Clinical trial NCT03186261
Conditions: Dental Caries
Sponsor: Cairo University
Start date: 2018-04-01; Completion date: 2018-12-28
Enrollment: 44 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
* The teeth of the patients that meet inclusion criteria will be anesthetized, isolated with a rubber dam.
* Cavity opened using conventional high-speed rotary instruments.
* The central cariogenic biomass and the superficial parts of the necrotic dentine will be removed with round burs.
* Caries lesion will be completely removed in the enamel/dentin junction.
* The excavation procedure will be terminated as soon as the soft and wet dentine was removed and the remaining tissue was leathery but not hard on exploring.
* A dentinal sample will then be collected from the base of the cavity using sterile spoon excavator as a baseline for bacteriological assessment.
* Then, application of either intervention or control agent, another dentinal sample will be collected using a sterile excavator and transferred into sterile tubes and transferred to the laboratory for microbiological analysis.
* Filling of the cavity with resin composite restoration.
* All clinical procedures will be carried out at the same visit.

Detailed description:
I.1. Study setting This clinical study will be held in the Clinic of Conservative Dentistry Department, Faculty of Dentistry, Cairo University, Egypt. The researcher will bear ultimate responsibility for all activities associated with the conduct of a research project including recruitment of patients, explaining and performing the procedures to them.

II.4.a Excavation protocol:

The teeth of the patients that meet inclusion criteria will be anesthetized, isolated with a rubber dam. Cavity opened using conventional high-speed rotary instruments. The central cariogenic biomass and the superficial parts of the necrotic dentine will be removed with round burs. Caries lesion will be completely removed in the enamel/dentin junction. The excavation procedure will be terminated as soon as the soft and wet dentine was removed and the remaining tissue was leathery but not hard on exploring. A dentinal sample will then be collected from the base of the cavity using sterile spoon excavator as a baseline for bacteriological assessment. Then, application of either intervention or control agent, another dentinal sample will be collected using a sterile excavator and transferred into sterile tubes and transferred to the laboratory for microbiological analysis.

II.4.b Assessment of Outcome:

Samples of carious dentine will be collected with sterile excavator before and after application of intervention/control agent. The dentine samples will be transferred to sterile container containing a 1mL thioglycollate medium used as a carrier, then this sterile container will be kept in an ice box and taken to the microbiology laboratory for processing, within an hour, by another examiner who is blinded to the type of agent applied after partial caries removal. Samples will be vortexed for two minutes, decimally diluted and 0.1 ml will be plated on Mitis Salivarius Bacitracin agar plates, these plates will be incubated anaerobically for 48 hour at 37ºC then aerobically for 24 hour at room temperature. The number of colonies will be expressed as colony-forming units (CFU/ml) compared before and after application of the antibacterial agent.

II.5. Participant timeline:

All clinical procedures will be carried out at the same visit.

II.6. Sample size calculation:

The aim of this study is to evaluate the antibacterial action Nano Silver Fluoride solution application in comparison to Chlorhexidine solution after partial caries removal in class I carious permanent molar. Based on a previous study by Mohan et al. 20165, the expected difference between two interventions is expected to be 2.1±2.2CFU/mL × 103. Using power 80% and 5% significance level we will need to study 18 in each group to be able to reject the null hypothesis that the population means of the experimental and control groups are equal. This number is to be increased to 22 patients in each group to compensate for possible losses during follow up. The sample size was calculated by PS program.

II.7. Recruitment:

Patients will be recruited from outpatient clinic of Conservative Dentistry Department in Faculty of Dentistry, Cairo University; after explaining the benefits/risks from the application of the interventions, then eligible patients will be recruited to fulfil the eligibility criteria according to participant timeline.

II.8.a. Recruitment Strategy:

The patients will be subjected to full examination and diagnosis using dental charts. Once the patients that are potentially eligible for this study are identified, they will be contacted by the researcher who will explain the study and ascertains the patient's interest. If interested, more detailed evaluations and preparations are made.

II.9. Randomization and assignment of interventions:

II.9.a. Allocation sequence generation:

The allocation sequence will be generated using (www.randomization.com).

II.9.b. Allocation concealment mechanism:

The randomization unit was the tooth, and the randomization procedure will be performed as follows. A number corresponding to each treatment group will be printed on pieces of paper and kept in dark containers. A paper will be selected from the container by a person other than the operator, and the treatment indicated will be performed (intervention/control). Blinding of the operator is not possible, because the color of intervention and control solution is different. The operator is blinded until randomization into groups, to avoid biases with regard to the application of antibacterial agent. Also, the examiner who will carry the microbiological analysis of dentin samples will be blinded to the type of agent applied during treatment. Finally, the treatment results will be assessed blindly by a statistician.

II.9.c. Implementation:

Dr. Mai Mamdouh (co-supervisor) will perform the allocation sequence and assign the participants to the intervention/Control treatment group.

II.9.d. Blinding:

The side to which interventions or control is assigned to will be recorded and all records of all patients will be kept with the main supervisor. Blinding of the operator is not possible, because the color of intervention and control solution is different. The operator is blinded until randomization into groups, to avoid biases with regard to the application of antibacterial agent. Also, the examiner who will carry the microbiological analysis of dentin samples will be blinded to the type of agent applied during treatment. Finally, the treatment results will be assessed blindly by a statistician.

II.10. Data collection methods:

\*Baseline data collection: For every patient medical history, dental history and examination charts will be filled by the operator. The report will be anonymous where patients identified by their serial numbers (the first letter of the first and last name and date of birth) only will be registered. Full detailed personal data of the patient will be written in separate sheet having the patient's serial number for further contact with patient.

\*Outcome data collection: The results will be converted into a table to facilitate the description of the results. The microbiological analysis will be performed blindly in relation to the type of solution applied after partial caries removal.

II.11.Data management:

The data will be entered and stored on a personal computer. Double data entry will be saved on an external hard disc to prevent loss of data.

II.12. 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. Categorical data will be explored for normality using Kolmogrov-Smirnov test and Shapiro-Wilk test. Comparisons between two groups for normally distributed numeric variables will be done using the Student's t-test while for non normally distributed numeric variables will be done by Mann-Whitney test. Comparisons between categorical variables will be performed using the chi square test. A p-value less than or equal to 0.05 will be considered statistically significant. All tests will be two tailed.

II.13. Monitoring:

II.13.a. Data monitoring:

The main supervisor will monitor this study. His role is to monitor any risk of bias could be done from participants, operator or assessors. Also to monitor blinding of the assessors and patient safety, outstanding benefits or harms.

II.13.b. Harms:

The main supervisor should inform participants about the possible harms, if present. Participants are allowed to contact the operator at moment through telephone. In case of accidental pulp exposure during excavation , emergency access cavity and pulp extirpation will be done by the operator and then the patient will be referred to Endodontic Department clinic to complete the rool canal treatment.

II.13.c. Auditing:

In the present trial, auditing will be done by the main and co-supervisors to assure quality of the research frequency procedures.

II.14. Ethics and dissemination

II.14.a. Research ethics approval:

Application forms for carrying out the clinical trial, checklist and informed consent of Research Ethics Committee (REC) Faculty of Dentistry, Cairo University will be retrieved and filled, then will be delivered for (REC) committee for approval. This is done to prevent any ethical problems during the study or any harm for any of the participants.

II.14.b. Protocol amendments:

If a new protocol will be used a protocol amendment will be submitted; containing a new copy of the new protocol and brief explanation about the differences between it and the previous protocols. If there is a change in the existing protocol that affects safety of subjects, investigation scope or scientific quality of the trial, an amendment containing a brief explanation about the change will be submitted. If a new author will be added to accomplish the study, an amendment including the investigator's data and qualifications to conduct the investigation will be submitted to prevent ghost authorship.

II.14.c. Consent:

The operator (Ali Mostafa Shamaa) is responsible for admitting and signing the written consents during the enrolment day.

II.15. Confidentiality:

Name, personal data and pictures of the participants will not appear on the protocol form and will be maintained secured for 10 years after the trial. This is done for protection of participants' privacy and civil rights.

II.16. Declaration of interests:

There is no conflict of interest, no funding or material supplying from any parties.

Access to final data will be allowed to the operator ,the main and co-supervisors of the study who are not involved in assessment of the outcome.

II.18. Ancillary and post-trial care:

Patients will be followed up after the application after 3, 6 months.

II.19. Dissemination policy:

Full protocol will be published online in www.clinicaltrials.gov to avoid repetition and to keep the integrity of the research work. Thesis will be discussed in front of judgment committee. The study will be published to report the results of this clinical trial.

Arms: arm 1: Nano silver fluoride solution (Prepared in Nanotech Co., Egypt) based on silver nanoparticles, chitosan and fluoride. Each tooth will receive two drops of NSF with a micro brush, equivalent to a dose of 10 mg of the solution. arm 2: Chlorhexidine digluconate 2 % solution (Cavity Cleanser, Bisco, USA). Each tooth will receive two drops of cavity cleanser with a micro brush, equivalent to a dose of 10 mg of the solution.
Interventions: intervention 1: Nano silver fluoride solution intervention 2: Cavity Cleanser
Intervention descriptions: intervention 1: Nano silver fluoride that composed of silver nanoparticles, chitosan and fluoride that combines preventive and antimicrobial properties. intervention 2: Moistens dentin surface after cavity preparation using a micro brush.

Protocol documents:
Prot_SAP_ICF_000.pdf: 
 
 
Evaluation of the Antibacterial Effect of Nano 
Silver Fluoride versus Chlorhexidine on Occlusal 
Carious Molars Treated with Partial Caries 
Removal Technique: A Randomized Clinical Trial 
 
 
 
 
Document date: 02/03/2021 
 
NCT number: 03186261 
 
 
 
 
 
 
 
 
 
 
 


2 
 
Evaluation of the Antibacterial Effect of Nano 
Silver Fluoride versus Chlorhexidine on Occlusal 
Carious Molars Treated with Partial Caries 
Removal Technique: A Randomized Clinical Trial 
 
Thesis 
Submitted to the Department of Conservative Dentistry 
Faculty of Dentistry Cairo University 
For Partial Fulfillment of the Requirements of Master Degree in 
 Conservative Dentistry 
 
By: 
Ali Mostafa Shamaa 
(B.D.S, Cairo University 2011) 
 
Supervised by: 
Prof. Dr. Randa Mohamed Hafez  
Professor, Conservative Dentistry Department, Faculty of Dentistry 
Cairo University 
 
 
Dr. Mai Mamdouh Mohamed 
Lecturer of Conservative Dentistry Department, Faculty of Dentistry 
Cairo University 
Faculty of Dentistry 
Cairo University 
(2019) 
 


3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Introductory 
 


4 
 
I. Administrative information: 
 
1. Trial registration: 
Trial will be registered in www.clinicaltrials.gov 
2. Protocol version:  
Mar-2017. 
3. Funding: 
The trial will be self funded by the main researcher. 
4. Roles and responsibilities 
 4.i. Ali Mostafa Shamaa 
Role: Operator, data enterer and writing the research. 
Affiliation: Master degree candidate in Conservative Dentistry Department, 
Faculty of Dentistry, Cairo University. 
 
4.ii. Prof. Dr. Randa Hafez. 
Role: Main supervisor, data monitoring, auditing  
Affiliation: Professor, Conservative Dentistry Department,  
Faculty of Dentistry, Cairo University. 
 
 4.iii. Dr. Mai Mamdouh. 
Role: Co-supervisor, data entry and auditing; 
Affiliation: Lecturer, Conservative Dentistry Department,  
Faculty of Dentistry, Cairo University. 
 
 


5 
 
4.iv .Bacteriological assessment: 
Department of  Microbiology, Faculty of Medicine, Cairo University. 
4.v. Mrs. Eman Desouky. 
Role: Sample size calculation; 
Affiliation: Statistician, Faculty of Dentistry, Cairo University, Egypt. 
 
4.vi. Research Ethics Committee (REC) 
Role: Protocol reviewer of the clinical trial in order to protect the right, 
safety, dignity and well-being of the participants; 
Affiliation: Faculty of Dentistry, Cairo University. 
 
 
 
 
 
 
 
 
 
 


6 
 
 
 
 
 
 
 
 
Introduction 
 
 
 
 
 
 
 
 


7 
 
▪ Scientific background: 
  Dental caries is a disease that affects both children and adults. Though it has a 
known etiology which can be prevented and controlled, it remains a major health 
concern in industrialized and developing communities. It is dependent on dental 
plaque stagnation on the tooth surface. The bacterial constituents of the dental 
plaque are responsible for the metabolic activity needed for the development of 
carious lesion and demineralization of tooth structure. However, caries can be 
arrested if the biofilm is controlled but that mainly occurs at the tooth surface, as in 
the cavitated lesions there is a difficulty in hindering or disorganizing the bacterial 
biofilm.1 
  Therefore, restorative intervention is needed which implicates the removal of 
carious tissue followed by restoration of tooth to regain surface integrity that 
enabling efficient removal of dental plaque.2 
  Traditional restorative techniques involve complete removal of carious dentin this 
is based on assumption that complete elimination of bacteria by complete caries 
removal is a must for the success of restoration. Also it may lead to extensive tooth 
loss and pulp exposure especially in deep caries teeth.3 
 
   Another technique is step wise excavation in which partial removal of carious 
dentin is made and the cavity sealed in the first visit till formation of tertiary dentin 
then a second visit is mandatory to remove the remaining carious tissue which adds 
additional cost and effort in the procedure.4 
 
  Moreover, there is several evidence indicating that total removal of carious tissue 
is not required to reach an arrested lesion state.2  Thereby partial caries removal 


8 
 
technique is approached which is less invasive and aims at leaving the deeper layer 
of carious dentin followed by permanent restorative material. The main target of 
this technique is that these deeper layer of carious dentin will be arrested and 
layoff of tertiary dentin is stimulated with subsequent dentinal sclerosis, thus either 
the possibility of pulp exposure or additional procedures during reentry of the 
cavity is eliminated.3  
  However, by leaving a layer of carious dentine which contain viable bacteria, 
there is a liability for caries to stay active and even progress further. In addition to 
the sealing achieved by the restorative material, an application of antibacterial 
agent that reduce bacterial load, slow or arrest the present lesion will ensure the 
success of the partial caries removal technique.5 
▪ Statement of the problem: 
The aim of partial caries removal technique is to eliminate the problems of 
complete caries removal and step-wise excavation by leaving the deeper layer of 
carious dentin. This will help in arresting the lesion with subsequent 
remineralization of the infected dentin. However; leaving bacterial infected dentine 
may lead to recolonization and subsequent recurrent caries.4  
Therefore, the application of an antibacterial agent prior to restoration of deep 
carious molar that eradicates viable bacteria in the remaining carious layer and aids 
in arresting carious lesion will ensure the success of the partial caries removal 
technique.5 Chlorhexidine is one of the most commonly used antibacterial agent. 
However, the use of chlorhexidine has some potential drawbacks like altered taste 
sensation, staining of teeth, and development of resistant bacteria that undermine its 
application.6  


9 
 
Several innovative approaches were made into developing antimicrobial agents 
that have bacteriostatic and bactericidal effects on the bacteria responsible for 
developing dental caries that may have a role in arresting carious lesions. A new 
agent which proven to be successful as antibacterial and in arresting existing caries 
is nano silver fluoride which is a new formulation that composed of silver 
nanoparticles, chitosan and fluoride that combines preventive and antimicrobial 
properties. This new formulation was proven to be safe and has shown good 
antimicrobial properties against cariogenic bacteria which helps in arresting 
carious lesion.7 
▪ Rationale: 
The purpose of this study is to evaluate the antibacterial effect of nano silver 
fluoride solution on the carious dentin. This new formula is a promising anti-
caries agent with low toxicity and ease of application in addition to its 
antimicrobial effect. This agent could help in caries arrest and decrease in the 
occurrence of secondary caries.  
 
➢ External validity: 
Benefits to patients/population: 
1. Preserve pulp vitality. 
2. Decrease the incidence of secondary caries. 
3. Time and cost effective methods. 
Benefits to practitioners/clinicians: 
1. Noninvasive and conservative. 
2. Avoid unnecessary removal of dentin tissues. 
3. Time and money saving. 
 


10 
 
➢ Objectives : 
The intention of this study is to evaluate the antibacterial action of nano silver 
fluoride solution application against oral streptococci mutans in comparison to 
chlorhexidine solution after partial caries removal in class I carious permanent 
molars. 
➢ Hypothesis: 
 The null hypothesis tested in this study, that there is no difference between nano 
silver fluoride and chlorhexidine application on the bacterial count of oral 
streptococci mutans after partial caries removal in class I carious permanent 
molars. 
➢ Trial design:  
This investigation will be a randomized controlled clinical trial (RCT).  
➢ Trial type:  
Parallel study design. 
➢ Trial framework:  
Equivalence trial 
 
➢ Allocation ratio:  
1:1. 
 
 
 


11 
 
 
 
 
 
 
 
 
Review of literature 
 
 
 
 
 
 
 
 
 


12 
 
➢ Search strategy: 
• Source:  
The results have been searched in two data bases:  
➢ PubMed database and Scopus database. 
• Date: 16\03\2017 
Index terms 
Mesh terms 
Deep carious lesion 
 
Dentin caries 
Carious dentin 
 
Nano silver fluoride 
 
Chlorhexidine 
 
Chlorhexidine 
Chlorhexidine gluconate 
Chlorhexidine Hydrochloride 
Chlorhexidine Acetate 
Chlorhexidine thymol 
Chlorhexidine bigluconate 
Chlorhexidine digluconate 
Bacterial count 
Bacterial count 
Bacterial counts 
Bacterial load 
Microbiological analysis 
Microbial colony count 
Microbial colony counts 
Microbial colony forming units 
assays 


13 
 
Microbial colony-forming units 
assays 
 
 
 
➢ Inclusion and exclusion criteria 
Inclusion Criteria 
Exclusion criteria 
• Articles based on clinical trials 
that report the antibacterial 
effect of nano silver particles.  
• Articles published in English 
• Articles published after 2006 
• Articles based on any other 
assessment method. 
• Any other antibacterial agent 
used. 
• Articles published in any other 
language. 
• Articles published before 2006 
 
 
 
 
 
 
 
 
Search terms 
Medline 
Scopus 
P 
Dentin caries OR Deep cavities OR 
52870 
4409 


14 
 
Deep caries lesion OR Carious dentin 
I 
Nano silver fluoride OR NSF OR 
Nano silver OR Silver nanoparticles 
OR Silver nanoparticle OR Silver 
nano  
19116 
178478 
C 
Chlorhexidine OR CHX 
OR Chlorhexidine gluconate 
OR Chlorhexidine Hydrochloride 
OR Chlorhexidine Acetate 
OR Chlorhexidine thymol 
OR Chlorhexidine bigluconate 
OR Chlorhexidine digluconate 
11640 
 
3250 
 
O 
Bacterial count OR Bacterialcounts 
OR Bacterial load OR 
Microbiological analysis OR 
Microbial colony count OR 
Microbial colony counts OR 
Microbial colony forming units 
assays OR Microbial colony-forming 
units assays 
 
553619 
 
1770 
P& I 
(((((Dentin caries) OR Deep cavities) 
OR Deep caries lesion) OR Carious 
dentin)) AND ((((((NSF) OR Nano 
silver) OR Silver nanoparticles) OR 
Silver nanoparticle) OR Silver nano) 
OR Nano silver fluoride) 
56 
 
94 


15 
 
P & C 
(((((Dentin caries) OR Deep cavities) 
OR Deep caries lesion) OR Carious 
dentin)) AND ((((((((Chlorhexidine 
thymol) 
OR 
Chlorhexidine 
digluconate) 
OR 
CHX) 
OR 
Chlorhexidine) 
OR 
chlorhexidine 
gluconate) 
OR 
Chlorhexidine 
Hydrochloride) 
OR 
Chlorhexidine 
Acetate) 
OR 
Chlorhexidine 
bigluconate) 
785 
0 
P &  I&O 
((((((Dentin caries) OR Deep cavities) 
OR Deep caries lesion) OR Carious 
dentin)) AND ((((((NSF) OR Nano 
silver) OR Silver nanoparticles) OR 
Silver nanoparticle) OR Silver nano) 
OR Nano silver fluoride)) AND 
((((((((Microbial colony counts) OR 
Microbial 
colony-forming 
units 
assays) OR Microbial colony forming 
units assays) OR Bacterial count) OR 
Bacterial counts) OR Bacterial load) 
OR Microbiological analysis) OR 
Microbial colony count) 
15 
3 
P&C&O 
((((((Dentin caries) OR Deep cavities) 
OR Deep caries lesion) OR Carious 
dentin)) AND ((((((((Chlorhexidine 
227 
0 


16 
 
thymol) 
OR 
Chlorhexidine 
digluconate) 
OR 
CHX) 
OR 
Chlorhexidine) 
OR 
chlorhexidine 
gluconate) 
OR 
Chlorhexidine 
Hydrochloride) 
OR 
Chlorhexidine 
Acetate) 
OR 
Chlorhexidine 
bigluconate)) AND ((((((((Microbial 
colony counts) OR Microbial colony-
forming units assays) OR Microbial 
colony forming units assays) OR 
Bacterial count) OR Bacterial counts) 
OR 
Bacterial 
load) 
OR 
Microbiological 
analysis) 
OR 
Microbial colony count) 
P& I& C& 
O 
(((((((Dentin caries) OR Deep cavities) 
OR Deep caries lesion) OR Carious 
dentin)) AND ((((((NSF) OR Nano 
silver) OR Silver nanoparticles) OR 
Silver nanoparticle) OR Silver nano) 
OR Nano silver fluoride)) AND 
((Chlorhexidine 
OR 
CHX 
OR 
Chlorhexidine 
gluconate 
OR 
Chlorhexidine 
Hydrochloride 
OR 
Chlorhexidine 
Acetate 
OR 
Chlorhexidine 
thymol 
OR 
Chlorhexidine 
bigluconate 
OR 
0 
0 


17 
 
Chlorhexidine digluconate))) AND 
((((((((Microbial colony counts) OR 
Microbial 
colony-forming 
units 
assays) OR Microbial colony forming 
units assays) OR Bacterial count) OR 
Bacterial counts) OR Bacterial load) 
OR Microbiological analysis) OR 
Microbial colony count) 
 
➢ Review of studies: 
dos Santos et al 20147, conducted a randomized controlled clinical trial to evaluate 
the effectiveness of a Nano Silver Fluoride (NSF), applied once a year to arrest 
caries in children. One hundred thirty decayed primary teeth were randomly 
divided into two groups: NSF as the experimental agent and water as the control 
group. Teeth were clinically diagnosed and treated by one masked examiner and 
followed up at seven days and five and 12 months by another calibrated examiner 
who was blinded to the type of treatment. The criteria of the ICDAS II were 
followed to determine the activity of lesion and the diagnosis of caries. The 
Pearson’s chi-square test was used to compare the groups during different follow-
up exams. At seven days, 81% of teeth in the NSF group exhibited arrested caries, 
whereas in controls, no teeth had arrested decay (p < 0.001). After five months, the 
NSF group had 72.7% with arrested decay, and the control group had 27.4% (p < 
0.001). At 12 months, 66.7% of the lesions treated with NSF were still arrested, 
while the control group had 34.7% remaining arrested ( p = 0.003). The number 
need to treat at five months was two, and at 12 months, the number was three. It 
was stated that  NSF formulation is effective to arrest active dentine caries and not 
stain teeth.  


18 
 
 
Fahadian et al 2016 8, evaluated the effect of silver nanoparticles incorporated 
into acrylic baseplates of orthodontic retainers on Streptococcus mutans colony-
forming units. The main outcome was to compare the number of S mutans 
colony-forming units between two groups of orthodontic patients at the debonding 
stage, in which one group wore orthodontic retainers which have silver 
nanoparticles incorporated into their acrylic baseplates while the other group 
received conventional orthodontic retainers. Swab samples were taken from the 
maxillary palatal side from the patient in the dental chair at retainer placement (T1, 
1 week after debonding the fixed orthodontic appliance) and T2, 7 weeks later. 
Twenty-nine patients in the control group and 32 in the intervention group were 
analyzed. At T1, the intervention group had higher S mutans colony counts relative 
to the control group.  Later, at T2 after 7 weeks there was a  significant reduction 
of colonies in the intervention group. Finally it was concluded that adding silver 
nanoparticles to the acrylic plate of retainers had a strong antimicrobial effect 
against S mutans under clinical conditions. 
 
Salas-López et al 20179, directed an experimental and clinical trial to evaluate the 
effects of pit and fissure sealant mixed with silver nanoparticles on dental caries, 
by means of monthly measurement of fluorescence with DIAGNOdent over six 
months. In the experimental phase, the adhesion and microleakage of the pit and 
fissure sealant experiment were evaluated. Two groups of 10 teeth, without serious 
carious lesions, were included. Group A received conventional Clinpro pit and 
fissure sealant (3M Espe, St. Paul, MN, USA), and group B received NNPs mixed 
with Clinpro pit and fissure sealant. For the clinical phase, a split-mouth study 
double blind clinical trial was performed on 40 children aged 6-10 years old with 
healthy, erupted permanent first molars. A conventional pit and fissure sealant or a 


19 
 
silver nanoparticle-mixed sealant was randomly placed. Repeated measures 
analysis was performed to detect presence of carious lesions was assessed using a 
pen-type laser fluorescence device, the DIAGNOdent pen (Kavo, Biberach, 
Germany). Conventional sealant presented an average microleakage of 30.6%, and 
the silver nanoparticle-mixed sealant showed 33.6% (P=NS). A three times greater 
reduction in fluorescence was found in the silver nanoparticles group compared to 
the conventional group (P<0.05). It was concluded by the author that silver 
nanoparticle-mixed sealant reduced tooth demineralization significantly and likely 
increased remineralization, compared to the conventional sealant. 
 
 
 
 
 
 
 
 
 
 
 
Aim of the study: 


20 
 
The aim of this study is to evaluate the antibacterial action of Nano Silver Fluoride 
solution application in comparison to Chlorhexidine solution after partial caries 
removal in class I carious permanent molars. 
PICOTS: 
P= Deep class I carious molars. 
I= Nano silver fluoride (Prepared in Nanotech Co., Egypt). 
C= Chlorhexidine2 % solution (Cavity Cleanser, Bisco, USA). 
O = Bacterial Count (Streptococcus Mutans) CFU/ml. 
T = T1 before application and T2 after application in the same visit. 
S= Randomized Controlled Trial, In-Vivo Comparative Study. 
 
 
Research question: 
Is the application of Nano Silver Fluoride solution after partial caries removal-
compared to the Chlorhexidine- will minimize the bacterial count in dentin in 
permanent class I carious molars? 
 
 
 
I. Materials: 
Measuring Device 
Measuring Unit 
Outcome Name 
Digital Colony Counter, Agar 
Diffusion test 
CFU/ml 
Bacterial count of 
(Strepto. Mutans) 
 


21 
 
1. Nano silver fluoride solution (Prepared in Nanotech Co., Egypt) based on silver 
nanoparticles, chitosan and fluoride. Each tooth will receive two drops of NSF 
with a micro brush, equivalent to a dose of 10 mg of the solution 
2. Chlorhexidine digluconate 2 % solution (Cavity Cleanser, Bisco, USA). 
Moistens dentin surface after cavity preparation using a micro brush.  
3. Rubber dam. 
4. Hand spoon excavators. 
5. High speed contra angle air-driven hand-piece. 
6. Round and round end cylinder carbide and diamond burs. 
7. Universal adhesive (Tetric® N-Bond Universal, Ivoclar Vivadent, Schaan, 
Liechtenstein). The adhesive wil be scrubbed into the tooth surface for at least 20 
seconds. Followed by dispersing with oil- and moisture-free compressed air until a 
glossy, immobile film layer results. Light-cure for 10 seconds using a light 
intensity of ≥ 500 mW/cm2.  
8. Bulk-fill resin composite (Tetric® N-Ceram Bulk Fill, Ivoclar Vivadent, Schaan, 
Liechtenstein) placed in increments of up to 4 mm and cured in 10 seconds (≥ 
1,000 mW/cm2 ) or 20 seconds (≥500 mW/cm2). 
9. Medium, fine and extra fine finishing diamond stones and rubber polishing 
disks. 
 
II. Methods: 


22 
 
II.1. Study setting 
This clinical study will be held in the Clinic of Conservative Dentistry Department,  
Faculty of Dentistry, Cairo University, Egypt. The researcher will bear ultimate 
responsibility for all activities associated with the conduct of a research project 
including recruitment of patients, explaining and performing the procedures to 
them. 
II.2. Eligibility criteria: 
II.2.a. Eligibility Criteria of participants: 
➢ Inclusion Criteria of participants:  
1. 18 – 40 years.  
2.  Not received antibiotic therapy since 1 month before sampling. 
3. Males or Females.  
4. Good oral hygiene.  
5. Co-operative patients approving the trial. 
 
➢ Exclusion criteria of participants:  
1. Pregnancy.  
2. Systemic disease or severe medical complications.  
3. Heavy smoking.  
4. Xerostomia.  
5. Lack of compliance.  
II.2.b. Eligibility Criteria of teeth: 


23 
 
➢ Inclusion Criteria of teeth: 
1. Class I deep caries lesions in permanent molar (reaching ≥ 1/2 of the dentin on 
radiographic examination). 
2. Absence of spontaneous pain; negative sensitivity to percussion; and absence of 
periapical lesions (radiographic examination). 
➢ Exclusion criteria of teeth: 
1. Class II caries lesion. 
2. Shallow or enamel caries. 
3. Cuspal loss or caries beneath the gingival margin. 
 
II.3. Variables of the study: 
Table (1) Variables of study 
 
 
II.4. Trial Description: 
            Variable 
  Symbol 
                    Refers to 
 
Intervention 
      I 
Nano silver fluoride solution 
      C 
Chlorhexidine 2 % 
Time of dentin sample      
collection 
      T1 
Before the application 
      T2 
After the application 


24 
 
II.4.a Excavation protocol: 
The teeth of the patients that meet inclusion criteria will be anesthetized, isolated 
with a rubber dam. Cavity opened using conventional high-speed rotary 
instruments. The central cariogenic biomass and the superficial parts of the 
necrotic dentine will be removed with round burs. Caries lesion will be completely 
removed in the enamel/dentin junction. The excavation procedure will be 
terminated as soon as the soft and wet dentine was removed and the remaining 
tissue was leathery but not hard on exploring. A dentinal sample will then be 
collected from the base of the cavity using sterile spoon excavator as a baseline for 
bacteriological assessment. Then, application of either intervention or control 
agent, another dentinal sample will be collected using a sterile excavator and 
transferred into sterile tubes and transferred to the laboratory for microbiological 
analysis. 
II.4.b Assessment of Outcome: 
Samples of carious dentine will be collected with sterile excavator before and after 
application of intervention/control agent. The dentine samples will be transferred 
to sterile container containing a 1mL thioglycollate medium used as a carrier, then 
this sterile container will be kept in an ice box and taken to the microbiology 
laboratory for processing, within an hour, by another examiner who is blinded to 
the type of agent applied after partial caries removal. Samples will be vortexed for 
two minutes, decimally diluted and 0.1 ml will be plated on Mitis Salivarius 
Bacitracin agar plates, these plates will be incubated anaerobically for 48 hour at 
37ºC then aerobically for 24 hour at room temperature. The number of colonies 
will be expressed as colony–forming units (CFU/ml) compared before and after 
application of the antibacterial agent. 


25 
 
II.5. Participant timeline: 
All clinical procedures will be carried out at the same visit.  
 
 
 
II.6. Sample size calculation: 
The aim of this study is to evaluate the antibacterial action Nano Silver Fluoride 
solution application in comparison to Chlorhexidine solution after partial caries 
removal in class I carious permanent molar. Based on a previous study by Mohan 
et al. 20165, the expected difference between two interventions is expected to be 
2.1±2.2CFU/mL × 103. Using power 80% and 5% significance level we will need 
to study 18 in each group to be able to reject the null hypothesis that the population 
means of the experimental and control groups are equal. This number is to be 
increased to 22 patients in each group to compensate for possible losses during 
follow up. The sample size was calculated by PS program. 
 
II.7. Recruitment: 
Cavity preparation
Chlorhexidine 2 %
Nano silver fluoride
Restoration of the 
cavity
Follow up
3 Month
6 Month
Diagnosis
Enrolment
1 month before clinical procedures 


26 
 
Patients will be recruited from outpatient clinic of Conservative Dentistry 
Department in Faculty of Dentistry, Cairo University; after explaining the 
benefits/risks from the application of the interventions, then eligible patients will 
be recruited to fulfil the eligibility criteria according to participant timeline. 
II.8.a. Recruitment Strategy: 
The patients will be subjected to full examination and diagnosis using dental 
charts. Once the patients that are potentially eligible for this study are identified, 
they will be contacted by the researcher who will explain the study and ascertains 
the patient’s interest. If interested, more detailed evaluations and preparations are 
made. 
II.9. Randomization and assignment of interventions: 
II.9.a. Allocation sequence generation: 
The allocation sequence will be generated using (www.randomization.com). 
II.9.b. Allocation concealment mechanism: 
The randomization unit was the tooth, and the randomization procedure will be 
performed as follows. A number corresponding to each treatment group will be 
printed on pieces of paper and kept in dark containers. A paper will be selected 
from the container by a person other than the operator, and the treatment indicated 
will be performed (intervention/control). Blinding of the operator is not possible, 
because the color of intervention and control solution is different. The operator is 
blinded until randomization into groups, to avoid biases with regard to the 
application of antibacterial agent. Also, the examiner who will carry the 
microbiological analysis of dentin samples will be blinded to the type of agent 


27 
 
applied during treatment. Finally, the treatment results will be assessed blindly by 
a statistician. 
 II.9.c. Implementation: 
Dr. Mai Mamdouh (co-supervisor) will perform the allocation sequence and assign 
the participants to the intervention/Control treatment group. 
II.9.d. Blinding: 
The side to which interventions or control is assigned to will be recorded and all 
records of all patients will be kept with the main supervisor. Blinding of the 
operator is not possible, because the color of intervention and control solution is 
different. The operator is blinded until randomization into groups, to avoid biases 
with regard to the application of antibacterial agent. Also, the examiner who will 
carry the microbiological analysis of dentin samples will be blinded to the type of 
agent applied during treatment. Finally, the treatment results will be assessed 
blindly by a statistician. 
 
II.10. Data collection methods: 
*Baseline data collection:  
For every patient medical history, dental history and examination charts will be 
filled by the operator. The report will be anonymous where patients identified by 
their serial numbers (the first letter of the first and last name and date of birth) only 
will be registered. Full detailed personal data of the patient will be written in 
separate sheet having the patient's serial number for further contact with patient. 
 


28 
 
*Outcome data collection: 
The results will be converted into a table to facilitate the description of the results. 
The microbiological analysis will be performed blindly in relation to the type of 
solution applied after partial caries removal. 
II.11.Data management: 
The data will be entered and stored on a personal computer. Double data entry will 
be saved on an external hard disc to prevent loss of data. 
II.12. 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. Categorical data 
will be explored for normality using Kolmogrov-Smirnov test and Shapiro-Wilk 
test. Comparisons between two groups for normally distributed numeric variables 
will be done using the Student’s t-test while for non normally distributed numeric 
variables will be done by Mann-Whitney test.  Comparisons between categorical 
variables will be performed using the chi square test. A p-value less than or equal 
to 0.05 will be considered statistically significant. All tests will be two tailed. 
II.13. Monitoring: 
II.13.a. Data monitoring: 
The main supervisor will monitor this study. His role is to monitor any risk of bias 
could be done from participants, operator or assessors. Also to monitor blinding of 
the assessors and patient safety, outstanding benefits or harms. 


29 
 
II.13.b. Harms: 
The main supervisor should inform participants about the possible harms, if 
present. Participants are allowed to contact the operator at moment through 
telephone. In case of accidental pulp exposure during excavation , emergency 
access cavity and pulp extirpation will be done by the operator and then the patient 
will be referred to Endodontic Department clinic to complete the rool canal 
treatment. 
II.13.c. Auditing: 
In the present trial, auditing will be done by the main and co-supervisors to assure 
quality of the research frequency procedures. 
II.14. Ethics and dissemination 
II.14.a. Research ethics approval: 
Application forms for carrying out the clinical trial, checklist and informed consent 
of Research Ethics Committee (REC) Faculty of Dentistry, Cairo University will 
be retrieved and filled, then will be delivered for (REC) committee for approval. 
This is done to prevent any ethical problems during the study or any harm for any 
of the participants. 
II.14.b. Protocol amendments: 
If a new protocol will be used a protocol amendment will be submitted; containing 
a new copy of the new protocol and brief explanation about the differences 
between it and the previous protocols. If there is a change in the existing protocol 
that affects safety of subjects, investigation scope or scientific quality of the trial, 


30 
 
an amendment containing a brief explanation about the change will be submitted. 
If a new author will be added to accomplish the study, an amendment including the 
investigator’s data and qualifications to conduct the investigation will be submitted 
to prevent ghost authorship. 
II.14.c. Consent: 
The operator (Ali Mostafa Shamaa) is responsible for admitting and signing the 
written consents during the enrolment day.  
II.15. Confidentiality: 
Name, personal data and pictures of the participants will not appear on the protocol 
form and will be maintained secured for 10 years after the trial. This is done for 
protection of participants’ privacy and civil rights. 
II.16. Declaration of interests: 
There is no conflict of interest, no funding or material supplying from any parties. 
II.17. Access to data: 
Access to final data will be allowed to the operator ,the main and co-supervisors of 
the study who are not involved in assessment of the outcome. 
II.18. Ancillary and post-trial care: 
Patients will be followed up after the application after 3, 6 months. 
II.19. Dissemination policy: 
Full protocol will be published online in www.clinicaltrials.gov to avoid repetition 
and to keep the integrity of the research work. Thesis will be discussed in front of 
judgment committee. The study will be published to report the results of this 
clinical trial. 


31 
 
 
 
 
 
 
 
 
References 
 
 
 
 
 
 
 
 


32 
 
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Raggio, D. P. (2014). Sealing versus partial caries removal in primary molars: a 
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Consent