Effect of Intracanal Cryotherapy and Metformin on Postoperative Pain in Teeth With Symptomatic Apical Periodontitis: A Randomized Clinical Trial
Phase: PHASE2
Conditions: Periapical Periodontitis, Chronic Nonsuppurative
Sponsor: Celalettin Topbaş

Effect of Intracanal Cryotherapy and Metformin on Postoperative Pain in Teeth with Symptomatic 
Apical Periodontitis: A Randomized Clinical Trial 
 
ClinicalTrials.gov Identifier 
NCT Number: Not yet assigned 
 
Document Title 
Study Protocol and Statistical Analysis Plan 
 
Document Type 
Study Protocol / Statistical Analysis Plan 
 
Version 
Version 1.0 
 
Document Date 
13 February 2023 
 
Responsible Party 
Principal Investigator 
 
Ethics Committee Approval 
Hamidiye Clinical Research Ethics Committee 
University of Health Sciences 
Approval No.: 13.02.2023–15184 
 
Study Description 
This document contains the complete study protocol and statistical analysis plan for a randomized, 
double-blind, 2×2 factorial clinical trial evaluating the effects of intracanal cryotherapy and intracanal 
medicaments on postoperative pain following root canal treatment. 
 
 
 

 
 
 
 
 
 
UNIVERSITY OF HEALTH SCIENCES 
HAMİDİYE CLINICAL RESEARCH ETHICS COMMITTEE 
 
 
 

 
 
 
 
 
 
 
Effect of Intracanal Cryotherapy and Metformin on Postoperative Pain in Teeth with Symptomatic 
Apical Periodontitis: A Randomized Clinical Trial 
 
Study Protocol: Effect of Intracanal Cryotherapy and Metformin on Postoperative Pain in Teeth with 
Symptomatic Apical Periodontitis 
1. Title 
Effect of Intracanal Cryotherapy and Metformin on Postoperative Pain in Teeth with Symptomatic 
Apical Periodontitis: A Randomized Clinical Trial 
 
2. Study Objective 
To evaluate the effects of intracanal cryotherapy and two intracanal medicaments (calcium hydroxide 
and metformin) on postoperative spontaneous and percussion pain in mandibular premolar teeth 
with symptomatic apical periodontitis. 
Null hypothesis: No significant difference exists in postoperative pain among the study groups. 
 
3. Study Design 
 
Randomized, double-blind, 2×2 factorial clinical trial 
 
Parallel-group design with four groups: 
1. Calcium hydroxide 
2. Metformin 
3. Cryotherapy + calcium hydroxide 
4. Cryotherapy + metformin 
 
Duration: Postoperative follow-up for 7 days 
 
 
 

4. Ethical Approval 
 
Approved by Hamidiye Clinical Research Ethics Committee, University of Health Sciences 
(Approval No.: 13.02.2023–15184) 
 
Written informed consent obtained from all participants 
 
5. Sample Size Calculation 
 
Software: G*Power 3.1 
 
Effect size: f = 0.40 
 
Power: 0.90 
 
Significance level: α = 0.05 
 
Total sample: 80 patients (20 per group) 
 
6. Participants 
Inclusion Criteria: 
1. Systemically healthy 
2. Mandibular premolars with pulpal necrosis requiring root canal treatment 
3. Periapical index score 0–2 
4. Age 18–70 years 
5. Able to understand and complete VAS assessments 
6. Provided written informed consent 
Exclusion Criteria: 
1. Systemic disease or pregnancy 
2. Analgesic use within 24 hours prior to treatment 
3. Severe bruxism 
4. Acute apical abscess or swelling 
5. Periodontal pocket >5 mm or tooth mobility > grade 1 
6. Unrestorable teeth 
7. Vital pulpal tissue or immature apices 
8. Radiographic evidence of root resorption, root fracture, perforation, or canal calcification 
 
7. Randomization and Blinding 

 
Random sequence generated using Randomizer.org 
 
Allocation concealment by independent investigator 
 
Operator and patients blinded to: 
o 
Type of intracanal medicament 
o 
Presence/absence of cryotherapy 
 
8. Intervention 
Root Canal Treatment Protocol: 
 
Local anesthesia: Articaine hydrochloride + epinephrine 
 
Rubber dam isolation 
 
Access cavity preparation 
 
Working length determination: Electronic apex locator + radiographic confirmation 
 
Canal instrumentation: ISO #40 apical size 
 
Irrigation: 
o 
2 mL 5.25% NaOCl per instrument change 
o 
5 mL 17% EDTA 
o 
Final rinse: 10 mL sterile saline over 5 min (4 °C in cryotherapy groups; 20 °C 
otherwise) 
 
Drying with sterile paper points 
Intracanal Medicament Placement: 
 
Powder-to-liquid ratio: 1:1 
 
Lentulo spiral, 800 rpm, 20 s, 2 mm short of working length 
 
Groups 1 & 3: Calcium hydroxide 
 
Groups 2 & 4: Metformin 
 
Temporary restoration of access cavity 
Cryotherapy Application: 
 
Final irrigation with sterile saline at 4 °C for cryotherapy groups (Groups 3 & 4) 
 
9. Outcome Measures 
Primary Outcome: 
 
Postoperative spontaneous pain (VAS 0–100 mm), recorded pre-op and daily for 7 days 

Secondary Outcome: 
 
Percussion pain (VAS 0–100 mm), assessed pre-op and post-op 
 
10. Statistical Analysis 
 
Software: R Statistical Software 
 
Significance: α = 0.05 
Analysis Plan: 
 
Spontaneous pain: Linear mixed-effects model for repeated measures 
 
Percussion pain: ANCOVA with preoperative pain as covariate 
 
Fixed effects: Time, cryotherapy, medicament type, and cryotherapy × medicament 
interaction 
 
Random effect: Patient 
 
11. Data Management 
 
Pain levels recorded by patients before any analgesic use 
 
Confidentiality maintained; individual patient data not shared publicly 
 
12. Follow-Up 
 
Pain assessment: Days 1–7 postoperatively 
 
No long-term follow-up in current study; suggested for future research 
 
13. Expected Results 
 
Intracanal cryotherapy expected to reduce postoperative pain 
 
Metformin effect on short-term pain expected to be comparable to calcium hydroxide 
 
No significant interaction anticipated between cryotherapy and medicament type 
 
14. Strengths and Limitations 
Strengths: 
 
Randomized, double-blind, factorial design 
 
Standardized treatment protocol 
 
Appropriate statistical methods for repeated measures 

Limitations: 
 
Short follow-up (7 days) 
 
Single-center study 
 
Subjective pain assessment (VAS)