Background
Tooth extraction is the most frequently administered treatment in dentistry. The healing of the tooth extraction wound consists of stages such as hematoma formation in the extraction cavity, organization of the clot, epithelization of the wound surface, formation of woven bone in the extraction cavity, replacement of woven bone with the trabecular bone, and reformation of the alveolar bone [ 1 ]. While this healing process is generally smooth in healthy individuals, it may be impaired or delayed in patients with a suppressed immune system for various reasons, diabetes, or metabolic bone disease. Therefore, many agents, which can be administered locally and systemically, such as bisphosphonate and parathyroid hormone (PTH), that control bone resorption and increase bone deposition are used [ 2 ].
Bisphosphonates are used in the treatment of breast, prostate and lung cancers with bone metastases, malignancies such as multiple myeloma, and systemic health problems such as osteoporosis, osteopenia, Paget’s disease, and osteogenesis imperfect [ 3 ]. Bisphosphonates have a direct effect on bone metabolism and inhibit osteoclast activity responsible for bone destruction and prevent its apoptosis. Furthermore, they also have antitumor effects such as inhibiting the involvement of tumor cells in the extracellular matrix and the tumor invasion, and providing apoptosis of tumor cells [ 4 ]. Bisphosphonates also have the activity to inhibit angiogenesis, which causes vascular obliteration [ 5 ].
Nowadays, bisphosphonates, which are frequently administered due to their therapeutic effects and known as effective and safe drugs, cause atrial fibrillation, acute phase reactions, renal failure, and undesirable clinical pictures such as osteonecrosis in the jaw bones especially in cases in which their nitrogen-containing derivatives are administered intravenously [ 6 ]. The first case report of bisphosphonate-related osteonecrosis of the jaw (BRONJ) was published by Marx et al. in 2003 [ 7 ]. An increasing number of case reports have been reported since that date.
The current treatment approaches reported for BRONJ are based on relieving or reducing complaints such as pain, swelling, inflammatory discharge, and tenderness, which affect the soft and hard tissues of the patient, eliminating findings such as bone necrosis, and improving the patient’s quality of life. These methods differ according to the stage of BRONJ and include medical or medical-surgical treatments. In addition to these treatment methods, some supportive treatment methods such as hyperbaric oxygen (HBO), platelet-rich plasma (PRP), platelet-rich fibrin (PRF), medical ozone, PTH and low-dose laser therapy (LDLT) have also begun to be accepted in recent years [ 8 – 10 ].
Since BRONJ is a negative picture that decreases the quality of life of the patient, the methods aimed at preventing the occurrence of osteonecrosis by reducing the risk factors have also become as important as its treatment. Nevertheless, there is no standard protocol in the treatment of BRONJ. Therefore, apart from the treatments administered in the treatment of BRONJ, new methods and agents that are considered to affect the bone and soft tissue healing positively should be investigated.
Melatonin is a hormone that is secreted from the pineal gland in the absence of light and affects the regulation of many biological functions such as sleep, circadian rhythm, immunity, and reproduction. Furthermore, melatonin also has anti-inflammatory, angiogenic, and bone-formation stimulating effects [ 11 ].
Melatonin reveals proangiogenic activity by increasing the expression of Vascular Endothelial Growth Factor (VEGF). It has been indicated that melatonin has positive effects on wound healing and angiogenesis and stimulates transforming growth factor-beta (TGF-β) and fibroblast proliferation [ 12 ].
In many studies, it has been demonstrated that melatonin is an important mediator in bone formation and stimulation and increases the proliferation of bone cells (osteoblastic cells) and the production of collagen from these cells [ 13 ]. Furthermore, it is also known that melatonin stimulates the gene expression of bone protein markers (alkaline phosphatase, osteocalcin) [ 14 ].
While the precise etiology of bisphosphonate-related osteonecrosis of the jaw is still not fully understood, several management strategies are currently used in clinical practice and are typically tailored to disease stage. These include conservative measures (e.g., antimicrobial mouth rinses, systemic antibiotics, and analgesia), surgical approaches (sequestrectomy, debridement, or resection), and adjunctive options such as hyperbaric oxygen, platelet-rich preparations (PRP/PRF), medical ozone, teriparatide, and low-level/low-dose laser therapy [ 15 – 18 ].
Nevertheless, there is no universally accepted gold-standard protocol, and outcomes remain variable. In this context, agents that may enhance bone and soft-tissue healing warrant investigation. Given melatonin’s pro-angiogenic and pro-osteogenic actions, we hypothesized that melatonin could mitigate BRONJ-like changes, and designed this study to evaluate its local and systemic effects.
Our study aimed to investigate the negative effects of bisphosphonates on wound healing in the jaw bones and to histologically and immunohistochemically evaluate the effectiveness of melatonin, which is administered locally and systemically, on these negative effects.
Methods
The ethics committee approval for the study was obtained from the Experimental Animals Ethics Committee of Inonu University, Faculty of Medicine, with the protocol number 2019/A-29. All experimental procedures complied with the Guide for the Care and Use of Laboratory Animals and the ARRIVE guidelines 2.0 [ 19 , 20 ]. The ARRIVE checklist is presented in supplementary material.
Experimental animals
Forty-four Wistar albino rats (female, 6–7 months old, 250–350 g) were obtained from the Inonu University Experimental Animal Research Center (Malatya, Türkiye). We selected adult (6–7-month-old) rats to ensure skeletal maturity (fully erupted molars and stable bone turnover) and adequate alveolar dimensions for standardized extractions; using a single sex and a narrow age window also reduces variance in healing endpoints and facilitates consistent dose normalization by body weight, as recommended in established BRONJ-like protocols [ 19 ]. These animals were bred and housed in the institutional facility, not privately owned, and therefore no owner consent was required. The subjects were kept at an average temperature of 24 °C and 12 h in light and 12 h in darkness. Standard food and water were placed in the cages in a way that the subjects could reach them easily. During the experimental process, the subjects were regularly evaluated for criteria such as changes in the skin, decrease in food/water intake, weakness, and jaw necrosis. To minimize potential confounding factors, several procedural and environmental controls were implemented. Animals were randomly allocated to experimental groups using tail-number–based simple randomization prior to the initiation of any intervention. All surgical procedures (tooth extraction, local treatments) and injections were performed following a standardized protocol by the same experienced operator, using identical instruments and anesthesia regimens, to reduce operator- and procedure-related variability. The order of surgical interventions and treatments was alternated across groups on each experimental day to minimize systematic bias related to treatment order or time of day. All animals were housed under identical environmental conditions (temperature, light–dark cycle, cage type, bedding, and access to food and water), and cage positions within the animal facility were rotated periodically to reduce potential effects related to cage location (e.g., light exposure or airflow differences). Outcome assessments, including histological and immunohistochemical evaluations, were performed under blinded conditions. Tissue samples and slides were coded by an independent investigator, and group allocation was concealed from observers during scoring and analysis. No formal stratification was performed based on cage clustering beyond random allocation, and no additional statistical adjustment for cage effects was applied. These factors are acknowledged as potential residual confounders.
The initial live weights of 44 adult female rats were measured, and they were randomly divided into four groups by numbering their tail parts. With a power analysis of α = 0.05 and 1-β (power) = 0.80, the minimum number of rats required in each subgroup to detect a significant difference between groups was determined to be 11.
Study groups
In the study, four groups, each of which consisted of 11 simple randomly selected animals, were classified as follows;
Control Group (C): No medical drug was administered for 8 weeks from the beginning of the experiment. On day 28 of the experiment, the lower left first molars were extracted, and the rats were sacrificed at the end of the experiment (day 56) (n:11). Bisphosphonate Group (BIF): Zoledronic acid was administered intraperitoneally once a week for 8 weeks from the beginning of the experiment. On day 28 of the experiment, the lower left first molars were extracted, and the rats were sacrificed at the end of the experiment (day 56) (n:11). Bisphosphonate and Local Melatonin Administered Group (BIF + LM): Zoledronic acid was administered intraperitoneally once a week for 8 weeks from the beginning of the experiment. On day 28 of the experiment, the lower left first molars were extracted. Local melatonin was administered in powder form into the tooth socket immediately after the extraction, and the rats were sacrificed at the end of the experiment (day 56) (n:11). Bisphosphonate and Systemic Melatonin Administered Group (BIF + SM): Zoledronic acid was administered intraperitoneally once a week for 8 weeks from the beginning of the experiment. On day 28 of the experiment, the lower left first molars were extracted. After the extraction, melatonin was administered intraperitoneally on a daily basis, the first one of which was administered immediately postoperatively, for 28 days. The rats were sacrificed at the end of the experiment (day 56) (n:11).
Bisphosphonate dosage and route of administration
The drug doses to be administered were calculated for the subjects, the weights of which were measured and recorded before the experiment. In the study, zoledronic acid (Zometa 4 mg/5 mL intravenous, Novartis Pharma AG, Basel, Switzerland) was diluted with normal saline and administered at a dose of 0.4 mg/kg in order to develop a BRONJ model. The body weights of subjects were re-weighed each week prior to injection, and the dose to be administered was standardized [ 21 , 22 ].
Tooth extraction method
On day 28, general anesthesia was achieved via intraperitoneal injection of xylazine hydrochloride (10 mg/kg; 2% Alfazyne, Alfasan, Netherlands) and ketamine hydrochloride (100 mg/kg; 10% Ketasol, Richter Pharma AG, Wels, Austria). In addition, 0.5 mL of 4% articaine with epinephrine (0.006 mg/mL) was administered locally at the surgical site for analgesia and hemostasis. Then, the teeth were extracted by performing luxation with a curved hemostat. Finally, the extraction sites were sutured with 4.0 Vicryl suture (Doğsan, Türkiye). No drug (antibiotic, etc.) was administered to the subjects after extraction. The study procedures could be generally tolerated by the subjects. However, since two subjects, including one subject from the BIF group and the other one from the BIF + LM group, died immediately after anesthesia, they were excluded from the study.
Melatonin dosage and administration
Local melatonin
The pure powder melatonin was weighed to be 1.2 mg on a precision scale and transferred to Eppendorf tubes. Powder melatonin in tubes was placed in the extraction socket, where the bleeding was controlled after tooth extraction, with a spoon curette. The region was sutured with 4.0 Vicryl suture to ensure the stabilization of melatonin administered in the socket [ 23 ].
Systemic melatonin
Pure powder melatonin (Sigma Aldrich, St. Louis, Missouri, USA, Cas no: 73-31-4) was weighed on a precision balance (Mettler Toledo, Columbus, Ohio, USA). The weighed melatonin was dissolved in an isotonic solution containing 0.9% sodium chloride in a vortex device (Falc Mix-20, Lurano, Italy). The tube was tightly sealed with a paraffin film to prevent air input. Each subject was weighed individually, and the amount of solution to be used was calculated and administered intraperitoneally [ 24 ].
Sacrification of the subjects and removal of the extraction socket site
On day 56 of the experiment, the animals were euthanized by exsanguination performed under surgical conditions following general anesthesia induced with ketamine and xylazine, and the left mandibles were subsequently removed for further analyses. Then, the excess soft tissues in the mandible removed were trimmed, and the materials were placed in 10% buffered formaldehyde. Mandibles were fixed in 10% neutral buffered formalin for 24–48 h at room temperature and then decalcified in 10% EDTA (pH ~ 7.2–7.4) with daily solution changes until radiographic/needle-prick testing confirmed complete decalcification. Specimens were rinsed in running water, dehydrated through graded ethanols, cleared in xylene, and paraffin-embedded with the extraction socket oriented in the frontal plane. Serial Sects. (4–5 μm) were prepared using a rotary microtome. The tissue sections taken from the subjects were examined at the Department of Histology and Embryology of Inonu University, Faculty of Medicine.
Histological techniques and analyses
Outcome assessment was performed under blinded conditions. Histological and immunohistochemical samples were coded by an independent investigator who was not involved in the experimental procedures. Observers responsible for microscopic evaluation and scoring were blinded to group allocation throughout the assessment process.
Data analysis was conducted using coded datasets. The investigator performing the statistical analyses was blinded to group identities until all analyses were completed and the primary results were finalized.
Histological Tissue Monitoring and Staining
This study was designed as a hypothesis-testing experiment. The primary outcome measure used to determine the sample size was the histopathological osteonecrosis score of the extraction socket, assessed on hematoxylin–eosin–stained sections using the predefined semi-quantitative scoring system.
Immunohistochemistry (IHC) scoring was conducted under blinded conditions using the same coding and dual-observer approach described above. Routine H-E staining was performed on deparaffinized sections to assess osteonecrosis, inflammatory cell infiltration, and epithelial coverage. For IHC, sections underwent heat-induced antigen retrieval in citrate buffer (10 mM, pH 6.0), endogenous peroxidase quenching (3% H₂O₂, 10 min), and protein blocking. Primary antibodies against osteocalcin and VEGF were applied (optimized dilutions; incubation 60 min at room temperature), followed by a polymer-HRP detection system and DAB chromogen. Slides were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. Negative controls were processed by omitting the primary antibody; positive control tissues known to express the respective targets were run in parallel.
Histological Analyses
All histological evaluations were performed blinded to group allocation. Slides were coded by an independent investigator, and two observers assessed each section independently; disagreements were resolved by consensus. Osteonecrosis was determined by the presence of more than eight empty lacunae (without osteocytes). Osteonecrosis level, inflammatory cell infiltration, and epithelization values were evaluated using a scale from 0 to 3 (Table 1 ). In immunohistochemical staining, scoring was performed semi-quantitatively from 0 to 3 based on the prevalence and severity of immunoreactivity. The total staining was obtained by calculating prevalence x severity. For each animal, three non-consecutive sections (≥ 100 μm apart) spanning the central third of the socket were analyzed. In H-E slides, five high-power fields (HPFs, ×400) centered on the apical, middle, and cervical thirds were assessed. Osteonecrosis was defined as > 8 empty osteocyte lacunae within trabeculae. Immunoreactivity for osteocalcin and VEGF was scored semi-quantitatively (0–3) for prevalence and 0–3 for intensity; Total Score = prevalence × intensity, as pre-specified. Two blinded observers independently scored all slides; disagreements were resolved by consensus, and inter-observer agreement (Cohen’s κ) was calculated. Representative images were acquired using a bright-field microscope with a digital camera under identical exposure and magnification settings. When needed, area measurements and DAB optical density were confirmed in ImageJ with standardized thresholds.
Table 1 Scoring criteria for histological and immunohistochemical analysis Score Osteonecrosis Inflammation Epithelization VEGF-Osteocalcin Prevalence Severity 0 - Normal Medium-thick epithelial tissue completely covering the extraction area 0–25% None 1 + Less than 25% change Thin epithelial tissue completely covering the extraction area 26–50% Mild 2 ++ Change between 25–50% Thin epithelial tissue only at the edges of the open surgical site 51–75% Moderate 3 +++ More than 50% change Absence of epithelial tissue on the open surgical site 76–100% Severe VEGF Vascular Endothelial Growth Factor
Table 1
Scoring criteria for histological and immunohistochemical analysis
VEGF Vascular Endothelial Growth Factor
| Score | Osteonecrosis | Inflammation | Epithelization | VEGF-Osteocalcin | |
| Prevalence | Severity | ||||
| 0 | - | Normal | Medium-thick epithelial tissue completely covering the extraction area | 0–25% | None |
| 1 | + | Less than 25% change | Thin epithelial tissue completely covering the extraction area | 26–50% | Mild |
| 2 | ++ | Change between 25–50% | Thin epithelial tissue only at the edges of the open surgical site | 51–75% | Moderate |
| 3 | +++ | More than 50% change | Absence of epithelial tissue on the open surgical site | 76–100% | Severe |
Statistical evaluation
Statistical analyses were performed using the statistical software program (WSSPAS: Web-Based Sample Size & Power Analysis Software) developed by Inonu University, Faculty of Medicine, Department of Biostatistics and Medical Informatics [ 25 ]. Normality was assessed using the Shapiro–Wilk test. In the study, the non-normally distributed variables ( p > 0.05) were evaluated by the Kruskal-Wallis analysis of variance. Intergroup comparisons were performed using the Mann-Whitney U test. The value of p < 0.05 was considered to be statistically significant. The data were expressed as median and minimum-maximum (med (min-max)) depending on the distribution.
Results
Evaluation of the general histological structure
In the control group, H–E stained sections showed bone trabeculae with regularly arranged osteocytes displaying basophilic nuclei and normal morphology at the apical and middle levels of the extraction socket. The cervical region was covered by granulation tissue with mild inflammatory cell infiltration. The extraction surface was lined by a continuous, medium-thick epithelial layer in most samples (Fig. 1 A).
Fig. 1 A : Control Group: Bone trabeculae (five-pointed stars), granulation tissue (four-pointed stars), epithelial tissue completely covering the extraction surface (arrows). H-E; 4x. B : BIF Group: Necrotic bone trabeculae (five-pointed stars), granulation tissue with inflammatory cell infiltration (four-pointed stars), extraction surface without the occurrence of epithelization (arrows). H-E; 4x. C : BIF + LM Group: Bone trabeculae (five-pointed stars), granulation tissue (four-pointed stars), thin epithelial tissue covering the extraction surface (arrows). H-E; 4x. D : BIF + SM Group: Bone trabeculae (five-pointed stars), granulation tissue with inflammatory cell infiltration (four-pointed stars), extraction surface without the occurrence of epithelization (arrows). H-E; 4x. C, Control; BIF, Bisphosphonate; LM, Local Melatonin; SM, Systemic Melatonin; H–E, Hematoxylin and Eosin
In contrast, the BIF group exhibited delayed healing characterized by necrotic bone trabeculae containing multiple empty osteocyte lacunae across all socket levels. Dense granulation tissue filled the intertrabecular spaces, accompanied by marked inflammatory infiltration. Epithelial coverage was largely absent in this group (Fig. 1 B).
In both melatonin-treated groups, tissue morphology showed clear improvement relative to BIF. In the BIF + LM and BIF + SM groups, necrotic areas and the density of empty lacunae were reduced, while viable osteocytes with distinct basophilic nuclei were frequently observed within the bone trabeculae. Granulation tissue persisted mainly in the cervical portion, but inflammatory infiltration was notably diminished. In the BIF + LM group, partial to nearly complete re-epithelization of the extraction surface was observed, whereas in the BIF + SM group epithelial regeneration remained incomplete (Fig. 1 C–D).
These histological observations are consistent with the quantitative findings summarized in Table 2 , where osteonecrosis and inflammation scores were significantly lower in the melatonin groups compared with BIF ( p < 0.05), and epithelization scores were improved particularly in the local melatonin group ( p < 0.05 vs. BIF and BIF + SM).
Table 2 Histopathological and immunohistochemical score results of each group Control med (min-max) BIF med (min-max) BIF + LM med (min-max) BIF + SM med (min-max) Osteonecrosis 0.0 (0.0–1.0) 3.0 (0.0–3.0) a 1.0 (0.0–3.0) a,b 1.0 (0.0–3.0) a,b Inflammation 1.0 (0.0–3.0) 2.0 (0.0–3.0) a 1.0 (0.0–3.0) b 1.0 (0.0–3.0) b Epithelization 1.0 (0.0–2.0) 3.0 (2.0–3.0) c 2.0 (0.0–3.0) d,e 3.0 (2.0–3.0) c VEGF 4.0 (0.0–12.0) 2.0 (0.0–9.0) c 6.0 (0.0–12.0) d 6.0 (0.0–12.0) d Osteocalcin 2.0 (0.0–9.0) 1.0 (0.0–9.0) c 3.0 (0.0–12.0) a,d,e 2.0 (0.0–12.0) d a A statistically significant increase compared to the control group b A statistically significant decrease compared to the BIF group c A statistically significant decrease compared to the control group d A statistically significant increase compared to the BIF group e A statistically significant increase compared to the BIF + SM group C, Control group; BIF, Bisphosphonate group; BIF + LM, Bisphosphonate + Local Melatonin group; BIF + SM, Bisphosphonate + Systemic Melatonin group; VEGF, Vascular Endothelial Growth Factor
Table 2
Histopathological and immunohistochemical score results of each group
a A statistically significant increase compared to the control group b A statistically significant decrease compared to the BIF group c A statistically significant decrease compared to the control group d A statistically significant increase compared to the BIF group e A statistically significant increase compared to the BIF + SM group C, Control group; BIF, Bisphosphonate group; BIF + LM, Bisphosphonate + Local Melatonin group; BIF + SM, Bisphosphonate + Systemic Melatonin group; VEGF, Vascular Endothelial Growth Factor
| Control med (min-max) | BIF med (min-max) | BIF + LM med (min-max) | BIF + SM med (min-max) | |
| Osteonecrosis | 0.0 (0.0–1.0) | 3.0 (0.0–3.0) a | 1.0 (0.0–3.0) a,b | 1.0 (0.0–3.0) a,b |
| Inflammation | 1.0 (0.0–3.0) | 2.0 (0.0–3.0) a | 1.0 (0.0–3.0) b | 1.0 (0.0–3.0) b |
| Epithelization | 1.0 (0.0–2.0) | 3.0 (2.0–3.0) c | 2.0 (0.0–3.0) d,e | 3.0 (2.0–3.0) c |
| VEGF | 4.0 (0.0–12.0) | 2.0 (0.0–9.0) c | 6.0 (0.0–12.0) d | 6.0 (0.0–12.0) d |
| Osteocalcin | 2.0 (0.0–9.0) | 1.0 (0.0–9.0) c | 3.0 (0.0–12.0) a,d,e | 2.0 (0.0–12.0) d |
Evaluation of VEGF and osteocalcin immunoreactivity
VEGF and osteocalcin immunoreactivity were observed as brownish staining in the osteocyte and granulation tissue. In the bisphosphonate group, VEGF and osteocalcin immunoreactivity generally had low prevalence and severity. On the other hand, it was observed that local and systemic melatonin administrations increased VEGF and osteocalcin immunoreactivity. In the local and systemic melatonin groups, while VEGF immunoreactivity was found to be similar, osteocalcin immunoreactivity was observed to be higher compared to the systemic melatonin group (Figs. 2 and 3 ).
Fig. 2 A : Control Group: VEGF immunoreactivity observed as brownish in osteocytes and granulation tissue (arrows). VEGF immunostaining; 20x. B : BIF Group: VEGF immunoreactivity (arrows). VEGF immunostaining; 20x. C : BIF + LM Group: VEGF immunoreactivity (arrows). VEGF immunostaining; 20x. D : BIF + SM Group: VEGF immunoreactivity (arrows). VEGF immunostaining; 20x. VEGF, Vascular Endothelial Growth Factor; C, Control group; BIF, Bisphosphonate group; BIF + LM, Bisphosphonate + Local Melatonin group; BIF + SM, Bisphosphonate + Systemic Melatonin group
Fig. 3 A : Control Group: Osteocalcin immunoreactivity observed as brownish in osteocytes and granulation tissue (arrows). Osteocalcin immunostaining; 20x. B : BIF Group: Osteocalcin immunoreactivity (arrows). Osteocalcin immunostaining; 20x. C : BIF + LM Group: Osteocalcin immunoreactivity (arrows). Osteocalcin immunostaining; 20x. D : BIF + SM Group: Osteocalcin immunoreactivity (arrows). Osteocalcin immunostaining; 20x. C, Control group; BIF, Bisphosphonate group; BIF + LM, Bisphosphonate + Local Melatonin group; BIF + SM, Bisphosphonate + Systemic Melatonin group
Statistical evaluation of the histological results
In our study, the score results of the histopathological evaluations of the groups in terms of osteonecrosis, inflammation, and epithelization and the immunohistochemical evaluations for VEGF and osteocalcin values are presented in Table 2 .
Discussion
In patients using bisphosphonate, surgical procedures have become a problem for dentists due to the side effects resulting from the action metabolism of the drug. The use of such drugs is known to affect bone metabolism negatively. Especially in cases where bone healing is disrupted, osteonecrosis leads to severe complications. Osteonecrosis is a term that is commonly used to describe the death of bone cells such as bone marrow stromal cells, preosteoblasts, mature osteoblasts, osteocytes, and osteoclasts [ 26 ]. Although osteonecrosis of the jaw is associated with the suppression of osteoclast activity and angiogenesis, osteoblast functions have also been demonstrated to be affected by bisphosphonates.
While it was observed that very low concentrations of bisphosphonates stimulated the osteoblasts [ 27 ], it was reported that their high concentrations decreased the migration capacity and cell viability of osteoblasts [ 28 , 29 ].
Bisphosphonates also inhibit bone proteins secreted by osteoblasts. In their recent study, Basso et al. indicated that the cytotoxic effects of bisphosphonates on osteoblasts played an important role in the development of oral osteonecrosis. In their in vitro study on osteoblasts, they observed that zoledronic acid decreased the osteoblast differentiation by inhibiting the expression of genes responsible for the production of alkaline phosphatase and osteocalcin in osteoblasts [ 30 ].
Bisphosphonates suppress angiogenesis, which leads to the inhibition of bone regeneration and delays in local tissue healing. In their study conducted in the cell culture, Basso et al. evaluated VEGF and FGF-b in the epithelial cells to which they administered zoledronic acid. As a result of the study, they reported that zoledronic acid directly prevented cell proliferation and decreased the expression of growth factors from epithelial cells by 34% for VEGF and 51% for FGF-b. The authors also indicated that the decrease in these growth factors might delay the local wound healing [ 31 ]. In their in vitro study, Ishtiaq et al. examined the effects of bisphosphonates on angiogenesis. In the study, VEGF and angiopoietin-1 production was also evaluated in osteoblast and osteocyte cell lines treated with zoledronic acid, and the inhibition effect of zoledronic acid on these proangiogenic proteins was revealed [ 32 ].
Melatonin can easily reach the intercellular matrix and all parts of the cells due to its high lipophilic property [ 33 ]. It is known that after melatonin enters the blood, it passes into the saliva and then into the oral cavity by passive diffusion [ 34 ]. In the literature, it has been reported that melatonin is safe for use, and unlike other antioxidants, it does not have a toxic effect even at very high doses (300 mg/day) and in the long-term use (such as 5 years). Mild side effects such as vertigo, headache, nausea and sleepiness were observed at levels close to placebo treatment [ 35 ].
In many studies, different systemic and local doses of melatonin were used in different animal models. While Acikan et al. administered systemic melatonin at two different doses, 25 mg/kg and 50 mg/kg [ 36 ], in the mandibular distraction osteogenesis model in rats, Li et al. administered systemic melatonin at a dose of 10 mg/kg [ 37 ] in rats with experimental pulpitis, and Koyama et al. administered systemic melatonin at doses of 1, 5, and 50 mg/kg in mice to evaluate its effect on bone density and mass [ 38 ]. 10 mg/kg dose of melatonin was preferred in many studies on rats [ 24 ]. Based on these studies, systemic melatonin was administered at a dose of 10 mg/kg/day in our study.
While Munoz et al. locally administered 1.2 mg lyophilized melatonin to the implant sockets in order to evaluate the formation of new bone in the dog jawbone [ 39 ], Cobo-Vazquez et al. locally administered 3 mg melatonin to the lower third molar sockets to examine its anti-inflammatory, analgesic, and osteogenic effect in 10 patients [ 40 ], and Cutando et al. locally administered 2 mg melatonin to the extraction sockets of 16 Beagle dogs to evaluate the antioxidant activity [ 41 ]. As can be understood from the difference of the data in the literature, there is no determining protocol for local dose administration. Within this information, in our study, 1.2 mg dose of melatonin was taken as a reference by considering the size of the extraction sockets [ 40 ].
The association between melatonin and bone metabolism was documented in many studies [ 14 , 34 , 38 ]. As a result of the study conducted by Roth et al. to examine the effects of pre-osteoblastic cells on osteogenic differentiation in vitro, it was reported that the differentiation time of new preosteoblasts transported from the bone marrow to the alveolar bed via the vascular system in the presence of melatonin, which is normally 21 days, decreased to 12 days and that its mineralization was accelerated [ 14 ]. In their study on pre-osteoblastic cells at melatonin concentrations of 0-250 µM, Son et al. observed that melatonin increased alkaline phosphatase, type 1 collagen and osteocalcin expression levels of cells depending on the concentration [ 42 ].
In many in vivo studies conducted with different models, as well as in vitro studies, the effects of melatonin, which was administered locally and systemically, on bone tissue were investigated. In their study conducted on 30 adult rats, Acikan et al. examined the effects of systemic melatonin (intraperitoneal) administration at different doses (25 and 50 mg/kg) during mandibular distraction osteogenesis on new bone formation, and at the end of the study, they reported that the numbers of osteoblasts, osteoclasts, osteopontin and VEGF levels were higher in the melatonin groups compared to the control group [ 36 ]. In their study, Satomura et al. administered 100 mg/kg melatonin intraperitoneally to the rats for 21 days and demonstrated that melatonin increased the newly formed cortical bone volume in the femur [ 43 ].
Osteocalcin, which is a non-collagen protein of the bone matrix, is specific to bone tissue, and its synthesis is performed by osteoblasts, odontoblasts, and chondroblasts. It is known as a specific indicator of osteoblastic activity in bone [ 44 ]. Therefore, osteocalcin in bone tissue was immunohistochemically examined to evaluate bone healing and new bone formation. The results of our study show that local and systemic melatonin application increases the production of osteocalcin suppressed by bisphosphonate in extraction sockets. Thus, it is considered that melatonin can induce bone formation by supporting osteoblast functions in BRONJ cases. The fact that the osteocalcin values were significantly higher in the BIF + LM group compared to the control group revealed that local melatonin administration accelerated the physiological bone formation process and increased the amount of new bone to be formed. Osteocalcin values in the BIF + LM group also increased statistically significantly compared to the BIF + SM group. It is considered that it may be due to the paracrine effect of the local administration of melatonin, which acts as a growth hormone in bone tissue, on bone cells. Furthermore, the amount of systemically administered melatonin reaching the bone tissue may be lower than the locally administered melatonin, which may be the reason for the difference. In the literature, there is no study in which osteocalcin values in bone tissue were compared with each other by administering melatonin locally and systemically. There is a need for further clinical studies with molecular and quantitative analyses in order to explain this difference.
In the studies, melatonin was shown to be effective in many steps of angiogenesis through various routes. It was reported that melatonin had its main effect on angiogenesis by increasing VEGF expression, which is known to be a mitogenic proangiogenic factor [ 45 ]. Moreover, it stimulates the proliferation of fibroblasts, which are known to play an important role in angiogenesis as the progenitors of endothelial cells, and thus has positive effects on angiogenesis [ 46 ]. In the studies, it was also reported that the increase in the number of mesenchymal stem cells (MSCs) was found to be associated with enhanced angiogenesis and cell proliferation. Melatonin protects MSCs from apoptosis and stimulates the expression of angiogenic and mitogenic factors (FGFs) involved in cell protection and repair from MSC [ 47 ].
Pugazhenthi et al. suggested that melatonin was an ideal candidate for the improvement of the wound healing process. In their study, they examined the VEGF165 values by administering 1.2 mg/kg melatonin to the wound edges in rats with incisions and observed significant increases compared to the control group. In conclusion, they reported that melatonin treatment accelerated the angiogenic process and increased the formation of new blood vessels [ 12 ]. Soybir et al. demonstrated that 0.4 mg/kg of melatonin, which was systemically administered in rats with incisions, significantly increased neovascularization and the number of vessels compared to the control group. As a result of the study, it was indicated that melatonin could have a positive effect on both angiogenesis and wound healing [ 46 ]. Ramirez-Fernandez et al. examined the effects of melatonin on angiogenesis in the repair of bone defects. In the study, two bone defects were created in rabbit tibiae, and 1.2 mg melatonin was administered to one of the defects while the other defect was left blank for control. As a result of the study, it was reported that the number of vessels in the group administered with melatonin increased significantly compared to the control group at the 1st and 2nd weeks after surgical intervention and that these accelerated angiogenic effects may increase bone cell proliferation [ 48 ].
VEGF, which is a highly specific mitogen for vascular endothelial cells, provides proliferation in endothelial cells, facilitates migration in cells and prevents cell apoptosis. In the studies, it was demonstrated that the penetration of bone marrow by new vessels enabled the migration of angiogenic and osteogenic cells to isolated areas. Since VEGF increases osteoblast differentiation, it plays a significant role in bone formation and repair during osteogenesis [ 49 ]. In our study, VEGF values were examined immunohistochemically because it was considered that the positive effects of melatonin on angiogenesis would prevent this vascular inhibition, which plays a significant role in the pathogenesis of BRONJ formation. The result of our study shows that melatonin induces angiogenesis suppressed by bisphosphonates during healing in extraction sockets. It is considered that these positive effects in angiogenesis support wound healing and thus lead to positive effects in the prevention of BRONJ formation.
Otto et al. argued that toxicity increased at the cellular level as the pH decreased and that the increase in cellular toxicity due to ambient acidity might have negative effects on the development of BRONJ [ 50 ]. Bisphosphonate that is released into the environment by separating from the bone tissue to which it is attached due to acidification to be caused by trauma following tooth extraction and inflammation in the residual alveolar bone leads to toxicity on soft tissue and macrophages, which results in the occurrence of areas of osteonecrosis and severe inflammation [ 51 ]. Melatonin is a powerful antioxidant. This antioxidant activity can struggle with the initiation and progression of inflammation, which is a process that causes the production of free oxygen radicals and the activation of prooxidant enzymes [ 52 ]. Furthermore, melatonin was reported to inhibit various adhesion molecules, proteolytic enzymes, cytokines, and the COX 2 enzyme, which is known to be involved in the inflammatory process [ 53 ]. Based on these literature data, inflammatory cell infiltration was examined to evaluate inflammation which revealed that melatonin administration reduced the inflammatory response. The lower level of inflammation in the BIF + LM and BIF + SM groups may prevent the tendency of pH to acidity. Thus, it may be possible to prevent toxicity occurring in the surrounding soft and hard tissues caused by the re-release of the bisphosphonate stored in the bone into the environment. Furthermore, it was considered that this anti-inflammatory and analgesic effect of melatonin might help reduce the pain that would occur in BRONJ.
The early phase of extraction-socket healing is characterized by a tightly regulated inflammatory response that orchestrates angiogenesis and osteogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs), especially non-selective COX inhibitors, can attenuate prostaglandin signaling and thereby influence early bone repair dynamics. In the present study, no postoperative NSAIDs were given to avoid this potential confounder [ 54 , 55 ]. However, because NSAID use is common in clinical practice, differences in drug class, dose, and timing may partly account for variability in BRONJ outcomes across studies. Future work comparing standardized analgesic regimens—including selective COX 2 inhibitors and non-NSAID options—could help define perioperative protocols that minimize adverse effects on socket healing.
Bisphosphonate to be released from the residual alveolar bone following the tooth extraction is expected to cause areas of osteonecrosis. As a result of the histological examinations in our study, osteonecrosis that occurred in the BIF group, in which zoledronic acid was administered in addition to tooth extraction, was found to be significantly higher compared to the control group with only tooth extraction without administering any medical agent. This result was consistent with the literature data indicating that tooth extraction may cause an adequate risk factor in the development of BRONJ after the use of zoledronic acid. It was observed that osteonecrosis was significantly lower in the BIF + SM and BIF + LM groups compared to the BIF group, which was considered to be due to the positive effects of melatonin on healing as a result of increasing new bone formation and angiogenesis. Nevertheless, osteonecrosis values were found to be statistically significantly higher in the BIF + LM and BIF + SM groups compared to the control group. In our study, local melatonin was administered as a single dose, and systemic melatonin was administered only in the postoperative period after tooth extraction. It is considered that repeated local doses of melatonin and systemic applications used for longer periods by starting simultaneously with the bisphosphonate will have more positive results on this resulting osteonecrosis picture.
In the studies, it was demonstrated that bisphosphonates had a direct cytotoxic effect on epithelial cells and also negative effects on epithelial growth factor [ 31 , 56 ]. As a result of our study, in parallel with these literature data, a significant decrease was observed in epithelization values in the BIF group. Nevertheless, the fact that the epithelization values in the BIF + LM group in our study were significantly higher compared to the BIF group reveals that local melatonin administration may have positive results in the prevention of epithelial damage caused by zoledronic acid. It is considered that melatonin provided these positive results in epithelial cells since it increased angiogenesis and thus healing and had cytoprotective effects on cells. Furthermore, in our study, it was found that epithelization values in the BIF + LM group were significantly higher compared to the BIF + SM group, which is considered to be due to the easy diffusion of melatonin, which was in contact with epithelial tissue during local administration, into these tissues around the socket due to its lyophilized structure. However, since there are no data to completely explain this situation in the literature, there is a need for clinical studies, including the relevant molecular mechanism and quantitative analyses.
There are in vitro studies on the cytoprotective effect of melatonin in different cell cultures treated with bisphosphonates In the literature, In their study, Camacho-Alonso et al. evaluated the cytoprotective effects of melatonin on human osteoblast cells treated with zoledronic acid. As a result of the study, the authors reported that melatonin had a cytoprotective effect on human osteoblasts treated with zoledronic acid and could be a promising protective alternative for patients at risk of BRONJ [ 57 ]. Rodriguez-Lozano et al. administered zoledronic acid and melatonin to the mesenchymal stem cells obtained from the periodontal ligament and bone marrow, and they evaluated the cytoprotective effects of melatonin. In the study, it was observed that especially high concentrations of zoledronic acid significantly decreased the number of cells in both cell types and that melatonin had a cytoprotective effect on these cells. The authors reported that the clinical use of cytoprotective agents such as melatonin in combination with bisphosphonate treatment might be a promising therapeutic alternative to avoid clinical complications such as BRONJ [ 58 ]. However there is no in vivo study in which the healing effect of melatonin on BRONJ is examined. Our study was also based on these literature data.
Future studies will incorporate RT-qPCR to quantify gene expression related to angiogenesis and bone formation/remodeling in peri-socket bone and soft tissues. Coupling these data with our histology/IHC outcomes will enable a more granular appraisal of melatonin’s effects on vascularization and bone turnover dynamics.
Study limitations
First, our analyses focused on histology and immunohistochemistry; we did not perform RT-qPCR, which would strengthen mechanistic interpretation of angiogenic and osteogenic pathways. In follow-up studies, we plan to collect fresh-frozen peri-socket tissues for RNA isolation and quantify targets with validated housekeeping genes. Second, although postoperative NSAIDs were not used here, differing perioperative anti-inflammatory/analgesic strategies in clinical care could affect early repair and limit direct generalizability. Third, our design used a single species, sex, and age window to reduce variability; this enhances internal validity but may limit external validity. Future studies incorporating molecular readouts, standardized analgesia comparisons, and broader biological strata are warranted.
Conclusions
Histopathological and immunohistochemical data obtained from our study have demonstrated that melatonin administration can reverse the negative effects caused by zoledronic acid by enhancing the formation of new bone in the extraction cavity and decreasing the amount of necrotic bone.
Supplementary Information
Supplementary Material 1.
Publisher’s note
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Authors’ contributions
CGK: Supervision, Conceptualization, Visualization, Data curation, Writing- Original draft preparation, Writing-Reviewing and Editing. HTA: Investigation, Data curation, Validation, Investigation, Methodology. AY: Investigation, Methodology, Visualization. OO: Supervision, Writing- Reviewing and Editing.
Funding
The drugs, the effect of which would be investigated, experimental animals, and the consumables to be used in the study were provided with the project support provided by the Scientific Research Projects Department of Inonu University (project number TDH-2019-1862).
Data availability
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and participate to participate
All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Experimental Animals Ethics Committee of Inonu University, Faculty of Medicine (Protocol No: 2019/A-29). The animals were obtained from the institutional Experimental Animal Research Center. As only institutionally bred animals were used, informed owner consent was not applicable.
Consent for publication
Not applicable.
Competing interests
All authors state that they have no conflicts of interest.
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