Effects of Application Injectable Platelet-Rich Fibrin (i-PRF) as Adjunct Therapy in the Initial Treatment of Periodontitis
Phase: PHASE4
Conditions: Periodontitis
Sponsor: University of Belgrade

Effect of Injectable Platelet-Rich Fibrin (i-PRF) in Initial Treatment of Chronic 
Periodontitis  
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NCT04178590 
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26 April, 2020 
 
The aim of the study  
The aim of the study was to determine the effects of SRP supplemented with the local i-
PRF application, in comparison with SRP alone, on periodontal disease course. 
 
Study population 
This study was a randomized controlled clinical trial with a split‐ mouth study design. Thirty 
systemically healthy chronic periodontitis (CP) patients, 13 male and 17 female, age 38.00±10.12 
years, range 22 – 64 years were recruited from the Department of Periodontology and Oral 
Medicine, School of Dental Medicine, University of Belgrade. The study protocol was approved 
by the Ethics Committee of the School of Dental Medicine, University of Belgrade (36/11) and is 
in compliance with Helsinki Declaration. After being informed of the research methods, all the 
participants submitted written consent for the enrolment in the study. The study was registered at 
ClinicalTrials.gov as “NCT04178590”. 
The patients were enrolled in the study according to the following inclusion criteria: adults (age 
between 20 and 75 years), without systemic and/or other oral disease or ongoing drug therapy, 
which might have an impact on the clinical signs and symptoms of periodontitis, the presence of 
minimum 6 teeth per quadrant, at least two teeth with periodontal probing depth (PPD) ≥ 5mm and 
minimum clinical attachment level (CAL) 3 mm in each quadrant, and a horizontal bone loss, no 
involvement of furcation degree II or III of both quadrants of the mandible or maxilla, confirmed 
by full-mouth radiograph images. 
Patients who underwent antibiotic therapy during the last 6 months or had initial and/or surgical 
periodontal treatment within last 12 months, patients with diabetes or in need of long-term use of 
anti-inflammatory and immunosuppressive medication, current or former smokers, as well as 
women who are pregnant or lactating, were excluded from the study. 
 

 
Periodontal examination 
Immediately before the first treatment, as well as at 1, 3 and 6 months, periodontal examinations 
were carried out by the same examiner. The examination was conducted on all teeth and tooth sites, 
except the third molars and the tooth sites associated with furcation involvements of II and III 
degree (Hamp et al.1975). To identify disease progression, certain variables were recorded from 
the mesio-buccal, mid-buccal, disto-buccal, disto-lingual, mid-lingual, and mesio-lingual surfaces 
of each tooth: clinical attachment level (CAL), gingival margin level (GML), periodontal probing 
depth (PPD), bleeding on probing (BOP), and plaque index (PI).  The examiner, a specialist in 
periodontology, performed and noted down all the recordings. The examiner had previously 
obtained the adequate level of competence and reproducibility skills according to the various 
clinical parameters and indices which were going to be utilized (Polson 1997). To avoid blood 
contamination, GCF collection was conducted one day after clinical examination, after non-
surgical periodontal treatment. 
Supragingival scaling and root planing and oral hygiene instructions  
At the beginning of the study, the patients were thoroughly instructed on self-performed oral 
hygiene consisting of: use of the modiﬁed Bass brushing technique, a soft manual toothbrush, a 
regular toothpaste twice a day, and use of inter-dental brushes once a day. A full-mouth SRP was 
conducted using ultrasonic scaler and hand instruments (Hu-Friedy, Chicago, IL) under local 
anesthesia in one or two sessions during the period of 24 hours. The standard of oral hygiene was 
checked at each examination visit and further instructions were provided according to individual 
requirements. Three months following the completion of the baseline treatment, all the patients 
were recalled for professional supragingival plaque control and reinforcement of oral hygiene. 
Additionally, reinstrumentation was conducted by using the ultrasonic device in all the sites with 
a remaining PPD of ≥ 5mm. 
Preparation of i-PRF 
Blood samples were taken into two 10 ml tubes and prepared for i-PRF preparation. The whole 
blood without anticoagulant was then centrifuged at 700 rpm for 3 min (60×g) at room temperature 

by a Duo Centrifuge (Process for PRF, Nice, France). The upper liquid layer was taken as i-PRF 
using a syringe.The i-PRF was applied in one quadrant (treatment group) of the chosen jaw 
(mandibula or maxilla), whereas the physiological saline was inserted in the opposite side (control 
group). Treatment allocation was decided by a toss of a coin. i-PRF was enabled to hold for a longer 
time by applying it into periodontal pockets through perforations at the point of interdental space 
on individually formed occlusal splints. After 15 minutes the splint was removed.  
Gingival crevicular fluid (GCF) sampling 
GCF was sampled at baseline and 1-, 3- and 6-month follow-ups, one day after clinical examination 
from two counter lateral periodontal pockets with PPD ≥ 5 mm, either in mandibula or maxilla. 
GCF was collected by absorbent paper strips (Periopaper, Amityville, NY, USA) after 
supragingival plaque had been removed, the tooth gently rinsed with water, air-dried and isolated 
with cotton rolls to avoid contamination from saliva. Four strips were inserted consecutively into 
the gingival sulcus up to 3 mm leaving them for 30s and pooled in two separate dried 
microcentrifuge tubes. The absorbed GCF volume was immediately estimated by using a calibrated 
instrument. Afterwards, the samples were stored at −70 °C pending the analyses. 
Protein quantification with ELISA 
Quantification of GCF biomarkers of periodontal disease (MMP -8, TNF- α and ALP) was 
performed using enzyme-linked immunosorbent assays (ELISA). GCF samples were defrosted by 
soaking the paper strips into 150 μl of 0,05% Tween 20 solution in sterile Phosphate-buffered saline 
(PBST) and eluted by 20 minutes of vortexing followed by 10 minutes of centrifugation at 10000 
× g at room temperature. Immediately after centrifugation the supernatant was collected and used 
to carry out the assays. Protein quantification was performed in duplicate using following ELISA 
kits: Human Total MMP-8 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA); 
Human Tumor Necrosis Factor Alpha (TNF-α) and Human Alkaline Phosphatase (ALP) ELISA 
kits (Elabscience Biotechnology, Wuhan, China) according to manufacturer’s recommendations. 
Optical density of each well was measured after color development at 450 and 570 nm wavelength 
using microplate reader (RT 2100C, Rayto Life and Analytical Sciences, Shenzhen, China). 
Concentrations of tested proteins were determined from generated standard curve.  
Quantification of microorganisms 

Bacterial DNA was isolated using a standard salting-out method. DNA extracts were stored at − 
70 °C prior to quantitative PCR (qPCR) analysis. The amplification of target region for 16S rRNA 
was performed as previously described (Brajović, Popović, Puletić, Kostić, & Milašin, 2016), using 
SensiFAST SYBR® Hi-ROX Kit (Bioline Reagents Ltd, London, UK) and the following primers: 
Fw 5′-TCCTACGGGAGCACAGT′-3 and Rv 5′GGACTACCAGGGTATCTAATCCTGTT-3′. 
Prevotella melaninogenica (ATCC 25845) was used as reference strain for standard curve analysis, 
and the results were obtained as total gene copy number.