The Effect of Silver Diamine Fluoride (SDF) on Bacteria Involved in Root or Cervical Carious Lesions Using HOMINGS Technology. A Clinical Study
Phase: PHASE4
Conditions: Caries
Sponsor: Heba Mitwalli

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 The effect of silver diamine fluoride (SDF) on bacteria involved in root or 
cervical carious lesions using HOMINGS technology. A clinical study 
 
Heba Mitwalli, DDS 
 
Version 2.0 
 
11 November 2016 
 
 
Thesis Committee: 
 
Margherita Fontana, DDS, PhD, Chair 
 
Joseph Dennison, DDS, MS 
 
Peter Yaman, DDS, MS 
 
Bruce Paster, PhD, Department of Microbiology, The Forsyth Institute, 
Cambridge, MA 
 
Introduction: 
The World Health Organization recommends that countries urgently need 
to adopt strategies for improving the oral health of elderly populations and 
recognizes that there is a universal need for prevention of oral diseases such 
as root caries.1 Root caries is common in institutionalized elders and 
periodontal patients with exposed root surfaces. Effective prevention 
methods are needed.2 The use of fluoride releasing agents as a means of 
preventing dental caries has long been recognized, and amongst these 
agents silver diamine fluoride (SDF) has received considerable attention as 
a caries-controlling agent of choice.3  
Clinical trials have demonstrated that silver diamine fluoride (SDF) can 
arrest caries.4-6 However, the mechanism of action is not yet fully known.7 
Previous studies on the mechanisms of SDF have mostly focused on limited 
antimicrobial effects and mineral changes in tooth structure.7-9 SDF is used 
in many countries and has been approved in the USA as “Advantage 
Arrest” manufactured by Elevate Oral Care company. 
SDF contains high concentrations of silver and fluoride ions which can 
inhibit the growth of multi-species cariogenic biofilms.10 The concentration 

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of 38% silver diamine fluoride (SDF) has been documented to arrest dental 
caries by reducing the demineralization process.11 -16 SDF is a clear liquid. 
Several studies in children reported that in rare cases SDF might cause 
gingival/mucosal irritation, which heals spontaneously within 48 hours.11,17-
19 Most studies recommend using SDF once a year to arrest caries, but 
applications every 6 months, or once every 2-3 years have also been tested. 
Furthermore, the need for maintaining salivary function and oral hygiene 
has been emphasized to avoid reactivation of treated caries. In 2016, 
Deutsch reported that caries lesions might reactivate within the year if 
salivary function and oral hygiene is poor.20 
A literature review of SDF suggested that 38% SDF can be an effective 
agent in preventing new caries and arresting dental caries in the primary 
teeth of children.21 Another review concluded that SDF is an effective, 
efficient, and equitable caries-control agent.22 Milgrom and Chi (2011) 
advocated SDF therapy as an important prevention-centered caries 
management strategy during critical early childhood periods.23 The clinical 
trial by dos Santos et al. (2012) concluded that 38 % SDF was better than 
interim restorative treatment with a glass ionomer for arresting cavitated 
lesions in early childhood caries (ECC).24 
A randomized clinical study used a 38% SDF solution among 
institutionalized elderly subjects and showed that it was more effective in 
preventing new root caries lesions than giving oral hygiene instruction 
(OHI) alone (Tan et al., 2010).3 Another clinical study reported for the first 
time that synergetic applications of SDF solution and oral health education 
(OHE) annually was more effective than giving OHI alone in preventing 
and arresting root caries among community-dwelling elderly subjects. 
There was a trend of less new root caries development among those who 
received synergetic applications of SDF and OHE compared to those who 
received SDF alone.25  
Since root caries is caused by plaque bacteria that adhere to exposed root 
surfaces (Berry et al., 2004), providing instructions on how to improve oral 
hygiene would theoretically be beneficial.26 Investigating the effects of SDF 
agents on plaque bacteria will allow better understanding of SDF’s 
mechanism of action on root caries arrest.  
The International Caries Detection and Assessment System (ICDAS) will 
be used to score carious lesions. The ICDAS criteria include early and later 
stage caries severity, and instructions for standard examination processes. 
ICDAS includes scores from 0 (sound) to 6 (extensive distinct cavity with 
visible dentin).  

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Human Oral Microbe Identification using Next Generation Sequencing 
(HOMINGS) utilizes the speed and efficiency of next generation 
sequencing combined with the refinement of species-level identification. 
Nearly 600 oral bacterial taxa are identified at the Species-level and Genus-
level identification of remaining sequences for 129 genera. HOMINGS 
allows identification and comparison of bacterial associations in oral health 
and disease, including different types of periodontitis, caries, gingivitis, 
ventilator-associated pneumonia, endodontic and odontogenic lesions, 
abscesses, and halitosis. Also, HOMINGS can determine the efficacy of 
therapies/treatments, e.g., mouth rinses, antibiotic treatment, scaling and 
root planning, and laser or periodontal surgery. HOMINGS will be utilized 
for microbial analysis in this study. 
Research Question: 
 
What is the mechanism of action of silver diamine fluoride (SDF) on 
bacteria involved in root or cervical carious lesions using human oral 
microbe identification microarray (HOMINGS) technology? 
 
Objective:  
 
To assess the effect of SDF treatment on a large array of microorganisms 
present in root or cervical carious lesions using HOMINGS technology. 
This will be accomplished by assessing microbial deposits on root or 
cervical caries lesions before and 1 month after SDF application.   
 
Rationale: 
Older people that have caries-active disease experience root caries at a rate 
which is at least as great as that of coronal caries among adolescents.27 
The use of fluoride releasing agents as a means of preventing dental caries 
has long been recognized, and amongst these agents silver diamine fluoride 
(SDF) has received considerable attention as a caries-arresting agent of 
choice.28 
Silver diamine fluoride outperforms other anticaries medicaments such as 
chlorhexine, NaOCl-based gel (Carisolv), and an enzyme-based gel 
(Papacarie) in killing cariogenic bacteria in dentinal tubules, and results in 
less aversive taste and texture responses than to fluoride varnish.29 
 
38% SDF arrests dental caries producing bacteria such as Streptococcus 
mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus 
rhamnosus and Actinomyces naeslundii by reducing the demineralization 
process. It minimizes the loss of mineral content and slows down collagen I 

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destruction. Furthermore, it contains high concentrations of silver and 
fluoride ions, which may inhibit the growth of multi-species cariogenic 
biofilms. 10 
Safety is an important aspect, which should be considered in the clinical use 
of SDF solution. An important review considered silver diamine fluoride 
(SDF) as a safe, effective, efficient, and equitable caries-preventive agent 
that appears to meet the criteria of the WHO Millennium Goals and the US 
Institute.28, 30 The effect of SDF in preventing dentine collagen degradation 
from bacterial collagenase challenge has also been well documented.31 The 
germicidal action of the SDF preparation in arresting active caries and 
contributing to disinfection of root canals has also been documented.32 
There are 3 components of SDF: silver, amine, and fluoride. Silver alone 
and the more complex silver nitrate have been used for millennia for 
medical applications. Fluoride is now widely used in a multitude of 
applications for caries prevention. Unlike the application of fluoride among 
children, the use of higher concentrations of fluoride among the elderly is 
acceptable and cannot cause fluorosis. 33 
Hypothesis: 
H0: there is no inhibitory effect of bacteria by 38% SDF  
HA: there is an inhibitory effect of bacteria by 38% SDF 
 
Materials and Methods: 
 
Setting: General Dentistry clinics of the University of Michigan School of 
Dentistry.  
 
Approval from the Internal Review Board Med (IRB) of the University of 
Michigan will be obtained prior to implementation.  
 
Study Subjects and Screening:  
 
Inclusion Criteria: 
 
● Generally healthy adult participants 18 years of age and older. 
● At least one tooth with active (soft) cavitated lesions of ICDAS score 
5 or 6 near the gingival margin (coronal cervical caries at the CEJ or 
exposed root surfaces with soft active carious lesions exposing 
dentin). 
 

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Exclusion Criteria: 
 
● Adult participants who suffer from serious life-threatening medical 
diseases that interfere with basic daily self care activities. 
● Patients taking antibiotics within the last two weeks. 
● Patients using chlorhexidine or fluoride mouth washes within the last 
two weeks. 
● Pregnant or breastfeeding women. 
● Teeth with arrested (hard) cervical or root caries lesions. 
● Teeth that have been diagnosed with an abscess or an irreversible 
pulpitis, or are mobile. 
 
Generally healthy adult participants 18 years of age and older with at least 
one tooth with active cavitated lesions of ICDAS score 5 or 6 near the 
gingival margin (coronal cervical caries at the CEJ or exposed root surfaces 
with soft active carious lesions exposing dentin) will be recruited by one of 
the researchers after the IRB approval in the General Dentistry Clinic at the 
University of Michigan. Other providers in the General Dentistry Clinic, or 
other departments, will be informed of the eligibility criteria by the study PI 
personally, via word of mouth, and via study flyers or emails, and can refer 
potential subjects to the study team. This could occur either at a patient 
appointment, or via a review of the patient chart (please see request for 
Waiver of Consent). The PI will approach potential subjects in person or 
via phone call to assess interest in study participation. If interested, a study 
appointment will be scheduled and Informed Consent performed. 
 
At the study appointment, patients will be provided with verbal and written 
information describing the purpose of the study and potential side effects 
(lesions will turn black). They will be informed that all information is 
confidential and that they can freely withdraw at any time during their 
participation. They will have the opportunity to ask questions related to the 
study. After obtaining written informed consent, study subjects will be 
examined by the study PI. Status of the cervical or root caries lesions will 
be assessed by visual inspection and aided by tactile detection using a 
probe. Decay- missing- filled teeth (DMFT) index will be assessed for each 
patient at the initial visit. We will also use ICDAS sensitivity and 
specificity criteria scores 5 and 6. Score 5 is a distinct cavity with visible 
dentin; frank cavitation involving less than half of a tooth surface. Score 6 
is an extensive distinct cavity with dentin; cavity is deep and wide 
involving more than half of a tooth surface. 
 

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Food particles obscuring visual inspection of root or cervical surfaces will 
be removed. Two surfaces per tooth (buccal and lingual) will be examined 
clinically for root surface caries. Individualized oral hygiene instructions 
will be provided to each participant, focusing on effective brushing with a 
manual toothbrush, and use of fluoride toothpaste will be recommended. 
 
Treatment: 
Eligible study subjects will have plaque collected from 1 randomly selected 
caries lesion that meets the inclusion criteria. Before applications of the 
study agent, a piece of gauze will be used to dry the teeth. Bacterial samples 
from subjects’ root or cervical carious lesions will be collected with sterile 
Gracey curettes via a gentle scraping movement.34, 35 Samples will be 
collected at the beginning of each appointment, but following consent and 
eligibility review. Samples will be frozen until analyses. The SDF solution 
will then be applied onto the exposed root surfaces of participants by means 
of a disposable microbrush following the manufacturer’s recommendation 
by using their specialized Advantage Arrest applicator. The applicator will 
not absorb the product as it applies, and there is no risk of leaving flock 
portions of the applicator on the site. The surface tension of the material 
being applied holds the liquid on the applicator until it contacts the 
application site. The participants will be instructed not to eat or drink for 
one hour after treatment. Patients will be asked to return 1 month later. At 
this time, the lesions will be assessed for changes in hardness and color, and 
then a second microbial sample will be collected as described below and 
frozen until analyses.  
 
Sample Size: 
A sample including 20 subjects is decided to constitute the study sample. 
Samples will be collected from each patient at the first visit and at the one 
month recall visit.  
 
An additional 2 test plaque samples will be collected for calibration 
purposes. These will be collected either from subjects who will also 
participate in the main project or 2 additional subjects. Plaque sampling on 
the two test subjects will be for calibration purposes, to ensure the sampling 
technique yields samples with sufficient volume to reliably extract bacterial 
DNA. 
 
 
 

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Patient Participation:  
Test Sample Collection (2 subjects): 
1/Consent form and Eligibility review 
2/Questionnaire 
3/Examination and diagnosis for root surface caries (photographs 
when possible) 
4/Bacterial sample collection 
1st appointment: 
1/Consent form and Eligibility review 
2/Questionnaire 
3/Examination and diagnosis for root surface caries (Pre-SDF 
photographs when possible) 
4/Bacterial sample collection 
5/SDF application 
6/Post-SDF application photograph 
2nd appointment (one month recall): 
1/Questionnaire 
2/Examination and diagnosis for root or cervical surface caries to 
confirm if caries still exist (Photographs when possible) 
(Baseline)  
Samples before 
and after   
SDF application 
Sample Size 
N=20  
1 month recall 
After SDF 
application 

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3/Bacterial sample collection 
Outcome Measures and its Assessment: 
Patients will be seen twice, at baseline and at one-month recall, to compare 
the microbial composition of bacterial samples at the two different stages. 
Also, questionnaires will be completed by the patients at baseline and after 
one month to collect information on tooth brushing behavior, use of 
fluoride toothpaste, frequency of sweet snack intake between meals. 
Patients will also be asked about the acceptability of the treatment. Two 
eligible subjects with multiple eligible lesions will be seen for plaque 
collection calibration to ensure the plaque sample technique yields adequate 
volume to extract bacterial DNA for analysis. These subjects can also 
participate in the baseline and one-month recall, if the subject elects. 
Incentives: 
1/Participants will be given a toothbrush and toothpaste at the initial visit, 
and $10 for their initial participation. We will pay for patient parking for 
the visit. 
2/Fifteen dollars at the one-month recall research clinical visit. We will pay 
for patient parking at the visit. 
3/At the one month recall, if active caries still exists or if the patient does 
not like the color, patients will be offered an appropriate restoration at our 
expense at the General Dentistry Clinic at the University of Michigan. 
 
4/Two subjects will also participate in a test plaque sample collection visit. 
These subjects will receive $18 for this visit and will receive compensation 
for patient parking. 
 
Collection and determination of the bacteria involved in the carious 
lesions: 
Gracey curettes will be used to collect plaque samples. Plaque samples will 
be coded, which will allow the samples to be connected to the overall study 
data. These samples will be used to identify the types of bacteria involved 
in the carious lesions and bacteria arrested after the application of the 
testing material after one month. The microbial composition of the samples 
will be assessed using human oral microbe identification microarray 
(HOMINGS). After the bacterial collection, the samples from the cervical 
or root carious lesions will be suspended in tubes containing 150 μl of TE 
(Tris, EDTA) Buffer. The bacterial samples will be stored at −80 °C. Prior 
to shipment to Forsyth for HOMINGS analyses, genomic DNA will be 

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purified using the Master Pure Gram Positive DNA Purification kit 
(Epicentre Biotechnologies, Madison, WI, USA), with modifications 
(http://mim.forsyth.org). DNA samples will be sent on dry ice to Forsyth 
Institute. Per Forsyth Institute and the University of Michigan Office of 
Technology Transfer, a Material Transfer Agreement (MTA) will not be 
required prior to sharing of the samples. 
HOMINGS: 
At the Forsyth Institute, microbial profiles will be generated from image 
files of scanned HOMINGS microarrays (https://homings.forsyth.org).  
In brief, concentration levels of approximately 600 oral taxa will be 
determined by microarray hybridization using a fluorescent readout reverse-
capture method.36 
Fluorescently labeled sample microbial DNA will be captured by 16S 
rRNA-based probes attached to glass slides. The fluorescent intensity for 
each probe will be scanned, normalized and scaled as previously reported.36 
Signals of 2× backgrounds will be considered to be negative and assigned a 
HOMINGS level score of 0. Positive hybridization signals will be 
categorized into five levels, with 1 indicating a signal that was just 
detectable, and 5 indicating maximum signal intensity.37 
Statistical Analysis: 
Data will be analyzed using SPSS Pc+ version 22.0 statistical software. 
Descriptive statistics (mean, standard deviation, frequencies and 
percentages) will be used to describe the quantitative and categorical 
variables. Appropriate parametric statistical tests (one way analysis of 
variance followed by Tukey’s test) and non-parametric statistical tests 
(Pearson’s chi-square and Krushal Wallis test) will be used depending on 
the type and distribution of variables. A p-value ≤ 0.05 will be used to 
report the statistical significance of results. 
Future Research: 
Data and any samples remaining following HOMINGS analysis may be 
retained for future analysis/research. Subjects will provide or deny 
permission for their samples and data from this project to be used in future 
research in the informed consent document. 
 

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