Effectiveness of biofilm-targeted therapy in managing and preventing dental caries: protocol for a systematic review and meta-analysis

Abstract

Background

Dental caries is a biofilm-dependent disease that continues to challenge conventional preventive strategies, such as fluoride application and mechanical plaque removal. Biofilm-targeted therapies (BTT), including probiotics, photodynamic therapy, enzymatic treatments, and natural compounds, represent promising alternatives to disrupt pathogenic biofilms effectively. However, the available evidence remains fragmented, with inconsistent methodologies and limited clinical data hindering comprehensive conclusions.

Methods

This systematic review and meta-analysis will synthesize evidence on the effectiveness of BTT in preventing and managing dental caries. A comprehensive search of PubMed (MEDLINE), Embase, and the Cochrane Library will identify randomized controlled trials (RCTs) and observational studies evaluating BTT interventions. To ensure methodological robustness, quantitative synthesis of intervention effectiveness will be restricted to RCTs, while observational studies will contribute to a structured narrative synthesis. Two independent reviewers will conduct study selection, data extraction, and risk of bias assessment using the RoB 2 tool for RCTs and ROBINS-I for observational studies. Where feasible, pairwise meta-analyses will be performed using random-effects models. If sufficient data are available, a network meta-analysis (NMA) will be considered. Subgroup and sensitivity analyses will explore the impact of age, caries risk level, and intervention type on treatment outcomes.

Discussion

This review anticipates providing evidence on the efficacy of BTT in reducing bacterial load, altering biofilm composition, and preventing caries progression. Variability in study design and outcome measures is expected, underscoring the need for standardized methodologies and more robust clinical trials. The findings aim to deliver evidence-based insights into the clinical applicability of BTT, addressing current knowledge gaps and guiding future research toward more effective caries prevention and management strategies.

Trial registration

Systematic review registration: PROSPERO CRD42024615568.

Introduction

Dental caries is a prevalent and multifactorial disease that affects individuals of all ages, making it one of the most significant public health challenges worldwide [1, 2]. This biofilm-dependent condition results from the demineralization of dental hard tissues caused by acidic byproducts of bacterial metabolism within a complex oral biofilm [3]. Despite advancements in preventive and therapeutic strategies, the management of dental caries continues to pose significant challenges due to its dynamic and multifactorial nature [1–3]. Conventional approaches, such as mechanical plaque removal and fluoride application, are effective but have limitations in addressing the underlying pathogenic biofilm responsible for caries progression [3, 4].

In recent years, biofilm-targeted therapies (BTT) have emerged as a promising alternative for caries management by disrupting or modifying the cariogenic biofilm [5]. Interventions such as probiotics, photodynamic therapy, enzymatic treatments, and natural compounds like essential oils, polyphenols, and cannabinoids have shown potential in reducing bacterial load, altering biofilm composition, and enhancing remineralization [5, 6]. While numerous studies have explored the efficacy of these therapies, the existing literature is fragmented, with limited clinical evidence and substantial variability in methodologies [7, 8].

Systematic reviews and meta-analyses have examined specific biofilm-targeted therapies, such as essential oils, polyphenols, and silver-based formulations, but their findings often lack generalizability due to methodological inconsistencies and heterogeneity across studies [7, 8]. Moreover, novel therapies, including arginine-based and cannabinoid interventions, remain underexplored in clinical settings [9, 10]. This protocol outlines a systematic approach to synthesizing the evidence on the effectiveness of diverse biofilm-targeted therapies for caries prevention and management. By addressing gaps in the current literature, this review aims to provide evidence-based insights into the clinical applicability of these therapies and identify directions for future research.

Methods

Study registration

This protocol is registered in the PROSPERO database under the registration number CRD42024615568. The protocol has been developed to ensure compliance with PRISMA-P (Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols) guidelines [11]. Additionally, the protocol adheres to the MOOSE (Meta-Analysis of Observational Studies in Epidemiology) guidelines [12], which are specific to the inclusion of observational studies in systematic reviews and meta-analyses.

Eligibility criteria

Types of studies

This review will include randomized controlled trials (RCTs) and observational studies (e.g., cohort, case–control) conducted in clinical, community health center, or school settings. However, only RCTs will contribute to the quantitative synthesis (meta-analysis) evaluating intervention effectiveness. Observational studies will be included for narrative synthesis to provide complementary insights into real-world applicability, safety, and long-term outcomes.

Types of participants

Eligible studies must involve human participants of any age group, including children, adolescents, adults, and elderly individuals, with or without a formal diagnosis of dental caries at baseline, provided the study evaluates either the prevention or management of dental caries. Studies focusing on participants with systemic conditions known to substantially alter caries risk, such as Sjögren’s syndrome or xerostomia, or those undergoing orthodontic treatment or with dental implants will be excluded to minimize confounding influences on biofilm characteristics and caries outcomes.

Types of interventions

This review will include studies evaluating biofilm-targeted therapies (BTT) specifically designed to disrupt, inhibit, or modify cariogenic biofilms. Eligible interventions include probiotics, antimicrobial peptides, photodynamic therapy (PDT), enzymatic treatments, biofilm-disrupting agents (such as chlorhexidine), natural antimicrobial compounds (such as essential oils, polyphenols, or cannabinoids), and novel biomaterials that exert anti-biofilm effects. Comparators may include conventional caries management strategies such as fluoride varnish, sealants, restorations, placebo, no treatment, or other BTTs in head-to-head comparison studies.

Studies that evaluate interventions based solely on mechanical plaque removal, such as professional scaling without the use of biofilm-targeted agents, or studies exclusively assessing fluoride-based interventions without a biofilm-targeted component, will be excluded. Additionally, in vitro studies, animal studies, and purely laboratory-based experimental models will not be considered.

Types of outcome measures

Eligible studies must report at least one of the following outcomes: the primary outcome will be the reduction in dental caries incidence or progression, measured using standardized indices such as the International Caries Detection and Assessment System (ICDAS) or the Decayed, Missing, and Filled Teeth (DMFT) index.

Secondary outcomes will include changes in biofilm composition, including bacterial diversity, virulence markers, and pathogenicity profiles; patient-reported outcomes such as oral health-related quality of life; and adverse effects or safety profiles associated with the intervention.

Studies that exclusively focus on populations with systemic conditions significantly modifying caries risk, such as those with salivary gland dysfunction or medical devices like orthodontic appliances or implants, will be excluded. In vitro studies, animal studies, and interventions limited to mechanical plaque removal or fluoride application without a biofilm-targeted component will also be excluded.

Search strategy

A comprehensive and systematic search will be conducted under the supervision of the principal investigator across the following electronic databases: Cochrane Library, Embase, and PubMed (MEDLINE). The search strategy will employ a structured combination of free-text keywords and controlled vocabulary terms (e.g., Medical Subject Headings [MeSH] in PubMed and Emtree terms in Embase) to ensure comprehensive identification of relevant studies.

Key search terms will include, but will not be limited to biofilm, biofilm-targeted therapy, dental caries, probiotics, photodynamic therapy, enzymatic treatments, natural antimicrobials, and oral health. Boolean operators such as AND, OR, and, where appropriate, proximity operators will be used to combine search terms effectively and refine the search. Truncation symbols and wildcards will be applied to capture variations in terminology.

No restrictions will be applied based on publication date. Studies published in languages other than English will be included if a reliable translation is available. In addition to database searching, the reference lists of all included studies, as well as relevant systematic reviews and meta-analyses, will be manually screened to identify additional eligible studies.

The complete, reproducible search strategy for each database will be developed with the assistance of an experienced information specialist and provided as a supplementary file to enhance transparency and replicability.

All identified records will be imported into Zotero reference management software for systematic organization. Duplicate records will be removed prior to the screening process.

Selection process

Two independent reviewers will screen titles, abstracts, and full texts to identify eligible studies. Discrepancies will be resolved through discussion, and if necessary, a third reviewer will be consulted. The study selection process will be documented and reported using a PRISMA flow diagram to ensure transparency and reproducibility.

Data collection process

Data from the included studies will be extracted independently by two reviewers using a standardized data extraction form. Key study characteristics will include author details, year of publication, study design, sample size, patient demographics, intervention details, comparators, outcomes assessed, follow-up duration, and reported complications. Discrepancies will be resolved through discussion or consultation with a third reviewer.

Assessment of risk of bias

Two independent reviewers will assess the methodological quality and risk of bias of all included studies. Any discrepancies between reviewers will be resolved through discussion, and, if necessary, by consulting a third reviewer. To ensure appropriate evaluation based on study design, different validated tools will be applied. For randomized controlled trials (RCTs), the Cochrane Risk of Bias 2 (RoB 2) tool will be used. For observational studies, the ROBINS-I (Risk Of Bias In Non-Randomized Studies of Interventions) tool will be applied. Risk of bias assessments for each study will be documented in detail, and a summary of the results will be presented using risk of bias tables and graphical representations.

Data synthesis and analysis

If sufficient homogeneous data are available, a meta-analysis will be conducted using random-effects models for the primary analysis to account for heterogeneity, with fixed-effects models considered as part of planned sensitivity analyses. Effect measures will include relative risk (RR), odds ratio (OR), and risk difference (RD) for dichotomous outcomes, and mean difference (MD) or standardized mean difference (SMD) for continuous outcomes.

Heterogeneity will be assessed using the I² statistic, which will be interpreted as follows: values between 0 and 25% will be considered indicative of low heterogeneity, 26 to 50% as moderate heterogeneity, 51 to 75% as substantial heterogeneity, and values exceeding 75% as considerable heterogeneity. Cochran’s Q test will also be applied to assess statistical heterogeneity.

Where sufficient direct comparison studies exist, pairwise meta-analyses will be performed within comparable groups of interventions. If the available data allow, a Network Meta-Analysis (NMA) will be considered to enable both direct and indirect comparisons across multiple biofilm-targeted therapies (BTTs).

Subgroup analyses will explore differences in intervention effectiveness based on participant age group (such as children or adults), type of intervention (including probiotics, photodynamic therapy, enzymatic agents, and biofilm-disrupting agents), caries risk level, and study setting (for example, clinical, community, or school-based environments). If at least 10 studies contribute to a given outcome, meta-regression will be performed to investigate potential sources of heterogeneity.

Sensitivity analyses will evaluate the robustness of the results by excluding studies at high risk of bias, excluding studies with small sample sizes, and applying alternative statistical models.

For outcomes with significant heterogeneity or where insufficient data preclude quantitative synthesis, a structured narrative synthesis will be performed. To ensure valid conclusions, quantitative synthesis of intervention effectiveness will be restricted to randomized controlled trials (RCTs). Observational studies will be included in the narrative synthesis to provide complementary insights into real-world application, safety, and long-term outcomes.

Assessment of publication bias

Publication bias will be assessed to determine whether the results of the included studies might be influenced by the tendency to publish studies with positive findings over those with null or negative results. Funnel plots will be used to visually evaluate asymmetry, where the distribution of effect sizes will be plotted against their standard errors. If the funnel plot suggests asymmetry, statistical tests such as Egger’s regression test or Begg’s rank correlation test will be performed to formally assess the presence of publication bias. For outcomes with fewer than 10 studies, visual inspection of the funnel plot may be inconclusive, and statistical tests will not be applied due to low power.

In cases where publication bias is detected, the potential impact on the meta-analysis results will be explored using sensitivity analyses or statistical adjustments, such as the trim-and-fill method. These steps will ensure that the findings are interpreted with caution and reflect the potential influence of missing or unpublished data.

Discussion

Existing systematic reviews and meta-analyses have explored various biofilm-targeted therapies for managing dental caries, highlighting promising yet inconsistent findings. While Freires et al. demonstrated the antibacterial potential of essential oils such as Eugenia caryophyllata and Lippia sidoides, Delimont and Carlson emphasized the role of grape seed extract in reducing biofilm formation and promoting remineralization [13, 14]. Similarly, Tharakan et al. found licorice to be effective in reducing Streptococcus mutans in children [15]. Photodynamic therapy (PDT) has also been a focus, with studies by Oliveira et al. and Santin et al. demonstrating microbial reduction, though variability in protocols limits its generalizability [16, 17]. In addition, Torabi et al. reviewed the inhibitory effects of cannabinoids on cariogenic biofilms [10], while García-Manríquez et al. and Schestakow et al. identified polyphenols, such as those in cranberries and grape seed extract, as promising agents for reducing biofilm biomass and enhancing remineralization [18, 19]. Novel biofilm agents, including arginine and silver-based formulations, have also shown potential [9, 20]. Despite these promising findings, Askar et al. and Maske et al. rightly highlight the persistence of challenges such as methodological variability, lack of standardization in biofilm models, and insufficient clinical data [7, 8]. Moreover, Veenman et al. and Chisini et al. emphasize the need for robust clinical trials linking biofilm characteristics to caries outcomes [21, 22]. This protocol seeks to address these gaps by systematically synthesizing evidence from clinical and high-quality observational studies to evaluate the effectiveness of diverse biofilm-targeted therapies across populations, settings, and caries risk levels.

This systematic review and meta-analysis is expected to provide a comprehensive synthesis of the effectiveness of biofilm-targeted therapies in managing and preventing dental caries. The review anticipates identifying evidence supporting the efficacy of interventions such as probiotics, photodynamic therapy, enzymatic treatments, and natural agents like essential oils and polyphenols in reducing cariogenic bacterial load, altering biofilm composition, and preventing caries progression. Additionally, therapies like silver-based formulations and cannabinoids are anticipated to demonstrate promising antimicrobial properties, though it is expected that clinical trials validating their use in real-world settings will be limited.

Substantial heterogeneity is expected across studies in terms of intervention protocols, target populations, and reported outcomes. This variability may highlight gaps in standardization and the need for more robust clinical trials. Subgroup analyses may reveal differences in therapy effectiveness based on factors such as age, caries risk levels, and treatment settings. Furthermore, this review is likely to uncover emerging biofilm-targeted agents that have shown potential in preclinical settings but remain underexplored in clinical practice.

Finally, the review anticipates identifying significant limitations in the existing literature, including inconsistent study designs, inadequate reporting of adverse effects, and a lack of long-term follow-up data. These findings are expected to inform future research directions, guide clinical decision-making, and provide evidence-based recommendations for integrating biofilm-targeted therapies into caries prevention and management strategies.

Several limitations are anticipated in this systematic review and meta-analysis. A key challenge is the expected heterogeneity across studies, including variability in study designs, intervention protocols, participant demographics, and outcome measures, which may complicate data synthesis and limit the generalizability of findings. Furthermore, while some therapies, such as photodynamic therapy, cannabinoids, and polyphenols, have shown promise, much of the existing evidence is derived from in vitro or preclinical studies, with limited clinical trial data to support their real-world application. Incomplete reporting of outcomes, particularly adverse effects, patient-reported measures, and long-term efficacy, may constrain the comprehensiveness of this review. Language barriers and publication bias could lead to the underrepresentation of non-English or negative-result studies. Variations in the quality and risk of bias across included studies, especially in observational research, may further impact the reliability of findings. A lack of standardized definitions for biofilm-targeted therapies and their mechanisms could introduce inconsistencies, while subgroup analyses, such as age-specific or risk-level-based comparisons, may be restricted by limited data or small sample sizes.

Lastly, given the rapid evolution of research in this field, newly published studies may emerge during or after the review’s completion, potentially affecting the currency of findings. These limitations underscore the importance of cautious interpretation and highlight the need for continued high-quality research in this area.

Dissemination

The search and screening process for the systematic literature review is anticipated to be completed by December 2024. Following this, data extraction, quality appraisal, and subsequent data synthesis will commence in January 2025. The overall review is expected to be finalized by January 2025. Efforts to publish the study results will begin immediately thereafter, with submissions planned for January 2025.

Author contributions

Venkata Suresh Venkataiah and Mohmed Isaqali Karobari made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

Nil.

Data availability

All data generated or analyzed during this study will be available from corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.