Effect of Remineralization Agents on Molar-Incisor Hypomineralization-Affected Incisors: A Randomized Controlled Clinical Trial

INTRODUCTION

Molar-incisor hypomineralization (MIH) is characterized by qualitative developmental defects of enamel of one or more first permanent molars (FPMs), with or without involvement of incisors [1] and different phenotypes of MIH affect different teeth to varying severity [2].

MIH-affected enamel shows increased amounts of proteins like serum albumin, type-I collagen, ameloblastin, alpha-I-antitrypsin and antithrombin-III, which were found to inhibit the growth of hydroxyapatite (HA) crystals and enzymatic activity during enamel maturation, resulting in an overall reduction of minerals and also has been shown to have increased porosity and disorganized prisms with loss of distinct boundaries between rods [3]. Concentration of the main chemical composition, primarly calcium (Ca) and phosphorus (P), is significantly reduced in hypomineralized enamel compared to healthy enamel.

Laser fluorescence (LF) most likely detects some fluorophores that are organic in nature. Farah et al showed a moderately strong Downloaded from correlation between the mechanical properties of MIH-enamel and LF scores [4].

Minimally invasive treatment approaches adopt a philosophy that integrates prevention, remineralization and minimal intervention in the treatment of teeth affected by MIH [5, 6]. Consequently, remineralization agents are often recommended for the management of MIH in order to increase the mineral content of the hypomineralized areas although scientific evidence of the effectiveness of this treatment is still limited [7].

Fluoride (F) has still remained gold standard as remineralization agent considering more than 50 years of clinical success. Calcium fluoride serves as a source of F for the formation of fluoroapatite, thereby inhibiting demineralization and enhancing remineralization [8]. Casein phosphopeptide amorphous calcium phosphate (CPP-ACP) complex is an appealing delivery system for bio-available Ca and phosphate (PO${}_4$) ions, maintaining an amorphous state of supersaturationwith respect to enamel, decreasing demineralization and promoting remineralization [9, 10]. In previous researches which were set out to determine the effect of remineralizing agents in MIH have reported that F-containing and non-fluoridated CPP-ACP derived agents both strengthened and remineralized enamel and reduced the porous structure at depth of lesion and affected surfaces of MIH [6, 9, 10, 11]. Studies since the 1930s have reported the importance of using Ca and PO${}_4$ derivatives for favouring the remineralization process in dental caries [12]. Calcium glycerophosphate (CaGP), which is used medically as a source of Ca and PO${}_4$, has demonstrated anticariogenic properties [12, 13]. CaGP is an organic PO${}_4$ capable of adsorption to enamel surfaces, leading to the release of Ca ions, which act on the remineralization process [14, 15, 16]. The results of demineralization tests confirm that it exerts a powerful protective action on surface of the enamel. This effect was independent from the action of F [15]. CaGP shows a protective effect by increasing the resistance of HA. Its effect in biofilms may be related to the enhance of Ca and PO${}_4$ levels, buffering capacity and reduced the mass of the biofilms [14]. It is thought that the effectiveness of caries prevention by increasing cumulative effect with F [15, 16].

Today, different detection and evaluation methods are used in remineralization researches. In the detection of demineralization and hypomineralization, it is very important to determine the activity, depth, and other characteristics of the lesion [17]. Researchers have shown an increasing interest in non-destructive methods for the assessment and longitudinal monitoring of mineral changes in enamel, such as LF. DIAGNOdent${}^{TM}$ is the application of LF method in dentistry, which is based on the principle of absorbing and scattering the laser beam at a different rate compared to the healthy structure with the demineralized/hypomineralized areas [18].

In current literature there is a lack of evidence for the effectiveness of remineralization agents to the MIH-affected teeth. The objective of this study was to evaluate the remineralization effect of two different mineral containing agents (CaGP and CPP-ACFP) on white/creamy and yellow/brown demarcated hypomineralized opacities in incisors with MIH in-vivo, to be used in addition to routine oral hygiene using LF. The hypothesis adopted in the current study is that the in-vivo use of commercially available remineralization agents, in addition to routine oral hygiene, does have an effect on and assess of remineralization of demarcated hypomineralized opacities over time by LF.

MATERIALS AND METHOD

The study was approved by the Clinical Researches Ethical Committee of the School of Dentistry, Marmara University under the protocol number 2019-297, and was conducted in accordance with the principles of medical research involving human subjects described by the Declaration of Helsinki. Remineralization of demarcated hypomineralized opacities in incisors with MIH was assessed during three months using by LF.

This trial was performed as a prospective, randomized, controlled trial. The allocation of subjects followed a randomization scheme with stratification for gender. No changes to the original protocol were made during or after the trial.

The subjects enrolled in the study were children with MIH who applied to the Department of Pediatric Dentistry, School of Dentistry, Marmara University. Informations about the study were explained to the parents and then the consent forms were signed.

This single-centre trial took from January 2019 to May 2019.

Randomization and Intervention Procedures

Participants, complying with the inclusion criteria as determined by one trained pediatric dentist (BS), were randomly assigned by BS to group the CaGP, CPP-ACFP or the control group, as determined by a computer-randomization scheme, created and locked before the start of the study. Participant allocation was kept separate from the data recording files in a locked closet. Data were collected and coded based on participants’ ID number and a sequential number in order of date of study visits. Data analysis was performed blind for group allocation.

Participants received (1) CaGP (R.O.C.S.® Medical Minerals Gel includes CaGP, magnesium and xylitol, R.O.C.S. Trading GmbH, Munich, Germany) (27 children), (2) CPP-ACFP (10% CPP-ACP plus 0.2% NaF; GC MI Paste Plus${}^{TM}$, GC Europe) (16 children), (3) Control (routine dental home care with Colgate Toothpaste including 1450 ppm F, Colgate Oral Pharmaceuticals, New York, NY) (ten children). (shown in Figure 1)

Figure 1: Flowchart

All patients received verbal and written instructions with respect routine oral hygiene procedures and product usage by researcher. They were advised how to brush properly using a fluoridated dentifrice (Colgate Total with 1450 ppm F) (i.e. at least twice a day, either with a hand toothbrush or an electric toothbrush for at least two minutes). In addition, participants were instructed to use their respective paste twice a day once in the morning after brushing and once in the evening immediately before going to bed. They should leave it on their teeth to slowly dissolve overnight. Children were asked to apply with a clean finger to the tooth surfaces in accordance with the manufacturer’s instructions, under the control of their parents. Parents were informed to apply at least a pea-size amount to the tooth surfaces in each arch using a clean, dry finger and keep the study product in the mouth for as long as possible. Participants were instructed not to rinse, eat or drink for minimum 30 minutes afterwards [11]. Compliance of the patients were checked by asking questions regarding product usage at each visit. Furthermore, parents were asked to bring the study product to each visit. The parents were informed not to administer additional F during this investigation. They were further asked to contact the study investigator if restorations were made.

RESULTS

From the 102 screened children with MIH; a total of 53 children [28 (53%) girls and 25 (47%) boys] ages eight to 12 years (9.34±1.4) with 401 demarcated hypomineralized teeth were evaluated according to different clinically diagnosed levels of MIH as white/creamy and yellow/brown demarcated lesions >one-mm in diameter but noncavitated teeth randomly assigned into three groups: 27 children with 197 teeth in Group 1 (CaGP), 16 children with 125 teeth in Group 2 (CPP-ACFP) and ten children with 79 teeth in Group 3 (F toothpaste, control) were evaluated by LF.

DISCUSSION

Preventive dentistry aims to prevent initial carious lesions known as white spot lesions, developmental enamel defects, and hypomineralized areas without cavitation [23].

Many different treatment approaches can be applied on incisors affected by MIH. The priority in the treatment of non-cavitated hypomineralized areas is remineralization protocols within the scope of minimally invasive and preventive dentistry [24]. Although it is known that the application of high concentrations of F provides a remineralization in the superficial layer of demineralized enamel, this effect is not been observed in deeper layers. For this reason, it is recommended to apply lower concentrations of F, which allows the absorbtion of Ca and F ions from saliva. In addition, researches on alternative remineralization agents to F are becoming common [25].

Improving the mineralization of MIH-teeth after eruption is possible according to some in-vivo and in-vitro studies; though, a complete resolution seems to be difficult due to the depth and/or the thickness of these lesions. In an effort to remineralize the MIH-teeth, the long-term use of products containing CPP-ACP is recommended especially at early stages where the surface enamel of newly erupted teeth is not completely matured [11, 26].

Some authors suggest that CPP-ACP may accelerate and increase the potential maturation of MIH-enamel structure, improving its mechanical properties after tooth eruption. Decreasing the surface porosity may reduce the caries risk and sensitivity due to thermal/tactile stimulation [6, 27]. In a study conducted by Baroni and Marchionni, it was hypothesized that the application of CPP did not alter the surface morphological characteristics of MIH-molars in-vivo [6]. The results suggested that the use of a mixture of Ca and PO${}_4$ ions carried by large molecules can be indicated in MIH-teeth where such minerals are deficient. CPP-ACP supplementation accelerates and increases the potential maturation of MIH-enamel structure, which may or may not take place over a much longer time. In conclusion, since improvements were found after the application of Ca-PO${}_4$ casein on MIH-molars, the results of the present study support the rejection of the tested hypothesis, warranting investigations of new products containing fluoridated CPP-ACP, and needing adjustments of administration protocols. Given the absolute lack of in-vivo studies and supplementation trials, protocols with CPP-ACP may help functional restorative techniques in treated MIH-molars and aesthetics in untreated incisors [6]. However, there is a lack of evidence as regards the long-term effectiveness and protocols of products containing CPP-ACP in MIH-lesions.

Restrepo et al. observed no favorable effect on the remineralization of MIH-lesions in anterior teeth after four applications of F varnish [7]. However in our study, it was observed that the use of fluoridated toothpaste provided remineralization in incisors affected by MIH.

Although the efficacy of CPP-ACFP for the prevention and regression of incipient lesions has been demonstrated in-vitro [28, 29] there is no reliable evidence for the treatment of MIH-lesions in-vivo and the long-term effect of this remineralizing agent is unclear.

The F ions contained in CPP-ACFP might slowed the penetration of the Ca and PO${}_4$ ions to the inner layers of the lesions and caused some early precipitation of these ions in the outer enamel surfaces [30]. In parallel with this information, it was determined that higher levels of remineralization in the CPP-ACFP group in LF scores less than 20, in our study.

In a study conducted by Bakkal et al, it was observed that CPP-ACP and CPP-ACFP were found to have achieved a significant decrease in LF scores in one-month [11]. This pilot study showed that using CPP-ACP and CPP-ACFP had a positive effect in reducing hypomineralization on enamel surfaces of MIH-teeth for one-month period [11]. Similarly, results of our study showed that CPP-ACFP could be an effective remineralization agent in incisors affected by MIH in the short treatment period.

CaGP is an organic polyphosphate also has the ability to adsorb to enamel surfaces through the PO${}_4$ radicals of its molecule. CaGP, however, acts at different levels of the demineralization process [31]. When this organic PO${}_4$ adsorbs to enamel surfaces, virtually all PO${}_4$ groups are neutralized by Ca and hydroxyl groups of HA molecules, so that Ca ions from CaGP are released next to enamel surfaces [31].

Research on CaGP and sodium monofluorophosphate interactions show that the ratio of monofluorophosphate to CaGP affects the demineralization of HA and enamel. It is known that use of sodium monofluorophosphate and CaGP separately is significantly less effective than a combination of both. Regardless of the presence and mechanism of F, CaGP has a strong protective effect [31]. In a study conducted by Grenby and Bull, pre-treating HA discs with NaF conferred a relatively long-lasting protective effect against demineralization [15]. According to this study, CaGP and F do not share the same mechanism of action, but their effects can be cumulative [15]. It has been stated that the CaGP interacted directly with the outer layers of the HA. Many possible mechanisms for the cariostatic properties of CaGP have been proposed. These include direct interaction with enamel, plaque pH-buffering, reduction in plaque mass, modified plaque metabolism, and elevation of Ca and PO4 levels in plaque [31]. CaGP alone has cariostatic properties and has repeatedly demonstrated anti-caries properties in-vivo. It would seem that these effects can be achieved readily but only if CaGP is applied frequently and at relatively high concentrations [31]. Further, F and CaGP have an additive effect and probably influence dissolution of enamel via different mechanisms. The potential anti-caries benefits of buffering of plaque acid are self-evident [31].

The low-F dentifrice with 0.25% CaGP demonstrated efficacy similar to that of the positive control (1,00 dentifrice) with respect to in-situ demineralization [32]. The results of a study about fluoridated toothpastes indicate that clinical efficacy of low-F toothpaste supplemented with the addition of CaGP leads to similar efficacy when compared to 1100 ppm F [33]. Zaze et al showed that low-F toothpaste (500 μg F/g) maintains the same efficacy as that of a 1100-μg F/g toothpaste when supplemented with 0.25 % CaGP and provides the best effect against in-vitro enamel demineralization [16]. In a study conducted by Rezende et al, remineralization of different F solutions on the enamel surface in the presence of CaGP (0.13%) was evaluated [34]. Although there was no statistically significant difference between groups with different levels of F containing agents, it was reported that agents containing CaGP increased remineralization significantly compared to the control group [34]. In parallel with these study results, in our study, it was observed that the hypomineralized areas on incisors affected by MIH was remineralized with CaGP. This study was the first to address these aspects.

In teeth with LF scores greater than 20, it was observed that CaGP performed statistically significantly more remineralization compared to CPP-ACFP and control group in comparing the mean values of intergroup change at the end of the three-month period (p<0.01). This can be explained by the CaGP due to the affinity of the enamel to the HA structure by exceeding the superficial remineralization layer made by F [31]. Despite the promising results reported for CaGP-containing toothpastes, no clinical evidence is still available to attest the anticaries effects with PO${}_4$ salts on the progression of caries lesions.

The hypomineralized teeth with high LF scores tend to have low mechanical properties of MIH-enamel suggest lower mineral content and the increased LF scores suggest increased organic content. The altered prismatic structure and the inhomogenity of hypomineralized enamel may have resulted in or contributed to the increased LF scores. Another factor that may contribute to the increased LF from MIH-enamel is its darker colour. In this study the higher LF scores recorded in MIH-lesions indicated a lower mineral content at baseline. High LF scores in non-carious teeth only indicate hypomineralized enamel. The increased scores in hypomineralised enamel may be related to proteins in the hypomineralized enamel and/or light scattering by the inhomogeneous enamel. Bacterial metabolites may not be the only fluorophore resulting in an increase in LF scores, especially in hypomineralized enamel. Regardless of the nature of the fluorophores in MIH-enamel, however, increased LF score indicates defective enamel [4]. Assuming that the increase in LF scores represents a real increase in organic substance across the all scale of the scores, it seems that beyond LF scores of around 20, the mechanical features are no longer that susceptible to the quantity of organic substance in hypomineralized enamel [4].

As a result, we can say that the application of CaGP and CPP-ACFP for three-months provides remineralization in incisors affected by MIH. Future investigations may increase the experimental time to determine effectiveness of remineralizing agents over time.

CONCLUSIONS

The use of CPP-ACFP and CaGP containing remineralization agents in children with MIH lesions improved these lesions over 3 months period of the study, as evaluated by LF measurements. Based on data obtained from our study, it was concluded that both CPP-ACFP and CaGP containing agents showed remineralization potential on hypomineralized enamel on incisors affected MIH by using LF. This investigation provided additional information for better understanding of the mechanisms of mineral containing agents, therefore being possible to accept the study’s hypothesis.

It may be advisable to conduct researches with different remineralizing agents and LF method, longer follow-up and more sample size to support these findings. Thus, clinical application of these agents will be a novel noninvasive patient-friendly and effective method to treat reversible carious lesions. However, further in-vivo studies are required to reassure the superiority of CaGP and CPP–ACFP. Further research may help assess that LF readings may indicate for evaluation of MIH tooth and also mineralising agents.