The response of salivary proinflammatory biomarkers to tooth extraction in individuals with type II diabetes mellitus

Twenty-seven patients were recruited from the University Dental Hospital of Sharjah between 1/10/2021 and 21/9/2022. Among them, 20 patients had type II DM, and seven were healthy control subjects. The study sample consisted of patients whose teeth were scheduled for extraction in accordance with the oral surgery protocol. The study included participants aged older than 18 years who had previously been diagnosed with type II DM for at least 1 year. Patients with type II DM had to have a recent glycated hemoglobin level (HbA1c) below 7.5% according to the glycated hemoglobin test and a random glucose level below 180 mg/dL according to glucometer-based measurements. In the DM group, all patients were well controlled and had no diabetes-related systemic disease. None of the patients in the control group had a history of systemic disease. The indications for extraction in both groups were nonrestorable necrotic teeth with no periodontal or apical lesions. Patients who smoked or used any type of tobacco were excluded, as were patients with immunological diseases or immunosuppressive treatments, antibiotic or anti-inflammatory therapy, acute infections or abscesses originating from the dental system, pregnant or breastfeeding women, those who needed prophylactic antibiotics before dental treatments, and those with any other systemic conditions or diseases that would influence current or previous inflammatory responses.

The sample size was calculated using the statistical software G*Power (ver. 3.1.9.7 software; University of Düsseldorf, Germany). Based on the information provided by Gutiérrez-Corrales et al. [10], we used an effect size of 0.5, an 80% power, and a 5% error, and 21 patients were required. Therefore, this study included a convenience sample of 27 patients. The purpose of the study was explained to the participants before an informed consent form was signed and obtained from each of them. The study was conducted in accordance with the guidelines of the Declaration of Helsinki. The University of Sharjah Human Research and Ethics Committee approved this study (REC-21-06-01-03-S).

A 5-ml sample of unstimulated whole saliva was collected from each patient three times: before extraction, 2 hours after extraction, and 2 days after extraction. Before sample collection, patients were asked not to eat or drink for 1 hour. The participants were asked to gently rinse their mouth with tap water for 2 minutes and then spit into the collection tube over a period of 5 minutes. To prevent disturbing blood clots, patients were instructed to allow passive saliva to flow into the collection tube after extraction. After being stored in an ice-cold box, all the samples were frozen at − 80 °C until analysis.

The TNF-α, IL-1β, IL-6, and IFN-γ concentrations in saliva were evaluated using a Luminex multiplex assay. All the collected samples were brought to room temperature and centrifuged at 4000 rcf for 15 minutes at 4 °C. The supernatant was transferred to a clean microcentrifuge tube, 75 µl of which was diluted with 75µl of calibrant diluent from the assay kit. The concentrations of the different cytokines were measured using a Human Magnetic Pre-Mixed Analyte Luminex Assay (R&D Systems, UK). The standard cocktail was prepared according to the manufacturer’s instructions, followed by 3-fold serial dilution to obtain 7 standards. After the preparation of all the standards and samples, 50 µl of the respective standards and samples were added to the respective wells of the 96-well plate, along with 50 µl of the premixed microparticle cocktail. The samples were incubated for 2 hours at room temperature on an orbital shaker at 800 rpm. Then, the wash buffer was diluted 25 times in distilled water and utilized for the washing steps performed using a Bio-Plex Washing Station (Bio-Rad Laboratories, USA). Next, 50 µl of the diluted biotin-antibody cocktail was added to the plate and left for 1 hour on an orbital shaker. Washing was repeated, followed by the addition of 50 µl of diluted streptavidin-PE for 30 minutes on an orbital shaker. Finally, the microparticles were resuspended in 100 µl of wash buffer, and fluorescence was detected using the Bio-Plex-200 system (Bio-Rad Laboratories, USA). Analysis of the data was performed using Bio-Plex Manager software (Bio-Rad Laboratories, USA). The standard curve was calculated using the 5-parameter logistic curve method. The respective fluorescence readings of the samples were converted to concentrations in pg/ml, which were subsequently multiplied by 2 to obtain the original concentrations of the samples.

The acute salivary proinflammatory cytokine response at baseline and after simple tooth extraction was investigated in 27 patients. Table 1 shows the demographic information of the participants.