Study setting
The study was conducted on 2023 in the animal household of the College of Veterinary Medicine/University of Mosul. The University Mosul Dental School’s research ethics committee reviewed and endorsed the research protocol (Approval No. UoM. Dent. 23/18 on 02.05.2023). A comprehensive examination was carried out by veterinarians on Awassi sheep to assess their general, periodontal, and dental health. The sheep were quarantined for weeks before the beginning of the experiment
(Henry et al., 2022). The following formula was performed to calculate the sample size:
n = DF/k + 1
Where,
n = Number of subjects per group,
DF= The between-subject error (that is, the within-subject DF) based on the acceptable range was set to 10.
k = Number of groups.
Following the necessary adjustments to the obtained value, the final sample size was calculated as the sample size per group.
Study design
The study comprised six adult male sheep (weight 55 kg; age 3 years). Each sheep possesses a total of eight permanent incisors, with four located on each side. To determine the effects of melatonin, a prospective randomized split-mouth experimental trial was conducted. The right and left sides of the sheep were randomly assigned to either the melatonin group (MG) or the control group (CG) based on a digitally produced sequence of random numbers. The study involved the second and fourth incisors on each side and the third incisor on each side was removed. Throughout the intended experiment, the sheep were anaesthetized numerous times with ketamine (22 mg/kg IM) and xylazine (0.2 mg/kg IM). Each sheep had its third incisors removed on both the right and left sides and the areas were permitted to heal for a week. The sheep were anaesthetized and the labial surfaces of the second and fourth incisors on each quadrant were polished using pumice powder (Produits Dentaires SA., Switzerland) and a dental polishing brush using low-speed handpiece (Coxo Medical Instrument CO., LTD., China). Then, teeth surfaces were etched with phosphoric acid 38% (Pulpdent Co., USA) for thirty seconds, washed with water for fifteen seconds and air dried. Standard 0.022 slot edgewise metal brackets of equilibrium® 2 series (Dentaurum GmbH and Co., Germany) were bonded using TrueBond LC (IOS Corp., USA) and cured by Eighteeth Curing Pen E (Changzhou Sifary Medical Technology Co., China) for twenty seconds from each direction. Each bracket was attached at the midpoint of the tooth’s long axis, in a mesiodistal direction. The brackets were all of equal height, to ensure there were no differences in vertical alignment among the bonded incisors. The two incisors on either side were then joined together using sectional 0.017´0.025 stainless steel orthodontic archwire and their ends were bent to make a non-traumatic end. An elastomeric chain (Orthometric, Marília, Brazil) was applied to the second and fourth incisors to achieve a starting force of 150 grams. Three times per week, the elastomeric chain was replaced until the second and fourth incisors had completely approximated. Then the brackets on the approximated teeth were passively ligated using stainless steel ligature wire 0.01 inch. (Dentaurum GmbH and Co., Germany) for four weeks (Fig 1).
Melatonin (N-Acetyl-5-methoxytryptamine) was prepared for injection by dissolving 10 mg melatonin powder (Chem-Impex INTL INC., USA) in 1 millilitre of 1% dimethyl sulfoxide (Chem-Impex INTL INC., USA) solution.
A melatonin injection was administered to a randomly selected side of each animal utilizing a disposable one-unit insulin syringe equipped with a 25-gauge microneedle. (Forlong Medical Corp., China). The injection was given adjacent and parallel to the mesial surface of the second incisor and the distal surface of the fourth incisor at the mucogingival junction, penetrating through the attached gingiva into the oral mucosa. The dose was divided, with 0.5 ml injected into the labial side and another 0.5 ml injected into the lingual side of the vestibular mucosa.
A volume of 1 millilitre of 1% dimethyl sulfoxide solution was injected on the opposite side using the same injection technique, serving as a control. The injection was performed two times weekly for four weeks.
Clinical assessment
After completing the injection procedure, the fixed orthodontic device was debonded utilizing bracket removal pliers (Dentaurum., Germany) and an intraoral impression Zhermack Zetaplus A Silicone impression Material (Badia Polesine (RO), Italy) loaded into a customized resin tray will be obtained immediately after the orthodontic appliances removal and 21, 42 days later of post-orthodontic relapse to create dental models, All impressions were poured with improved die stone (Elite Rock Dental Stone; Zhermack, Badia Polesine, Italy). All stone models were scanned with an E1 lab scanner (3Shape Co., USA) to generate three-dimensional models in stl. file format. Utilizing the Viewbox software (V 4.0.1.7; dHal Software, Greece), The models were aligned according to the mandibular occlusal plane, which was determined by the position of the incisal edge of the lower incisors. Two planes were constructed at right angles to the occlusal plane of the mandible. The first plane was initially sketched on the distal surface of the second incisor, precisely at the location of the furthest contact region. A second plane was sketched to make contact with the most mesial contact area of the mesial surface of the fourth incisor. The post-orthodontic relapse is quantified by measuring the linear distance, which runs parallel to the mandibular occlusal reference plane, between the two constructed planes. Measurements were obtained utilizing the ruler tool in the Viewbox software, Fig (2,3).
On 21 and 42 days of orthodontic appliance removal 3 sheep were slaughtered, and a dense diamond saw (Cadence Inc., USA) was utilized to precisely cut the anterior portion of each mandible 5 mm distal to the fourth incisor. A sterile 4-mm diameter trephine drill (NTI-Kahla GmbH, Germany) using a low-speed handpiece (COXO Medical Instrument Co., Ltd., China) and profuse Sodium Chloride 0.9% Irrigation Solution (Jedux Parenteral Private Limited, India) was employed to remove bone and root tissue from the distal aspect of the fourth incisor of all samples parallel to the visible inclination of the fourth incisor root through full thickness of the alveolar bone. Then, the removed tissues were placed in sterile micro-centrifuge tubes containing 1.5 ml of DNA/RNA shield lysis solution (Zymoresearch, Irvine, USA) and stored at -20°C for further RNA extraction and qRT-PCR analysis
Histological assessment
The specimens were fixed for three days in a 10% neutral buffered formalin solution, followed by decalcification for five to six weeks in an 8% hydrochloric and 8% formic acid solution. Histological section were treated based on standard protocol.
RNA extraction and cDNA synthesis
Samples in micro-centrifuge tubes were crushed using a pestle and mortar and subjected to ultrasonic cell disruption using Vibra-Cell 500 processor (Sonics and Materials, Inc., Newtown, USA). RNA was extracted from the homogenate according to the manufacturer’s protocol using AddPrep Total RNA Extraction Kit (Add Bio Inc., Yuseong-gu, South Korea). Then, the extracted RNA concentration was quantified spectrometrically using a Nanodrop 2000 (Thermofisher Scientific Inc., USA). 2 µl of extracted RNA was used for the synthesis of complementary DNA (cDNA) using AddScript cDNA Synthesis Kit (Add Bio Inc., Yuseong-gu, South Korea). Thermal cycler steps of cDNA Reverse Transcription were 25oC for five minutes, then 42oC for thirty minutes and lastly 5
oC for five minutes. The resultant cDNA was stored at -20
oC until further qRT-PCR was carried out.
Gene selection and primer sequence
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the housekeeping gene and Runt-related transcription factor 2 (RUNX2) as the target gene (Table 1) (
Dash and Singh, 2024).
Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)
The mRNA expression levels of the reference and target genes were determined using Sybr green quantitative reverse transcriptase polymerase chain reaction (qRT PCR) assays as per manufacturer instruction (Add Bio Inc., Yuseong-gu, South Korea) (Table 2)
(Panigrahy et al., 2023; Vyas et al., 2022).
Statistical analysis
Statistical analysis was conducted using SPSS V26 (Statistical Package for Social Science, IBM Inc., USA). The data were tested for their normal distribution by using the Shapiro-Walk test. Comparison between the Melatonin group (MG) and Control group (CG) regarding relapse distance, periodontal ligament width and new bone formation area was done on 21 and 42 days respectively after removal of the orthodontic appliance using independent sample t-test or Mann Whitney U test depending on a normal distribution of data. A significance level of 0.05 (two-tailed) was used to determine statistical significance for all analyses.