The Clinical, Microbiological, and Immunological Effects of Probiotic Supplementation on Prevention and Treatment of Periodontal Diseases: A Systematic Review and Meta-Analysis

(1) Background: Periodontal diseases are a global health concern. They are multi-stage, progressive inflammatory diseases triggered by the inflammation of the gums in response to periodontopathogens and may lead to the destruction of tooth-supporting structures, tooth loss, and systemic health problems. This systematic review and meta-analysis evaluated the effects of probiotic supplementation on the prevention and treatment of periodontal disease based on the assessment of clinical, microbiological, and immunological outcomes. (2) Methods: This study was registered under PROSPERO (CRD42021249120). Six databases were searched: PubMed, MEDLINE, EMBASE, CINAHL, Web of Science, and Dentistry and Oral Science Source. The meta-analysis assessed the effects of probiotic supplementation on the prevention and treatment of periodontal diseases and reported them using Hedge’s g standardized mean difference (SMD). (3) Results: Of the 1883 articles initially identified, 64 randomized clinical trials were included in this study. The results of this meta-analysis indicated statistically significant improvements after probiotic supplementation in the majority of the clinical outcomes in periodontal disease patients, including the plaque index (SMD = 0.557, 95% CI: 0.228, 0.885), gingival index, SMD = 0.920, 95% CI: 0.426, 1.414), probing pocket depth (SMD = 0.578, 95% CI: 0.365, 0.790), clinical attachment level (SMD = 0.413, 95% CI: 0.262, 0.563), bleeding on probing (SMD = 0.841, 95% CI: 0.479, 1.20), gingival crevicular fluid volume (SMD = 0.568, 95% CI: 0.235, 0.902), reduction in the subgingival periodontopathogen count of P. gingivalis (SMD = 0.402, 95% CI: 0.120, 0.685), F. nucleatum (SMD = 0.392, 95% CI: 0.127, 0.658), and T. forsythia (SMD = 0.341, 95% CI: 0.050, 0.633), and immunological markers MMP-8 (SMD = 0.819, 95% CI: 0.417, 1.221) and IL-6 (SMD = 0.361, 95% CI: 0.079, 0.644). (4) Conclusions: The results of this study suggest that probiotic supplementation improves clinical parameters, and reduces the periodontopathogen load and pro-inflammatory markers in periodontal disease patients. However, we were unable to assess the preventive role of probiotic supplementation due to the paucity of studies. Further clinical studies are needed to determine the efficacy of probiotic supplementation in the prevention of periodontal diseases.


Introduction
Periodontal disease is a growing public health concern, affecting approximately 750 million individuals worldwide [1]. The burden of this disease is expected to continue to grow as the global population ages [2,3]. Periodontal disease is preventable and reversible in its early stages; however, it can progress to chronic, irreversible states with significant destruction of the tooth-supporting tissues [4]. The cause of periodontal disease is multifactorial with modifiable risk factors, including smoking, unhealthy diet (e.g., a western diet with high sugars and saturated fats), poor oral hygiene, hormonal changes, stress, various medications, and poorly managed comorbidities (e.g., type 2 diabetes), while non-modifiable risk factors include age, sex, and genetics [5]. Periodontal disease, when left untreated, can have local and/or systemic consequences, leading to poor oral and systemic health and quality of life [5,6]. The underlying link of periodontal disease with other chronic systemic diseases likely results from the dissemination of periodontopathogens into the bloodstream, endotoxin release, and the associated imbalanced inflammatory response to periodontopathogens [7,8].
Periodontal disease is an inflammatory progressive multi-stage disease of the periodontium (which includes the gingiva, periodontal ligament, alveolar bone, and cementum); this disease is triggered in response to periodontopathogens in the biofilm of the dental plaque on tooth surfaces located near the gingiva ( Figure 1) [9][10][11]. The first stage and mildest form of periodontal disease is known as gingivitis [4]. Gingivitis is a reversible condition, and, if untreated, may progress to periodontitis, which is the advanced stage of periodontal disease [4,12,13]. Gingivitis is characterized by redness, swelling, mild irritation and inflammation of the gingival tissue, and mild bleeding on brushing or flossing, while periodontitis is characterized by deep inflammation and loss of alveolar bone and connective tissue between the gingiva and tooth root [9,14]. The progression of periodontal disease is associated with dynamic shifts in the subgingival bacterial counts and composition in the periodontal pocket [15][16][17][18].
Periodontal disease is a growing public health concern, affecting approximately 750 million individuals worldwide [1]. The burden of this disease is expected to continue to grow as the global population ages [2,3]. Periodontal disease is preventable and reversible in its early stages; however, it can progress to chronic, irreversible states with significant destruction of the tooth-supporting tissues [4]. The cause of periodontal disease is multifactorial with modifiable risk factors, including smoking, unhealthy diet (e.g., a western diet with high sugars and saturated fats), poor oral hygiene, hormonal changes, stress, various medications, and poorly managed comorbidities (e.g., type 2 diabetes), while nonmodifiable risk factors include age, sex, and genetics [5]. Periodontal disease, when left untreated, can have local and/or systemic consequences, leading to poor oral and systemic health and quality of life [5,6]. The underlying link of periodontal disease with other chronic systemic diseases likely results from the dissemination of periodontopathogens into the bloodstream, endotoxin release, and the associated imbalanced inflammatory response to periodontopathogens [7,8].
Periodontal disease is an inflammatory progressive multi-stage disease of the periodontium (which includes the gingiva, periodontal ligament, alveolar bone, and cementum); this disease is triggered in response to periodontopathogens in the biofilm of the dental plaque on tooth surfaces located near the gingiva ( Figure 1) [9][10][11]. The first stage and mildest form of periodontal disease is known as gingivitis [4]. Gingivitis is a reversible condition, and, if untreated, may progress to periodontitis, which is the advanced stage of periodontal disease [4,12,13]. Gingivitis is characterized by redness, swelling, mild irritation and inflammation of the gingival tissue, and mild bleeding on brushing or flossing, while periodontitis is characterized by deep inflammation and loss of alveolar bone and connective tissue between the gingiva and tooth root [9,14]. The progression of periodontal disease is associated with dynamic shifts in the subgingival bacterial counts and composition in the periodontal pocket [15][16][17][18].  . Both modifiable and nonmodifiable risk factors impact the oral microbiome composition and disrupt homeostasis between the host and microbiome. Modifiable risk factors include diet, smoking, oral hygiene, and comorbidities (such as type 2 diabetes), while genetics, age, and sex are nonmodifiable risk factors. (B). Disrupted homeostasis provides appropriate conditions for the growth of periodontopathogens and biofilm formation on the tooth surfaces extending sub-gingivally. There are no clinical symptoms in this stage. (C). These bacteria penetrate and grow in the gingival epithelium. Host-bacteria interactions cause a chemotactic gradient that attracts innate immune cells, including neutrophils, macrophages, and NK cells, to the affected sites. In addition, the outgrowth of bacteria progressively destroys the tissue and provides enough nutrients for more pathogen growth, followed by increased activity of innate immune cells and the secretion of pro-inflammatory cytokines, including IL-1, IL-8, and TNF. Early clinical symptoms in this stage are redness, swelling, mild inflammation, and bleeding of the gingiva, which are diagnosed by measuring the PlI, GI, and BOP. (D). Then, Antigen-Presenting Cells (APC), including dendritic cells, present bacterial antigens to lymphocytes and trigger adaptive immune system activity and antibody and cell-mediated immune responses, resulting in a pro-inflammatory response with high expression of IL-4, 6, 8, 10, 12, TGF-β, and IFN-γ. (E). High levels of these inflammatory mediators stimulate more inflammatory mediators, causing periodontal tissue destruction and leading to the loss of the gingival attachment to the tooth, and causing deep pockets around the teeth that provide appropriate conditions for the growth and colonization of other anaerobic periodontopathogens. Untreated, these pathophysiological changes can lead to alveolar bone resorption and, ultimately, tooth loss in the most advanced stage of the disease. (F). Probiotics may have therapeutic benefits in periodontal disease treatment when used as an adjuvant to standard periodontal care. Various mechanisms of action have been considered for the role of probiotics in periodontal disease improvement. Probiotics interact directly with periodontopathogens through colonization resistance, which includes competition for binding sites and nutrients, and the production of antibacterial agents inhibiting pathogen growth. Probiotics can play a role in periodontal disease improvement indirectly via the modulation of innate and adaptive immunity and through the gut-oral microbiome axis. PlI, Plaque Index; GI, Gingival Index; BOP, Bleeding on Probing; IL, Interleukin; TGF-β, Tumor Growth Factor-β; and IFN-γ, Interferon-γ.

Etiology of Periodontal Disease
The primary etiology of periodontal disease is an imbalanced subgingival microbiome population developing progressively over time due to an increasing relative abundance of periodontal disease-associated bacteria and a corresponding decrease in health-associated bacteria, leading to the disruption of the microbiota-host homeostasis [16,[19][20][21]. This gradual phenomenon begins with the early adherence, growth, and colonization of Gramnegative and Gram-positive bacteria on the tooth surface extending sub-gingivally [22]. This provides appropriate conditions for the growth of and colonization by other anaerobic Gram-negative orange and red-complex bacteria [20]. The orange complex consists of Prevotella intermedia, Parvimonas micra, and Fusobacterium nucleatum, while Porphyromonas gingivalis, Tanerella forsythia, and Treponema denticola are components of the red complex [20]. These bacteria are highly pathogenic and have the ability to release bacterial collagenases and other proteases, leading to the stimulation of the pro-inflammatory response and periodontal tissue damage [9,10,20] (Figure 1).

Periodontal Disease Assessment, Diagnosis, and Therapy
The clinical assessment of periodontal disease includes the evaluation of patientreported outcomes (i.e., bleeding while brushing or flossing, receding gums, halitosis, sensitive teeth, pain during mastication, and loose teeth) [4] and visual assessment of the distance between the base of the periodontal pocket and gingival margin or cementoenamel junction, the amount of plaque in the gingival margin on the tooth surface, and gingival bleeding [9,23]. All of these factors are measured by standard procedures and defined as specific indexes, including: the plaque index (PlI), gingival index (GI), periodontal pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), gingival recession (REC), and gingival crevicular fluid (GCF) volume [23][24][25][26][27].
The clinical diagnosis of periodontal diseases is based on the periodontal exam, radiography, and patient's oral/dental and medical history [28]. The American Academy of Periodontology (AAP) classifies periodontitis into four stages based on severity, complexity, and extent (stage I, II, III, and IV) and three grades based on the evidence of the disease's progression and its rate (slow, moderate, and rapid) [29,30].
Periodontal therapy consists of the removal of the supra-and subgingival plaque from tooth surfaces using Scaling and Root Planing (SRP) [31,32]. To improve disease outcomes, SRP can be incorporated into surgical procedures or adjunctive antibiotics can be administered [3,[31][32][33]. However, antibiotics may cause adverse side effects, or can be contraindicated in some situations; therefore, there is a need for alternative approaches [14].

Periodontal Diseases and Probiotics
Increasing attention has been devoted to probiotic supplementation as a therapeutic adjuvant/alternative to improve oral health [34]. Probiotics are live organisms (usually bacteria) administered to provide health benefits in the prevention or clinical management of different diseases [35,36]. Probiotics have been traditionally accepted in the medical field as an adjuvant treatment of gastrointestinal disorders [37]. Moreover, probiotics are recommended to patients who take antibiotics for the prevention and treatment of Antibiotics-Associated Diarrhea (AAD) [38]. They have also been considered in the clinical management of other conditions, including respiratory tract infections [39]. Furthermore, probiotics may have a therapeutic benefit in dental caries prevention by decreasing the number of cariogenic bacteria, such as Streptococcus mutans [40]. Probiotics may function through various mechanisms, including the production of antimicrobial metabolites, immunomodulation, mucosal barrier enhancement, and microbial flora shift through competition for cell adhesion with pathogenic strains [34,41]. The use of probiotics for the clinical management of periodontal diseases is an active area of research. There have been conflicting results based on several individual studies assessing the effects of probiotics on gingival inflammation [42,43]. Furthermore, previously published systematic reviews have had limited findings with conflicting results when examining the clinical efficacy of probiotics on periodontal diseases. For instance, Akram et al. showed no improvement in PlI and GI in patients with gingivitis after probiotic use, while other reviews concluded that probiotics could improve PlI and GI [44][45][46]. A previous systematic review was unable to assess the immunological benefits of probiotic supplementation due to limited studies; however, the included individual studies indicated an immunomodulatory effect of probiotics [47]. Microbiological findings suggested that probiotic supplementation reduced periodontopathogens in subgingival plaque samples [47]; however, there is conflicting evidence in the literature [48].
The purpose of our systematic review and meta-analysis was to combine results from randomized clinical trials involving adults with periodontal diseases or healthy volunteers receiving probiotic supplementation (control groups did not receive probiotic supplementation) to assess the effects on the clinical, microbiological, and immunological outcomes related to periodontal disease prevention and management.
English language, peer-reviewed studies published since 2000, which were either open access or accessible to the researchers (via the University of Saskatchewan Library, inter-library loan, or through Google scholar), were included. Detailed information about the inclusion and exclusion criteria are available in Supplemental Table S1.

Information Sources, Search Strategy, and Study Selection
Six databases were searched without restrictions, including PubMed, MEDLINE, EM-BASE, CINAHL, Web of Science, and Dentistry and Oral Science Source. A combination of keywords and MeSH terms related to the following search domains were used: 1. Periodontal diseases; 2. Clinical, microbiological, and immunological outcomes; 3. Probiotics. These three domains were combined with the "AND" operator. Details of the search strategy are included in the Supplementary Materials (Supplemental Table S2).
This systematic review and meta-analysis was registered with the International Prospective Register of Systematic Reviews (PROSPERO) (registration number: CRD42021249120).
After calibration, two screeners (ZG and AH) independently conducted dual screening (title and abstract, and full-text), and if consensus was not reached, a third author (RM) provided a tie-breaker vote.

Data Items and Collection Process
Two authors (ZG and AH) independently conducted data extraction, then compared the extracted data, and, in case of disagreement, they referred to the publication. The researchers used a Microsoft Excel (Microsoft Inc., Redmond, WA, USA) spreadsheet to record data pertaining to the study design, sample size, age of participants, health status, periodontal disease stage, dose and probiotic strain, treatment and follow-up durations, oral hygiene instructions, mode of probiotic delivery, clinical measurements (PlI, GI, PPD, CAL, BOP, GCF, and REC), oral bacterial count (P. gingivalis, F. nucleatum, T. forsythia, P. intermedia, A. actinomycetemcomitans, S. mutans, and Lactobacillus species), immunological outcomes (MMP-8, IL-6, IL-1β, IL-8, IL-10, and TNF-α), and key findings of each study.

Risk of Bias within Studies
The risk of bias within studies was assessed independently by two authors (ZG and AH) using the Cochrane risk-of-bias assessment tool version 2 designed for randomized trials [49]. This tool evaluates within-study bias by assessing the randomization process, deviations from intended interventions, missing outcome data, measurement of the outcome, and selection of the reported result. Studies were categorized as follows: low risk, some concerns, or high risk of bias.

Summary Measures and Synthesis of Results
Comprehensive meta-analysis version 3.3070 (Biostat Inc., Englewood, NJ, USA) was used for the meta-analysis. The meta-analysis was conducted using the mean and standard deviations from the studies at baseline until the immediate follow-up after the end of probiotic treatment. Hedge's g standardized mean difference (SMD) was calculated with a 95% confidence interval (CI) for each individual study and pooled in the meta-analysis using random-effects models. SMD can be interpreted as follows: a value of 0 indicates that there is no statistically significant difference in the effect of treatment compared to the control; values greater or less than 0 indicate a difference in effect. The strength of the effect can be roughly interpreted as small (SMD = 0.2), medium (SMD = 0.5), and large (SMD = 0.8) [50]. Heterogeneity was assessed using I 2 , which can be interpreted as low (25%), moderate (50%), or high (75%) [51].
The meta-analysis used the following steps: 1. Pooled random-effects models examining the effect of probiotic supplementation on clinical, microbiological, and immunological outcomes in periodontal disease patients compared to controls (without probiotics); 2. Pooled random-effects models examining the potential preventive effect of probiotic use on clinical, microbiological, and immunological outcomes in healthy individuals (without periodontal diseases) compared to controls (without probiotics); 3. Subgroup analysis (see Section 2.7). The results of the meta-analysis were reported as the SMD, 95% CI, heterogeneity score (I 2 ), and p-value.
Studies lacking data on the mean, standard deviation, and sample size for treatment and/or control were excluded from the meta-analysis, unless these values could be calculated.

Risk of Bias across Studies
Publication bias and small study effects were evaluated using visual inspection of funnel plots and Egger's regression test. If publication bias/small study effects were detected, we used Duval and Tweedie's trim and fill methodology to correct for funnel plot asymmetry [52].

Additional Analysis (Subgroup Analysis and Investigation of Heterogeneity)
The following subgroup analyses were conducted: 1. Type of periodontal disease (gingivitis, periodontitis); 2. Disease severity; 3. Probiotic treatment duration; 4. Mode of probiotic delivery; 5. Type of probiotic strain; 6. Type of lactobacillus species; and 7. Oral hygiene instructions (yes/no). In addition, subgroup analyses assessed the effects of probiotics compared to a control group receiving antibiotics. Disease severity was categorized as moderate (PPD = 4-6 mm) or deep (PPD > 6 mm) periodontal pockets. The treatment duration was categorized as follows: 1. Up to one month; 2. More than one month to two months; or 3. More than two months. The mode of probiotic delivery was categorized as follows: 1. Oral (i.e., toothpaste, mouthwash, sachet applied orally, mouth spray, or oil drops); 2. Oral and ingestion (i.e., lozenges, tablets, or chewing gum); 3. Ingestion (i.e., sachet dissolved in water to drink, capsules, yogurt, or fermented milk/Yakult); or 4. Local application (i.e., subgingival delivery as a paste or gel). The type of probiotic strain was classified as follows: 1. Lactobacillus (Lactobacillus species only); 2. Mixed (Lactobacillus species and other bacterial species); and 3. Other (not lactobacillus species). The type of Lactobacillus was classified as follows: 1. Lactobacillus Reuteri; and 2. Other Lactobacillus spp. (any Lactobacillus species other than L. Reuteri). The oral hygiene instructions were categorized as follows: 1. Yes (when providing specific oral hygiene instructions); or 2. No (when asking participants to maintain their regular oral hygiene habits).

Study Selection
A total of 1883 publications were identified through the electronic search of the six databases. After removing duplicates, two reviewers conducted title and abstract screening on 994 publications, and 890 were excluded. In the next step, 104 publications underwent full-text screening. Finally, 64 publications remained for the systematic review and 47 were included in the meta-analysis (17 studies were removed due to insufficient/incompatible data for meta-analysis) ( Figure 2).

Study Characteristics
There was a total of 64 studies eligible for the systematic review and 47 studies fo the meta-analysis. Table 1 presents the detailed individual study characteristics includin periodontal disease status, sample size, probiotic strain, treatment duration and immed ate follow-up, mode of probiotic delivery, other treatments, oral hygiene instructions, ou comes investigated, and key findings. The probiotic treatment duration varied from on day to four months. The sample sizes varied from 10 to 120 individuals. The most commo probiotic formulation was composed of Lactobacillus reuteri. The majority of studies ha periodontitis patients.

Study Characteristics
There was a total of 64 studies eligible for the systematic review and 47 studies for the meta-analysis. Table 1 presents the detailed individual study characteristics including: periodontal disease status, sample size, probiotic strain, treatment duration and immediate follow-up, mode of probiotic delivery, other treatments, oral hygiene instructions, outcomes investigated, and key findings. The probiotic treatment duration varied from one day to four months. The sample sizes varied from 10 to 120 individuals. The most common probiotic formulation was composed of Lactobacillus reuteri. The majority of studies had periodontitis patients.

Risk of Bias within Studies
Using the Cochrane risk-of-bias assessment tool version 2, the majority of studies included were classified as having a low risk of bias (n = 38). Additionally, 13 studies were classified as having some concerns, and another 13 studies were classified as having a high risk of bias. The majority of concerns were due to questions related to the randomization process domain. There were minimal concerns regarding the missing outcome data domain. The results are presented in Supplemental Table S3.

Synthesis of Results
This meta-analysis used the Hedge's g standardized mean difference (SMD) to report effect sizes. Forest plots depicting the pooled meta-analysis examining the effects of probiotic supplementation on clinical, microbiological, and immunological outcomes are presented in Figures 3-5, respectively. The overall measures of effect are summarized in Supplemental Table S4. The results of subgroup analysis are presented in Table 2 with additional information available in the Supplemental File. The forest plots for the subgroup analysis of clinical parameters are depicted in Supplemental Figure S1.

Favors Control Favors Probiotics
Favors Control Favors Probiotics  Figure 3. Pooled meta-analysis examining the effects of probiotic supplementation on clinical outcomes. 1. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the plaque index (P1I) using a random-effects model. Note that we detected publication bias and/or small study effects, and the adjusted Hedge's g SMD = 0.557, 95% CI: 0.228, 0.885, and p-value ≤ 0.05. 2. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the mean plaque percentage (MPP) using a random-effects model. 3. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the gingival index (GI) using a random-effects model. 4. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the probing pocket depth (PPD) using a random-effects model. 5. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the clinical attachment level (CAL) using a random-effects model. 6. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on bleeding on probing (BOP) using a random-effects model. Note that we detected publication bias and/or small study effects, and the adjusted Hedge's g SMD = 0.841, 95% CI: 0.479, 1.200, and p-value ≤ 0.05. 7. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the gingival crevicular fluid (GCF) using a random-effects model.

Favors Control Favors Probiotics
Favors Control Favors Probiotics Figure 3. Pooled meta-analysis examining the effects of probiotic supplementation on clinical outcomes. 1. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the plaque index (P1I) using a random-effects model. Note that we detected publication bias and/or small study effects, and the adjusted Hedge's g SMD = 0.557, 95% CI: 0.228, 0.885, and p-value ≤ 0.05 [14,41,43,53,58,60,62,67,69,70,80,86,91,105]. 2. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the mean plaque percentage (MPP) using a random-effects model [54,66,68,72,77,82,88,89,92,107,109]. 3. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the gingival index (GI) using a random-effects model [14,41,43,53,58,60,69,70,75,80,89,91,92,105,110]. 4. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the probing pocket depth (PPD) using a random-effects model [   . Pooled meta-analysis examining the effects of probiotic supplementation on microbiological outcomes in periodontal disease patients. 1. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the subgingival P. gingivalis bacterial count using a random-effects model [14,43,76,77,102]. 2. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the subgingival F. nucleatum bacterial count using a random-effects model [14,43,76,77,102]. 3. Forest plot of the Hedge's g SMD comparing the effects of probiotic supplementation to control groups on the subgingival T. forsythia bacterial count using a random-effects model [14,43,76,102].   The effect of probiotic supplementation on the plaque index (PlI) indicated a statistically significant decrease with probiotic supplementation compared to controls in patients with periodontal diseases (SMD = 0.483, 95% CI: 0.163, 0.803, I 2 = 67.044, p-value ≤ 0.05, and n = 13 studies). Upon visual inspection of the funnel plot and confirmation through Egger's regression test (p-value = 0.040), there was evidence of publication bias/small study effects. Duval and Tweedie's trim and fill method gave an adjusted pooled estimate (SMD = 0.557, 95% CI: 0.228, 0.885, I 2 = 75.716, p-value ≤ 0.05, and n = 13 studies) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Mean Plaque Percentage (MPP)
A change in the mean plaque percentage (MPP) was reported in 11 studies. The pooled SMD showed a statistically significant decrease in the MPP with probiotic supplementation compared to controls (SMD = 0.879, 95% CI: 0.308, 1.450, I 2 = 87.544, p-value ≤ 0.05, and n = 11 studies). There was no evidence of publication bias or small study effects upon visual inspection of the funnel plot and confirmation through Egger's regression test (p-value > 0.05) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Gingival Index (GI)
The pooled SMD using data from patients with periodontal disease indicated a statistically significant reduction in the gingival index (GI) with probiotic supplementation compared to controls (SMD = 0.920, 95% CI: 0.426, 1.414, I 2 = 86.027, p-value ≤ 0.05, and n = 14 studies). There was no evidence of publication bias or small study effects upon visual inspection of the funnel plot and confirmation through Egger's regression test (p-value > 0.05) (Figure 3).
Pooled Meta-Analysis Examining the Effects of Probiotics on Probing Pocket Depth (PPD) The pooled SMD indicated a statistically significant decrease in the probing pocket depth (PPD) with probiotic supplementation in patients with periodontal disease compared to controls (SMD = 0.578, 95% CI: 0.365, 0.790, I 2 = 62.840, p-value ≤ 0.05, and n = 27 studies). There was no evidence of publication bias or small study effect upon visual inspection of the funnel plot, and this was confirmed by Egger's regression test (p-value > 0.05) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Clinical Attachment Level (CAL)
The pooled meta-analysis of the effect of probiotic supplementation on the clinical attachment level (CAL) was assessed in patients with periodontitis (n = 19 studies). Metaanalysis was not conducted to assess the effects of probiotics on CAL in healthy participants and those with gingivitis due to a lack of studies.
The pooled SMD indicates that there was a statistically significant CAL gain with probiotic supplementation compared to controls in patients with periodontitis (SMD = 0.413, 95% CI: 0.262, 0.563, I 2 < 0.001, p-value ≤ 0.05, and n = 19 studies). There was no evidence of publication bias and small study effects upon visual inspection of the funnel plot, which was confirmed by Egger's regression test (p-value > 0.05) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Bleeding on Probing (BOP)
The effect of probiotic supplementation on bleeding on probing (BOP) indicated a statistically significant decrease with probiotic supplementation compared to controls in patients with periodontal disease (SMD = 0.756, 95% CI: 0.381, 1.130, I 2 = 83.699, p-value ≤ 0.05, and n = 20 studies). Inspection of the funnel plot for potential publication bias or small study effects suggested a right skew. This was confirmed by Egger's regression test (p-value = 0.020). After adjusting with Duval and Tweedie's trim and fill method, the adjusted SMD remained significant with high heterogeneity (SMD = 0.841, 95% CI: 0.479, 1.20; and I 2 = 86.750) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Gingival Crevicular Fluid (GCF)
The effect of probiotic supplementation on the gingival crevicular fluid (GCF) volume indicated a statistically significant decrease in the GCF with probiotic supplementation compared to controls in patients with periodontal disease (SMD = 0.568, 95% CI: 0.235, 0.902, I 2 < 0.001, p-value ≤ 0.05, and n = four studies). There was no evidence of publication bias or small study effects upon visual inspection of the funnel plot and confirmation by Egger's regression test (p-value > 0.05) (Figure 3).

Pooled Meta-Analysis Examining the Effects of Probiotics on Gingival Recession (REC)
There was no statistically significant evidence that probiotic supplementation improved gingival recession (REC) compared to controls in patients with periodontitis (p-value = 0.741, and n = 5 studies). There was no evidence of publication bias or small study effects upon visual inspection of the funnel plot and confirmation by Egger's regression test (p-value > 0.05).

Associations between Probiotic Supplementation and Microbiological Outcomes in Periodontal Disease Patients Pooled Meta-Analysis Examining the Effects of Probiotics on Subgingival Porphyromonas Gingivalis Count
The pooled SMD indicated a statistically significant decrease in the subgingival P. gingivalis count with probiotic supplementation compared to controls in patients with periodontal disease (SMD = 0.402, 95% CI: 0.120, 0.685, I 2 = 10.769, p-value ≤ 0.05, and n = five studies). There was no evidence of publication bias or small study effects through visual funnel plot inspection and Egger's regression test (p-value > 0.05) (Figure 4).

Pooled Meta-Analysis Examining the Effects of Probiotics on Subgingival Fusobacterium nucleatum Count
The pooled SMD using data from periodontal disease patients indicated a statistically significant decrease in the subgingival F. nucleatum count with probiotic supplementation compared to controls (SMD = 0.392, 95% CI: 0.127, 0.658, I 2 < 0.001, p-value ≤ 0.05, and n = 5 studies). There was no evidence of publication bias or small study effects through visual inspection of the funnel plot and Egger's regression test (p-value > 0.05) (Figure 4).

Pooled Meta-Analysis Examining the Effects of Probiotic on Subgingival Tannerella forsythia Count
The pooled SMD indicated a statistically significant decrease in the subgingival T. forsythia count with probiotic supplementation compared to controls in patients with periodontal disease (SMD = 0.341, 95% CI: 0.050, 0.633, I 2 < 0.001, p-value ≤ 0.05, and n = four studies). There was no evidence of publication bias or small study effects upon visual inspection of the funnel plot and with Egger's regression test (p-value > 0.05) (Figure 4).

Pooled Meta-Analysis Examining the Effects of Probiotics on Subgingival Counts of Other periodonthopathogenes
The meta-analyses conducted to assess the effects of probiotic supplementation on the subgingival bacterial counts of P. intermedia (p-value = 0.193, and n = four studies) and A. actinomycetemcomitans (p-value = 0.164, and n = 5 studies) were not statistically significant when compared to controls. The GCF Interleukin-6 (IL-6) levels showed statistically significant decreases with probiotic supplementation compared to controls in patients with periodontal disease (SMD = 0.361, 95% CI: 0.079, 0.644, I 2 < 0.001, p-value ≤ 0.05, and n = three studies). There was no evidence of publication bias or small study effects through visual funnel plot inspection and Egger's regression test (p-value > 0.05) ( Figure 5).

Associations between Probiotic Supplementation and Immunological Outcomes in
Pooled Meta-Analysis Examining the Effects of Probiotics on Other Immunological Biomarkers in the Gingival Crevicular Fluid (GCF) The meta-analyses conducted to assess the effects of probiotic supplementation on GCF levels of IL-1β (p-value = 0.393 and n = three studies), IL-8 (p-value = 0.434 and n = two studies), IL-10 (p-value = 0.902 and n = two studies), and TNF-α (p-value = 0.495 and n = three studies) in periodontal disease patients were not statistically significant when compared to controls.

Discussion
The aim of this systematic review and meta-analysis was to examine if probiotic supplementation is associated with preventive and therapeutic benefits in terms of improvement of clinical, microbiological, and immunological outcomes in patients with periodontal disease. In summary, the findings of this analysis are promising and indicate that probiotic supplementation significantly improved clinical outcomes, decreased certain periodontopathogen counts, and reduced the levels of specific pro-inflammatory biomarkers in patients with periodontal disease.
The overall results of this meta-analysis showed statistically significant improvements in all clinical parameters (PlI, MPP, GI, PPD, CAL, BOP, and GCF volume) in patients with periodontal disease after probiotic supplementation compared to control groups who did not receive probiotics. When examining the microbiological outcomes, the subgingival periodontopathogen counts of P. gingivalis, F. nucleatum, and T. forsythia showed statistically significant reductions with probiotic supplementation. Furthermore, there was a statistically significant reduction in the GCF levels of inflammatory mediators (MMP-8 and IL-6) with probiotic supplementation.
In addition to the pooled meta-analysis, the effects of probiotic supplementation on clinical outcomes were further assessed through subgroup analysis based on: 1.
Type of periodontal disease, which indicated that probiotic supplementation improved clinical outcomes in patients with periodontitis, but not in those with gingivitis or healthy individuals. However, the GCF volume had statistically significant reductions in both gingivitis and periodontitis patients; 2.
Probiotic formulations consisting of Lactobacillus species and, more specifically, L. reuteri were associated with statistically significant improvements in all clinical outcomes in patients with periodontal disease; 3.
Probiotic treatment duration, which showed that probiotic supplementation resulted in statistically significant improvements in the clinical outcomes after one month of supplementation in periodontal disease patients; 4.
Mode of probiotic delivery, which indicated that probiotic supplementation through the "oral and ingestion" mode was associated with statistically significant improvements in all clinical outcomes in periodontal disease patients.

5.
Oral hygiene instructions along with probiotic supplementation improved PlI and BOP, while GI, PPD, and CAL improved with probiotic supplementation, irrespective of the presence or absence of oral hygiene instructions.
Periodontal disease stability refers to a state of successful treatment where clinical indicators do not progress in severity and is characterized by a minimized BOP score and improved PPD and CAL scores [111,112]. The results of our meta-analysis suggest that probiotic supplementation is a promising adjuvant to the current standard of care (SRP) to improve prognosis and clinical outcomes in patients with periodontitis. Given the improvements in the clinical, immunological, and microbiological outcomes in periodontal disease patients, it seems plausible that probiotics may contribute to reaching periodontal stability.

Probiotics and Severity of Periodontal Disease
Our subgroup analysis did not have significant findings in gingivitis patients, which may be partially attributed to: 1. Gingivitis being an early stage of periodontal disease, and, therefore, the clinical aspects of disease and consequently the effects of probiotic supplementation may be less notable; 2. Some studies on gingivitis did not use probiotics along with the standard of care; and 3. These results could be due to the lack of eligible studies and the moderate to high levels of heterogeneity in our analysis. However, the statistically significant improvements that our meta-analysis found in all clinical parameters at the late/severe disease stages (periodontitis) adds evidence to the existing literature on the clinical benefits of probiotic supplementation in the management of periodontal disease.

Probiotics vs. Antibiotics
The current non-surgical standard of care for periodontal disease includes SRP, which is sometimes followed by antibiotic treatment [31,33]. However, previous studies have reported that antibiotic use, particularly long-term administration, may increase the risk of oral and gut dysbiosis, antibiotic-resistant bacteria, allergic reactions, and other side effects [42]. Probiotics are a promising adjuvant/alternative approach that can be taken safely for longer periods of time. Our meta-analysis comparing probiotic supplementation to antibiotics as adjuvants to SRP did not show any statistically significant differences in CAL and PPD in periodontitis patients. (Note: The other clinical outcomes were not assessed due to insufficient studies). This finding suggests that probiotic supplementationmay be as effective as antibiotics in improving clinical outcomes. Previous studies found similar results when either one is administered as an adjuvant to SRP [61,83]. Furthermore, a more recent study concluded that probiotics are a more effective adjuvant with SRP than antibiotics [113]. However, there is evidence indicating that, when probiotics or antibiotics were administered independently, neither one was more efficacious than SRP alone [10,61]. Although these conclusions warrant more research, our meta-analysis indicated improved clinical outcomes, and it is plausible from a mechanistic perspective that probiotic supplementation as an adjuvant to SRP may be a safer and more effective long-term therapeutic option in the management of periodontal disease compared to antibiotics.

Probiotic Formulation and Duration
Our findings also indicated that probiotic formulation and the type of probiotic strain have clinical relevance. Specifically, our subgroup analysis examining the type of probiotic strain indicated that Lactobacillus probiotic formulations improved all clinical outcomes. Further analysis into the types of Lactobacillus species identified that L. reuteri-containing probiotic formulations improved all clinical outcomes. Lactobacillus species may play a role in plaque control through direct interaction via colonization resistance, which includes competition for binding sites and nutrients or antimicrobial agent production to inhibit other oral bacteria [114]. Additional evidence suggests that L. reuteri may also influence the composition of the oral microbiome and is correlated with reduced periodontopathogens, including F. nucleatum [115,116]. As oral microbiome dysbiosis is one of the root causes of periodontal disease, the shift induction by L. reuteri may result in improvements in periodontal disease clinical outcomes. The only study without Lactobacillus included in our meta-analysis with statistically significant reduction of PPD and CAL, but not PlI, used the subgingival delivery of Saccharomyces Boulardii [58]. S. Bouldarii is a yeast probiotic that is non-colonizing [117], which likely caused nonsignificant changes in plaque accumulation [58]. However, the improvements in other clinical parameters (PPD and CAL) may be due to its role in immunomodulation [118].
Two out of eleven studies with L. reuteri probiotic formulations in patients with periodontitis did not result in significant reductions in plaque accumulation [62,119]. Elsadek et al. recruited periodontitis patients with type 2 diabetes, and the most immediate followup data available for our analysis was nine weeks after ending the probiotic treatment [62]. Non-significant plaque reduction could be due to the fast turnover of the oral microbiome after probiotic treatment completion [116], although a recent meta-analysis conducted to assess the long-term efficacy of probiotics found that clinical improvements persisted for up to eleven months after the completion of probiotic treatment [47]. Our subgroup analysis based on probiotic treatment duration suggested that the beneficial effects of probiotics on clinical outcomes may occur within a month. However, the optimal treatment duration cannot be inferred from our meta-analysis because there were few studies of participants with a treatment duration of more than one month to two months.

Probiotic Mode of Delivery
Studies have suggested that probiotics may have different mechanisms of action to improve periodontal disease, including direct interaction with oral bacteria and/or indirect action in the oral cavity through the modulation of innate and adaptive immunity [114] and the gut-oral microbiome axis [120]. It has also been suggested that probiotics need to have enough contact time with the oral environment to become part of the biofilm and fight against pathogens through competition for binding sites, nutrients, and the production of anti-microbial substances [114,121]. Probiotics can also interact with immune cells, altering the production of inflammatory mediators, thereby modulating the immune response in the oral cavity [114]. Probiotics are also often administered to balance the gut microbiome [122]. A well-balanced gut microbiome regulates overall immune homeostasis [123,124]. The high count of periodontopathogens in periodontal disease may translocate to the gut resulting in gut microbiome dysbiosis [120]. Thus, it is plausible that probiotic supplementation may improve periodontal disease severity, at least in part, via this indirect mechanism linking the gut and oral microbiomes. Therefore, different modes of probiotic delivery may improve clinical outcomes based on the type of administration (oral, ingestion, or a combination of both). Interestingly, our meta-analysis indicated that probiotic administration via 'ingestion' only improved signs of tissue destruction (PPD and CAL). However, all clinical outcomes showed improvements with 'oral and ingestion' probiotic administration, potentially due to the combined direct and indirect mechanisms of action.

Probiotic Supplementation and Oral Hygiene Instructions
Poor oral hygiene leads to periodontal disease progression, recurrence, and treatment failure [125]. Oral hygiene instructions (OHI) play a key role in all stages of periodontal disease management. Educating patients on the importance of oral hygiene practices and techniques to ensure motivation to adhere to oral hygiene routines are critical components of OHI [125]. Our analysis showed that, irrespective of the oral hygiene instructions, probiotic supplementation improved GI, PPD, and CAL, but not plaque accumulation. Poor plaque control is a key determinant of periodontal disease [126]. Effective OHI can control and reduce plaque accumulation and improve the gingival health status, independent of professional prophylactic care [127,128]. Supporting this evidence, our findings indicated that probiotic supplementation combined with OHI resulted in statistically significant improvements in all clinical outcomes, including plaque accumulation. This suggests that probiotics may synergistically improve clinical outcomes with OHI, stressing the importance of OHI in effectively preventing/reducing plaque accumulation.

Probiotic Supplementation and GCF Volume
In the healthy gingival sulcus, the GCF flow and volume are lower compared to those of the inflamed gingival sulcus [25]. Increased GCF flow is a host defense mechanism to flush bacteria and their metabolites from the gingival sulcus [25]. Furthermore, the GCF content, including the microbial composition and concentration of inflammatory mediators, differs between periodontal disease and healthy individuals, as well as during disease progression and during and after treatment [129]. This is in line with findings from a study by Teles et al., who found a greater GCF volume and inflammatory mediators when comparing clinically healthy oral sites in periodontitis patients compared to healthy individuals. Their results suggested a higher risk of periodontal disease initiation and progression from healthy sites in patients with periodontitis [130]. Altogether, these findings indicate the potential clinical importance of the GCF volume and its level of inflammatory mediators as an early diagnostic test for periodontal disease prior to the onset of clinical manifestations. Based on our findings, the GCF volume was significantly decreased after probiotic supplementation in both gingivitis and periodontitis patients, and other clinical outcomes also improved in these patients [67,72,74,106]. Although our study did not find statistically significant differences between probiotic supplementation and GCF volume in healthy participants, it should be noted that each individual study included in our analysis reported that the GCF volume increased without probiotic supplementation, but not with probiotic supplementation [42,78]. Probiotics may potentially be of therapeutic value for the prevention and early treatment of periodontal disease. Due to the limited number of studies included in our analysis, our results need to be interpreted with caution until further studies fully ascertain the preventive role of probiotics in periodontal disease.

Probiotic Supplementation and Microbiological Outcomes
Our meta-analysis examined the effects of probiotic supplementation on subgingival bacterial counts in periodontal disease. Our results found statistically significant reductions in the periodontopathogens P. gingivalis and T. forsythia from the red complex and F. nucleatum from the orange complex after probiotic supplementation. Teles et al. reported that subgingival plaque samples from healthy sites in patients with periodontitis had a higher proportion of orange and red-complex bacteria compared to healthy individuals (without periodontitis), suggesting a higher risk for these sites to be colonized by periodontopathogens [130]. Hence, probiotics, by reducing periodontopathogens, may be beneficial in inhibiting the progression of periodontal diseases to healthy sites. Orange-complex bacteria create favorable conditions for colonization by red-complex bacteria, which are strictly anaerobic [131]. F. nucleatum plays a crucial role as a bridging organism between early and late colonizers, including red-complex bacteria [132]. Red-complex pathogens are higher in number in plaques found in deeper periodontal pockets and advanced lesions [20]. Although, in our meta-analysis, probiotic supplementation resulted in statistically significant improvements in PPD for both moderate and deep pockets, this improvement was more pronounced in deeper pockets. These findings and their potential ramifications may imply that probiotic supplementation has the potential to alter the relative abundance of orange and red-complex bacteria, thus reducing oral tissue destruction and leading to conditions favoring periodontal healing/stability.

Probiotic Supplementation and Immunological Outcomes
Our analysis of the effects of probiotic supplementation on immunological outcomes indicated that the GCF levels of MMP-8 and IL-6 were reduced after probiotic supplementation in patients with periodontal disease. Previously, MMP-8 and IL-6 were identified as salivary biomarkers that increased in periodontitis [133,134]. MMP-8 is the main collagenase involved in periodontal disease, with the highest collagenolytic activity in GCF [135]. A recent meta-analysis reported significantly higher levels of salivary MMP-8 in periodontitis patients compared to healthy participants [135]. Similarly, a study found that the GCF in the healthy sites of periodontitis patients had elevated inflammatory mediators, including MMP-8, when compared to healthy individuals [130], suggesting that inflammatory mechanisms may occur before they are clinically diagnosed or symptomatic. Moreover, additional evidence indicates that higher GCF levels of MMP-8 may predict periodontal disease progression [136], and that MMP-8 can also be measured as a grading biomarker to classify disease stage and progression in periodontitis [137].
IL-6 is an inflammatory mediator induced by pathogens and other pro-inflammatory cytokines, and has been reported to be elevated in periodontitis [138]. A recent metaanalysis concluded that the GCF levels of IL-6 are significantly increased in patients with chronic periodontitis [139]. However, there is conflicting evidence in the literature, with other studies suggesting that there is no correlation between the IL-6 levels and periodontal disease [140,141]. The proposed role of IL-6 in periodontal disease pathogenesis is through the stimulation of MMP production and the activation of pathways involved in inflammation [142]. Accordingly, there may be a link between the IL-6 levels and clinical parameters of periodontal disease. The pooled results of our meta-analysis showed statistically significant reductions in the GCF levels of IL-6 after probiotic supplementation, despite individual studies having differing results. Twetman et al. indicated that the use of L. reuteri probiotic gum reduced the GCF levels of IL-6 and numbers of sites with bleeding on probing [106]. Keller et al. did not observe any significant change in the GCF levels of IL-6 with other strains of Lactobacillus probiotics; however, BOP improved significantly compared to the baseline in both the probiotic and control groups [72]. Shetty et al. found a significant reduction in the GCF levels of IL-6 after non-Lactobacillus-containing probiotic supplementation in periodontitis patients compared to controls, but not clinical outcomes [95]. Further research is required to determine the potential effects of different probiotic formulations on inflammatory molecular pathways and their impact on the clinical aspects of periodontal diseases.

Probiotic Supplementation and Periodontal Disease Prevention
When examining the potential preventive role of probiotic supplementation in periodontal disease in healthy individuals, the results of our meta-analysis did not find any statistically significant improvements in PlI and GI after probiotic supplementation. There were insufficient studies to analyze the effects of probiotic supplementation on other outcomes in healthy participants to clearly assess the preventive potential of probiotics in periodontal diseases.

Strengths and Limitations
This is the first study examining the influence of probiotic formulation, mode of delivery, treatment duration, and the impact of oral hygiene instructions on periodontal diseases using subgroup analysis. Another strength of this study is that it used an up-todate analysis of the literature, used a systematic approach, and examined emerging factors that may assist in the prevention, diagnosis, and treatment of periodontal diseases.
A limitation of this meta-analysis was that the number of studies examining microbiological and immunological outcomes was scarce; thus, some of the results need to be interpreted with caution. We used Hedge's g standardized mean difference to provide better accounting for small study sample sizes. A further limitation was that, due to the aggregated data, we were unable to examine the effects of probiotic supplementation on periodontal diseases stratified by risk factors, such as sex, age, smoking status, and comorbidities. We were also unable to investigate the correlation between periodontopathogens and inflammatory biomarkers before and after probiotic treatment.

Conclusions
This systematic review and meta-analysis highlights the potential therapeutic benefits of probiotic supplementation in the treatment of periodontal disease. The results indicate that probiotic supplementation improved the clinical parameters, reduced the subgingival bacterial counts of specific periodontopathogens, and reduced the GCF levels of some proinflammatory mediators in periodontal disease patients. The impact of probiotic supplementation on clinical outcomes is affected by the probiotic formulation, mode of delivery, treatment duration, and the type of periodontal disease. More research is needed to better assess the therapeutic and preventive value of probiotic supplementation in patients with gingivitis (early disease), as well as in healthy (without periodontal disease) individuals.  Table S4. Overall measure of effects of probiotic supplementation on clinical, immunological, and microbiological outcomes. Subgroup analysis of the associations between probiotic supplementation and clinical outcomes in periodontal diseases patients. Supplemental Figure S1. Subgroup analysis-forest plots examining the effects of probiotic supplementation on clinical outcomes: 1. Plaque Index (PlI); 2. Mean Plaque Percentage (MPP); 3. Gingival Index (GI); 4. Probing Pocket Depth (PPD); 5. Clinical Attachment Level (CAL); 6. Bleeding on Probing (BOP); and 7. Gingival Crevicular Fluid (GCF).