Antibiofilm Property of Silver Diamine Fluoride Against Elected Periodontal Pathogens: An In Vitro Study

Abstract

Objective

This in vitro study aimed to evaluate the antibiofilm effects of silver diamine fluoride (SDF) against key periodontal pathogens.

Methods

Single-species biofilms of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans, as well as their consortium biofilm, were established on glass coverslips by anaerobic incubation for 72 hours. Mature biofilms were subsequently treated with 38% SDF solution for three minutes. Biofilm viability was assessed using confocal laser scanning microscopy (CLSM). Biofilm morphology and ultrastructure were examined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. 0.2% chlorhexidine gluconate (CHX) served as a positive control, while phosphate-buffered saline (PBS) served as a negative control.

Results

CLSM showed that the percentage of viable cells in biofilms treated with SDF, CHX and PBS was 9.67% ± 1.53%, 31.33% ± 4.73%, and 89% ± 3.61% for Porphyromonas gingivalis; 6.33% ± 0.58%, 33.33% ± 4.73%, and 75.67% ± 8.74% for Fusobacterium nucleatum; 6.67% ± 1.53%, 29.33% ± 4.04% and 83.33% ± 7.37% for Aggregatibacter actinomycetemcomitans, and 8% ± 1%, 31.33% ± 2.52%, and 74.67% ± 6.66% for the multi-species biofilm (p < .001; SDF<CHX<PBS). SEM images confirmed that SDF markedly disrupted biofilm morphology, leading to greater reductions in biofilm density than CHX. TEM analysis further demonstrated that SDF induced severe ultrastructural damage, including abnormal membrane curvature and disruption of cytoplasmic and cell wall integrity.

Conclusion

38% SDF shows stronger antibiofilm activity than 0.2% CHX against key periodontal pathogens, markedly reducing biofilm viability and disrupting structural integrity.

Clinical significance

These in vitro findings support further evaluation of SDF as an adjunct to conventional periodontal therapy, particularly in patients at high risk of biofilm-mediated periodontal disease.

Introduction

Periodontal disease represents a significant global public health challenge, recognized as a prevalent chronic inflammatory condition primarily initiated and sustained by a dysbiotic microbial community within subgingival biofilms.¹ The disease affects over 40% of the global adults with its severe forms associated with increased risks of systemic conditions such as cardiovascular diseases, diabetes, and inflammatory bowel disease.^2,3 The pathogenesis of periodontitis is closely linked to the persistence of polymicrobial biofilms, which are complex communities dominated by anaerobic pathogens like Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans).⁴ These bacteria evade host immune responses and sustain tissue-destructive inflammation through the release of virulence factors.⁵

The cornerstone of periodontal therapy remains mechanical debridement via scaling and root planning (SRP), which aims to remove calculus and disrupt the subgingival biofilm.⁶ However, complete eradication of pathogenic bacteria is often challenging, especially in deep periodontal pockets or areas with complex anatomy and surface irregularities.⁷ Furthermore, patient compliance with optimal oral hygiene and supportive periodontal maintenance is variable, leading to a high risk of disease recurrence. To address residual pathogens, adjunctive antimicrobial therapies are commonly employed. Systemic antibiotics face the mounting crisis of antimicrobial resistance, with studies indicating significant resistance rates among periodontal pathogens.⁸ Locally delivered antimicrobials, such as chlorhexidine and doxycycline gels, offer targeted action but present challenges including precise application, potential for allergic reactions, tooth staining, and disruption of the commensal oral microbiome.⁹ These limitations highlight the urgent need for alternative adjunctive agents that are effective, easy to apply, minimally invasive, and capable of penetrating and disrupting pathogenic biofilms.

Silver diamine fluoride (SDF) has gained widespread attention for its potent antimicrobial and remineralizing properties.¹⁰ It is a colourless alkaline solution containing approximately 24.4%-28.8% silver, 5.0%-5.9% fluoride, and 8.0%-9.8% ammonia, which is originally and extensively used for arresting dental caries and managing dentin hypersensitivity.¹¹ Despite reported adverse effects like soft tissue irritation, metallic taste, and tooth staining in caries management, SDF remains simple, painless, and cost-effective, making it valuable for paediatric, geriatric, and special needs populations.¹² The antimicrobial action of SDF is primarily attributed to silver ions (Ag⁺), which exert broad-spectrum activity by disrupting bacterial cell membranes, inactivating enzymes, and damaging DNA.¹³

Beyond caries control, preliminary evidence suggested that SDF may also benefit periodontal health. A clinical observation reported that SDF application reduced gingival bleeding and plaque scores.¹⁴ In vitro studies have demonstrated concentration-dependent bactericidal activity of SDF against key periodontal pathogens. For example, P. gingivalis showed greater resistance than A. actinomycetemcomitans, necessitating higher concentrations for complete inhibition.¹⁵ Ex vivo experiments using subgingival plaque from periodontitis patients further showed that 19% or 38% SDF significantly reduced total bacterial load and nearly eliminated key pathogens, including P. gingivalis, F. nucleatum, and Tannerella forsythia. ¹⁶ Collectively, these findings indicate that SDF possesses potent antimicrobial properties against periodontal bacteria. However, the specific effects of SDF on the structured and mature biofilms of primary periodontal pathogens remain poorly understood. This knowledge gap impedes the rational exploration of SDF as a targeted periodontal adjunct. Therefore, this in vitro study was designed to rigorously investigate the direct antibiofilm effects of 38% SDF on single- and multi-species biofilms of P. gingivalis, F. nucleatum and A. actinomycetemcomitans. We hypothesized that SDF would exhibit potent activity against these periodontal pathogen biofilms. By exploring SDF’s potential as an adjunctive, non-invasive therapy, this research seeks to inform future strategies for improving periodontal health.

Materials and methods

Biofilm strains

Three key periodontal pathogens, including P. gingivalis (ATCC 33277), F. nucleatum (ATCC 25586), and A. actinomycetemcomitans (ATCC 29523) were obtained from the Central Research Laboratory, Faculty of Dentistry, The University of Hong Kong. Each strain was initially revived and cultured anaerobically at 37°C in its respective recommended broth medium until the mid-logarithmic phase of growth was reached, as determined by optical density measurements.

Biofilm culture

For biofilm formation, bacterial suspensions were adjusted to an optical density (OD₆₀₀) of 0.5 to standardize the inoculum. Single-species biofilms were established by inoculating 1 mL of the adjusted suspension per well into sterile 24-well polystyrene plates containing sterile glass coverslips (13 mm diameter). The multi-species biofilm consisted of P. gingivalis, F. nucleatum, and A. actinomycetemcomitans in a 1:1:1 ratio.¹⁷ All wells were supplemented with 1 mL of brain-heart infusion (BHI) broth, enriched with 5 µg/mL hemin and 1 µg/mL vitamin K to support the growth of fastidious organisms. The plates were then incubated under anaerobic conditions at 37°C for 72 hours to allow for the development of mature, structured biofilms. The culture medium was carefully aspirated and replaced with fresh, pre-reduced medium every 24 hours to replenish nutrients and maintain anaerobic conditions without mechanically disturbing the developing biofilm.

Biofilm viability

After the incubation period, the mature biofilms were gently washed twice with sterile phosphate-buffered saline (PBS, pH 7.4) to remove non-adherent planktonic cells. The biofilms were then randomly allocated to one of three treatment groups (n = 6 independent biological replicates per group per bacterial type):

Test Group: 38% silver diamine fluoride solution (Saforide, Japan).

Positive Control Group: 0.2% chlorhexidine gluconate (CHX) solution (Corsodyl, UK).

Negative Control Group: Phosphate-buffered saline (PBS).

One millilitre of the respective treatment agent was applied directly onto the biofilm-covered coverslips and left in contact for 3 minutes. Following treatment, the agents were aspirated, and the biofilms were washed twice with PBS to terminate the action. Biofilm viability was immediately assessed using the LIVE/DEAD® BacLight Bacterial Viability Kit (Invitrogen, USA) according to the manufacturer’s instructions. The stained samples were visualized using a confocal laser scanning microscopy. Quantitative analysis was performed using Image J software to calculate the ratio of the green fluorescence area (live bacteria) to the total fluorescence area (green + red), expressed as the percentage of viable cells.

Biofilm morphology

Scanning electron microscopy (SEM) was used to evaluate the architectural integrity and surface topography of the biofilms post-treatment. Biofilm samples on coverslips were fixed with 2.5% glutaraldehyde overnight at 4°C. After fixation, samples were subjected to a graded series of ethanol dehydrations (50%, 70%, 80%, 90%, 95%, and 100% v/v), with 15 minutes per step. The coverslips were then mounted on aluminum stubs and sputter-coated with gold. The specimens were examined using a field-emission scanning electron microscope. Representative images from randomly selected fields were captured to assess biofilm density and surface characteristics.

Bacterial ultrastructure

To investigate the ultrastructural changes induced by the treatments, biofilms on coverslips were processed for transmission electron microscope (TEM) to achieve nanometre-scale resolution. After treatment and washing, biofilm samples were primarily fixed with 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide, both in 0.1 M phosphate buffer. Subsequent dehydration was performed using a graded ethanol series, followed by infiltration and embedding in epoxy resin. The sections were stained with uranyl acetate and lead citrate before being examined under a transmission electron microscope. Images were captured to assess morphological alterations in bacterial cell walls and cytoplasmic membranes following each treatment.

Sample size calculation and statistical analysis

To detect a minimum difference of 20% in viable cell percentage between groups, with a standard deviation of 10%, a significance level (α) of 0.05, and a power (1–β) of 0.80, it was determined that at least three independent biological replicates per group were necessary. Data were analysed using SPSS software (version 27.0, IBM Corp.). Results are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed. A P value of less than .05 was considered statistically significant for all tests.

Results

Biofilm viability assessment

The confocal laser scanning microscopy analysis provided a clear and quantitative assessment of the antimicrobial efficacy of the different treatments. The PBS group exhibited dense, confluent biofilms with a high percentage of viable (green) cells, confirming robust biofilm health after the 72-hour incubation period (Figure 1). Treatment with 0.2% CHX resulted in a significant reduction in biofilm viability across all bacterial species and the multi-species biofilm. This was evidenced by a marked increase in red fluorescence (dead/damaged cells) and a corresponding decrease in the calculated percentage of live cells. Notably, 38% SDF-treated showed markedly fewer viable bacteria across all groups, indicating a near-total loss of viable bacteria. For P. gingivalis, the live cell percentage was 89.00 ± 3.61% in the PBS control, which was significantly reduced to 31.33% ± 4.73% by CHX (p < .001) (Table 1). SDF treatment drove this down to a mere 9.67% ± 1.53%, a reduction significantly greater than that achieved by CHX (p < .001). An identical and highly significant pattern (p < .001 for all inter-group comparisons) was observed for F. nucleatum (SDF: 6.33% ± 0.58%; CHX: 33.33% ± 4.73%; PBS: 75.67% ± 8.74%), A. actinomycetemcomitans (SDF: 6.67% ± 1.53%; CHX: 29.33% ± 4.04%; PBS: 83.33% ± 7.37%) and the multi-species biofilm (SDF: 8% ± 1.00%; CHX: 31.33% ± 2.52%; PBS: 74.67% ± 6.66%). The statistical post-hoc test (Tukey) confirmed the efficacy hierarchy for every biofilm type: SDF < CHX < PBS.

Fig. 1.

CLSM images of the P. gingivalis, F. nucleatum, A. actinomycetemcomitans, and multi-species biofilms treated with SDF, CHX and PBS. The CLSM images show that the bacterial biofilms treated with SDF and CHX had a higher proportion of red to green fluorescence than that treated with PBS. Red fluorescence indicated dead bacteria; green fluorescence indicated live bacteria. Scale: 50 μm.

Table 1.

The proportion of live cells in the biofilms across the 3 groups.

Group	SDF	CHX	PBS	p value	Tukey test
P. gingivalis	9.67 ± 1.53	31.33 ± 4.73	89.00 ± 3.61	<.001	SDF<CHX < PBS
F. nucleatum	6.33 ± 0.58	33.33 ± 4.73	75.67 ± 8.74	<.001	SDF<CHX < PBS
A. actinomycetemcomitans	6.67 ± 1.53	29.33 ± 4.04	83.33 ± 7.37	<.001	SDF<CHX < PBS
Multi-species	8.00 ± 1.00	31.33 ± 2.52	74.67 ± 6.66	<.001	SDF<CHX < PBS

Biofilm morphology assessment

The effects of SDF, CHX, and PBS on the structure of mature biofilms are illustrated in Figure 2. The PBS group biofilms presented as dense, intricate three-dimensional structures with a smooth, confluent appearance. Biofilms treated with 0.2% CHX showed clear signs of disruption. The dense, continuous architecture was compromised, appearing porous and ragged. Bacterial cells were visibly damaged, with evidence of cell shrinkage and lysis. The extracellular matrix appeared degraded, though remnants of the biofilm structure and some apparently intact cells, particularly of F. nucleatum in the multi-species model. Notably, biofilms treated with 38% SDF were almost completely eradicated. The biofilm architecture was not merely damaged but was substantively absent, indicating a profoundly destructive effect that extended beyond simple bactericidal action to comprehensive biofilm matrix disruption.

Fig. 2.

SEM images of the P. gingivalis, F. nucleatum, A. actinomycetemcomitans, and multi-species bacteria within biofilms with SDF, CHX, and PBS. The SEM images show complete inhibition growth of P. gingivalis, F. nucleatum, A. actinomycetemcomitans, and multi-species treated with SDF and CHX, whereas a confluent growth of bacteria treated with PBS.

Bacterial ultrastructure assessment

TEM images elucidated the mechanism of SDF's action at the cellular level in Figure 3. Bacterial cells from the PBS control groups displayed classic, intact ultrastructure: a clearly defined, smooth cell wall. In A. actinomycetemcomitans, SDF caused notable disruption of the cytoplasmic membrane, including membrane rupture and irregularities. Similar morphological changes were observed in P. gingivalis and F. nucleatum, with alterations such as abnormal membrane curvature and increased transparency of the cytoplasmic zones. Additionally, the presence of high electron-density particles was present within the cytoplasm. These ultrastructural alterations suggest that SDF exerts its antimicrobial effect by disrupting bacterial membrane integrity and interfering with intracellular processes, ultimately leading to cell death.

Fig. 3.

TEM images of periodontal pathogens treated with SDF or PBS. SDF treated bacteria showed aberrant membrane curvature and breakdown of the cytoplasmic membrane, while PBS treated bacteria showed an intact cell wall. Red arrow indicated cytoplasmic clear zone; yellow arrow indicated electron-dense granular deposits. Scale for P. gingivalis and A. actinomycetemcomitans: 200 μm; Scale for F. nucleatum: 1 μm.

Discussion

The present study provides compelling in vitro evidence that 38% SDF possesses potent antibiofilm activity against primary periodontal pathogens, surpassing the efficacy of 0.2% CHX. The superior efficacy was corroborated at structural and ultrastructural levels, revealing SDF's unique capacity to disrupt mature biofilm architecture and cause severe cellular damage of periodontal pathogens.

In the absence of a universal gold standard for in vitro periodontal multispecies biofilm models, a defined combination of three keystone pathogens—P. gingivalis, F. nucleatum, and A. actinomycetemcomitans—is frequently employed. P. gingivalis (red complex) acts as a keystone pathogen that disrupts host immunity and tissue homeostasis.¹⁸ F. nucleatum (orange complex) serves as a critical structural bridge, binding early and late colonizers to enhance biofilm structural integrity.¹⁹ A. actinomycetemcomitans (green complex), an early colonizer, produces leukotoxins and invades host tissues.²⁰ Together, they form a synergistic pathogenic network that effectively models the polymicrobial etiology of periodontitis. Notably, these Gram-negative pathogens exhibit heightened susceptibility to metal ions compared to their Gram-positive counterparts, providing a relevant therapeutic target.²¹

A central challenge in treating these infections arises because pathogens embedded within the extracellular polymeric substance (EPS) matrix can be up to 1000 times more resistant to antimicrobials than their planktonic counterparts. The EPS barrier physically impedes the penetration of bactericidal agents and hampers immune recognition, making biofilms exceedingly difficult to eradicate completely.²² Consequently, this intrinsic resilience is a key driver of disease chronicity and a major contributor to the failure of conventional periodontal therapies.²³ Despite the structural complexity of multispecies biofilms, our findings demonstrate that SDF effectively penetrates mature EPS matrices to exert broad-spectrum antibacterial effects. This suggests that SDF possesses favourable diffusivity and infiltration capacity, which are crucial for targeting bacteria embedded deep within biofilms.²⁴

The antimicrobial efficacy of SDF is primarily attributed to the sustained release of silver ions, which have demonstrated superior potency against periodontal pathogens compared to other metal ions like copper or zinc.²¹ Silver ions exert a multi-pronged attack: they have a high affinity for sulphur-containing proteins, disrupting cell membrane integrity and inactivating essential metabolic enzymes.²⁵ Crucially, SDF also causes the generation of reactive oxygen species, leading to oxidative stress, and ultimately inducing bacterial cell death.²⁶ This concerted mechanism of action makes the development of bacterial resistance to silver exceedingly rare compared to conventional antibiotics.

TEM analysis revealed that SDF induced substantial morphological damage to bacterial cell membranes, supporting the notion that silver ions penetrate the bacterial cell membrane, enter the cytoplasm and contribute to cellular damage. The electron-dense granules visualized are likely aggregates of silver-sulphur or silver-phosphate complexes, serving as physical testaments to this intracellular interaction. This ability to cause profound structural damage suggests that SDF could help control residual bacteria following scaling and root planning, thereby reducing the risk of disease recurrence.

Clinical evidence from caries management demonstrates that the efficacy of SDF increases with application time. While a 16-second application of 38% SDF can arrest 80% of caries lesions, its maximal effect is achieved with application times up to 3 minutes.²⁷ This time-dependent antibacterial activity has prompted investigation into its potential benefits beyond caries control. In a randomized clinical trial involving elderly patients, weekly professional applications of 38% SDF—even without concomitant professional mechanical plaque removal—significantly reduced gingival index scores and plaque accumulation.²⁸ Furthermore, case reports have documented reductions in gingival inflammation following SDF application as an adjunct to periodontal scaling. Although standardized treatment protocols for SDF in periodontal practice are not yet established, these cumulative findings indicate that its efficacy extends beyond in vitro conditions and may translate into tangible clinical benefits. This supports the investigation of SDF as a promising therapeutic adjunct for periodontal management.

Despite its potent antibiofilm property, concerns have been raised regarding the cytotoxicity of SDF. In vitro studies have shown potential adverse effects on human cells.²⁹ For example, exposure to even low concentrations (0.394%) caused immediate necrosis of human gingival fibroblasts.¹⁵ Furthermore, a 3-minute exposure to 38% SDF damaged artificial gingival tissue by disrupting epithelial layers and reducing intercellular cohesion.³⁰ However, SDF exhibited limited damage in three-dimensional human skin equivalent models, causing only tissue destruction within 50 μm of the superficial connective tissue.¹⁵ Current clinical applications of SDF in periodontal therapy involve superficial application to the buccal surfaces of teeth rather than direct placement into periodontal pockets. Consequently, there is a lack of direct in vivo evidence regarding SDF’s therapeutic efficacy against periodontal pathogens within the pocket environment. Given these considerations, further clinical research is essential to establish safe and effective protocols for SDF application, evaluate its long-term safety, and confirm its efficacy as an adjunctive an adjunctive therapeutic agent in the management of periodontal diseases.

Several limitations of this study should be acknowledged. First, as an in vitro investigation, the experimental conditions cannot fully replicate the complex oral microenvironment, including the dynamic flow of saliva, host immune responses, and the presence of diverse microbial communities. Second, the biofilms were grown on glass coverslips rather than on clinically relevant substrates such as tooth roots, which may influence biofilm architecture and treatment response. Accordingly, future studies using more clinically relevant models are needed to validate these findings.

Conclusion

In conclusion, 38% SDF exhibits significantly stronger antibiofilm activity against key periodontal pathogens than 0.2% CHX in vitro. SDF effectively disrupts the structural integrity of both single- and multi-species biofilms and induces severe ultrastructural damage to bacterial cells. These findings support the potential of SDF as an adjunctive agent to conventional mechanical debridement. However, further in vivo studies are needed to evaluate its clinical efficacy and safety in periodontal applications.

Author contributions

Jing Zhang: Writing review & editing, Writing original draft, Methodology, Formal analysis, Data curation. Zelda Ziyi Zhao: Writing review & editing, Methodology, Data curation. Chun Hung Chu: Editing, Supervision, Conceptualization.

Funding

This work was supported by the HKU Seed Fund for Basic Research (No.109900105).

Conflict of interest

None disclosed.