Preparation of tea tree oil gel
Tea tree oil 5% gel (Sigma Aldrich® Steinheim, Germany) for local sub gingival application was prepared by the Department of Pharmaceutics, Faculty of Pharmacy.
The gelling agent; Carbopol 940 (1% w/v) was first soaked in distilled water for 2 h then TTO dissolved in an appropriate amount of propylene glycol was added to the Carbopol dispersion. Methyl paraben 0.2% w/v dissolved in preheated water was used as a preservative. The gel mixture was then magnetically stirred for 30 min. Finally, pH was adjusted using 1 N NaOH added dropwise with gentle stirring with a spatula until the desired pH value (6.5–7) was reached. The gel was sterilized by autoclaving at 110 °C for 20 min.
Study population
A six-month, parallel randomized controlled clinical trial was conducted on thirty patients at the Department of Oral Medicine, Periodontology, Diagnosis and Oral Radiology, Faculty of Dentistry, Alexandria University between November 2019 and August 2020. Ethical approval was obtained from the Research Ethics Committee at the Faculty of Dentistry, Alexandria University, Egypt (IRBNO: 00010556- IORG:0,008839). The study was registered at clinicaltrials.gov NCT04769271, on 24/2/2021.
The objective of the study and the methods used in it were described to the patients and all of them signed an informed consent. The study was performed in accordance with the Helsinki declaration.
Inclusion criteria
Patients of both sexes with age range 25–50 participated in the study if diagnosed with stage 2, grade B periodontitis according to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions [19].
This diagnosis was confirmed by the presence of CAL 3–4 mm, BOP and radiographic horizontal bone loss related to the coronal third of the root (15%-33%). The patients also showed no teeth loss due to periodontitis. On assessing the severity and distribution of the disease, these patients had CAL 3–4 mm < 30% of the teeth involved with periodontitis. Patients with CAL caused by non-periodontal causes were excluded from the study.
Moreover, the grade of periodontitis was assessed by radiographic bone loss/age % which was 0.25–1% [37]. Radiographic bone loss was assessed from the tooth showing the most bone destruction. Only patients having CAL 3–4 mm and BOP in proximal tooth surface and who could maintain an O'Leary plaque index ≤ 10% proceeded into the study [38].
Exclusion criteria
Patients were excluded if they had any systemic disease that may affect the treatment outcomes, or if the patients were smokers, pregnant, or receiving contraindicated medications, chemotherapy, or radiotherapy in the previous year [10]. Patients were screened against these criteria during the study period and recruited if they were eligible.
Sample size estimation
Sample size was estimated assuming alpha error = 5% and study power = 80%. Mean ± SD pentraxin levels were = 0.12 ± 0.03 when tea tree oil was used after SRP, and = 0.35 ± 0.24 when tea tree oil was not used [23]. MMP-8 is a periodontal inflammatory mediator, and is assumed to be more predictive in diagnosing periodontitis than pentraxin [39]. Based on comparison of means, sample size was calculated [31] to be 10 per group, increased to 11 to compensate for loss during follow up. The minimum total sample size = 22.
Grouping and randomization
Thirty patients with stage 2 periodontitis were equally divided into 2 groups: Group I (control group, mean age28.9 ± 6.3 years) was treated with SRP only and Group II (test group, mean age 30.5 ± 5.6 years) was treated with SRP combined with intrapocket application of TTO gel. Patients complying with the inclusion criteria were randomly assigned using a computer-generated list of random numbers to one of the two groups [40] in blocks of four and the allocation sequence was handed to an assistant not directly involved with the study. Patients were sequentially numbered in a list and the number of each patient was written on an opaque envelope that included identical pieces of paper that were folded after adding the group to which the patient was assigned. On the day of the intervention, the assistant retrieved the envelope carrying the patient number, opened it and the intervention assigned to the patient was provided.
Treatment
After completion of baseline measurements for both clinical and biochemical evaluation, the two groups received full mouth SRP using hand instruments and ultrasonic scalers and oral hygiene instructions.
For test group only, partial isolation was performed for the tested site followed by drying with air. A bent, blunt-end needle syringe was used to inject the TTO gel in the pocket assuring that its tip reaches the deepest point in the pocket. As the gel appeared at the gingival margin the needle was removed slowly from the pocket (as shown in Fig. 1). After application of approximately 0.5 mL of the gel, excess gel was removed by a sterile gauze in order to avoid any spillover effect or possible systemic effect by swallowing the gel.
Patients were instructed to follow strict oral hygiene measures during the study period. They were also asked not to use the toothbrush at selected site after TTO gel application for 24 h and not to chew hard or sticky foods at the gel placement sites. On the subsequent recall visit, the clinical parameters and any adverse effects were recorded.
Clinical assessment
Periodontal clinical parameters were recorded at 3 and 6 months after treatment, including: PPD, CAL, GI [41] and BOP [42] which was assessed within 15 s after probing, using a dichotomous scoring system (+ and −) for presence or absence, respectively). All measurements were recorded at tested sites with graduated William's probe by a single blinded pre-calibrated clinician who carried out double assessments for the clinical examination. Intraexaminer reliability was for probing depth 0.380 per test group and 0.250 per control group. For CAL was 0.392 per test group and − 0.142 per control group as personal coefficient test.
Biochemical assessment
In all patients, GCF sample was taken from the area showing the deepest pocket depth around the area received the treatment, from study and control sides. The samples were collected at baseline, and after 1, 3 and 6 months following treatment. For each site GCF sample was collected by using prefabricated paper points [43], (as shown in Fig. 2) which were inserted into the pockets after drying and complete isolation of saliva using cotton rolls and high suction unit, until resistance is felt, and kept there for 30 s [43]. Any paper point with blood contamination was discarded. The samples were diluted in phosphate buffer saline (PBS) up to 1 ml [44]. After waiting for 15 min, the paper points were removed and the samples were frozen at − 20 °C for analysis of MMP-8 [44].
Human Matrix metalloproteinase 8/Neutrophil collagenase (MMP-8) ELISA Kit (Biovision Company., Ltd, Shanghai, China) was used and analysis was done according to the manufacturer instructions.
The method used was Sandwich-ELISA technique. The Microelisa stripplate present in this kit had been pre-coated with an antibody specific to MMP-8. Samples were added to the appropriate Microelisa stripplate wells and combined to the specific antibody. Then a Horseradish peroxidase (HRP)-conjugated antibody specific for MMP-8 was added to each Microelisa stripplate well and incubated, which was followed by washing away of the free components. Then Tetramethylbenzidine (TMB) substrate solution was added to each well. Only those wells that contain MMP-8 and HRP conjugated MMP-8 antibody appeared blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 nm. The OD value was proportional to the concentration of MMP-8. The concentration of MMP-8 in the samples was calculated using a standard curve.
Statistical analysis
Data were sent to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The patient was the unit of analysis for both clinical and biochemical parameters at baseline and follow-up assessments. Qualitative data were in percent number. The Kolmogorov–Smirnov test was used to measure the distribution and normality. Quantitative data were in range (minimum and maximum), mean, standard deviation, median and interquartile range (IQR). Comparison of gender and age between the two study groups were done using chi-square and Student t- tests, respectively. Comparisons of periodontal parameters and MMP-8 level between the two study groups were done using Mann–Whitney U test. Spearman correlation was performed to assess the relation between different periodontal parameters and MMP-8 level. Significance of the obtained results was judged at the 5% level.
