Abstract
Objectives
The management of demineralized dentin resulting from dental caries or acid erosion remains an oral healthcare clinical challenge. This paper investigates, through a range of studies, the ability of colloidal silica and hydroxyapatite (HA) nanoparticles to infiltrate the collagen structure of demineralized dentin.
Methods
Dentin samples were completely demineralized in 4 N formic acid. The remaining collagen matrix of the dentin samples was subsequently infiltrated with a range of nano-particulate colloidal silica and HA solutions. The effectiveness and extent of the infiltration was evaluated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS).
Results
Silica nanoparticles have the ability to penetrate dentin and remain embedded within the collagen matrix. It is suggested that particle size plays a major role in the degree of dentin infiltration, with smaller diameter particles demonstrating a greater infiltrative capacity. The infiltration of demineralized dentin with sol–gel HA nanoparticles was limited but was significantly increased when combined with the deflocculating agent sodium hexametaphosphate. The use of acetone as a transport vehicle is reported to enhance the infiltration capacity of sol–gel HA nanoparticles.
Significance
Collagen infiltrated with HA and silica nanoparticles may provide a suitable scaffold for the remineralization of dentin, whereby the infiltrated particles act as seeds within the collage matrix and given the appropriate remineralizing environment, mineral growth may occur.
1
Introduction
Dentin demineralization results from exposure to acids, from bacterial origin (caries) or dietary/gastric sources (dental erosion). Among the diseases and chronic conditions affecting populations throughout the world, dental caries is among the foremost in prevalence; the consequences of which involve not only physical health but also the economic, social, and psychological well-being of individuals. Dental erosion is a more modern affliction, causing gradual but irreversible loss of tooth structure. It affects primarily the adolescent population worldwide with a reported international incidence of up to 20% of the population and greater national incidences in specific regions .
The management of dental caries involves a range of different strategies, all of which have their inherent limitations: (a) prevention by controlling dietary habits, oral hygiene, plaque control and the use of topical fluoride; dependent on total patient compliance, (b) chemical or mechanical caries removal techniques; that are destructive of tooth tissue, and (c) techniques that enhance remineralization of the mineral content of the tooth; aimed at preserving the demineralized collagen matrix, but at an early stage of development and use.
This paper concerns itself with the latter of these three strategies; the remineralization of dentin. Remineralization of dentin affected by caries or dietary/gastric acid is the process of restoring minerals to the tooth structure. A recent study of caries pathology using synchrotron-based X-ray scattering, confirms that while bacterial acids dissolve the inorganic structure of dentin; the collagen network remains unaffected, enabling the development of future caries treatments that re-mineralize the dentin . Different dentin remineralization strategies have been investigated, most of which focus on the use of bioactive glass , fluoride-releasing materials , casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) complexes , artificial saliva solutions , calcium hydroxide and Portland cement . An alternative strategy, that is becoming the focus of much research in this field, is the use of nanoparticles for the management of dental caries. Nano-sized calcium fluoride (n-CaF 2 ) , nano-particulate hydroxyapatite , nano-sized carbonated apatite (n-CAP) , carbonate-hydroxyapatite nano-crystals (CHA) and nano-particulate bioactive glass are among those nano-materials that seem to increase the mineral content of enamel and/or dentin. A significant challenge with the use of these materials is to achieve an effective and deep infiltration of the intact demineralized dentin collagen with the particles, while avoiding precipitation on the surface. This paper investigates the novel use of nanoparticles (synthetic calcium hydroxyapatite and colloidal silica) to infiltrate the collagen structure of demineralized dentin as part of a dentin remineralization strategy.
Synthetic calcium hydroxyapatite (HA) [Ca 10 (PO 4 ) 6 (OH) 2 ] is chemically and biologically similar to the mineral component of human bone and teeth. It is one of few materials that are classed as bioactive , meaning that it will support bone in-growth and osseo-integration without breaking down or dissolving, when used in orthopedic, dental and maxillofacial applications. Dentin is comprised of 70% inorganic materials, 20% organic materials, and 10% water by weight. The inorganic phase of dentin includes trace amounts of calcium carbonate, zinc, fluoride and magnesium but HA is principal inorganic component . Thus, synthetic HA is considered a logical mineral compound to substitute the natural mineral constituent of dentin.
Colloidal silica consists of a stable dispersion of very fine silica particles which are amorphous rather than crystalline. They remain in a colloidal suspension due to their low mass and the electrostatic interactions between the particles and the dispersion medium . The particles are practically insoluble in the dispersing medium which is usually distilled water. The application field of colloidal silica is fairly broad and includes use in the refractory industry, high temperature binders, investment casting, catalysts, abrasion resistant coatings, carbonless paper, increasing friction, as an abrasive, anti-soiling, surfactant or absorbent. Silica is also used in the food industry as a food additive (e.g. anti-caking agent E551). The colloidal nature of the silica solution makes it an attractive mineral for collagen infiltration with the hope that it will penetrate into the demineralized collagen matrix without precipitating on the surface .
The aim of this study was to assess the ability of HA and silica nano-particulate solutions to infiltrate demineralized dentin, that retains its collagen structural integrity. Our hypothesis is based on the premise that nanoparticles in a colloidal suspension may better infiltrate the demineralized collagen network of dentin and not precipitate on the surface. Thereafter, once the particles [hydroxyapatite (HA) and silica] have infiltrated the demineralized dentin they will remain embedded in the subsurface collagen matrix that acts as a scaffold retaining the particles. Subsequently, given the right environment, the particles may act as a seed for the remineralization of the dentin.
2
Methods and materials
The materials investigated were HA and silica nanoparticles. Two different nano-HA solutions were used; one made by the research group using the sol–gel technique and a commercially available formulation manufactured according to the precipitation technique (sintering temperature: 1100–1200 °C; batch number: P275S, Plasma Biotal Ltd., UK). The second is referred to as ‘control nano-HA’. Two colloidal silica solutions were used; one electronegative (Ludox ® HS-30, Grace Davison, UK) and the other electropositive (Ludox ® CL, Grace Davison, UK) due to an alumina surface coating (Al 2 O 3 ). Both colloidal silica solutions contain 12 nm particles and are commercially available products characterized by the manufacturer. The specifications for the two colloidal silica solutions are shown in Table 1 .
| Specifications | Ludox ® CL | Ludox ® HS-30 |
|---|---|---|
| Lot number | 2007850480 | 2006850568 |
| Particle charge | Positive | Negative |
| Particle size (nm) | 12 | 12 |
| Silica (as SiO 2 ) (wt%) | 30.0 a | 30.1 |
| pH (25 °C) | 3.9 | 9.8 |
| Stabilizing counter ion | Chloride | Sodium |
The in-house nano-HA solution was made from a powder that was synthesized using the sol–gel technique, as mainly described by Liu et al. . The final product was characterized with X-ray diffraction (XRD) using a Philips PW1825/00 X-ray diffractometer; the data acquired were analyzed with STOE WinXPOW 2.1 and Traces 4.0 software ( Fig. 1 ). In the powder form the HA nanoparticles have a natural tendency to form agglomerates, which were broken down by ball milling with zirconia balls. The resulting HA powder was mixed with distilled water (DW) and then DW was allowed to evaporate in an electric chamber furnace at 90 °C. HA powder was then separated from the zirconia balls and sieved using a micro-sieve with aperture size 106 μm. The sieved HA was diluted in ethanol (≥99.5%, Sigma–Aldrich), as ethanol is believed to disperse the HA agglomerates . The HA–ethanol mixture was left in a volatile chamber furnace overnight to remove the ethanol and subsequently was left to dry. This final HA product is referred to hereafter as ‘sol–gel nano-HA’. A range of sol–gel nano-HA solutions were prepared by mixing the HA powder with DW or other solvents at varying concentrations as detailed in Table 1 . The solutions were stirred to achieve a homogeneous dispersion of the solute. In an attempt to de-agglomerate further the HA particles, for some of the solutions sol–gel nano-HA was mixed with the deflocculant agent sodium hexametaphosphate (SHMP) (laboratory reagent, Fisher Scientific) at a ratio of 1:3. Sodium hexametaphosphate was selected as it is widely used as a food additive (E number E452i) to prevent precipitation of particles in beverages (e.g. fruit pulp in juice), as a water softener, in detergents and as a dispersing agent to break down clay and other soil types. The different solutions used in this investigation are referred to herein as solutions A, B, C, etc., and described in detail in Table 2 .
| Infiltration solution | Description |
|---|---|
| Solution A | Ludox ® CL (Grace Davison, UK) |
| Solution B | Ludox ® HS-30 (Grace Davison, UK) |
| Solution C | 0.03% control nano-HA (Plasma Biotal Ltd., UK) in DW |
| Solution D | 0.03% sol–gel nano-HA in DW |
| Solution E | 0.03% sol–gel nano-HA mixed with 0.01% SHMP powder in a 50/50 DW/acetone solution (≥99.5% acetone, Sigma–Aldrich) |
| Solution F | 15% control nano-HA (Plasma Biotal Ltd., UK) solution in DW |
| Solution G | 15% sol–gel nano-HA in DW |
| Solution H | 15% sol–gel nano-HA in a 50/50 DW and acetone solution (≥99.5% acetone, Sigma–Aldrich) |
| Solution I | 15% sol–gel nano-HA mixed with 5% SHMP in a 50/50 DW/acetone solution |
2.1
Materials characterization in solution
The HA and silica nanoparticles were further characterized with transmission electron microscopy (TEM) ( Fig. 2 ). Particle sizes were determined using a novel TEM technique that enabled embedding the particles in solution in a photo-polymerizable resin; direct particle measurements were made from TEM images of slices taken from the resin-embedded particles. For the characterization of the HA products (control nano-HA, sol–gel nano-HA and sol–gel nano-HA mixed with SHMP) 0.03% (w/v) solutions were prepared. This low concentration of HA proved to be ideal as it enabled distinct visualization of primary particles while avoiding clustering. TEM examination of the silica solutions was carried out with the solutions as provided by the manufacturer. The particle size analysis was performed using the ImageJ 1.44p software (Rasband W, National Institute of Health, Bethesda, MD) ( Table 3 ). The particle size distribution for each material is illustrated in Fig. 3 .
| Solution A | Solution B | Solution C | Solution D | Solution E | |
|---|---|---|---|---|---|
| Mean circular diameter (nm) | 15.73 ± 2.36 | 13.61 ± 2.47 | 27.23 ± 23.96 | 80.71 ± 16.82 | 3.51 ± 0.87 |
| Maximum diameter (nm) | 20.96 | 20.80 | 192.65 | 128.63 | 6.36 |
| Minimum diameter (nm) | 9.87 | 8.12 | 5.99 | 37.94 | 1.67 |
2.2
Infiltration of dentin specimens
Oblong shape dentin blocks (5 mm × 1 mm × 1 mm) obtained from human premolar teeth, extracted for orthodontic purposes, were prepared. Ethical approval for the use of extracted human teeth was obtained in accordance with guidelines from the University of Sheffield, UK. Specimens were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer. The specimens were then fully demineralized by immersion in a 4 N formic acid solution for 48 h. The endpoint of complete demineralization was confirmed radiographically and by EDS. The infiltration protocols are described in detail in the following paragraphs in accordance with the analytical techniques employed: TEM, scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analysis.
2.3
TEM specimen preparation
Eighteen dentin specimens were randomly divided in three groups of six specimens each in order to examine the specimens in triplicates. All specimens were fixed and demineralized as previously described. Of the dentin blocks, one sample from each group was assigned as non-infiltrated control (NIC) whereas the five remaining blocks were infiltrated with the two colloidal silica solutions (solutions A and B) and a series of 0.03% (w/v) nano-HA solutions (solutions C–E) as shown in Table 2 . The nano-solutions used for the infiltration of dentin were prepared at the same concentrations at which they had been previously characterized. All infiltrated samples were stored separately in glass vials containing 5 ml of the infiltration solutions. Specimens were fully immersed and the vials were kept under continuous slow speed rotation (4 rpm) to ensure that the particles within the solutions remain in suspension, thus avoiding precipitation. The infiltration period was 24 h for all specimens. Samples were then prepared for TEM following the routine processing protocol. Ultra-thin sections were obtained using a Leica EM UC6 ultramicrotome. TEM images were acquired using FEI Tecnai Spirit G2 120KV.
2.4
SEM/EDS specimen preparation
Twenty-four human dentin blocks were randomly divided in three equal groups in order to examine the specimens in triplicates. One block from each group was assigned as sound control and the remaining specimens were fixed and demineralized as previously described. One demineralized specimen from each group was then assigned as NIC while the six remaining specimens of each group were infiltrated for 24 h with the two colloidal silica solutions (solutions A and B) and a range of 15% (w/v) nano-HA solutions (solutions F–I) as described in Table 2 . Each of the specimens was fully immersed in 5 ml of the infiltration solutions. The use of acetone as a potential carrier vehicle for the sol–gel nano-HA particles was investigated in solutions H and I. Solution H was used as a control to discriminate from the similar solution (solution I) that also incorporated the deflocculant SHMP. The effect of acetone was analyzed using EDS. The low concentration nano-HA solutions (0.03%, w/v) previously used for the TEM experiments were deemed inadequate for visualization of particles in the dentin collagen matrix with SEM. Also higher levels of nano-HA within the collagen matrix were required to generate a strong signal for the EDS analysis. As the aim of this particular experiment was to establish whether infiltration of dentin collagen matrix with nanoparticles occurred, a concentration matched to that of control nano-HA product (Plasma Biotal Ltd., UK) was employed (15%, w/v HA).
Following infiltration, dentin specimens were sectioned in halves. One half was assessed with SEM and the other half with EDS Standard dehydration process involves ascending ethanol steps. However, immersion into a number of solutions could potentially displace the nanoparticles within the demineralized dentin matrix by partially or completely washing them away. Thus, specimens were left to dry at room temperature instead.
The depth of penetration was assessed by sectioning the specimens transversely using a fine surgical scalpel so that the outer 1 mm thick layer was removed leaving exposed the collagenous surface lying underneath. The internal surface area which was then revealed was subjected to SEM and EDS examination. Specimens that were prepared for SEM were gold coated whereas specimens prepared for EDS were carbon coated. SEM and EDS examination were performed using FEI Inspect F high resolution SEM device equipped with an EDS detector. EDS data acquisition and spectra analysis was achieved with EDAX Genesis 5.21 software.
2
Methods and materials
The materials investigated were HA and silica nanoparticles. Two different nano-HA solutions were used; one made by the research group using the sol–gel technique and a commercially available formulation manufactured according to the precipitation technique (sintering temperature: 1100–1200 °C; batch number: P275S, Plasma Biotal Ltd., UK). The second is referred to as ‘control nano-HA’. Two colloidal silica solutions were used; one electronegative (Ludox ® HS-30, Grace Davison, UK) and the other electropositive (Ludox ® CL, Grace Davison, UK) due to an alumina surface coating (Al 2 O 3 ). Both colloidal silica solutions contain 12 nm particles and are commercially available products characterized by the manufacturer. The specifications for the two colloidal silica solutions are shown in Table 1 .
| Specifications | Ludox ® CL | Ludox ® HS-30 |
|---|---|---|
| Lot number | 2007850480 | 2006850568 |
| Particle charge | Positive | Negative |
| Particle size (nm) | 12 | 12 |
| Silica (as SiO 2 ) (wt%) | 30.0 a | 30.1 |
| pH (25 °C) | 3.9 | 9.8 |
| Stabilizing counter ion | Chloride | Sodium |
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