex multispecies oral microbial community, focused primarily on microbial outcomes, which may not fully replicate the diverse and complex oral environment encountered in clinical peri-implantitis cases. In vitro models do not account for the dynamic interplay of host factors, such as immune responses, saliva flow, and tissue interactions, all of which can significantly influence the treatment outcomes. Therefore, the efficacy observed in vitro may differ in clinical scenarios with more diverse biofilm compositions and host dynamics. Additionally, while this study assesses the short-term effects of treatments on biofilm viability (24 h after treatment), long-term outcomes were not evaluated. The potential for biofilm recolonization and the recurrence of peri-implantitis over time is a critical factor that could impact the overall success of treatments. Future research should aim to address these gaps by investigating both microbial and host responses to treatment modalities, providing a more comprehensive understanding of their efficacy in managing peri-implantitis. Long-term studies that assess outcomes such as tissue regeneration, implant stability, and biofilm recurrence will be crucial for developing more effective and holistic treatment strategies. Conclusion The investigation into biofilm removal from implant surfaces via diverse treatment modalities offers substantial scientific insights. Our findings demonstrated the significant efficacy of chlorhexidine (CHX) and Terramycin® in reducing biofilm viability, reinforcing their well-established roles in the management of periodontal and peri-implant diseases. Notably, the Galvosurge® system also demonstrated considerable effectiveness, achieving reductions in viable biofilm microbial load comparable to those observed with antimicrobial treatments. These results position electrolytic cleaning methods, such as GalvoSurge®, as promising strategies for biofilm control on implant surfaces. However, it is important to note that not all electrolytic cleaning systems perform equally. While the GalvoSurge® system showed strong biofilm removal capabilities, the X-Implant® system exhibited significantly lower efficacy in reducing viable biofilm bacteria. This disparity highlights the variability in performance among different electrolytic systems and underscores the need for further optimization of the different systems to enhance their effectiveness. Clinical trials and randomized controlled studies are crucial to validate the long-term efficacy in clinical settings. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests All the authors declare no conflict of interest Funding information This study was supported by grants from the Research Foundation-Flanders (FWO G0B2719N). M.G.S. is a Strategic Basic Research Ph.D. fellow at the Research Foundation-Flanders (FWO-Vlaanderen) (grant number 1SHFK24N). Author Contribution A.B.C. contributed to the experimental design, data analysis and interpretation, and critically revised the manuscript, J.H. carried out the experiments, contributed to data acquisition, and wrote the manuscript, M.G.S. and K.S. contributed to the data acquisition and revised the manuscript, K.B. and A.B. contributed to the data analysis, and critically revised the manuscript, N.Z. contributed to the experimental design, carried out the experiments, data acquisition, analysis, and interpretation, wrote, and critically revised the manuscript. Acknowledgement We would like to thank Dr. Martine Pauwels for the support with the v-qPCR measurements. Data Availability Data supporting the current study are available from the corresponding author on reasonable request. References Herrera D, Berglundh T, Schwarz F, Chapple I, Jepsen S, Sculean A, et al. Prevention and treatment of peri-implant diseases—The EFP S3 level clinical practice guideline. J Clin Periodontol. 2023;50:4–76. Heitz-Mayfield LJ, Salvi GE. Peri‐implant mucositis. J Clin Periodontol. 2018;45:S237–45. Derks J, Tomasi C. Peri-implant health and disease. A systematic review of current epidemiology. J Clin Periodontol. 2015;42:S158–71. dos Santos Corpas L, Jacobs R, Quirynen M, Huang Y, Naert I, Duyck J. Peri-implant bone tissue assessment by comparing the outcome of intra‐oral radiograph and cone beam computed tomography analyses to the histological standard. Clin Oral Implants Res. 2011;22(5):492–9. Costa J, Mendes J, Salazar F, Pacheco J, Rompante P, Câmara M. Analysis of peri-implant bone defects by using cone beam computed tomography (CBCT): an integrative review. Oral Radiol. 2023;39(3):455–66. Song D, Shujaat S, de Faria Vasconcelos K, Huang Y, Politis C, Lambrichts I, et al. Diagnostic accuracy of CBCT versus intraoral imaging for assessment of peri-implant bone defects. BMC Med Imaging. 2021;21:1–8. Romandini M, Laforí A, Pedrinaci I, Baima G, Ferrarotti F, Lima C, et al. Effect of sub-marginal instrumentation before surgical treatment of peri‐implantitis: A multi‐centre randomized clinical trial. J Clin Periodontol. 2022;49(12):1334–45. Baima G, Citterio F, Romandini M, Romano F, Mariani GM, Buduneli N, et al. Surface decontamination protocols for surgical treatment of peri-implantitis: A systematic review with meta‐analysis. Clin Oral Implants Res. 2022;33(11):1069–86. Ramanauskaite A, Schwarz F, Cafferata EA, Sahrmann P. Photo/mechanical and physical implant surface decontamination approaches in conjunction with surgical peri-implantitis treatment: a systematic review. J Clin Periodontol. 2023;50:317–35. Del Pozo JL, Rouse MS, Mandrekar JN, Steckelberg JM, Patel R. The electricidal effect: reduction of Staphylococcus and Pseudomonas biofilms by prolonged exposure to low-intensity electrical current. Antimicrob Agents Chemother. 2009;53(1):41–5. Mohn D, Zehnder M, Stark WJ, Imfeld T. Electrochemical disinfection of dental implants–a proof of concept. PLoS ONE. 2011;6(1):e16157. Zayed N, Ghesquière J, Kamarudin N, Bernaerts K, Boon N, Braem A, et al. Oral biofilm cryotherapy as a novel ecological modulation approach. J Dent Res. 2023;102(9):1038–46. Zayed N, Boon N, Bernaerts K, Chatzigiannidou I, Van Holm W, Verspecht T, et al. Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms. J Periodontal Res. 2022;57(1):52–62. Zayed N, Munjaković H, Aktan M, Simoens K, Bernaerts K, Boon N, et al. Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro. J Dent Res. 2024;103(3):243–52. Zhao H, Hu J, Zhao L. Adjunctive subgingival application of Chlorhexidine gel in nonsurgical periodontal treatment for chronic periodontitis: a systematic review and meta-analysis. BMC Oral Health. 2020;20:1–12. Ramanauskaite E, Machiulskiene V. Antiseptics as adjuncts to scaling and root planing in the treatment of periodontitis: a systematic literature review. BMC Oral Health. 2020;20:1–19. Dommisch H, Hoedke D, Valles C, Vilarrasa J, Jepsen S, Pascual La Rocca A. Efficacy of professionally administered chemical agents as an adjunctive treatment to sub-marginal instrumentation during the therapy of peri‐implant mucositis. J Clin Periodontol. 2023;50:146–60. Liu S, Li M, Yu J. Does chlorhexidine improve outcomes in non-surgical management of peri-implant mucositis or peri-implantitis? a systematic review and meta-analysis. Medicina oral patología oral y cirugía bucal. 2020;25(5):e608. Wilensky A, Shapira L, Limones A, Martin C. The efficacy of implant surface decontamination using chemicals during surgical treatment of peri-implantitis: A systematic review and meta‐analysis. J Clin Periodontol. 2023;50:336–58. Armitage GC, Xenoudi P. Post-treatment supportive care for the natural dentition and dental implants. Periodontol 2000. 2016;71(1):164–84. Zimmerli W, Sendi P, editors. Pathogenesis of implant-associated infection: the role of the host. Seminars in immunopathology. Springer; 2011. Slots J. Systemic antibiotics in periodontics. J Periodontol. 2004;75(11):1553–65. Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 2001;65(2):232–60. Herrera D, Matesanz P, Martín C, Oud V, Feres M, Teughels W. Adjunctive effect of locally delivered antimicrobials in periodontitis therapy: A systematic review and meta-analysis. J Clin Periodontol. 2020;47:239–56. Teughels W, Seyssens L, Christiaens V, Temmerman A, Castro AB, Cosyn J. Adjunctive locally and systemically delivered antimicrobials during surgical treatment of peri-implantitis: A systematic review. J Clin Periodontol. 2023;50:359–72. Schneider S, Rudolph M, Bause V, Terfort A. Electrochemical removal of biofilms from titanium dental implant surfaces. Bioelectrochemistry. 2018;121:84–94. Schlee M, Naili L, Rathe F, Brodbeck U, Zipprich H. Is complete re-osseointegration of an infected dental implant possible? histologic results of a dog study: a short communication. J Clin Med. 2020;9(1):235. Assunção MA, Botelho J, Machado V, Proença L, Matos AP, Mendes JJ, et al. Dental implant surface decontamination and surface change of an electrolytic method versus mechanical approaches: a pilot in vitro study. J Clin Med. 2023;12(4):1703. Schlee M, Wang H-L, Stumpf T, Brodbeck U, Bosshardt D, Rathe F. Treatment of periimplantitis with electrolytic cleaning versus mechanical and electrolytic cleaning: 18-month results from a randomized controlled clinical trial. J Clin Med. 2021;10(16):3475. Schlee M, Rathe F, Brodbeck U, Ratka C, Weigl P, Zipprich H. Treatment of peri-implantitis—electrolytic cleaning versus mechanical and electrolytic cleaning—a randomized controlled clinical trial—six-month results. J Clin Med. 2019;8(11):1909. Bosshardt DD, Brodbeck UR, Rathe F, Stumpf T, Imber J-C, Weigl P, et al. Evidence of re-osseointegration after electrolytic cleaning and regenerative therapy of peri-implantitis in humans: a case report with four implants. Clin Oral Invest. 2022;26(4):3735–46. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5402056","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391404141,"identity":"f740254b-e27c-4885-8ae2-34fecaf94033","order_by":0,"name":"Ana. B. Castro","email":"","orcid":"","institution":"University Hospitals Leuven","correspondingAuthor":false,"prefix":"","firstName":"Ana.","middleName":"B.","lastName":"Castro","suffix":""},{"id":391404142,"identity":"6b05c536-e083-4ef8-bc26-6dbc350f973f","order_by":1,"name":"Jill Hadisurya","email":"","orcid":"","institution":"University Hospitals Leuven","correspondingAuthor":false,"prefix":"","firstName":"Jill","middleName":"","lastName":"Hadisurya","suffix":""},{"id":391404143,"identity":"b3e282a4-1d09-433b-988c-dde679c4783d","order_by":2,"name":"Kenneth Simoens","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Simoens","suffix":""},{"id":391404144,"identity":"7451191f-aa82-4aaa-ab9a-3668bd0d3f1e","order_by":3,"name":"Mrinal Gaurav Srivastava","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Mrinal","middleName":"Gaurav","lastName":"Srivastava","suffix":""},{"id":391404145,"identity":"ed94155a-88c4-4c98-a777-6a9ae0872314","order_by":4,"name":"Kristel Bernaerts","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Kristel","middleName":"","lastName":"Bernaerts","suffix":""},{"id":391404146,"identity":"c89ed592-e72a-40b9-b567-4752e070ef2a","order_by":5,"name":"Annabel Braem","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Annabel","middleName":"","lastName":"Braem","suffix":""},{"id":391404147,"identity":"a0e26943-0fd7-45bb-8eca-cdc3a8e51f45","order_by":6,"name":"Naiera Zayed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIie3OMUvDQBjG8bcU4nKQjhdO0q9wIaCWFvJVPALJcqBjB6EXCulSnPNxTg7icuB6YJcQyFwRiiKIQYrocJhR8H7TO9yfewAc508aia8T7yWcHm85LAkqCWhA8g1BQ5LzE1W0yxtI/Cq9e1xohXzMo/YadqEtmW3ZOtI1jLHJ0jk3CgUVj+MKutiWUMnKQHjggeFnhO8VoibLCALFhC15aDav4h3Q1FwdyEWfJCbL3/pkZU0MK0dFCZga7hHoh1Gc1uM+ubQOM806KG4xjXQXz7Y6R1i3iiDaRfZhefMkDoskvE8b81LPQ3/Dime03E1tvxxhgMmPJfSX4JMvh7xyHMf5jz4A0zhaVUDtlX4AAAAASUVORK5CYII=","orcid":"","institution":"KU Leuven","correspondingAuthor":true,"prefix":"","firstName":"Naiera","middleName":"","lastName":"Zayed","suffix":""}],"badges":[],"createdAt":"2024-11-06 10:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5402056/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5402056/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73899392,"identity":"beea121b-e4c3-4f6e-9428-32cc6b46bdee","added_by":"auto","created_at":"2025-01-15 17:07:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1614534,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of biofilm growth steps on Ti implants surfaces using bioreactor-derived multispecies oral microbial communities. The implants were incubated for 48 h under microaerophilic conditions to allow biofilm formation; (a) Top view of a 96-well implant incubation plate; and (b) side view of the 96-well plate. Figure created with Biorender.com.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/6075504f8f3dcd9b7b8a25aa.jpg"},{"id":73899398,"identity":"c94c177c-969d-463b-b111-a4978b67ab1b","added_by":"auto","created_at":"2025-01-15 17:07:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":632059,"visible":true,"origin":"","legend":"\u003cp\u003eElectrolytic cleaning with two different systems according to the corresponding manufacturer’s instructions; (a) Galvosurge® cleaning system operated with the corresponding solution, and (b) X implant® cleaning system operated in a dry approach.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/942fdb2503d99fbf0377ba0e.jpg"},{"id":73900314,"identity":"c11512dd-7fab-4b4f-8275-57c19c108a16","added_by":"auto","created_at":"2025-01-15 17:15:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228839,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots of the percentage (%) of the remaining viable biofilms on the implant surfaces after 24 h of incubation for recovery following treatment with various cleaning methods. A significant reduction of biofilm is observed after treatment with CHX, Terramycin\u003csup\u003e®\u003c/sup\u003e and Galvosurge\u003csup\u003e®\u003c/sup\u003e. No significant difference was observed after treatment with the X-Implant\u003csup\u003e®\u003c/sup\u003e system. * indicates a significant reduction compared to PBS control.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/8dc22e49c5c2687905faec18.jpg"},{"id":73899401,"identity":"aa2ea12f-6489-4896-bf29-6e4e674a98dd","added_by":"auto","created_at":"2025-01-15 17:07:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3664439,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscope (SEM) visualization of control, CHX-, Terramycin®-, Galvosurge®-, and X-Implant®-treated implant surfaces after 24 h of biofilm recovery following treatment. (a) Control biofilm characterized by prevalence of both cocci- and rod-shaped cells, (b) CHX-treated implants showing less dense biofilms than the control but more than the Galvosurge®- and the Terramycin®- treated surfaces, (c) Terramycin®-treated implant surface, showing less detected bacterial accumulation, (d) Galvosurge®-treated implant surface showing obvious less attached cells, (e) X-Implant®-treated implant surface showing prevalence of both cocci and rod-shaped cells in comparable quantities to the control group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/78ced9267a1ac5119587cb0f.jpg"},{"id":73899400,"identity":"ee6e89b7-624c-425f-83f5-c3b439927600","added_by":"auto","created_at":"2025-01-15 17:07:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1244359,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal laser scanning microscope (CLSM) visualization of control and differently-treated implant surfaces after 24 h of biofilm recovery following treatment. Green pixels indicate intact live bacteria, red pixels indicate damaged bacteria, and yellow pixels indicate co-uptake of both dyes by the cells. (a) Control PBS-treated implant surfaces showing the colonizing biofilm structure, (b) CHX-treated implants showing dominance of red signals indicating a higher proportion of damaged bacteria, (c) Terramycin®-treated implant surface, showing decreased green fluorescence signals compared to control biofilms, indicating less live bacterial cells (d) Galvosurge®-treated implant surface showing noticeably decreased green and red fluorescence signals compared to control, suggesting a lower incidence recovered live bacteria post-treatment, (e) X-Implant®-treated surfaces showing high green fluorescent signals compared to control, indicating week biofilm decontamination potential.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/0abc3626fa39a37bae44e636.jpg"},{"id":77165753,"identity":"f86ccb5b-09c4-44aa-8385-2731fb11a033","added_by":"auto","created_at":"2025-02-25 19:46:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8137233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5402056/v1/b727c46e-6dcd-4ac2-9dcd-960537e70737.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Advancing Biofilm Removal: Evaluating Electrolytic Methods for Decontaminating Dental Implants In Vitro","fulltext":[{"header":"Background","content":"\u003cp\u003eAccording to the S3-level clinical practice guidelines by the European Federation of Periodontology (EFP), peri-implantitis is defined as an inflammatory condition triggered by the peri-implant accumulated biofilms (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). This condition manifests as either peri-implant mucositis or peri-implantitis. Peri-implant mucositis refers to inflammation confined to the peri-implant mucosa without marginal bone loss (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), while peri-implantitis involves both the soft tissues and the surrounding bone. The reported prevalence of peri-implantitis and peri-implant mucositis varies across studies. A systematic review commissioned at the XI European Workshop of Periodontology in 2014 reported a patient-level prevalence of 43% for peri-implant mucositis and 22% for peri-implantitis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The accumulation of peri-implant plaque biofilm is the primary etiological factor for the development and progression of peri-implantitis. In addition, a history of severe periodontitis, inadequate plaque control, and the lack of a rigorous maintenance regimen, are significant risk factors for peri-implantitis and peri-mucositis development. As to the growing numbers of dental implants, the prevalence of infected implants is also rising. Clinical manifestations include bleeding on probing (BoP), increased periodontal pocket depth (PPD), as well as inflammation signs such as erythema, swelling, and/or suppuration. In more advanced cases, marginal bone loss can take place, resulting in crater-like defects around the affected implants, which are often more severe than periodontitis bone loss (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). A radiographic examination is essential for confirming the diagnosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of peri-implantitis treatment is to alleviate inflammation via eliminating microbial biofilms and to prevent any further loss of the supporting bone. As outlined in the EFP S3 level clinical practice guidelines, managing peri-implantitis involves two distinct phases: an initial non-surgical treatment phase, which may be followed by surgical intervention depending on the observed outcomes (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Even though non-surgical treatment, particularly sub-marginal instrumentation, plays a critical role in the initial phase of peri-implantitis management, its effectiveness remains a subject of ongoing research. Non-surgical debridement can help reduce inflammation and microbial load; however, its efficacy is often limited, particularly in advanced peri-implantitis cases where significant bone loss has already occurred (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This limitation emphasize the necessity of subsequent surgical intervention, to allow direct access to the implant contaminated surface for thorough surface decontamination.\u003c/p\u003e \u003cp\u003eVarious techniques have been employed in peri-implantitis treatment, including mechanical subgingival debridement with hand instruments or ultrasound-driven curettes, as well as the use of brushes, lasers, pellets, cold plasma, and air-powder sprays, which may be used alone or combined with disinfectants or antibiotics. However, many current treatment modalities have shown limited efficacy in the majority of peri-implantitis cases (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The ineffectiveness of these treatments can be attributed to several factors, including the complex morphology of bone defects, limited device access, suboptimal angles, and the implant surface design. Additionally, oversized cleaning particles often fail to reach and remove smaller bacteria from textured implant surfaces. Ultimately, the progression of the peri-implant bone loss may necessitate explantation and replacement of the implant, resulting in substantial costs and patient discomfort. This underscores the urgent need for more effective strategies to decontaminate infected implants and improve peri-implantitis outcomes.\u003c/p\u003e \u003cp\u003eIn 2008, the concept of electrochemical disinfection was introduced as a groundbreaking approach for sanitizing titanium (Ti) dental implant surfaces, providing a minimally invasive technique for biofilm eradication (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). A prominent system and an emerging one utilizing this technology for disinfecting Ti dental implants are GalvoSurge\u0026reg; and X-Implant, respectively. These systems are designed to tackle the limitations of traditional mechanical and chemical cleaning methods, offering a more comprehensive biofilm removal and microbial debris. They aim to enhance the long-term success rates of implants affected by peri-implantitis, providing a solution that is safe for the implant surface and efficient in biofilm eradication. This study aimed to evaluate the microbiological outcomes of the electrolytic cleaning of the GalvoSurge\u0026reg; system, developed by Straumann, that is specifically designed for screw-retained superstructures and the X-Implant system, developed by LED SpA, that also applies electrochemical principles to ensure effective biofilm eradication without damaging the implant surface. The study compared those two different electrolytic cleaning systems compared to treatments utilizing chlorhexidine (CHX) 0.2%, local antibiotics (Terramycin\u0026reg; gel), or phosphate-buffered saline (PBS), as a negative control, on implants contaminated with multispecies biofilms.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Bacterial strains, media, and culture conditions\u003c/h2\u003e \u003cp\u003eDifferent oral bacterial strains representing various bacterial complexes (peri-implantitis associated, peri-implant health associated, and core microbiome bacteria) were included in the study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). They were maintained using blood agar supplemented with hemin (5.0 mg/L), menadione (1.0 mg/L), and 5% sterile horse blood. Brain Heart Infusion (BHI) broth supplemented with 0.04% L-cysteine HCl (BHIC), or only BHI were used to prepare broth cultures (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial strains included in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterial complex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacterial species\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeri-implantitis associated bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePrevotella intermedia\u003c/em\u003e ATCC\u003csup\u003e\u0026dagger;\u003c/sup\u003e 25611\u003c/p\u003e \u003cp\u003e\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e ATCC 33277\u003c/p\u003e \u003cp\u003e\u003cem\u003eFusobacterium nucleatum\u003c/em\u003e DSM\u003csup\u003e\u0026dagger;\u003c/sup\u003e 20482\u003c/p\u003e \u003cp\u003e\u003cem\u003eAggregatibacter actinomycetemcomitans\u003c/em\u003e ATCC 43718\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeri-implant health associated bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus sanguinis\u003c/em\u003e LM 14657\u003c/p\u003e \u003cp\u003e\u003cem\u003eActinomyces viscosus\u003c/em\u003e DSM 43327\u003c/p\u003e \u003cp\u003e\u003cem\u003eActinomyces naeslundii\u003c/em\u003e ATCC 51655\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCore microbiome bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus mutans\u003c/em\u003e ATCC 20523\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptococcus sobrinus\u003c/em\u003e ATCC 20742\u003c/p\u003e \u003cp\u003e\u003cem\u003eVeillonella parvula\u003c/em\u003e DSM 2008\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptococcus gordonii\u003c/em\u003e ATCC 49818\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptococcus oralis\u003c/em\u003e DSM 20627\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptococcus mitis\u003c/em\u003e DSM 12643\u003c/p\u003e \u003cp\u003e\u003cem\u003eStreptococcus salivarius\u003c/em\u003e TOVE-R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003csup\u003e\u0026dagger;\u003c/sup\u003e ATCC; American Type Culture Collection, DSM; Deutsche Sammlung von Mikroorganismen.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Multispecies oral microbial communities:\u003c/h3\u003e\n\u003cp\u003eA multispecies oral microbial community was established using a BIOSTAT\u0026reg; B TWIN bioreactor (Sartorius, Germany). Briefly, 750 mL of Brain Heart Infusion-2 (BHI-2) broth supplemented with 5.0 mg/L hemin, and 1.0 mg/L menadione was used to grow the microbial communities. The medium was pre-reduced over 24 hours at 37\u0026deg;C and pH 6.7 by bubbling 100% N\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e in the medium under continuous stirring at 100 rpm. After 24 h, overnight cultures of the involved species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were added at an equal optical density (OD) to the bioreactor medium. During the first 48 h, the medium was not replaced, and the different species were allowed to grow in harmony under controlled temperature and pH conditions. After that, the medium was refreshed at a rate of 200 mL/24 h.\u003c/p\u003e\n\u003ch3\u003e3. Biofilm growth on the Ti implant surfaces:\u003c/h3\u003e\n\u003cp\u003eBioreactor-derived biofilms were allowed to grow in BHI-2 medium with extra L-cysteine HCL (BHI-2C) on the surface of sterile Ti implants, 4 mm in diameter and 8 mm height (Ossean\u0026reg; surface implant, Intra-Lock), for 48 h in 96-well plates under microaerophilic conditions while shaking at 170 rpm according to Zayed et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The contaminated Ti implants were divided into five experimental groups. Five independent biological replicates were performed for each group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e4. Implants decontamination\u003c/h3\u003e\n\u003cp\u003eAfter 48 h of microaerophilic incubation for biofilm development, the following decontamination approaches were applied to the different experimental groups for biofilm removal:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eElectrolytic cleaning with Galvosurge\u0026reg; system utilizing the corresponding cleaning solution according to manufacturer instructions for 2 min (system provided by Straumann\u0026reg;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eElectrolytic cleaning with X-Implant system\u0026reg; according to manufacturer instruction (system provided by LED SpA\u0026reg;), utilizing the appropriate size electrode to the tested implants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eRinsing with chlorhexidine 0.2% for 2 min at room temperature (positive control group).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eApplication of Terramycin\u0026reg; gel as a local antibiotic approach (positive control group) for 2 min. Terramycin\u0026reg; includes a combination of antibiotics: oxytetracycline and polymyxin B sulfate.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eRinsing with sterile phosphate-buffered saline (PBS) for 2 min at room temperature (negative control group).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAfter applying the different decontamination approaches, all the implants were PBS-washed to remove any detached biofilms or remaining cleaning materials, and re-incubated for 24 h in fresh sterile media to investigate the possible biofilm survival and recovery.\u003c/p\u003e\n\u003ch3\u003e5. Biofilm collection, viability DNA extraction, and quantitative polymerase chain reaction (V-qPCR)\u003c/h3\u003e\n\u003cp\u003eAfter 24 h of incubation of the decontaminated implants in fresh sterile medium, biofilms were collected from the implants\u0026rsquo; surfaces (n\u0026thinsp;=\u0026thinsp;3 for each experimental group) using trypsin for 45 min at 37\u0026deg;C with shaking at 250 rpm. Viability DNA extraction and quantitative polymerase chain reaction (v-qPCR) were performed using propidium monoazide xx (PMAxx) and strain-specific primers and probes (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6. Scanning electron microscopy imaging of the decontaminated implant surfaces\u003c/h2\u003e \u003cp\u003eFollowing the 24-h recovery time, decontaminated implants (n\u0026thinsp;=\u0026thinsp;1 for each condition) were collected for imaging using a scanning electron microscope (SEM) under high vacuum conditions and a 10-keV acceleration voltage (FEI XL30-FEG; FEI) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Prior to imaging, the implants were rinsed with PBS and the biofilms on their surfaces were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 30 min. Excess fixing solution was removed by washing with PBS. Dehydration was achieved by sequential incubation in ethanol solutions of increasing concentrations (30%, 50%, 70%, 90%, and 100%) for 20 min each. The implants were then air-dried and coated with a Pt/Pd (80/20) layer using a sputtering device (Quorum Technologies) before SEM imaging.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e7. CLSM visualization\u003c/h3\u003e\n\u003cp\u003eA confocal laser scanning microscope (CLSM) was utilized to visualize the viability of any surviving biofilm bacteria following the 24-h recovery time from the applied decontamination approaches (n\u0026thinsp;=\u0026thinsp;1 each). Biofilms were rinsed with Milli-Q\u0026reg; water, stained with LIVE/DEAD BacLight\u0026reg; fluorescent dye (Invitrogen Life Technologies) for 20 min at room temperature in darkness (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The remaining biofilms were imaged fully hydrated using a Leica TCS SP8 inverted CLSM with a 63\u0026times; glycerol immersion objective (Leica Lasertechnik). Z-stacks of 8-bit, 1,024 \u0026times; 1,024-pixel resolution were acquired from at least three different locations for each decontamination condition, with a 1 \u0026micro;m z-step size. ImageJ software was employed to visualize the acquired image stacks and Imaris viewer was used for creating a 3D view of the stacks.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e8. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using R (v4.0.3). Residual normality was evaluated through the Shapiro\u0026ndash;Wilk test and normal quantile plots, while Levene\u0026rsquo;s test was employed to assess homogeneity of variances across groups. For nonparametric data, the Kruskal\u0026ndash;Wallis test, followed by Dunn multiple comparisons post hoc analysis, was applied to discern significant differences between treatment conditions and the control group. For parametric data, analysis of variance (ANOVA) test with Tukey honestly significant difference multiple comparisons analysis was employed. A confidence level of 95% was applied to all analyses. CLSM image processing was performed using ImageJ and Imaris viewer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of various treatments on biofilm removal from implant surfaces: v-qPCR quantification\u003c/h2\u003e \u003cp\u003eThe % of the 24-h biofilm recovery was calculated for the decontaminated implants (CHX, Terramycin\u0026reg;, Galvosurge\u0026reg;, or X-Implant groups) relative to the control PBS-treated biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A significant reduction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the biofilm viable biomass on the implant surfaces was observed for the CHX- and the Terramycin\u0026reg;-treated implant group (98.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1% and 99.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% respectively). On the other hand, the electrolytic cleaning results varied greatly depending on the employed system. For the Galvosurge\u0026reg;-decontaminated implants, a significant reduction (93.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6%) in the viable biofilm microbial load was detected (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which did not significantly differ from the CHX- or the Terramycin\u0026reg;-treated groups. Contrastingly, the X-implant\u0026reg;-treated group did not significantly diverge from the control PBS-treated group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.89), showing only 5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16% reduction in the biofilm viable biomass on the treated implants surfaces compared to the control PBS-treated ones. The biofilm reduction due to the X-implant\u0026reg; treatment was significantly less (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than the other employed cleaning techniques (CHX, Terramycin\u0026reg; or Galvosurge\u0026reg;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSEM visualization of recovered biofilms on implant surfaces\u003c/h2\u003e \u003cp\u003eScanning electron micrographs were taken for the implant surfaces after 24-h recovery time to examine the microstructure of biofilms on their surfaces in all the control and experimental groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-a, the control group (PBS-rinsed) exhibited predominant biofilms, with abundant rod-shaped cells and filaments of elongated rods as well as cocci-shaped bacterial cells. Similarly, the surface of the X-implant\u0026reg;-treated group showed a comparable biofilm structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-e). On the other hand, both the Terramycin\u0026reg; and Galvosurge\u0026reg; treatments resulted in an obvious decreased bacterial count on the implant surfaces compared to the control group, indicating a diminished presence of the biofilms (Fig.\u0026nbsp;\u0026lt;link rid=\"fig4\"\u0026gt;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u0026lt;/link\u0026gt;\u003c/span\u003e-c and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-d). Remarkably, the CHX decontaminated implant exhibited a higher cell density on the implant surface compared to the preceding experimental groups, with cocci- and rod-shaped cells and filaments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCLSM visualization of biofilms\u003c/h2\u003e \u003cp\u003eCLSM was employed to visualize the viability of bacterial cells within the remaining biofilms after 24 h of recovery. The 3D CLSM stacks of the CHX-, Galvosurge\u0026reg;- and Terramycin\u0026reg;- treated groups showed a remarkable reduction in the density of bacterial cells within the biofilm compared to the control group, with some differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The stacks of the CHX-treated implant surfaces showed dominance of red signals, indicating incidence of dead or compromised cells, while the stacks of the Terramycin\u0026reg;-treated implant surfaces showed more dominance of green signals, indicative for viable cells. On the other hand, the total observed signals after treatment with the Galvosurge\u0026reg; was less compared to the CHX- or the Terramycin\u0026reg;-treated groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe investigations of this study compared the efficacy of different electrolytic decontamination systems to conventional approaches for biofilm removal from implant surfaces, revealing notable insights into the differential effects of different systems and methods. With the exception of the X-Implant\u0026reg; group, all the tested groups (CHX, Terramycin\u0026reg;, and Galvosurge\u0026reg;) demonstrated significant reductions in biofilm viability.\u003c/p\u003e \u003cp\u003eThe remarkable reduction in the viable biofilm bacteria on the implant surfaces treated with CHX highlights the efficacy of antiseptic treatments in controlling biofilms on implant surfaces, corroborating their established roles in periodontal and peri-implant disease management. Chlorhexidine (CHX) is widely regarded as the \"gold standard\" for oral antiseptics and is frequently included in studies as a positive control. In periodontitis, the adjunctive use of CHX formulations with subgingival debridement has been shown to result in a slightly greater reduction in probing pocket depth (PPD) compared to scaling and root planing (SRP) alone (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, despite these benefits, several studies found no improved outcomes in the (non-)surgical management of peri-implant mucositis and peri-implantitis with adjunctive CHX use (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Consequently, the current EFP S3 level clinical guidelines do not recommend the adjunctive use of chlorhexidine for treating peri-implant diseases beyond (non-)surgical management (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the v-qPCR analysis revealed a significant reduction in the viable bacterial population on implant surfaces following treatment with CHX. Nevertheless, SEM visualization of the implant surfaces showed a notable discrepancy compared to the aforementioned v-qPCR results. Additionally, the CLSM 3D stacks of the CHX-treated group displayed dominance of red signals, indicating the incidence of dead or compromised cells. This suggests that the bacteria visualized through SEM are presumably remnants of compromised or deceased cells. These findings imply that while CHX treatment effectively targets and compromises bacterial cells within the biofilm, reducing the overall biofilm viability on implant surfaces, it is not necessarily removing all the biofilm remnants from the surface. However, even if bacteria are no longer viable, their remnants can still trigger immune responses, hindering healing and promoting inflammation around the implant (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTerramycin\u0026reg;, a well-known tetracycline antibiotic, demonstrated the highest reduction in viable bacterial population, achieving a 99.6% decrease in viable biofilm bacteria. This antibiotic functions by inhibiting bacterial protein synthesis, exhibiting bacteriostatic properties. The resurgence of localized antibiotic treatments, such as Terramycin\u0026reg;, stems from concerns over the side effects associated with systemic antibiotics (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The Terramycin\u0026reg; group showed a significant reduction in the viable bacterial population, indicating effective biofilm control. SEM visualization revealed disruptions in the biofilm matrix and bacterial cell morphology following treatment. CLSM visualization was employed to assess the viability of bacterial cells within the remaining biofilm, showing a significant reduction in bacterial density in the Terramycin\u0026reg; group compared to the control. CLSM visualization in the Terramycin\u0026reg; group revealed a remarkable reduction in the density of bacterial cells within the biofilm compared to the control group. The predominance of green signals, suggesting viable cells, implies the bacteriostatic effect of the Terramycin\u0026reg; (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). According to Herrera et al., the adjunctive use of local antibiotics (e.g. doxycycline, tetracycline, and minocycline) with subgingival instrumentation in periodontitis has proven efficacy, yielding significant improvements in PPD (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, despite these positive findings, similar to CHX, the benefits of locally administered antibiotics in treating of peri-implant diseases remain limited, and current guidelines do not recommend their use (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe lack of standardized and effective treatment highlights the significance of advancements in controlling or treating implant-related infections. One such advancement is electrochemical disinfection technology, which has recently shown promise in implant cleaning. A leading example is the GalvoSurge\u0026reg; cleaning system by Straumann\u0026reg;, designed specifically for implants with screwed superstructures. This system utilizes electrolytic cleaning by leveraging the electrical conductivity of the Ti implant surface to disinfect and eliminate adherent biofilms (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The process involves applying controlled low voltage to the Ti implant while simultaneously irrigating it with an electrolyte solution. This generates small hydrogen bubbles at the implant surface, creating mechanical forces that aid in detaching biofilms and debris. The GalvoSurge\u0026reg; system demonstrated promising results in this study, resulting in a substantial reduction (93.3%) in viable biofilm microbial load, comparable to the reductions observed with CHX and Terramycin\u0026reg; treatments. SEM visualization provided further insights, revealing a marked decrease in biofilm cell counts on the implant surface, indicating effective biofilm removal. This, in turn, supports the potential for re-osseointegration of the affected implant, which is a key objective of such treatments. In an animal study by Schlee et al., complete re-osseointegration was observed histologically following electrolytic cleaning, further demonstrating the efficacy of GalvoSurge\u0026reg; system in treating peri-implantitis (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Furthermore, CLSM provided additional perspectives on biofilm viability following Galvosurge\u0026reg; treatment in this study. The reduction in total observed signals compared to CHX and Terramycin\u0026reg; indicates greater biofilm removal effect, rather than just aiming to kill the biofilm bacteria. Consistent with our findings, Ratka et al. demonstrated a significant reduction in bacterial cell counts on contaminated implant surfaces following electrolytic cleaning. Further supporting evidence has been provided by Assun\u0026ccedil;\u0026atilde;o et al., who conducted an \u003cem\u003ein vitro\u003c/em\u003e study comparing electrolytic decontamination of dental implant surfaces to various mechanical approaches, revealing the effectiveness of the Galvosurge\u0026reg; treatment in removing \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e biofilms from implants (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Recent clinical trials have provided valuable insights into the efficacy of electrolytic cleaning for managing peri-implantitis, confirming effective surface decontamination (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In a randomized clinical trial conducted by Schlee et al., promising findings regarding the clinical application of Galvosurge\u0026reg; in patients with peri-implantitis were reported. Significant improvements in clinical parameters, such as bleeding on probing (BoP), suppuration, and probing pocket depth (PPD), were observed post-surgery, at 6 months following augmentation, and between 6 to 12 months after restoration replacement (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Statistically significant radiographic bone fill was noted 18 months after therapy. The study also compared the effects of electrolytic cleaning alone to electrolytic cleaning combined with powder spray, finding no statistically significant difference between the two groups. This led to the conclusion that additional mechanical cleaning via powder spray following electrolytic cleaning is unnecessary. Further clinical evidence has been provided by Bosshardt et al., who observed similar improvements in BoP, PPD, and radiographic bone gain (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Their study confirmed that re-osseointegration of contaminated implant surfaces following electrolytic cleaning was possible. Nevertheless, not all electrolytic cleaning approaches perform equally. Despite the promising outcomes seen with the Galvosurge\u0026reg; system, the efficacy of the X-Implant\u0026reg; system was notably limited in this study.\u003c/p\u003e \u003cp\u003eThe X-Implant\u0026reg; system, developed by LED SpA, employs a distinct method compared to the Galvosurge\u0026reg;. While the Galvosurge\u0026reg; utilizes an electrolyte solution in combination with a small electric current, the X-Implant\u0026reg; system operates without any solution, employing a dry technique. In this system, the active electrode is applied to the implant collar, while the patient holds the ground electrode. The X Implant\u0026reg; decontaminator is configured for treating peri-implantitis with an established specific program, delivering currents according to predefined durations and methods, with high-frequency electromagnetic waves facilitating the debonding and destruction of the bacterial biofilm. Despite its innovative approach, the X-Implant\u0026reg; system could not significantly reduce viable biofilm bacteria on implant surfaces compared to the control group, indicating potential challenges and limitations in its use for peri-implantitis management.\u003c/p\u003e \u003cp\u003eSEM and the CLSM observations following treatment with the X-Implant\u0026reg; system revealed biofilm structure and bacterial viability similar to the control group. These findings underscores the need for further investigation into the optimization of the X-Implant\u0026reg; system's protocols or the exploration of complementary strategies to enhance its efficacy. This study emphasizes that not all electrolytic cleaning methods perform equally or effectively in decontaminating dental implant surfaces, highlighting the importance of ongoing research and refinement in this area.\u003c/p\u003e \u003cp\u003eEven though the promising findings, several limitations of this study must be acknowledged. First, the body of evidence supporting the use of the GalvoSurge\u0026reg; device remains relatively limited compared to more established treatments like chlorhexidine (CHX) and Terramycin\u0026reg;. Although early studies and clinical trials have demonstrated promising results, the current scarcity of research specifically addressing the electrolytic cleaning approaches restricts the ability to draw comprehensive conclusions regarding its long-term efficacy. Future research should prioritize extensive, long-term clinical studies to validate these preliminary findings and fully explore the potential of electrolytic cleaning in implant dentistry.\u003c/p\u003e \u003cp\u003eSecond, this study was conducted \u003cem\u003ein vitro\u003c/em\u003e using a semi-complex multispecies oral microbial community, focused primarily on microbial outcomes, which may not fully replicate the diverse and complex oral environment encountered in clinical peri-implantitis cases. \u003cem\u003eIn vitro\u003c/em\u003e models do not account for the dynamic interplay of host factors, such as immune responses, saliva flow, and tissue interactions, all of which can significantly influence the treatment outcomes. Therefore, the efficacy observed \u003cem\u003ein vitro\u003c/em\u003e may differ in clinical scenarios with more diverse biofilm compositions and host dynamics. Additionally, while this study assesses the short-term effects of treatments on biofilm viability (24 h after treatment), long-term outcomes were not evaluated. The potential for biofilm recolonization and the recurrence of peri-implantitis over time is a critical factor that could impact the overall success of treatments.\u003c/p\u003e \u003cp\u003eFuture research should aim to address these gaps by investigating both microbial and host responses to treatment modalities, providing a more comprehensive understanding of their efficacy in managing peri-implantitis. Long-term studies that assess outcomes such as tissue regeneration, implant stability, and biofilm recurrence will be crucial for developing more effective and holistic treatment strategies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe investigation into biofilm removal from implant surfaces via diverse treatment modalities offers substantial scientific insights. Our findings demonstrated the significant efficacy of chlorhexidine (CHX) and Terramycin\u0026reg; in reducing biofilm viability, reinforcing their well-established roles in the management of periodontal and peri-implant diseases. Notably, the Galvosurge\u0026reg; system also demonstrated considerable effectiveness, achieving reductions in viable biofilm microbial load comparable to those observed with antimicrobial treatments. These results position electrolytic cleaning methods, such as GalvoSurge\u0026reg;, as promising strategies for biofilm control on implant surfaces. However, it is important to note that not all electrolytic cleaning systems perform equally. While the GalvoSurge\u0026reg; system showed strong biofilm removal capabilities, the X-Implant\u0026reg; system exhibited significantly lower efficacy in reducing viable biofilm bacteria. This disparity highlights the variability in performance among different electrolytic systems and underscores the need for further optimization of the different systems to enhance their effectiveness. Clinical trials and randomized controlled studies are crucial to validate the long-term efficacy in clinical settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll the authors declare no conflict of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding information\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the Research Foundation-Flanders (FWO G0B2719N). M.G.S. is a Strategic Basic Research Ph.D. fellow at the Research Foundation-Flanders (FWO-Vlaanderen) (grant number 1SHFK24N).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.B.C. contributed to the experimental design, data analysis and interpretation, and critically revised the manuscript, J.H. carried out the experiments, contributed to data acquisition, and wrote the manuscript, M.G.S. and K.S. contributed to the data acquisition and revised the manuscript, K.B. and A.B. contributed to the data analysis, and critically revised the manuscript, N.Z. contributed to the experimental design, carried out the experiments, data acquisition, analysis, and interpretation, wrote, and critically revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Dr. Martine Pauwels for the support with the v-qPCR measurements.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData supporting the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHerrera D, Berglundh T, Schwarz F, Chapple I, Jepsen S, Sculean A, et al. Prevention and treatment of peri-implant diseases\u0026mdash;The EFP S3 level clinical practice guideline. J Clin Periodontol. 2023;50:4\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeitz-Mayfield LJ, Salvi GE. Peri‐implant mucositis. J Clin Periodontol. 2018;45:S237\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDerks J, Tomasi C. Peri-implant health and disease. A systematic review of current epidemiology. J Clin Periodontol. 2015;42:S158\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003edos Santos Corpas L, Jacobs R, Quirynen M, Huang Y, Naert I, Duyck J. Peri-implant bone tissue assessment by comparing the outcome of intra‐oral radiograph and cone beam computed tomography analyses to the histological standard. Clin Oral Implants Res. 2011;22(5):492\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosta J, Mendes J, Salazar F, Pacheco J, Rompante P, C\u0026acirc;mara M. Analysis of peri-implant bone defects by using cone beam computed tomography (CBCT): an integrative review. Oral Radiol. 2023;39(3):455\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong D, Shujaat S, de Faria Vasconcelos K, Huang Y, Politis C, Lambrichts I, et al. Diagnostic accuracy of CBCT versus intraoral imaging for assessment of peri-implant bone defects. BMC Med Imaging. 2021;21:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomandini M, Lafor\u0026iacute; A, Pedrinaci I, Baima G, Ferrarotti F, Lima C, et al. Effect of sub-marginal instrumentation before surgical treatment of peri‐implantitis: A multi‐centre randomized clinical trial. J Clin Periodontol. 2022;49(12):1334\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaima G, Citterio F, Romandini M, Romano F, Mariani GM, Buduneli N, et al. Surface decontamination protocols for surgical treatment of peri-implantitis: A systematic review with meta‐analysis. Clin Oral Implants Res. 2022;33(11):1069\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamanauskaite A, Schwarz F, Cafferata EA, Sahrmann P. Photo/mechanical and physical implant surface decontamination approaches in conjunction with surgical peri-implantitis treatment: a systematic review. J Clin Periodontol. 2023;50:317\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDel Pozo JL, Rouse MS, Mandrekar JN, Steckelberg JM, Patel R. The electricidal effect: reduction of Staphylococcus and Pseudomonas biofilms by prolonged exposure to low-intensity electrical current. Antimicrob Agents Chemother. 2009;53(1):41\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohn D, Zehnder M, Stark WJ, Imfeld T. Electrochemical disinfection of dental implants\u0026ndash;a proof of concept. PLoS ONE. 2011;6(1):e16157.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZayed N, Ghesqui\u0026egrave;re J, Kamarudin N, Bernaerts K, Boon N, Braem A, et al. Oral biofilm cryotherapy as a novel ecological modulation approach. J Dent Res. 2023;102(9):1038\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZayed N, Boon N, Bernaerts K, Chatzigiannidou I, Van Holm W, Verspecht T, et al. Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms. J Periodontal Res. 2022;57(1):52\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZayed N, Munjaković H, Aktan M, Simoens K, Bernaerts K, Boon N, et al. Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro. J Dent Res. 2024;103(3):243\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Hu J, Zhao L. Adjunctive subgingival application of Chlorhexidine gel in nonsurgical periodontal treatment for chronic periodontitis: a systematic review and meta-analysis. BMC Oral Health. 2020;20:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamanauskaite E, Machiulskiene V. Antiseptics as adjuncts to scaling and root planing in the treatment of periodontitis: a systematic literature review. BMC Oral Health. 2020;20:1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDommisch H, Hoedke D, Valles C, Vilarrasa J, Jepsen S, Pascual La Rocca A. Efficacy of professionally administered chemical agents as an adjunctive treatment to sub-marginal instrumentation during the therapy of peri‐implant mucositis. J Clin Periodontol. 2023;50:146\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Li M, Yu J. Does chlorhexidine improve outcomes in non-surgical management of peri-implant mucositis or peri-implantitis? a systematic review and meta-analysis. Medicina oral patolog\u0026iacute;a oral y cirug\u0026iacute;a bucal. 2020;25(5):e608.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilensky A, Shapira L, Limones A, Martin C. The efficacy of implant surface decontamination using chemicals during surgical treatment of peri-implantitis: A systematic review and meta‐analysis. J Clin Periodontol. 2023;50:336\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmitage GC, Xenoudi P. Post-treatment supportive care for the natural dentition and dental implants. Periodontol 2000. 2016;71(1):164\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmerli W, Sendi P, editors. Pathogenesis of implant-associated infection: the role of the host. Seminars in immunopathology. Springer; 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlots J. Systemic antibiotics in periodontics. J Periodontol. 2004;75(11):1553\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 2001;65(2):232\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerrera D, Matesanz P, Mart\u0026iacute;n C, Oud V, Feres M, Teughels W. Adjunctive effect of locally delivered antimicrobials in periodontitis therapy: A systematic review and meta-analysis. J Clin Periodontol. 2020;47:239\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeughels W, Seyssens L, Christiaens V, Temmerman A, Castro AB, Cosyn J. Adjunctive locally and systemically delivered antimicrobials during surgical treatment of peri-implantitis: A systematic review. J Clin Periodontol. 2023;50:359\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider S, Rudolph M, Bause V, Terfort A. Electrochemical removal of biofilms from titanium dental implant surfaces. Bioelectrochemistry. 2018;121:84\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlee M, Naili L, Rathe F, Brodbeck U, Zipprich H. Is complete re-osseointegration of an infected dental implant possible? histologic results of a dog study: a short communication. J Clin Med. 2020;9(1):235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAssun\u0026ccedil;\u0026atilde;o MA, Botelho J, Machado V, Proen\u0026ccedil;a L, Matos AP, Mendes JJ, et al. Dental implant surface decontamination and surface change of an electrolytic method versus mechanical approaches: a pilot in vitro study. J Clin Med. 2023;12(4):1703.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlee M, Wang H-L, Stumpf T, Brodbeck U, Bosshardt D, Rathe F. Treatment of periimplantitis with electrolytic cleaning versus mechanical and electrolytic cleaning: 18-month results from a randomized controlled clinical trial. J Clin Med. 2021;10(16):3475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlee M, Rathe F, Brodbeck U, Ratka C, Weigl P, Zipprich H. Treatment of peri-implantitis\u0026mdash;electrolytic cleaning versus mechanical and electrolytic cleaning\u0026mdash;a randomized controlled clinical trial\u0026mdash;six-month results. J Clin Med. 2019;8(11):1909.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosshardt DD, Brodbeck UR, Rathe F, Stumpf T, Imber J-C, Weigl P, et al. Evidence of re-osseointegration after electrolytic cleaning and regenerative therapy of peri-implantitis in humans: a case report with four implants. Clin Oral Invest. 2022;26(4):3735\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dental Implants, Peri-Implantitis, Oral Biofilms, Infection, Decontamination","lastPublishedDoi":"10.21203/rs.3.rs-5402056/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5402056/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study evaluated the decontaminating efficacy of two electrolytic cleaning systems on titanium implants contaminated with multispecies biofilm, compared to conventional treatments like 0.2% chlorhexidine (CHX) or local antibiotics (tetracycline), with phosphate-buffered saline (PBS) as a negative control.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eA 14-species oral microbial community, developed using a bioreactor system, was used to grow biofilms on dental implant surfaces. Implants were then treated with two electrolytic systems, CHX, tetracycline, or phosphate-buffered saline (PBS). After cleaning, the implants were reincubated for 24 h. Biofilm viability was assessed through viability DNA extraction and quantitative PCR (v-qPCR). Additionally, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze biofilm structure and viability respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCHX and tetracycline treatments significantly reduced viable biofilm bacteria by 98.8% and 99.6%, respectively, compared to the negative control. The effect of the two electrolytic systems varied, with one reducing biofilm by 93.3%, similar to the positive controls, while the other showing only a 5.7% reduction in biofilm viability. SEM and CLSM imaging confirmed the distinct effects of the treatments on biofilm structure and viability.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe findings of this study highlight the potential of electrolytic cleaning as an effective minimally invasive approach for peri-implantitis management. However, this promising efficacy of the electrolytic cleaning systems alongside traditional antimicrobial agents in biofilm removal from dental implant surfaces greatly depend on the system. Further research is warranted to optimize different electrolytic cleaning protocols and validate their clinical efficacy in preventing and treating peri-implantitis.\u003c/p\u003e","manuscriptTitle":"Advancing Biofilm Removal: Evaluating Electrolytic Methods for Decontaminating Dental Implants In Vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 17:07:11","doi":"10.21203/rs.3.rs-5402056/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50fe0a31-0ad0-4cc6-b2fd-0f663af2f285","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-25T19:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-15 17:07:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5402056","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5402056","identity":"rs-5402056","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}