Evaluation of the GCF Periostin, IL-17A and IL-17E Levels of Smoking and Non-smoking Periodontitis Patients Received Periodontal Treatment: Short-term Follow-up Study
Phase: NA
Conditions: Periodontitis, Smoking
Sponsor: Ondokuz Mayıs University

Evaluation of the GCF periostin, IL-17A and IL-17E levels of smoking and non-smoking  
periodontitis patients received periodontal treatment: short-term follow-up study 
 
Şule Özdoğan1 , Müge Lütfioglu1 
Ondokuz Mayıs University Faculty of Dentistry, Department of Periodontology, Samsun, 
Turkey 
Contact mail address: suleozdgn@gmail.com 
 
 
Study Protocol and Statistical Analysis Plan 
Before the study, One Way ANOVA test with a significance level of 5% was used for 
the sample size calculation. Based on this analysis, the sample size was determined as at least 
11 people in each group and a total of 44 subjects to detect a difference between groups. This 
cross-sectional study was approved by Ondokuz Mayıs University Clinical Research Ethics 
Committee (OMUKAEK) Samsun, Turkey (protocol number; 2019/324). 
Fourty four individuals, 22 males and 22 females who were older than 18 were enrolled 
in the study. The study groups were selected according to the following criteria from individuals 
who applied to the Ondokuz Mayıs University Faculty of Dentistry Periodontology Department 
Clinic for treatment or control, accepted to participate in the study, were systemically healthy, 
did not take any medication in the previous 6 months, and did not receive periodontal treatment 
in the previous 6 months: 
Group 1: (SP) Stage 3 Grade C patients (n:11) 
Group 2: (P) Stage 3 Grade A patients (n:11) 
Group 3: (SH) Periodontally healthy smokers (n:11) 
Group 4: (H) Non-smokers with periodontally healthy (n:11) 
Individuals were included as smokers if they had smoked ≥10 cigarettes/day for ≥5 
years, whereas individuals were included as nonsmokers if they had no previous history of 
smoking. The periodontitis stage III patients were evaluated clinically and radiographically, 
and were selected to have teeth with CAL ≥5 mm in at least 30% of them. Exclusion criterias 
were as follows; patients under the age of 18, pregnant or lactating women, patients who have 
a history of with systemic disease or cancer or chemotherapy and radiotherapy or ongoing drug 
therapy who might be affect the clinical characteristics of periodontitis, patients who smoke 
less than 10 cigarettes/day or who quit smoking.  
In order to standardize the study, all radiological and clinical examinations were 
performed by a single investigator. In clinical examination, Plaque Index (PI), Gingival Index 
(GI), bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL) 
scores were measured and recorded from 6 regions of each tooth (buccomesial, midbuccal, 

buccodistal, linguo/palatomesial, midingual/palatal, linguo/palatodistal regions) with using 
using millimetric (mm) calibrated University of North Carolina (UNC) (Hu-Friedy, Chicago, 
Illinois, USA). And up-to-date panoramic radiographs were taken from all patients on the day 
of the examination to determine periodontal status. After the clinical and radiographic 
evaluations, the participants were asked to come for GCF sample and treatment 1 day after the 
clinical evaluations, since bleeding may occur during probing.  
From the patients in the SP and P groups, GCF samples were obtained from the 5 
deepest pockets before the treatment, on the 15th and 30th days after the treatment. GCF 
samples were obtained taken from 5 randomly selected teeth with GI=0 PI=0 and SPD ≤3 
from healthy individuals in the SH and H groups.  
GCF samples were acquired by intrasulcular technique from selected pockets. Before 
sampling, the sites were isolated with cotton rolls, saliva and supragingival plaque were 
removed by cotton pellets, if present. Standardized filter paper strips (Periopaper® Oraflow 
Inc. New York, USA) were placed in the pocket for 30 seconds. Strips contaminated with 
blood or saliva were discarded. The absorbed GCF volume was measured with a precalibrated 
Periotron 8000 instrument (PeriotronR 8000, Pro Flow Inc., Amityville, NY, USA). Then, the 
strips were placed in sterile 0.5 ml Eppendorf (Eppendorf AG, Hamburg, Germany) tubes. 
Eppendorf tubes were stored at -80°C in Ondokuz Mayıs University Faculty of Medicine 
Department of Biochemistry until ELISA analysis. GCF samples were converted to an actual 
volume (μL) by reference to a standard curve. 
Treatment and Follow-up Protocol 
Patients in the SH and H groups were advised to come for routine controls by not 
applying any periodontal treatment. However for patients in the SP and P groups, non-surgical 
periodontal treatment was applied as full mouth debridement protocol in a single session, after 
the GCF samples were collected from selected pockets.  
The periodontitis patients were called again on the 15th and 30th days after the 
treatment. First of all, the GCF sampling process was repeated from the teeth whose GCF 
sample was collected before. Then, clinical examination parameters (PI, GI, BOP, PD and 
CAL) values were measured again and recorded.  
Determination of GCF Periostin, IL-17A, IL-17E Amount 
GCF Periostin, IL-17A, IL-17E levels were analyzed by the Sandwich ELISA method 
with the commercially available Human Periostin ELISA kit (Bioassay Technology Laboratory, 
Cat No. E3226Hu, Zhejiang, China), Human IL-17A ELISA kit (Bioassay Technology 
Laboratory, Cat No. E0047Hu, Zhejiang, China), Human Interleukin-25 ELISA kit (Bioassay 

Technology Laboratory, Cat No. E0054Hu, Zhejiang, China) in Ondokuz Mayıs University 
Faculty of Medicine, Department of Medical Biochemistry Research Laboratory. The study 
data were converted to pg/mL30sec and statistical analyzes were made. 
Statistical analysis 
SPSS for Windows Ver. for statistical analysis and graphics. 22.0 (SPSS Inc., Chicago, 
ILL, USA) and MS-Excel 2016 programs were used. Statistical significance level was accepted 
as p<0.05. 
The normal distribution of the data in our study was evaluated with the Shapiro-Wilks 
test. The mean±standard deviation was used for the descriptive statistics for the normally 
distributed variables, and the median (min-max) value for the non-normally distributed 
variables. While examining the distribution of the gender variable in the groups, the result of 
the Chi-Square test was evaluated. 
OneWay ANOVA test was applied to the variables showing normal distribution in the 
comparison of the data between groups. In post-hoc analyses, homogeneously distributed data 
according to Levene variance homogeneity test were evaluated with Tukey test, and data that 
were not homogeneously distributed were evaluated with Tamhane T2 test. Variables that did 
not show normal distribution were evaluated with the Kruskal-Wallis test. Bonferroni corrected 
Mann-Whitney U test was used in order to determine the group that caused the difference in 
the variables with a significant difference as a result of the Kruskal-Wallis test. 
Paired-Sample T Test and Repeated Measured Analysis of Variance were used for the 
variables showing normal distribution in the evaluation of the within-group changes according 
to time. Significant differences as a result of analysis of variance were examined with 
Bonferroni, one of the multiple comparison tests. Variables that did not show normal 
distribution were analyzed with the Friedman test. The Wilcoxon test was used to determine 
the group that caused the difference in the variables with a significant difference as a result of 
the Friedman test. 
RESULTS 
Demographic Findings 
The distributions of gender and age of the groups were as follows: SP (seven male and 
four female subjects; mean age: 40,9 ± 9,28 years); P (three male and eight female subjects; 
mean age: 41 ± 12.30 years); SH (seven male and four female subjects; mean age: 30.27 ± 9.99 
years); H (three male and eight female subjects; mean age: 26.36 ± 4.20 years).  
The study groups were evaluated in terms of age and gender, and a significant difference 
was found between the groups in terms of mean age (=16.567; p=0.001). There was no 

significant difference in terms of gender ( =0.364; p=0.546). The mean age of the H group 
was lower than the mean age of the patients in the SP (Z=18,000; p=0.006) and P groups 
(Z=16.682; p=0.014). It was determined that the mean age of the individuals was similar 
(Z=3,682; p=1,000) between group H and group SH.  
 
Clinical Parameters 
Full Mouth Clinical Findings 
When the pre-treatment (day 0) data were evaluated, a significant difference was found 
between the groups in all clinical parameters (PI, GI, BOP, PD and CAL). The measurements 
of SP and P groups were higher than the healthy groups (p<0.05).   
When the results of the in-group treatment were evaluated, the data before the treatment 
(day 0), the 15th day and the 30th day after the treatment were compared and it was observed 
that there was a statistically significant decrease in all clinical parameters (PI, GI, BOP, PD, 
CAL) in both the SP group and the P group. 
There was no statistically significant difference between the periodontitis groups (SP-
P) and healthy groups (SH-H) on the 0th, 15th and 30th days in terms of smoking (p>0.05). 
 
Sample Teeth Clinical Findings 
GCF was taken from the 5 deepest pockets measured before the treatment from the 
patients in the SP and P groups of the study groups before the treatment, on the 15th and 30th 
days after the treatment. GCF was taken from 5 randomly selected teeth with GI=0 PI=0 and 
PD ≤3 from individuals in SH and H groups.  
All clinical parameters (PI, GI, BOP, PD, CAL and GCF volume) of these selected teeth 
were analyzed statistically. There was a significant difference between the groups in all clinical 
parameters before treatment. The measurements of SP and P groups were higher than the 
healthy groups (p<0.05).   
 There was a significant difference between the groups in terms of BOP, PD, CAL and 
GCF volumes on the 15th and 30th days after treatment. 
There was no statistically significant difference between the periodontitis groups (SP-
P) and healthy groups (SH-H) on the 0th, 15th and 30th days in terms of smoking (p>0.05).  
 
Biochemical Parameters 
GCF Periostin levels  
 

When the GCF Periostin data were examined, the difference between the groups was 
found to be statistically significant at the beginning (p=0.003). In terms of periodontal disease, 
the amount of GCF Periostin in the P group was found to be significantly lower than in the H 
group (p=0.007), while there was no statistically significant difference between the SP group 
and the SH group. 
When evaluated in terms of smoking, it was observed that the GCF Periostin amount of 
the SP group was significantly higher than the P group (p=0.021). There was no significant 
difference between the SH and H groups (p>0.05). 
A statistically significant difference was found between the groups on the 15th day 
(p<0.001). When examined in terms of periodontal disease in the post-hoc analysis, it was found 
that the amount of GCF Periostin in the P group was significantly lower than in the H group, as 
at the beginning (p=0.002). It was determined that there was no statistically significant 
difference between the SP group and the SH group. When evaluated in terms of smoking, it 
was observed that the GCF Periostin amount of the SP group was significantly higher than the 
P group (p=0.003). 
However, on the 30th day, it was determined that there was no statistically significant 
difference between the groups. 
In the in-group evaluation of the change with treatment on a time basis, it was found 
that the GCF Periostin level in the SP group decreased over time (p<0.001). In post-hoc 
analyzes, this decrease was observed to be significant between all times (p=0.009(0-15.days, 
respectively), p=0.001). (day 0-30) and p=0.003(day 15-30)). In the P group, there was no 
statistically significant difference in the evaluations of the 0th day, 15th day and 30th day within 
the group (p>0.05). 
 
GCF IL-17A levels 
In the analysis of GCF IL-17A values, no statistically significant difference was found 
between the groups in terms of both periodontal disease and smoking at any time (p>0.05). 
In the evaluation of the change with non-surgical periodontal treatment according to 
time, no statistically significant difference was found in the GCF IL-17A levels of the SP group 
between the baseline, the 15th day and the 30th day (p>0.05). only in the P group, it was found 
that the GCF IL-17A level increased on the 30th day, with a statistically significant difference 
(p<0.05). Significant differences were found between day 0 and day 30, and day 15 and day 30 
in post-hoc analyzes (p=0.009, p=0.005, respectively). 
 

GCF IL-17E levels 
In the analysis of GCF IL-17E levels, there was no statistically significant difference 
between the groups in terms of both periodontal disease and smoking at any time (p>0.05). 
 In the evaluation of the change with non-surgical periodontal treatment, there was no 
statistically significant change in GCF IL-17E levels at any time in both the SP group and the 
P group (p>0.05).