Clinical trial NCT04178590
Conditions: Periodontitis
Keywords: periodontitis therapy Platelet Rich Fibrin Nonsurgical
Sponsor: University of Belgrade
Start date: 2019-10-02; Completion date: 2020-07-09
Enrollment: 32 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Current treatments of periodontitis have limited efficacy since they fail to suppress microorganisms satisfactorily over time. The aim of present study was to investigate whether there are differences between initial treatment of chronic periodontitis (SRP) and SRP in conjunction with injectable platelet-rich fibrin (i-PRF) application.

Detailed description:
Current treatments of periodontitis have limited efficacy since they fail to suppress microorganisms satisfactorily over time. The aim of present study was to investigate whether there are differences between initial treatment of chronic periodontitis (SRP) and SRP in conjunction with injectable platelet-rich fibrin (i-PRF) application.

Thirty patients with chronic periodontitis who had at least two sites with perriodontal pocket depth (PPD) ≥ 4 mm on contralateral side will be involved in the study. Using a split-mouth design, patients will be treated with SRP + I-PRF -(test group) or SRP only-(control group). Gingival crevicular fluid (GCF) and subgingival plaque will be collected with paper points (DentsplyMaillefer, Tulsa, Oklahoma, USA) at baseline and 1, 3 and 6 months after the treatment. The presence and concentrations of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia(Tf) will be analyzed by real-time polymerase chain reaction (qPCR). Periodontal parameters, including bleeding on probing (BOP), probing pocket depth (PPD) and the clinical attachment level (CAL), will be recorded on both sides, as well as concentration of TNF-alpha (tumor necrosis factor alpha), ALP(alkaline phosphatase) and MMP-8 (matrix-metalloproteinase-8).

Arms: arm 1: Scaling and root planing in conjunction with Injectable Platelet-Rich Fibrin arm 2: Scaling and root planing in conjunction with saline
Interventions: intervention 1: SRP + Injectable Platelet-Rich Fibrin intervention 2: SRP + placebo
Intervention descriptions: intervention 1: Injectable Platelet-Rich Fibrin application as adjunct to scaling and root planing intervention 2: Saline application during scaling and root planing

Protocol documents:
ICF_002.pdf: 
Department of Periodontics 
School of Dental Medicine 
University of Belgrade 
 
Effect of Injectable Platelet-Rich Fibrin (i-PRF) in Initial Treatment of Chronic Periodontitis 
NCT04178590 
26 April, 2020 
Participant Information Sheet 
You are invited to participate in the clinical trial called: “Effect of Injectable Platelet-Rich Fibrin (i-PRF) 
in Initial Treatment of Chronic Periodontitis”. The research includes the assessment of clinical 
parameters, microbiological and biochemical research of gingival crevicular fluid (GCF) before and after 
chronic periodontitis therapy: scaling and root planing (SRP) or scaling and root planing (SRP) with 
application Injectable Platelet-Rich Fibrin (i-PRF). The study protocols was approved by the Ethics 
Committee of the School of Dental Medicine, University of Belgrade (36/11) and is in compliance with 
Helsinki Declaration.  
The aim of the study is to establish the way in which the parameters change before and after the 
conventional therapy with or without adding i-PRF, and to show whether there is difference in the 
treatment.  
The patients will be 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, and the presence of minimum 6 
teeth per quadrant. The patients who have not received any antibiotic, anti-inflammatory and 
immunosuppressive therapy in the previous 6 months can be included in this trial; as well as those who 
have not had any periodontal treatments within the last 12 months. 
Current or former smokers, and women who are pregnant or lactating cannot be included in the trial.  
After clinic examination and establishing certain parameters patients will be thoroughly instructed on 
self-performed oral hygiene consisting of: the use of the modiﬁed Bass brushing technique, a soft manual 
toothbrush, a regular  toothpaste twice a day, and the use of inter-dental brushes once a day. After taking 
GCF samples with paper points, safe and  painless procedure, a full-mouth SRP will be 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. It will be also necessary to take two 10 ml tubes of blood and 
centrifuge it for 3 minutes for i-PRF preparation. I-PRF application will be performed only once at the 
beginning of the therapy in the periodontal pocket of one side of the jaw, while saline will be applied on 
the opposite side. The standard of oral hygiene will be checked at each examination after 1, 3 and i 6 
mounts, along with taking GCF samples. 


 
Inform consent  
I confirm that I have read and understood the information about the project as provided in the Participant 
Information Sheet. 
I confirm that I have had the opportunity to ask questions and the investigator has answered all the 
questions about the study. 
I understand that my participation is voluntary and that I am free to withdraw from the project at any time. 
Failure to participate in this study will not affect the therapy required and thus I will not be denied my 
legal rights as a patient. With my voluntary consent to participate in this research, I will not gain any 
personal or material gain. 
 
Participant:                                                                              Principal Investigator: 
_______________________________                                   ________________________________ 
Signature of Participant                                                            Signature of Principal Investigator 
 
 
 
Date: ___ (dd) ___ (mm), 20__ 

Prot_000.pdf: 
Effect of Injectable Platelet-Rich Fibrin (i-PRF) in Initial Treatment of Chronic 
Periodontitis  
- 
NCT04178590 
- 
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.  
 

SAP_001.pdf: 
Effect of Injectable Platelet-Rich Fibrin (i-PRF) in Initial Treatment of Chronic 
Periodontitis  
- 
NCT04178590 
- 
26 April, 2020 
 
Statistical analysis  
Statistical Package for Social Science (SPSS software package, version 26.0; SPSS Inc., Chicago, 
IL, USA) was used for all the data analyses. For numerical data, mean values, medians, standard 
deviations (SD) and ranges were used for description. The distributions of all outcome values were 
examined using the Kolmogorov–Smirnov normality test and depending on the distribution, 
measurements at baseline and at each follow-up between two treatment groups were compared 
using paired Student’s t-test or Wilcoxon signed-rank test. Comparisons within each treatment 
group for all the measurements were done by repeated measures ANOVA with Bonferroni 
correction or Friedman and Wilcoxon signed-rank test.  Univariate and multivariate logistic 
regression models were used to assess the predictors of difference between treatment groups. 
Univariate and multivariate linear regression models were for the predictors of primary outcome 
(CAL) at 6 months. The differences were considered as significant when the p value was <0.05. 
Study power was calculated using DSS Research, for primary outcome (CAL) at 6 months and it 
was above 90%.