Clinical trial NCT05221749
Conditions: Primary Teeth Microbial Colonization Caries Dental Caries in Children
Keywords: Nano silver fluoride silver diamine fluoride antibacterial caries arrest microbiology primary teeth pediatric clinical trial nanosilver SDF
Sponsor: Nour Ammar, BDS
Start date: 2022-03-15; Completion date: 2022-06-15
Enrollment: 50 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
To assess the antibacterial effect of Nanosilver Fluoride (NSF) in relation to caries activity in dentin caries lesions of primary teeth in comparison to Silver Diamine Fluoride (SDF).

Detailed description:
Fifty children aged 4 to 6 years old with active dentin caries lesions (score 5 according to International Detection and Assessment System (ICDAS II) criteria) will be enrolled in the study. They will be equally and randomly allocated into 2 groups: a group receiving NSF and a control group receiving SDF treatment. Microbiological samples will be collected from the carious lesions and from unstimulated saliva at the baseline and at the 1 and 3 months' follow-up appointments. Bacterial counts will be assessed using Mitis Salivarius agar (selective culture media for S. mutans) and Rogosa agar (selective culture media for lactobacilli), and the results will be expressed in colony-forming units. Data regarding the children's oral health will be collected and their dmf index will be scored. The arrest of active carious lesions will be measured at the follow-up appointments according to ICDAS II criteria.

Arms: arm 1: These active caries lesions will receive silver diamine fluoride treatment arm 2: These active caries lesions will receive nanosilver fluoride treatment
Interventions: intervention 1: Nanosilver Fluoride (NSF) intervention 2: Silver diamine fluoride
Intervention descriptions: intervention 1: Optimized synthesis of Nanosilver Fluoride intervention 2: Silver Diamine Fluoride

Protocol documents:
Prot_SAP_001.pdf: 
1 
 
Official title: The Antibacterial Effect of Nanosilver Fluoride 
in Relation to Caries Activity in Primary Teeth: a Randomized 
Controlled Clinical Trial 
 
NCT number: NCT05221749 
 
Date uploaded: 25th July, 2023 
 
 
 
 
 


2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
THE ANTIBACTERIAL EFFECT OF NANOSILVER 
FLUORIDE IN RELATION TO CARIES ACTIVITY IN 
PRIMARY TEETH: 
A RANDOMIZED CONTROLLED CLINICAL TRIAL 
 
 
 
 
 
2021/2022
 
 
 


3 
 
ABSTRACT
 
Background: Dental caries is among the most prevalent chronic diseases in children. 
Minimally invasive dentistry is a highly convenient and efficient method of managing caries 
in pediatric patients. Recently, Nanosilver fluoride (NSF) has emerged as a promising topical 
fluoride agent with a potent cariostatic and antibacterial potential. This new anticaries agent is 
used as an alternative to overcome the drawbacks of topical fluoride and silver diamine fluoride 
(SDF) in caries prevention. 
Objective: To assess the antibacterial effect of NSF in relation to caries activity in dentin caries 
lesions of primary teeth in comparison to SDF after 1 month. 
Methods: Fifty children aged 4 to 6 years old with active dentin caries lesions according to 
ICDAS score 5 will be recruited from the Pediatric Dentistry clinic at the Faculty of Dentistry, 
Alexandria University. The children will be equally and randomly allocated into 2 groups; the 
first group will be treated using NSF, while the second group will be the control group receiving 
SDF treatment. Microbiological assessment using a microbrush will be done at baseline and 
follow-up appointments to record the bacterial counts using the suitable culture media, the 
results will be expressed in colony forming units (CFU). Data regarding the children’s oral 
health will be collected and their dmf index will be scored. The arrest of active carious lesions 
will be measured after 1 month according to ICDAS criteria and a microbiological assessment 
will be completed. 
Results: The relation between bacterial counts (CFU) and lesion activity for both interventions 
will be assessed. The data collected will be statistically evaluated with the suitable tests and 
tabulated. 
 
Keywords: Nanosilver fluoride, silver diamine fluoride, antibacterial, caries arrest, 
microbiology, primary teeth, pediatric, clinical trial


4 
 
INTRODUCTION 
 
Dental caries is a chronic disease, ranking among the most highly 
prevalent diseases in children. According to the Global Burden of Disease study in 
2017,1 untreated dental caries in the deciduous dentition was the 10th most prevalent 
condition globally, affecting 532 million children worldwide.2 It is one of the most 
common infectious diseases of childhood which is evident from the fact that in 
children, it is 7 times more common than hay fever or allergic rhinitis and 5 times 
more common than asthma.3 Under severe conditions, the pain and discomfort 
associated with dental caries can affect the quality of life of children and may lead 
to acute and chronic infections, changed eating and sleeping habits, and an increased 
risk of hospitalization.4,5 
 
Among a variety of chemotherapeutic agents that have been tested for 
preventing and arresting caries. Silver diamine fluoride (SDF) has a proven 
antibacterial action6 and releases fluoride ions onto the applied tooth surfaces, 
therefore, assisting in enamel remineralization7. Recently, the promising results of 
published clinical trials have popularized the use of SDF throughout the world.8,9 
Today, it is available for arresting caries as a simple, non-invasive, low-cost 
medication.8,10 The major drawback of SDF is the staining of carious tissue to dark 
black due to the oxidation of the ionic silver in its formulation, along with reversible 
slightly painful lesions in oral mucosa caused by accidental contact of the SDF 
solution with soft tissues, these lesions usually disappear within 48 hours.11  
 
New formulations of silver nanoparticles (AgNP) show potent 
antimicrobial properties against a wide variety of microorganisms.12–14 Haghgoo et 
al.15 suggested the incorporation of nanosilver in varnishes and reported increased 
antimicrobial properties against cariogenic microorganisms such as streptococcus 
mutans and streptococcus salivarius. Nanosilver fluoride (NSF) is an experimental 
formula obtainable as a yellow solution containing uniformly dispersed silver 


5 
 
nanoparticles and fluoride. It was developed to be an effective anti-caries agent that 
does not stain the porous dental tissues black as does SDF.16  
 
Interestingly, it is unknown if the reduction in caries activity brought 
about the use of nanosilver fluoride is due to its remineralizing action, antibacterial 
action, or due to a combination of both.17,18 There is a need to understand the 
interaction of topical caries arresting agents like NSF and SDF with the oral 
microbiome associated with dental caries, since the in vitro findings cannot be 
extrapolated to the clinical use of NSF.19  
 
To our knowledge, no publication has reported the in vivo antimicrobial 
efficiency of NSF on the primary dentition in comparison to SDF. Therefore, this 
study will be undertaken to evaluate and compare the clinical antibacterial and 
cariostatic effectiveness of NSF compared to that of SDF in 4-6 year old children. 
The null hypothesis of this study is that no statistically significant difference will be 
detected regarding the antibacterial effect of nanosilver fluoride in relation to caries 
activity in dentin caries lesions of primary teeth, in comparison to silver diamine 
fluoride, after one month. 
 
 


6 
 
AIM OF THE STUDY 
 
General aim 
 
To assess the antibacterial effect of nanosilver fluoride in relation to 
caries activity in dentin caries lesions of primary teeth in comparison to silver 
diamine fluoride after one month. 
 
Specific aims 
• To compare the change in Streptococcus mutans and Lactobacilli counts in 
dentin caries lesions of primary teeth after one month of the application of 
nanosilver fluoride or silver diamine fluoride. 
• To compare the effect of nanosilver fluoride to that of silver diamine fluoride 
on caries lesion activity in dentin caries lesions of primary teeth after one 
month of application according to ICDAS criteria. 
 
 


7 
 
MATERIALS AND METHODS 
Study Design 
This will be a randomized, parallel, two-arm clinical trial, with a 1:1 
allocation ratio. It will be set up and reported according to the CONSORT 
guidelines.20  
The PICOT question is: will children (4-6 years old) with an active carious 
lesion (ICDAS code 5) (population; P) using nanosilver fluoride varnish 
(Intervention; I) in comparison to silver diamine fluoride (comparison; C) show 
a greater percentage decrease in bacterial counts in the caries lesion (outcome; O) 
after 1 month follow-up (time; T)? 
 
Study Setting and Location 
The study will take place in the Department of Pediatric Dentistry and 
Dental Public Health at the Faculty of Dentistry and the Department of Medical 
Microbiology and Immunology at the Faculty of Medicine, Alexandria 
University, Alexandria, Egypt. 
 
Sample 
Sample size estimation 
The minimum sample size was calculated based on the results of a previous 
study.6 Adopting a power of 80% and a statistical significance level of 5% 
(=0.05), 24 children in each group will be needed to generate a statistically 
significant result. The sample size will be increased to 25 children of both genders 
per group to compensate for possible drop-out participants. Total sample size= 
25x2=50. The sample size estimation was based on Rosner’s method21 and 
calculated using G*Power 3.1.9.7.22 


8 
 
Eligibility criteria 
The participants enrolled in this study will be selected after fulfilling the 
following criteria: 
 
Inclusion Criteria 
• Children aged 4-6 years old. 
• The presence of at least one active carious lesion on a primary tooth, with a 
score of 5 according to the International Detection and Assessment System 
(ICDAS), detected by visual-tactile inspection to assess lesion severity and 
activity.23 (Appendix I, Tables 1 and 2 ) 
• Completion of an informed consent to participate in the study. 
 
Exclusion criteria  
• Children reporting spontaneous or elicited pain from caries or showing any 
signs of pulpal infection, swelling, abscess, obvious discoloration of the tooth, 
or premature mobility.11  
• Reported usage of local or systemic antibiotics, chlorhexidine or fluoride 
mouthwashes within the last 2 weeks.24 
• Children presenting with special health care needs or undergoing medical 
treatment for chronic or acute diseases affecting salivary flow. 
• Allergy or sensitivity to silver or any of the materials included in the study.11 
• Child weight less than 10 Kg (to avoid concerns for toxicity).25  
 
 


9 
 
Randomization technique and allocation26 
Subjects complying with the inclusion criteria will be randomly assigned 
using a computer-generated list of random numbers to either the NSF or the SDF 
group (www.random.org). Allocation will be performed by a trial independent 
individual and the allocation ratio is intended to be equal (1:1). 
Allocation concealment27 
An assistant will be responsible for giving each participant a serial number 
that will be used for his/her allocation. A duplicate of this number will be kept in 
an opaque envelope indicating to which group the patient belongs. This envelope 
will be kept by a trial independent individual who will be assigned the role of 
opening it only at the time of intervention; so that the group to which the child is 
allocated is concealed from the investigator. 
Grouping 
Participants will be randomly and equally allocated to one of the two treatment 
groups: 
• Test group (NSF): Twenty-five children meeting the eligibility criteria will 
receive Nanosilver fluoride. 
• Control group (SDF): Twenty-five children meeting the eligibility criteria 
will receive 38% Silver diamine fluoride. 
 
Blinding 
           The investigator will not be blinded to the treatment type as the SDF 
solution has a bluish tint and will stain the active caries lesions black, while the 
NSF solution has a yellowish tint and is not expected to stain the lesions. 
However, the participants, the statistician, and the microbiologist will be blinded 
to the treatment group.  


10 
 
Materials 
PEG-coated nanosilver fluoride (50 nm average particle size) will be prepared in 
the Center of Excellence for Research in Regenerative Medicine and its 
Applications (CERRMA) at the Faculty of Medicine, Alexandria University).  
Once all of the above characterization tests and cytotoxicity assays yield 
satisfactory results, fluoride will then be added. Ten ml of 22,600 ppm of 5% 
Sodium fluoride varnish will be added to the nanosilver particles in a light proof 
brown bottle, vigorous stirring will be performed to achieve uniform dispersion 
of the nanosilver particles.  
• 38% silver diamine fluoride: Advantage Arrest, Elevate Oral care (8 mL 
bottle).*28 
• Gauze 
• Mirror  
• Cotton rolls and pelllets 
• Tweezers 
• Ball ended probe 
• Plastic dappen dish 
• Petroleum jelly 
• Microbrush 
 
Materials for the microbiological assessment 
• Microbrush 
• Mitis Salivarius Bacitracin (MSB) agar (selective culture media for S. 
mutans), prepared according to the manufacturer’s instructions.** 
• Rogosa SL agar plates (selective culture media for lactobacilli), prepared 
according to the manufacturer’s instructions.*** 


11 
 
• Saline as a transport medium. 
• Sterile test tubes.  
• Sterile plastic Petri dishes. 
• Anaerobic gas jar. 
• Incubator. **** 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
__________________________________________________ 
  
*      Elevate Oral Care, LLC, West Palm Beach, FL, USA.  
**    Difco Laboratories Inc, NJ, USA. 
***  Himedia Laboratories, Mumbai, India. 
****Red line by binder  


12 
 
Method 
First visit: baseline examination 
After obtaining the informed consent from the care giver/parent, the 
researcher will provide oral hygiene instructions to each of the study participants 
and will inform them about the importance of maintaining good oral hygiene 
and a proper diet.29 The patients and caregivers will be informed about the 
therapeutic intervention to be applied, the possible outcomes, and side effects in 
detail. All of the examinations and interventions will be done by one calibrated 
examiner. All of the lesions in the children’s oral cavity indicated for treatment 
with NSF or SDF will be treated and sampled for microbiological analysis.30 
On the day of the intervention, the children will be asked to refrain from 
tooth brushing in the morning, as well as eating and drinking (except water) for 
at least two hours before the appointment. The dmf index will be recorded for 
each patient. 
For caries treatment in all groups, no caries or unsupported enamel will be 
removed. The patients will be instructed to rinse with a cup of water, then any 
gross debris will be gently removed with a piece of gauze. The tooth will then be 
partially isolated using cotton rolls and a saliva ejector. Additionally, petroleum 
jelly will be applied to the gingiva for added protection. Before applying the 
intervention, a 2-mm wide microbrush will be rubbed on the carious lesion for 4 
seconds to collect the baseline microbiological sample.6 The microbrush will then 
be inserted in a sterile test tube containing 1 mL of saline and transported to the 
microbiology laboratory within 1-2 hours.31 
Intervention 
Test group (NSF): 
• 1 drop of NSF will be dispensed into a sterile plastic dappen dish.  


13 
 
• Affected tooth surfaces will be dried with a gentle flow of air.  
• The solution will be applied to the carious lesions using a microbrush. 
• No more than one drop of NSF will be used for each patient. 
• The NSF solution will be left in contact with the tooth surface for 1 minute.32 
• Any excess will be removed with a cotton pellet to minimize systemic 
absorption. 
Control group (SDF): 
It will be applied according to the manufacturer’s instructions and the AAPD 
guidelines for using SDF.28,30 
• One drop of 38% SDF will be dispensed into a sterile plastic dappen dish. 
• Affected tooth surfaces will be dried with a gentle flow of air.  
• A microbrush will be dipped into the SDF and dabbed on the side of the 
dappen dish to remove excess liquid before applying the solution to only the 
affected tooth surface. 
• No more than one drop of SDF will be used for each patient. 
• The SDF will be allowed to dry on the lesion for 1 minute.33 
• Any excess will be removed with a cotton pellet to minimize systemic 
absorption. 
 
After the application of fluoride agents, all of the children will be 
instructed not to drink or eat for 1 hour after the appointment, their parents will 
be asked to observe this instruction.24 Additionally, for ethical considerations, 
teeth not treated with SDF or NSF or those showing signs of failure or any new 
carious lesions that will appear during follow-up period will be appropriately 
treated.  


14 
 
Microbiological Procedure  
 
1. Sample dilution 
All samples will be dispersed by vortexing for 30 seconds then 10-fold serially 
diluted using sterile saline. A measure of the dilution will then be used for 
traditional plate culturing methods.34,35 
2. Culture 
Aliquots of 10 ml of each dilution will be inoculated into freshly prepared 
Mitis Salivarius agar and Rogosa agar media respectively, using a 
micropipette.  
• Mitis Salivarius agar plates will be incubated anaerobically in an 
atmosphere containing 10% CO2 at 37°C for 72 hours to detect 
Streptococcus mutans counts. 
• Rogosa agar plates will be incubated aerobically at 37 C for 48 hours 
to detect Lactobacilli count. 
3. Isolation and Enumeration 
Following the predetermined incubation period, colonies grown on the 
specified media will be counted and represented as (CFU/ml). Streptococcus 
mutans will be identified based on their characteristic morphology on Mitis 
Salivarius agar plates. Similarly, Lactobacilli will be identified biochemically 
and microscopically on the basis of their morphology.   
4. Colony count  
The number of colonies will be determined and expressed as colony forming 
units using the following equation.36 
 
CFU/ml =                               n of colonies × dilution factor 
                                                   Volume taken in ml (1) 
 


15 
 
STUDY OUTCOME 
 
Treatment effect will be evaluated after one month by comparing: 
 
• Microbiological assessment of colony forming units (CFU) of Streptococcus 
mutans and Lactobacilli. 
• Caries activity according to ICDAS criteria. 
 
 


16 
 
Follow up 
All patients will be recalled after one month. On the day of the recall 
appointment, patient preparation will be done as mentioned before. 
Outcome assessment 
• Microbiological assessment will be done as mentioned before and CFU counts 
will be recorded. 
• Lesion activity will be assessed according to the ICDAS criteria. Using a blunt 
ended probe, if the cavity is found to be shiny and feels hard on gentle probing 
of the dentin, it will be recorded as an inactive lesion. While if it remains soft 
or leathery on gentle probing, it will be recorded as an active lesion.  
 
 
 


17 
 
STUDY PLAN 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participants assessed for eligibility 
Excluded participants  
• Not meeting inclusion criteria 
• Declined to participate 
Enrollment 
Random allocation 
N=50 
Group II 
38% SDF n= 25 
Baseline 
Assessment 
• Microbiological assessment. 
• WHO questionnaire on oral health of the child. 
• dmf score. 
 
Group I 
NSF n= 25 
• Microbiological assessment. 
• Caries activity using ICDAS criteria.  
Analysis 
Intervention 
Test group 
NSF application 
Control group 
38% SDF application 
1 month follow-up 
Follow-up 
Figure: flow chart of the study plan 


18 
 
STATISTICAL ANALYSIS 
 
The statistical software SPSS version 27.0 for Windows will be used for 
data analysis. The outcome variable is the number of colony forming units (CFU) 
of streptococcus mutans and lactobacilli, and the number of arrested caries 
lesions after 1 month in both the SDF and NSF groups. The collected data will be 
recorded and statistically evaluated. 
The Chi square test will be used to compare the difference between the two 
groups regarding change caries lesion activity, the T-test or the Mann-Whitney U 
test will be used to analyze the differences between the microbiological samples, 
Wilcoxon signed rank will be used to assess the changes over time in bacterial 
counts. Regression analysis will be used to assess the potential confounders such 
as gender, social status, and dietary habits. The data will be graphically presented 
with suitable graphs. The level of statistical significance will be set at 5%. 


19 
 
ETHICAL CONSIDERATIONS IN SUBJECTS 
INVOLVING HUMAN SUBJECTS 
 
1. This research protocol will be approved by the Research Ethics Committee of 
Alexandria University Faculty of Dentistry (IORG 0008839-IRB No. 0359-
12/2021). prior to any research-related activities.  
 
2. All research activities involving human subjects will abide by the Declaration 
of Helsinki37 and other ethical guidelines adopted by the Research Ethics 
Committee of Alexandria University Faculty of Dentistry.  
 
Benefits: 
• It will provide a caries arresting treatment for young children with multiple 
active carious lesions using minimally invasive atraumatic procedures. 
• It will allow overcoming the disadvantages of silver diamine fluoride 
including black staining of the teeth and any clothes it contacts, soft tissue 
injury, unpleasant taste for children. 
• Support the use of nanosilver fluoride as a more convenient and more 
economical substitute to silver diamine fluoride for caries arrest in the 
primary dentition. 
 
 
Harms/Risks:  
Nothing more than the standard of care and the risk of black discoloration 
with SDF treatment. 
 
 
Privacy and confidentiality:  
Each participant will be provided with a serial number that will only be 
accessible to the principal investigator. No data regarding the identity of 
the participants will be shared under any circumstances. All participants or 
their guardians must provide a written informed consent prior to any 
procedures. 
 
 
3. 
4. 
5. 


20 
 
DURATION OF THE STUDY 
 
Study Activity 
Month 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Purchase material 
✓ 
✓ 
 
 
 
 
 
 
 
Patient recruitment 
✓ 
✓ 
✓ 
 
 
 
 
 
 
Treatment procedures 
 
 
 
✓ 
✓ 
 
 
 
 
Evaluation 
Patients will be examined for 
caries activity and samples will 
be collected for microbiological 
assessment 
 
 
 
 
✓ 
✓ 
 
 
 
Data collection and statistical 
analysis 
 
 
 
 
 
✓ 
✓ 
 
 
Thesis submission 
 
 
 
 
 
 
 
✓ 
✓ 


21 
 
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26 
 
Appendix I
 
 
Table (1): ICDAS severity criteria38 
Score 
Criteria 
0 
No change in enamel translucency with prolonged air drying 
1 
First visual change in enamel (after prolonged air drying) 
2 
Distinct visual change in enamel 
3 
Localized enamel breakdown or discolored enamel with no visible dentine 
involvement 
4 
Underlying dark shadow from dentine 
5 
Distinct cavity with visible dentine 
6 
Extensive distinct cavity with visible dentine 
 
 
Table (2): ICDAS caries activity criteria38 
 
ICDAS code 
Characteristics of Lesion 
Active Lesion 
Inactive Lesion 
1, 2 or 3 
Surface 
of 
enamel 
is 
whitish/yellowish opaque with loss 
of luster; feels rough when the tip of 
the probe is moved. Lesion is in a 
plaque stagnation area, i.e.: pits and 
fissures, near the gingival and 
approximal 
surface 
below 
the 
contact point. 
Surface 
of 
enamel 
is 
whitish, 
brownish or black. Enamel may be 
shiny and feels hard and smooth when 
the tip of the probe is moved. For 
smooth surfaces, caries lesion is 
typically located at some distance 
from the gingival margin 
4 
Probably active 
5 or 6 
Cavity feels soft or leathery on 
gently probing the dentin 
Cavity may be shiny and feels hard on 
gently probing the dentin. 


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