Highlights
- •
Demineralization of dentin causes a decrease in the surface free energy.
- •
Acetone added to a nano-HA solution increases the surface wetting of dentin.
- •
Acetone facilitates the infiltration of dentin collagen with HA nanoparticles.
-
Mineral recovery of demineralized dentin is higher in the presence of acetone.
- •
Demineralized dentin absorbs nano-HA solutions faster due to increased permeability.
Abstract
Objectives
This study investigates the role of acetone, as a carrier for nano-hydroxyapatite (nano-HA) in solution, to enhance the infiltration of fully demineralized dentin with HA nanoparticles (NPs).
Methods
Dentin specimens were fully demineralized and subsequently infiltrated with two types of water-based nano-HA solutions (one containing acetone and one without). Characterization of the dentin surfaces and nano-HA particles was performed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The surface wettability and infiltration capacity of the nano-HA solutions were quantified by means of contact angle measurements and energy dispersive X-ray spectroscopy (EDS), respectively. Contact angle measurements were taken at baseline and repeated at regular intervals to assess the effect of acetone. The P and Ca levels of infiltrated dentin specimens were measured and compared to sound dentin and non-infiltrated controls.
Results
The presence of acetone resulted in an eight-fold decrease in the contact angles of the nano-HA solutions recorded on the surface of demineralized dentin compared to nano-HA solutions without acetone (one-way ANOVA, p < 0.05). Perfect wetting of the demineralized dentin surface was achieved 5 min after the application of the nano-HA solution containing acetone. Infiltration of demineralized dentin with the nano-HA solution containing acetone restored the lost mineral content by 50%, whereas the mean mineralization values for P and Ca in dentin treated with the acetone-free nano-HA solution were less than 6%.
Significance
Acetone was shown to act as a vehicle to enhance the capacity to infiltrate demineralized dentin with HA NPs. The successful infiltration of dentin collagen with HA NPs provides a suitable scaffold, whereby the infiltrated HA NPs have the potential to act as seeds that may initiate heterogenous mineral growth when exposed to an appropriate mineral-rich environment.
1
Introduction
A number of different dentin remineralization strategies have been reported, most of which involve the use of bioactive glass , fluoride-releasing materials , casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) complexes , artificial saliva solutions , calcium hydroxide and Portland cement . The use of engineered nanoparticles (ENPs) has also been examined as an alternative strategy for the management of dental caries and has become the focus of much research in this field. Nano-sized calcium fluoride (n-CaF 2 ) , nano-hydroxyapatite (nano-HA) , nano-sized carbonated apatite (n-CAP) , carbonate-hydroxyapatite nano-crystals (CHA) , nano-particulate bioactive glass and silica nanoparticles (NPs) are among those ENPs that have demonstrated an increase in the mineral content of enamel and/or dentin. However, a significant challenge when using these materials is to achieve an effective and deep infiltration of the demineralized dentin collagen matrix with the ENPs, while avoiding precipitation of the particles on the surface.
In adhesive restorative dentistry, the infiltration of partly demineralized dentin with resin-based hydrophilic dentin adhesives is also a significant problem. The dentin surface to be infiltrated is kept moist to prevent the surface collagen network from collapsing and thus creating a pathway for the hydrophilic monomers to penetrate. The deep infiltration of the resin monomers into the intra-fibrillar spaces of the collagen matrix network is further aided by the inclusion of volatile solvents in the adhesive that displace the water from the dentin surface . Acetone is a clear, colorless, low-boiling, flammable and volatile liquid characterized by rapid evaporation and a faintly aromatic, sweetish odor. It readily mixes with most organic solvents and mixes completely with water. It is naturally produced and disposed of in the human body as a result of normal metabolic processes and is considered a safe ingredient that is commonly found in a range of commonly used products ranging from cosmetics to processed and unprocessed foods; it has been rated as Generally Recognised As Safe (GRAS) substance when present in beverages, baked foods, desserts, and preserves at concentrations ranging from 5 to 8 mg l −1 . The successful application of this strategy in dentin bonding systems forms the basis for our hypothesis that HA NPs suspended in a water–acetone solution should work in a similar manner and aid a higher concentration and deeper infiltration of the particulates into the collagen network of the demineralized dentin.
Previous work by this group has reported a strategy to increase the mineral content of dentin that has been fully demineralized by acid . We have investigated the potential of infiltrating and embedding NPs in the collagen matrix and thus create nucleation seeds for subsequent mineral growth by means of heterogenous deposition of interfibrillar calcium phosphate minerals. The aim of this present study is to investigate the role of acetone, which has been used successfully in dentin bonding systems, to increase the infiltration potential of nano-HA solutions into the demineralized dentin. The hypothesis is that acetone will act as a carrier, enhancing the depth of penetration of HA NPs into the demineralized dentin matrix.
2
Methods and materials
2.1
Experimental design and specimen preparation
The experimental design involved exposing sound and fully demineralized dentin to two types of nano-HA solutions (one containing acetone and one without) and a distilled-deionised water (DW) solution with no added NPs, which served as a control. After application of the test solutions to the dentinal surfaces, surface wettability was quantified by contact angle measurements to aid interpretation of the role of acetone. Demineralized dentin specimens were also infiltrated with the test solutions. Following a 24 h infiltration, the infiltration capacity of the two nano-HA solutions was tested by means of energy dispersive X-ray spectroscopy (EDS) to determine whether the presence of acetone facilitates the penetration of NPs to the collagen matrix.
Dentin specimens were prepared in the form of discs (1 mm thick) and blocks (length × width × height: 5 mm × 1 mm × 1 mm) from the crowns of sound human premolar teeth that had been extracted for orthodontic purposes as part of routine dental care. Ethical approval for the use of the extracted human teeth was obtained. A total of forty dentin specimens were sectioned (18 discs and 22 blocks) using a low-speed precision blade saw (VC-50, Leco, Michigan, USA) equipped with a diamond wafering blade (Buehler, Dusseldorf, Germany). All dentin specimens were sonicated for 10 min to remove cutting debris and then allocated for morphological characterization of the surface by scanning electron microscopy (SEM), contact angle measurements and EDS analysis. A number of sound dentin specimens were stored in DW at 4 °C and the remaining specimens were fully demineralized in formic acid, in accordance with the demineralization protocol reported by Besinis et al. . In brief, specimens were initially fixed overnight at 4 °C with 3% glutaraldehyde in 0.1 M cacodylate buffer and then rinsed (3 × 3 min) with 0.1 M cacodylate buffer to remove glutaraldehyde before rinsing with DW. Specimens were then fully submersed in 4 N formic acid for 48 h. When the demineralization process was complete, specimens were rinsed with DW (3 × 3 min) to remove the acid and subsequently stored in DW at 4 °C. Fixation of dentin was essential to ensure that the collagen substructure would maintain its morphology during the demineralization process and that would resist shrinkage and any other deformational forces due to the dehydration process . Glutaraldehyde stabilizes the collagen fibrils in biological tissues and induces intra- and intermolecular crosslinks, including crosslinking of the extracellular proteins of the dentin collagen matrix .
2.2
SEM morphological characterization
Sound ( n = 3) and fully demineralized dentin ( n = 3) blocks were prepared for morphological surface characterization by SEM. All specimens were dehydrated with ascending ethanol grades and hexamethyldisilazane (HMDS) to prevent tissue shrinkage prior to being gold sputtered. Examination was performed using a FEI Inspect F high resolution SEM instrument.
2.3
Preparation of the nano-HA solutions
A nano-HA powder was synthesized by the research group according to the sol-gel technique as previously described by Besinis et al. . The morphology and particle size of the sol-gel HA NPs were assessed using a novel transmission electron microscopy (TEM) technique consisting of embedding the particles in a clear photopolymerisable resin ( Fig. 1 A) , thus allowing the characterization of non-agglomerated individual particles in suspension. The particle size was measured from multiple TEM images using ImageJ 1.46r software (Rasband W, National Institute of Health, Bethesda, MD) on more than one hundred particles. The particle size distribution is shown in Fig. 1 B (average particle diameter: 79.0 ± 16.3 nm; maximum particle diameter: 133.8 nm; minimum particle diameter: 43.5 nm).
Two types of nano-HA solutions were prepared; one containing sol–gel HA NPs in DW (nano-HA in DW) and a second using DW and acetone as solvent (≥99.5% acetone, Sigma–Aldrich Ltd, Dorset, UK) mixed together at a 1:1 ratio (nano-HA in DW-Ac). The prepared solutions contained HA NPs at 3 and 15% (w/v). The contact angle measurements were performed using 3% (w/v) nano-HA solutions as a preliminary study suggested that some particle sedimentation occurred within 5 min at higher concentrations, which could potentially affect the measurements (contact angles were recorded for 5 min, see below). The dentin infiltration and the EDS analysis were performed using 15% (w/v) nano-HA solutions because higher levels of nano-HA within the collagen matrix were required to generate a strong signal for the EDS analysis, in accordance with previous infiltration studies .
2.4
Contact angle measurements
The role of acetone in determining the surface wettability of dentin and the infiltration capacity of demineralized dentin with NPs was evaluated by measuring the contact angles of a 3% (w/v) nano-HA in DW solution on the flat surface of sound and fully demineralized dentin discs compared to a 3% (w/v) nano-HA in DW-Ac solution. A third DW solution, with no added NPs, was used as control to investigate whether the presence of the HA NPs affects the contact angle measurements. Each experiment was performed in triplicate (three separate dentin discs) for each type of dentin. The contact angle measurements were carried out using a Ramé-hart NRL model 100-00 contact angle goniometer following the sessile droplet technique. The hydration state of the dentin specimens was carefully controlled by removing the excess water, which remained on the surface as the specimens had been previously stored in DW, immediately prior to conducting the contact angle measurements. Although excess water was removed, dentin was not desiccated, leaving a moist and slightly glossy surface. A 2 μl droplet from the solutions was gently rested on the surface of the dentin discs using a Hamilton microsyringe (Hamilton Co., Reno, NV, USA). Contact angle measurements were taken immediately after the solutions droplets were placed on the surface of the dentin discs, and measurements were repeated after 1 min, 2 min, 3 min, 4 min and 5 min ( Fig. 2 ). The decrease of the contact angle over time as a percentage of the initial contact angle was calculated according to the equation: C d = (1 − θ t / θ 0 ) × 100% ( Fig. 3 ), where C d refers to the contact angle decrease, θ t is the contact angle for the droplet at a given time t and θ 0 is the contact angle for the droplet at baseline ( t = 0).
2.5
Energy dispersive X-ray spectroscopy
EDS was employed as a chemical microanalysis technique to determine the degree and depth of infiltration with HA NPs by analysing dentin specimens infiltrated with the nano-HA in DW and nano-HA in DW-Ac solutions, and comparing them with sound and fully demineralized dentin. Three sound dentin blocks were assigned as ‘sound controls’ and thirteen fully demineralized dentin blocks were randomly divided in three groups. The specimens of the first group ( n = 3) were assigned as ‘non-infiltrated controls’, whereas the specimens in the remaining two groups ( n = 5) were infiltrated with 15% (w/v) nano-HA in DW and 15% (w/v) nano-HA in DW-Ac solutions, respectively, for 24 h, in accordance with the protocol reported by Besinis et al. . Each of the specimens to be infiltrated was stored separately in a glass vial containing 5 ml of the infiltration solutions. Specimens were fully submersed and the vials were kept under continuous slow speed rotation (4 rpm) to ensure that the particles within the solutions remained in suspension, thus avoiding precipitation. The infiltrated dentin specimens were left to dry at room temperature for 72 h in a dust-free protected environment. Infiltrated specimens were prepared for EDS by transversely cutting the infiltrated dentin blocks to 1 mm thick sections across the longitudinal axis of the specimens using an ultra-thin skin-graft blade, in a manner analogous to a loaf of bread. The end slice was removed and not tested as this would have been directly exposed to the infiltration solutions. Each slice was laid on its side and EDS analysis was performed on the central area of the newly exposed sub-surface collagen matrix. EDS examination of the sound and non-infiltrated specimens was performed using the initial blocks as their composition was homogenous and thus there was no need to be further sectioned. All specimens were carbon coated prior to EDS analysis. All operating conditions, scanning parameter settings and specimen positioning were kept identical for all EDS scans to standardize the analysis method, increase results repeatability and allow comparison between samples as previously described by Besinis et al. . The spot size was set at 3.0 and the voltage at 15 kV. Accuracy was further enhanced by selecting the full frame scan mode instead of the spot scan mode. The surface area examined in each scan was 900 μm 2 and the scan duration was 180 s for all samples.
The extent of infiltration was assessed by recording the net intensity (intensity of a peak minus the background after deconvolution) for phosphorus (P) and calcium (Ca) in the infiltrated specimens after exposure to the infiltration solutions. Calcium hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] is the mineral phase of dentin and fully demineralized dentin lacks both P and Ca. Thus, an increase in the levels of P and Ca suggests that infiltration of dentin with HA NPs was successful. The mean intensity values for P and Ca in the sound controls were used as a reference (100% mineralized dentin) and all results were expressed as a percentage of the P and Ca levels in sound dentin (mean mineralization values). EDS examination was performed using a FEI Inspect F high resolution SEM instrument equipped with an EDS detector. Data and spectra analysis was achieved using EDAX Genesis 5.21 software.
2.6
Surface free energy
The reactivity of a surface is measured in terms of surface free energy, which is defined as “the work required to increase the area of a substance by 1 cm 2 ” . When applied to solid materials, the term “surface energy” acquires a different meaning and is considered as an adhesive parameter characterising the affinity of the solid surface to other materials. The surface free energy can be determined by measuring the contact angle formed using several diverse approaches . In this study, free energy values of the dentin surfaces before and after demineralization with formic acid were calculated according to the Neumann method taking advantage of the equation of state, which is expressed as (1 + cos θ ) γ L = 2( γ L γ S ) 0.5 exp[− β ( γ L − γ S ) 2 ], where θ is the contact angle, γ L is the liquid’s surface tension, β is an empirical constant and γ S is the surface energy to be determined. The surface tension used was that of DW (72.80 mN m −1 ), whereas the value for the β constant was 1.057 × 10 −4 .
2.7
Statistics
All data are presented as mean ± standard deviation and were analyzed using version 19.0 of IBM SPSS Statistics software for Windows (IBM Corp. Armonk, NY, USA). The differences in the contact angle measurements between the experimental and control groups within each end point (treatment effect), as well as the differences between each of the end points and baseline (time effect) were evaluated using one-way analysis of variance (one-way ANOVA, Tukey post hoc test). The differences in the contact angles measurements between sound and demineralized dentin were tested using two-way ANOVA with Tukey’s post hoc test. The differences in the mean mineralization values between the infiltrated groups and the non-infiltrated controls for each of the elements under investigation (P and Ca) were tested using one-way analysis of variance (one-way ANOVA, Tukey post hoc test). Demineralized dentin infiltrated with nano-HA in DW was tested against demineralized dentin infiltrated with nano-HA in DW-Ac (in relation to P and Ca levels together) using two-way ANOVA with Tukey’s post hoc test. The differences in the surface free energy between sound and demineralized dentin, as well as the differences between the Ca/P ratios, were evaluated using independent-samples t -test. All statistical analysis used a 95% confidence limit, so that p values equal to or greater than 0.05 were not considered statistically significant.
2
Methods and materials
2.1
Experimental design and specimen preparation
The experimental design involved exposing sound and fully demineralized dentin to two types of nano-HA solutions (one containing acetone and one without) and a distilled-deionised water (DW) solution with no added NPs, which served as a control. After application of the test solutions to the dentinal surfaces, surface wettability was quantified by contact angle measurements to aid interpretation of the role of acetone. Demineralized dentin specimens were also infiltrated with the test solutions. Following a 24 h infiltration, the infiltration capacity of the two nano-HA solutions was tested by means of energy dispersive X-ray spectroscopy (EDS) to determine whether the presence of acetone facilitates the penetration of NPs to the collagen matrix.
Dentin specimens were prepared in the form of discs (1 mm thick) and blocks (length × width × height: 5 mm × 1 mm × 1 mm) from the crowns of sound human premolar teeth that had been extracted for orthodontic purposes as part of routine dental care. Ethical approval for the use of the extracted human teeth was obtained. A total of forty dentin specimens were sectioned (18 discs and 22 blocks) using a low-speed precision blade saw (VC-50, Leco, Michigan, USA) equipped with a diamond wafering blade (Buehler, Dusseldorf, Germany). All dentin specimens were sonicated for 10 min to remove cutting debris and then allocated for morphological characterization of the surface by scanning electron microscopy (SEM), contact angle measurements and EDS analysis. A number of sound dentin specimens were stored in DW at 4 °C and the remaining specimens were fully demineralized in formic acid, in accordance with the demineralization protocol reported by Besinis et al. . In brief, specimens were initially fixed overnight at 4 °C with 3% glutaraldehyde in 0.1 M cacodylate buffer and then rinsed (3 × 3 min) with 0.1 M cacodylate buffer to remove glutaraldehyde before rinsing with DW. Specimens were then fully submersed in 4 N formic acid for 48 h. When the demineralization process was complete, specimens were rinsed with DW (3 × 3 min) to remove the acid and subsequently stored in DW at 4 °C. Fixation of dentin was essential to ensure that the collagen substructure would maintain its morphology during the demineralization process and that would resist shrinkage and any other deformational forces due to the dehydration process . Glutaraldehyde stabilizes the collagen fibrils in biological tissues and induces intra- and intermolecular crosslinks, including crosslinking of the extracellular proteins of the dentin collagen matrix .
2.2
SEM morphological characterization
Sound ( n = 3) and fully demineralized dentin ( n = 3) blocks were prepared for morphological surface characterization by SEM. All specimens were dehydrated with ascending ethanol grades and hexamethyldisilazane (HMDS) to prevent tissue shrinkage prior to being gold sputtered. Examination was performed using a FEI Inspect F high resolution SEM instrument.
2.3
Preparation of the nano-HA solutions
A nano-HA powder was synthesized by the research group according to the sol-gel technique as previously described by Besinis et al. . The morphology and particle size of the sol-gel HA NPs were assessed using a novel transmission electron microscopy (TEM) technique consisting of embedding the particles in a clear photopolymerisable resin ( Fig. 1 A) , thus allowing the characterization of non-agglomerated individual particles in suspension. The particle size was measured from multiple TEM images using ImageJ 1.46r software (Rasband W, National Institute of Health, Bethesda, MD) on more than one hundred particles. The particle size distribution is shown in Fig. 1 B (average particle diameter: 79.0 ± 16.3 nm; maximum particle diameter: 133.8 nm; minimum particle diameter: 43.5 nm).
