Does melatonin gel improve alveolar preservation? A triple-blind randomized controlled clinical trial

Abstract

Background:

Preserving ridge volume after tooth extraction is essential for predictable implant outcomes. Various techniques and biomaterials have been proposed to minimize post-extraction dimensional changes. Melatonin, a pineal hormone, is an important mediator of new bone formation. The effect of melatonin gel incorporated into a porcine xenogeneic collagen sponge in post-extraction sockets has not been evaluated in humans, making this trial novel.

Purpose:

To assess the effect of melatonin gel incorporated into a collagen sponge on intra-socket bone formation and tissue characteristics in post-extraction sockets.

Materials and methods:

Twenty participants requiring extraction of premolars or molars with adjacent teeth were randomly allocated to test (n = 10) and control (n = 10) groups. After extraction, sockets were filled with xenogenic collagen sponge containing either melatonin gel (1.2 mg; test) or placebo gel (control). Ninety days later, during implant placement, bone samples were harvested using a trephine drill. Samples were processed for histomorphometric analysis of newly formed bone and connective tissue. Secondary outcomes included descriptive microscopic histology, quantitative microcomputed tomography (µCT) analyses and intra-alveolar volumetric analysis.

Results:

Histomorphometry revealed significantly greater bone density in the test group (44.0%) than in the control group (35.4%; p = 0.0099), with a mean difference of 8.6% ± 3.2%. Microscopic evaluation showed increased osteoblastic activity in the test group. Quantitative µCT analysis confirmed more new bone formation in the test group, although qualitative bone characteristics did not differ between groups. The intra-alveolar bone volume formed was numerically higher in the test group than in the control group.

Conclusion:

Incorporating melatonin gel into a collagen sponge enhanced bone repair in human post-extraction sockets over 90 days. This clinical trial is registered at the Brazilian Clinical Trials Registry (ReBEC; https://ensaiosclinicos.gov.br): RBR-67jkxsj.

Keywords

melatonin new bone formation placebo-controlled randomized clinical trial

Introduction

Maintaining ridge volume after tooth extractions is crucial for achieving success and predictability in dental implant treatments. The alveolar preservation procedure, performed at extraction, aims to minimize alveolar ridge resorption and maximize bone tissue formation within the socket.¹

Numerous techniques and biomaterials, with or without biological barriers, have been proposed to mitigate dimensional changes in ridges after extractions.^2–7 Studies indicate that the complete preservation of alveolar dimensions is unlikely after tooth extraction.⁷ While many techniques and materials can limit or reduce dimensional changes, the quality of the new tissue formed within the socket can vary greatly.²

Melatonin is a hormone synthesized by pinealocytes in the pineal gland. Its precursor is the essential amino acid tryptophan, which is transformed into N-acetylserotonin through an enzymatic cascade and then into melatonin (N-acetyl-5-methoxytryptamine) by hydroxyindole-O-methyltransferase (HIOMT).⁸ As an antioxidant, melatonin detoxifies free radicals produced during osteoclastogenesis, inhibiting bone resorption.⁹ It also promotes the formation of new bone, as it can suppress the activity of the receptor activator of nuclear factor kappa-B ligand (RANKL) pathway by downregulating RANKL-mediated osteoclastic formation and activation.^10–13 Moreover, it directly affects osteoblasts by increasing type I collagen secretion and the expression of bone sialoprotein, alkaline phosphatase, and osteocalcin.¹³

Previous in vitro studies have shown melatonin to be an important mediator of bone formation and stimulation, promoting osteoblast differentiation,¹⁴ and in vivo studies have demonstrated melatonin’s ability to induce new bone formation.^{9,11,12,15–17}

Applying melatonin-impregnated collagen sponges in post-extraction sockets of dogs accelerated bone tissue formation in the early stages of healing, stimulating bone maturation. Post-extraction sockets that received melatonin exhibited increased osteoblast proliferation, and early cell differentiation significantly accelerated the synthesis and mineralization of the osteoid matrix.¹² Two preclinical studies have examined the effectiveness of locally applying melatonin in post-extraction sockets to induce new bone formation and reduce oxidative stress.^12,15 In one study, the upper and lower premolars and molars were extracted in 16 Beagle dogs under general anesthesia. The post-extraction sockets and gingival tissue surrounding the surgical area were treated with 2 mg of melatonin (powder) in eight dogs and left untreated in the other eight dogs. In blood samples collected 24 h after the surgical procedure, oxidative stress was lower in the melatonin-treated socket group, suggesting the direct action of melatonin as a free radical neutralizer.¹⁵ In the other study in dogs, the post-extraction sockets filled with collagen sponges impregnated with melatonin gel showed significantly greater new bone formation than the unfilled post-extraction sockets. This study concluded that the topical application of melatonin stimulated greater bone maturation over the 60-day observation period.¹²

Another preclinical study in dogs demonstrated that locally applying a layer of lyophilized melatonin powder (1.2 mg) before implant placement increased new bone formation compared to no prior treatment (after 8 weeks: 90.71% vs 88.08%, p < 0.05).⁹ In a randomized clinical study, locally applying melatonin gel (1.2 mg/mL) before implant placement also showed favorable bone formation and growth with no reported side effects.¹⁸

Both preclinical¹⁹ and clinical^20–22 trials have examined the effect of melatonin in periodontal disease when associated with non-surgical treatment. Applying the melatonin gel in intraosseous defects provided better clinical and radiographic results,¹⁹ and daily melatonin supplementation reduced probing depth, clinical attachment loss,^21,22 and dental root exposure.¹⁹

Two randomized clinical studies have verified the osteogenic action of local melatonin application (3 mg in 2 mL of hydroxyethylcellulose gel 2%) in post-extraction sockets of third molars, showing no significant difference in bone density compared to the control group at experimental periods of 60 days²³ and 6 months.²⁴

The use of melatonin gel impregnated in a porcine xenogeneic collagen sponge in post-extraction tooth sockets has never been studied in humans. Therefore, this clinical trial aimed to verify, through clinical and quantitative analysis of micro-computed tomography (μCT) and histomorphometry, the dimensional changes and newly formed tissues in post-extraction tooth sockets filled with melatonin gel impregnated in a collagen sponge. Its results are novel, contributing to the limited evidence from clinical studies on the impact of melatonin on bone formation in post-extraction tooth sockets.

Materials and methods

Study design and setting

This triple-blind randomized controlled trial was approved by the Ethics Committee of the Galeão Air Force Hospital (Rio de Janeiro, RJ, Brazil), Brazilian Air Force (FAB), through the Brazil Platform System (judgment number: 4.868.960). It adhered to the principles in the Declaration of Helsinki and is reported according to the Consolidated Standards of Reporting Trials (CONSORT) guidelines to ensure its quality and transparency.²⁵ All research participants voluntarily signed the informed consent form. The study flowchart is shown in Figure 1.

Figure 1.

Flowchart diagram of the study protocol according to Consort guideline.

Study population

This trial included healthy individuals aged >18 years who had an indication for the extraction of premolar or molar teeth from any arch with immediately adjacent teeth. The exclusion criteria were: (i) acute periapical/periodontal infection (abscesses); (ii) severe systemic diseases or the use of medications such as chemotherapy, anticoagulants, corticosteroids, antiresorptive drugs (bisphosphonates or RANKL modulators), and immunosuppressive drugs; (iii) systemic use of melatonin; (iv) uncontrolled chronic diseases (e.g. hypertension, diabetes, rheumatologic, renal, and liver diseases); (v) metabolic bone diseases (osteomalacia, hypocalcemia, and hypercalcemia); (vi) any motor dysfunction that impairs or hinders oral hygiene; (vii) smokers, with ex-smokers required to have been smoke-free for at least 6 months; (viii) pregnant women; and (ix) radiotherapy within the last 5 years.

Randomization and formulation of test and control gels

The randomization method used was a simple coin toss (heads or tails): heads determined treatment with Gel A, and tails determined Gel B. Randomization was performed for each participant after eligibility confirmation and informed consent signature. The randomization code linking gel labels (A or B) to melatonin or placebo was held exclusively by a single researcher who was not involved in surgical procedures, patient recruitment, or outcome assessments. This researcher maintained code confidentiality until completion of all data collection and statistical analyses.

The operators (I.C.C.K. and R.B.), the researchers involved in the μCT and histomorphometric analyses, and the study participants were blinded to the gel type (A or B) used in the sockets.

Triple-blind procedures were implemented as follows: (1) Gel preparation and labeling were performed by an independent compounding pharmacy (Bem Viver); purity analysis was conducted at National Institute of Metrology, Quality, and Technology (INMETRO; Rio de Janeiro, RJ, Brazil). (2) Blinding was maintained at all stages: surgeons (I.C.C.K. and R.B.) and participants were blinded during the surgical phase; the evaluator performing histomorphometric analysis (I.C.C.K.) was blinded; the evaluator performing microscopic descriptive analysis (A.T.N.N.A.) was blinded; and the evaluators performing µCT analysis were blinded (C.M.S.F.F.S.). (3) Pre-coded syringes containing 0.1 mL of gel were used. During outcome assessment, samples and imaging files were identified only by sequential participant number (P1–P20) without group designation. Unblinding occurred only after completion of all data collection and statistical analyses.

The melatonin gel (Gel A) was prepared by mixing lyophilized melatonin powder at 1.2 mg^18,26 with 1.5% carboxymethylcellulose (carrier agent). This mixture achieved a fluidity that allowed it to be easily incorporated into the collagen sponge. The control gel (Gel B) comprised only the carrier agent (1.5% carboxymethylcellulose). The gels were formulated in a single pharmacy (Bem Viver Homeopathy and Compounding Pharmacy, Niterói, RJ, Brazil; National Health Surveillance Agency authorization no. 7.32332-0) and subjected to purity analysis at the INMETRO. The gels had identical color, texture, and viscosity, identified only as Gels A and B, ensuring the blinding of the gel contents to the patients and researchers involved in the surgical procedure and evaluations, establishing a triple-blind trial.

Clinical procedures

All participants, recruited according to the eligibility criteria, underwent basic periodontal therapy and oral hygiene instructions. None had risk factors for bacterial endocarditis. The tooth extractions were performed under local anesthesia (2% mepivacaine with epinephrine (1:100,000, Mepiadre); Nova DFL, Rio de Janeiro, RJ, Brazil) and in a minimally traumatic manner to preserve the alveolar bone ridges and periodontal tissues as much as possible. The sockets were carefully inspected and curetted to remove any possible fibrosis, granulation, and/or soft tissue, followed by copious irrigation with sterile saline solution.

After the atraumatic extractions, the sockets were filled with xenogeneic collagen sponges (Hemospon^® Cube; Maquira, Maringá, PR, Brazil) impregnated with Gel A (1.2 mg in 0.1 mL) or Gel B (0.1 mL), following the randomization. Once the sockets were filled with the impregnated xenogeneic collagen sponges, an “X” suture was performed using 5.0 synthetic absorbable thread (Vycril; Johnson & Johnson, New Brunswick, NJ, USA) to promote hemostasis and keep the collagen sponge in place.

The postoperative medication regimen consisted of amoxicillin (875 mg) with clavulanic acid (125 mg) every 12 h for 7 days (Amoxicillin Trihydrate + Potassium Clavulanate; Eurofarma, Itapevi, SP, Brazil). For the one participant allergic to penicillin, clindamycin (300 mg) was prescribed every 8 h for 7 days (Clindamycin Hydrochloride (300 mg); TEUTO, Anápolis, GO, Brazil). All participants were instructed to use a 0.12% chlorhexidine digluconate solution (PerioGard^®; Colgate Palmolive Industrial, São Bernardo do Campo, SP, Brazil) without vigorous rinsing, twice daily, half an hour after brushing, for 15 days, and to maintain a diet free of hard or sticky foods. Follow-up appointments were scheduled in the first week for suture removal and the second and fifth weeks thereafter.

Sample preparation

Ninety days after the dental extractions prior to implant placement (Epikut^® or Epikut Plus^®; SIN Implant System, São Paulo, SP, Brazil), a bone cylinder measuring 2 × 6 mm was removed using a 2 mm diameter trephine bur (WF Cirúrgicos, Barueri, SP, Brazil).

Intra-alveolar volumetric assessment

Cone-beam computed tomography (CBCT) scans were obtained at two time points using the same device and standardized parameters (Orthophos SL, Sirona Dental Systems, Charlotte, NC, USA; 85 kVp, 7 mA): T1 (immediately post-extraction, within 7 days) and T2 (90 days post-extraction, prior to implant placement).

Images were exported in DICOM format, and intra-alveolar volume was determined by semi-automatic segmentation using ITK-SNAP (version 3.8; Cognitica, Philadelphia, PA, USA). The region of interest was delimited to the smallest square area containing the entire socket, respecting limits observed in axial, sagittal, and coronal planes. The “Segmentation 3D” tool generated a 3D model, and the volume (mm³) was obtained using the “Volumes and Statistics” function. The bone volume formed was calculated as the difference between intra-alveolar volumes at T1 and T2.

To verify measurement reliability, 15% of participants (n = 3) had their CBCT scans (T1 and T2) evaluated three times by the same blinded operator (I.C.C.K.), with 1-week intervals between measurements. Variation was <10%, confirming acceptable intra-examiner reliability. Two CBCT files (one per group) were corrupted during data transfer and could not be recovered, reducing the volumetric analysis to n = 9/group.

Histomorphometric and histology analyses

The bone cylinders obtained were preserved individually in 3.7% formaldehyde for 48 h and then submitted for standard histological processing at the Applied Biotechnology Laboratory (LABA-Histology) of the School of Dentistry at the Fluminense Federal University (FOUFF). After dehydration and paraffin embedding, 5 µm sections were cut and stained with hematoxylin and eosin (HE). For histomorphometric analysis, images were captured using a 20× objective on a binocular bright-field light microscope (Olympus BX43; Olympus Corporation, Tokyo, Japan). The selected images were digitized with a digital camera using CellSens software (Olympus Corporation, Tokyo, Japan) and transferred to the Image Pro-Plus software (version 7; Media Cybernetics, Rockville, MD, USA). Quantitative histomorphometric analysis of newly formed bone tissue and connective tissue was performed based on the area of the evaluated field, expressed as a percentage per unit area (volume density). A counting grid containing 200 points was superimposed on the images, and the results were transferred to Microsoft Excel^® (Seattle, WA, USA) for statistical analysis. The same blinded evaluator (I.C.C.K.) performed all quantitative histomorphometric analyses. Histological descriptions and analyses were conducted to observe the cells in the samples, including inflammatory infiltrate, osteoblastic lining, multinucleated giant cells, newly formed bone tissue, connective tissue, granulation reaction, and necrotic areas. The same blinded evaluator (A.T.N.N.A.) conducted the microscopic analyses at the Associated Laboratory for Clinical Research in Dentistry (LPCO, FOUFF).

X-ray μCT

The μCT scans were performed using a SkyScan 1275 micro-CT scanner (Bruker, Kontich, Belgium) with settings of 65 kV, 80 mA, and isotropic pixel size of 8.5 μm (0.0085 mm), following published guidelines.²⁷ In order to obtain the tomographic slices of interest, the sample’s images were repositioned along their longitudinal axis in the DataViewer software (Bruker) until its entire extent was fully visible. Then, the sagittal slice view was exported to the CTAn software (Bruker), where the three-dimensional region of interest (ROI) involving only the area of new bone formation was selected. Quantitative morphometric analysis used the following parameters (Figure 2): bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N).

Figure 2.

(a–d) 3D sequence of the selection of the ROI involving only the area of new bone formation (in red).

Sample size and statistical methods

An a priori sample size calculation for a superiority design with a continuous primary outcome was performed using the Sealed Envelope online platform (https://www.sealedenvelope.com/power/continuous-superiority/). Estimates were derived from Kutkut et al.,²⁸ who evaluated histomorphometric new bone formation in post-extraction sockets. Parameters used were: significance level (α) = 0.05 (two-sided), power (1 − β) = 95%, expected mean in control group = 38.3%, expected mean in experimental group = 65.5%, and standard deviation (σ) = 15. The required sample size was eight participants per group (total n = 16). To account for potential dropout, a 10% attrition adjustment was applied, yielding a final target of 10 participants/group (total n = 20). Effect sizes were calculated using Hedges’ g, which accounts for small-sample bias, along with 95% confidence intervals.

Results

Participants’ characteristics and interventions

All participants were recruited from the Aeronautical Health System of the FAB, and the procedures were performed at the Santos-Dumont Aeronautical Dental Clinic (FAB, Rio de Janeiro, RJ, Brazil) by two blinded operators (I.C.C.K. and R.B.). Of the 20 participants (11 men and nine women) selected, none dropped out. They were aged 28–74 years (mean: 50.75 years). Of the 20 teeth involved (eight premolars, 12 molars), 13 were in the maxilla, and seven were in the mandible (Table 1). There were no postoperative complications, and tissue healing proceeded within normal limits. All 20 participants had implants (Epikut^® CM or Epikut Plus^® CM; SIN Implant System, São Paulo, SP, Brazil) placed, and the reopening to install the healing screw was performed at the appropriate time. All participants are either fully rehabilitated or in the final stages of definitive crown placement.

Table 1.

List of research participants according to gender, age, tooth, and experimental group.

Participant (P)	Gender	Age	Tooth	Group
1	Female	26	Upper left first molar^a	Test
2	Female	62	Upper right second premolar^b	Control
3	Male	49	Upper right first molar^a	Control
4	Male	49	Lower right second premolar^a	Control
5	Female	28	Lower left first molar^a	Control
6	Male	50	Upper left first premolar^a	Control
7	Male	39	Upper right first molar^c	Test
8	Male	31	Upper left first molar^a	Control
9	Female	56	Upper left first molar^a	Test
10	Female	34	Upper left first premolar^a	Test
11	Female	59	Lower left second molar^a	Test
12	Male	58	Upper left first molar^a	Test
13	Male	74	Lower left first molar^c	Control
14	Female	51	Lower left second premolar^a	Test
15	Male	55	Upper right first dental^b	Control
16	Female	37	Upper left first premolar^a	Test
17	Male	66	Lower left first molar^a	Control
18	Male	55	Lower right first molar^b	Test
19	Female	69	Upper right second premolar^b	Test
20	Male	51	Upper right second premolar^b	Control

Caries.

Tooth fracture.

Root resorption.

Intra-alveolar volumetric analysis

The intra-alveolar bone volume formed (T1−T2 difference) was numerically higher in the test group (242.9 ± 143.7 mm³) than in the control group (210.5 ± 118.2 mm³). However, due to high standard deviations, attributable to anatomical variability between single-rooted and multi-rooted teeth and between maxillary and mandibular sites, this difference did not reach statistical significance. The volumetric data are presented as a secondary, supportive outcome alongside the primary histomorphometric analysis.

Histomorphometric analysis

Bone density was significantly greater in the test group (44.0% ± 8.1%) than in the control group (35.4% ± 6.7%; p = 0.0099), with a mean difference of 8.6% (95% CI: 2.3%–14.9%). The standardized effect size was large (Hedges’ g = 1.14; 95% CI: 0.18–2.10). In contrast, connective tissue volume density was significantly greater in the control group (64.6%) than in the test group (56.1%; p = 0.0098), with a mean difference of 8.6% ± 3.2%. For connective tissue, the effect size was similarly large (Hedges’ g = 1.13; 95% CI: 0.17–2.09). These findings suggest that, under these experimental conditions, the melatonin gel incorporated into Hemospon^® collagen sponges promoted an ~8.6% increase in bone tissue (p = 0.0099) compared to the inert gel (Figure 3 and Table 2).

Figure 3.

Effect of melatonin gel incorporated into Hemospon^® (collagen sponge) on bone formation in the sockets 90 days after tooth extraction compared to bone formation with the gel without melatonin incorporated into Hemospon^®: bone tissue density in the test group (melatonine) was significantly higher (44%) compared to the control group (35.4%), p = 0.0099** and connective tissue density in the control group was significantly higher (64.6%) compared to the test group (melatonine) (56.1%), p = 0.0098**.

Table 2.

Histomorphometry data. Percentage of bone tissue density and connective tissue density in the control and test groups.

Tissue volume density (%)
Bone tissue		Connective tissue
Control group	Test group	Control group	Test group
39	35	61	65
28.5	61.5	71.5	38.5
31.5	46	68.5	54
30.5	47	69.5	53
39	25	61	75
22	39.5	79	60.5
11	44.5	89	55.5
20.5	84	79.5	16
20	57.5	80	42.5
54.5	33.5	45.5	66.5
31	40.5	69	59.5
17	19.5	83	80.5
35.5	37	64.5	63
47	37	53	63
40.5	46.5	59.5	53.5
41.5	57	58.5	43
42.5	24.5	57.5	75.5
41.5	39.5	58.5	60.5
48	55.5	52	44.5
57	58	43	42
16.5	47.5	83.5	52.5
44	13	56	87
7.5	45	92.5	55
45	40.5	55	59.5
37	54.5	63	45.5
42.5	28.5	57.5	71.5
11	53.5	89	46.5
43.5	49.5	56.5	50.5
59	55	41	45
40	54.5	60	45.5
45.5	51	54.5	49
30.5	72	69.5	28
44	41	56	59
45	37.5	55	62.5
37.5	24.5	62.5	75.5
27.5	37	72.5	63
-	31.5	-	68.3
Mean (SD): 35.4% (13)	Mean (SD): 44% (14.4)	Mean (SD): 64.6% (13.1)	Mean (SD): 56.1% (14.4)

SD: standard deviation.

p = 0.0099. p = 0.0098.

Histological analysis

Microscopic evaluation revealed that the test group exhibited enhanced osteoblastic activity and a higher osteocyte count (Figures 4 and 5), findings that were further supported by histomorphometric analysis. At the 90-day follow-up, both groups predominantly exhibited woven bone, with minimal lamellar bone formation, reflecting the expected stage of bone maturation. No inflammatory infiltrate was observed in any specimen, indicating a favorable tissue response and good biocompatibility of the materials used. No residual biomaterial was identified in either group, which is consistent with the complete physiological resorption of the xenogeneic collagen sponge (Hemospon^® Cube; Maquira, Brazil) that occurs within 15 days.

Figure 4.

Photomicrograph of the control group: (A) 10× objective and (B) 20× objective. ON, with abundant osteocytes (black arrows), interspersed with delicate TC. Minimal osteoblastic activity is observed (yellow arrows).

Figure 5.

Photomicrograph of the test group: (C) 10× objective and (D) 20× objective. ON, with abundant osteocytes (black arrows), interspersed with delicate TC. Intense osteoblastic activity is observed (yellow arrows).

μCT analysis

Due to fragmentation of bone cylinders during retrieval from the trephine bur lumen, only six intact samples were available for μCT analysis (n = 3/group). Sample inclusion was based exclusively on structural integrity, with no additional subjective criteria applied. Given this limited sample size, μCT results are presented as exploratory and descriptive data only; no confirmatory statistical analysis was performed, and no inferential conclusions should be drawn from these findings. The μCT bone volume percentages were generally consistent with histomorphometric measurements from the identical specimens (Table 3), providing complementary microstructural characterization.

Table 3.

Bone volume percentage: μCT versus histomorphometry.

	Test group			Control group
Participant (P)	P10	P16	P19	P6	P17	P2
BV/TV%	35.565	47.815	38.534	51.574	43.859	44.967
H%	33.5	48.2	32.625	46.166	40.00	32.75

BV/TV%: percent bone volume in µCT; H%: percent tissue bone in histomorphometry.

Discussion

This randomized clinical trial demonstrated for the first time that melatonin gel (1.2 mg/0.1 mL) incorporated into a collagen sponge promoted greater new bone formation in post-extraction tooth sockets than an inert gel (bone density: 44% in the test group vs 35.4% in the control group, p = 0.0099). All participants had Epikut^® line (SIN Implant System, São Paulo, SP, Brazil) implants placed in the healed extraction socket, which feature a nanometric layer of hydroxyapatite (HA) crystals. Almeida et al. have already shown that melatonin does not interfere with the biocompatibility and osteoconduction of HA based on a lack of effect of melatonin (1.2 mg) on nanostructured carbonated HA (CHA) spheres in post-extraction tooth sockets of Wistar rats.²⁹

The scientific basis for evaluating the effect of melatonin on bone formation in post-extraction tooth sockets in preclinical and clinical studies is still limited. Most studies used melatonin gel or powder before implant placement. In a preclinical study on dogs, Cutando et al. introduced melatonin powder (1.2 mg) at the instrumented bone osteotomy site before dental implant placement. After 2 weeks of healing, the evaluated histomorphometric parameters were significantly higher in the melatonin-treated (test) group than in the non-melatonin-treated (control) group: bone-to-implant contact (BIC: 38.73% ± 1.46% vs 25.05% ± 2.43%, p < 0.0001), bone formation between the implant threads (36.3% ± 2.73% vs 25.08% ± 3.47%, p < 0.05), and new bone formation (35.18% ± 0.31% vs 28.65% ± 1.92%, p < 0.0001).¹⁶ In rabbit tibiae, BIC was also better when melatonin powder (3 mg) was directly inserted into the osteotomy site before implant placement (24.61% ± 2.87%) compared to no melatonin (13.62% ± 1.44%, p < 0.01).³⁰ In a preclinical study on dogs, implants that received porcine bone graft mixed with melatonin powder (5 mg) showed higher BIC (84.5% ± 1.5%) than implants with only the bone graft (67.17% ± 1.2%) after 12 weeks.¹¹ These results suggest that using melatonin with implant placement improves BIC and may thus reduce the osseointegration period, improving patients’ quality of life.

In a preclinical study with dogs, a histomorphometric evaluation conducted 60 days after tooth extraction showed significantly greater new bone in the post-extraction tooth sockets filled with collagen sponges impregnated with melatonin (1.2 mg; 81.23% ± 0.13%) than in the unfilled sockets (65.89% ± 0.45%). However, while the melatonin-treated group maintained a higher amount of new bone at 90 days, it did not differ significantly from the control group. Optical microscopy analyses showed no difference and similar results in both groups, characterized by lamellar bone tissue.¹²

In our study, the histomorphometry results corroborated the μCT findings, as both showed similar percentages of bone volume (Table 3 and Figure 6). These results were also consistent with the descriptive microscopic analysis, which revealed greater osteoblastic activity in the test group (Figures 4 and 5). Moreover, the μCT analyses revealed that the number and thickness of the trabeculae and the distance between them were similar between the test and control groups (Table 4), indicating compatibility with 90-day lamellar bone. This finding is consistent with Calvo-Guirado et al., who also observed that the melatonin group exhibited lamellar bone characteristics (greater cortical width and length) similar to those of the control group (without melatonin) after 90 days.¹²

Figure 6.

Histological (H&E stain) and micro-CT images (µCT) of the bone after 90 days of healing. 1A: H&E stain of test group; 2A: µCT reconstruction of test group; 1B: H&E stain of control group; 2B: µCT reconstruction of control group.

Table 4.

Quantitative and qualitative analysis of μCT.

	Test group			Control group
BV/TV%	35.565	47.815	38.534	51.574	43.859	44.967
Tb.Th (mm)	0.079	0.107	0.153	0.104	0.101	0.122
Tb.N (1/mm)	4.495	4.458	2.513	4.919	4.329	3.660
Tb.Sp (mm)	0.146	0.127	0.254	0.104	0.172	0.208

BV/TV: bone volume/tissue volume; BV/TV%: percent bone volume; Tb.N: trabecular number; Tb.Sp: trabecular separation; Tb.Th: trabecular thickness.

In a triple-blind, split-mouth randomized clinical trial, the osteogenic effect of melatonin (3 mg in 2 mL of 2% hydroxyethylcellulose gel) in post-extraction sockets of third molars was not observed when evaluating bone density, measured in Hounsfield Units (HU), using the quantitative radiographic subtraction method over a 60-day observational period (melatonin-treated vs untreated: 561.98 ± 105.92 vs 598.82 ± 209.03 HU).³⁰ Similar results were observed in another randomized controlled trial with a 6-month observational period (melatonin-treated vs untreated: 978.5 vs 965.8 HU).³¹ These studies shared not only the same concentration and formulation of melatonin (3 mg of melatonin in 2 mL of 2% hydroxyethylcellulose gel) but also the method of applying the gel directly into the socket without using any mechanism, besides sutures, to ensure better retention in the intra-alveolar compartment. The absence of a device, such as a collagen sponge in our study, may have contributed to the early loss of the gel, thus leading to similar bone density results between the test and control groups.

One of the limitations of this study was the exact location of bone sample extraction inside healed socket. It might be associated to the collagen sponge used (Hemospon^®) which is completely absorbed in 90 days, leaving no traces at the implantation site.

Another limitation of this study concerns the micro-computed tomography (µCT) analysis. Complete retrieval of specimens from the trephine bur lumen was achieved in only six cases, as fragmented samples were considered unsuitable for µCT evaluation due to the risk of compromising morphological accuracy. This restricted number of analyzable specimens substantially reduced the statistical power of this secondary analysis. To minimize potential selection bias, sample inclusion for µCT was based exclusively on structural integrity, with no additional subjective criteria applied.

In conclusion, our randomized clinical trial suggests that, under its experimental parameters and within its limitations, melatonin gel promoted bone repair in post-extraction tooth sockets, proving to be safe and without adverse effects. Future research should explore additional melatonin concentrations to better determine their influence on new bone formation. Moreover, because the present study focused on early-stage healing, the 90-day follow-up is a relatively short interval for evaluating complete bone remodeling. Longer observation periods of 6–9 months are needed to provide a more comprehensive assessment of bone remodeling and maturation. In accordance with antimicrobial stewardship principles, future investigations should also incorporate selective antibiotic protocols, taking into account biomaterial-specific requirements to optimize clinical outcomes.

An important consideration in this study is that registration in the Brazilian Clinical Trials Registry (RBR-67jkxsj) was completed retrospectively. The timeline was as follows: ethics approval was obtained in August 2021 (judgment no. 4.868.960), first participant enrollment occurred in September 2021, and trial registration was completed in December 2021. Although this delay occurred due to an administrative oversight, we acknowledge that prospective registration is strongly recommended by CONSORT and ICMJE guidelines. Critically, the primary outcome (histomorphometric bone density), all secondary outcomes, sample size calculation parameters, and statistical analysis plan remained unchanged from protocol version 1.0 to final analysis. No post hoc modifications were made, thereby preserving the trial’s methodological rigor and scientific integrity.

Additionally, while coin-toss randomization is a valid method for sequence generation, we acknowledge that computer-generated randomization with centralized allocation or sequentially numbered, sealed, opaque envelopes would have provided stronger methodological rigor. Nevertheless, the combination of third-party code custody and complete blinding of all clinical personnel substantially mitigated the risk of selection bias. Notably, the CBCT-based volumetric assessment measured intra-socket tissue formation (T1−T2 volume difference) rather than external ridge dimensional changes (width/height). Therefore, our radiographic dimensional outcome reflects new tissue formation within the socket compartment and should not be interpreted as a measure of external ridge contour preservation.

Footnotes

Acknowledgements

The authors would like to thank the participating surgeons and the staff of the following institutions: Santos-Dumont Aeronautical Dental Clinic of the Brazilian Air Force; National Institute of Metrology, Quality, and Technology; School of Dentistry at the Fluminense Federal University (Associated Laboratory of Clinical Research in Dentistry and Applied Biotechnology Laboratory).

ORCID iDs

Ingrid Chaves Cavalcante Kischinhevsky

Caio Márcio Sorrentino de Freitas Farias dos Santos

Rafael Bonato

Suelen Cristina Sartoretto

Carlos Fernando Mourão

José Mauro Granjeiro

Mônica Diuana Calasans-Maia

Ethical considerations

Author contributions

The concept and design of this study was performed by Mônica Diuana Calasans Maia and Ingrid Chaves Cavalcante Kischinhesvky. All surgical procedures were performed by Ingrid Chaves Cavalcante Kischinhevsky and Rafael Bonato. Data was collected by Ingrid Chaves Cavalcante Kischinhesvky . Data analysis and interpretation of data was performed by Ingrid Chaves Cavalcante Kischinhesvky, Mônica Diuana Calasans-Maia, Suelen Cristina Sartoretto Lorenzi, Adriana Alves, Caio Márcio Sorrentino de Freitas Farias dos Santos, and Carlos Fernando Mourão. Statistics was executed by José Granjeiro. Writing and critical revision was performed by all authors.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Trial registry

This clinical trial is registered at the Brazilian Clinical Trials Registry (ReBEC; ): RBR-67jkxsj.

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