Efficacy of Access Flap Surgery on Subgingival Peridontopatgogens in Individuals With Periodontitis

The participants in the present study were individuals diagnosed with stage III grade B periodontitis, as determined by the criteria established in the 2017 World Workshop for the Classification of Periodontal Diseases and Conditions.

Non-surgical periodontal treatment was administered to all quadrants in patients scheduled to participate in the study, using periodontal hand instruments (Gracey curettes, Hu-Friedy®; Chicago, IL) in conjunction with an ultrasonic device (Cavitron Plus, Dentsply®, Duisburg, Germany). Each patient underwent the first step of non-surgical periodontal treatment, which involved the mechanical removal of supragingival deposits, motivation, and personalized oral hygiene instructions, including interdental plaque control and toothbrushing using the modified Bass technique. In the first week following the initial session, the second step of the non-surgical periodontal treatment, which encompassed subgingival instrumentation (SI), was performed on all four quadrants in a single appointment administered by the same blinded operator. Patients did not receive any prescribed medication or mouthwash. At the one-month follow-up visit after SI (14 days pre-surgery), patients were clinically re-examined, and their compliance with the inclusion/exclusion criteria was reassessed. Patients requiring access flap surgery, indicated by the presence of deep residual pockets (PD ≥ 6 mm), were identified and underwent the randomization procedure. Different sites of participating patients scheduled for periodontal surgery were assigned as either access flap surgery sites (test group) or untreated sites (control group) through a randomization procedure. Other patients determined not to need access flap surgery were not included in the study and were instead enrolled in repeat subgingival instrumentation or a supportive periodontal care program.

Patients were assigned consecutive ascending numbers during the enrollment visit. Prior to the commencement of the study, the order of treatment quadrants was randomized by an investigator not directly involved in the examination or treatment process, using random sequences generated by a computer random-number generator. Allocation concealment was achieved using opaque sealed envelopes, sequentially numbered. Based on this process, the teeth on the right or left site were designated as the test group while those on the opposite site were assigned to the control group. The allocation remained concealed from the examiner throughout the periodontal examination and recordings, and the envelope was not opened by the operator until the first treatment visit.

Each patient who met the study criteria underwent one of the following: one side of the mouth was treated with AFS, while the contralateral quadrants served as the control. All the access surgeries were conducted by the same blinded periodontist Following local anesthesia, mucoperiosteal flap was elevated and handled with microsurgical instruments (Blades, scalpel, needle holder, scissor, and tweezer, Carl Martin GmbH, Solingen, Germany) in accordance with the simplified papilla preservation flap (SPFF) technique. Granulation tissue attached to the alveolar bone was excised to ensure complete access and optimal visibility of the root surfaces. In addition, instrumentation was performed using a combination of ultrasonic and hand instruments, and no alveolar bone modification was undertaken during the flap procedure. Subsequently, the mucoperiosteal flap was repositioned and primarily closed with proximal simple sutures employing monofilament suture material (5/0 Propilen, Dogsan, Türkiye). Patients were given post-operative recommendations; however, no medication was prescribed.

Periodontal measurements including probing depth (PD) (mm), clinical attachment level (CAL) (mm), bleeding on probing (BOP) (±), gingival index (GI), and plaque index (PI)were assessed at two distinct time intervals by a calibrated periodontist who was blinded to the experimental sites.20 These measurements were conducted on six different aspects of each tooth (disto-buccal/labial, mid-buccal/labial, mesio-buccal/labial, disto-lingual/palatinal, mid-lingual/palatinal, and mesio-lingual/palatinal) using a manual periodontal probe (Williams probe, Hu-Friedy®; Chicago, IL), and the average value was computed. To evaluate the healing of periodontal tissues, detailed periodontal scores were recorded prior to and four weeks after access flap surgery.

Among the periodontal scores recorded for each patient, four teeth (two for the treatment group and two for the control group) demonstrating the highest scores (PD ≥6 mm, CAL ≥5 mm) were designated as donor sites for subgingival plaque sampling. Sample collection was conducted before and one month after surgery by the investigator responsible for performing clinical measurements. Sampling sites were isolated using sterile cotton rolls, followed by the supragingival plaque removal from the tooth surface. Subsequently, sterilized paper points (Paperpoint #50, EndoPoint®, Paraiba do Sul, RJ, Brazil) were delicately inserted into the designated periodontal pockets, exerting gentle pressure until encountering mild resistance. The paper points were retained within the periodontal pocket for 20 seconds before being carefully transferred to sterile microcentrifuge tubes, securely capped for storage. The samples were stored in a -80°C freezer at Trakya University Technology Research and Development Application Center.

The molecular genetic identification and quantification of subgingival periodontopathogens were performed by an operator who was blinded to group distribution. The PureLink® Genomic DNA Mini Kit (INVITROGEN, Massachusetts, USA) was employed for DNA isolation. Samples within 1.5 ml centrifuge tubes were thawed at room temperature for dissolution after being retrieved from the -80°C freezer. DNA isolation proceeded according to the manufacturer's instructions, involving sequential steps of cell lysis, DNA binding to filter membranes, washing, and elution. Subsequently, the tubes containing the samples underwent centrifugation at 21,300 x g for 1 minute to procure pure genomic DNA of microorganisms. These isolated DNA samples were preserved at -20˚C until the PCR stage.

Within the Real-time quantitative PCR methodology, TaqMan® Probes (Applied Biosystems™, CA, USA) labeled with fluorochromes (fluorescent substances) at the 5' and 3' termini were utilized. The TaqMan Multiplex Master kit is a convenient pre-mixture of all components necessary, aside from primers and probes, to perform qPCR. It contains DNA polymerase, dNTPs (adenosine triphosphate, guanosine triphosphate, thymidine triphosphate, cytidine triphosphate), KCL or MgCL2, and polymerase buffer. The purpose of this technology is to detect known genomic, plasmid or complementary DNA (cDNA) sequences both qualitatively and quantitatively.

For PCR analyses, 20 μl of master mix (Applied Biosystems™, CA, USA) was placed in 96-well microplates. Then, 5 μl of the DNA sample taken from each case was added to some of these wells to serve as a test, while 5 μl of the sample taken from 3 healthy patients, which were not included in the study, was added to the remaining wells to serve as an internal control. These mixtures, each with a total volume of 25 μL, were run on the PCR machine (ABI 7500 Fast Real-Time PCR System, Applied Biosystems™, CA, USA) The qPCR study protocol included the following steps: a polymerase activation step (1 cycle at 95 °C for 3 min), a denaturation step (40 cycles at 95 °C for 15 s), and an annealing/extension step (40 cycles at 5 °C for 30 s).

In the context of the qPCR method, an increase in the number of cycles corresponds to a concomitant increase in the amount of DNA detected by the fluorescent dye. The threshold cycle value (Ct) refers to the cycle number at which the fluorescence radiation surpasses a predetermined threshold during amplification. In the present study, the Ct values obtained from the analysis results of subgingival plaque samples were integrated into the 2-ΔCt formula to calculate the difference in fold change occurring in the quantities of microorganisms.The disparity between the target bacteria detected at baseline and on the 1st month was computed utilizing the following formula:

ΔCt: [Ct_target bacteria in 1st month] - [Ct_target bacteria in baseline] The fold change in quantities of the target bacteria on 1st month compared to baseline: 2-Δ∆Ct All analyses were conducted using the SPSS version 23 software package (IBM Corp, Armonk, NY). Two groups, test and control, were defined with a substantial effect size (0.4) difference in microbial quantities and a significance level of 0.05. To assess correlations in repeated measurements with a correlation coefficient of 0.5, a sample size of 40 cases (20 test - 20 control) was determined using G*Power 3.1.9.2 software. The significance level was set at p < 0.05.

Continuous variables were assessed for normality using the Shapiro-Wilk test. Subsequently, based on the normal distribution assumption, comparisons between independent groups were conducted using either the Student's t-test or the Mann-Whitney U test. For comparisons between dependent groups, the analyses employed either the Paired t-test or the Wilcoxon test.

The correlation between continuous variables was investigated through Spearman correlation analysis. Continuous variables were described in terms of mean ± standard deviation (SD), median (interquartile range (IQR)), and range (minimum-maximum), while categorical variables were represented by frequency and percentage.