Assessment of the Effects of Enamel Remineralization After Treatment with Hydroxylapatite Active Substance: SEM Study

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The current investigations suggest that Mentadent Professional Resilience could be beneficial as a remineralizing agent.

Abstract

This study aimed to investigate the remineralization processes of dental enamel via scanning electron microscopy and observe the changes induced in the microstructure by oral hygiene products. More specifically, the effectiveness of Mentadent Professional Resilience was analyzed for its ability to demineralize erosion-affected enamel surfaces. This involved the sectioning of some teeth to preserve enamel integrity, followed by dehydration and preparation for SEM analysis. SEM observations were made at various magnifications to detect differences in enamel morphology after treatment with the product. These observations provide valuable insights into the mechanisms of action of dental care products and their potential to protect enamel. The study makes a contribution to our understanding of remineralization processes and describes the importance of microscopic analysis for evaluating and developing effective dental products.

1. Introduction

Dental enamel is the outermost layer of the tooth crown and the hardest tissue in the body. The tooth enamel acts as a protective strip against mechanical, chemical, and microbial attacks, which are the processes generated by the following: physical forces, chemical erosion and microbial attack, respectively [1]. Hydroxyapatite (HA) constitutes the primary component of enamel, representing 96% of its composition. This makes enamel the most mineralized surface in the human body [2,3,4,5,6,7,8]. The mineral component of enamel plays an essential role in protecting and preserving the innermost tooth structures, such as dentin and pulp, from mechanical stress induced by mastication, chemical erosion, and the presence of bacteria in the oral cavity [2,9,10]. Even while enamel is resilient, it is not regenerative; thus, without treatment, any structural damage or mineral loss brought on by demineralization is irreparable [11,12].

Demineralization is a slow process that weakens tooth structure and causes erosion by dissolving calcium and phosphate ions from the HA crystals in the enamel with acids from food or bacterial metabolism [13].

Under the acidic circumstances caused by bacterial metabolism or food, H+ ions attach to the carbonate and phosphate groups in the hydroxyapatite, releasing the previously bonded anions and dissolving the mineral structure. Because hydroxyapatite contains many carbonates, which react with a small amount of H+ ions, it is especially vulnerable to acid attack. The integrity of the enamel is weakened over time by this acid-driven process, which produces an acidic chemical reaction [11,14,15].

Although demineralization is often challenging to diagnose in its early stages, it becomes visible as a change in enamel translucency and color as the structural changes affect optical properties. Demineralized dental tissues are weak regions that open the door to possible cavity formation and are more prone to fracture [15,16,17,18,19].

In 2010, caries was the most common disease in adults and the 10th most frequent in children: around 2.4 billion people had untreated caries in permanent teeth, while 621 million children had the same problem in their primary teeth [20,21,22]. Miller’s chemical–parasitic theory postulates that oral bacteria convert sugars derived from carbohydrate consumption into acids. This pH lowering demineralizes teeth and results in caries [23].

There are factors affecting the two crucial processes of demineralization and remineralization. The main factors contributing to enamel demineralization are cariogenic bacteria, fermentable carbohydrates, and alterations in salivary composition, which facilitate the dissolution of the apatite crystal lattice and calcium and phosphate ions. However, minerals such as fluoride, an adequate level of saliva, antimicrobials, and specific local ions in the oral environment promote enamel remineralization. Although both of these interconnected processes create a dynamic balance within the oral cavity [19,24,25,26], the natural remineralization process or redeposition of minerals within the enamel is essential to the maintenance of dental health by preventing caries.

Fluoride is a substance particularly known for its remineralizing properties. The presence of fluoride promotes the deposition of calcium (Ca²⁺) and phosphate (PO₄³⁻) ions on the enamel surface and creates a compound, fluorapatite, that is more resistant to acidic conditions than hydroxyapatite [24,27,28]. However, one must always consider the amount of fluoride present, because too much can lead to fluorosis, a pathological condition that causes enamel hypomineralization and aesthetic problems. This is highly visible in children. Thus, the challenge has arisen to find more cost-effective remineralizing agents that act independently or synergistically with fluoride in promoting this repair process.

In recent years, there has been a growing interest in understanding how different dental care products contribute to this balance, particularly in terms of promoting remineralization [12,24,29]. Dentistry now emphasizes non-invasive treatment options for early caries lesions, mainly through remineralizing products [19]. Previous research has primarily focused on the chemical and physical properties of dental care products and their impact on enamel hardness and caries prevention [18,30,31,32,33,34,35].

The use of casein calcium amorphous phosphopeptide (CPP–ACP), which is derived from milk protein, has been shown to be an effective means of remineralization, as it provides stable sources of bioavailable calcium and phosphates, thus allowing minerals to deposit more quickly on the enamel surface. Studies have revealed that CPP–ACP can decrease lesion depth, enhance enamel microhardness, and boost demineralization resistance. As a result, it is considered moderately beneficial for patients who are at a high risk of caries or have conditions that hinder natural remineralization [29,31,36].

Another important innovation is nano-hydroxyapatite (nHA), a synthetic form of hydroxyapatite that more closely resembles natural enamel in microstructure. nHA can penetrate micro- or nano-scale defects on the enamel surface, providing deeper levels of remineralization. Recently, research has shown the ability of nHA-based formulations to restore enamel hardness and resistance to acidic erosion [25,37].

Bioactive glass (BAG) has also gained attention due to its ability to release calcium and phosphate ions upon contact with saliva, forming an HA-like layer on enamel. This feature is particularly beneficial for individuals at high caries risk, such as orthodontic patients, as BAG provides sustained ion release even under acidic conditions, thereby preventing white spot lesions around brackets [37,38].

Technological advancements in fluoride delivery, such as fluoride iontophoresis (FI), have expanded treatment options for high-risk individuals. FI involves a mild electric current to enhance fluoride penetration into the enamel, allowing deeper ion integration and improved acid resistance. Studies have shown that FI increases fluoride retention and strengthens acid resistance, which is especially useful in patients requiring intensified fluoride therapy without the side effects of frequent high-dose applications [16].

Another biomimetic approach is to use phosphorylated chitosan–calcium phosphate complexes (Pchi–ACP). These complexes replicate the natural formation of enamel by organizing calcium and phosphate ions in a structured pattern, enhancing enamel hardness, and supporting subsurface lesion repair. Given their high biocompatibility and effectiveness, Pchi–ACP complexes are suitable for patients with sensitivities to other agents, and their efficacy has been confirmed in vitro through techniques such as scanning electron microscopy (SEM) [37,39].

Advanced imaging techniques such as SEM and atomic force microscopy (AFM) are invaluable for assessing remineralization efficacy. These high-resolution methods provide insights into the enamel’s microscale structure, validating the effects of agents such as nHA and CPP–ACP on enamel microhardness and surface integrity [40,41,42,43,44]. These techniques allow quantitative assessment of remineralization [24].

The study evaluates the remineralizing ability of Mentadent Professional Resilience, a toothpaste based on nano-hydroxyapatite, as a non-invasive solution for enamel demineralization. These technologies enable precise measurements of remineralization, offering additional support for evidence-based choices in preventive and restorative dentistry. Using SEM for high-resolution analysis, this research aims to quantify mineral deposition and morphological changes in demineralized enamel treated with nHA. By positioning nHA as a viable alternative to fluoride, this study aligns with the broader movement toward minimally invasive dentistry, which prioritizes preserving natural tooth structure and enhancing enamel resilience through biomimetic approaches [45,46,47].

2. Materials and Methods

This study utilized two human teeth extracted from a patient for orthodontic reasons, free from any visible signs of caries or structural damage.

The samples were thoroughly cleaned and stored in saline until further processing. Each tooth was sectioned near the cementoenamel junction (CEJ) using a precision diamond saw (Buehler IsoMet—Lake Bluff, IL, USA), ensuring that the enamel portion remained intact. After the cut, the dental surfaces were decontaminated using 0.2% chlorhexidine.

2.1. Sample Preparation

The samples were dehydrated before the scanning electron microscope (SEM) analysis. This was achieved by placing the samples in a digitally controlled stove set at approximately 35 °C for 24 h. To ensure that detections were consistently made in the same areas, the LEGO^® technique for SEM analysis was employed. The sample holder was constructed using LEGO pieces attached with epoxy adhesive. The process involved attaching a first piece of LEGO to a stub, on top of which a second piece was placed orthogonally. An additional stub, devoid of the requisite mounting support, was affixed to the structure via an epoxy adhesive. Finally, a small hole was drilled in the bottom stub to accommodate a tinned copper wire, establishing an electrical connection between the top and bottom stubs. The specimen was then positioned and attached to the upper stub using a conductive silver adhesive. In addition to maintaining precise positioning along the z-axis, minimizing bias, and producing repeatable and dependable results, the system was built to recognize and align features throughout several repositioning cycles [48].

Subsequently, the samples were affixed to aluminum SEM stubs using a conductive silver adhesive (Electron Microscopy Science—Silver conducted paint 503—Hatfield, PA, USA) to guarantee electrical conductivity throughout the SEM imaging process [49]. A 14 nm thick layer was applied to the samples using an Emitech K550 Sputter Coater (Emitech—Montigny-le Bretonneux—France). Then, the experimental and the control samples were analyzed using the FEI XL-30 FEG (now Thermo Fisher, Waltham, MA, USA) scanning electron microscope (SEM), operating at an accelerating voltage of 15 kV to evaluate the normal enamel surface.

The samples were then demineralized in vitro by applying 37% orthophosphoric acid for 30 s on the entire and dried surface.

2.2. Application of the Product

The study focused on Mentadent Professional Resilience, which was applied following the manufacturer′s instructions. The gel contains aqua, silica, CI 17200, glycerin, propylene glycol, xylitol, CI 42090, calcium carbonate, hydroxyapatite, perlite, phenoxyethanol, sodium dehydroacetate, mentha piperita oil and sodium fluoride. The products that have the greatest effect on remineralization are hydroxyapatite and calcium carbonate.

The product was tested on both intact and demineralized teeth. A soft-bristled toothbrush (Mentadent Expert Toothbrush Sensitive, Rotterdam, Holland) was used to apply the product, simulating the brushing process. According to the instructions, the product was left on the enamel surface for two minutes. Samples were rinsed gently in distilled water to remove any residual agent.

2.3. SEM Analyses

A secondary electron imaging mode was employed to obtain high-resolution images of the enamel surface. Images were captured at various magnifications (ranging from 500× to 10,000×) to observe the overall enamel surface and detailed microstructural changes. The teeth were observed before any treatment, after the demineralization process had been induced, and after the remineralization procedure with Mentadent Professional Resilience had been completed.

A qualitative analysis of the SEM images was performed to examine the overall morphology, surface texture, and any visible cracks or lesions. The null hypothesis stated that there would be no significant difference in enamel remineralization after applying the test remineralizer compared to the control.

The null hypothesis is that applying the tested remineralizing agent does not cause any significant difference in enamel remineralization compared to the control, as observed through scanning electron microscopy (SEM) analysis

3. Results

The SEM analysis produced high-resolution images that showed clear differences in the enamel surface morphology between the non-demineralized and demineralized tooth treated with this product. All samples showed characteristic enamel prisms, but variations were observed in their surface textures post-treatment. The demineralized sample showed significant damage, with enamel modifications and prism degradation. The enamel surfaces treated with Mentadent Professional Resilience exhibited a notable reduction in surface irregularities compared to the control samples. The SEM images at higher magnifications (up to 10,000×) revealed a smoother surface with fewer pits and fissures (Figure 1).

Samples treated with demineralization and subsequent application of Mentadent Professional Resilience showed a specific pattern. The enamel surfaces appeared more intact, with a slight increase in surface irregularity compared to the other samples. These surface irregularities appeared due to the deposition of a layer that SEM analysis suggested was remineralization. The control samples, which did not receive treatment, maintained their original surface morphology, characterized by natural enamel wear and irregularities (Figure 2). These samples served as a baseline for comparing the effects of the dental care products.

4. Discussion

The findings of this study offer significant insights into the microstructural effects of Mentadent Professional Resilience on dental enamel, as observed through SEM analysis. The results indicate that the product affects enamel morphology. This could be attributed to the product’s formulation, which may facilitate the deposition of minerals onto the enamel surface, thus filling micro-pits and fissures [50]. The treated samples exhibited a pronounced remineralization effect. The increased surface irregularities and reduced visibility of microfractures and pits suggest more substantial mineral deposition. This could indicate an effective remineralization process, potentially protecting against enamel erosion and caries development. In particular, a key role could be played by the presence of hydroxyapatite in the product [34].

The remineralizing efficacy of nano-hydroxyapatite (nHA), but more generally of hydroxyapatite (HA) in its nano form (nHA), is highlighted by this study. This is mainly attributable to the fact that the structure of HA is very similar to the mineral structure of enamel, which allows it to penetrate micro defects. Filling them in and creating a stronger surface provides restorative and protective benefits. This analysis agrees with the findings of Lacruz et al., who showed that the HA matrix of enamel can be effectively fortified using HA-based treatments, thus improving microhardness and reducing susceptibility to future demineralization [2].

Fluoride is widely used in remineralization treatments, especially for remineralizing carious enamel lesions [51]. This is explained by fluoride’s ability to generate fluorapatite, which is more resistant to acid attack due to its exchange with the hydroxyl group in hydroxyapatite. It is, therefore, logical that the more fluoride there is, the more numerous and larger the resulting crystals will be [16,24,39,52,53,54,55,56,57,58]. This chemical mechanism is one of the pillars of dental caries prevention, especially in the early stages of demineralization. Traditional fluoride treatments work by binding fluoride ions with enamel hydroxyapatite to form a fluorapatite surface layer that resists acid dissolution. In this scenario, tooth polishing is key and should be carefully selected based on patients’ needs. Tooth polishing allows you to remove plaque, biofilm, stains, and acquired pellicle, but excessive use of this procedure could lead to the wear of the superficial tooth structure, the loss of the fluoride-rich layer, and more accumulation of residues on the dental surface [59,60]. Excessive polishing can compromise the protective function of the enamel, making it more susceptible to future erosion. Some studies, including those by Bizhang et al., show that fluoride’s remineralizing effect is primarily superficial, with limited ability to penetrate deeper subsurface lesions [61]. By contrast, nHA’s biomimetic properties allow it to integrate more thoroughly into the enamel, addressing subsurface demineralization. This is especially beneficial for repairing initial carious lesions that fluoride alone cannot access. Our results align with these observations, as HA treatments significantly increased enamel microhardness, suggesting surface-level and potential subsurface repair. This comprehensive action of HA reduces the need for fluoride, particularly in populations at risk for fluorosis, as suggested by Featherstone and Doméjean [12]. Pepla et al. [37]. analyze how nano-hydroxyapatite crystals are fundamental to remineralizers on initial enamel lesions, which are superior to traditional fluorides used to date for this purpose. According to the existing literature, hydroxyapatite prevents caries by forming a protective barrier on enamel and reducing bacterial plaque adhesion. It also acts as a desensitizer by occluding exposed dentinal tubules and as a bleaching agent, proving a key component in preventive and dental care products. The study of Gore et al. [62]. evaluated the remineralizing efficacy of four different types of products on artificially demineralized elements by evaluating surface microhardness. From their evaluations they conclude that the nano-hydroxyapatite compound creates a resistant surface by remineralizing residual enamel.

These observations are consistent with previous studies that have emphasized the importance of fluoride and other remineralizing agents in dental care [16,36,63].

Casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) is widely used for stabilizing calcium and phosphate ions on enamel surfaces, creating a supersaturated environment that promotes mineral deposition. However, research by Thimmaiah et al. and De Menezes Oliveira et al. indicates that, while CPP–ACP effectively remineralizes outer enamel, its penetration into deeper lesions is limited compared to nHA [41,64]. This study found HA treatments to yield results comparable to CPP–ACP in terms of improving surface hardness but with the added benefits of deeper remineralization, making HA a practical option for managing both early-stage and advanced demineralization with minimal invasiveness. The analyses by Vitiello et al. [43] are consistent with our results. Also, SEM studies indicate that, following treatment, the enamel structure was nearly fully remineralized, resembling smooth, sound enamel in both the CPP–ACP and synthetic nHA groups. Another study [42] also utilized SEM analysis to evaluate the remineralizing efficacy of CPP–ACP by assessing morphological changes in artificially demineralized dental enamel. The findings evaluated how the CCP–ACP paste regenerated a homogeneous and compact surface layer, which agreed with our SEM analysis despite using another product.

Erosive wear, often exacerbated by acidic diets and lifestyle factors, poses a significant risk to enamel integrity, especially in younger individuals or patients with gastroesophageal reflux disease (GERD). Wiegand et al. observed that acidic environments accelerate enamel loss, particularly when combined with mechanical wear [65]. HA-based treatments offer a unique solution by forming a resistant layer on the enamel surface that mitigates acid attacks and mechanical wear. In this study, HA-treated enamel displayed smoother surfaces, indicating enhanced resistance to erosive wear. This aligns with findings by Green, who demonstrated that nHA forms a barrier against acid erosion, thus preserving enamel integrity over time [18].

Aging impacts the enamel’s mineral composition, reducing its resilience and increasing susceptibility to demineralization. Carvalho et al. and Atsu et al. observed that aging enamel exhibits reduced thickness and mineral density, which can accelerate demineralization due to lower fluoride content [13,66]. By preventing caries and erosion-related complications, HA’s ability to replenish minerals offers a preventive strategy suitable for all age groups, aligning with the Caries Management by Risk Assessment (CAMBRA) philosophy, which advocates for minimally invasive, risk-based caries management [26,67].

Saliva plays a crucial role in natural remineralization by supplying essential ions that buffer pH and promote mineral deposition on enamel. Abou Neel et al. reported that saliva’s buffering capacity is essential for maintaining a neutral pH conducive to remineralization [11]. However, natural salivary remineralization may be insufficient to reverse advanced lesions, as its effect is mostly limited to surface enamel. HA-based treatments can enhance the function of saliva by supplying extra calcium and phosphate ions that penetrate the enamel, helping to restore its mineral density. This study indicates that HA works harmoniously with saliva, especially benefiting patients who experience reduced salivary flow, such as those with xerostomia.

Minimally invasive dentistry focuses on preserving tooth structure through preventive and therapeutic methods that avoid invasive procedures. HA’s ability to remineralize enamel without changing its natural structure makes it perfect for non-invasive caries management.

Philip highlighted that biomimetic materials such as nHA can prevent caries progression and strengthen enamel without extensive restorations [24]. The findings of this study support HA’s role in minimally invasive dentistry by providing natural enamel repair, particularly for high-risk populations such as children, elderly individuals, and patients with compromised enamel. Wiegand et al. observed that enamel wear is significantly exacerbated under erosive conditions, particularly with antagonistic ceramic materials, which exert high mechanical forces on softened enamel [65]. In this study, HA-treated enamel demonstrated reduced wear and improved microhardness under simulated acidic conditions, suggesting that HA can mitigate enamel wear while preserving structure. This aligns with the protective effects observed by Green, who noted that HA-based applications provide resistance to acidic conditions and mechanical wear [18]. Comparisons with glass ionomer cement also indicate that HA, unlike fluoride-releasing materials, does not degrade over time, making it an effective long-term solution. Incorporating HA-based products into routine oral hygiene can shift preventive dentistry’s focus from solely controlling acid production to actively restoring and preserving enamel integrity.

The preventive benefits of HA are increasingly relevant given the global rise in caries prevalence and dietary habits contributing to erosion. Regular HA-based treatments offer a proactive approach to maintaining enamel health, reducing the need for restorations, and extending the functional life of natural teeth. This study’s findings provide valuable insights into the microstructural effects of Mentadent Professional Resilience on enamel as observed through SEM analysis [16,25,29,31,37,46,57,68].

This study’s findings have practical implications for selecting dental care products. Mentadent Professional Resilience demonstrated a high degree of remineralization, as suggested by the SEM analysis, which might effectively prevent dental caries and maintain enamel integrity. This is particularly relevant for individuals at higher risk of dental erosion and caries [69,70].

These findings can guide consumers and dental professionals in making informed choices about oral care products, ultimately contributing to better dental health outcomes. A conscious choice of these products could be decisive for reducing the risk of long-term dental diseases, improving patients’ quality of life, and reducing the costs associated with dental care. Furthermore, this research adds to the growing body of knowledge regarding the microstructural aspects of enamel health, offering new perspectives for dental researchers and clinicians.

While this study provides valuable insights, it is not without limitations. The in vitro nature of the study means that the results may not fully replicate the complex conditions of the oral environment. Future research could involve in vivo studies to confirm these findings and explore the long-term effects of these products on dental health. Additionally, investigating the specific chemical mechanisms behind the observed remineralization effects could further enhance our understanding of how different formulations impact enamel health.

5. Conclusions

In conclusion, while the immediate effects of hydroxyapatite in Mentadent Professional Resilience are promising, the long-term benefits and mechanisms require further investigation. In addition to demonstrating HA’s capacity to replenish mineral density in regions susceptible to erosion, this study underscores the necessity for in vivo clinical investigations to ascertain the optimal utilization of hydroxyapatite and its potential influence on dental well-being on a macro scale.