Effects of Melatonin in the Non-Surgical Treatment of Periodontitis: A Systematic Review

Featured Application: Melatonin is a powerful antioxidant; it can bring numerous advantages to periodontal diseases, pursuing homeostasis, such as restoration of the concentration of antioxidants, reduction of periodontal inﬂammation with the regulation of inﬂammatory cytokines, reduction of oxidative stress, and signiﬁcant reduction of bone resorption through the modulation of osteoclastic and osteoblastic activities. It was considered a reliable and feasible option as an adjunctive to the classical NSPT, obtaining a signiﬁcative improvement of the periodontal parameters (PD, CAL, BOP, PI, and GI), a signiﬁcative reduction of the pro-inﬂammatory proteins (IL-1b, IL-6, and TNF-α ), and a better response for other biomarkers. Abstract: Background: Melatonin is a hormone produced by the pineal gland, an endocrine gland located at the base of the brain. It acts as a powerful antioxidant; it can bring numerous advantages to periodontal diseases, pursuing homeostasis, such as restoration of the concentration of antioxidants, reduction of periodontal inﬂammation with the regulation of inﬂammatory cytokines, reduction of oxidative stress, and signiﬁcant reduction of bone resorption through the modulation of osteoclastic and osteoblastic activities. Then, the goal of this integrative review was to evaluate the literature to better understand whether the use of melatonin is feasible to improve the non-surgical treat-ment of periodontitis. Methods: The integrative review was based on PICO strategy and PRISMA methodology. The focus question was: “Are there signiﬁcant beneﬁts in applying melatonin for the non-surgical treatment of periodontitis?” The PubMed, B-On, and Cochrane Library databases were enrolled, using the keywords melatonin, periodontal therapy, non-surgical treatment, and periodontitis, as associated with the Boolean connectors. The inclusion criteria were (i) CCT or RCT, (ii) adult population, (iii) full-text articles available, and (iv) in the last 10 years (2012–2022). The exclusion criteria were (i) animal studies, (ii) systematic review, and (iii) no other languages than English, Spanish, Portuguese, and Italian. A risk of bias was performed to assess the articles. Results: Initially, 2705 articles were identiﬁed. However, only six articles were included. From a total of 228 patients (109F and 119M) diagnosed with periodontitis, 22 patients dropped out (9.65%). The follow-up period varied between 8 weeks, 3 months, and 6 months. For clinical and molecular parameters, the melatonin group had signiﬁcant and greater improvement (intragroup) and better data than the control group, which also had favorable results. There was low risk of bias for all studies. Conclusions: Within the limitation of this study, melatonin is a reliable and feasible option as an adjunctive to the classical NSPT, obtaining a signiﬁcative improvement of the periodontal parameters (PD, CAL, BOP, PI, and GI), a signiﬁcative reduction of the pro-inﬂammatory proteins (IL-1b, IL-6, and TNF-α ), and a better response for other biomarkers.

The anti-inflammatory property of melatonin allows us, in fact, to eliminate ROS (exogenous and endogenous reactive oxygen species) and RNS (reactive nitrogen species), which are the causes of tissue damage [3]. In addition, melatonin also stimulates the synthesis of type I collagen fibers through the receptor located in pre-osteoblasts, which leads to the production, in these cells, of bone sialoprotein, alkaline phosphatase (ALP), osteopontin, and osteocalcin, significantly reducing the time required for their differentiation into mature osteoblasts, from 21 to 12 days [27].
Thus, the goal of this systematic review was to evaluate the literature, in order to verify if the use of melatonin improves the non-surgical treatment of periodontitis. The positive hypothesis was that there would be an improvement in the periodontal parameters when using melatonin for non-surgical periodontal therapy. In contrast, the null hypothesis was that no difference would be found using melatonin.

Protocol and Focus Question
This systematic study was conducted following the PRISMA guidelines, diagram methodology for article selection [28,29], with the focus question: "Are there significant benefits in applying melatonin for the non-surgical treatment of periodontitis?" PICO strategy was applied and presented according to Table 1, which shows the population (P), intervention (I), comparison (C), and outcomes (O).

Search Strategy
Based on the methodological objectives outlined, the electronic research was performed between 16 December 2021 and 20 February 2022 and carried out through three databases: Medline/PubMed, On-Line Knowledge Library (B-On), and Cochrane Library. It applied specific keywords associated with the Boolean connectors "AND" and "OR" to combine them, achieving a more significant number of articles: melatonin, methoxytryptamine, "periodontal therapy", "periodontal treatment", "non-surgical treatment", periodontitis, "clinical attachment level", "pocket depth", and "bleeding on probing".

Inclusion and Exclusion Criteria
The articles included were (i) clinical controlled trials and randomized controlled trials, (ii) adult population (females and males), (iii) full-text articles available, and (iv) within the last 10 years (2012-2022). The exclusion criteria were (i) in vitro and animal studies, (ii) systematic or narrative review, (iii) reports based on questionaries, interviews, and case series/report, and (iv) no other languages than English, Spanish, Portuguese, and Italian.

Selection of Articles and Data Extraction
The analysis was carried out independently by two reviewers (SP and FPO), and a third reviewer (FCC) helped in case of disagreement in selecting the articles to include. The results obtained were discussed by integrating the inclusion/exclusion criteria, analyzing the title and abstract, and after reading the full text. Duplicate articles were removed.
The main domains extracted from the included articles will be the following: author, type study, follow-up, sample size and sample characterization, clinical features, treatment done, adverse effects observed, and results found. All studies were randomized or controlled clinical trials that treated patients with NSPT and melatonin. A total of 228 patients (109F and 119M) diagnosed with periodontitis were included. Twenty-two patients dropped out (9.65%). According to the systemic conditions, two studies included only healthy patients, two others included patients with type-2 diabetes, and another included individual with primary insomnia, which is not related to any other systemic causes or drug intake, according to the International Classification of Sleep Disorders (ICSD-3).

Groups of Treatment: Control vs. Test
There were two groups of treatments in all studies, the control group and the test group. All patients received NSPT as a treatment modality. In all studies, the control group (CG) used a placebo [30][31][32][33][34][35]. Javid et al. [33] applied 250 mg per day of placebo tablets with the ingredients containing cellulose, silicon dioxide, magnesium stearate, All studies were randomized or controlled clinical trials that treated patients with NSPT and melatonin. A total of 228 patients (109F and 119M) diagnosed with periodontitis were included. Twenty-two patients dropped out (9.65%). According to the systemic conditions, two studies included only healthy patients, two others included patients with type-2 diabetes, and another included individual with primary insomnia, which is not related to any other systemic causes or drug intake, according to the International Classification of Sleep Disorders (ICSD-3).   Non-surgical periodontal therapy (NSPT); body mass index (BMI); clinical attachment loss (CAL); plaque index (PI); gingival index (GI); full mouth bleeding score (FMBS %); full mouth plaque score (FMPS %); Total Antioxidant Capacity (TAC); matrix of metalloproteinase (MMP); interleukin (IL), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx); high-sensitivity C-reactive protein (hs-CRP).      Table 5. Biochemical parameters found in the included studies (Part I).   Table 6. Biochemical parameters found in the included studies (Part II).

hc-CRP (mg/L) Final p-Value
Ahmed et al.

Groups of Treatment: Control vs. Test
There were two groups of treatments in all studies, the control group and the test group. All patients received NSPT as a treatment modality. In all studies, the control group (CG) used a placebo [30][31][32][33][34][35]. Javid et al. [33] applied 250 mg per day of placebo tablets with the ingredients containing cellulose, silicon dioxide, magnesium stearate, starch, and a few tastes of peppermint oil matching with the melatonin tablets for shape, color, and size, always administered one hour before bedtime for 8 weeks. Ahmed et al. [30] used placebo gel administration, whereas El-Sharkawy et al. [35] used a capsule, Tinto et al. [34] used 1 mg oral placebo capsules, and Bazyar et al. [32] used 6 mg placebo (2 capsules, 1 per/day).
All test groups received NSPT as a treatment associated with melatonin. Ahmed et al. [30] applied a 5% melatonin gel format for treatment. Bazyar et al. [32] and Javid et al. [33] used 250 mg daily, two tablets of sodium starch glycolate, magnesium stearate, and 3 mg net melatonin. The other three studies used capsules: Tinto et al. [34] treated with 1 mg of oral melatonin capsules; likewise, El-Sharkawy et al. [35] used 10 mg of oral melatonin capsules once daily before bedtime. Anton et al. [31] provided two melatonin tablets (250 mg) containing 3 mg of melatonin for 8 weeks.
For the control groups (CG), placebo, also there was a decrease in PD; therefore, the reduction was lower, compared with the test group: Tinto et al. [34] reported a similar statistical significance (p = 0.02) between groups, and the CG had 0.73 mm of reduction after 6 months; Ahmed et al. [30] had 0.9 mm after 3 months, a decrease of 22.5%; Anton et al. [31] and Bazyar et al. [32] had a similar result, with the reduction of 0.13 mm (p = 0.12), with statistical significance found between groups (p < 0.001) in 8 weeks and with 0.18 mm (p = 0.1), and with high difference, compared with the test group after 8 weeks; El-Sharkawy et al. [35] also had significant reduction of PD in 3 months of 1.3 mm (p < 0.001) and in 6 months of 1.4 mm (p < 0.001), and compared with a test group, a significant result was found with greater reduction for melatonin group, the difference in 3 and 6 months was 0.7 mm (p < 0.01).
CAL was not described by Tinto et al. [34]. Nevertheless, this parameter had significant and higher results for the melatonin group, with a reduction in improvement varying by 1.2-2.2 mm (p < 0.001). The differences found for the control group were lower, but significant, between 0.04-1.3 mm.

Biomarkers
The main biomarkers associated with periodontal diseases were also evaluated. IL-1beta was only reported by Javid et al. [33]. There was a statistically significant result only for the test group (p = 0.008), with values from 2.41 ± 0.55 pg/mL (baseline) to 2.06 ± 0.48 pg/mL after 8 weeks. IL-6 was reported by Bazyar et al. [32], also with significant results found in the test group: 2.0 ± 0.92 pg/mL to 1.42 ± 0.73 pg/mL (p = 0.008). Bazyar et al. [32] also reported the analysis of tumoral necrosis factor-alpha (TNF-α), with significative results reported only for the test group, 9.05 ± 3.56 pg/mL at baseline and 8.24 ± 3.45 pg/mL after 8 weeks (p = 0.1). Another important parameter found was reported by Ahmed et al. [30], who assessed the levels of the matrix of metalloproteinases-9 (MMP-9); the authors found significant reductions for both groups, the melatonin, and the placebo, with a significant statistical result (p < 0.001). Other biomarkers were reported and detailed in Tables 5 and 6.
One article [35] included patients with an Athens insomnia scale (AIS) score ≥ 6, provided that individuals have primary insomnia, which is not related to any other systemic causes or drug intake, according to the International Classification of Sleep Disorders (ICSD-3); AIS is an anatomically-based injury severity scoring system.

Risk Assessment
The risk of bias was evaluated, and all studies had a low level of bias. Only two unclear data were found in Anton et al. [31] and one in Ahmed et al. [30], which did not interfere with the final result ( Figure 2). The low level for RoB found can be interpreted as the included studies transmit trustworthy and reliable data.

Risk Assessment
The risk of bias was evaluated, and all studies had a low level of bias. Only two unclear data were found in Anton et al. [31] and one in Ahmed et al. [30], which did not interfere with the final result ( Figure 2). The low level for RoB found can be interpreted as the included studies transmit trustworthy and reliable data.

Discussion
This integrative review aimed to analyze the most robust literature (including just controlled and randomized clinical trials) to ground the use of melatonin as an adjuvant in NSPT. Six articles were included to answer the focus question, "are there significant benefits in the application of melatonin for the non-surgical treatment of periodontitis?" Therefore, one of them [33] had the same cohort of patients as Bazyar et al. (2019) [32]; nonetheless, it was considered, due to reports of different findings.
Periodontal medicine terminology has stimulated research to improve the connection between oral and systemic conditions, bringing the interdisciplinary medical approach to patients with systemic and periodontal diseases [36]. Thereby, melatonin has been considered a relevant antioxidant, anti-inflammatory, and immunomodulatory function, beyond exerting a powerful endogenous effect as a free-radical scavenger [37], being a key molecule for periodontal protection. In addition, melatonin can protect cells from oxidative stress and neutralize up to ten molecules of reactive oxygen species (ROS), in contrast to classical antioxidants that neutralize only one molecule [38], which can be hypothesized as a reduction of oxidative stress through its application, which would be effective in the treatment of diabetes and periodontal diseases [39]. Moreover, it plays a crucial role in periodontal homeostasis by preventing and preempting periodontal destruction [14,26]. Additionally, melatonin administration can either be topical (mouthwash or gel) or systemic.

Proteins Analysis
Bone resorption enrolls osteoclasts that secrete various molecular agents for bone degradation, mainly TNF-a, IL-1b, MMP, and free radicals. Furthermore, monocytes, macrophages, neutrophils, and lymphocytes also accumulate on the site, due to chronic inflammatory processes, which are stimulated to produce free radicals and liberate many types of mediators. Then, melatonin might be an adequate adjunctive therapy associated with NSPT to bring and improve treatment, limiting tissue destruction [14]. In addition, Arabaci et al. [26] obtained excellent results in the melatonin group studying alveolar bone resorption in rats with periodontitis.
It was reported that melatonin reduces the synthesis of prostaglandins (PGE-2) and the up-regulation of a variety of pro-inflammatory cytokines [40], inhibits the adhesion of leuko-cytes to endothelial cells [41], and attenuates transendothelial cell migration [42]. It also stimulates the release of IL-2 [43] and IL-6 [44]. Additionally, melatonin can inhibit acute inflammatory reactions [42]. Various proteins were analyzed in the included studies, such as IL-6, TNF-α, hs-CRP, MMP-9, IL-1b, MDA, and others. Regarding El-Sharkawy et al.'s [35] assessment, TNF-α level and AIS scores, at 3 and 6-month, a statistically significant reduction in these two parameters was observed in the melatonin group (p < 0.01), which is in agreement with Cutando et al.'s study [45], who obtained a reduction (in serum) of TNF-α and other pro-inflammatory cytokines, following topical application of melatonin; in contrast, Bazyar et al.'s study [32] did not report statistically significant results for TNF-α reduction; there was no improvement, probably due to different samples/period of administration. Bazyar et al.'s [32] values coincided with studies conducted by Kara et al. [46] and Köse et al. [47].
On the other hand, Bazyar et al. [32] obtained a significant reduction in the inflammatory biomarkers IL-6 and hs-CRP (p = 0.008 and p = 0.017, respectively) in the intervention group. At baseline, IL-6 for the control and intervention groups were, respectively, 2.16 ± 0.91 pg/mL and 2.0 ± 0.92 pg/mL, in contrast to the results obtained after 8 weeks in the intervention group, which showed a statistically significant decrease of 1.42 ± 0.73 pg/mL, compared to the control group 2.08 ± 0.87 pg/mL, which had no statistical significance.
Ahmed et al. [30] considered the level of MMP-9 (collagenase), which has significantly involved in connective tissue destruction. After melatonin treatment, compared with the control site, there was a statistically significant reduction in the MMP-9 of −39.25 ± 16.46% (p < 0.001). That result was supported by Rudra et al. [48], which highlighted the effect of melatonin on MMP-9, in association with other diseases, such as rheumatoid arthritis, atherosclerosis, gastric ulcer, tumor growth, and cancer metastasis, proving significant inhibition of MMP-9 activity in a dose-and time-dependent manner. Other approaches to inhibit MMP-9 expression have been studied, such as developing reversible peptidomimetic hydroxamate inhibitors, concluding with negative results, due to a lack of specificity and toxic effects in physiological systems. This obstacle can be overcome through the medical delivery of melatonin.
Javid et al. [33] evaluated the intervention group's serum levels of IL-1b and MDA. Compared with the baseline results, there were decreased statistically significances (IL-1b with p = 0.008 and MDA with p < 0.001, respectively). At the same time, serum levels of SOD, GPx, CAT, and TAC significantly increased in the melatonin group. A significant increase in TAC (p < 0.001) was also observed by Ahmed et al. [30]. There is a close relationship between diabetes and periodontitis; diabetes increases the oxidative stress level that affects insulin secretion and action, accelerates the progression of the periodontitis, and, consequently, increases ROS and MDA.

Clinical Parameters
Practically all studies reported statistically significant results on the effect of melatonin on periodontitis after NSPT. All six included studies that were considered the basic clinical parameters (PD and CAL). Some studies also considered GI, PI, and BOP. There was a positive overall result with different melatonin concentration levels and follow-up, thus improving the periodontal parameters for the control and melatonin groups, with statistically significant results for the test group.
The systemic use of melatonin was used by El-Sharkawy et al. [35], Bazyar et al. [32], Tinto et al. [34], Javid et al. [33], and Anton et al. [31]. According to Balaji et al. [38], the systemic administration of melatonin was more effective than topical (mouthwash or gel). This may be explained by the GCF (gingival crevicular fluid, periodontal defense fluid) presence, which is an exudate composed of plasma and transports molecules from the bloodstream. The clinical results obtained were better in these studies, supporting the use of melatonin capsules as an auxiliary in treating periodontitis.
For all studies, PD levels statistically significantly decreased, and a considerable gain in CAL was also obtained. Tinto et al. [34] observed that the results were significant for the melatonin groups for 4-5 mm and >5 mm PD sites (p < 0.001).
Anton et al. [31] evaluated and correlated BOP and HbA1c. Plaque index (PI) and BOP decreased, after systemic administration of melatonin (patients received two melatonin tablets 250 mg that contained 3 mg of melatonin), showing significantly lower values. There was also a significant reduction in HbA1c during the studies, which was suggested to be matched to the NSPT. Still, a significant decrease was shown in the melatonin-treated group, compared to the control group.
A couple of studies used patients with T2DM and periodontal disease [31][32][33]. Ostadmohammadi et al. [49], in addition to the HbA1c, also detected the value of serum insulin levels. The results on the effectiveness in glycemic control were also exposed in the study. Thereby, Anton et al. [31] confirmed the veracity of the previous studies, showing that melatonin acted on periodontal parameters and had a relevant effect on glycemic control. Some divergent results from the studies can probably be attributed to the sample studied, follow-up, and the different dosages of melatonin. Javid et al. [33] analyzed the effect of melatonin, in addition to NSPT in patients with T2DM and periodontal disease. The intervention group consisted of 25 patients who received 2 tablets daily of 250 mg for 8 weeks. The results showed that melatonin intake significantly changed various clinical parameters.
All the studies included supported that the use of melatonin helped the NSPT. In contrast, Konečná et al.'s study [51] instructed patients to use 20 mL of melatonin, 5 mg/mL solution, to rinse the oral cavity before sleep and after brushing their teeth for 14 days, without having been subjected to the NSPT. The patients were observed one year after the two-week melatonin treatment and showed no improvement. In fact, the scored parameters and oxidative stress markers did not change. The probable causes of the study failure may depend on various factors, such as the presence of refractory periodontitis that was not assessed, the type of administration (oral rinse), the duration of treatment, and the concentration of melatonin administered. Mouthwash administration is a topical treatment; consequently, its effect depends on the volume and speed of absorption of melatonin from the oral mucosa and the time the patients take to rinse their mouths. In that study, the better and correct way of using mouthwash was not reported to the patients. For the mouthwash acts, the duration of rinsing and the contact time with the oral mucosa must allow the melatonin to be adequately absorbed by the tissues and, thus, produce effects. Indeed, further studies should be carried out to confirm the efficacy of melatonin in the mouthwash because a simple rinse that lasts on average 30 to 40 s or less can probably never give the best time to absorb all melatonin [52].
In another study by Ahmed et al. [30], the test group was treated by NSPT, followed by intrapocket application of 5% melatonin gel (topical administration) with a plastic disposable syringe. The results were positive, in terms of improvement of periodontal parameters, with a reduction in PD of −32.56% (test group) vs. −22.5% (control), while the percentage of CAL was −25.53% in TG and −13.95% in GC, demonstrating that melatonin treatment had significative effects. Although the administration was topical in this study, it was denoted that the intrapocket application gel had a good molecule propagation into the tissue, probably due to the melatonin's direct contact with the affected area, allowing it to have optimal diffusion. Otherwise, other studies that used systemic melatonin also had significant results and a high percentual of improvement, which did not permit us to say that one method is superior to another.
Regarding the bone-melatonin relationship, studies conducted by Montero et al. [53], Calvo-Guirado et al. [54], Gómez-Moreno et al. [55], Arabaci et al. [26], and Cutando et al. [6], in addition to the report of bone formation improvement, found increased osteoblast differentiation. In particular, Cutando et al. [6] showed that, at 2 weeks of melatonin placement (1.2 mg lyophilized powdered melatonin) in implants, all parameters of osseointegration significantly increased: percentage of bone-implant contact, total peri-implant bone, and percentage of new bone formation. Furthermore, osteoclasts generate high levels of superoxide anions during bone resorption that contribute to the bone degradative process. Melatonin, an important free radical scavenger and a strong antioxidant, eliminate osteoclasts' free radicals during bone resorption by protecting cells from oxidative attacks [55][56][57].

Adverse Effects
The use of melatonin has led researchers to verify whether the melatonin regimen is safe, with minimum or no adverse effects. Only two out six articles studied found mild adverse effects: Tinto et al. [34] reported sleepiness (20%) and headache (10%), which were resolved spontaneously after a few days; and El-Sharkawy et al. [35] also had mild adverse reactions, presenting headache, dizziness, nausea, constipation, diarrhea, or abdominal cramp. Otherwise, El-Sharkawy et al. [35] concluded that daily oral melatonin intake before bedtime improved sleep quality and daily life activities for insomniac patients by using melatonin.

Limitations of the Study
A low number of articles (controlled or randomized) were found, reducing the impact of the study. Additionally, there was a standard of the parameters analyzed, which did not permit the development of a meta-analysis. Some studies included patients with the disease, such as type-2 diabetes, which brought a variable that might impair the possibility of comparison with healthy patients. Moreover, there was no standard for posology and administration pathway, nor a standard to recall the patients and period of intake or use of the melatonin.

Conclusions
Within the limitation of this study, melatonin is a reliable and feasible option as an adjunctive to the classical NSPT, obtaining a significative improvement of the periodontal parameters (PD, CAL, BOP, PI, and GI) and a significative reduction of the pro-inflammatory proteins (IL-1b, IL-6, and TNF-α) and better response for other biomarkers. Therefore, further clinical studies must be performed, following a standard period and dosage, with a higher number of patients, in order to demonstrate and confirm the significant results found.

Conflicts of Interest:
The authors declare no conflict of interest.