Nobio In Situ Clinical Study - Pilot Study
Phase: NA
Conditions: Dental Caries, Denture, Partial, Removable
Sponsor: University of California, San Francisco

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Nobio Clinical Study - Demineralization Prevention With a New Antibacterial 
Restorative Composite 
 
NCT04059250 
 
September 10th, 2018 
 
 

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Protocol PROPOSAL 
1. 
TITLE PAGE 
Study Title: 
Nobio In Situ Clinical Study – Pilot Study 
Protocol Number: 
Nobio-2018-08 – September 10th, 2018 
Trial registration 
ClinicalTrials.gov – registration is not necessary 
Investigational Product: Nobio quaternary ammonium silica dioxide  
antimicrobial nanoparticles in composite resins 
Indication: 
Antibacterial – antibiofilm effect on enamel adjacent to composite 
Funding: 
 
Source of Funding: 
 
Secondary Sponsor: 
 
Development Phase: 
Clinical in situ study, pilot 
Sponsor's Responsible 
Officer: 
 
Sponsor Signatory: 
 
Effective Date: 
 
 

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2. 
SYNOPSIS 
Study Title: Nobio In Situ Clinical Study – Pilot Study 
Protocol number: Nobio-2018-08 
Principal Investigator: Peter Rechmann, PhD, DMD, Prof. Dr. med. dent.  
Department of Preventive and Restorative Dental Sciences,  
School of Dentistry, UCSF 
Sponsored Research, PI initiated 
Study center: University of California at San Francisco 
Study period: 10-2018 to 06-2019 
Phase of development:  Clinical In Situ Study, Pilot  
 
Background 
In research the use of experimental intra-oral caries models dealing with caries prevention 
has increased. The most realistic experimental model is the in vivo model that uses living 
teeth, followed by the in situ model using specimens with natural surfaces held in the mouth 
during the experimental period [1]. In situ models have the potential to study both fundamental 
aspects of the caries process as well as more applied research problems in caries prevention in 
human subjects without actually causing caries in the natural dentition [2]. In situ models are 
able to produce statistically significant differences to compare the use of different fluoride 
toothpastes and controls [3-9], fluoride mouth rinses [10], antimicrobial releasing agents [11], 
varnishes [12], and other fluoride releasing products [13-16]. In situ studies also may deal with 
the effect of biomaterials and food on demineralization and remineralization [17, 18]. A few in 
situ studies have been done related to root caries and its prevention [19-22]. 
Regarding the study design numerous in situ models have been described [2]. Orthodontic 
brackets have been used as a plaque trap on enamel [23, 24], as well as a carrier for slabs [25]. 
Interproximal models [26] with partial dentures for testing the effect of fluoride on enamel 
have also been used.  
As a major issue in dentistry, it has been stated that two-thirds of all restorative dentistry 
involves the replacement of failed restorations [27]. Secondary caries indicates a lesion 
developing at the margins of an existing restoration and is the major reason for replacement of 
amalgam and composite resin restorations in operative dentistry [28, 29]. 
Two regions have been described when considering the process of secondary caries; the 
surface lesion, which develops perpendicular to the tooth surface and can be considered a 
primary lesion developing next to a restoration, and the wall lesion, which develops 
perpendicular to the tooth/restoration interface [30]. To simulate a failed interface between a 
tooth and a restoration, a gap can be formed between the two during restoration prior to a 
cariogenic challenge [31]. An in situ study with four different gap sizes (50 μm, 100 μm, 200 
μm, and 400 μm and 1 non-bonded interface without a gap) showed no trend for a relationship 
between the lesion depth and the gap size [32]. 

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A systematic review about simulation of secondary caries in in situ models lately reported 
that three main groups of in situ models could be identified by sample placement; 68% of the 
models placed samples palatally in the upper jaw, and the lower jaw model could be divided 
into the buccal (26%) and approximal areas (6%, only in edentulous patients). Average lesion 
progression in enamel per day next to composites was 4.3 +/- 2.8 μm (range1.1-8.8 μm/day). It 
was found that studies conducted with palatal models showed caries progression rates 2-5 
times higher than the estimated clinical progression rates.[33] Up to eight dippings a day with 
concentration of 20% sucrose were used. Fluoride-containing toothpastes were used in 48% of 
the studies, and 42% had their volunteers use fluoride-free toothpaste. Lesion development was 
measured using microradiography techniques (TMR or transversal wavelength independent 
microradiography [TWIM]) in 36% of the studies, 48% used cross-sectional microhardness. 
Two main trends were observed, with 4 studies showing a rate of ≤2 μm/day and 7 studies 
showing a rate of >4 μm /day. These 7 studies with a high progression rate were all palatal 
models, used mesh for plaque promotion, and experiment durations of only 2 weeks. It was 
also observed that samples with a natural surface, a surface not polished flat, slowed down 
initial lesion formation [33].  
The influence of dental materials to inhibit caries development has been tested evaluating 
for instance the caries inhibition effect of glass ionomer cement [34, 23], and the effect of 
glutaraldehyde on secondary caries [35]. It was shown that fluoridated composites might 
reduce or inhibit secondary caries. In an in situ model investigation the effect of F-releasing 
composites on enamel demineralization around an artificial gap of 200 microns width was 
quantified after 1 month [35]. In the gap, all fluoridated composites reduced the enamel 
demineralization significantly with respect to the non-fluoridated controls [35]. The reduction 
of the lesion depth for three different materials was 44, 46 and 27%, respectively, while the 
mineral loss reduction was 56, 54 and 25%, respectively [35]. 
There is an increasing clinical need to design novel dental materials that combine 
regenerative and antibacterial properties. A recently developed sol-gel-derived bioactive glass 
ceramic containing silver ions (Ag-BG) in the resin composite induced remineralizing and 
showed antibacterial properties [36]. The total bond strength of the new Ag-BGCOMP 
composites was not significantly affected [37]. 
As stated, secondary caries and in addition hybrid layer degradation are the two major 
challenges encountered in long-term resin-dentin bond stability. As a link between resin and 
dentin, adhesives that possess both antimicrobial and anti-proteolytic activities are in demand 
for eliminating bacteria-induced secondary caries and preventing hybrid layers from 
degradation. Lately a study reported that a new quaternary ammonium methacryloxy silane 
(QAMS) prepared from sol-gel chemistry was incorporated into experimental adhesives to 
examine their antimicrobial effect and anti-proteolytic potential [38]. Around the world several 
groups are researching the possibility of using composite restorative materials that incorporate 
antibacterial agents to overcome the described problems [37, 39-43]. 
Nobio has developed dental restorative materials with long term antibacterial properties in 
order to fight recurrent decay around restorations. These composites with incorporated non-
leaching antibacterial agents might overcome the vicious circle of newly developed cavities 
around freshly placed fillings.  The ongoing caries activity due to bacteria around the filling 
may be interrupted by quaternary ammonium silica dioxide (QASi) particles incorporated in 
the test composite at 1.2% (by weight). These particles are members of the Nobio 

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Antimicrobial Particle (NAP) family (Nobio, Israel). QASi are synthesized to form a high 
concentration of antimicrobial groups that are covalently-bound onto a carrier core, such as 
silica. The resulting micro or nano-sized QASi particles are mixed with other fillers of the 
dental composite material, during manufacturing. Following in situ placement and light-
initiated polymerization, QASi particles are permanently retained in the 
final dental restoration. Laboratory studies have shown that the quaternary ammonium silica 
particles are potent antibacterial agents, do not leach out in contrast to other antimicrobials or 
caries preventive active substances, inhibit the breakdown of the composite, and maintain 
antibacterial activity over time [39-42]. 
Nevertheless, the key experimental parameters for a successful and valid in situ study are 
the panelists selection and appliance design, the type of hard tissue substrate and the method of 
assessing mineral status, and the overall study design and clinical protocol [2, 18, 44, 45].  
____________________________________________________ 
Objective 
The overall objective of this clinical pilot study is to prove in an in situ model how effective 
Nobio composites with antimicrobial activity reduce caries in enamel adjacent to the 
composite restorative material.  
_________________________________________________________________________ 
Hypothesis 
The hypothesis to be tested is whether composites with Nobio’s QASi antibacterial 
particles significantly reduce demineralization in adjacent enamel in comparison to a 
conventional composite in an in situ clinical study, with mineral loss evaluated by cross 
sectional microhardness testing in the laboratory.  
Product under Investigation 
The product under investigation is Nobio’s light-cured composite restorative material, suitable 
for all applications from Class I through Class VI. 
Study Design & Outcomes 
The study is designed as an in situ study. 
Primary outcome 
1. Mineral loss DZ: Significant reduction in mineral loss DZ in the Nobio group compared to 
the control is expected. 
Secondary outcome 
2. Microbial plaque pH - level: A pH-indicator used on the composite slab immediately after 
the wearing period will check for the pH level of the microbial plaque on top of the composite 
slab after wearing; a neutral pH on the Nobio composite slabs in comparison to the control 
composite slabs is expected. Prior to pH-measurements the denture might be rinsed with 2% 
dextrose. 

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Methodology 
The study is planned as an in situ study. Subjects, wearing partial dentures with acrylic flanges 
on both sides of the mouth will be recruited. On each side of the denture an enamel slab will be 
placed next to a composite, separated by a tiny gap. Placing a thin metallic matrix band  
(38 µm) between the enamel slab and the composite, before curing the composite, will create 
the gap. After curing, the matrix band will be removed. On one side of the denture the 
composite will be Nobio’s antibacterial composite, on the other side a regular composite (a 
Nobio composite w/o particles or another commercial composite (3M / Dentsply) will serve as 
control material). Enamel slab and composite will be recessed into the flange, allowing 
microbial plaque to accumulate on top of it and especially in the gap. After a specific period of 
wearing the dentures with the slabs, decalcification - DZ mineral loss - in the enamel slabs 
adjacent to the gap will be determined by cross-section microhardness testing in the laboratory. 
Average DZ mineral loss will be calculated for the Nobio group and the control group, and 
difference will be tested for statistical significance. DZ mineral loss of 670 ±195 Vol%xµm for 
the active Nobio-composite and DZ of 1,200±450 Vol%xµm might be expected for the inactive 
control side. These predictions are based upon former studies related to fluoride use, but they 
are predictions based on a best guess as to what might happen. 
 
A pre-study will be conducted, in which 3 to 5 subjects will be selected and will wear dentures 
with enamel slabs incorporated, as described above, to determine whether a wearing period of 
four weeks is enough to achieve sufficient demineralization of the enamel slab. Depending on 
the results, the data from these subjects might be included in the final data set. If the 4 week 
test period proves to be sufficient in this pre-study and the same protocol is used for the main 
study this will allow the pre-study subjects to be included in the main results. It is possible that 
a second pre-study will be needed, depending on the results of the first one. The second pre-
study may potentially ask the same subjects to be involved, again.  
___________________________________ 
 
Study Participants and Procedures 
A total of about 20 subjects will be recruited into the study (20 subjects will allow for 2-3 
drop-outs). Subjects will be recruited from the UCSF Dental Center and will be partial denture 
wearers with denture flanges on both sides of the mouth. Subjects will be selected according to 
specific inclusion and exclusion criteria (see below). 
Subjects’ age range will be from 18 to 80 years. We will select subjects, who are already 
wearing a lower removable partial denture with lateral teeth replacement. The natural dentition 
must have at least 8 remaining teeth to ensure typical caries bacteria colonization. The subjects 
must be healthy, and have healthy oral conditions. They are not allowed to show active caries 
lesions but should have a recent history of former caries. Subjects with acute periodontal 
diseases are excluded. The stimulated saliva flow rate will be measured. Subjects exhibiting 
saliva flow rates below 0.7 ml/min for stimulated saliva will be excluded (subjects with a 
saliva flow rate below 0.7ml/min are at extreme caries risk and are in need of special caries 
prevention efforts). 
Subjects start their study involvement with a 2-week washout period using an Over-The-
Counter (OTC) 1,100 ppm F-toothpaste to brush their teeth (there will be no professional tooth 

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cleaning at the study start in order to not interrupt biofilm activities). After the 2-week washout 
period, the slabs will be mounted into the denture flanges (test side: enamel slab adjacent to 
Nobio antimicrobial test composite; control side with enamel slab adjacent to inactive 
composite), and subjects will wear their denture for 4 weeks. After 4 weeks plaque pH-levels 
on top of the composite will be checked, enamel samples will be removed, and samples will be 
prepared for the laboratory cross sectional microhardness testing to determine the DZ mineral 
loss. 
The participants will be instructed to brush their teeth twice daily for one minute with the 
provided OTC 1,100 ppm Fluoride toothpaste. The participants will be asked to remove the 
dentures from the mouth before brushing their teeth. They will be asked to brush the inside of 
the denture and the denture teeth but not the area with the slabs. 
 
The subjects will be asked to fill in a log of their daily tooth-brushing schedule, and tubes of 
toothpaste for subject’s use only will be distributed and weighed before and after each trial 
period to crosscheck compliance. The study coordinator will telephone the households 
approximately once a week to assist with compliance. 
 
Recruitment and Consenting 
Flyers posted in the UCSF Dental Center will be used to recruit patients and study 
investigators (and/or staff) will recruit their own patients directly in person (outside the direct 
treatment area). To achieve adequate participant enrolment to reach targeted sample size, 
advertisements will be placed in the Dental Clinic areas as well as in other patient areas of the 
UCSF School of Dentistry, Dental Center.  
When patients are seen by their dentist and the treating dentist believes that the patient 
might fit into the study, he/she will ask the potential participant whether she/he might be 
interested in being in a dental study. If the subject is interested, the treating dentist will ask the 
potential participant whether she/he gives permission to disclose her/his name to the 
researchers. If the patient agrees, the Principal Investigator (PI) (Peter Rechmann) and/or the 
Clinical Study Coordinator (CSC) (Beate Rechmann) will ask the potential subject whether 
she/he is interested in participating in this study. If so, the Investigator/Clinical Study 
Coordinator will explain the whole study and will further check for eligibility. 
Consenting all study subjects will take place prior to entering the study (written 
informed consent). Potential participants will be consented and enrolled by the PI and the CSC, 
respectively.  
Blinding 
The doctor performing the treatment and the study coordinator will not be blinded to which 
side of the denture will carry the test and which is the control slab. The participant will be 
blind to the treatment/control allocation side. For blinding of the laboratory person doing the 
microhardness testing  – please, see the below section “Blinding in the laboratory”. 
Source of teeth: We will use teeth that have been previously extracted for clinical reasons (not 
research purposes) in the Dental Clinics of the School of Dentistry. Correct collecting, storing, 

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handling etc. of human teeth under biosafety aspects is covered under our BUA approval 
(BUA 2308-BU-01-INC) and a UCSF IRB exempt approval for collecting extracted teeth. 
Test samples 
Extracted teeth (molars) will be stored in 0.1% thymol solution in deionized water and 
sterilized with gamma irradiation (Cs 137) at a dose above 173 krad overnight. Following 
sterilization, the collection media will be replaced with fresh deionized water and thymol. The 
tooth roots will be removed below the cemento-enamel junction. The dental crowns will be cut 
in halves. 
At least 60 enamel samples from 20 extracted molars will be prepared. The test surfaces will 
be cleaned, and the surface will be flattened by serial polishing (600 grit silicon carbide 
polishing paper followed by 6 µm diamond polishing suspension/or 1,200 grit polishing paper, 
sterilized). The smear layer will be removed by sonication. Three standard slabs approximately 
3 x 2 x 2 mm will be cut from each tooth crown. One will be retained in the laboratory in a 
moist atmosphere in a sealed vial as an untreated control, and the adjacent two will be used in 
the subjects' mouths. All teeth will be sterilized with gamma irradiation before use in the 
mouth. 
 
Testing and Analysis methods  
The analysis method used will be cross sectional microhardness by detailed “scatter pattern” of 
indentation. At the end of the 4-week in situ wearing period treatment slabs and the control 
slabs will be removed from the denture flanges. Both slabs will be embedded and used for 
microhardness testing as described below.  
Microhardness measurements 
After the 4-week wear period the slabs will be embedded in epoxy resin (Ladd Research 
Industries), leaving the flat section surface uncovered. After serial polishing, the exposed flat 
surface will be indented (25 g weight) to test for microhardness cross-sectionally using a 
Buehler Microhardness tester (Buehler, Germany) and microscopic examination (Featherstone 
et al., 1983)[46]. The first indent will be placed 15 µm from the resin interface and 100 µm 
from the edge of the lesion. Subsequent indents are placed in 5 µm increments to a final depth 
of 50 µm in the underlying enamel; implementing a V-shaped pattern prevents interaction and 
interference between the indents. 
Additional indents will be placed at 25µm intervals into underlying sound enamel following a 
straight line perpendicular to the outer surface to a depth of 300 µm. The volume percent 
mineral for each indent is normalized based on sound underlying enamel (100-300 µm) set at 
85%. This is an internal calibration of the measurements that exists within the ΔZ formula, 
which allows for normalization of the microhardness data on a per-tooth basis, such that tooth-
to-tooth variability is eliminated. All data will be verified for reproducibility of the 
measurement method and quality assurance by repeating any outlier measurements. 
Measurement of indentation lengths / demineralization will be calculated with the aid of Image 
pro plus 4.0 software which is used for capturing and measuring the image through a 
microscope (Olympus BX50, Melville, NY) at 500X magnification.  
The overall relative mineral loss, ΔZ, for each sample is calculated by creating a hardness 
profile curve by plotting normalized volume percent mineral against distance from the outer 

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enamel surface. The area under the curve that represents ΔZ (µm x vol % mineral) is calculated 
using Simpson's integration rule. The individual ΔZ values for each lesion in each group will 
be combined to give a mean ΔZ and standard deviation for each of the groups (test, control).  
Blinding in the laboratory 
The technician measuring the indentation lengths will be blinded to the group assignment - test 
and control group, respectively. Slabs will be delivered to the laboratory technician in number 
coded vials. The CSC will have an appropriate list for coding and encoding.  
Statistical methods – sample size calculation 
The Nobio composite with antibacterial nanoparticles has not been tested so far in a clinical or 
in an in situ study.  Consequently, there is no way to calculate what the effective sample size 
would be and how much demineralization/remineralization will occur.  
We assume that based upon our previous experience a sample size of 15 per treatment group 
will give 80% power to separate demineralization in the test enamel slabs from the control 
slabs.   
Each sample will exhibit a relative mineral loss value (∆Z).  Means and standard deviations for 
each group are calculated and the groups are compared statistically by a two-tailed, paired t-
test for significance at P<0.05. 
Updated sample size calculation after performing the pe-study with 5 subjects 
After having performed the planned pre-study with 5 subjects, we calculated the sample size 
for paired differences based on the ∆Z mineral loss results obtained from these 5 subjects. The 
calculation took into account the mean difference between pairs and the standard deviation of 
the differences. Based on these data, we assumed that the study would require a sample size of 
17 (number of pairs – 17 subjects, each wearing control and Nobio samples) to achieve a 
power of 80% and a level of significance of 5% (two sided), for detecting a mean of the ∆Z 
differences of 383 between pairs, assuming the standard deviation of the differences to be ∆Z 
507. To account for attrition, we were planning to recruit 20 additional subjects into the study. 
__________________________________________________ 
Eligibility Criteria for Participants 
Inclusion criteria 
• 
aged between 18 and 80 years, 
•  
have at least eight natural teeth remaining and have a recent history of dental caries 
• 
wearing lower partial denture (with replaced teeth on both sides of the mouth) 
• 
willing to wear their denture during the night 
• 
are in good health, of either gender 
• 
are in good current oral health with no active caries or periodontal disease (but with a  
 
history of caries) 
• 
have an understanding of the study  
• 
have saliva flow within the normal range  
 
(stimulated saliva flow rate of greater than 0.7 ml/minute) 
• 
no antibiotics for the last three months 
• 
willing to comply with all study procedures and protocols,  
• 
residing in San Francisco or other nearby locales with community water fluoridation  
 
(to eliminate water fluoridation as a potential confounding variable),  
• 
able to give written consent themselves  
• 
must be able to read and understand English, 

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• 
willing to sign the “Authorization for Release of Personal Health Information and Use of 
 
Personally Unidentified Study Data for Research” form; data will only be used for 
 
research. 
 
Exclusion criteria 
• 
subjects who have less than 8 natural teeth remaining 
• 
subjects who have used a 5,000 ppm fluoride toothpaste in the last 6 months 
• 
subjects who have used Chlorhexidine or other any other antimicrobials 
 
(cetylpyridinium etc.) in the last 6 months  
• 
show evidence of extremely poor oral hygiene 
• 
subjects suffering from systemic diseases, significant past or medical history with  
 
 
conditions that may affect oral health or oral flora (i.e. diabetes, HIV, heart  
 
conditions that require antibiotic prophylaxis),  
• 
taking medications that may affect the oral flora or salivary flow (e.g. antibiotic use  
 
in the past three months, drugs associated with dry mouth / xerostomia),  
• 
other conditions that may decrease the likelihood of adhering to study protocol,  
• 
in-office fluoride treatment within the last three months,  
• 
subjects who will leave the area and are unable to complete the study 
 
IRB approval - ClincalTrials.gov registration 
Investigational Review Board approval (UCSF Committee on Human Research - CHR) 
will be achieved before the study will be started. Since the pilot study is an in situ study it does 
not need to be registered with ClincalTrials.gov. 
 
Study Duration: anticipated end date June 2019 
  
 
 
 
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