Remineralization of Molar Incisor Hypomineralization (MIH) With a Hydroxyapatite Toothpaste - an in Situ Study
Phase: NA
Conditions: Molar Incisor Hypomineralization
Sponsor: The University of Texas Health Science Center at San Antonio

21-570H, Amaechi, Form BB, 01-05-22, NS.doc 
 
 
 
 
Official Study Title: Remineralization of Molar Incisor 
Hypomineralization (MIH) with a Hydroxyapatite Toothpaste – an in 
situ study 
 
 
NCT number: NCT 05485662 
 
IRB Approval Date: 01/05/2022 
 
Unique Protocol ID: HSC20210570H 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Remineralization of Molar Incisor Hypomineralization (MIH) with a Hydroxyapatite Toothpaste – 
an in situ study 
 
 
Institution: 
University of Texas Health San Antonio  
7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA 
 
 
 
Investigator and Affiliations: 
Bennett T. Amaechi, BDS, MS, MFDS RCPS (Glasg), PhD, FADI 
Professor and Director of Cariology  
Department of Comprehensive Dentistry 
University of Texas Health San Antonio 
7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA 
 
 
 
 
Representatives of Sponsoring Company: 
Dr. Joachim Enax and Dr. Frederic Meyer 
Senior Scientists Oral Care 
DR. KURT WOLFF GMBH & CO. KG  
Johanneswerkstr. 34-36  
33611 Bielefeld 
Germany 

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A. Null Hypothesis 
1. Hypomineralized enamel treated with hydroxyapatite toothpaste would not regain up to 20% mineral 
density relative to healthy enamel. 
2. The effectiveness of hydroxyapatite toothpaste in remineralizing MIH would not be significantly different 
from that of 1450 ppm fluoride toothpaste.  
B. Specific Aims 
The objective of this in situ clinical study is to determine whether a toothpaste formulation containing 
hydroxyapatite is effective in promoting remineralization of molar incisor hypomineralization (MIH), in 
comparison to a toothpaste formulation containing 1450 ppm fluoride. Effectiveness will be declared if the MIH 
lesion regain up to 20% mineral density relative to healthy enamel. 
C. Significance / Background 
MIH is a qualitative enamel defect observed on at least one first permanent molar (FPM) with or without affecting 
permanent maxillary incisors1. Clinically, it’s diagnosed when a FPM presents with a demarcated opacity, larger 
than 1 mm, varying in color from white to yellow and even brown2,3. Additionally, post-eruptive enamel 
breakdown, atypically sized, shaped, and extended restorations, or a history of extraction of FPMs in an otherwise 
sound dentition, are also diagnostic criteria of MIH. The location, asymmetry and varying severity of the defects, 
distinguishes them from other enamel mineralization defects such as fluorosis4. The severity of the affection can be 
divided into 2 categories, mild and moderate/severe. These categories are differentiated clinically by the presence 
or absence of enamel breakdown, the degree of hypersensitivity and the esthetic concerns3. It is also correlated with 
reduced levels of mineral density (MD) of the affected enamel to 2.24 g/m3 in mild cases and 1.93 g/m3 in 
moderate/severe cases in comparison to a 2.51 g/m3 average MD of normal enamel5.  
Hypomineralization can affect both primary and permanent teeth, but the molar-incisor model is the most common 
with a reported prevalence between 10% to 20%6,7. As for their etiology, MIH defects can result from genetic or 
systemic environmental factors. Perinatal and postnatal factors appear to be the most strongly associated with its 
occurrence8. In fact, premature birth and associated complications, hypoxia, dioxin exposure and other childhood 
illnesses such as ear or respiratory infections and antibiotic use are implicated in an increased incidence of MIH. If 
these conditions develop in the early childhood, up to age 3, when the crowns of the FPMs and incisors are 

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undergoing mineralization, they predispose the child to MIH3,9. Consequently, the time of onset, span and severity 
of the conditions dictate the pattern and intensity of the lesions. 
Management of MIH defects is complicated by the penetration of bacteria through the often-exposed dentinal 
tubules10. This process causes subclinical inflammation of the pulp and hypersensitivity that lead to a higher 
anesthetic threshold and negligence of oral hygiene by the child subsequently increasing the need for dental 
treatment11. Additionally, esthetic and functional concerns are frequent in these patients and are associated with a 
lower oral health related quality of life12. The treatment of teeth with hypomineralization defects is twofold, 
preventive and/or interventional. Therapeutic interventions must take place when the teeth display severe defects, 
enamel breakdown, decay or signs of pulpal inflammation/infection13. This approach includes restoration with glass 
ionomer, composite resin or preformed metallic crowns for posterior teeth and micro-abrasion with resin infiltration 
and composite restoration for anterior teeth and extraction with space management for non-restorable teeth3,14. 
However, when MIH diagnosis is established early, the prevention of the progression of the defect is recommended 
by using sealants and promoting remineralization. In fact, increasing the mineral content of the enamel to a 
threshold close to the norm can improve its physical properties, hence improving the esthetic and functional 
outcomes. Several techniques pertaining to remineralization of MIH lesions have been described in the literature 
including the use of low-level laser therapy or the application of pastes and varnishes containing arginine, fluoride 
(with or without tricalcium phosphate), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) or casein 
phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP)13. Toothpastes containing nanohydroxyapatite 
(HAP) were shown to have comparable effects to fluoride toothpastes in terms of remineralization of incipient 
carious lesions and prevention of decay, while excluding the risk of fluorosis15. The synthesized particles of HAP 
are structurally similar to the crystals found in enamel and dentin, have analogous properties and can readily 
penetrate enamel surface16. However, their use as a mean of increasing mineral density in MIH defects has not been 
investigated. Thus, the objective of this study is to determine the effectiveness of a toothpaste containing HAP in 
promoting remineralization of teeth with MIH defects. 
D. Experimental Design 
Production of Samples 
30 freshly extracted permanent teeth with MIH will be collected from the pediatric clinics of the University of 
Texas Health San Antonio (UTHSA). These teeth were extracted as part of the patient’s standard of care and 

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donation will be done based on the patients willing ness. If the patient wishes to donate the teeth, they will be 
placed in a container within the pediatric dental clinics and will be donated without in identifiable information 
attached to them. The teeth will be cleaned of debris/stains and examined with a transilluminator. Teeth with MIH 
but free of caries or other malformations will be selected and cleaned with pumice to remove the pellicle. Using a 
water-cooled diamond wire saw, 3 blocks, two from MIH affected area and one from a healthy area will be 
produced from each tooth, with each block measuring approximately 2 mm length x 2 mm width x 1 mm thickness. 
The cut will take place so that the different layers from the outer surface to the inner surface present in each block 
are: outer enamel surface, enamel, dentino-enamel junction (DEJ) and dentin.  
Measurement of baseline mineral density of the tooth blocks 
Micro computed X-ray tomography (µ-CT) is a high resolution, non-destructive radiographic method of analysis 
that generates qualitative images and quantitative analysis of the mineral density (MD) in tooth samples. For this 
reason, images will be collected from the samples to measure the mineral density of the MIH enamel lesions before 
(MDb) and after (MDt) remineralization treatment as well as the mineral density (MDs) of healthy enamel samples. 
The µ-CT scanning and analysis procedure will be performed as described in our previous publications.17 
Study Population/Participants 
Fifteen subjects (8 females, 7 males) will be recruited into the study. The subjects to be included should be 18 to 60 
years old in good general health with no known history of allergy to personal care/consumer products. They must 
have a minimum of 20 natural uncrowned teeth (excluding third molars), no active unrestored cavities and a normal 
salivary flow rate (stimulated and unstimulated flow of ≥0.7 ml/min and ≥0.2 ml/min respectively) ascertained from 
a preliminary sialometry test. They also should give written informed consent, be available throughout entire study, 
and be willing to wear intra-oral appliance 24 hours per day and use only assigned products for oral hygiene 
throughout the duration of the study.  
The exclusion criteria are having advanced periodontal disease, a medical condition that requires premedication 
prior to dental visits/procedures, an impaired salivary function, wearing an orthodontic retainer(s), not having 
enough teeth to secure the oral appliance, having diseases of the soft or hard oral tissues, using drugs that can affect 
salivary flow, using antibiotics one month prior to or during this study, participating in another clinical study one 
week prior to the start of the washout period or during this study period, using tobacco products, having an allergic 

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history to common toothpaste ingredients, and having a compromised immune system (HIV, AIDS, immuno-
suppressive drug therapy) as determined by review of medical history 
Recruitment of Study Subjects 
This is a double-blind, randomized, crossover, single center, controlled clinical trial. The study will be blinded to 
both the study team and the subjects. Recruitment of potential subjects will be from the local San Antonio area. The 
subjects will be identified with code numbers (CP01 to CP15). The approval of the UTHSA IRB will be obtained. 
This study will be conducted in compliance with International Conference on Harmonization (ICH) Good Clinical 
Practice Guidelines. At the screening visit, each subject will complete a medical / dental history and read and sign 
an informed consent form. Following consent, a visual oral health exam will be performed by a licensed dentist. 
Subjects will be enrolled based on all the inclusion and exclusion criteria and other screening evaluations such as 
preliminary sialometry, and then randomly assigned to one of two treatment sequences. 
Construction of the in-situ appliance 
The 3 blocks from each tooth will be used, as follows: two blocks with MIH lesions will be used for 
remineralization assessment and one healthy tooth block will serve as control for the MD of healthy enamel. The 
MIH-bearing blocks will be covered with polyester gauze (Bard Peripheral Vascular, Inc., USA), which will 
facilitate plaque retention on the surface of the tooth blocks on intra-oral exposure. Following recruitment, an 
impression of the subject’s lower dentition will be taken using an alginate impression material. A dental technician 
will fabricate a lower removable intra-oral appliance. Each of the MIH-lesion-bearing blocks will be mounted on 
each side within the acrylic portion of the removable appliance, using Intermediate Restorative Material (IRM) 
cement (fluoride–free). All appliances are sterilized with ethylene oxide prior to delivery to the subject.  
Test Product Description and Labeling 
The toothpastes to be used for treatments are 10% HAP toothpaste, 1450 ppm NaF fluoride toothpaste, and non-
fluoride toothpaste (washout). The 2 test products will be coded by the manufacturing/packaging company, who 
will retain the code until the completion of the study and data interpretation. The experiment will consist of two 
distinct treatment phases, Phase 1 and Phase 2, during which subjects will be exposed to one of the 2 products in a 
randomized crossover design, with each phase lasting for 2 weeks. 
Study Procedure and Patient Instructions 

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Prior to each 2-week treatment phase, subjects will complete a 1-week washout period. This period allows for 
attenuation of any residual effect of the subject's previously used toothpaste. During this period, no appliance will 
be worn, and subjects will be using only the provided washout toothpaste and a soft-bristled toothbrush twice daily. 
Following the washout period, the intra-oral appliance will be fitted to each subject by a qualified dentist. 
Immediately after fitting of the first appliance (on day 1 of the first treatment phase), each subject will receive a soft 
bristled manual toothbrush for use throughout the duration of the study and a toothpaste according to the treatment 
phase. They will make the first use of the test product under the supervision of the study coordinator. For the 
remainder of the study, subjects will complete the procedure at home and as instructed. 
The subjects will be instructed to brush their teeth with the appliance in the mouth, two times daily, for 3 minutes 
on each brushing episode, in the morning after breakfast and last thing before bed, then rinsing with 10 ml of water. 
In dispensing the toothpaste onto the toothbrush, the subjects should fill the toothbrush surface from end to end but 
no more than one ribbon of toothpaste. The subjects will be advised not to brush directly on the blocks but rather to 
brush around the blocks to prevent disruption of the plaque. Subjects should neither eat nor take any drink for at 
least 30 minutes after brushing, they will be also instructed to remove the appliance while eating. A timer and 
measuring cup will be provided to each subject. To monitor product usage and compliance, a diary will be provided 
to each subject and checked at every visit, to record the number of tooth-brushings performed each day and the time 
it was done. Further, subjects will be instructed to return the remaining washout or test toothpaste after each 
treatment phase. The weight of toothpaste will be measured before and after each treatment phase. Over the study 
period, all subjects are to maintain their normal dietary habits.  
On day 15, the subject, without using the product that morning, will arrive at the clinic, and the tooth block will be 
harvested and sent to the laboratory for µCT analysis. Then the subject will be given washout toothpaste and a soft-
bristle toothbrush to undergo another 7-day washout period without an appliance. After completion of the second 
washout period, subjects will return to the clinic, and the appliance, with another MIH-bearing tooth blocks 
mounted, will be fitted to the subject for the phase 2 treatment period. This procedure will then be repeated until the 
2-week treatment phase is completed, and each subject has gone through the two arms of the study. 
Upon completion of the treatment phases, the post-remineralization mineral density (MDt) of all the MIH-lesion-
bearing blocks exposed intra-orally for remineralization will be assessed with µCT. For the lesion-bearing blocks, 

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this process will yield the following information: the pre-test (MDb) and post-test (MDt) mineral density of the 
lesions and their associated images, while for the healthy tooth blocks, the MDs and the µCT images. 
At every visit, the dental examiner will visually examine the soft and hard tissues of the oral cavity and peri-oral 
area using a dental light and dental mirror. Additionally, subjects will be asked about and examined for any adverse 
events. Subjects may call and request a visit for any concern of potential adverse event.  
 
Data Handling 
Using the µCT images, the pattern and the extent of remineralization produced within each lesion by each treatment 
product will be examined and described. This will be clearly shown by comparing the pre- and post-test images 
side-by-side. For all calculations, the absolute MD will be measured and used. For the remineralization blocks, the 
values of the MDb and MDt for each test product will be compared using paired t-test to determine any significant 
change (remineralization) made by the test product (intra-group comparison). However, to make comparisons 
between the two products (intergroup comparison), percentage change in MD calculated relative to the control will 
be determined for each test product as such:  % Change in MD (%∆MD) = [(MDb – MDt)/MDb] x 100 
The percentage MD recovery following remineralization will be calculated as follows: 
% MD recovery (%MDR) = [(B-T)/(B-S)] x 100, with S = mean MDs of healthy enamel blocks, B = mean MDb of 
MIH enamel blocks before remineralization, and T = mean MDt of MIH enamel blocks after intra-oral exposure. 
Sample Size Calculation 
The power analysis and sample size calculation were performed using nQuery Advisor software (Statistical 
Solutions, Cork, Ireland). The sample size for this study is based on the primary efficacy parameter, % MDR 
following 2 weeks of treatment. A sufficient number of subjects will be screened to randomize approximately 15 
subjects with the intention that approximately 15 subjects complete all study treatments and be evaluable for the 
efficacy analysis. With these conditions met, the study will have 80% power at the 5% significance level, using 
two-sided testing, to detect a mean treatment difference in %MDR of approximately 8.1% assuming a within 
subject standard deviation of approximately 12.6%. In the absence of available 2 week data from previous mineral 
density studies, the within subject standard deviation has been estimated using 14-day post-treatment data from a 
previous results obtained by this group in a demineralization and remineralization study using HAP toothpaste15. In 
that study the mean % change in mineral loss was equal to 30.3 with a standard deviation equal to 16.3.  

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Statistical Analysis 
After assumptions of normality and homogeneity of variances have been verified by normal probability plots, 
comparisons of the mean values of MD, %ΔMD and %MDR between the two treatment groups as well as the intra-
group comparisons will be performed using the paired Student’s t-test (STATA version 10.0, StataCorp LP, USA) 
at a significance level of 5%. The primary endpoints of the present study are expected to be enhanced 
remineralization of MIH lesion. 
E. References 
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meeting on MIH held in Athens, 2003. European Journal of Paediatric Dentistry. 2003 Sep 1;4:110-4. 
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