Assessment of Antimicrobial Efficacy of Nano Chitosan, Chlorhexidine, Chlorhexidine/ Nano Chitosan Combination Versus Sodium Hypochlorite Irrigation in Patients With Necrotic Mandibular Posterior Teeth: A Randomized Clinical Trial
Phase: NA
Conditions: Dental Pulp Necrosis
Sponsor: Cairo University

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Assessment of Antimicrobial Efficacy of Nano Chitosan, Chlorhexidine, Chlorhexidine/ 
Nano Chitosan Combination versus Sodium Hypochlorite Irrigation in Patients with 
Necrotic Mandibular Premolars: A Randomized Clinical Trial 
 
تقييم الفعالية المضادة للميكرو بات للنانو شيتوزان, الكلورهيكسيدين, و المزيج بين النانوشيتوزان والكلورهيكسيدين
 مقارنة بالكلور في ري قنوات اللب عند المعالجة اللبية للمرضي المصابين بتنخر اللب في
الضواحك 
 :السفلية 
.تجربة إكلينيكية باإلنتقاء العشوائي 
 
Submitted for partial fulfillment of the Ph. D requirements in Faculty of Dentistry 
Cairo University 
By 
Maha Nasr Morsy 
B.D.Sc (2009), M.Sc (2016) 
 
Prof. Dr. Alaa Diab 
Professor of Endodontics, Faculty of Dentistry, Cairo University 
 
Dr. Nehal Nabil 
Lecturer of Endodnotics, Faculty of Dentistry, Cairo University 
 
Dr. Amira Farouk Hussein 
Lecturer of Clinical Pathology, Faculty of medicine, Kasr Al Ainy, Cairo University. 
 
Department of Endodontics 
Faculty of Dentistry -Cairo University 
2018 
 
 
 
 
              Supervisors’ signature:                       Head of the department signature: 
1. 
2. 
3.                  
Code: ENDO 5:2 

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Administrative information 
1. Title: Assessment of Antimicrobial Efficacy of Nano Chitosan, Chlorhexidine, 
Chlorhexidine/ Nano Chitosan Combination versus Sodium Hypochlorite Irrigation in 
Patients with Necrotic Mandibular Premolars: A Randomized Clinical Trial 
 
2. Trial registration: This study was registered on Protocol Registration and                         
Results System (PRS). 
 
- 
Website: www.clinicaltrails.gov  
- 
ClinicalTrail.gov ID NCT03668899. 
- 
Release Date: September 15, 2018. 
 
3. Protocol version:    version 1. 
4. Funding:                 Totally Self-funding 
Sponsor:                  Cairo University, Faculty of Dentistry. 
 
5. Trial roles and responsibilities:  
 Maha Nasr Morsy Abo shanab 
Assistant lecturer of Endodontics, Faculty of Dentistry, ERU University. The main 
investigator; will be responsible for funding the study, clinical part of the study, screening 
and recruitment of sample, follow up the patients, writing the thesis, interpretation of results 
and drawing conclusions. 
Prof. Dr. Alaa Diab  
Professor of Endodontics, Faculty of Dentistry, Cairo University. Main Supervisor, 
Guidance throughout the clinical part, responsible for reviewing the thesis and drawing 
conclusions. 
Dr. Nehal Nabil 
Lecturer of Endodontics, Faculty of Dentistry, Cairo University. Co-supervisor, she 
will guide throughout the clinical part, analysis of the collected data, proof reading of the 
thesis and drawing conclusions. 
Dr. Amira Farouk Hussein 
          Lecturer of Clinical Pathology, Faculty of medicine, Kasr Al Ainy, Cairo University. 
Co-supervisor, she will be responsible for the microbiology part. 
 
 
 
 
 
 
 

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6. Introduction 
a. Description of the research question: 
PICOS 
P - Adult patients with necrotic mandibular premolars. 
I- Interventions  
Irrigation with nano chitosan
: 
1I
 
Irrigation with chlorhexidine
: 
2I
 
Irrigation with Chlorhexidine/Chitosan combination
: 
3I
 
C- Irrigation with Sodium Hypochlorite. 
O- Primary Outcome:  Antimicrobial efficacy of the different irrigants. 
     Secondary Outcome: Postoperative pain. 
S- Randomized controlled trial. 
 
Formulated question:  
In necrotic mandibular posterior teeth, would irrigating the canals using either Nano 
chitosan, Chlorhexidine or a combination of Nano chitosan and Chlorhexidine versus Sodium 
Hypochlorite have different effects on canal disinfection and postoperative pain? 
 
Statement of the problem:  
Bacteria play a major role in the pathogenesis of apical periodontitis; therefore, 
success of endodontic treatment is dependent on its complete eradication before obturation1.  
Sodium hypochlorite (NaOCl); the gold standard for irrigation has many 
disadvantages including irritation of the periapical tissues and burning of surrounding 
tissues2,3. Therefore, the present study aims to explore new irrigating solutions probably more 
effective and less irritating to the periapical tissues than NaOCl.  
 
Rationale for carrying out the trial: 
          Many studies have been conducted to assess the antimicrobial efficacy of sodium 
hypochlorite, (as a gold standard) compared to other suggested alternatives such as 
Chlorhexidine (CHX), both in vitro4,5 and in-vivo6,7.  
 
          Zandi et al 2016, evaluated the antimicrobial efficacy of 2% Chlorhexidine compared 
to 1% Sodium hypochlorite irrigation in treating teeth with apical periodontitis and found that 
bth solutions are similarly effective in reducing bacterial cell counts6.  The results of this 
study agreed with the results of a previous study that was conducted by Ercan et al on 2004 
where the antimicrobial efficacy of 2% CHX was compared to that of 5.25% NaOCl in 
treating necrotic teeth with periapical pathosis and concluded that both solutions were 
significantly effective to reduce the microorganisms in the teeth with necrotic pulp, periapical 
pathologies, or both, and could be used successfully as an irrigant solution7. 
 
         Regarding postoperative pain, Almeida et al 2012, evaluated postoperative pain after 
single visit root canal treatment using either 2% CHX or 5.25% NaOCl as irrigating solutions 

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and found a low incidence of postoperative pain in both groups with no statistically 
significant difference between them8. 
        A recent study by Farzaneh et al evaluated the effects of two different concentrations of 
NaOCl ,2.5% and 5.25%, on postoperative pain after single visit endodontic treatment of 
molars with symptomatic irreversible pulpitis and found that 5.25% NaOCl irrigation resulted 
in significantly less postoperative pain in the first 72 hours postoperatively9. 
 
         The current available evidence has inconclusive results when comparing the 
antimicrobial efficacy of NaOCl to CHX as suggested by a recent systematic review 
conducted by Goncalves et al 2016, they included only 5 clinical studies out of 172 reviewed 
articles and found that the results of the studies were inconsistent and recommended further 
clinical trials to reach conclusive results10. 
 
         Recent studies has shed the light on a wide range of natural substances like Chitosan, 
Propolis, and other herbal solutions that are claimed to be as effective against bacteria as 
NaOCl, less toxic and less irritant2,3,11 ,12,13,14 and also introduced nanoparticles with their 
known antimicrobial capabilities as endodontic irrigants15,16.  
                           
         An in vitro study in 2017 claimed that the combination of 2% Chlorhexidine and 2% 
Chitosan is as effective as NaOCl against E. Faecalis in biofilms3. 
Since within the scope of our search no study evaluated the antimicrobial effects of 
Nano chitosan, Chlorhexidine/Chitosan combination as possible natural irrigants to those of 
NaOCl for irrigation in-vivo. 
The Aim of the current study is to compare the antimicrobial effects of Nano 
chitosan, chlorhexidine, and their combination to NaOCl in patients with necrotic mandibular 
Premolars. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Review of literature: 
 
Chemo-mechanical preparation: 
 
It is well established that the most probable cause failure of endodontic treatment is 
the presence of a persisting infection. Therefore, it is important that chemo-mechanical canal 
preparation be directed towards complete eradication of all bacteria from the root canal 
system so that the best possible prognosis can be achieved in endodontic treatment1. 
 
“Only a few studies have evaluated the effect of infection at the time of root ﬁlling on 
the prognosis of treatment. These studies have shown that the success rate of endodontic 
treatment is approximately 10-15% lower for teeth which yield a positive culture before 
obturation than for teeth which yield a negative culture”1. 
 
Dunavant et al 20064, compared the efficacy of sodium hypochlorite to ,Smear Clear, 
Chlorhexidine, REDTA and BioPure MTAD against E. Faecalis. They submerged an in vitro 
grown E. Faecalis biofilm in wells containing either 1% NaOCl, 6% NaOCl, Smear Clear, 
2% CHX, REDTA or BioPure MTAD for one and five minutes. Phosphate buffered saline 
was used as a negative control while 6% NaOCl was used as a positive control. After that the 
biofilms were re-cultured on THB agar plates and the number of colony-forming units 
(CFUs) was count. There was no significant difference between 1% NaOCl and 6% NaOCl 
yet, there was a significant difference between 1% and 6% NaOCl and all other tested 
irrigants with BioPure MTAD being the least effective solution. 
 
Perochena et al 201516, studied the chelating and antibacterial effects of chitosan 
nanoparticles (CNPs) on bovine dentin. Five groups of bovine dentin sections (20 per group) 
were used in the study. They used 2.5% sodium hypochlorite (NaOCl) for 20 min, 17% 
ethylenediaminetetraacetic acid (EDTA) for 3 min and 1.29 mg/mL CNPs for 3 min as 
irrigating solutions. The groups were irrigated with distilled water as acontrol, NaOCl alone, 
NaOCl then EDTA, NaOCl then EDTA then CNPs or NaOCl then CNPs. After irrigation, 
half of the samples were used to assess the chelating effect of the solutions using scanning 
electronic microscopy, while the other half were infected intra-orally to examine the post-
treatment bacterial biofilm forming capacity under confocal laser scanning microscopy. The 
results of this study showed that smear layer removal was significant in all of the groups 
except the control and NaOCl groups and that the samples treated with CNPs were resistant 
to biofilm formation significantly more than other treatment groups. Therefore, they 
concluded that CNPs irrigation was superior to NaOCl in that as well as inhibiting bacterial 
growth it also can remove the smear layer effectively. 
 
Rocas et al 201617, evaluated the antibacterial effects of 2% chlorhexidine  and 2.5 % 
NaOCl irrigation during rotary preparation of contaminated root canals. A total of 50 single 
rooted teeth with infected necrotic pulps were included in the study (two groups n=25). All 
root canals were prepared using BioRace rotary file system. Irrigation was done using either 
2. NaOCl in one group or 2% CHX in the other group. Two samples were collected from 

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each canal S1 before chemomecanical preparation and S2 after chemomechanical 
preparation. In this study, the number of viable bacteria was determined by a quantitative 
polymerase chain reaction all initial samples (S1) were contaminated while in NaOCl group 
44% of the treated canals were still contaminated and in CHX group 40% of the treated 
canals were contaminated.  This difference was not statistically significant.  
 
Zandi et al 20166, evaluated the antimicrobial efﬁcacy of 2% chlorhexidine compared 
to 1% sodium hypochlorite used as irrigants in the retreatment of endodontically treated teeth 
with apical periodontitis. A total of 67 patients were randomly assigned to either 1% NaOCl 
group (n=29) or 2% CHX group (n=38). Three samples were taken from each tooth; S1 
before preparation, S2 after preparation and irrigation with either of the tested irrigants and 
S3 after application of calcium hydroxide intracanal medication. The amount of bacterial 
reduction was evaluated using 16s ribosomal RNA gene-based polymerase chain reaction. 
The results of this study suggested that both solutions are similarly effective in reducing 
bacterial cell count. 
 
Jaiswal et al 20173, evaluated antibacterial efficacy of Chitosan, Chlorhexidine, 
Propolis and Sodium hypochlorite on E. Faecalis biofilm invitro, Ninety single rooted 
mandibular premolars were included in the study. The root canals were then instrumented 
with rotary ProTaper instruments to an apical size of F3. 2 ml of 5% NaOCl was used as an 
irrigant during preparation. Teeth then vertically sectioned into two halves and divided into 
nine experimental groups with 20 samples each. irrigated with 3 ml of each irrigant for 10 
minutes. Group 1: 5% NaOCl, group 2: 2% Chlorhexidine, group 3: 1% Acetic acid, group 4: 
Propolis, group 5: 0.2% Chitosan, group 6: 0.2%Chitosan+2%Chlorhexidine, group 7: 1% 
Chitosan+1%Chlorhexidine, group 8: 2%Chitosan+2%Chlorhexidine and group 9: Saline 
(negative control). The results of this study showed that Chitosan/ Chlorhexidine 
combination, Chlorhexidine and Propolis were as effective as sodium hypochlorite, so they 
concluded that their use as natural alternatives for NaOCl could be advantageous to overcome 
the disadvantages of NaOCl. 
 
Yadav et al 201712, evaluated antibacterial effects and cytotoxicity of 0.25% 
Chitosan, 0.5% Chitosan, 2% chlorhexidine and 3% sodium hypochlorite against E. Faecalis 
and C. Albicans. C. albicans and E. faecalis cultures were prepared in vitro, then the 
antimicrobial activity of the tested solutions (0.25% Chitosan, 0.5% Chitosan, 2% CHX and 
3% NaOCl) against them was evaluated using agar diffusion, microdilution and biofilm 
susceptibility tests. Saline was used as a negative control. For the cytotoxicity evaluation 
fresh blood was centrifuged then plasma was abstracted and packed cell volume of red blood 
corpuscles was obtained. Then 1 ml of packed RBCs was added to 4 ml of saline. 100µl of 
this diluted RBCs was distributed to 18 test tubes to obtain 3 groups (Chitosan, Chlorhexidine 
and hypochlorite), 6 test tubes each. For all groups, the first test tube was kept as a control in 
which no irrigant was added. In the second test tube 10 μl of the irrigant was integrated. 20 μl 
was integrated to the third test tube, 30 μl to the fourth test tube, 40 μl to the fifth test tube 
and 50 μl to the test tube. Tubes were incubated for 3 minutes then hemoglobin percentage 
after hemolysis was detected utilizing an automated hemoanalyzer. The results of this study 

6
 
 
suggested that chitosan could be used as a non-toxic natural alternative to NaOCl since the 
antibacterial activity of the all chitosan groups was comparable to 3% NaOCl and 2% 
Chlorhexidine and that the chitosan showed no cytotoxicity at 3mg/ml. 
 
6. b. Choice of comparators: 
Sodium hypochlorite (NaOCl) was considered the most efficient of all available 
solutions for its superior antimicrobial effects and tissue dissolving abilities16. Therefore, as a 
gold standard for irrigation; it was chosen as a comparator in the current study. 
 
7. Objectives: 
- 
Research hypothesis: 
There is no difference in the antimicrobial efficacy among Nano chitosan, Chlorhexidine, 
Chlorhexidine/Nano Chitosan and Sodium hypochlorite in root canal treatment. 
- 
Primary objective: 
The primary objective of this study is to assess the antimicrobial efficacy of Nano-chitosan, 
chlorhexidine and Chlorhexidine/Nano Chitosan combination versus NaOCl. 
- 
Secondary objective: 
The secondary objective is to assess the incidence and severity of postoperative pain after 
root canal treatment using either Nano-chitosan, CHX, Chitosan/CHX combination or NaOCl 
as a final flush before obturation. 
PICOS 
P - Adult patients with necrotic mandibular Premolars. 
I- Interventions  
Irrigation with nano chitosan
: 
1I
 
Irrigation with chlorhexidine
: 
2I
 
Chlorhexidine/Chitosan combination
Irrigation with 
: 
3I
 
C- Irrigation with 5.25% sodium hypochlorite. 
O1- Antimicrobial efficacy of the different irrigants. 
O2- postoperative pain. 
S- Randomized controlled trial. 
 
8. Trial design: 
- 
A Randomized controlled clinical trial.  
- 
Unicenter: A trial will be carried out by one person in one hospital.  
- 
Randomization double blinded: Laboratory assessor and participant will not know the 
method of treatment. 
- 
Equal randomization: participants with equal probabilities for intervention.  
- 
Positive controlled: All groups receiving treatment. 
- 
"Parallel group study: Each group of patients receives a single treatment 
simultaneously". 
- 
Two assessors will assess the results.  
 

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III. Methods 
 
A) Participants, interventions & outcomes 
 
9.  Study setting:   
- 
This study will be carried out on patients attending outpatient clinics in the 
Department of Endodontics, Faculty of Dentistry; Postgraduate Endodontics clinic 7th 
floor new section. Cairo University, Egypt. Adec 200 U.S.A dental units. 
10.  Eligibility criteria:  
10. a. Inclusion criteria  
1. 
Patient's age between 22-42 years. 
2. 
Both male and female. 
3. 
Patient who are medically free and with good health 
4. 
Necrotic Mandibular Premolars. 
10. b. Exclusion criteria:   
1. 
Pregnant females. 
2. 
Teeth with vital inflamed pulps, symptomatic periapical abscess and facial 
cellulitis, periodontally diseased and hopeless teeth. 
3. 
Teeth with previous fillings and/ or previous endodontic treatment. 
 
11. Interventions:  
 
11. a. General operative procedures: 
- 
Eligible patients will be randomly divided into equal groups (Nano Chitosan group), 
(Chlorhexidine group), (Chlorhexidine/Chitosan combination group) and the control 
group (Sodium Hypochlorite group). 
- 
Preoperative measures (for all groups): 
• Medical and dental history will be obtained from all patients participating in this 
research. Clinical and radiographic evaluation for each tooth included in this study 
will be recorded. 
• Pain scale chart will be given to each patient to rate his /her pain level before 
endodontic treatment as preoperative reading on an NRS scale.  
• Tooth will be anaesthetized using 1.8 ml Mepivacaine HCl 2% - Levonordefrin 
1:20000 (Carpule Mepecaine-L, Alexandria Company for Pharmaceuticals and 
Chemical Industries, Egypt). 
• The tooth will be isolated with rubber dam to maintain aseptic field, after isolation 
the tooth and surrounding field will be disinfected by a protocol using 3% 
hydrogen peroxide 2.5% sodium Hypochlorite before and after coronal access 
cavity preparation17. 
• The initial sample S1 will be collected from root canals before preparation using 
sterile # 15 paper points. 

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•  Sterile saline solution will be injected in the canals followed by the insertion of a 
#15 K-file (Mani, Japan) to at least 1 mm short of the apex as confirmed by apex 
locator (Root ZX, J.Morita USA, Irvine, CA)17 to facilitate the insertion of the 
paper points to collect the samples. 
• Each paper point will remain in place for at least 1 minute then immediately placed 
in test tubes containing reduced transport fluid and sent to the Microbiology 
Laboratory, Faculty of Medicine, Cairo University for microbiologic processing7. 
• Root canals will be mechanically prepared in a crown-down approach using the 
rotary files (M-Pro file system) in an endodontic motor (X-Smart, Dentsply 
Maillefer, USA) according to the manufacture instructions. 
The following sequence will be used for all groups: 
 
▪ # 18 file taper (orifice opener) 0.09 will be used to prepare two thirds of 
estimated working length. 
▪ # 20 file taper 0.04 will used to full working length. 
▪ #25 file taper 0.06 will used to full working length. 
▪ #35file taper 0.04 will used to full working length. 
• The canals will be thoroughly irrigated for 1 minute between every two subsequent 
files using 2 ml of 2.5% NaOCl. 
• After complete preparation all canals will be flushed with EDTA followed by sterile 
saline.  
- 
Intervention for test group A  
• After complete preparation, the canals will be flushed using 5 ml of Nano chitosan 
(Nano tech, Egypt) in a plastic syringe using Navitip needles (Ultradent, South 
Jordan,UT). 
- 
Intervention for test group B 
• After complete preparation, the canals will be flushed using 5 ml of Chlorhexidine 
(CHX-Plus, Cosmopack, Egypt) in a plastic syringe using Navitip needles 
(Ultradent, South Jordan,UT). 
- 
Intervention for test group C 
• After complete preparation, the canals will be flushed using 5 ml of a combination 
of Chlorhexidine and nano chitosan in a plastic syringe using Navitip needles 
(Ultradent, South Jordan,UT). 
- 
Intervention for control group 
• After complete preparation, the canals will be flushed using 5 ml of 5.25% sodium 
hypochlorite (Clorex, Egypt Ltd, Egypt) in a plastic syringe using Navitip needles 
(Ultradent, South Jordan,UT). 
➢ After flushing the canals with each irrigating solution all canals are to be flushed with 10 
ml sterile saline and the final sample S2 is to be collected in the same manner as S1. 
➢ After dryness, root canals will be obturated using lateral compaction technique by 
selection of master cone corresponding to the same size as the master apical file, and then 
a finger spreader will be used to allow space for auxiliaries. All canals will be sealed with 
a resin sealer (ADSEAL, META Biomed CO., LTD Chungbuk. Korea). 

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➢  After obturation, a cotton pellet will be placed in the pulp chamber and the access cavity 
will be closed with a temporary filling. 
Post-operative care:  
 
• All patients will receive postoperative instructions, in case of moderate and severe pain, 
patients will be allowed to take Ibuprofen (400mg), and instructed to record number of 
Ibuprofen tablets. 
 
11. b. Strategies to improve adherence to intervention protocol:  
 
- 
Face to face adherence reminder session, phone calls and text message reminders will 
tack place to stress on the post-operative instructions to record postoperative pain 
levels.  
 
12. Outcomes: 
 
Primary outcome: 
• Antibacterial efficacy: will be assessed by counting the number of colony forming units 
(CFU) before and after irrigation (S1 and S2) for each patient18. Collected samples will 
be cultured in serial dilution on blood agar plates, incubated for 24 hours, and then the 
number of colony forming units (CFUs) will be counted per ml. 
Outcome 
Tool 
Unit 
Time 
antibacterial efficacy 
 
Number 
of 
CFUs17 
Numerical data  
 
Samples collected before 
and after preparation and 
transferred to the laboratory 
for immediate cultivation. 
 
Secondary outcome: 
• Postoperative pain: will be recorded on a Numerical Rating scale (NRS), pain intensity 
will be recorded preoperatively and postoperatively after 6, 12, 24, 48, 72 hours and 7 
days. The NRS consists of a10-cm line anchored by two extremes "No pain" and "pain 
as bad as could be", patient will be asked to choose the mark that represent their level of 
pain. Pain level will be assigned to one of 4 categorical scores: 1, None(0); 2,Mild(1-3); 
3,Moderate(4-6); 4,Severe (7-10)19. 
Outcome  
Tool  
Unit  
Time  
Postoperative 
pain  
Numerical 
rating 
scale NRS18 
Categorial data 
Immediately 
after 
treatment and Up to 7 
days 
after 
endodontic 
treatment 

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13. Participants time line 
 
STUDY PERIOD 
Time point 
T0 
T1 
F1 
F2 
F3 
F4 
F5 
F6 
F7 
Activity 
 
 
 
 
 
 
 
 
 
Enrollment 
 
 
 
 
 
 
 
 
 
Eligibility criteria 
x 
 
 
 
 
 
 
 
 
Informed consent 
 
x 
 
 
 
 
 
 
 
Allocation 
 
x 
 
 
 
 
 
 
 
Intervention 
 
 
 
 
 
 
 
 
 
Access  
 
x 
 
 
 
 
 
 
 
Initial sample 
 
x 
 
 
 
 
 
 
 
Cleaning and shaping 
 
x 
 
 
 
 
 
 
 
Final sample 
 
x 
 
 
 
 
 
 
 
Obturation 
 
x 
 
 
 
 
 
 
 
Assessment 
 
 
 
 
 
 
 
 
 
Intracanal bacterial count 
 
x 
 
 
 
 
 
 
 
Postoperative pain 
 
 
x 
x 
x 
x 
x 
x 
x 
 
T0: Enrollment of the patient after diagnosis and radiographic evaluation. 
T1: Signing the informed consent and allocation of the patient to either intervention or 
control group. Starting the procedure, taking the first root canal sample before cleaning 
and shaping, second root canal sample after cleaning and shaping, obturation. 
F1: Patients will be asked to evaluate their postoperative pain levels using a numerical 
rating scale (NRS) 
F2: similarly, at 6 hours. 
F3: similarly, at 12 hours. 
F4: similarly, at 24hours. 
F5: similarly, at 48 hours. 
F6: similarly, at 72 hours. 
F7: similarly, at 7 days. 
 
 
14. Sample Size: 
- 
The aim of the current study is to compare the antimicrobial effects of Nano chitosan, 
Chlorhexidine, Chlorhexidine/ Nano Chitosan combination to NaOCl in patients with 
necrotic mandibular posterior teeth. Based on a previous study by Shingare & 
Chaugule 20112 the difference in bacterial count between at least 2 groups is 75±75. 

11
 
 
Using power 80% and 5% significance level we will need to study 17 in each group. 
This number is to be increased to a sample size of 20 to compensate for losses during 
follow up. Sample size calculation was achieved using PS:  Power and Sample Size 
Calculation Software Version 3.1.2 (Vanderbilt University, Nashville, Tennessee, 
USA). 
 
15. Recruitment strategy: 
- M.N. will select patients from the outpatient clinic of the Department of Endodontics, 
Faculty of Dentistry, Cairo University, Egypt. 
- "Screening of patients will be carried on until the target population is achieved"20. 
- 
"Identifying and recruiting potential subjects is achieved through patient database"20. 
 
16.  Allocation 
16.a. Randomization: 
Sequence generation:  using computer-generated random numbers. 
- 
List will be created on (https://www.random.org/), the patients will be randomly 
classified into four groups:  
▪ "Group A: Irrigation with Nano chitosan". 
▪ "Group B: Irrigation with Chlorhexidine 
▪ "Group C: Irrigation with Chlorhexidine/Nano Chitosan combination". 
▪ "Group D: Irrigation with 5.25% Sodium Hypochlorite". 
- 
Allocation ratio will be 1:1. 
- 
This method will be done by the co-supervisor. 
 
16.b.  Allocation – concealment mechanism:  
- 
According to the allocation sequence obtained from the computer software, the 
numbers that generate randomly from the software will be written in small folded 
opaque papers then insert into Envelope. (Opaque sealed envelope and sign across 
sea). 
- 
All those papers will be ready before conducting any procedure. This method will be 
done by the co-supervisor. That Envelops will be placed in a container (box), each 
participant will grasp one envelope blindly before operation and will be assigned 
accordingly. The number of papers will decrease as each patient picks his number and 
so on. 
 
16. c. Allocation - Implementation: 
- 
Principle investigator (M.N) will referee all participants’ number to the co-supervisor 
who will generate the random sequence, and assign the patients for intervention or 
control group. Neither the assessor nor the patients know the criteria of allocation. 
 
17. Blinding: 
- 
"Blinded participants: The patient will be informed of the steps of the treatment (as 
mentioned in the consent form) without getting into details". 

12
 
 
- 
"Blinded Assessors: Blinding to the laboratory assessors is done by not involving 
them in sequence generation or allocation concealment or treatment options". 
 
C) Data collection, management, and analysis: 
 
18.  Data collection methods: 
- 
Base line data will be recorded on a personal history chart prior to clinical 
examination.   
- 
Antimicrobial efficacy will be measured by counting the number of colony forming 
units CFU of bacteria after cultivating the collected samples18. 
- 
Paper based pain scale (VAS) assessments will be filled by the patient preoperatively, 
and postoperatively after 6, 12, 24, 48,72 hours and after7 days15. 
19. Data management: 
- 
Patient files will be stored in numerical order and stored in secure and accessible 
place. M.N will assignee each patient a folder containing all their data including 
primary and secondary outcomes, patient’s data recorded as a hard copy with the 
signed consent. "Also, a soft copy of the data will be kept on operators' laptops and 
will be backed up on an external hard disk". 
- 
All data will be maintained in storage for 1 year following completion of the study. 
20. Statistical methods: 
- 
Data will be analyzed using IBM SPSS advanced statistics (Statistical Package for 
Social Sciences), version 21 (SPSS Inc., Chicago, IL). Numerical data will be 
described as mean and standard deviation or median and range. Data will be explored 
for normality using Kolmogrov-Smirnov test and Shapiro-Wilk test. Comparisons 
between 4 groups for normally distributed numeric variables will be done using the 
ANOVA while for nonnormally distributed numeric variables will be done by 
Kruskal Wallis test. A p-value less than or equal to 0.05 will be considered 
statistically significant. All tests will be two tailed. 
- 
Categorical data will be described as numbers and percentages and comparisons will 
be done by chi square test or fisher exact as appropriate  
 
D) Data monitoring: 
- The data will be monitored by the main supervisor (A.D) for any modification of the 
trial if needed 
21. Harms: 
- Any temporary or permanent adverse effect will be recorded and documented and 
treated. 
22. Auditing: 
- 
The study supervisors: Prof Dr. Alaa Diab and, dr Nehal Nabil will regularly assess 
the trial process and documents. "Adherence to trial interventions and policies to 
protect participants, including reporting of complications and their treatments. "20,21.  
Auditing of the study design will be done by the evidence based committee. 
 
 

13
 
 
 
 
IV. Ethics and dissemination 
 
23. Research ethics approval: 
- 
"This protocol and the template informed consent form will be reviewed by the Ethics 
Committee of Scientific Research - Faculty of Dentistry, Cairo University". 
24. Protocol amendments: 
- 
"Any amendment to the protocol which may affect the conduct of the research, 
potential benefit of the patient or may have impact on patient safety, inclusive 
changes of study objectives, study design, sample sizes, study procedures, or 
significant administrative aspects will demand a formal modification to the 
protocol"16, 17. Such modifications will be agreed upon by the Council of the 
Department of Endodontics. 
 
25. Consent: 
- 
The treatment plan will be fully explained to the patient and after educating the 
patient with all the needed data and complications that could occur, an Arabic consent 
form will be signed by patients who are willing to participate. 
26. Confidentiality:  
- 
"All study- associated information will be maintained securely. All participant data 
will be stored in locked lockers in areas with restricted entrance. All experimental 
specimens, reports, data gathering process, and administrative charts will be 
recognized by a coded ID number only to keep participant exclusiveness. All 
documentation that comprise personal identifiers including name, age, sex, 
identification number, address, phone number, work address will be secured 
disparately from study records identified by code number. All available databases will 
be secured with countersign- preserved entry systems"16, 17.  
27. Declaration of interests: 
- 
This study is a part of a PhD degree in Endodontics, Faculty of Dentistry; Cairo 
University. No financial conflict of interests is confirmed. The study is self-funded by 
the principal investigator. 
28. Access to data: 
- 
"All Principal investigators will be given entrance to the data sets. All data sets will 
be kept secure. To ensure confidentiality, data dispersed to project team members will 
be blinded of any recognizing participant data"16, 17. 
29. Dissemination policy: 
- 
Study results will be published as partial fulfillment of the requirements for PHD 
degree in Endodontics. 
- 
Topics proposed for presentation or publication will be expanded to the authors. 
 
V. Appendices:  
 
 

14
 
 
 
 
VI: Statement of originality: 
- 
Within the scope of our search no study evaluated the antimicrobial effects of Nano 
chitosan, Chlorhexidine, Chlorhexidine/ Nano Chitosan combination to those of 
NaOCl for irrigation in endodontic treatment of necrotic teeth in-vivo; therefore, this 
study is an original study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

15
 
 
 
 
 
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