CDEWorld > Courses > Reconsidering Remineralization Strategies to Include Nanoparticle Hydroxyapatite

Reconsidering Remineralization Strategies to Include Nanoparticle Hydroxyapatite

V. Kim Kutsch, DMD; John C. Kois, DMD, MSD; Yada Chaiyabutr, DDS, DSc, MSD; and Graeme Milicich, BDS

March 2013 Issue - Expires March 31st, 2016

Compendium of Continuing Education in Dentistry

Abstract

Dental caries is a transmissible biofilm-mediated disease of the teeth that is defined by prolonged periods of low pH resulting in net mineral loss from the teeth. Hydroxyapatite, fluorapatite, and the carbonated forms of calcium phosphate form the main mineral content of dental hard tissues: enamel, dentin, and cementum. Active dental caries results when the biofilm pH on the tooth surface drops below the dissolution threshold for hydroxyapatite and fluorapatite. The clinical evidence of this net mineral loss is porosity, whitespot lesions, caries lesions, and/or cavitation. The potential to reverse this mineral loss through remineralization has been well documented, although previous remineralization strategies for dental hard tissues have focused on the use of fluorides and forms of calcium phosphate. This in-vitro study documented the deposition of nanoparticle hydroxyapatite on demineralized enamel surfaces after treatment with an experimental remineralization gel. This finding supports consideration of an additional approach to remineralization that includes pH neutralization strategies and nanoparticle hydroxyapatite crystals.

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Dental caries is a transmissible biofilm-mediated disease of the teeth that is defined by prolonged periods of pH below the dissolution pH of hydroxyapatite and fluorapatite, resulting in net mineral loss from the teeth. Currently, there is a global increase in dental caries despite the dental profession’s attempts to prevent and treat the disease.1 Prevention of dental caries has traditionally focused on the use of fluoride, and treatment has been based on the surgical model of restoring caries lesions. Marsh was the first to recognize that the prolonged periods of low pH in the biofilm, not sugar availability, was responsible for the selection pressure favoring acidogenic/aciduric organisms in the biofilm.2 The ecological shift in the biofilm to more acidogenic and aciduric bacteria favors a lower pH in the biofilm, which leads to demineralization.

In a healthy individual, there is an ongoing balance between episodes of demineralization and remineralization, with no resulting mineral loss from the teeth. After an acid challenge, buffering agents and bacteria in the saliva and biofilm return the pH above the solubility pH (5.5 for hydroxyapatite), and remineralization occurs. This normal pH cycling in the mouth is a dynamic stability, where mineral is lost during periods of low pH and returns to the teeth during periods of higher pH. Conversely, dental caries results from a loss of this balance, with prolonged periods of low pH favoring demineralization.

Takahashi and Nyvad noted that the behavior of the biofilm was as critical as its bacterial composition. Commensal early colonizers, which were previously thought to be healthy and desirable organisms, are now known to adapt in an acidic environment and are capable of producing acid as readily as Mutans streptococci, and could be described as facultative aciduric bacteria.3-5

Dental caries today is best assessed, diagnosed, and managed by identifying disease indicators and risk factors and examining the balance between pathogenic and protective risk factors.6-10 The current disease model and approach—caries management by risk assessment (CAMBRA)—may appear complex, but it is actually a simple model: active disease results when low pH leads to net mineral loss. In this sense, dental caries is a simple “pH-specific” disease. It is this disease model that provides the backdrop for understanding the demineralization process and the attempts to reverse mineral loss by remineralization.

Enamel Formation and Stability

Enamel forms when single hydroxyapatite crystals assemble within the amelogenin protein matrix and form hydroxyapatite nanocrystals. The body maintains the tooth mineral content by replacing the lost hydroxyapatite and fluorapatite as soon as the pH rises above the critical solubility pH. The critical solubility pH is 5.5 for hydroxyapatite and 4.5 for fluorapatite. In the presence of fluoride, there is a window between pH 4.5 and 5.5 where the hydroxyapatite is dissolving and fluorapatite is forming. It is this pH window that explains enamel maturation, where weaker elements of the enamel are lost and replaced by stronger, more acid-resistant constituents.

Intraorally, teeth are maintained because they are bathed in saliva that is supersaturated with both hydroxyapatite and fluorapatite. However, it is pH that determines whether remineralization occurs. Additionally, as the saliva flow increases, the degree of salivary apatite supersaturation increases as well, which is important in maintaining enamel mineral content, thus protecting the integrity of the teeth. Hydroxyapatite and fluorapatite are the most bioavailable forms of mineral, and they probably play the most significant role in remineralization. This is a prerequisite condition for teeth to exist in the mouth. If the saliva were unsaturated with respect to apatite, the teeth would simply dissolve. Furthermore, the available salivary apatite may lead to deposition and repair of enamel damaged by caries or erosion. As a nanoparticle crystallite, hydroxyapatite is the most thermodynamically stable form of calcium phosphate11; it resists dissolution and also plays a role in remineralization. Remineralization of enamel lesions requires partially demineralized apatite crystals that can grow as a result of exposure to the supersaturated levels of apatite in saliva. It appears that the subsurface remineralization of these apatite crystals occurs by the addition of nanohydroxyapatite crystals, not amorphous calcium phosphate.12 Development of entirely new enamel crystals is rare; rather, nanocrystals of apatite attach to existing enamel crystals. The building blocks of repair are formed when nanosized hydroxyapatite crystals develop into intermediate structural nanorods.

Demineralization

Exposed tooth surfaces are covered by a protein-based pellicle, and the bacterial biofilm attaches to this pellicle. As the bacteria in the biofilm metabolize any substrate introduced into the mouth, the pH drops within the biofilm, and at the critical pH of solubility, the enamel begins to dissolve. Mineral is lost in the form of hydroxyapatite and fluorapatite. After rapid surface demineralization, the dissolution rate decreases, resulting in hollow enamel cores and nanoparticle remaining as crystals, which are resistant to further dissolution.

Recent evidence points to nanoparticle hydroxyapatite being the basic structural unit of enamel lost during occlusal wear, and it may play an important role in remineralization.13,14 The microscopic appearance on scanning electron microscope (SEM) images of unetched enamel compared to acid-etched enamel revealed that the mineral loss and gain in enamel could be in the form of nanoparticle crystallites (Figure 1 and Figure 2). The acid-etched enamel displays hydroxyapatite nanocrystals that are becoming separated from the structure.

As the acids penetrate deeper into the enamel, more minerals are lost from below the surface, the enamel becomes more porous, and there is a change in the enamel’s appearance because light diffracts differently between sound and porous enamel. At a microscopic level, the loss of interprismatic enamel exposes the enamel rods, and the nanoparticle infrastructure of the enamel becomes apparent (Figure 1).

Remineralization

Remineralization is a natural component of the dynamic stability of teeth, and it is also the foundation for prevention and nonsurgical therapeutic strategies for dental caries. Remineralization is a complex process that is influenced by the anatomy and physiology of the existing tooth structure, quantity and quality of saliva, the content and behavior of the bacteria biofilm, the presence or bioavailability of fluoride, and fluctuations of biofilm pH. Most scientific evidence on therapeutic remineralization involves the use of fluoride, and numerous studies demonstrate the value of fluoride in the remineralization process.15-18 While it has been proposed that fluoride concentration affects the remineralization dynamics within a lesion, it is apparent that lesions remineralize from the surface first, regardless of the fluoride concentration.19,20 Additional evidence indicates that xylitol also influences remineralization by transporting calcium ions deeper into the lesion.21 Xylitol has been recognized as an effective anticaries agent22 and also potentiates the effects of even small amounts of fluoride.23

Fluoride alone as a remineralization strategy has not been shown to be sufficient to prevent dental caries at the population level. More recently, various forms of calcium phosphate have been included with fluoride in the therapeutic strategies. Tricalcium phosphate, amorphous calcium phosphate, and casein phospho-peptide-coated amorphous calcium phosphate (CPP/ACP) have all been added to different oral care products.24-26 The scientific reports evaluating calcium phosphate remineralization strategies are mixed; some studies conclude there is a lack of evidence or no benefit, while others demonstrate improved results.27-32

Nanoparticle hydroxyapatite as a biomimetic remineralizing agent has been recently studied.33-35 In comparing remineralization effects, fluoride ions generate a surface modification of the enamel apatite crystals, increasing their degree of crystallinity and generally increasing the mechanical and acid resistance of enamel, while the addition of nanoparticle hydroxyapatite results in deposition of a new apatitic layer of mineral on the enamel surface.33 Nanoparticle hydroxyapatite has been shown to restore luster to enamel damaged by bleaching agents.34 Particles deposited on the microscopic structure of the demineralized enamel surface appear to form new surface layers and, therefore, might have the potential to remineralize initial enamel lesions.35 In addition, nanoparticles in the 20-nm size have been described both as biomimetic for the building blocks of natural enamel and effective as an enamel repair material and anticaries agent.36 As biomimetic building blocks, these nanoparticle crystallites might promote remineralization and physiological biofilm management at the tooth surface.13 Recently, the application of nanoscaled hydroxyapatite particles has been shown to inhibit oral biofilm formation. Biomimetic approaches based on these crystals should allow adsorbed particles to interact with bacterial adhesins, reduce bacterial adherence, and decrease biofilm formation.37 When nanoparticle hydroxyapatite was added to a dentifrice, mineral loss and lesion depth—as measured by microradiography—compared favorably to fluoride and amine fluoride treatment.38,39 Moreover, the addition of the nanoparticle hydroxyapatite did not increase the abrasiveness of a dentifrice to tooth structure.40

In-Vitro Study of Hydroxyapatite Nanoparticles as Remineralization Agents

This study evaluated the artificial demineralization lesion on enamel surface using energy-dispersive x-ray spectroscopy (EDS) and scanning electron microscopy (SEM) after the application of hydroxyapatite nanocrystals.

Materials and Methods

Extracted human third molars were selected based on criteria establishing that they were intact: there was no evidence of caries, no restorations, and there were no cracks or fractures in the crown. From the time of extraction, these teeth were kept hydrated in distilled water at room temperature. The coronal parts of each tooth were sectioned, and enamel surfaces were cleaned and polished using water and fluoride-free pumice with a prophylaxis brush at a slow speed. Each tooth was cut inciso-cervically into three parts using double-sided diamond disks (n = 8 in each group). One tooth section received no treatment (control), while the other two parts received an artificial demineralization lesion. An 18.2% hydrogen peroxide solution was used to create artificial demineralization lesions. The solution was applied with a microbrush for 1 minute, and then rinsed with water for 30 seconds. One of the artificial demineralization lesions received the experimental remineralization gel, which was a solution of 0.1% nanoparticle hydroxyapatite and 1.1% sodium fluoride. The gel was applied with a microbrush on the enamel surface. All the experimental specimens were stored in artificial saliva for 24 hours prior to the surface analysis.

Specimens were analyzed using a SEM (FEI Sirion SEI, EDAX Inc., www.edax.com), and the morphology of the enamel surface was observed after 24 hours. The specimens were attached to SEM stubs with cyanoacrylate cement and sputter coated with gold-palladium (Technics Hummer® Sputter Coater, Anatech, www.anatechusa.com) for 60 seconds. To determine the element present on the enamel surface with or without the surface treatment, an EDS (FEI Sirion SEM, NPGS v.9 [EDAX Falcon]) was performed on a separate set of teeth. In each measurement area, the intensity profile of the major elements present was analyzed. The accelerating voltage was set at 7 KV with a working distance of 15 mm. The x-ray detector was set at 5 cm throughout the experiment under secondary electron (SE) mode at x500 magnification. Concentrations were determined after calculating the average percentage of the weight of a particular element at each spot.

Results

From the 2-dimensional (2-D) SEM images, microporosities on the enamel surface with some exposed enamel prisms were found in the group of artificial demineralization, while the untreated enamel surface exhibited a smooth surface structure (Figure 3). Some remaining nanoparticle hydroxyapatite crystals were found on the enamel surfaces of the group that received remineralization gel (Figure 4). The average composition of calcium (Ca) was 31.71% (3.43% standard deviation [SD]) by weight for the group with untreated enamel surfaces, which was similar to measured quantities of Ca for the group of artificial demineralization enamel (34.84% [3.38% SD]) and the group that received the remineralization gel (37.02% [6.78% SD]). The average composition of phosphorous (P) of the untreated enamel group was 20.22% (2.55% SD) by weight, while phosphorous composition for the group with artificial demineralization was 21.54% (1.44% SD) and the group that received remineralization gel was 21.48% (2.18% SD). Within the limitation of the present study, there was no difference in percent by weight of calcium (P = 0.109) and phosphorus (P = 0.384) among the untreated enamel surface, an artificial demineralization enamel, and enamel surface that received remineralization agents. Nanoparticle hydroxyapatite crystals were detected on enamel surface of teeth in the treatment group after teeth were stored in artificial saliva for 24 hours.

Conclusions

Remineralization is a natural component of the dynamic stability of enamel mineral through the pH cycling that occurs in the mouth. It is now known to be a very complex process involving the anatomy and physiology of the existing tooth structure, quantity and quality of saliva, the content and behavior of the bacteria biofilm, the presence or bioavailability of fluoride, and biofilm pH fluctuations. Remineralization has also become an integral component of clinical therapeutic strategies to repair demineralized tooth structure to prevent the need for restorative dentistry.

Historically, fluoride has been the primary agent used in remineralization therapies, although in recent years, there has been growing interest in the role of calcium phosphate mineral in this repair process. Scientific evidence from a broad range of sources points toward enamel behaving as a nanosized crystalline system, and current research has demonstrated significant remineralization results with the use of biomimetic nanoparticle hydroxyapatite crystals. This in-vitro study demonstrates the presence of nanohydroxyapatite crystals on the enamel surface of teeth treated with a remineralization agent containing nanohydroxyapatite crystals. The results of these studies suggest that therapeutic strategies in remineralization should be expanded to include nanoparticle hydroxyapatite.

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Related Content:

A CE article, Proactive Intervention Dentistry: A Model for Oral Care through Life, is available from CDEWorld at dentalaegis.com/go/cced337

About the Authors

V. Kim Kutsch, DMD
Private Practice
Albany, Oregon

Chief Executive Officer
Oral Biotech
Albany, Oregon

John C. Kois, DMD, MSD
Affiliate Professor
Department of Restorative Dentistry
School of Dentistry
University of Washington
Seattle, Washington

Private Practice
Seattle, Washington

Yada Chaiyabutr, DDS, DSc, MSD
Affiliate Instructor
Department of Restorative Dentistry
School of Dentistry
University of Washington
Seattle, Washington

Kois Center
Seattle, Washington

Graeme Milicich, BDS
Private Practice
Hamilton, New Zealand

SEM micrograph of normal enamel demonstrates the nanoparticle apatitic infrastructure (x60000 magnification).

Figure 1

Figure 2  SEM micrograph of acid-etched enamel. The nanoparticle apatitic infrastructure is more apparent (x76500 magnification).

Figure 2

Fig 3. SEM micrograph of normal enamel surface (untreated), exhibiting a smooth surface structure (x40000 magnification).

Figure 3

Figure 4  SEM micrograph of enamel after application of nanoparticle hydroxyapatite gel. Note that some nanoparticle hydroxyapatites remain on the enamel surface (x40000 magnification).

Figure 4

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SOURCE: Compendium of Continuing Education in Dentistry | March 2013

Learning Objectives:

  • discuss dental caries as a transmissible biofilm-mediated disease of the teeth
  • describe principles of enamel formation and stability, as well as demineralization and remineralization
  • discuss the proposed approach to remineralization that includes pH neutralization strategies and nanoparticle hydroxyapatite crystals