Evaluation of IL-2, IL-5 and IL-17 Expression Levels in Saliva of Patients Treated With Stainless Steel Crowns
Conditions: Saliva, Dental Crown, Nickel Sensitivity
Sponsor: Yuzuncu Yil University

Study Protocol with Statistical Analysis Plan 
 
Title: 
Evaluation of IL-2, IL-5, and IL-17 Levels in the Saliva of Patients Treated with Stainless Steel 
Crowns 
 
NCT Number: NCT06439095 
 
Date: May 1,2025 
 
 
This prospective before-and-after clinical study was approved by the Local Ethics Committee 
of Van Yüzüncü Yıl University in accordance with the Declaration of Helsinki (Approval No: 
2024/01-03). The study protocol was registered on ClinicalTrials.gov (NCT06439095). All 
parents/guardians were informed about the procedures and potential outcomes and were asked 
to sign a written informed consent form prior to participation. Children whose parents declined 
consent were excluded from the study. 
 
Selection of Participants 
 
Participants were selected among 30 patients who applied to the Pediatric Dentistry Clinic of 
Van Yüzüncü Yıl University Faculty of Dentistry. The study was conducted on 20 observations 
from 10 pediatric patients undergoing general anesthesia. Data were collected at two time points: 
baseline (before treatment) and seven days after treatment. All treatment plans followed routine 
pediatric dental protocols, and no modifications were made for study purposes. Therefore, the 
study posed no harm to the patients. 
 
Inclusion criteria: 
 
• 
Children aged 3–10 years 
• 
Voluntary participation 
• 
Clinical indication for stainless steel crowns (SSC) without need for additional crowns 
in the next month 
• 
Adequate unstimulated saliva volume in the morning 
• 
No need for space maintainers in the following month 
• 
Candidates for treatment under general anesthesia 

Exclusion criteria: 
 
• 
History of previous dental treatment 
• 
History of systemic disease or syndromes 
• 
History of allergic reactions 
• 
History of using imitation jewelry 
• 
Presence of metal restorations or orthodontic appliances 
• 
Patients not undergoing general anesthesia 
 
This was a pilot/exploratory study, and no formal sample size calculation was performed. As 
no previous studies were available evaluating IL-2, IL-5, and IL-17 gene expression after SSC 
placement, the primary aim was to obtain preliminary data and provide a basis for future large- 
scale research. 
 
Study Design 
 
The study was performed under general anesthesia in children with material loss requiring SSC 
treatment. Patients were selected among those needing at least four posterior SSCs. To avoid 
delays and ensure no new SSC needs arose during the study period, all treatments were 
completed in a single session following standard pediatric protocols. Of 30 children evaluated, 
12 met inclusion criteria and were enrolled. 
 
Only patients receiving SSC or SSC combined with composite restorations were included. 
Those receiving other metal restorations, such as amalgam or space maintainers, were excluded. 
 
Dental Treatments 
 
The proximal surfaces of the teeth planned for SSCs were contoured with a #69L round bur on 
a high-speed handpiece. Occlusal surfaces were reduced by ~1 mm. Caries removal was 
performed using a round bur on a low-speed handpiece. In case of pulp exposure, an access 
cavity was prepared with an 008 fissure bur, and appropriate pulp therapy was conducted. SSCs 
(Kids Crown, Shinhung, Korea) were selected and cemented with nickel- and chromium-free 
glass ionomer cement (Meron, Voco, Germany). 
 
Saliva Sampling 

Saliva samples were collected at two time points: before treatment (control) and seven days 
after treatment. Parents were advised to follow a low-nickel diet and avoid nickel-containing 
materials until saliva collection was completed. The diet excluded canned foods, acidic foods 
cooked in stainless steel, herring, oysters, asparagus, beans, mushrooms, onions, corn, spinach, 
tomatoes, peas, whole grain flour, pears, rhubarb, tea, cocoa, chocolate, and baking powder. 
The use of fluoride-containing mouth rinses was also prohibited. Approximately 5 mL of 
unstimulated saliva was collected into polyethylene tubes free of nickel/chromium, washed with 
acetone and distilled water. Saliva was collected in the morning before surgery and at follow-
up, without prior rinsing. 
 
Clinical Examination 
 
At follow-up, a pediatric dentist examined the oral mucosa and soft tissues for signs of allergic 
reactions, inflammation, or tissue changes using a dental mirror and light. Parents and children 
were asked about any discomfort or changes over the previous seven days. 
 
RNA Isolation 
 
Tissue samples were carefully collected with a sterile spatula, weighed at 25 mg, and placed in 
RNase-free tubes with 700 μL TRIzol reagent. Samples were homogenized, incubated at room 
temperature for 5 minutes, mixed with chloroform, and centrifuged at 12,000 × g, 4°C for 15 
minutes. The upper phase was transferred, mixed with isopropanol, incubated, and centrifuged. 
The RNA pellet was washed with 75% ethanol, centrifuged, and air-dried. Pellets were 
dissolved in RNAse-free water or buffer. 
 
cDNA Synthesis 
 
cDNA synthesis was performed on a cold plate at +4°C using the All-in-One cDNA synthesis 
kit (Solver). 1 μL of RNA (10 ng/μL) was used in a 20 μL reaction, incubated at 42°C for 60 
minutes and 80°C for 10 minutes. 
 
Gene Expression Analysis 
 
cDNA products were analyzed using RotorGene Q. β-actin was used as the reference gene, and 
primers were designed for IL-2, IL-5, and IL-17. The PCR mixture included 12 μL SYBR Green 
qPCR Master Mix, 2 μL forward primer, 2 μL reverse primer, 5 μL water, and 4 μL cDNA 

(total volume 25 μL). PCR cycling conditions were: 95°C for 15 min, followed by 45 cycles of 
95°C for 30 s and 60°C for 30 s. 
 
Statistical Analysis Plan 
 
CT values obtained from Real-Time PCR were normalized using the Bio-Q online 
bioinformatics tool. Gene expression levels were calculated using the 2−ΔΔCT method. Group 
differences were analyzed using one-way ANOVA, and the Tukey HSD post-hoc test was used 
to identify significant differences. ΔCT values were calculated by subtracting housekeeping 
gene CT values from target gene CT values, and expression differences between groups were 
visualized using bar graphs. 
 
 
 
Key Results Summary 
 
Patients included were aged 3–6 years. Of the 12 enrolled, two were excluded due to loss to 
follow-up, leaving six girls and four boys. IL-2, IL-5, and IL-17 gene expression changes were 
assessed. Control group expression levels were low and homogeneous; follow-up samples 
showed marked increases, particularly in IL-5 and IL-17. IL-2 expression increased slightly 
without statistical significance. IL-5 increased moderately and reached statistical significance 
(p < 0.05), while IL-17 showed a sixfold increase and the highest variability, also reaching 
statistical significance. Heatmap and Venn diagram analyses illustrated expression changes and 
potential interactions among the genes. 

Informed Consent Form for Participation in a Research Study 
 
Dear participant, 
 
Van Yüzüncü Yıl University, Department of Pediatric Dentistry, is conducting a research study 
titled “Evaluation of IL-2, IL-5, and IL-17 Levels in the Saliva of Patients Treated with Stainless 
Steel Crowns.” This study aims to evaluate the release of nickel ions from pediatric stainless 
steel crowns on days 1 and 7, assess pH levels, and compare cytokine levels in saliva before 
and after the procedure to understand the body’s reaction. 
 
We invite you to participate in this research. Please note that your participation is entirely 
voluntary. Before making your decision, we will inform you about the study. After reading and 
understanding this information, if you agree to participate, please sign this form. 
 
The purpose of this research is to determine the nickel and chromium levels in the saliva of 
patients, considering the possibility that components of stainless steel crowns interact with 
saliva and may release heavy metals. We will also assess the body’s response using cytokine 
levels. This study will be conducted in collaboration with the Departments of Pediatric 
Dentistry and Oral Diagnosis and Radiology at Van Yüzüncü Yıl University, Faculty of 
Dentistry. Your participation is important for the success of the research. 
 
If you agree to participate, you or your child will be examined by the research team, and findings 
will be recorded. If eligible, approximately 10 mL of saliva will be collected before the 
procedure. Additional saliva samples may be collected during follow-up. Saliva samples will 
be analyzed for metal ion release and cytokine levels such as IL-2, IL-5, and IL-17. 
 
There is no risk from saliva collection. Participation is free of charge, and no additional payment 
will be provided. All medical information will be kept confidential and may only be reviewed 
by study monitors, ethics committees, or official authorities if necessary. Participation is 
voluntary, and refusal will not affect medical care. You have the right to withdraw consent at 
any time. 
 
I have been informed about the study and have been invited to participate. I understand that 
confidentiality will be maintained, and that the research results may be used for educational and 
scientific purposes without revealing personal identity. 

I understand that I can withdraw from the study at any time without providing a reason. I 
understand the researcher may exclude me from the study if necessary. I will not bear any 
financial responsibility for expenses related to the study and will not receive payment. 
 
I have been assured that any health issues arising from the study will be addressed without 
financial burden to me. I understand that I am under no obligation to participate and have not 
been coerced. Refusal to participate will not affect my medical care or relationship with my 
healthcare providers. 
 
I have read (or heard) and understood the above information. I voluntarily consent to participate 
(or allow my child to participate) in this study and consent to the use, presentation, and 
publication of the collected information in accordance with confidentiality rules. A signed copy 
of this form will be provided to me. 
 
I want to receive information about the results related to me in this study. ( ) 
I do not want to receive information about the results related to me in this study. ( ) 
 
Participant Name Surname Date of Birth Address/Phone Signature 
 
 
 
Parent/Guardian Name Surname Date of Birth Address/Phone Signature 
 
 
 
Witness (if any) Name Surname Date of Birth Address/Phone Signature 
 
 
 
Dentist: 
 
Title, Name Surname: 
 
Address/Phone: 
Signature: