Voltage-Tunable Plasma-Activated Water: A Strategy for Combating Peri-Implantitis via Dual-Path Biofilm Disruption and Vascular Regeneration

Voltage-Tunable Plasma-Activated Water: A Strategy for Combating Peri-Implantitis via Dual-Path Biofilm Disruption and Vascular Regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Voltage-Tunable Plasma-Activated Water: A Strategy for Combating Peri-Implantitis via Dual-Path Biofilm Disruption and Vascular Regeneration Yiyuan Lang, Han Mei, Jingyun Zhang, Chenxi Hu, Zixu Pang, Yan Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7612573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Clinical Oral Investigations → Version 1 posted 12 You are reading this latest preprint version Abstract Objectives: Peri-implantitis therapy requires both effective biofilm eradication and subsequent tissue regeneration. This study aimed to develop a voltage-gated plasma-activated water (PAW) system with switchable bioactivities to address these dual needs and evaluate its efficacy for potential application in peri-implantitis management. Materials and Methods: PAW was generated using a dielectric barrier discharge system across a voltage spectrum of 33–46 kV. Its antibacterial activity was assessed against Porphyromonas gingivalis and multi-species biofilms on titanium surfaces, quantified via colony-forming unit (CFU) counts and visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The pro-angiogenic potential was evaluated through endothelial cell viability, migration, and tubule formation assays. Results: High-voltage PAW (46 kV, 30 min) achieved a >6.0-log reduction of P. gingivalis and induced structural collapse of multi-species biofilms. In contrast, low-voltage PAW (36 kV, 15 min) significantly enhanced endothelial cell viability to 118.3 ± 5.2%, and accelerated both cell migration and tubulogenesis. Conclusions: This study demonstrates that PAW's biological functionality can be precisely tuned via modulation of activation voltage. The same platform can be switched between a potent antibacterial state and a pro-regenerative state, enabling adaptive bioactivity suited to the staged therapeutic requirements of peri-implantitis. Clinical Relevance: The voltage-gated PAW system presents a promising, affordable strategy for dual-phase peri-implantitis therapy. It offers clinicians a potential non-antibiotic tool to first disinfect implant surfaces and then promote vascular healing, which is critical for improving clinical outcomes in implant dentistry. plasma-activated water peri-implantitis biofilm disruption vascular regeneration reactive nitrogen and oxygen species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Titanium implants are widely regarded as the gold standard for prosthetic tooth replacement due to their superior biocompatibility and manufacturability [ 1 , 2 ]. However, peri-implantitis remains a predominant inflammatory complication in implant dentistry [ 3 ]. Similar to periodontitis, peri-implantitis is primarily caused by microbial biofilm accumulation, which triggers a disproportionate host immune reaction [ 4 , 5 ]. The open and pathogen-rich oral environment further increases long-term infection risks. Current evidence demonstrates the impairment of host cell regenerative differentiation under bacterial co-culture conditions underscores the critical necessity of antimicrobial resistance as a fundamental prerequisite for peri-implantitis prevention [ 6 ]. Peri-implantitis-associated biofilms comprise bacterial species analogous to periodontitis pathogens, including Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) [ 7 ]. These biofilms adhere tenaciously to implant surfaces [ 8 ] and exhibit recalcitrance to treatment through dual mechanisms: physical protection via extracellular polymeric matrices and adaptive antimicrobial tolerance [ 9 , 10 ]. Conventional antimicrobials often fail to penetrate biofilms or selectively target embedded pathogens [ 11 ], underscoring an urgent need for innovative strategies to eradicate biofilm-mediated infections without exacerbating resistance. Blood vessels, in charge of supplying cells with sufficient oxygen and nutrients, as well as disposal of metabolic waste, are a prerequisite for cell survival and viability [ 12 , 13 ]. To promote angiogenesis, direct injection of angiogenic stimulators (such as vascular endothelial growth factor (VEGF)) or related genes has been implemented [ 14 , 15 ]. However, these methods are not only expensive but also lack sustained efficacy. Hypoxia conditions, specific and non-specific acting factors, and some cytokines, chemokines, active oxygen and active nitrogen substances can affect the formation of neovascularization [ 16 , 17 ]. Given the limitations of current pro-angiogenic strategies and the recognized role of specific RONS in vascularization, we propose a novel therapeutic strategy for peri-implantitis that concurrently addresses the dual challenges of bacterial biofilm eradication and vascular regeneration using PAW. This approach leverages the inherent bioactivity of RONS generated by plasma in a unique, tunable, and cost-effective aqueous medium. Plasma is characterized by a rich array of highly reactive chemical species. Within this classification, non-thermal plasma (NTP), alternatively termed cold a atmosphere plasma (CAP), has emerged as a biologically compatible technology for medical applications due to its operation at near-ambient temperatures (typically 25–40°C) [ 18 , 19 ]. CAP delivers a complex cocktail of RONS—including hydroxyl radicals (•OH), superoxide (O₂•⁻), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), nitric oxide (•NO), and peroxynitrite (ONOO⁻)—alongside charged particles and UV photons, which collectively drive potent antimicrobial effects [ 20 – 22 ]. Transferring these reactive species to aqueous media generates PAW. Compared to direct plasma application, PAW offers enhanced uniformity of bioeffects, extended storage stability, and improved clinical practicality for targeting complex anatomical sites [ 23 – 25 ]. This study leverages the tunable chemistry of PAW by strategically modulating generation parameters (voltage, exposure time) to achieve two critical therapeutic outcomes: targeted eradication of pathogenic biofilms and promotion of vascular regeneration. 2. Materials and Methods 2.1Preparation of plasma-activated water The plasma-activated water was generated using a custom dielectric barrier discharge (DBD) reactor system (Fig. 1 A), comprising a high-voltage pulse generator with adjustable output, a cylindrical reactor with a stainless-steel mesh electrode (304 grade, 80 mesh) concentrically surrounding a borosilicate glass tube (outer diameter(OD): 15 mm) housing a central tungsten rod electrode (Ø 2 mm; discharge gap: 5 mm), a peristaltic pump circulating ultrapure water at 100 mL/min, and a digital oscilloscope monitoring discharge characteristics. For each experimental condition, 150 mL of water was circulated through the reactor for 15 minutes under four optimized voltages (33 kV [breakdown threshold], 36 kV, 42 kV, and 46 kV), with PAW collected immediately after activation. The Fig. 1 shows a typical voltage-current waveform diagram, in which the power density gradually increases as the voltage rises. The pH was adjusted before subsequent biological assays were performed. 2.2Antibacterial Function Evaluation Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC25586) and Streptococcus sanguinis (ATCC10556) were used for they associated with the peri-implantitis [ 26 ]. Titanium discs (1 cm in diameter) were placed in 1 mL of bacterial suspension (107 CFU mL − 1) and cultured for 72 h at 37°C under anaerobic conditions to allow biofilm formation. To evaluate the antimicrobial activity of PAW, the biofilms were treated and then analyzed. The contact killing effect was assessed by: (1) culturing and CFU counting using Petrifilm and (2) live/dead staining using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, USA). The sterilization ability of PAW was evaluated by the Log Reduction, calculated as follows: LogReduction = Log(CFUcontrol/CFUtreated). For the staining, after the 72 h biofilm formation and treatment, the discs were washed with PBS and stained following the manufacturer's protocol, then imaged by confocal laser scanning microscopy (CLSM). Additionally, scanning electron microscopy (SEM) was used to observe structural and morphological changes of bacteria after PAW treatment. 2.3 Culture and Treatment of HUVECs Human umbilical vein endothelial cells (HUVECs) were donated by the Nanjing Medical University. 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) with 1% penicillin (100 U/mL)-streptomycin (100 µg/mL) mixed solution (Gibco, USA) was cultured in ECM culture medium (ScienCell, USA). Cultures were maintained at 37°C/5% CO₂ with medium changes every 48 h, and cells at passages 3–5 were used for all experiments. 2.4 CCK8 Cytotoxicity Assay HUVECs were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere for 24 h. The medium was then replaced with medium containing the respective PAW solutions generated at 0 kV (control), 33 kV, 36 kV, 42 kV, or 46 kV. Three technical replicates were performed per condition. 2.5 Tube Formation Assay To assess angiogenic potential, HUVECs were trypsinized and seeded (8 × 10⁴ cells/well) onto 24-well plates pre-coated with 20 µl of growth factor-reduced Matrigel. Cells were then incubated at 37°C under 5% CO₂ in low-serum (2% FBS) medium containing the respective PAW treatment groups (0kv for control, 33kv, 36kv, 42kv and 46kv). Cellular network formation was visualized and imaged under an inverted phase-contrast microscope (Olympus IX73) at three critical time points: immediately after seeding (0 h), and following 6 h and 24 h of incubation. Tube formation was quantified by measuring three key parameters per field of view: (1) the total number of branch points, (2) the total number of enclosed tubular structures, and (3) the total tube length (µm) using image analysis software ( ImageJ with the Angiogenesis Analyzer plugin). 2.6 Wound Healing Assay HUVECs were seeded into a 6-well plate and cultured for 24 hours. The original medium was then removed, and the cells were gently washed three times with PBS to remove detached cells. Subsequently, low-serum (2%) medium containing the different PAW treatment groups was added. Images of the scratch wound area were captured under a microscope at 0, 6, and 24 hours. Scratch width was measured or cell-covered area was calculated to compare wound healing and cell migration rates between groups. 2.7 Statistical analysis Data are expressed as means ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5.01, employing one-way ANOVA with appropriate post-hoc testing for multi-group comparisons and Student's *t*-test for two-group comparisons. Statistical significance was defined as p < 0.05. 3. Results 3.1. Characterization of PAW As shown in Table 1 and Fig. 2 , immediately post-treatment, the key physicochemical properties including pH, oxidation reduction potential (ORP), and reactive species concentrations were quantified. ORP serves as a critical metric for solution oxidative capacity, directly correlating with activity and concentration. ORP surges from mildly oxidative to highly oxidative at 46 kV. Concurrently, spectrophotometric measurements confirmed increasing concentrations of nitrate (NO₃⁻) and nitrite (NO₂⁻) anions with higher activation voltages. These collective trends demonstrate that elevated voltages drive significantly higher concentrations of reactive nitrogen/oxygen species (RNS/ROS). Table 1 Electrochemical Properties Control 33kv 36kv 42kv 46kv pH 7.2 ± 0.1 3.5 ± 0.2 1 2.8 ± 0.3 1 2.6 ± 0.2 1 2.4 ± 0.2 1 ORP(mV) + 150 ± 15 + 780 ± 25 1 + 950 ± 30 1 + 1020 ± 35 1 + 1100 ± 40 1 Conductiity(uS/cm) 0.5 ± 0.1 320 ± 18 1 450 ± 25 1 580 ± 30 1 690 ± 32 1 1 Data: mean ± SD, n = 3; p < 0.05 vs control 3.2. Antimicrobial Activity 3.2.1 Time-dependent inactivation of Porphyromonas gingivalis by PAW at different voltages PAW was generated via 15-minute plasma activation at four discharge voltages: 33 kV, 36 kV, 42 kV, and 46 kV. The bactericidal efficacy against P.g suspensions was assessed after exposure to PAW for 5, 10, 15, and 30 minutes. Voltage- and time-dependent inactivation was observed (Fig. 3 ). At 46 kV, a 6.0-log₁₀ reduction occurred, corresponding to 99.99% inactivation (p < 0.001 vs. control; ANOVA-Tukey). Crucially, no viable colonies were detected in: 42 kV groups ( 15/30 minute) and the 46 kV (10/15/30 minute) groups, confirming the complete bactericidal eradication rate. 3.2.2 Morphological Alterations in Tri-Species Biofilm Induced by Plasma-Activated Water: Scanning Electron Microscopy Scanning electron microscopy provided visual evidence of PAW's bactericidal efficacy against the tri-species biofilm. Figure 4 illustrates the substantial morphological damage inflicted by a 10-minute treatment with 42 kV PAW to three types of bacterial biofilms. Following exposure, the biofilm transitioned from its original intact structure to a state characterized by widespread cell rupture, surface collapse, and severe shrinkage.The biofilm thickness of both P.g and S.s also decreased significantly. Due to its biological characteristics as a “bridge bacterium,” F.n has a relatively weak ability to independently form mature biofilms, a significant reduction in its numbers was also observed [ 27 , 28 ]. 3.2.3 PAW effect on Porphyromonas gingivalis viability: Live/Dead Staining Visualized by Confocal Laser Scanning Microscopy The viability of bacteria was determined by Baclight LIVE/DEAD assay kit. As shown in Fig. 5 , untreated control biofilms exhibited dense green fluorescence indicating abundant live cells, whereas PAW treatment at 42 kV for 10 minutes caused a dramatic reduction in green-fluorescent viable bacteria alongside red fluorescence from dead cells with compromised membranes. Concurrently, biofilm architecture underwent substantial disruption with visibly reduced thickness. These morphological and viability changes directly align with the previously documented greater than 6.0-log CFU reduction, confirming PAW's capacity to induce lethal cellular damage and degrade biofilm integrity. 3.4. Biphasic Cellular Response to PAW Voltage As shown in Fig. 6 A, scratch assays demonstrated voltage-dependent wound closure: 33 kV significantly enhanced closure compared to control (p < 0.01), 36 kV elicited maximal closure, while voltages more than 42 kV exhibited cytotoxicity (p < 0.001 vs 36 kV). Complementary CCK-8 assays confirmed a biphasic response: 36 kV showed peak viability (p < 0.01 vs control), whereas voltages ≥ 42 kV demonstrated cytotoxicity (p < 0.001 vs 36 kV). (ANOVA: F = 185.6, p < 0.0001; n = 6). This functional decline corresponded to an 84.3% reduction in tube formation at 42 kV, with 107 branches versus 684 branches at 36 kV, and near-complete angiogenic failure at 46 kV with only 85 branches. These effects establish a narrow therapeutic window at 33–36 kV for PAW’s voltage-dependent bioactivity. 4. Discussion The observed voltage-dependent electrochemical properties of PAW can be mechanistically interpreted through the lens of discharge physics and aqueous reaction kinetics. The stepwise increase in conductivity directly reflects the elevated ionic content resulting from plasma-water interactions. As the applied voltage increases, the energy input into the DBD system rises, leading to enhanced electron impact dissociation of N₂, O₂, and H₂O vapour in the discharge zone. This generates higher densities of reactive precursors such as •OH, O•, NO•, and excited species, which subsequently hydrolyse or oxidise in water to form stable ions including NO₃⁻, NO₂⁻, H⁺, and H₂O₂. The consequent rise in ionic strength directly accounts for the increasing conductivity. Simultaneously, the surge in oxidative species concentration—particularly HNO₂/NO₂⁻ and peroxynitrite (ONOO⁻)—drives the pronounced drop in pH (to 2.4 ± 0.2 at 46 kV) and the sharp positive shift in ORP (from + 150 ± 15 mV to + 1100 ± 40 mV). The ORP, serving as an integrative indicator of the solution's redox potential, confirms the transition into a highly oxidative state at 46 kV, dominated by the accumulation of short-lived reactive oxygen and nitrogen species (RONS) with high redox potential. This electrochemical framework directly establishes voltage as a biological switch for plasma-activated water, enabling on-demand transition between two therapeutic modes: regenerative mode (36 kV ): Promotes angiogenesis through endothelial migration (96.3 ± 1.5% wound closure, p < 0.001 vs control) and accelerated tubulogenesis and antibacterial mode achieves more than 6-log biofilm eradication via rapid, cascadic ROS/RNS burst causing membrane peroxidation and DNA fragmentation in bacteria. Critically, the 42 kV threshold marks a functional tipping point that triggers coordinated collapse of viability, migration and tubulogenesis (Fig. 6 ). The dual nature of RONS critically influences peri-implantitis outcomes. Biofilm infection initiates a vicious cycle: pathogen-associated molecular patterns (e.g., P. gingivalis LPS) trigger excessive inflammatory responses, leading to uncontrolled endogenous RONS production. Damages vascular endothelia through eNOS uncoupling and VE-cadherin degradation [ 29 ], impairing angiogenesis; activates the NLRP3 inflammasome via mitochondrial ROS (mtROS) overproduction [ 30 ]. Conversely, PAW-engineered RONS reprogram pathological microenvironments: liquid plasma promotes angiogenesis by inducing extracellular matrix metabolism through upregulation of endothelial nitric oxide synthase (eNOS) [ 31 ]. Unlike uncontrolled oxidative damage in peri-implantitis, PAW-engineered RONS deliver “redox messages” that mimic physiological wound healing and turn the double-edged sword into a precision surgical tool. Compared to current peri-implantitis treatments, voltage-switchable PAW offers several advantages: Peri-implantitis requires not only eradicating pathogens but also restoring damaged peri-implant bone and soft tissues. Antibiotics focus solely on sterilisation but do not promote healing [ 32 ]; mechanical debridement may further disrupt fragile regenerative environments [ 33 ]; and growth factors, while pro-healing, lack inherent antimicrobial properties, risking re-infection [ 34 – 36 ]. Besides, a major drawback of antibiotic-based treatments is the emergence of drug-resistant pathogens, which complicates retreatment and increases failure risks. PAW's antimicrobial mechanism relies on oxidative damage to bacterial cell membranes and DNA via ROS/RNS—pathways bacteria rarely develop resistance to, as they cannot mutate to evade non-specific oxidative stress [ 37 , 38 ]. This study has several limitations: First, while our study employed mono-species biofilms (P. g, S.s, F. n), clinical peri-implantitis involves complex polymicrobial communities [ 39 ]. Second, short NO₂⁻ half-life necessitates point-of-care PAW generation, current protocols require bedside plasma devices [ 31 , 40 ]. Third, absence of in vivo validation in mammalian models limits translational prediction of host-microbe dynamics. We have developed PAW as a voltage-responsive liquid biomaterial. Its physical properties undergo distinct transformations at critical electrical thresholds, enabling it to perform specific biological functions. This electro-metabolic switching behavior—where application of 36 kV promotes tissue regeneration, while 46 kV triggers potent biocidal activity—demonstrates potential for precisely controlled biointerfaces. Beyond initial applications in dentistry, this technology shows promise for managing chronic wounds, particularly in contexts where infection and compromised vascularization are concurrent challenges. 5. Conclusions We demonstrate that voltage-controlled PAW delivers dual therapeutic functions for peri-implantitis management. PAW at 46 kV eradicates pathogenic biofilms, while PAW at 36 kV promotes angiogenesis, enhancing endothelial cell migration and function. Critically, these bioeffects are voltage-specific, with a cytotoxic threshold emerging at more than 42 kV. This tuning strategy addresses the dual challenge of biofilm elimination and tissue regeneration. Although clinical translation requires further investigation, PAW shows significant potential as a targeted therapy for implant-related tissue damage. Abbreviations The following abbreviations are used in this manuscript: PAW Plasma-activated water Pg Porphyromonas gingivalis Fn Fusobacterium nucleatum VEGF Vascular endothelial growth factor NTP Non-thermal plasma CAP Cold a atmosphere plasma DBD Dielectric barrier discharge CLSM Confocal laser scanning microscopy SEM Scanning electron microscopy HUVECs Human umbilical vein endothelial cells SD Standard deviation ORP Oxidation reduction potential RNS/ROS Reactive nitrogen/oxygen species mtROS Mitochondrial reactive oxygen species eNOS Endothelial nitric oxide synthase Declarations Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding: This study was funded by a grant from the Science and Technology Department of Jiangsu Province, grant number BE2020707. Author Contribution Conceptualization, methodology and writing—original draft preparation, Y.L.; data curation, H.M.; writing—review and editing, J.Z and C.H.; supervision and funding acquisition, C.X.; visualization, Z.P. and Y.C; . All authors have read and agreed to the published version of the manuscript. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References Brånemark, P.I.; Adell, R.; Breine, U.; Hansson, B.O.; Lindström, J.; Ohlsson, A. Intra-Osseous Anchorage of Dental Prostheses. I. Experimental Studies. Scand J Plast Reconstr Surg 1969 , 3 , 81–100. Koizumi, H.; Takeuchi, Y.; Imai, H.; Kawai, T.; Yoneyama, T. Application of Titanium and Titanium Alloys to Fixed Dental Prostheses. J Prosthodont Res 2019 , 63 , 266–270. Roccuzzo, A.; Stähli, A.; Monje, A.; Sculean, A.; Salvi, G.E. Peri-Implantitis: A Clinical Update on Prevalence and Surgical Treatment Outcomes. J Clin Med 2021 , 10 , 1107. Berglundh, T.; Armitage, G.; Araujo, M.G.; Avila-Ortiz, G.; Blanco, J.; Camargo, P.M.; Chen, S.; Cochran, D.; Derks, J.; Figuero, E.; et al. Peri-Implant Diseases and Conditions: Consensus Report of Workgroup 4 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Clin Periodontol 2018 , 45 Suppl 20 , S286–S291. Schwarz, F.; Derks, J.; Monje, A.; Wang, H.-L. Peri-Implantitis. J Clin Periodontol 2018 , 45 Suppl 20 , S246–S266. Li, D.; Tan, X.; Zheng, L.; Tang, H.; Hu, S.; Zhai, Q.; Jing, X.; Liang, P.; Zhang, Y.; He, Q.; et al. A Dual-Antioxidative Coating on Transmucosal Component of Implant to Repair Connective Tissue Barrier for Treatment of Peri-Implantitis. Adv Healthc Mater 2023 , 12 , e2301733. De Bruyn, H.; Raes, S.; Ostman, P.-O.; Cosyn, J. Immediate Loading in Partially and Completely Edentulous Jaws: A Review of the Literature with Clinical Guidelines. Periodontol 2000 2014 , 66 , 153–187. Caton, J.G.; Armitage, G.; Berglundh, T.; Chapple, I.L.C.; Jepsen, S.; Kornman, K.S.; Mealey, B.L.; Papapanou, P.N.; Sanz, M.; Tonetti, M.S. A New Classification Scheme for Periodontal and Peri-Implant Diseases and Conditions - Introduction and Key Changes from the 1999 Classification. J Clin Periodontol 2018 , 45 Suppl 20 , S1–S8. Flemming, H.-C.; van Hullebusch, E.D.; Neu, T.R.; Nielsen, P.H.; Seviour, T.; Stoodley, P.; Wingender, J.; Wuertz, S. The Biofilm Matrix: Multitasking in a Shared Space. Nat Rev Microbiol 2023 , 21 , 70–86. Stewart, P.S.; Costerton, J.W. Antibiotic Resistance of Bacteria in Biofilms. Lancet 2001 , 358 , 135–138. Darby, E.M.; Trampari, E.; Siasat, P.; Gaya, M.S.; Alav, I.; Webber, M.A.; Blair, J.M.A. Molecular Mechanisms of Antibiotic Resistance Revisited. Nat Rev Microbiol 2023 , 21 , 280–295. Sun, F.; Lu, Y.; Wang, Z.; Shi, H. Vascularization Strategies for Tissue Engineering for Tracheal Reconstruction. Regen Med 2021 , 16 , 549–566. Tian, T.; Zhang, T.; Lin, Y.; Cai, X. Vascularization in Craniofacial Bone Tissue Engineering. J Dent Res 2018 , 97 , 969–976. Kamran, N.; Kadiyala, P.; Saxena, M.; Candolfi, M.; Li, Y.; Moreno-Ayala, M.A.; Raja, N.; Shah, D.; Lowenstein, P.R.; Castro, M.G. Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy. Mol Ther 2017 , 25 , 232–248. Skipper, M. Targeting the Worm. Nat Rev Genet 2004 , 5 , 883–883. Isenberg, J.S.; Ridnour, L.A.; Espey, M.G.; Wink, D.A.; Roberts, D.D. Nitric Oxide in Wound-Healing. Microsurgery 2005 , 25 , 442–451. Kieran, M.W.; Kalluri, R.; Cho, Y.-J. The VEGF Pathway in Cancer and Disease: Responses, Resistance, and the Path Forward. Cold Spring Harb Perspect Med 2012 , 2 , a006593. VON Woedtke, T.; Schmidt, A.; Bekeschus, S.; Wende, K.; Weltmann, K.-D. Plasma Medicine: A Field of Applied Redox Biology. In Vivo 2019 , 33 , 1011–1026. Yamabe, C.; Takeshita, F.; Miichi, T.; Hayashi, N.; Ihara, S. Water Treatment Using Discharge on the Surface of a Bubble in Water. Plasma Processes & Polymers 2005 , 2 , 246–251. Tresp, H.; Hammer, M.U.; Winter, J.; Weltmann, K.-D.; Reuter, S. Quantitative Detection of Plasma-Generated Radicals in Liquids by Electron Paramagnetic Resonance Spectroscopy. J. Phys. D: Appl. Phys. 2013 , 46 , 435401. Yang, Y.; Zheng, M.; Yang, Y.; Li, J.; Su, Y.-F.; Li, H.-P.; Tan, J.-G. Inhibition of Bacterial Growth on Zirconia Abutment with a Helium Cold Atmospheric Plasma Jet Treatment. Clin Oral Investig 2020 , 24 , 1465–1477. Zhang, Q.; Liang, Y.; Feng, H.; Ma, R.; Tian, Y.; Zhang, J.; Fang, J. A Study of Oxidative Stress Induced by Non-Thermal Plasma-Activated Water for Bacterial Damage. Applied Physics Letters 2013 , 102 , 203701. Arda, G.; Hsu, C. Preservation of Reactive Species in Frozen Plasma-Activated Water and Enhancement of Its Bactericidal Activity Through pH Adjustment. Plasma Chem Plasma Process 2023 , 43 , 599–618. Harley, J.C.; Suchowerska, N.; McKenzie, D.R. Cancer Treatment with Gas Plasma and with Gas Plasma-Activated Liquid: Positives, Potentials and Problems of Clinical Translation. Biophys Rev 2020 , 12 , 989–1006. Qiao, D.; Li, Y.; Pan, J.; Zhang, J.; Tian, Y.; Wang, K. Effect of Plasma Activated Water in Caries Prevention: The Caries Related Biofilm Inhibition Effects and Mechanisms. Plasma Chem Plasma Process 2022 , 42 , 801–814. Pérez-Chaparro, P.J.; Duarte, P.M.; Shibli, J.A.; Montenegro, S.; Lacerda Heluy, S.; Figueiredo, L.C.; Faveri, M.; Feres, M. The Current Weight of Evidence of the Microbiologic Profile Associated With Peri-Implantitis: A Systematic Review. J Periodontol 2016 , 87 , 1295–1304. Kolenbrander, P.E.; Andersen, R.N.; Blehert, D.S.; Egland, P.G.; Foster, J.S.; Palmer, R.J. Communication among Oral Bacteria. Microbiol Mol Biol Rev 2002 , 66 , 486–505, table of contents. Periasamy, S.; Kolenbrander, P.E. Aggregatibacter Actinomycetemcomitans Builds Mutualistic Biofilm Communities with Fusobacterium Nucleatum and Veillonella Species in Saliva. Infect Immun 2009 , 77 , 3542–3551. Ye, X.; Ma, T.; Blais, J.E.; Yan, V.K.C.; Kang, W.; Chui, C.S.L.; Lai, F.T.T.; Li, X.; Wan, E.Y.F.; Wong, C.K.H.; et al. Association between BNT162b2 or CoronaVac COVID-19 Vaccines and Major Adverse Cardiovascular Events among Individuals with Cardiovascular Disease. Cardiovasc Res 2022 , 118 , 2329–2338. Zhou, R.; Yazdi, A.S.; Menu, P.; Tschopp, J. A Role for Mitochondria in NLRP3 Inflammasome Activation. Nature 2011 , 469 , 221–225. Kang, S.U.; Kim, H.J.; Ma, S.; Oh, D.-Y.; Jang, J.Y.; Seo, C.; Lee, Y.S.; Kim, C.-H. Liquid Plasma Promotes Angiogenesis through Upregulation of Endothelial Nitric Oxide Synthase-Induced Extracellular Matrix Metabolism: Potential Applications of Liquid Plasma for Vascular Injuries. Cell Commun Signal 2024 , 22 , 138. Lio, P.A.; Kaye, E.T. Topical Antibacterial Agents. Infect Dis Clin North Am 2004 , 18 , 717–733. Enevold, C.; Nielsen, C.H.; Christensen, L.B.; Kongstad, J.; Fiehn, N.E.; Hansen, P.R.; Holmstrup, P.; Havemose-Poulsen, A.; Damgaard, C. Suitability of Machine Learning Models for Prediction of Clinically Defined Stage III/IV Periodontitis from Questionnaires and Demographic Data in Danish Cohorts. J Clin Periodontol 2024 , 51 , 1561–1573. Ghatnekar, O.; Persson, U.; Willis, M.; Odegaard, K. Cost Effectiveness of Becaplermin in the Treatment of Diabetic Foot Ulcers in Four European Countries. Pharmacoeconomics 2001 , 19 , 767–778. Lepperdinger, U.; Angwin, C.; Milnes, D.; Sobey, G.; Ghali, N.; Johnson, D.; Brady, A.F.; Kammin, T.; Bowen, J.M.; Gröbner, R.; et al. Oral Characteristics in Adult Individuals with Periodontal Ehlers-Danlos Syndrome. J Clin Periodontol 2022 , 49 , 1244–1252. Werner, S.; Grose, R. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 2003 , 83 , 835–870. Cabiscol, E.; Tamarit, J.; Ros, J. Oxidative Stress in Bacteria and Protein Damage by Reactive Oxygen Species. Int Microbiol 2000 , 3 , 3–8. Dharmaraja, A.T. Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria. J Med Chem 2017 , 60 , 3221–3240. Falcao, A.; Bullón, P. A Review of the Influence of Periodontal Treatment in Systemic Diseases. Periodontol 2000 2019 , 79 , 117–128. Wende, K.; Williams, P.; Dalluge, J.; Gaens, W.V.; Aboubakr, H.; Bischof, J.; von Woedtke, T.; Goyal, S.M.; Weltmann, K.-D.; Bogaerts, A.; et al. Identification of the Biologically Active Liquid Chemistry Induced by a Nonthermal Atmospheric Pressure Plasma Jet. Biointerphases 2015 , 10 , 029518. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Clinical Oral Investigations → Version 1 posted Editorial decision: Revision requested 17 Oct, 2025 Reviews received at journal 11 Oct, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers invited by journal 18 Sep, 2025 Editor assigned by journal 16 Sep, 2025 Submission checks completed at journal 16 Sep, 2025 First submitted to journal 14 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7612573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521676975,"identity":"e97201b2-9a20-4130-aa75-725ffe55bac0","order_by":0,"name":"Yiyuan Lang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiyuan","middleName":"","lastName":"Lang","suffix":""},{"id":521676976,"identity":"453b2d53-860b-47f7-9120-6e667dd63c7a","order_by":1,"name":"Han Mei","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Mei","suffix":""},{"id":521676977,"identity":"4998dc72-7c14-4523-b07e-e55290cffc10","order_by":2,"name":"Jingyun Zhang","email":"","orcid":"","institution":"Nanjing University of Aeronautics and Astronautics","correspondingAuthor":false,"prefix":"","firstName":"Jingyun","middleName":"","lastName":"Zhang","suffix":""},{"id":521676978,"identity":"1916ceba-a524-4276-bf86-dad93b81d0fb","order_by":3,"name":"Chenxi Hu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Hu","suffix":""},{"id":521676979,"identity":"13423baa-e4bd-43d4-a4fc-0ad7524de92a","order_by":4,"name":"Zixu Pang","email":"","orcid":"","institution":"Nanjing University of Aeronautics and Astronautics","correspondingAuthor":false,"prefix":"","firstName":"Zixu","middleName":"","lastName":"Pang","suffix":""},{"id":521676980,"identity":"152c429e-111a-49eb-88ea-7944bef3c6fb","order_by":5,"name":"Yan Chen","email":"","orcid":"","institution":"Nanjing first hospital","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Chen","suffix":""},{"id":521676981,"identity":"71eecfac-9573-4d97-a31e-1425b070a765","order_by":6,"name":"Changao Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYJACZih1gEECzEggWgtbAslaeAygfAJaDG4kH/xcUHPHrr+959sDy5zDDPzsOQYMP3Nwa5GckZYsPePYs+QZZ85uN5DcdphBsueNAWPvNtxa+CVyzJh52A4nM9zI3SYB0mJwI8eAmRGPFjaJ/G/MPP8OJ8vfyHkG1mJPSAvQFjZm3rbDdkDD2SC2SBDQItnzzFiat+9wguGZY2ZALek8EmeeFRzE5xeD48kPP/N8O2wvd7z5mbTkNms5/vbkjQ9+4tECA4kNQIIZGJU8IN4BwhoYGOxBBOMHYpSOglEwCkbBiAMAfeVO8ZtRkWUAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Changao","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2025-09-14 12:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7612573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7612573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00784-026-06749-3","type":"published","date":"2026-04-24T15:59:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92422500,"identity":"af5a92ea-32ab-4603-a11d-60e0cec93ceb","added_by":"auto","created_at":"2025-09-29 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14:38:55","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104115,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/b4f402a4d838173f0025605e.html"},{"id":92422501,"identity":"0005adb1-b8a5-43cb-915d-59d9b1529dab","added_by":"auto","created_at":"2025-09-29 14:38:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":289720,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic diagram of the device (B) Typical voltage-current diagram (C) Power density at different voltages\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/2bd010ef873c7bf9c63ab00d.png"},{"id":92422496,"identity":"dbc17899-6141-46e8-8bca-2e10afa2d5d6","added_by":"auto","created_at":"2025-09-29 14:38:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32075,"visible":true,"origin":"","legend":"\u003cp\u003eReactive Species Quantification.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/f8ec1baf3adcdb6451b3f34b.png"},{"id":92422483,"identity":"f34a392c-14d6-4999-a5af-26a3840d7293","added_by":"auto","created_at":"2025-09-29 14:38:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":953752,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Antibacterial activity of PAW against \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e on blood agar. (B) Quantitative analysis of PAW's bactericidal efficacy against \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e biofilm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/4ae10cb898dd54d716d2d56a.png"},{"id":92422480,"identity":"62fe5401-b25b-4d73-b8fc-580e10da3254","added_by":"auto","created_at":"2025-09-29 14:38:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1090345,"visible":true,"origin":"","legend":"\u003cp\u003eSEM validation of Post-PAW treatment (42 kV, 10 min) showing structural collapse; Scale bars: 1 μm . Samples fixed with 2.5% glutaraldehyde and gold-sputtered prior to imaging (Hitachi SU8010, 5 kV).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/9c57c4b51058d5d79ece30e4.png"},{"id":92422495,"identity":"8797b9bc-792e-4b30-b4a4-1ab667dcc7d9","added_by":"auto","created_at":"2025-09-29 14:38:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436435,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Live; (\u003cstrong\u003eb\u003c/strong\u003e) Dead; (\u003cstrong\u003ec\u003c/strong\u003e) Merge of live/dead channels; (\u003cstrong\u003ed\u003c/strong\u003e) 3D-rendered reconstruction of biofilm viability. (PI: red SYTO9: green).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/17614abc6b62cee7d7c683bb.png"},{"id":92422505,"identity":"6a66d64b-fe70-4a38-9aa4-97ca616be64c","added_by":"auto","created_at":"2025-09-29 14:38:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":688732,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Scratch closure at 0,6,24h; (B) Quantitative closure rate; (C) Tube networks at 24h; (D) Number of tubes; (E) Viability by CCK-8.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/83333fc39e5101eb989242a0.png"},{"id":107928604,"identity":"4aa10747-6fee-4559-b0e7-3d5a65352875","added_by":"auto","created_at":"2026-04-27 16:11:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3864108,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7612573/v1/0418e416-6855-46a1-a4fd-d455f33ce986.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Voltage-Tunable Plasma-Activated Water: A Strategy for Combating Peri-Implantitis via Dual-Path Biofilm Disruption and Vascular Regeneration","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTitanium implants are widely regarded as the gold standard for prosthetic tooth replacement due to their superior biocompatibility and manufacturability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, peri-implantitis remains a predominant inflammatory complication in implant dentistry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Similar to periodontitis, peri-implantitis is primarily caused by microbial biofilm accumulation, which triggers a disproportionate host immune reaction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The open and pathogen-rich oral environment further increases long-term infection risks. Current evidence demonstrates the impairment of host cell regenerative differentiation under bacterial co-culture conditions underscores the critical necessity of antimicrobial resistance as a fundamental prerequisite for peri-implantitis prevention [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePeri-implantitis-associated biofilms comprise bacterial species analogous to periodontitis pathogens, including \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (Pg) and \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e (Fn) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese biofilms adhere tenaciously to implant surfaces [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and exhibit recalcitrance to treatment through dual mechanisms: physical protection via extracellular polymeric matrices and adaptive antimicrobial tolerance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Conventional antimicrobials often fail to penetrate biofilms or selectively target embedded pathogens [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], underscoring an urgent need for innovative strategies to eradicate biofilm-mediated infections without exacerbating resistance.\u003c/p\u003e\u003cp\u003eBlood vessels, in charge of supplying cells with sufficient oxygen and nutrients, as well as disposal of metabolic waste, are a prerequisite for cell survival and viability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To promote angiogenesis, direct injection of angiogenic stimulators (such as vascular endothelial growth factor (VEGF)) or related genes has been implemented [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, these methods are not only expensive but also lack sustained efficacy. Hypoxia conditions, specific and non-specific acting factors, and some cytokines, chemokines, active oxygen and active nitrogen substances can affect the formation of neovascularization [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Given the limitations of current pro-angiogenic strategies and the recognized role of specific RONS in vascularization, we propose a novel therapeutic strategy for peri-implantitis that concurrently addresses the dual challenges of bacterial biofilm eradication and vascular regeneration using PAW. This approach leverages the inherent bioactivity of RONS generated by plasma in a unique, tunable, and cost-effective aqueous medium.\u003c/p\u003e\u003cp\u003ePlasma is characterized by a rich array of highly reactive chemical species. Within this classification, non-thermal plasma (NTP), alternatively termed cold a atmosphere plasma (CAP), has emerged as a biologically compatible technology for medical applications due to its operation at near-ambient temperatures (typically 25\u0026ndash;40\u0026deg;C) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CAP delivers a complex cocktail of RONS\u0026mdash;including hydroxyl radicals (\u0026bull;OH), superoxide (O₂\u0026bull;⁻), singlet oxygen (\u0026sup1;O₂), hydrogen peroxide (H₂O₂), nitric oxide (\u0026bull;NO), and peroxynitrite (ONOO⁻)\u0026mdash;alongside charged particles and UV photons, which collectively drive potent antimicrobial effects [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Transferring these reactive species to aqueous media generates PAW. Compared to direct plasma application, PAW offers enhanced uniformity of bioeffects, extended storage stability, and improved clinical practicality for targeting complex anatomical sites [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This study leverages the tunable chemistry of PAW by strategically modulating generation parameters (voltage, exposure time) to achieve two critical therapeutic outcomes: targeted eradication of pathogenic biofilms and promotion of vascular regeneration.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1Preparation of plasma-activated water\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe plasma-activated water was generated using a custom dielectric barrier discharge (DBD) reactor system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), comprising a high-voltage pulse generator with adjustable output, a cylindrical reactor with a stainless-steel mesh electrode (304 grade, 80 mesh) concentrically surrounding a borosilicate glass tube (outer diameter(OD): 15 mm) housing a central tungsten rod electrode (\u0026Oslash; 2 mm; discharge gap: 5 mm), a peristaltic pump circulating ultrapure water at 100 mL/min, and a digital oscilloscope monitoring discharge characteristics. For each experimental condition, 150 mL of water was circulated through the reactor for 15 minutes under four optimized voltages (33 kV [breakdown threshold], 36 kV, 42 kV, and 46 kV), with PAW collected immediately after activation. The Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows a typical voltage-current waveform diagram, in which the power density gradually increases as the voltage rises. The pH was adjusted before subsequent biological assays were performed.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2Antibacterial Function Evaluation\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (ATCC 33277), \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e (ATCC25586) and \u003cem\u003eStreptococcus sanguinis\u003c/em\u003e (ATCC10556) were used for they associated with the peri-implantitis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Titanium discs (1 cm in diameter) were placed in 1 mL of bacterial suspension (107 CFU mL\u0026thinsp;\u0026minus;\u0026thinsp;1) and cultured for 72 h at 37\u0026deg;C under anaerobic conditions to allow biofilm formation.\u003c/p\u003e\u003cp\u003eTo evaluate the antimicrobial activity of PAW, the biofilms were treated and then analyzed. The contact killing effect was assessed by: (1) culturing and CFU counting using Petrifilm and (2) live/dead staining using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen, USA). The sterilization ability of PAW was evaluated by the Log Reduction, calculated as follows:\u003c/p\u003e\u003cp\u003eLogReduction\u0026thinsp;=\u0026thinsp;Log(CFUcontrol/CFUtreated).\u003c/p\u003e\u003cp\u003eFor the staining, after the 72 h biofilm formation and treatment, the discs were washed with PBS and stained following the manufacturer's protocol, then imaged by confocal laser scanning microscopy (CLSM). Additionally, scanning electron microscopy (SEM) was used to observe structural and morphological changes of bacteria after PAW treatment.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Culture and Treatment of HUVECs\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were donated by the Nanjing Medical University. 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) with 1% penicillin (100 U/mL)-streptomycin (100 \u0026micro;g/mL) mixed solution (Gibco, USA) was cultured in ECM culture medium (ScienCell, USA). Cultures were maintained at 37\u0026deg;C/5% CO₂ with medium changes every 48 h, and cells at passages 3\u0026ndash;5 were used for all experiments.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 CCK8 Cytotoxicity Assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHUVECs were seeded in 96-well plates at a density of 5 \u0026times; 10\u0026sup3; cells/well and allowed to adhere for 24 h. The medium was then replaced with medium containing the respective PAW solutions generated at 0 kV (control), 33 kV, 36 kV, 42 kV, or 46 kV. Three technical replicates were performed per condition.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Tube Formation Assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo assess angiogenic potential, HUVECs were trypsinized and seeded (8 \u0026times; 10⁴ cells/well) onto 24-well plates pre-coated with 20 \u0026micro;l of growth factor-reduced Matrigel. Cells were then incubated at 37\u0026deg;C under 5% CO₂ in low-serum (2% FBS) medium containing the respective PAW treatment groups (0kv for control, 33kv, 36kv, 42kv and 46kv). Cellular network formation was visualized and imaged under an inverted phase-contrast microscope (Olympus IX73) at three critical time points: immediately after seeding (0 h), and following 6 h and 24 h of incubation. Tube formation was quantified by measuring three key parameters per field of view: (1) the total number of branch points, (2) the total number of enclosed tubular structures, and (3) the total tube length (\u0026micro;m) using image analysis software ( ImageJ with the Angiogenesis Analyzer plugin).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Wound Healing Assay\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHUVECs were seeded into a 6-well plate and cultured for 24 hours. The original medium was then removed, and the cells were gently washed three times with PBS to remove detached cells. Subsequently, low-serum (2%) medium containing the different PAW treatment groups was added. Images of the scratch wound area were captured under a microscope at 0, 6, and 24 hours. Scratch width was measured or cell-covered area was calculated to compare wound healing and cell migration rates between groups.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eData are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5.01, employing one-way ANOVA with appropriate post-hoc testing for multi-group comparisons and Student's *t*-test for two-group comparisons. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of PAW\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, immediately post-treatment, the key physicochemical properties including pH, oxidation reduction potential (ORP), and reactive species concentrations were quantified. ORP serves as a critical metric for solution oxidative capacity, directly correlating with activity and concentration. ORP surges from mildly oxidative to highly oxidative at 46 kV. Concurrently, spectrophotometric measurements confirmed increasing concentrations of nitrate (NO₃⁻) and nitrite (NO₂⁻) anions with higher activation voltages. These collective trends demonstrate that elevated voltages drive significantly higher concentrations of reactive nitrogen/oxygen species (RNS/ROS).\u003c/p\u003e\u003c/div\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\u003eElectrochemical Properties\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33kv\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36kv\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42kv\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46kv\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003epH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eORP(mV)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e+\u0026thinsp;150\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e+\u0026thinsp;780\u0026thinsp;\u0026plusmn;\u0026thinsp;25 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e+\u0026thinsp;950\u0026thinsp;\u0026plusmn;\u0026thinsp;30 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e+\u0026thinsp;1020\u0026thinsp;\u0026plusmn;\u0026thinsp;35 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e+\u0026thinsp;1100\u0026thinsp;\u0026plusmn;\u0026thinsp;40 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eConductiity(uS/cm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e320\u0026thinsp;\u0026plusmn;\u0026thinsp;18 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e450\u0026thinsp;\u0026plusmn;\u0026thinsp;25 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e580\u0026thinsp;\u0026plusmn;\u0026thinsp;30 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e690\u0026thinsp;\u0026plusmn;\u0026thinsp;32 \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003e Data: mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs control\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Antimicrobial Activity\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Time-dependent inactivation of Porphyromonas gingivalis by PAW at different voltages\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePAW was generated via 15-minute plasma activation at four discharge voltages: 33 kV, 36 kV, 42 kV, and 46 kV. The bactericidal efficacy against P.g suspensions was assessed after exposure to PAW for 5, 10, 15, and 30 minutes. Voltage- and time-dependent inactivation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At 46 kV, a 6.0-log₁₀ reduction occurred, corresponding to 99.99% inactivation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. control; ANOVA-Tukey). Crucially, no viable colonies were detected in: 42 kV groups ( 15/30 minute) and the 46 kV (10/15/30 minute) groups, confirming the complete bactericidal eradication rate.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Morphological Alterations in Tri-Species Biofilm Induced by Plasma-Activated Water: Scanning Electron Microscopy\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eScanning electron microscopy provided visual evidence of PAW's bactericidal efficacy against the tri-species biofilm. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the substantial morphological damage inflicted by a 10-minute treatment with 42 kV PAW to three types of bacterial biofilms. Following exposure, the biofilm transitioned from its original intact structure to a state characterized by widespread cell rupture, surface collapse, and severe shrinkage.The biofilm thickness of both P.g and S.s also decreased significantly. Due to its biological characteristics as a \u0026ldquo;bridge bacterium,\u0026rdquo; F.n has a relatively weak ability to independently form mature biofilms, a significant reduction in its numbers was also observed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 PAW effect on Porphyromonas gingivalis viability: Live/Dead Staining Visualized by Confocal Laser Scanning Microscopy\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe viability of bacteria was determined by Baclight LIVE/DEAD assay kit. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, untreated control biofilms exhibited dense green fluorescence indicating abundant live cells, whereas PAW treatment at 42 kV for 10 minutes caused a dramatic reduction in green-fluorescent viable bacteria alongside red fluorescence from dead cells with compromised membranes. Concurrently, biofilm architecture underwent substantial disruption with visibly reduced thickness. These morphological and viability changes directly align with the previously documented greater than 6.0-log CFU reduction, confirming PAW's capacity to induce lethal cellular damage and degrade biofilm integrity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Biphasic Cellular Response to PAW Voltage\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, scratch assays demonstrated voltage-dependent wound closure: 33 kV significantly enhanced closure compared to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 36 kV elicited maximal closure, while voltages more than 42 kV exhibited cytotoxicity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs 36 kV). Complementary CCK-8 assays confirmed a biphasic response: 36 kV showed peak viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs control), whereas voltages\u0026thinsp;\u0026ge;\u0026thinsp;42 kV demonstrated cytotoxicity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs 36 kV). (ANOVA: F\u0026thinsp;=\u0026thinsp;185.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; n\u0026thinsp;=\u0026thinsp;6). This functional decline corresponded to an 84.3% reduction in tube formation at 42 kV, with 107 branches versus 684 branches at 36 kV, and near-complete angiogenic failure at 46 kV with only 85 branches. These effects establish a narrow therapeutic window at 33\u0026ndash;36 kV for PAW\u0026rsquo;s voltage-dependent bioactivity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe observed voltage-dependent electrochemical properties of PAW can be mechanistically interpreted through the lens of discharge physics and aqueous reaction kinetics. The stepwise increase in conductivity directly reflects the elevated ionic content resulting from plasma-water interactions. As the applied voltage increases, the energy input into the DBD system rises, leading to enhanced electron impact dissociation of N₂, O₂, and H₂O vapour in the discharge zone. This generates higher densities of reactive precursors such as \u0026bull;OH, O\u0026bull;, NO\u0026bull;, and excited species, which subsequently hydrolyse or oxidise in water to form stable ions including NO₃⁻, NO₂⁻, H⁺, and H₂O₂. The consequent rise in ionic strength directly accounts for the increasing conductivity. Simultaneously, the surge in oxidative species concentration\u0026mdash;particularly HNO₂/NO₂⁻ and peroxynitrite (ONOO⁻)\u0026mdash;drives the pronounced drop in pH (to 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 at 46 kV) and the sharp positive shift in ORP (from +\u0026thinsp;150\u0026thinsp;\u0026plusmn;\u0026thinsp;15 mV to +\u0026thinsp;1100\u0026thinsp;\u0026plusmn;\u0026thinsp;40 mV). The ORP, serving as an integrative indicator of the solution's redox potential, confirms the transition into a highly oxidative state at 46 kV, dominated by the accumulation of short-lived reactive oxygen and nitrogen species (RONS) with high redox potential. This electrochemical framework directly establishes voltage as a biological switch for plasma-activated water, enabling on-demand transition between two therapeutic modes: regenerative mode (36 kV ): Promotes angiogenesis through endothelial migration (96.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% wound closure, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs control) and accelerated tubulogenesis and antibacterial mode achieves more than 6-log biofilm eradication via rapid, cascadic ROS/RNS burst causing membrane peroxidation and DNA fragmentation in bacteria. Critically, the 42 kV threshold marks a functional tipping point that triggers coordinated collapse of viability, migration and tubulogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe dual nature of RONS critically influences peri-implantitis outcomes. Biofilm infection initiates a vicious cycle: pathogen-associated molecular patterns (e.g., P. gingivalis LPS) trigger excessive inflammatory responses, leading to uncontrolled endogenous RONS production. Damages vascular endothelia through eNOS uncoupling and VE-cadherin degradation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], impairing angiogenesis; activates the NLRP3 inflammasome via mitochondrial ROS (mtROS) overproduction [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Conversely, PAW-engineered RONS reprogram pathological microenvironments: liquid plasma promotes angiogenesis by inducing extracellular matrix metabolism through upregulation of endothelial nitric oxide synthase (eNOS) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Unlike uncontrolled oxidative damage in peri-implantitis, PAW-engineered RONS deliver \u0026ldquo;redox messages\u0026rdquo; that mimic physiological wound healing and turn the double-edged sword into a precision surgical tool.\u003c/p\u003e\u003cp\u003eCompared to current peri-implantitis treatments, voltage-switchable PAW offers several advantages: Peri-implantitis requires not only eradicating pathogens but also restoring damaged peri-implant bone and soft tissues. Antibiotics focus solely on sterilisation but do not promote healing [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; mechanical debridement may further disrupt fragile regenerative environments [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; and growth factors, while pro-healing, lack inherent antimicrobial properties, risking re-infection [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Besides, a major drawback of antibiotic-based treatments is the emergence of drug-resistant pathogens, which complicates retreatment and increases failure risks. PAW's antimicrobial mechanism relies on oxidative damage to bacterial cell membranes and DNA via ROS/RNS\u0026mdash;pathways bacteria rarely develop resistance to, as they cannot mutate to evade non-specific oxidative stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study has several limitations: First, while our study employed mono-species biofilms (P. g, S.s, F. n), clinical peri-implantitis involves complex polymicrobial communities [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Second, short NO₂⁻ half-life necessitates point-of-care PAW generation, current protocols require bedside plasma devices [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Third, absence of in vivo validation in mammalian models limits translational prediction of host-microbe dynamics.\u003c/p\u003e\u003cp\u003eWe have developed PAW as a voltage-responsive liquid biomaterial. Its physical properties undergo distinct transformations at critical electrical thresholds, enabling it to perform specific biological functions. This electro-metabolic switching behavior\u0026mdash;where application of 36 kV promotes tissue regeneration, while 46 kV triggers potent biocidal activity\u0026mdash;demonstrates potential for precisely controlled biointerfaces. Beyond initial applications in dentistry, this technology shows promise for managing chronic wounds, particularly in contexts where infection and compromised vascularization are concurrent challenges.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe demonstrate that voltage-controlled PAW delivers dual therapeutic functions for peri-implantitis management. PAW at 46 kV eradicates pathogenic biofilms, while PAW at 36 kV promotes angiogenesis, enhancing endothelial cell migration and function. Critically, these bioeffects are voltage-specific, with a cytotoxic threshold emerging at more than 42 kV. This tuning strategy addresses the dual challenge of biofilm elimination and tissue regeneration. Although clinical translation requires further investigation, PAW shows significant potential as a targeted therapy for implant-related tissue damage.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePAW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003ePlasma-activated water\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003ePg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003ePorphyromonas gingivalis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eFusobacterium nucleatum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eVEGF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eVascular endothelial growth factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eNTP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eNon-thermal plasma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eCold a atmosphere plasma\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eDBD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eDielectric barrier discharge\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCLSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eConfocal laser scanning microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eScanning electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eHUVECs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eHuman umbilical vein endothelial cells\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eStandard deviation\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eORP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eOxidation reduction potential\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eRNS/ROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eReactive nitrogen/oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003emtROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMitochondrial reactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eeNOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eEndothelial nitric oxide synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was funded by a grant from the Science and Technology Department of Jiangsu Province, grant number BE2020707.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, methodology and writing\u0026mdash;original draft preparation, Y.L.; data curation, H.M.; writing\u0026mdash;review and editing, J.Z and C.H.; supervision and funding acquisition, C.X.; visualization, Z.P. and Y.C; . All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBr\u0026aring;nemark, P.I.; Adell, R.; Breine, U.; Hansson, B.O.; Lindstr\u0026ouml;m, J.; Ohlsson, A. Intra-Osseous Anchorage of Dental Prostheses. I. Experimental Studies. \u003cem\u003eScand J Plast Reconstr Surg\u003c/em\u003e \u003cstrong\u003e1969\u003c/strong\u003e, \u003cem\u003e3\u003c/em\u003e, 81\u0026ndash;100.\u003c/li\u003e\n\u003cli\u003eKoizumi, H.; Takeuchi, Y.; Imai, H.; Kawai, T.; Yoneyama, T. Application of Titanium and Titanium Alloys to Fixed Dental Prostheses. \u003cem\u003eJ Prosthodont Res\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e63\u003c/em\u003e, 266\u0026ndash;270.\u003c/li\u003e\n\u003cli\u003eRoccuzzo, A.; St\u0026auml;hli, A.; Monje, A.; Sculean, A.; Salvi, G.E. Peri-Implantitis: A Clinical Update on Prevalence and Surgical Treatment Outcomes. \u003cem\u003eJ Clin Med\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 1107.\u003c/li\u003e\n\u003cli\u003eBerglundh, T.; Armitage, G.; Araujo, M.G.; Avila-Ortiz, G.; Blanco, J.; Camargo, P.M.; Chen, S.; Cochran, D.; Derks, J.; Figuero, E.; et al. Peri-Implant Diseases and Conditions: Consensus Report of Workgroup 4 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e45 Suppl 20\u003c/em\u003e, S286\u0026ndash;S291.\u003c/li\u003e\n\u003cli\u003eSchwarz, F.; Derks, J.; Monje, A.; Wang, H.-L. Peri-Implantitis. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e45 Suppl 20\u003c/em\u003e, S246\u0026ndash;S266.\u003c/li\u003e\n\u003cli\u003eLi, D.; Tan, X.; Zheng, L.; Tang, H.; Hu, S.; Zhai, Q.; Jing, X.; Liang, P.; Zhang, Y.; He, Q.; et al. A Dual-Antioxidative Coating on Transmucosal Component of Implant to Repair Connective Tissue Barrier for Treatment of Peri-Implantitis. \u003cem\u003eAdv Healthc Mater\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e, e2301733.\u003c/li\u003e\n\u003cli\u003eDe Bruyn, H.; Raes, S.; Ostman, P.-O.; Cosyn, J. Immediate Loading in Partially and Completely Edentulous Jaws: A Review of the Literature with Clinical Guidelines. \u003cem\u003ePeriodontol 2000\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e66\u003c/em\u003e, 153\u0026ndash;187.\u003c/li\u003e\n\u003cli\u003eCaton, J.G.; Armitage, G.; Berglundh, T.; Chapple, I.L.C.; Jepsen, S.; Kornman, K.S.; Mealey, B.L.; Papapanou, P.N.; Sanz, M.; Tonetti, M.S. A New Classification Scheme for Periodontal and Peri-Implant Diseases and Conditions - Introduction and Key Changes from the 1999 Classification. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e45 Suppl 20\u003c/em\u003e, S1\u0026ndash;S8.\u003c/li\u003e\n\u003cli\u003eFlemming, H.-C.; van Hullebusch, E.D.; Neu, T.R.; Nielsen, P.H.; Seviour, T.; Stoodley, P.; Wingender, J.; Wuertz, S. The Biofilm Matrix: Multitasking in a Shared Space. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, 70\u0026ndash;86.\u003c/li\u003e\n\u003cli\u003eStewart, P.S.; Costerton, J.W. Antibiotic Resistance of Bacteria in Biofilms. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e2001\u003c/strong\u003e, \u003cem\u003e358\u003c/em\u003e, 135\u0026ndash;138.\u003c/li\u003e\n\u003cli\u003eDarby, E.M.; Trampari, E.; Siasat, P.; Gaya, M.S.; Alav, I.; Webber, M.A.; Blair, J.M.A. Molecular Mechanisms of Antibiotic Resistance Revisited. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, 280\u0026ndash;295.\u003c/li\u003e\n\u003cli\u003eSun, F.; Lu, Y.; Wang, Z.; Shi, H. Vascularization Strategies for Tissue Engineering for Tracheal Reconstruction. \u003cem\u003eRegen Med\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 549\u0026ndash;566.\u003c/li\u003e\n\u003cli\u003eTian, T.; Zhang, T.; Lin, Y.; Cai, X. Vascularization in Craniofacial Bone Tissue Engineering. \u003cem\u003eJ Dent Res\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e97\u003c/em\u003e, 969\u0026ndash;976.\u003c/li\u003e\n\u003cli\u003eKamran, N.; Kadiyala, P.; Saxena, M.; Candolfi, M.; Li, Y.; Moreno-Ayala, M.A.; Raja, N.; Shah, D.; Lowenstein, P.R.; Castro, M.G. Immunosuppressive Myeloid Cells\u0026rsquo; Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy. \u003cem\u003eMol Ther\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e25\u003c/em\u003e, 232\u0026ndash;248.\u003c/li\u003e\n\u003cli\u003eSkipper, M. Targeting the Worm. \u003cem\u003eNat Rev Genet\u003c/em\u003e \u003cstrong\u003e2004\u003c/strong\u003e, \u003cem\u003e5\u003c/em\u003e, 883\u0026ndash;883.\u003c/li\u003e\n\u003cli\u003eIsenberg, J.S.; Ridnour, L.A.; Espey, M.G.; Wink, D.A.; Roberts, D.D. Nitric Oxide in Wound-Healing. \u003cem\u003eMicrosurgery\u003c/em\u003e \u003cstrong\u003e2005\u003c/strong\u003e, \u003cem\u003e25\u003c/em\u003e, 442\u0026ndash;451.\u003c/li\u003e\n\u003cli\u003eKieran, M.W.; Kalluri, R.; Cho, Y.-J. The VEGF Pathway in Cancer and Disease: Responses, Resistance, and the Path Forward. \u003cem\u003eCold Spring Harb Perspect Med\u003c/em\u003e \u003cstrong\u003e2012\u003c/strong\u003e, \u003cem\u003e2\u003c/em\u003e, a006593.\u003c/li\u003e\n\u003cli\u003eVON Woedtke, T.; Schmidt, A.; Bekeschus, S.; Wende, K.; Weltmann, K.-D. Plasma Medicine: A Field of Applied Redox Biology. \u003cem\u003eIn Vivo\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e33\u003c/em\u003e, 1011\u0026ndash;1026.\u003c/li\u003e\n\u003cli\u003eYamabe, C.; Takeshita, F.; Miichi, T.; Hayashi, N.; Ihara, S. Water Treatment Using Discharge on the Surface of a Bubble in Water. \u003cem\u003ePlasma Processes \u0026amp; Polymers\u003c/em\u003e \u003cstrong\u003e2005\u003c/strong\u003e, \u003cem\u003e2\u003c/em\u003e, 246\u0026ndash;251.\u003c/li\u003e\n\u003cli\u003eTresp, H.; Hammer, M.U.; Winter, J.; Weltmann, K.-D.; Reuter, S. Quantitative Detection of Plasma-Generated Radicals in Liquids by Electron Paramagnetic Resonance Spectroscopy. \u003cem\u003eJ. Phys. D: Appl. Phys.\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e46\u003c/em\u003e, 435401.\u003c/li\u003e\n\u003cli\u003eYang, Y.; Zheng, M.; Yang, Y.; Li, J.; Su, Y.-F.; Li, H.-P.; Tan, J.-G. Inhibition of Bacterial Growth on Zirconia Abutment with a Helium Cold Atmospheric Plasma Jet Treatment. \u003cem\u003eClin Oral Investig\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e24\u003c/em\u003e, 1465\u0026ndash;1477.\u003c/li\u003e\n\u003cli\u003eZhang, Q.; Liang, Y.; Feng, H.; Ma, R.; Tian, Y.; Zhang, J.; Fang, J. A Study of Oxidative Stress Induced by Non-Thermal Plasma-Activated Water for Bacterial Damage. \u003cem\u003eApplied Physics Letters\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e102\u003c/em\u003e, 203701.\u003c/li\u003e\n\u003cli\u003eArda, G.; Hsu, C. Preservation of Reactive Species in Frozen Plasma-Activated Water and Enhancement of Its Bactericidal Activity Through pH Adjustment. \u003cem\u003ePlasma Chem Plasma Process\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e43\u003c/em\u003e, 599\u0026ndash;618.\u003c/li\u003e\n\u003cli\u003eHarley, J.C.; Suchowerska, N.; McKenzie, D.R. Cancer Treatment with Gas Plasma and with Gas Plasma-Activated Liquid: Positives, Potentials and Problems of Clinical Translation. \u003cem\u003eBiophys Rev\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e, 989\u0026ndash;1006.\u003c/li\u003e\n\u003cli\u003eQiao, D.; Li, Y.; Pan, J.; Zhang, J.; Tian, Y.; Wang, K. Effect of Plasma Activated Water in Caries Prevention: The Caries Related Biofilm Inhibition Effects and Mechanisms. \u003cem\u003ePlasma Chem Plasma Process\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e42\u003c/em\u003e, 801\u0026ndash;814.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez-Chaparro, P.J.; Duarte, P.M.; Shibli, J.A.; Montenegro, S.; Lacerda Heluy, S.; Figueiredo, L.C.; Faveri, M.; Feres, M. The Current Weight of Evidence of the Microbiologic Profile Associated With Peri-Implantitis: A Systematic Review. \u003cem\u003eJ Periodontol\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e87\u003c/em\u003e, 1295\u0026ndash;1304.\u003c/li\u003e\n\u003cli\u003eKolenbrander, P.E.; Andersen, R.N.; Blehert, D.S.; Egland, P.G.; Foster, J.S.; Palmer, R.J. Communication among Oral Bacteria. \u003cem\u003eMicrobiol Mol Biol Rev\u003c/em\u003e \u003cstrong\u003e2002\u003c/strong\u003e, \u003cem\u003e66\u003c/em\u003e, 486\u0026ndash;505, table of contents.\u003c/li\u003e\n\u003cli\u003ePeriasamy, S.; Kolenbrander, P.E. Aggregatibacter Actinomycetemcomitans Builds Mutualistic Biofilm Communities with Fusobacterium Nucleatum and Veillonella Species in Saliva. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e2009\u003c/strong\u003e, \u003cem\u003e77\u003c/em\u003e, 3542\u0026ndash;3551.\u003c/li\u003e\n\u003cli\u003eYe, X.; Ma, T.; Blais, J.E.; Yan, V.K.C.; Kang, W.; Chui, C.S.L.; Lai, F.T.T.; Li, X.; Wan, E.Y.F.; Wong, C.K.H.; et al. Association between BNT162b2 or CoronaVac COVID-19 Vaccines and Major Adverse Cardiovascular Events among Individuals with Cardiovascular Disease. \u003cem\u003eCardiovasc Res\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e118\u003c/em\u003e, 2329\u0026ndash;2338.\u003c/li\u003e\n\u003cli\u003eZhou, R.; Yazdi, A.S.; Menu, P.; Tschopp, J. A Role for Mitochondria in NLRP3 Inflammasome Activation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e469\u003c/em\u003e, 221\u0026ndash;225.\u003c/li\u003e\n\u003cli\u003eKang, S.U.; Kim, H.J.; Ma, S.; Oh, D.-Y.; Jang, J.Y.; Seo, C.; Lee, Y.S.; Kim, C.-H. Liquid Plasma Promotes Angiogenesis through Upregulation of Endothelial Nitric Oxide Synthase-Induced Extracellular Matrix Metabolism: Potential Applications of Liquid Plasma for Vascular Injuries. \u003cem\u003eCell Commun Signal\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e, 138.\u003c/li\u003e\n\u003cli\u003eLio, P.A.; Kaye, E.T. Topical Antibacterial Agents. \u003cem\u003eInfect Dis Clin North Am\u003c/em\u003e \u003cstrong\u003e2004\u003c/strong\u003e, \u003cem\u003e18\u003c/em\u003e, 717\u0026ndash;733.\u003c/li\u003e\n\u003cli\u003eEnevold, C.; Nielsen, C.H.; Christensen, L.B.; Kongstad, J.; Fiehn, N.E.; Hansen, P.R.; Holmstrup, P.; Havemose-Poulsen, A.; Damgaard, C. Suitability of Machine Learning Models for Prediction of Clinically Defined Stage III/IV Periodontitis from Questionnaires and Demographic Data in Danish Cohorts. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e51\u003c/em\u003e, 1561\u0026ndash;1573.\u003c/li\u003e\n\u003cli\u003eGhatnekar, O.; Persson, U.; Willis, M.; Odegaard, K. Cost Effectiveness of Becaplermin in the Treatment of Diabetic Foot Ulcers in Four European Countries. \u003cem\u003ePharmacoeconomics\u003c/em\u003e \u003cstrong\u003e2001\u003c/strong\u003e, \u003cem\u003e19\u003c/em\u003e, 767\u0026ndash;778.\u003c/li\u003e\n\u003cli\u003eLepperdinger, U.; Angwin, C.; Milnes, D.; Sobey, G.; Ghali, N.; Johnson, D.; Brady, A.F.; Kammin, T.; Bowen, J.M.; Gr\u0026ouml;bner, R.; et al. Oral Characteristics in Adult Individuals with Periodontal Ehlers-Danlos Syndrome. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e49\u003c/em\u003e, 1244\u0026ndash;1252.\u003c/li\u003e\n\u003cli\u003eWerner, S.; Grose, R. Regulation of Wound Healing by Growth Factors and Cytokines. \u003cem\u003ePhysiol Rev\u003c/em\u003e \u003cstrong\u003e2003\u003c/strong\u003e, \u003cem\u003e83\u003c/em\u003e, 835\u0026ndash;870.\u003c/li\u003e\n\u003cli\u003eCabiscol, E.; Tamarit, J.; Ros, J. Oxidative Stress in Bacteria and Protein Damage by Reactive Oxygen Species. \u003cem\u003eInt Microbiol\u003c/em\u003e \u003cstrong\u003e2000\u003c/strong\u003e, \u003cem\u003e3\u003c/em\u003e, 3\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eDharmaraja, A.T. Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria. \u003cem\u003eJ Med Chem\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e60\u003c/em\u003e, 3221\u0026ndash;3240.\u003c/li\u003e\n\u003cli\u003eFalcao, A.; Bull\u0026oacute;n, P. A Review of the Influence of Periodontal Treatment in Systemic Diseases. \u003cem\u003ePeriodontol 2000\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e79\u003c/em\u003e, 117\u0026ndash;128.\u003c/li\u003e\n\u003cli\u003eWende, K.; Williams, P.; Dalluge, J.; Gaens, W.V.; Aboubakr, H.; Bischof, J.; von Woedtke, T.; Goyal, S.M.; Weltmann, K.-D.; Bogaerts, A.; et al. Identification of the Biologically Active Liquid Chemistry Induced by a Nonthermal Atmospheric Pressure Plasma Jet. \u003cem\u003eBiointerphases\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 029518.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"plasma-activated water, peri-implantitis, biofilm disruption, vascular regeneration, reactive nitrogen and oxygen species","lastPublishedDoi":"10.21203/rs.3.rs-7612573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7612573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003ePeri-implantitis therapy requires both effective biofilm eradication and subsequent tissue regeneration. This study aimed to develop a voltage-gated plasma-activated water (PAW) system with switchable bioactivities to address these dual needs and evaluate its efficacy for potential application in peri-implantitis management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods: \u003c/strong\u003ePAW was generated using a dielectric barrier discharge system across a voltage spectrum of 33–46 kV. Its antibacterial activity was assessed against Porphyromonas gingivalis and multi-species biofilms on titanium surfaces, quantified via colony-forming unit (CFU) counts and visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The pro-angiogenic potential was evaluated through endothelial cell viability, migration, and tubule formation assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e High-voltage PAW (46 kV, 30 min) achieved a \u0026gt;6.0-log reduction of P. gingivalis and induced structural collapse of multi-species biofilms. In contrast, low-voltage PAW (36 kV, 15 min) significantly enhanced endothelial cell viability to 118.3 ± 5.2%, and accelerated both cell migration and tubulogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study demonstrates that PAW's biological functionality can be precisely tuned via modulation of activation voltage. The same platform can be switched between a potent antibacterial state and a pro-regenerative state, enabling adaptive bioactivity suited to the staged therapeutic requirements of peri-implantitis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance: \u003c/strong\u003eThe voltage-gated PAW system presents a promising, affordable strategy for dual-phase peri-implantitis therapy. It offers clinicians a potential non-antibiotic tool to first disinfect implant surfaces and then promote vascular healing, which is critical for improving clinical outcomes in implant dentistry.\u003c/p\u003e","manuscriptTitle":"Voltage-Tunable Plasma-Activated Water: A Strategy for Combating Peri-Implantitis via Dual-Path Biofilm Disruption and Vascular Regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 14:38:30","doi":"10.21203/rs.3.rs-7612573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-17T09:26:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T23:33:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T18:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166643973444029877941984938407680875264","date":"2025-09-20T07:16:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T10:49:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36349100248353418243518957386140579560","date":"2025-09-18T10:11:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111498234673077807776709700722789092682","date":"2025-09-18T09:15:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215656430259686019357285233619004085927","date":"2025-09-18T06:49:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-18T06:32:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T10:40:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-16T10:40:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2025-09-14T12:19:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"51b4a6c7-6a06-4382-8518-f3c67a07a2a9","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:09:32+00:00","versionOfRecord":{"articleIdentity":"rs-7612573","link":"https://doi.org/10.1007/s00784-026-06749-3","journal":{"identity":"clinical-oral-investigations","isVorOnly":false,"title":"Clinical Oral Investigations"},"publishedOn":"2026-04-24 15:59:48","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-09-29 14:38:30","video":"","vorDoi":"10.1007/s00784-026-06749-3","vorDoiUrl":"https://doi.org/10.1007/s00784-026-06749-3","workflowStages":[]},"version":"v1","identity":"rs-7612573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7612573","identity":"rs-7612573","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}