In this single-blind, randomized controlled, prospective clinical study, individuals with gingival complaints between August 2021 and March 2022 were diagnosed with chronic inflammatory gingival enlargement as a result of clinical and radiographic examinations. This study was conducted in accordance with the revised Declaration of Helsinki and was approved for human subjects by the Ethical Committee of Afyonkarahisar Health Sciences University (permit number 2021/338). The Afyonkarahisar Health Sciences University Scientific Research Projects Unit supported the study as project number 21.DUS.005. All individuals who participated in the study were informed about the objective and methods of the study and signed informed consent forms. The study was registered at in the US National Institutes of Health Clinical Trials Registry (NCT05871190).
Determination of Sample Size and Study Design
Previous studies on this subject were reviewed, when α = 0.05 (95% confidence interval), effect size (effect size) was d = 0.8, and power (1-β) = 0.85 (80%) was taken in power analysis, in evaluating the effects of I-PRF applied following gingivectomy and gingivoplasty on wound healing. It was calculated that a minimum 21 patients should be included in the study for the change in the deepithelized surface area to be 0.60 units in the I-PRF group compared to the control group. Accordingly, considering that there may be missing data, it was decided to select 23 individuals for each group. The study was completed with 46 patients. (23 females, 23 males) ages 13–28 years (mean: 16.98 years). The inclusion criteria were as follows: (a) systemically healthy patients ages 13–30 years, (b) pregnancy or lactation, (c) patients with chronic inflammatory gingival overgrowth in the mandibular and maxillary anterior area(11,12,13,21,22,23,31,32,33,41,42,43), (d) no clinical attachment and bone loss, (e) not using immunosuppressive agents, systemic corticosteroids, chemotherapy, and/or radiotherapy drugs taken or prescribed 2 months before the study attempt, which may affect the study results, wound healing, and coagulation mechanism, and (f) patients with adequate oral hygiene. The exclusion criteria were as follows: (a) patients who had a history of scaling and root planning in the last 6 months, (b) smokers and alcohol users, © those who used drugs that may cause gingival enlargement in the last 6 months, (c) patients with poor communication, and (d) patients who did not attend controls at designated times.
All patients received initial periodontal therapy (IPT) and oral hygiene instructions. The patients were called again 2 weeks later for control, and gingivectomies were planned for patients with adequate oral hygiene. Gingival overgrowth was graded according to the following indices: The buccolingual direction of gingival enlargement was classified according to the index defined by Seymour and later modified by Miranda et al. (MB index) [17]. The vertical direction of gingival overgrowth was also measured according to the index described by Angelopoulos and Goaz and later modified by Miller et al. (GOİ index) [18]. Patients with grades other than 0 in both indices were included in the study. A total of two groups, one test and one control, were planned in the study.
Test group: I-PRF is placed on the wound site after a gingivectomy with the conventional method (the procedure of gingivectomy with a #15 scalpel ) and closed with a periodontal dressing
Control group: closure of the wound site with only a periodontal dressing after a gingivectomy with the conventional method.
İnvestigators’s primary aim in this study was to confirm investigators’ hypothesis that I-PRF accelerates wound healing. Our secondary aim was to see the effect of I-PRF on biochemical parameters.
Randomization
Sequentially numbered, opaque, sealed envelopes were used for the allocation [19]. An allocation array was created using a computer-generated random list, and sealed, opaque envelopes including the procedures were randomly divided into two groups for each patient by an independent examiner (NCK) with arrays generated using a computer-assisted randomization table (www.randomizer.org/Copyright© 1997–2011 by Geoffrey C. Urbaniak and Scott Plous). Until the first treatment visit, the physician (ŞÇB) who was applying and recording the clinical periodontal measurements throughout the study was blinded. All participants were blinded during the practice and control sessions.
Gingivectomy and Gingivoplasty Operation
Considering the results of many studies, it was decided to prefer the conventional method in our study because the cost is lower, the recovery is faster, and the use is practical. After the surgical procedure was completed, the control areas were left to heal spontaneously. I-PRF was applied to the test areas. Surgical areas in the control and test sites were covered with a periodontal dressing (Coepak, Isip, IL, USA).
Postoperative Care and Suggestions
Patients were advised to avoid very cold or hot food and beverages to protect the wound area from trauma and to consume very soft foods. They were warned that the periodontal dressing should be in the mouth until the next control day (3rd day) and that if the periodontal dressing was damaged during that period, they should come to our clinic without delay. The patients were prescribed mouthwash containing 0.12% chlorhexidine and analgesic containing paracetamol (Parol 500 mg). Patients were advised not to brush their teeth in the wound area until the end of the periodontal dressing process (3 days). They were told to provide oral hygiene in the areas where the operation was not performed. Brushing the operation area with a soft toothbrush after the periodontal dressing was removed was recommended.
Clinical Measurements
All operations were performed by a single clinician (Ş.Ç.) to maintain standardization and apply the single-blind protocol throughout the study. All clinical parameters were measured from the six regions (distobuccal, buccal, mesiobuccal, distopalatinal/lingual, midpalatinal/lingual, mesiopalatinal/lingual) of each tooth using a 0.5 mm diameter Williams periodontal probe. Clinical measurements included the Löe and Silness gingival index (GI), plaque index (PI) [20], bleeding of probing (BOP) [21], and probing depth (PD). Measurements were made at the baseline (t0) and on 14 (t1) and 21 (t2) days after the gingivectomy operation.
Preparation of I-PRF
The blood samples taken from the patients were collected in 10 ml plastic tubes without anticoagulants. The tubes were centrifuged for 3 minutes at 2300 rpm (RCF-max (relative centrifugal force) = 509.53 g) in a centrifuge device ( Intraspin centrifugation device with a 33° rotor angulation with a radius of 53 mm at the clot and 86 mm at the max (PC-O2, Process for PRF, Nice, France) [16, 22]. After centrifugation, the I-PRF in the upper part of the tube was collected via an injector and transferred to a metal godet. It was left for 15–20 minutes for polymerization of the I-PRF [16]. Afterward, the polymerized I-PRF was applied to the secondary wound, and the operation area was covered with a periodontal dressing (Fig. 1).
Gingival Crevicular Fluid Sampling
Before Gingival Crevicular Fluid (GCF) samples were taken, saliva contamination was avoided, and the area was isolated with cotton roll pads. The GCF samples were then collected in sterile paper strips. The strips (Periopaper®, Interstate Drug Exchange) were placed in the sulcus until a moderate resistance was felt and left for 30 seconds [23]. Paper strips contaminated with blood or saliva were discarded. When the patients were called for the gingivectomy operation, the GCFs on the control and test teeth (VEGF from the buccal of 11 and FGF-10 from the buccal of 21, or VEGF from the buccal of 41 and FGF-10 from the buccal of 31) were sampled before the gingivectomy operation. This procedure was repeated in the same regions before the operation (baseline) and on days 14 and 21. Samples were stored in Eppendorf tubes at − 20°C until further processing was carried out [23]. Then, the obtained samples were prepared using the ELISA test. The test followed the manufacturer's instructions, and the mean VEGF and FGF-10 levels were calculated for each operation site.
Clinical Follow-up of Patients
The patients were called for control on 3, 7, 14, and 21 days after the gingivectomy and gingivoplasty operations. The parameters evaluated in the control sessions were:
1. Evaluation of wound epithelialization with a Mira-2 tone solution.
2. LTH wound healing index (valuation of soft tissue healing).
3. MMS scale (evaluation of soft tissue healing).
4. Assessment of pain.
Taking Photos of Operation Areas and Evaluation of Epithelialization of the Wound Area
Mira-2 tone solution, which is a plaque-disclosing agent, was used to measure wound area epithelialization on postoperative days 0, 3, 7, 14, and 21 (Fig. 2). This solution has been used in studies where epithelization is lacking. The areas of abrasion and the level of epithelialization during post-operative healing were evaluated. Wound surface epithelialization in the obtaining photographs was evaluated using the ImageJ software program (National Institutes of Health, USA ImageJ 1.48V) (Fig. 3). Photo size was scaled using a 10mm Williams periodontal probe during photographing. To ensure standardization, all photographs were taken by the same person with the same camera at the same angle, distance (20 cm), and light values (ISO-800) (Nikon D7500). The actual intraoral dimensions (mesial to distal) of the maxillary right central incisor in each patient were measured and compared with the dimensions of the right central incisor in the images, and the ratio between actual and photographic size was used to calibrate the images. In addition, wound surface epithelialization area measurements were repeated at one-week intervals over photographs of 10 patients who had undergone gingivectomy and gingivoplasty and were not included in the study for the calibration of the person who made the measurements [24].
LTH Index
Wound healing was evaluated by using the LTH index, which classifies the healing period according to redness, granulation tissue, presence of bleeding, suppuration, and epithelialization. In this index, recovery is scored between 1 (very poor recovery) and 5 (excellent recovery) [25]. It was evaluated in both the control and test groups on days 3, 7, 14, and 21.
MMS Scale
The MMS scale was used to evaluate the contour of the wound area, distortion status, and color match. A low total score indicates weak healing, and a high score indicates good healing [26]. It was evaluated in both the control and test groups on postoperative days 3, 7, 14, and 21.
Assessment of Pain
İnvestigators’s study used a Visual Analog Scale to measure pain levels during the post-operative recovery period. Patients were asked to score their current pain status between 0 and 10 on the same days, and on days 3 and 7 after the operation (0 = no pain, 10 = worst pain) [27].
Biochemical Analysis of GCF Samples
GCF samples of the test and control groups were placed in Eppendorf tubes and stored until the study day at − 20°C. A phosphate buffer saline (PBS) solution (ph: 7.4) of 0.2 ml was added to the GCF samples. One minute ultra-sonication at 20,000 rpm was performed with a Hielscher (Germany) sonicator.
The GCF samples were then centrifuged at 10,000 g for 15 minutes, and the supernatant was used. FGF-10 and VEGF levels were studied at 450 nm with ELISA (Bioassay Technology Laboratory, Shanghai/China) kit and Thermo Scientific Multiskan FC Microplate (Thermo Fisher Scientific Instruments Co. Ltd. Shanghai/China) reader at 450 nm. The results were expressed as ng/L.
Statistical Analysis
IBM SPSS.25 program was used in all statistical analyses. Normality assumptions were examined with the Shapiro-Wilk test. An independent sample t-test was used for inter-group statistical comparisons of the parameters showing normal distribution, and the Mann–Whitney U test was used for inter-group statistical comparisons of parameters showing non-normal distribution. In intragroup comparisons, the one-way analysis of variance or Friedman test was used for repeated measurements according to the assumption of normality, and post hoc tests with Bonferroni correction were used in case of significant differences. Correlations between categorical variables were examined by chi-square analyses.