Therapeutic Efficacy of Floating Electrode–Dielectric Barrier Discharge Plasma in Experimental Periodontitis: A Pilot Study

Abstract

Periodontitis is a chronic inflammatory disease characterized by dysbiotic biofilms and host-mediated destruction of periodontal tissues. This study evaluated the efficacy of a novel needle-shaped floating electrode–dielectric barrier discharge (FE-DBD) plasma probe in treating experimental periodontitis. Using a split-mouth design in a rat model of ligature-induced periodontitis, subgingival microbiome changes were analyzed via 16S rRNA sequencing, while gene expression of inflammatory mediators and osteoclastogenic factors was quantified by qRT-PCR. Histopathological evaluation and osteoclast activity were assessed through H&E and TRAP staining, respectively. FE-DBD treatment significantly shifted the subgingival microbiome by reducing pathobionts such as Bacteroidota and Fusobacteriota and increasing health-associated taxa including Proteobacteria and Actinobacteriota. The therapy also exerted immunomodulatory effects by suppressing pro-inflammatory genes (TNF-α, ICAM-1, CCL2) and elevating anti-inflammatory IL-10 expression. Moreover, FE-DBD favorably modulated bone remodeling by downregulating RANK and RANKL, upregulating OPG, and raising the OPG/RANKL ratio 3.72-fold, accompanied by reduced inflammatory infiltration and osteoclast numbers. These findings demonstrate that FE-DBD plasma effectively ameliorates periodontitis by simultaneously targeting pathogenic biofilms, host inflammation, and osteoclastogenesis, highlighting its potential as a multifaceted adjunctive therapy for periodontal disease.

1. Introduction

Periodontitis is a chronic inflammatory disease characterized by the progressive destruction of tooth-supporting structures, primarily driven by dysbiotic microbial biofilms and host immune responses [1]. Although conventional treatment based on scaling and root planing, often combined with antimicrobial agents, remains the clinical standard, its effectiveness is limited by incomplete biofilm removal, restricted access to deep periodontal pockets, and concerns regarding antimicrobial resistance [1,2,3].

In response to these challenges, the field of regenerative dentistry has witnessed continuous and dynamic research aimed at developing adequate solutions for rehabilitation procedures. Significant advancements have been made in the development of biocompatible and multifunctional biomaterials for surgeries such as guided bone regeneration (GBR) and periodontal regeneration. These materials are designed to interact with the tissue environment to activate specific biological responses [4]. Alongside these material-based advancements, novel physical modalities are also emerging as promising adjunctive tools. Among them, non-thermal plasma (NTP), especially atmospheric pressure plasma, has gained significant attention in dentistry for its broad-spectrum antimicrobial and biofilm-disrupting effects. Cold atmospheric plasma (CAP) has potential therapeutic effects on periodontitis through three mechanisms: antimicrobial effect, inflammation attenuation, and tissue remodeling [5]. Among NTP devices, plasma jet devices have been extensively studied for periodontal applications [6,7,8,9]. Although plasma jet devices have demonstrated promising antimicrobial effects, and recent advancements in micro-plasma jets and compact translational devices have significantly improved their portability, the requirement for an external supply of noble gases (e.g., helium or argon) remains a logistical consideration [10]. While the cost of gases like argon is relatively low, the necessity for gas tanks, tubing, and flow regulation systems can add complexity to routine clinical setups. Furthermore, the reliance on directional gas flow can sometimes limit uniform treatment within the complex geometries of deep, narrow periodontal pockets. In this context, the compact FE-DBD system presented here offers a distinct alternative to conventional plasma jets. By utilizing a floating electrode configuration, this system generates a stable, uniform discharge directly above the biological surface without the need for external gas supplies. This design not only simplifies deployment in routine dental settings but also enables more consistent and controllable application across the complex topographies of the oral cavity. Biologically, FE-DBD-enhanced wound healing was demonstrated in a full-thickness mouse skin model, showing faster re-epithelialization, collagen maturation, and suppressed inflammation [11]. However, in vivo evidence supporting the application of FE-DBD in periodontal therapy remains scarce, with no studies to date having employed FE-DBD in established periodontitis models.

This study aims to evaluate the therapeutic potential of FE-DBD plasma in a rat model of ligature-induced periodontitis, with the following objectives: to assess the antimicrobial and ecological effects on subgingival biofilm via 16S rRNA sequencing; to quantify anti-inflammatory effects through gingival gene expression of cytokines and bone remodeling mediators via qRT-PCR; and to evaluate impacts on osteoclast activity and tissue histology by TRAP and H&E staining. A specially designed needle-shaped FE-DBD probe (10 mm in length, 0.8 mm in diameter) was developed to facilitate treatment of deep, narrow periodontal pockets. This study provides preclinical evidence for FE-DBD plasma as a novel, non-invasive adjunctive therapy for periodontitis.

2. Materials and Methods

2.1. Animals and Experimental Design

A total of eight 4-week-old Sprague-Dawley rats (weighing 90–130 g, with an equal sex distribution) obtained from SPF (Beijing) Biotechnology Co., Ltd., Beijing, China, were used in the experiment. Animals were maintained with a 12/12 h light/dark cycle at 22 ± 2 °C with ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of China-Japan Friendship Hospital (Issue No. ZRDWLL250048).

Rats were anesthetized via intraperitoneal injection of 10% chloral hydrate (0.3 mL/100 g body weight). Following anesthesia, 0.2 mm orthodontic stainless-steel wires were inserted submarginally around the bilateral maxillary first molars for 14 days to induce bacterial biofilm formation and alveolar bone loss. Figure 1 shows a representative image of the ligature-induced periodontitis model. The study employed a split-mouth design to minimize inter-individual variability. The left and right maxillary molars were randomly assigned to two groups (n = 8 per group): the FE-DBD plasma treatment group (FE-DBDG) and the control group (CG, receiving no plasma treatment).

2.2. FE-DBD Plasma Device and System Optimization

The FE-DBD plasma device and its operating principle are illustrated in Figure 2. As shown in Figure 2A, the system employs a floating electrode configuration. Unlike conventional DBD devices, the probe utilizes a single high-voltage electrode encapsulated within a dielectric barrier, with the treated biological tissue serving as the counter-electrode. Specifically, the device utilizes a needle-shaped working terminal (length: 10 mm; diameter: 0.8 mm; Figure 2B(i)) designed to facilitate insertion into narrow and deep periodontal pockets. This geometry ensures a uniform glow discharge across the entire working length (Figure 2B(ii)). To ensure standardized treatment, the probe is designed to adapt to varying periodontal pocket depths. Consequently, the effective discharge surface area corresponds to the insertion depth, reaching a maximum of approximately 25 mm² (calculated as A = π × D × L) to ensure consistent plasma exposure covering the entire inflammatory zone.

To ensure efficient plasma generation and operational safety, the dielectric layer utilizes a heat-resistant epoxy resin (up to 120 °C) with a thickness of 0.8 mm, while the discharge gap is maintained between 1 and 2 mm. The dielectric barrier layer limits the current density (<1 mA/cm²) to prevent tissue damage. The power supply is a custom-designed AC source employing a Zero Voltage Switching (ZVS) inverter circuit and a step-up transformer to effectively reduce switching losses and achieve high-efficiency energy transfer. A compact, highly integrated PCB design was adopted to meet deployment requirements in confined spaces.

To determine the optimal operating parameters, discharge characteristics were evaluated at peak voltages of 2.7 kV, 2.9 kV, 3.1 kV, and 3.3 kV. At 2.7 kV; the discharge was non-uniform. Conversely, voltages ≥ 3.1 kV resulted in excessive internal dielectric loss and thermal accumulation. Consequently, a peak voltage of 2.83 kV (frequency: 16–17 kHz) was selected to achieve an optimal balance of discharge stability, efficiency, and safety.

For electrical diagnostics, plasma discharge images were captured using a digital camera (Canon, EOS 650D, Canon, Tokyo, Japan). A voltage probe (Tektronix P6015A, Tektronix Inc., Beaverton, OR, USA) and a current coil (Tektronix P6016, Beaverton, OR, USA) were used to monitor voltage and current waveforms, respectively, which were recorded by a digital oscilloscope (Tektronix, DPO3034, Beaverton, OR, USA). Power determination was performed using the Lissajous figure method via the oscilloscope, incorporating an external 47 pF capacitor and calculated based on the formula:

P = S × f × C_m × K_x × K_y × N

where S is the area of the Lissajous figure, f is the discharge frequency, C_m is the integration capacitor value, K_x and K_y are the horizontal and vertical scaling factors of the oscilloscope, and N is the transformation ratio of the current coil.

Optical emission spectroscopy (OES) was performed to characterize the reactive species generated by the plasma. To ensure signal stability and reproducibility, spectra were acquired during the interaction of the FE-DBD with a grounded stainless-steel plate in an ambient air environment. This reference setup was selected to avoid interference caused by the variable surface properties of biological tissues. Spectra were recorded using a fiber-optic spectrometer (Ideaoptics FX2000, Shanghai Ideaoptics Corp., Shanghai, China; resolution of 0.39 nm, integration time of 100 ms), with the fiber probe positioned approximately 2 mm from the discharge region.

2.3. FE-DBD Plasma Treatment

Following periodontitis induction, the FE-DBD group received plasma treatment for 3 min once every other day over a 14-day period, whereas the control group received no therapeutic intervention. The treatment protocol was standardized as follows: (1) The FE-DBD handpiece was held in a pen-grip manner to ensure precise control during intra-pocket delivery. (2) The needle-shaped plasma probe (10 mm in length, 0.8 mm in diameter) was gently inserted into the periodontal pocket until resistance was met at the base of the pocket (confirmed via periodontal probing depth measurement). (3) To ensure uniform plasma exposure across the pathogen-colonized root surface and gingival epithelium, a slow, continuous motion was employed, moving the probe coronally to apically in overlapping strokes for 30 s per site. (4) No mechanical debridement was performed post-plasma to isolate the specific antimicrobial effects of FE-DBD. Molars in the control group (CG) were subjected to a sham treatment using the same manipulation protocol but with the plasma device switched off. Following the 2-week treatment period, animals were humanely euthanized for tissue harvesting.

2.4. Microbiological Analysis: Antibiofilm Efficacy

Subgingival plaque was collected using sterile #25 paper points (Dentsply Sirona, Charlotte, NC, USA) inserted for 30 s into the gingival sulcus at baseline (pre-treatment) and 2 weeks post-FE-DBD treatment. Samples were immediately placed in 200 μL of reduced transport fluid (RTF) and stored at −80 °C until further processing.

The bacterial 16S rRNA gene was amplified via PCR using barcode-specific primers and TransStart FastPfu DNA Polymerase (TransGen Biotech, Beijing, China; Cat. No. AP221-02), with optimized low-cycle conditions to minimize amplification bias and ensure reproducibility. The resulting amplicons were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). Sequencing libraries were constructed through ligation of Y-shaped adapters, followed by purification with magnetic beads to remove adapter dimers and subsequent PCR-mediated enrichment. The final libraries were denatured to generate single-stranded DNA and subjected to paired-end sequencing on an Illumina platform employing bridge PCR and sequencing-by-synthesis chemistry. High-quality sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using the UPARSE algorithm. Taxonomic annotation of representative OTU sequences was performed using the RDP classifier with the SILVA database. Alpha diversity was assessed by calculating the Richness, Chao1, and Shannon indices. Microbial community composition at the phylum level was visualized using relative abundance bar plots, and differences between the FE-DBD treatment group (FE-DBDG) and the control group (CG) at baseline and 2 weeks post-treatment were compared. Linear discriminant analysis effect size (LEfSe) was employed to identify differentially abundant microbial taxa (LDA score > 2.0), and cladograms were generated to visualize taxa that were significantly enriched at the phylum, class, order, and family levels.

2.5. Gene Expression Analysis of Inflammatory and Osteoclastogenic Factors

The inflammatory response and bone remodeling activity were assessed by measuring gene expression levels. At the end of the treatment period (2 weeks), gingival tissues were collected and stored at −80 °C.

Total RNA was extracted from gingival tissue samples using the TRIzol-chloroform method, with tissue homogenization performed in liquid nitrogen to ensure complete cell lysis and RNA release. The RNA quality was rigorously assessed by both spectrophotometric analysis (nanodrop measurements showing A260/A280 ratios between 1.8 and 2.1 indicated high purity) and electrophoretic separation on 1% agarose gels to confirm RNA integrity through clear 28S and 18S ribosomal RNA bands.

For cDNA synthesis, 1–2 μg of high-quality total RNA was reverse-transcribed using the ExonScript RT SuperMix with the dsDNase kit (ExonScript Biotechnology, Beijing, China). The protocol included an initial genomic DNA elimination step at 25 °C for 10 min followed by reverse transcription at 55 °C for 15 min and enzyme inactivation at 85 °C for 5 min, ensuring complete cDNA synthesis while removing potential DNA contamination. Quantitative real-time PCR analysis was conducted in 20 μL reaction volumes containing 1 μL of cDNA template, 10 μL of SYBR Green qPCR master mix, 0.4 μL each of forward and reverse primers (10 μM stock concentration), and 8.2 μL of nuclease-free water. Amplification was performed on a Q2000B Real-Time PCR System (LongGene Instruments, Hangzhou, China) under optimized cycling conditions: initial denaturation at 95 °C for 5 min to activate the polymerase, followed by 40 cycles of denaturation at 95 °C for 10 s, primer annealing at 58 °C for 20 s, and extension at 72 °C for 20 s. A final melting curve analysis (95 °C to 60 °C to 95 °C) was performed to verify amplification specificity.

The expression levels of target genes (including inflammatory cytokines CCL2, ICAM-1, IL-1β, TNF-α, and IL-10 and bone remodeling markers RANK, RANKL, and OPG) were normalized to the housekeeping gene β-actin and relative quantification was performed using the comparative 2^−ΔΔCt method. All experiments included appropriate no-template controls and inter-run calibrators to ensure reproducibility.

2.6. Histomorphometric Analysis

Following sacrifice, the maxillae were harvested, fixed in 10% formalin, and decalcified using 10% EDTA for 4 weeks to facilitate sectioning. The tissues were subsequently embedded in paraffin and sectioned into 4 μm thick slices. Sections were stained with hematoxylin and eosin (H&E) following standard protocols. Qualitative assessment of periodontal tissues was performed under light microscopy to evaluate histological changes in the gingival epithelium, collagen fiber organization, and inflammatory cell infiltration.

Tartrate-resistant acid phosphatase (TRAP) staining was performed using a commercial kit. Osteoclasts were defined as TRAP-positive multinucleated cells (≥3 nuclei) exhibiting purple–red staining, situated in the interdental alveolar bone between the first and second molars. Histomorphometric analysis was performed by blinded investigators. Osteoclast activity was quantified as the number of TRAP-positive cells per square millimeter (cells/mm²) of the interdental alveolar bone area.

2.7. Statistical Analysis

Data are expressed as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism (version 10.5.0, GraphPad Software, San Diego, CA, USA). The Shapiro–Wilk test was used to assess normality. Given the split-mouth study design, comparisons between the FE-DBDG and CG were performed using the paired Student t-test for parametric data or the Wilcoxon signed-rank test for non-parametric data. Microbiome biomarker discovery was performed using LEfSe (R package lefser, version 1.21.7) with an LDA score threshold of >2.0. A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. Electrical and Optical Characteristics of the FE-DBD System

Figure 3A,B display the electrical characteristics of the optimized FE-DBD system. The device was excited by an AC power supply with a peak voltage of 2.83 kV and a frequency of 16–17 kHz. Under these conditions, the peak current was maintained below 10 mA, strictly complying with the medical electrical equipment safety standard IEC 60601-1 [12]. The measured discharge power was 0.325 W. Furthermore, infrared thermal imaging confirmed that the probe surface temperature remained below 30 °C during operation (Figure 2B(iii)), ensuring thermal safety for periodontal tissues.

Spectral analysis (Figure 3C) reveals that the most significant emission peaks are concentrated in the 300–430 nm band, primarily attributed to the transitions of the second positive system (C³Πu → B³Πg) of nitrogen molecules in the air. Typical peak positions include 337.1 nm, 357.7 nm, and 380–400 nm. The high intensity of these peaks suggests that N₂ molecules underwent strong electron impact excitation during the discharge process. Furthermore, a significant sharp peak observed near 391 nm is attributed to the first negative system (B²Σu⁺ → X²Σg⁺) (0–0) of N₂⁺. This feature indicates the presence of a significant ionization process in the discharge region, reflecting characteristics of high local electron energy and transient strong electric fields, which are consistent with the physical characteristics of filamentary micro-discharges in atmospheric pressure non-equilibrium plasmas.

Regarding the hydroxyl radical (OH), the characteristic emission band typically observed at 307–309 nm was not prominent in our measured spectra. This is primarily attributed to the experimental conditions: the spectra were recorded in ambient air with relatively low humidity compared to the aqueous environment of the oral cavity. Since OH radicals are predominantly generated through the dissociation of water molecules, their density is limited in dry air discharge. However, in clinical periodontal applications, the presence of gingival crevicular fluid and saliva is expected to facilitate significant OH radical generation.

Notably, although the overall intensity in the visible light region is relatively weak, a distinct narrow peak attributed to atomic oxygen is observed at 630 nm. This indicates that the discharge induced significant O₂ dissociation and the generation of high-density atomic oxygen. It is worth noting that this emission wavelength falls precisely within the red light spectrum, which corresponds to the therapeutic window typically associated with photobiomodulation (PBM).

3.2. Microbiological Results: Antibiofilm Efficacy

To evaluate the impact of FE-DBD plasma treatment on the alpha diversity of the periodontal pocket microbiota, we analyzed species richness (Richness index), community diversity (Shannon index), and community richness (Chao1 index). No statistically significant differences (p ≥ 0.05) in the Richness, Shannon, or Chao1 indices were observed between the FE-DBD and control groups at either baseline or two weeks post-treatment, although the p-value for the Richness index approached marginal significance at 0.05 (Figure 4). Notably, all three indices showed a declining trend in the FE-DBD group, in contrast to an upward trend in the control group.

Analysis of the 16S rRNA sequencing data at the phylum level revealed structural shifts in the microbial community composition. As shown in Figure 5, after two weeks of treatment, the FE-DBDG exhibited a decrease in the relative abundance of Bacteroidota and Fusobacteriota, which are known pathogenic phyla associated with periodontitis. In contrast, an increase was observed in phyla typically associated with a healthy microenvironment, specifically Proteobacteria and Actinobacteriota.

To identify distinct microbial biomarkers before and after FE-DBD treatment, we further performed LEfSe analysis (LDA score > 2.0) for comparison. The cladogram in Figure 6 illustrates significantly different taxa at multiple taxonomic levels between the pre-treatment (disease state) and post-treatment (recovery state) groups. The pre-treatment group was primarily enriched with Bacteroidota-related taxa (e.g., f__Bacteroidaceae, f__Rikenellaceae), sulfate-reducing bacteria such as Desulfovibrionaceae (e.g., f__Desulfovibrionaceae, o__Desulfovibrionales), Gram-positive Firmicutes (e.g., f__Lactobacillaceae, f__Streptococcaceae), and candidate phylum-level taxa (e.g., f__Saccharimonadaceae). Following treatment, the biomarker profile shifted to Actinobacteriota-related taxa (e.g., c__Actinobacteria, o__Micrococcales), other Gram-positive bacteria (f__Aerococcaceae), and specific Proteobacteria families (e.g., f__Pectobacteriaceae, f__Yersiniaceae).

3.3. Anti-Inflammatory and Osteoprotective Gene Expression Analysis

Quantitative real-time PCR analysis revealed distinct expression patterns of inflammatory and bone metabolism markers between the FE-DBD plasma treatment and control groups.

3.3.1. Inflammatory Cytokine Expression

The FE-DBD group exhibited significant downregulation of key pro-inflammatory mediators (Figure 7). Specifically, CCL2 expression was reduced by 39% (0.61-fold, p < 0.01) and ICAM-1 by 40% (0.60-fold, p < 0.05), and TNF-α showed the most pronounced decrease of 57% (0.43-fold, p < 0.01). Although IL-1β levels were numerically lower by 51% (0.49-fold) in the FE-DBD group, the difference did not reach statistical significance (p > 0.05). In contrast, the anti-inflammatory cytokine IL-10 displayed a 66% upregulation (1.66-fold, p < 0.001), representing the sole cytokine showing increased expression.

3.3.2. Bone Metabolism Markers

Analysis of osteoregulatory factors revealed coordinated changes in bone remodeling pathways (Figure 8). RANK expression decreased by 47% (0.53-fold, p < 0.05), while its ligand RANKL was downregulated by 41% (0.59-fold, p < 0.05). Conversely, osteoprotegerin (OPG) expression increased substantially by 105% (2.05-fold, p < 0.0001). This reciprocal regulation resulted in a 3.72-fold elevation in the OPG/RANKL ratio (p < 0.0001), indicating a pronounced shift toward an osteoprotective environment favoring bone preservation.

3.4. Histomorphometric Analysis and Quantification of Osteoclasts

3.4.1. Histological Assessment

Histological examination of H&E-stained sections revealed substantial improvement in periodontal tissues after FE-DBD treatment (Figure 9). The control group exhibited severe pathological alterations, including gingival epithelial hyperplasia, elongated rete pegs, epithelial vacuolization, intraepithelial angiogenesis, and dense inflammatory cell infiltration. In contrast, the FE-DBD treatment group showed notable reduction in epithelial vacuolization and hyperplasia, substantially diminished inflammatory cell infiltration, and improved collagen fiber organization in the connective tissue.

3.4.2. Osteoclast Quantification

Histomorphometric analysis of TRAP-stained tissues demonstrated significant inhibition of osteoclastogenesis in the FE-DBD group (Figure 10). The number of TRAP-positive multinucleated osteoclasts was significantly lower in the FE-DBD treatment group (4.55 ± 2.15 cells/mm²) compared to the control group (11.40 ± 3.43 cells/mm²), representing an approximately 60% reduction (p < 0.05). This direct histological evidence aligns with the molecular findings of the suppressed RANK/RANKL pathway and the elevated OPG/RANKL ratio, confirming the osteoprotective effect of FE-DBD treatment.

4. Discussion

This study demonstrates the multifaceted therapeutic potential of floating electrode–dielectric barrier discharge (FE-DBD) plasma in an experimental periodontitis model. Its mechanism of action appears to involve the synergistic modulation of the subgingival microbiome, host inflammatory response, and bone homeostasis. Regarding the microecological impact, we confirmed that FE-DBD can effectively reverse the dysbiotic state of the periodontal microbiome. While extensive in vitro studies have established the potent antibacterial efficacy of CAP against key periodontal pathogens (e.g., P. gingivalis, A. actinomycetemcomitans, F. nucleatum) [13,14,15,16], current research remains predominantly focused on plasma jet systems, leaving alternative plasma generation methods relatively unexplored. Although a single in vivo study in a rat model reported a reduction in specific pathogens following CAP intervention [6], the considerable interspecies differences in subgingival microbiota between humans and rats, alongside the inherent challenges in establishing stable colonization of human-derived periodontal pathogens in animal models [17], necessitate a more comprehensive investigation into the ecological shifts induced by alternative plasma sources.

To address this need, the present study utilized 16S rDNA high-throughput sequencing to systematically compare the subgingival plaque microbial communities between a control group and an FE-DBD treatment group in a rat model of periodontitis. Consistently with the premise that increased microbial diversity marks periodontitis progression, the results demonstrated that FE-DBD treatment exhibited a notable decreasing trend in the alpha diversity indices (Richness, Chao1, and Shannon) of the microbial community, whereas these indices showed an increasing trend in the control group. Although these specific changes did not reach statistical significance (p ≥ 0.05), likely due to the limited sample size in this pilot study, the observed pattern is consistent with the characteristic decrease in diversity following successful treatment [18]. Crucially, despite the lack of statistical significance in alpha diversity, FE-DBD treatment significantly restructured the microbial communities in ligated molars, consistently with established microbiome patterns that differentiate periodontal health and disease [19,20]. The results demonstrate a reversal of disease-associated microbial features: Bacteroidota and Bacillota (formerly Firmicutes) decreased, while Proteobacteria and Actinobacteriota increased (Figure 4). This shift pattern is consistent with previous reports in periodontitis studies [21,22]. LEfSe analysis further demonstrated that FE-DBD intervention significantly reduced the relative abundance of multiple pathogenic taxa strongly associated with oral dysbiosis and periodontal inflammation, including Bacteroidota (such as Bacteroidaceae and Rikenellaceae) and specific Gram-positive bacteria within Firmicutes (e.g., Bacilli and Erysipelotrichaceae). This finding aligns with recent rat model studies [23], further confirming the crucial role of these taxa in the pathological state of periodontitis. Concurrently, Desulfovibrionaceae and other sulfate-reducing bacteria were again validated as characteristic microorganisms of periodontitis [24,25]. Post-treatment microbial communities were predominantly composed of restorative taxa, particularly Actinobacteria (e.g., Actinobacteria class, Micrococcales order), which have shown significant association with periodontal health [26], supporting their potential value as biomarkers for periodontal health. These findings suggest that FE-DBD facilitates the transition of periodontal tissue from a diseased to a healthy state by selectively modulating the oral microbial community structure.

At the host level, FE-DBD demonstrated potent immunomodulatory capabilities, confirmed by both molecular and histological analyses. Our qPCR results showed significant downregulation of pro-inflammatory mediators (TNF-α: 57%; ICAM-1: 40%; CCL2: 39%) and marked upregulation of the anti-inflammatory cytokine IL-10 (66%). This aligns with previous findings using CAP jet therapy [6,9,27,28], suggesting that different plasma modalities share common mechanisms in balancing the cytokine network. These molecular changes were visually corroborated by histological examination: the significant reduction in inflammatory cell infiltration and improved collagen organization observed in H&E staining perfectly corresponded with the cytokine profile shifts detected by qPCR.

Periodontitis progression is strongly correlated with an upregulation of bone remodeling markers (RANK and RANKL) coupled with decreased OPG expression [29,30]. This molecular imbalance triggers a pathological cascade: RANKL-RANK binding stimulates osteoclast differentiation and bone resorption, while diminished OPG levels exacerbate bone loss by reducing its natural inhibition of RANKL [31]. Our experimental results demonstrate that FE-DBD treatment significantly downregulates RANK (47%) and RANKL (41%) expression, while upregulating OPG by 105% (2.05-fold), resulting in a 3.72-fold increase in the OPG/RANKL ratio. These findings align with studies using alternative plasma technologies (e.g., CAP jet), which similarly suppress osteoclastogenesis by modulating the RANKL/RANK/OPG axis [6,9,27]. This molecular regulation translated directly into cellular outcomes: TRAP staining revealed a 60% reduction in multinucleated osteoclasts in the treatment group (Figure 10). This finding aligns with reports on other cold plasma technologies. For instance, Zhang et al. [6] observed fewer osteoclasts with plasma-assisted SRP than with SRP alone, while Choi et al. [9] noted a time-dependent decrease in osteoclasts with increased plasma application, correlating with RANKL downregulation and OPG upregulation. The anti-osteoclastogenic effect of FE-DBD is likely intertwined with its anti-inflammatory action. In our study, osteoclast reduction coincided with lowered pro-inflammatory cytokines and an elevated OPG/RANKL ratio, suggesting that FE-DBD mitigates bone loss by concurrently suppressing osteoclast activity and modulating inflammation. Furthermore, FE-DBD appears to rapidly regulate key osteogenic factors, with its precise reach within periodontal pockets enhancing its potential to curb disease progression in confined anatomic spaces.

Mechanistically, we hypothesize that these therapeutic effects are mediated through a “dual-modal” pathway involving both reactive chemical species and photobiomodulation (PBM). First, regarding chemical species, plasma-generated reactive oxygen and nitrogen species (RONS) are known to suppress the NF-κB signaling pathway and activate MAPK pathways, thereby reducing pro-inflammatory cytokines (TNF-α, IL-1β) and promoting osteogenic differentiation [32,33]. Although our OES analysis showed negligible OH radical emissions due to the dry ambient air test conditions, a distinct atomic oxygen peak was observed at 630 nm. As a potent oxidant, atomic oxygen ensures bactericidal efficacy even in the absence of a prominent OH signal. Second, and notably, this 630 nm emission falls precisely within the optical window for PBM. Previous studies have demonstrated that 630–635 nm red light can not only improve clinical attachment levels by regulating oxidative stress and accelerating fibroblast proliferation [34] but also alleviate inflammation by inhibiting nitric oxide production in LPS-stimulated macrophages [35]. Therefore, unlike devices relying solely on chemical species, our FE-DBD system likely operates via synergistic oxidative bacteriostasis (driven by atomic oxygen) and anti-inflammation (mediated by red light radiation).

Despite these promising findings, several limitations of this pilot study must be acknowledged. First, the small sample size (n = 8) and short 14-day duration restrict statistical power and the assessment of long-term stability. Second, alveolar bone loss was evaluated via 2D histology rather than 3D Micro-CT volumetric quantification. Future investigations should incorporate longitudinal monitoring, 3D imaging, and deeper mechanistic studies to fully elucidate specific signaling pathways. Clinical Translation Potential: The results of this pilot study highlight the FE-DBD system as a promising candidate for clinical translation in periodontics. Unlike conventional gas-fed plasma jets, the FE-DBD device operates directly in ambient air, eliminating the need for external gas tanks and regulators. This significantly reduces the device footprint and operating costs, making it highly practical for chairside use in general dental practices. The needle-shaped probe design specifically addresses the anatomical constraints of periodontal pockets, allowing for site-specific treatment that is difficult to achieve with broad-beam physical therapies. From a maintenance perspective, the detachable and sterilizable probe design aligns with infection control protocols. Future development will focus on optimizing the dosage parameters (frequency and duration) for human tissues and integrating the power supply into a cordless handpiece to further enhance ergonomic utility.

5. Conclusions

In summary, this study systematically elucidates the multiple beneficial mechanisms of FE-DBD plasma in the treatment of experimental periodontitis. The therapy collectively promotes the restoration of periodontal tissue health by reshaping the oral microbial community, balancing the host inflammatory response, and inhibiting osteoclastic bone destruction. These findings provide a solid experimental basis for establishing FE-DBD as a novel and efficient adjunctive therapeutic strategy for periodontal disease. Future research should focus on clinical translation, including conducting human clinical trials to validate its safety and efficacy, and optimizing treatment parameters (such as dose, frequency, and duration) to facilitate the transition of this technology from the laboratory to clinical application.