Participants

This cross-sectional, observational study conducted at Alanya Alaaddin Keykubat University Faculty of Dentistry Department of Periodontology and Biochemistry. The study began after approval was granted by the Akdeniz University Faculty of Medicine Clinical Research Ethics Committee (dated 05.12.2018, protocol# 859). The participants were selected from individuals who applied to the periodontology clinic due to periodontal problems or for routine cleanings from September 2019 to January 2020. The study’s objectives and extent were thoroughly explained, along with comprehensive information about the potential complications of participating. Written informed consent was acquired from every participant. They were then categorized into two groups based on their periodontal health status [17].

Control group: Individuals without periodontal disease.

Test group: Patients diagnosed with stage III and stage IV, grade B generalized severe chronic periodontitis.

Generalized severe chronic periodontitis was defined as individuals who experied bleeding from over 30% of their teeth on probing, with probing depth equal to or greater than 7 mm, clinical attachment loss equal to or greater than 5 mm, and radiographic bone loss of above 30%. The control group consisted of oeriodontally healthy individuals.

The inclusion criteria were:

1.

Aged 30 to 65

2.

Controlled or no systemic diseases

3.

Nonsmoker.

The exclusion criteria were:

1.

Uncontrolled systemic diseases

2.

Body mass index ≥ 40 kg/m2

3.

Pregnant

4.

Immunosuppressant drug use

5.

Smokers and alcohol user

6.

Receiving chemotherapy, radiotherapy or corticosteroid, anticoagulant, antiplatelet, or nonsteroidal anti-inflammatory drugs

7.

Drug user

8.

Received antibiotics treatment in the last 6 months

9.

Received surgical or nonsurgical periodontal therapy for periodontitis within 2 years [18].

Preparation for PRF

After the demographic data were collected and a periodontal examination was conducted, the patients were provided with follow-up numbers, and the i-PRF collection tubes were labeled with tracking numbers. Two blood samples were collected from the antecubital veins of each participant. One tube was used to prepare the i-PRF, and the other was designated for whole blood, containing WBCs and platelets.

A total of 12 mL of whole blood was collected. Of this, 3 mL were used in the complete blood count analysis, and 9 mL were used for i-PRF centrifugation. The time needed to fill the sample tubes with blood was measured with a timer during collection. The blood extraction procedure was completed in a maximum of 25 s. The blood samples were promptly deposited in centrifuge equipment (within 60 s).

The blood was transferred to plastic i-PRF tubes containing no anticoagulant. The sample was subjected to fixed-angle centrifugation in a tabletop centrifuge (ElektroMag M 415P, Istanbul, TR) at 700 rpm (60 g) for 3 min. After centrifugation, the blood formed two distinct layers. The lower stratum comprised red blood cells, and the top stratum comprised plasma, platelets, and coagulation factors. The separated plasma and platelets were pale yellow. A Pasteur pipette was used to meticulously extract the uppermost layer. This aspirate is a partially active injectable type of platelet-rich fibrin. The i-PRF samples were transferred to Eppendorf tubes. The sample was kept at -80 °C until the day of analysis.

Evaluation of the complete blood count

Blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA). Automated hematology analyzers were used to measure the complete blood count, which quantifies the various blood cells within a specified volume of blood (Sysmex xn-1000, Japan). We analyzed WBCs and platelets.

Molecular analysis of biological substances

The levels of TGF-β, human IGF-1, human PDGF-BB, human EGF, and human VEGF-A in the samples were analyzed in duplicate using the ELISA method (Elabscience, Texas, USA [19, 20]. After the completion of the reactions, absorbance measurements were conducted with a microplate reader instrument (Biotek Synergy H1, VT, USA). A concentration vs. optical density plot was generated with the seven-level standard for each parameter. The concentrations of TGF-β, human IGF-1, human PDGF-BB, human EGF, and human VEGF-A in the samples were determined with this graph. The kit specifications for each parameter, including sensitivity, evaluation range, specificity, and repeatability, were: TGF-β: 0.1 ng/mL; 1.16–10 ng/mL, 100% specificity, and coefficient of variation (CV) less than 10%; human IGF-1: 0.94 ng/mL, 1.56–100 ng/mL, 100% specificity, and CV less than 10%; human PDGF-BB: 18.75 pg/mL; 31.2–2000 pg/mL, 100% specificity, and CV less than 10%; human EGF: 2.35 pg/mL, 3.91–250 pg/mL, 100% specificity, and CV less than 10%; and human VEGF-A: 18.75 pg/mL; 31.2–2000 pg/mL, 100% specificity, and CV less than 10%.

Data analysis

The statistical analyses were conducted with SPSS 19.0 (IBM Inc., IL, USA). The normality of the distribution was assessed with the Kolmogorov–Smirnov and Shapiro–Wilk tests. A significance level of 0.05 was employed. Independent t-tests were used to compare the groups because the data adhered to a normal distribution.

We performed Pearson’s correlation analysis to explore the associations between the growth factor levels and the WBC and platelet counts. The correlation were compared between the test and control groups.

According to the power analysis, with a sample size of 30 in each group, a two-tailed t-test, a significance level of 0.05, and an effect size of 0.75, we could expect around 80% power to detect the effect. This indicates a high probability that the study would detect a difference between the means of the two groups if such a difference existed.