Prevention of Enamel Demineralization in Fixed Appliance Orthodontic Patients Using UDMA-K18 Sealant to Prevent Microbial Attachment Compared to a UDMA Control and no Sealant, a Randomized Split Mouth Clinical Trial
Phase: NA
Conditions: Dental Caries
Sponsor: University of Colorado, Denver

 
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w 
COMIRB Protocol 
 
PROTOCOL #: 
17-0633  
  Version Date:  12-14-2017 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title:  Prevention of enamel demineralization in fixed appliance orthodontic patients using 
UDMA-K18 sealant to prevent microbial attachment. A randomized split mouth clinical 
trial. 
Principal Investigator:  Dr. Clifton Carey, Professor and Director of Translational Research 
Orthodontics Residents: Dr. Shivani Patel and Dr. Megan Fellows 
 
Dr. Carey is the research mentor for University of Colorado, School of Dental Medicine, Department of 
Orthodontics residents Drs. Patel and Fellows.  Drs. Patel and Fellows will be performing all the sealant 
and bracket placements.  Dr. Carey will assist in the histology and evaluation of the extracted teeth. 
 
 
  
 
I. 
Hypotheses and Specific Aims:  The hypothesis is that UDMA-K18 containing smooth 
surface sealant will be more effective at reducing enamel demineralization than the UDMA 
control or no treatment.  This study will assess the effect of a quaternary ammonium 
methacrylate (QAM) called K18 antimicrobial compound tethered to urethane dimethacrylate 
resin (UDMA-K18). This resin will be tested against UDMA sealant and no sealant. This study 
will evaluate effectiveness of a UDMA-K18 sealant to prevent biofilm attachment to tooth 
surfaces thereby eliminating the possibility for the tooth to be demineralized.   
II. 
Background and Significance: Far too frequently orthodontic patients are unable to perform 
sufficient oral hygiene to remove all of the bacterial biofilm surrounding orthodontic brackets.  
When the bacterial biofilm is not removed, tooth demineralization is possible.  The incidence 
of new tooth demineralization, often called white spot lesions (WSL), has been studied at the 
University of Colorado Department of Orthodontics.  Forty-four orthodontic patients ages 
between 11 and 21 years of age were assessed over the course of their therapy for the 
development of new WSL.  This study reported that 84.6 % of the patients developed new 
WSL, and the total area of WSLs also increased from 16.6 ± 23.6 mm2 per patient to 42.6 ± 
50.7 mm2 per patient.  The period of therapy where the greatest increase in WSL occurred 
was between initial banding and 1 year of therapy. The area most susceptible to WSLs during 
treatment is the cervical third, and both anterior and posterior teeth are equally susceptible to 
the development of new lesions.  (Faranesh et al., 2017).  Given that the bacterial biofilm is 

 
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hard to remove due to compromised access to all of the tooth surfaces by orthodontic 
appliances (brackets and wires) a new strategy that limits the ability of bacterial to attach to 
tooth surfaces may have a protective capacity preventing WSL. The UDMA-K18 dental resin 
is designed to protect enamel from bacterial attachment and biofilm associated acid 
demineralization which can lead to the formation of white spot lesions (WSL) on the surface 
on the tooth.  The composition of the experimental sealant is K18, a quaternary ammonium 
methacrylate linked to a siloxane backbone covalently bonded to branched organic urethane 
dimethacrylate (UDMA) monomers.  See Figure 1 for the chemical structure. These UDMA or 
UDMA-K18 monomers are mixed with camphorquinone photoinitiators, which when placed 
on a tooth surface can be polymerized by exposure to a dental blue light. 
Figure 1. Structure of UDMA-K18. 
III. 
Preliminary Studies/Progress Report: This tethered K18 anti-attachment compound has 
been tested in clinical studies for antimicrobial accumulation on orthodontic retainers (as well 
as  antimicrobial and cytotoxic activity in clinical split mouth study (Liu et al., 2016) and for 
cytotoxic and carcinogenic activity (Gong et al, 2012).  
a. Liu et al., 2016 studied the antimicrobial activity of a quaternary ammonium methacryloxy 
silicate-containing acrylic resin in a double blind randomized clinical trial. The quaternary 
amine (QAM) was synthesized with a sol-gel reaction between a tetraalkoxysilane and 

 
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two trialkoxysilanes, creating a methacryloxy functional group and long C-18 carbon 
chain. Liu et al, mentioned that in previous studies QAMs at a concentration of 4 % to 6 
% have been shown to have immediate contact killing antimicrobial properties. Cell 
viability assays have also been previously completed and the acrylic resin has been 
deemed noncytotoxic and received 510(K) clearance from the US Food and Drug 
Administration. This in-vivo experiment of 32 patients reviewed the antimicrobial efficacy 
of quaternary ammonium methacrylate (QAMs) containing orthodontic acrylic added to 
orthodontic retainers. There was a statistically significant difference between the QAMs 
containing side and the control side in the percentage kill in the biovolume. The study 
found that the QAMs-containing acrylic demonstrates favorable antimicrobial activity 
against biofilms in vivo experiments. 
b. Gong et al., (2012) studied the possible cytotoxicity by use of the MTT assay on a mouse 
fibroblast cell line (L929).  They determined the effects of increasing molar feed ratios of 
3-(Trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (SiQAC, a starting 
material) on the cytotoxicity of a series of silsesquioxane-silica hybrids (SqSHs, which 
include K18).  The results of the cell viability assays indicated that the QAMS-containing 
orthodontic acrylic resin is relatively non-cytotoxic Therefore, the QAMS-containing 
orthodontic acrylic has received 510(K) clearance for marketing by the U.S. Food and 
Drug Administration (FDA) .     
c. K18-UDMA has been tested in the Principal Investigator’s laboratory for anti-attachment 
of biofilm (antimicrobial activity) (Stricker et al., 2016).  2.6 x 10‐6 All of these studies 
show that tethered K18 to UDMA or acrylates prevents attachment of microbial biofilms to 
surfaces. 
d. The methods for determination of lesion depth and mineral density have been tested in 
the Principal Investigator’s laboratory (Lee et al., 2015; and Schmuck & Carey, 2010). In 
the Lee study a total of 64 extracted teeth were cleaned and prepared with an orthodontic 
bracket.  The teeth were randomly divided into 8 experimental groups of 8 samples to 
evaluate the protection from erosion efficacy of commercial and experimental smooth 

 
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surface sealants.  The outcome was the amount of erosion that the teeth experienced, 
which was measured by determining the lesion (tooth loss) depth as seen in the cross-
section through the orthodontic bracket.  The results of the demineralization were 
between 44.7 ± 21.9 µm for the negative control to 1.1 ± 4.4 µm lost for one of the 
fluoride containing smooth surface sealants.  The erosive loss for the Transbond XT 
smooth surface sealant that this study proposes to use as a bonding agent was 29.3 ± 
9.6 µm which is significantly less erosion than the control and significantly more erosion 
than the commercially available fluoride containing smooth surface sealant (p < 0.05). 
e. The methods described in the paper by Schmuck and Carey (2010) will be used for the 
transverse microradiography (TMR) of the samples in this study.  The extracted teeth will 
be sectioned and digital images will be taken with incident and transmitted polarized light 
to visualize any lesion in the sample.  TMR will be used to determine surface lost.  TMR 
determination of lesion depth will be used to compare the measurement via polarized 
light microscopy. 
IV. 
Research Methods: 
a. Outcome Measures: mineral density profile and lesion depth of extracted teeth in these 
experiments.  Mineral density will be measured by transverse x-ray microradiography 
(Schmuck and Carey, 2010), lesion depth will be measured by polarized light microscopy 
(Wefel and Harless, 1984; Darvishiabyaneh and Carey 2016; and for orthodontic 
evaluations Lee et al., 2015). 
b. Description of Population to be Enrolled: 
Research participants will be between 12 and 89 years of age who are subjects 
undergoing orthodontic treatment at the Department of Orthodontics Clinic, School of 
Dental Medicine, University of Colorado. The following inclusion criteria will be applied: 
comprehensive fixed orthodontic treatment with brackets from at least second premolar 
to second premolar, treatment planned already for the extraction of at least 3 bicuspids 
(any combination), patient at the University of Colorado School of Dental Medicine 
Orthodontic Clinic, can place bracket base on teeth to be extracted for at least 1 month 

 
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prior to extractions, no previous white spot lesions, caries or restorations present on the 
facial surface of the teeth being studied, adequate oral hygiene.  
In the recruitment process the attending resident will provide information and answer any 
questions about the study to the potential study participant.  If there is interest on behalf 
of the patient then the consent form will be reviewed and after presentation the form will 
be signed by all who wish to join the study.  The consent form is attached as a separate 
document.  There is no benefit to the patient to participate other than participating in a 
study that may find a beneficial treatment for others in the future.  There will be no effect 
on the patients treatment or experience should they decide not to participate or to 
participate. 
The requirement of at least 3 biscupids to be extracted is to meet the requirements of the 
matched pair experimental design.  Because each patient is different and their oral 
biology is different treating and analyzing teeth that do not have matched pairs in the 
same patient are not useful for this study therefore the inclusion criteria cannot include 
patients with less than 3 planned extractions. 
The inclusion of minors aged 12 and older is because minors are more likely to develop 
white spot lesions and should this sealant provide protection then the whole orthodontic 
population will benefit.  Also there is no risk associated with the procedures outlined in 
this trial so there is no reason to exclude minors from this study. 
Currently the Department of Orthodontics enrolls 50 to 75 patients per month.  The 
number of patients that meet the above inclusion criteria will be about 30 % of those 
enrolled.  It is anticipated that a minimum of 1 patients per month can reasonably be 
enrolled into the study.  Thus, the study should receive full enrollment in 4 months. 
c. Study Design and Research Methods (see CONSORT diagram on last page) 
i. The study has a split mouth clinical design where each of two controls and one 
experimental sealant will be in each person.   
1. Each patient will have all three experimental sealant systems including: no 
sealant (negative control), UDMA sealant (control) and K18-UDMA sealant 

 
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(experimental group).  The number of patients required for this study was 
calculated from studies that had a similar design (de Moura et al., 2006; Uysal et 
al., 2010).  De Moura et al. studied the effect of a fluoridated antiplaque dentifrice 
on the development of caries lesions adjacent to orthodontic brackets.  Fourteen 
orthodontic patients with planned extractions were divided into two groups of 7.  
The patients used experimental or control toothpastes 3 times per day for 28 
days.  The teeth were extracted and the mineral loss was determined in the 
cross-sections by use of microhardness.  The results showed that there was a 
statistically significant (p < 0.05) improvement with the experimental antiplaque 
toothpaste for the first 10 µm of depth.  There was no significant difference at 
deeper distances from the surface.  A calculation of their experimental power 
shows that an n of 7 per group is insufficient ot achieve 80 % power at an α of 
0.05.  Additionally, the microhardness method is less sensitive to mineral density 
loss than the TMR methods to be used in this study.  Balancing the increased 
sensitivity via TMR with the insufficient power in the de Moura study to make 
conclusions about the efficacy of the antiplaque toothpaste to prevent tooth loss; 
we opt to be more conservative and use a larger n per group.   Based on the 
previously observed variations in mineral density and clinically relevant minimum 
difference of 10 % mineral density in the first 50 microns of tooth surface 
between the negative control and the experimental K18-UDMA sealant, the 
number of subjects to achieve 80 % power at an alpha of 0.05 is 20 subjects.  It 
is anticipated that up to 30 patients may be recruited to this study, with 20 
needed for statistical power to answer our research question.  
2. If the patient has a fourth bicuspid is planned for extraction, a random treatment 
will administered to this last tooth.  This will provide additional information on the 
effect of location (upper or lower jaw) on the protective ability of the experimental 
sealant. 

 
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ii. Teeth planned for extraction will be bonded with a bracket pad using one of three 
systems: no treatment (negative control), UDMA sealant (control) and K18-UDMA 
sealant (experimental group).  The bracket pad does not have the bracket protruding 
from the base.  
1. Blinding:   
a. The patients will be blinded as to which teeth receive the various treatments.  
All the teeth to be extracted will receive a bracket pad and which will serve to 
blind the patient. 
b. The dentists will be partially blinded as they will not be able to identify the 
UDMA from the UDMA-K18 sealants.  These are provided to the dentists as 
Sealant A or Sealant B.  The identity of Sealant A and Sealant B will be in a 
sealed envelope stored in the desk of the Chair of the department of 
Orthodontics.  This drawer is kept locked.   
c. The tooth extraction, preparation of the extracted teeth for analysis and the 
statistical evaluation will be blinded to the identity of the sealants until the 
evaluations are completed. 
d. When the patient is to receive their orthodontic bands, the attending dentist 
(either Dr. Patel or Fellows) will randomly draw an envelope from a container 
of envelopes that contains a 3 x 5 card which indicates which teeth will 
receive Sealant A, Sealant B or no treatment.  The attending dentist will write 
the patient number and date of sealant placement on the card and place that 
card into a study file for this information.  The placement allocation indicated 
on the cards will be determined via computer randomization.  
2. Bonding may occur then there is referral for extractions.  The bonding protocol 
will be the standard protocol used in the orthodontic clinic for bonding orthodontic 
brackets to teeth.   
3. The differences will be that instead of bonding a full orthodontic bracket to the 
bicuspids planned for extraction, a bracket pad will be bonded. The use of a 

 
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bracket pad in place of a full bracket is to reduce the possibility of the bracket 
from interfering with the orthodontic archwires attached to the other teeth as part 
of the standard orthodontic therapy. 
4. The treatment to be used on each of the planned to be extracted bicuspids will 
be determined by selecting an envelope from a container that has within it a card 
indicating which teeth are to receive which treatments.  The patient ID number 
will be recorded on the card which will be kept by the study coordinator in a 
secure location. 
iii. All patients will be given standard hygiene instructions. 
iv. When the patient reports to the oral surgeon for extractions, the extracted teeth will be 
placed in Tooth Storage Solution which is antimicrobial and does not alter the surfaces 
or composition of the teeth.  The container will have the patient number and date of 
extraction written on the label. 
v. There is no difference related to the procedures used to extract the teeth that are to be 
extracted for orthodontic therapy.  The standard of care is the same for all patients 
whether there is a sealant or not on the teeth to be extracted.  
vi. These teeth will be returned to the laboratory for histological evaluation of mineral 
density and lesion depth.  
vii. The original location of the extracted teeth can be easily made from the tooth 
morphology.  The source of the extracted tooth (original location and patient number) 
will be recorded in the laboratory notebook, and when data evaluation is conducted will 
be used to identify the experimental group for that the tooth.  
d. Description, Risks and Justification of Procedures and Data Collection Tools: 
i. Description: These experiments utilize bicuspids that are treatment planned for 
extraction to allow for an approximant 30-day period where the experimental treatment 
is exposed to the environment of the mouth in exactly the same manner as all other 
teeth in the patients mouth. 

 
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1. About 30 % of the patients who receive treatment planning for orthodontic 
therapy will require extraction of bicuspids.   
2. The extractions are usually scheduled to occur at about 30 days after bracket 
bonding to the other teeth.  
3. The experimental design requires that each patient will have all experimental and 
control conditions (on separate teeth).  This split mouth paired samples design 
increases the power of the experiment and allows for fewer patients to be 
required for sufficient statistical power.  Paired analyses within an individual 
renders greater sensitivity to experimental effects than population based 
analyses. 
4. Bonding bracket pads instead of full brackets removes the protruding portion of 
the bracket so that it does not interfere with the orthodontic wires that are placed 
for the therapy.   
5. The use of smooth sealants on the teeth to be extracted is not unique as many 
patients have a smooth surface sealant placed after the orthodontic brackets are 
placed.  There are several commercial products that release fluoride specifically 
for the purpose of prevention of WSL.  It is unfortunate that there are few well 
designed studies about the use of these sealants. 
6. Should the patient decide to withdraw from planned orthodontic therapy prior to 
extractions, the bracket pads can be removed following the standard procedures 
for removing brackets at the same time as the brackets are removed.  The 
sealants will come off at the same time because the standard procedure includes 
polishing the tooth surfaces when brackets are removed. 
7. Patient identification is only to identify which tooth received the specific 
treatments so that when the teeth are extracted by the oral surgeon the groups 
can be identified.  No patient information is associated with the outcome 
measures.  

 
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ii. Justification of Procedures: There are no procedures delivered to the patient that are 
not within the scope of the standard procedures for orthodontic patients.  All data is 
gathered from the extracted teeth in the laboratory. 
iii. Data Collection Tools: Mineral density and lesion depth will be determined in the 
laboratory through histological examination of the teeth.  Briefly, the teeth are cross-
sectioned through the bracket pad to generate a 1 mm thick slab from the middle of the 
tooth.  This slab is observed under polarized light microscopy to measure the lesion 
depth (if any) from the surface, and subjected to x-ray to determine the mineral density 
across the lesion. 
e. Potential Scientific Problems: None are anticipated. 
f. 
Data Analysis Plan 
i. Statistical analysis will include graphical evaluations, along with descriptive statistics 
(mean, standard deviation), and one-way ANOVA for the three groups for both mineral 
density and lesion depth outcomes. If appropriate, there will be follow-up ranked 
comparison testing at a significance of P ≤ 0.05.  There will be paired-t tests between 
UDMA and negative controls, UDMA-K18 and negative controls, and paired-t tests 
between UDMA and UDMA-K18 sealants on mineral density and lesion depth 
outcomes.  Should there be data from a 4th tooth (2 teeth will receive the same 
treatment) the data from the 2 teeth with the same treatment will be averaged prior to 
comparison to the controls. 
ii. The possibility of regional variations (left vs. right, upper vs. lower) has been taken into 
account in the calculation of the size of the patient cohort.  The sample number 
calculation was based on differences and variations that are relatively large, and an 
additional 10 (50 % number) patients were added to the cohort to give the strongest 
probability of being able to make statistically based conclusions. 
iii. Secondary analyses may be possible to assess the ability of microbial biofilm to cause 
demineralization differences between upper and lower arches.  If a patient has a fourth 
bicuspid that is to be extracted (which is usually the case) the assignment of one of the 

 
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three treatments will be to generate a pair (left vs. right or upper vs. lower)  for 
comparison.  The statistical analyses will include paired-t tests for the differences that 
may be observed within each treatment group. 
g. Summarize Knowledge to be Gained: Whether the use of UDMA-K18 sealant prevents 
tooth demineralization during orthodontic therapy.   
i. This is not a pilot study, and is intended to provide the clinical efficacy of the UDMA-
K18 sealant to prevent white spot lesions around orthodontic brackets.  Unless the 
variation is much larger than anticipated there will be sufficient power in the 
experimental design to make statistically based conclusions about the sealant efficacy.  
One of the experimental design features is that each patient will have all the controls 
and the experimental sealant so paired differences within each patient will be 
determined.  This sort of paired difference experimental design is one of the most 
sensitive   
ii. The data generated and the conclusions achieved will be useful for the dental industry 
to decide whether to commercialize the sealant.   
 

 
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References: 
Darvishiabyaneh S, Carey CM. Stereomicroscope based method to assess enamel and dentin erosion. J 
Dent Res 2016;95 Spec Iss A:1089 
Gong S-Q, Niu L-N, Yiu CKY, Rhou H, Qi Y-P, Blissasrd JD, Nikonov S, Brackett MG, Messer RLW, Wu 
DC, Jao J, Brister LB, Rueggeberg FA, Arola DD, Pashley DH, Tay FR. Quaternary ammonium 
silane-functional, methacrylate resin composition with antimicrobial activities and self-repair 
potential. Acta Biomaterialia 2012;8:3270-3282. 
Faranesh S, Shellhart D, Newman S, Tilliss T, Minick G, Carey CM. The incidence of white spot lesions in 
a university orthodontic clinic patient population: an observational clinical cohort study. 2017; 
MSD Thesis, University of Colorado Denver, School of Dental Medicine, Department of 
Orthodontics. 
Lee H, Newman SM, Oesterle LJ, Shellhart WC, Minick GT, Tillis T, Carey CM: In vitro investigation of 
primers and RMGIC on bracket retention and resistance to erosion, 2015; MSD Thesis, University 
of Colorado Denver, School of Dental Medicine, Department of Orthodontics. 
de Moura MS, Simplicio AH, Cury JA. In-vivo effects of fluoridated antiplaque dentifrice and bonding 
material on enamel demineralization adjacent to orthodontic appliances. Am J Ortho Dentofacial 
Orthoped 2006;130:357-363. 
Liu SY, Tonggu L, Niu L-N, Gong S-Q, Fan B, Want L, Zhao J-H, Huang C, Pahsley DH, Tay FR. 
Antimicrobial activity of a quaternary ammonium methacryloxy silicate-containing acrylic resin: a 
randomized clinical trial. Nature Sci Rpts 2016a;6:21882. DOI: 10.1038/srep21882 
Stricker B, Johnston AD, Carey CM. Assessment of tethered UDMA‐K18 antimicrobial efficacy on biofilm 
activity, J Dent Res 2016;95 Spec Iss A:1019. 
Schmuck BD, Carey CM. Improved contact x-ray microradiographic method to measure mineral density of 
hard dental materials. J Res Natl Inst Stand Tech 2010;115:75-83. 
Uysal T, Amasyali M, Ozcan S, Koyuturk AE, Akyol M, Sagdic D. In vivo effects of amorphous calcium 
phosphate-containing orthodontic composite on enamel demineralization around orthodontic 
brackets. Aust Dent J 2010;55:285-291. 
Wefel JS, Harless JD. Comparison of artificial white spots by microradiography and polarized light 
microscopy. J Dent Res 1984;63:1271-1275. 
 
 

 
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Experimental Diagram. 
 
 

 
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