In vitro remineralization of primary teeth with a mineralization-promoting peptide containing dental varnish

Abstract Mineralization-promoting peptides are attractive candidates for new remineralization systems. In previous studies, peptides have been applied as aqueous solutions, which is not a clinically relevant form. Objective This study aims to investigate the efficiency of a mineralization-promoting peptide, applied in varnish, on remineralizing artificial caries on primary teeth. Methodology 55 primary molars were collected. Specimens were immersed in a demineralizing solution for 7 days and then, divided into 7 groups: Baseline: No-remineralization, Placebo: Blank colophony, F: Colophony 5% fluoride, P: Colophony 10% peptide, P+F: Colophony 5% fluoride and 10% peptide, Embrace: Embrace™ varnish, Durashield: Durashield™ varnish. A mixture of 35% w/v colophony varnishes were prepared in ethanol and applied accordingly. Specimens were immersed in a remineralization solution for 4 weeks and it was evaluated using PLM and SEM. Lesion depth reduction was examined by one-way ANOVA. Results There was no significant difference in mean lesion depths between baseline (147.04 ± 10.18 µm) and placebo groups (139.73 ± 14.92 µm), between F (120.95 ± 12.23 µm) and Durashield (113.47 ± 14.36 µm) groups and between P (81.79 ± 23.15 µm) and Embrace (90.26 ± 17.72 µm) groups. Lesion depth for the P+F group (66.95±10.59 µm) was significantly higher compared to all other groups. All groups contained samples with subsurface demineralized regions. Number of subsurface demineralized regions were higher in fluoride-containing groups. Conclusions We conclude that the mineralization-promoting peptide (MPP3) is effective in this in vitro study and the peptide shows benefits over fluoride as it yields less subsurface demineralized regions.


Introduction
Caries formation is a result of a shift towards d e m i n e ra l i z a t i o n i n t h e d e m i n e ra l i z a t i o n / remineralization cycle. When demineralization becomes dominant, carious lesions appear in certain teeth regions. 1 Epidemiological data indicate that dental caries is the single most common chronic childhood disease, and its prevalence is increasing in most industrialized countries, with a 60 to 90% prevalence among children in school age. 2 The demineralization process involves dissolution of hydroxyapatite, mainly caused by acidic attacks originating from the fermentation of sugars by microbial for remineralization, such as sufficient calcium and phosphate concentration, increased oral pH, agents that promote remineralization, should be provided. 3 Until this date, fluoride has been the most widely used agent to prevent and to treat incipient caries.
Fluoride works by partially replacing hydroxyl ions in the hydroxyapatite and thus forming fluorapatite, a more acid-resistant mineral. 4 Due to the decades-long evidence on its caries-preventive effects, use of fluoride is recommended by the regulatory and scientific bodies.
The doses used in dental applications does not pose a risk of acute poisoning. However, prolonged exposure to low doses has been shown to cause dental and skeletal fluorosis, 5 development of subsurface lesions [6][7][8][9] and change in enzymes activities, such as casein kinase II and alkaline phosphatase. 10 Non-fluoride containing remineralization products are also available in the market. These products mostly act by increasing the soluble calcium and phosphate ions in the saliva via stabilized forms of metastable calcium phosphate phases. Upon contact with saliva, metastable calcium phosphate is dissolved, and calcium and phosphate ions are released to the oral cavity. 3 However, clinical and in vitro studies report that increasing intra-oral calcium and phosphate concentration alone does not provide better results compared to standard oral hygiene regimes. 11,12 Therefore, there is still a need for new remineralization systems especially for pediatric population, which is more sensitive to fluoride.
Peptide agents that promote peptide mineralization have emerged as attractive candidates for such new remineralization systems. [13][14][15][16] Several peptides have been proposed for improving remineralization. Acidic fragments of naturally occurring biomineralization proteins or peptides designed or selected via experimental and computational tools have been shown to promote hydroxyapatite mineralization, therefore, they are suggested as agents for remineralizing the enamel. Self-assembling peptides have also been proposed as scaffolds to control the orientation of newly formed mineral, to resemble natural 3D architecture of the enamel. 17,18 In vitro 15, 19 and in vivo 20 studies have yielded promising results for the use of peptides as remineralizing agents in dentistry. However, in these studies peptides have been applied as aqueous solutions. Aqueous solutions are challenging in a clinical setting as they require more patient's cooperation and they do not provide slow continuous release. 21,22 The potential of these peptide agents has not yet been investigated as a real or prototype product that is more relevant to a clinical setting. To the best of our knowledge, no studies have been conducted using mineralization-promoting peptides in a real or prototype product format, especially focusing on primary teeth. This in vitro study aims to assess whether a mineralization-promoting peptide, applied in a colophony varnish preparation, is effective on remineralizing artificial carious lesions formed on primary teeth. We have compared the efficacy of peptide-containing, in-lab made varnishes, with two commercially available varnishes.

Methodology
This study was approved by the "Research Ethics Helsinki. All parents/guardians of the patients have signed a consent form following a written and verbal explanation of the study. A general outline of the study design is shown in Figure 1.
Collection of the teeth: An a priori power analysis was conducted using G*power to test the difference between seven independent group means using oneway ANOVA test, medium effect size of d=0.50 based on similar studies 19,23 and an alpha value of 0.05. Result showed that a total sample size of n=91 with seven equal sized groups (n=13) was required to achieve a power of 0.80. Two additional samples were included in each group, resulting in seven equal sized groups of n=15.
In total, 55 primary molars, extracted due to exfoliation or orthodontic reasons from children aged     The enamel thickness in primary teeth is nearly half the size of permanent teeth and the mineral content is lower compared to permanent teeth, resulting in weaker mechanical properties compared to permanent teeth. 26 Therefore, the conditions for the restorative or remineralization therapies for primary teeth differ from those of permanent teeth. 28 In this study, we have

Conclusions
Within the limitations of this in vitro study, we conclude that MPP3 is effective in varnish formulations for remineralization of enamel lesions.
We also conclude the peptide-containing varnish shows benefits over fluoride-containing varnish as it results in less demineralized subsurface regions.