Evaluation of the effects of melatonin on the post-extraction sockets

of wistar rats exposed to a therapeutic dose of alendronate

Abstract

Objectives: The objective was to evaluate the effects of melatonin administration on the post-
extraction alveoli of rats exposed to alendronate.
Materials and methods: This was a pre-clinical cohort study of adult male Wistar rats, conducted
over a period of 5 months in an animal facility suitable for animal experiments.
Rats in groups 1 and 2 received melatonin and vitamin D respectively per os at a rate of 1
mg/kg/week and 0.25 mg/kg twice a week for 4 weeks. Group 3 received saline subcutaneously.
All rats received alendronate at a dose of 0.05 mg/ kg twice/week for 8 weeks. Four weeks after
drug administration, a dental extraction was performed along with blood sampling to determine
serum levels of RANKL and OPG. At eight weeks, the rats were sacrificed and the jaw blocks
were removed for CT and histological examination.
Results: Overall, three groups of 12 rats were formed. From the fourth to the eighth week, the
frequency of stage 3 osteonecrotic lesions in the post-extraction sockets had increased in the
control group from 8.33% to 66.6%. In the melatonin and vitamin D groups, the frequency of
corresponding stage 3 osteonecrotic lesions increased from 0 to 17% and 0 to 8.33%
respectively.
Conclusion: Oral administration of melatonin prior to tooth extraction in alendronate-exposed
rats reduces the risk of developing osteonecrotic jaw lesions.
Keywords: melatonin, post-extraction socket, alendronate, Wistar rat
 1. Introduction
Oral hygiene, patient education and appropriate oral care are essential for the prevention of drug-
related osteonecrosis of the jaws (MRONJ), formerly known as bisphosphonate-related
osteonecrosis of the jaws (BRONJ) [1]. However, the usefulness of a preventive drug holiday
during tooth extraction for patients receiving bisphosphonate remains controversial. Although
there is little evidence that temporary discontinuation of bisphosphonates(BP) is effective in
preventing BRONJ [ [1], others suggest that discontinuation of BP therapy for about 2 months
should be considered in patients at high risk of BRONJ who have been receiving BP for more
than 4 years, provided that they are also at low risk of fracture [2].
Several studies do not support the principles of drug discharge. Based on the physicochemical
properties of BPs, which deposit and persist in bone over a long period of time[3], it is unlikely
that short-term BP drug withdrawal can prevent BRONJ. Furthermore, discontinuation of
treatment may worsen osteoporosis by decreasing bone mineral density and increasing the risk of
fractures[4,5]. Given the extremely low incidence of BRONJ in patients with osteoporosis, the
benefits of BPs for fracture prevention outweigh the risks of BRONJ[5]. Furthermore, the key
elements in the diagnosis of BRONJ are, in order of importance, bisphosphonate exposure,
clinical signs and very little mention is made of CT, histological and biochemical features.
It is therefore urgent to design prevention protocols adapted to real clinical situations. We
therefore propose melatonin because it acts as a protector in osteoporosis, osteoarthritis and
periodontitis by exerting multiple effects [6].
The present study therefore aimed to evaluate the clinical, CT, histological and biochemical
effects of melatonin on the post-extraction sockets of wistar rats exposed to a therapeutic dose of
alendronate
2. Materials and methods
2.1 study design
This was a single preclinical blind study in male rats (Rattus norvegicus albinus). The study was
conducted from March 2021 to July 2021, a five-month period in an animal facility adapted to
animal and experimental procedures. The experimental procedures were approved by an ethics
committee and conducted according to the principles of the 'ARRIVE' guidelines [7].
Rats of 12 weeks of age and body weight ≥ 230 g were housed at standard temperature and
humidity and with a 12-hour light/dark cycle. Food and filtered water were delivered as much as
possible. Body weight was measured weekly.
2.2 Experimental design and surgical procedures
After a 2-week acclimatisation period, the animals were divided into strata according to body
weight, melatonin intake (group I), vitamin D intake (group II or positive control) and negative
control group (group III). (see table I)
Melatonin was administered between 4 and 5 pm, when its blood concentration was considered
minimal[8].
Saline solution (0.9%) was chosen as a wound control because it is an isotonic solution that does
not interfere with the normal healing process, damage tissue, cause sensitization or allergies or
alter the normal bacterial flora of the skin [9]. It was used subcutaneously because oral NaCl 3%
(0.513 mmol/mL) syringes (injectable solution used for the oral route) were not available.
During the 4th experimental week, blood samples were taken to determine serum levels of
receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG),
accompanied by a clinical examination for possible lesions. In addition, the maxillary and left
mandibular first molars of each rat were extracted under anaesthesia [10]. The animal was
placed in dorsal recumbency with the oral cavity held open with retractors. The tooth was
removed using a suitable forceps and the post-extraction socket sutured as shown in Figure 1.
After surgery, all animals were given a daily dose of 25 mg/kg ampicillin and 0.075 mg/ kg
Tramadol for three days.
The time of each surgical procedure was recorded
2.3 CT analysis
Four weeks after tooth extraction, a second blood sample was taken to determine the RANKL
and OPG markers. The rats were then sacrificed and their jaws were removed in their totality by
decapitation to be inserted in the polyacrylic resin. They were scanned. Projection images of the
raw data were reconstructed and analyzed using dentascan.
A circular area of predefined size (0.001 to 0.002) was selected as the region of interest in the
two-dimensional images of the post-extraction socket of each rat. The slice level varied from
0.625 to 1 mm. Trabecular and cortical regions were defined with four phantoms using a
histogram volume with a threshold value of 690 mg and 1452 mg HA\ cm3.
Cortical and trabecular volumetric bone mineral density (vBMD; mg HA/cm 3), and
morphometric indices of the post-extraction zone were measured on axial, coronal and sagittal
sections.
2.4 Histopathological analysis
The jaw specimens previously used for CT analysis were stained with hematoxylin and eosin.
Several histological parameters were assessed:
 The number of apoptotic osteoblasts and osteoclasts
 Foci of osteonecrosis: defined as 8-10 adjacent empty lacunae (without
 osteocytes) in the alveolar bone;
 Number of fibroblasts: number of fibroblasts within the alveoli near the surface of the
alveolar bone was counted and each was classified as
- Grade 0 (less than 30 cells), -Grade 1 (31-50 cells),
- Grade 2 (51-75 cells), - Grade 3 (more than 76 cells) [11].
 Inflammation intensity: the severity of inflammation was measured by counting the number
of lymphoplasmocytes on the surface of the alveolar bone around the alveoli and classified as
- Grade 0 (no inflammation), -Grade I (less than 10 lymphoplasmocytes),
- Grade II (11-25 lymphoplasmocytes), -Grade III (26-50 lymphoplasmocytes)
- Grade IV (more than 50 lymphoplasmocytes) [11].
2.5 Statistical methods
Friedman's test, one-way ANOVA were used to compare medians and means respectively.
Tukey's post hoc test to define significantly different groups. A chi-square test was used to
compare frequencies.
The significance level was set at 0.05
The summary of the experimental procedure is shown in Figure 2.
3. Results
3.1 Characteristics of the rats included in the study
Eight weeks after the start of the trial, rats in the melatonin and control groups had lost weight,
but none had lost more than 10%. In addition, the mean body weight values of group 2 increased
slightly in the fourth week compared to the baseline measurements shown in figure 3.
The animals tolerated medication, anaesthesia and dental extractions well. Indicators of surgical
trauma (extraction time and frequency of alveolar fractures) did not differ between groups (p=
0.1). (See Figure 4).

3.2 Establishment of an animal model for the prevention of alendronate-associated

osteonecrosis of the jaw by melatonin.
In the clinical evaluation, before the first administration of the drugs, none of the animals in the
three groups showed any lesions of the oral mucosa.
At eight weeks, the incidence of stage 3 osteonecrotic lesions had increased in the control group
by 58.33%, i.e. 7 more rats. However, in the melatonin and vitamin D groups, the incidence of
stage 3 osteonecrotic lesions was increased by 17% and 8.33% respectively. The following table
(table II) shows the results of the fourth and eighth week assessment and the representative
photographs in figure 5.
3.3 Comparative evaluation of melatonin and vitamin D on trabecular volumetric bone
mineral density in the jaws of rats exposed to alendronate.
The mean values of trabecular bone mineral densities were higher for the melatonin and vitamin
D exposed groups of rats (899.25± 351.3 mg HA/cm 3 ; 1088.9± 395.5mg/cm3 ) respectively than
in the control group (426.5± 591.8 mg HA /cm3) (p =0,024). (See figure 6)
The following graph also shows that the median trabecular BMDs of the melatonin and vitamin
D exposed rat groups were above 1000 mg HA/cm3.
The Dentascan results show a homogenization of the trabecular mineralization in relation to the
high values of the bone mineral densities. However, the low values of vBMD in the control
group are related to a heterogeneous appearance of trabecular mineralization as presented in
figure 7.

3.4 Characterization of morphometric indices of drug-exposed rat groups

3.4.1 For trabecular thickness (Tb.Th)

The mean values were 0.79±0.24 mm in G1, 0.97±0.57 mm in G2 and 0.31±0.18 mm in G3.
For all three study groups, there was a significant correlation between trabecular thickness and
trabecular vBMD. (G1: r1'=0.609, p=0.035; G2: r2'=0.724, p=0.008; G3: r3'= -0.37 p=0.000)
3.4.2 For the number of trabeculae (N. Tb)
Ten rats (83.3%) exposed to melatonin and 8 rats (66.6%) exposed to vitamin D had a number of
trabeculae greater than or equal to 2. In the control group, two rats (16.6%) had a number of
trabeculae greater than or equal to 2. (p=0.01)

3.4 .3 For the connectivity density (Conn D)

The following figure shows that in the test groups, the mean values for the connectivity
density were significantly higher than in the control group with a significant difference
(p<0.001). (see figure 8)

On observation of the oblique sagittal sections as shown in figure 9, the trabeculae of the
melatonin and vitamin D exposed rat groups had a regular figure eight shape interconnected by
bony trabeculae with a higher and more homogeneous grey level. In contrast, in the control
group the trabeculae were separated by locally hypodense bony trabeculae sometimes
accompanied by a break in the intertrabecular bony septa.
3.4.4 Comparison of osteoblast number and necrosis level of the study rat groups

Osteonecrosis was observed in 8.3%, 0% and 66.6% of the first, second and third groups,
respectively, and a chi-square test showed a significant difference between the 3 groups (P =
0.008).
The mean number of osteoblasts was 8.91± 4.85, 10.91± 6 and 9.45± 10 in the first, second and
third group, respectively, and a Friedman test showed no significant difference between the 3
groups (P = 0.45). (See figure 10)
3.4.5 Other histological features related to melatonin administration in rats exposed to
alendronate

More than half of the rats exposed to melatonin and vitamin D had grade 2 vascularization, only
33.3% of the rats in the control group had grade 2 vascularization. The rest of the histological
features are presented in the following table (table III).
3.4.6 Effect of melatonin on serum expression of the cytokines RANKL and OPG in rats after
prolonged treatment with alendronate
Between the fourth and eighth experimental weeks, the decrease in serum RANKL
concentrations was greater in the control group (14.8±5.4 pg/ml) than in the melatonin and
vitamin D groups, 9.46±14.64 pg/ml and 2±2.5 pg/ml respectively (p=0.01). (p = 0,01). (See
table IV)
At the fourth week, the lowest mean OPG value was found in the control group, 58.29± 7.3
pg/ml, compared to the values of the melatonin and vitamin D exposed groups, 93.8 ± 12.8 pg/ml
and 85.02± 13.8 pg/ml (p = 0.008). (See table V)
The RANKL/OPG ratio was greater in the melatonin (1.17) and vitamin D (1.12) exposed rats
than in the controls (0.97) (p < 0.01).
4. Discussion
4.1 Forces
The main strength of this work is the reliability of the study design and methodology, which can
be easily replicated. osteonecrosis of the jaw (ONJ) was successfully reproduced in Group 3, as
described in previous studies, with the same doses of alendronate as those administered to
humans.
4.2 Limits
In this part of the work the sample sizes were small, the follow-up period was only 8 weeks and
the population was rats, so the results may not be generalizable to clinical populations at this
time, but recommendations for next steps in the research could be made. In our study the
resolution of the multi-bar scanner used was in the range of 300-500 μm in-plane, which was still
insufficient to analyze trabeculae individually.
4.3 Melatonin prevents the occurrence of alendronate-related osteonecrosis of the jaw in
wistar rats
We investigated the preventive effect of oral administration of melatonin and vitamin D on the
incidence of ONJ after tooth extraction.
The greater amount of osteonecrosis in the third group compared to the first and second group
was predictable and similar to some studies [12,13]. Tuba Develi et al. reported a rate of 0% in a
group of rats exposed to relaxin and 90% in the group exposed to zoledronate[14]. This high
incidence of ONJ in the control group was probably due to the dose, type of bisphosphonate and
duration of administration[14]. The lower incidence of ONJ in the melatonin-treated rats
suggests the likelihood of effectiveness of this drug in preventing the development of ONJ in
patients taking bisphosphonates.
To date, no studies (human and animal) have examined ONJ after administration of melatonin at
-1
1 mg-kg per week. However, previous studies indicate that melatonin has antioxidant
properties and is a free radical scavenger [15,16], and according to a study by Cutando et al.
topical application of melatonin to tooth extraction sockets eliminated both oxidative stress and
its effects and accelerated healing of the tooth sockets[16]. On the other hand, melatonin
increases and accelerates the differentiation of osteoblast precursor cells [15]. These properties
may partly explain the reduction in the occurrence of osteonecrosis in the dental sockets of
melatonin-treated rats.
4.4 Melatonin promotes bone mineralization in rats exposed to long-term alendronate
treatment
In our study, the mean trabecular mineral density measured in rats treated with melatonin and
vitamin D was significantly higher than in the control group. At the trabecular level this
difference was greater with vitamin D (662.4 ± 196.3 mg HA/cm3) than with melatonin (472.75
± 240.5 mg HA/cm3). These values are similar to those obtained previously comparing test
groups exposed to alendronate (G2= 486.22±94.76 mg HA/cm 2; G3= 584.9±42.47 mg HA/cm2;
G1= 421.61± 125.81 mg HA/cm2) with the control group [17]. Yi Zhou et al. reported similar
results for the effect of melatonin at the same dose on femurs of ovariectomized mice[18].
It is also important to remember that fully mineralized bone has a vBMD of 1200 mg.
HA/cm3[19,20]. Therefore, the medians found in the test groups above 1000 mg HA/cm 3 confirm
the influence of melatonin as well as vitamin D on trabecular mineralization in rats exposed to
alendronate for 8 weeks. These observations are confirmed by high values for trabecular
morphometric indices in the test groups.
Indeed, for both groups, trabecular BMD was positively correlated with trabecular thickness and
number of trabeculae. This was also the case for the density connectivity (ConnD), which counts
the number of nodes in the trabecular network, i.e. the number of intersections of trabeculae with
each other, and then relates this to the total volume [21]. Therefore, a strong ConnD indicates
that the trabecular network is structured and connected. All the above results suggest that
melatonin and vitamin D may inhibit the osteonecrosis process.
While alendronate has the effect of decreasing the frequency of activation of bone remodeling
and thus decreasing the likelihood of a BSU (Bone Structural Unit) being resorbed before it
reaches a maximum degree of demineralization during osteoporosis[22], the situation is different
in the post-extraction socket. Indeed, the environment is quite different, as shortly after a tooth
extraction a high degree of resorption takes place within the socket to remove necrotic bone and
bone debris [23,24]. This will result in a very large release of alendronate which will bind to
osteoclasts, exposing osteocytes to an environment rich in bacterial toxins, inflammatory
cytokines and oxidative stress [23,25]. As a result, although the rate of remodeling will be
reduced, the BSU will inevitably have already been exposed to significant demineralization,
thereby limiting its reminéralisation, hence the reduction in vBMD in the control group.
In contrast, bone loss and microstructure disorders in rats exposed to alendronate were reversed
by melatonin, due to its anabolic and antiresorptive effects [26].
4.5 Melatonin prevents the occurrence of osteonecrotic defects by promoting osteogenesis
without osteoclast apoptosis
The mean value of apoptotic osteoclasts in the melatonin and vitamin D groups was significantly
lower than in the control group. This trend was also observed with the mean values of the
number of empty lacunae representing osteonecrosis. The differences were statistically
significant (P = 0.0001). The high values of apoptotic osteoclasts in the control group and low
values in the test groups are well documented and in agreement with previous studies [23,27].
But it was the study by Afshin Yadegari et al. that reported values most similar to ours. The
mean values of apoptotic osteoclasts in the melatonin and zoledronate-exposed rats were 0.5 and
5 in the zoledronic acid-exposed group [8].
The decrease in the number of apoptotic osteoclasts can be attributed to melatonin. Melatonin
inhibits osteoclast activation by preventing the binding of RANKL produced by osteoblasts to
RANK from osteoclasts[25]. However, it does not induce apoptosis of osteoclasts as observed in
our study.
According to a study by Dayisoylu et al, the release of bisphosphonates from the hydroxyapatite
of the bone at the site of dental extraction causes apoptosis of target cells including osteoclasts
and requires inflammation and inflammatory mediators [11]. We found that none of the rats
exposed to melatonin had an inflammatory grade greater than 2, i.e. greater than 25
lymphoplasmocytes. Thus, melatonin has anti-inflammatory, anti-free radical and anti-oxidant
effects on stress[15] and may be effective in preventing the induction of apoptosis in osteoclasts
by bisphosphonates. This suggests that melatonin may reduce rather than eliminate the bone
resorption activity of osteoclasts, allowing bone destruction to be controlled more selectively and
circumventing the negative effects of general osteoclast suppression.
4.6 Melatonin regulates RANKL and OPG expression in rats exposed to prolonged
alendronate treatment
The RANKL/RANK/OPG system is one of the most important signaling pathways regulating
bone resorption and osteogenesis [28]. RANKL is primarily present on the surface of osteoblast
precursors and binds to the RANK receptor on the surface of osteoclast precursors, activating the
signal transduction pathway to promote osteoclast precursor differentiation and maturation and
enhance bone resorption[28]. OPG is a key cytokine for osteoblast precursors to inhibit bone
resorption [28]. It can compete with RANKL to bind to RANK and inhibit osteoclast formation
and activation, thereby inhibiting bone resorption by osteoclasts.
In the current study, 4 weeks after extraction, we found less inhibition of RANKL expression
and a significant increase in OPG expression in the test groups compared with the controls. This
is also reflected in a higher RANKL/OPG ratio in the melatonin-treated group of rats than in the
vitamin D-exposed groups and the negative control. From these facts, it can be said that
melatonin down-regulates osteoclast activation by preventing the binding of osteoblast RANKL
to osteoclast RANK [38,39]. However, it does not induce apoptosis of osteoclasts as observed in
our study. Thus, the maturing osteoclasts will be able to phagocytose the necrotic mineral matrix,
indirectly promoting the differentiation of osteoblasts [40,41] and the formation of a new
mineralized matrix [40].
5. Conclusion
At the end of this research, we show that inflammatory conditions leading to osteonecrosis of
jaw and triggered by a combination of dental extraction and treatment with alendronate are
inhibited by the administration of melatonin without interruption of alendronate. In addition,
trabecular volumetric bone mineral densities and morphometric indices of melatonin-treated rats
showed a significant difference from controls, evidence of melatonin’s contribution to preserving
the mineralization of the bone matrix in exposed rats. This work also highlighted the interest of
RANKL cytokines and OPG in the management of osteonecrosis of the jaw before and after
dental extraction.
Funding: This research has not received any specific grants from funding agencies in the public,
commercial or non-profit sectors. Institutional Review Board Statement. The study was
conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics
Committee of the Faculty of Medicine and Biomedical Sciences of Yaoundé.

Acknowledgements: my thanks to all the research team

Disclosures: The authors declare that they have no competing interest.

Patient Permission/Patient Consent statement: obtained from study patients

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