Highlights
- •
Monitoring by VSI of topographical evolutions occurring on enamel surfaces.
- •
Quantification of demineralization/remineralization rates of enamel surfaces.
- •
New figures of merit to establish the therapeutic performance of dental treatments.
Abstract
Objective
Remineralization and demineralization are processes that compete in the oral environment. At this time, numerous therapeutic agents are being developed to promote remineralization (precipitation) or suppress demineralization (dissolution). To evaluate the relative efficacy of such treatments, there is a need for non-invasive, real-time, high-resolution quantifications of topographical changes occurring during demineralization and remineralization.
Methods
Vertical scanning interferometry (VSI) is demonstrated to be a quantitative method to assess reactions, and topographical changes occurring on enamel surfaces following exposure to demineralizing, and remineralizing liquids.
Results
First, the dissolution rate of enamel was compared to that of synthetic hydroxyapatite (HAP) under acidic conditions (pH = 4). Second, VSI was used to compare the remineralization effects of F − -based and CCP-ACP agents. The former produced a remineralization rate of ≈349 nm/h, similar to simulated body fluid (SBF; concentration 4.6×) while the latter produced a remineralization rate of ≈55 nm/h, corresponding to 1.7× SBF. However, the precipitates formed by the CCP-ACP agent are found to demineralize 2.7× slower than that produced by its F − -counterpart.
Significance
Based on this new VSI-based data, a remineralization factor (RF) and demineralization (DF) factor benchmarked, respectively, to 1× SBF and the demineralization rate of human enamel are suggested as figures of merit of therapeutic performance of dental treatments. Taken together, the outcomes offer new insights that can inform clinicians and researchers on the selection of remineralization strategies.
1
Introduction
Remineralization and demineralization are dynamic processes that compete in oral environments. While caries is associated with bacterial activity in dental plaque , erosion involves intrinsic and extrinsic acidic substances . Both conditions result from imbalances that favor tissue demineralization. Demineralized hydroxyapatite (HAP) surfaces are more susceptible to other damage, e.g., abrasion, and the combination of acid dissolution and mechanical stresses promotes further enamel removal by exposing underlying tissues to successive acid attack . As a result, there is significant interest in favoring remineralization and preventing demineralization. Besides enforcing dietary changes to reduce acidic food intake and hence demineralization , the routine use of topical agents can enhance remineralization .
Demineralization and/or remineralization of dental tissues has been widely studied using scanning electron microscopy , transmission electron microscopy , atomic force microscopy , indentation , microradiography , electron probe microanalysis , light induced fluorescence , secondary ion mass spectroscopy , confocal microscopy , electrochemical impedance spectroscopy , ultrasonic measurements , iodide permeability test and contact profilometry . However, microscopy techniques only offer qualitative 2D means for assessment . Other techniques, e.g., microradiography and transmission electron microscopy are destructive and do not provide real-time evaluation. Further, the experimental conditions can also be limiting—e.g., in the case of microradiography the alignment and geometry of the X-ray beam can limit imaging precision at the sample edge . While methods such as atomic force microscopy offer nanoscale resolution, the measurements are time-consuming and of limited statistical relevance, as only small lateral areas can be scanned in realistic durations.
Recently, non-contact profilometry (e.g., laser scanning or white light interferometry, WLI) has been proposed as a new method to study dental tissues . By building on past studies, this paper establishes procedural details for full-field quantitative assessments of reactions (i.e., dissolution or precipitation) occurring on dental tissue surfaces in contact with a solution using vertical scanning interferometry (VSI). VSI allows quantifications of surface topographies with a vertical resolution of ≈0.1 nm and a lateral resolution of around 500 nm. VSI uses interferometric objectives consisting of an objective lens, a reference mirror and a beam-splitter (see Fig. 1 ). The most common interferometric objectives differ in terms of the mirror location, such that the mirror can be located between the objective lens and the beam-splitter (Mirau objective) or it can be placed in another position because of its larger size (a Michelson objective). In both cases, a source directs a light beam onto the sample surface through the interferometric objective, where the beam-splitter separates the light into two beams. One beam is reflected back by the reference mirror, while the other travels along the optical axis and interacts with the sample. This latter beam is reflected by the sample’s surface. This results in an optical path difference between the two light beams and, a pattern of interference fringes forms when the beams are recombined.
This interference pattern is composed of light and dark bands: when the two beams are in phase their amplitudes are summed and a light band forms, whereas when the beams are out of phase their amplitudes are subtracted and a dark band of zero amplitude results. The interference fringes are sampled by a CCD (charge-coupled device) sensor and the signal is digitized and processed to obtain 3D topographical maps of the sample’s surface. VSI offers distinct advantages over other techniques, including: (i) non-destructive evaluation, (ii) nanoscale resolution of surface profiles (≈0.1 nm in the z -direction) with the ability to scan large lateral areas on the order of 10 s of mm 2 , in real time, and (iii) the ability to render statistically relevant quantifications of true reaction (dissolution or precipitation) rates, while duly accounting for the exposed (surface) area of the solid. Therefore, this paper uses VSI to quantify the dissolution rate of enamel and synthetic HAP at pH 4. Further, the remineralizing effects of fluoride and CPP–ACP toothpastes are measured with subsequent analysis of demineralization behavior of the remineralized enamel surfaces.
The first aim of this study is to establish VSI as a quantitative tool to evaluate demineralization and remineralization rates of dental tissues. The main motivation is to use real-time, continuous, high-resolution, quantitative data to rationalize the contradictory observations in the literature. For instance, Prabhakar et al. reported fluoride-based toothpastes to be a superior remineralizing agent compared to CCP-ACP treatments, while Somasundaram et al. noted opposite findings. Elsayad et al. stated that fluoride-based and CCP-ACP toothpastes can have a “synergistic effect” on remineralization if used together; while Huang et al. and Balan et al. reported that there is no significant difference in the effectiveness of these toothpastes. Besides minor differences in the treatment protocol, the studies also relied on analytical techniques that did not provide real time quantitative demineralization/remineralization rates. The second aim of this paper is to identify and benchmark therapeutic treatments that can mitigate acid attack of hard dental tissues.
2
Material and methods
2.1
Sample preparation
Caries-free, extracted human molars were collected from local oral surgery clinics. The teeth were sectioned axially using a precision slow speed diamond saw (IsoMet™ 1000, Buehler Inc.). The sections comprised the buccal, lingual, mesial and distal walls of the tooth and had a size on the order of 5 mm × 5 mm × 5 mm. After sectioning, the specimens were embedded in a cold-cured, inert, epoxy resin (EpoxiCure Resin, PN 203430128: Buehler Inc.), with the enamel portion facing outward and polished to a uniform smoothness using SiC abrasive sheets of grit levels ranging from 400-to-1200. The polished sections were fixed to a glass slide using an inert adhesive (double sided tape 3M 950, supplied by Uline) to facilitate handling. It should be noted that the samples are polished flat to remove natural variations in the surface of tooth sites that may result in different responses to acid attack .
Powdered synthetic hydroxyapatite (HAP: Ca 5 (PO 4 ) 3 (OH); molecular weight of 502.31 g/mol) was sourced from Acros Organics™ (CAS 1306-06-5). For dissolution analysis, powder particles with median diameter d 50 = 6.5 μm were fixed on a glass slide using inert double sided tape (3M 950, supplied by Uline) prior to further analysis.
2.2
De-/re-mineralizating treatment protocol
VSI was used to monitor the enamel surfaces over three steps: (Step 1) demineralization, (Step 2) remineralization, and (Step 3) demineralization of the newly remineralized surface ( Fig. 2 ).
Step 1 : Demineralization is carried out at room temperature (25 ± 3 °C) using a pH 4 buffer solution (Fisher Scientific CAS 50-00-0: water 98.91%, 1,2-benzenedicarboxylic acid, monopotassium salt 1.0%, formaldehyde 0.05%, methyl alcohol 0.02%, fluorescein, 2′,4′,5′,7′-tetraiodo, disodium salt 0.02%, all percentages written as mass %). A few drops of buffer solution (50–75 μL) were applied on the sample surface using a micropipette to obtain a liquid-to-solid ratio ( l / s , by mass) between 50,000–75,000 to approximate the dilute limit. This elevated ratio hindered solution saturation with ions and prevents precipitation, if any. After 20 min of solvent contact, the sample is rinsed using distilled water and gently dried with compressed air. The dry sample is analyzed by VSI over fifteen demineralization cycles resulting in a solution contact time of 5 h per sample. For comparison, the dissolution rate of synthetic hydroxyapatite (HAP) was also determined under the same experimental conditions.
Step 2 : Enamel remineralization was carried out at room temperature using 2 commercial agents: (i) PreviDent5000 (Colgate-Palmolive, New York, USA) containing 1.1% NaF, and (ii) MI Paste (GC Corporation, Tokyo, Japan) whose active ingredient is Recaldent™ (CPP–ACP: casein phosphopeptide, CPP, and amorphous calcium phosphate, ACP). Each toothpaste was applied on the enamel surface for a contact time of 60 min. This procedure was repeated 9 times (i.e., for a total application duration of 9 h). Between each remineralization treatments, the samples were rinsed thoroughly and then dried prior to VSI analysis.
Besides comparing the toothpastes’ remineralization efficacy against each other, it would be more beneficial to compare them to standard benchmark remineralization agents. Simulated body fluid (SBF) was selected as a benchmark agent because of its two advantages over artificial saliva:
- (i)
SBF is widely used and its composition (and ionic strength) can be closely controlled, and,
- (ii)
for the purposes of this study, i.e., to demonstrate the ability to detect varying degrees of mineralization, it is important to select an easily reproducible protocol that can be replicated by other investigators around the world.
SBF is a well-established agent (ISO 23317) that is suitable for use in international standardized protocols to evaluate biomineralization . In contrast, it is difficult to replicate the unique properties of human saliva because this complex fluid is a mixture of fluids secreted by several salivary glands. Each of these individual saliva producers make highly variable saliva in terms of viscosity, ionic strength, etc. according to the time of day, diet, and many physiologic factors. For example, Gal et al. investigated several different artificial saliva formulations and found that several were arbitrary formulations which yielded highly variable properties. While some of the more recent artificial saliva formulations contain similar ions as SBF, they do not allow simple additions of calcium and phosphates species to alter ionic strength, without producing insoluble precipitants. On the other hand, SBF is a well-designed system that allows easy manipulation of ionic strength without precipitation in most hands.
Therefore, three SBF concentrations (1×, 2× and 5×) were prepared, and their remineralization rates were used to provide data against which the commercial toothpastes could be compared. After demineralization at pH 4 (Step 1), remineralization is carried out by contacting the enamel surface with 50–75 μL of SBF for 30 min. After each SBF application, the sample is rinsed with distilled water and dried with compressed air prior to VSI analysis. The remineralization step was repeated 14 times for a total contact time of 7 h.
Step 3 : Similar to Step 1, acid exposure (pH 4) was carried out to ascertain the demineralization rates of the “deposited or remineralized” tissue surfaces, as noted in Step 2.
2.3
Vertical scanning interferometry (VSI)
A Zygo NV 8200 vertical scanning interferometer fitted with a 50× Mirau objective (N.A. = 0.55) was used in the analyses. This setup permitted visualization of a scanning area of 152.27 μm × 141.86 μm per image field. Since images are recorded in stitched mode, using a P × Q grid ( P rows × Q columns) composed of PQ tiles, the full-image field is on the order of (152.27 × P ) μm × (141.86 × Q ) μm wherein P and Q values ranged between 2–5. A back-scan length of 145 μm was used, which allows peak-to-valley height differences up to 290 μm to be sampled. The acquisition time of the tiled image field, depending on grid size, ranged between 300-to-1200 s.
For any given sample, a scanning area was selected to monitor the topography of the surface (i.e., enamel or HAP), while establishing the embedment resin (for the enamel) or the polymer substrate (for the HAP), as a reference plane. Both the resin and the polymer used are inert to the solutions applied (see Section 3.1 ). After the images are captured, a data processing procedure consisting of several steps is applied:
- (i)
filtering is carried out to include or exclude specific characteristics of the surface. For the data presented herein, a median filter is chosen to reduce noise.
- (ii)
Form removal is carried out, if needed, to minimize alignment errors and to remove any background tilt that could mask details of the surface. To perform such leveling, mathematical functions are chosen to describe a geometrical form (e.g., a plane) that can be fit to, and then subtracted from the acquired data.
- (iii)
Masking is used to select and analyze a specific region in detail, while excluding (masking) the rest of the image field from the analyses. With the use of multiple masks placed over different regions of a given image-field, topographical parameters spanning a large area can be acquired, and, as such, a statistically significant analysis can be performed.
- (iv)
Topographical parameters are calculated using the Mx software (Ver. 6.1.0.4, distributed by Zygo Corporation) to quantify the changes in surface height. The parameter used in this study is the area averaged height ( h ) that is computed by averaging the height profile over M × N pixels as follows :
h = 1 M N ∑ x = 0 M − 1 ∑ y = 0 N − 1 Z ( x , y ) − Z ¯
where M and N are the number of pixels along the x and y directions, Z is the height of the sample (nm) at the location of the pixel located at ( x , y ), and <SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='Z¯’>Z¯¯¯Z¯
Z ¯
is the height of the reference plane (i.e., XY plane with respect to fixed origin in 3D space; in this case the epoxy in which the enamel is embedded). The difference between Z and <SPAN role=presentation tabIndex=0 id=MathJax-Element-3-Frame class=MathJax style="POSITION: relative" data-mathml='Z¯’>Z¯¯¯Z¯
Z ¯
, therefore, is a measure of the height of a given feature on the sample with respect to the reference plane.
- (v)
The demineralization or remineralization rates ( D R , or R R ) is computed as:
D R ( m / s ) = Δ h Δ t
where Δ h = h ( i ) − h ( i + 1) is the change in the average height (nm) of the exposed material with respect to the reference plane between step ( i ) and its successive step ( i + 1) measured over a period of time Δ t (s). The highest uncertainty in dissolution and precipitation rates, quantified in this manner is on the order of 10% . The reproducibility of the experimental results is examined by repeating each experiment three times under the same conditions.
2.4
Scanning electron microscopy (SEM)
SEM images of select enamel samples were obtained before and after demineralization and after subsequent remineralization. Imaging was carried out using secondary electrons (SE) using a FEI Nova NanoSEM 230 (10 kV, 80 pA). The images allow for qualitative comparison of the pristine enamel surfaces before the application of any treatment, and following demineralization (Step 1, t = 5 h) and remineralization (Step 2, t = 14 h).
2.5
X-ray diffraction (XRD)
XRD patterns were obtained on demineralized enamel surfaces treated with a F − -based and CPP–ACP toothpastes using a Bruker D8 diffractometer with θ – θ “Bragg-Brentano” geometry, using Cu-Kα radiation ( λ = 1.5406 Å), 40 kV accelerating voltage, and 40 mA beam current. The samples were scanned on a rotating stage over 5–75° 2 θ range with a scan step of 0.02° (2 θ ) and a step time of exposure time of 0.15 s per step.
2
Material and methods
2.1
Sample preparation
Caries-free, extracted human molars were collected from local oral surgery clinics. The teeth were sectioned axially using a precision slow speed diamond saw (IsoMet™ 1000, Buehler Inc.). The sections comprised the buccal, lingual, mesial and distal walls of the tooth and had a size on the order of 5 mm × 5 mm × 5 mm. After sectioning, the specimens were embedded in a cold-cured, inert, epoxy resin (EpoxiCure Resin, PN 203430128: Buehler Inc.), with the enamel portion facing outward and polished to a uniform smoothness using SiC abrasive sheets of grit levels ranging from 400-to-1200. The polished sections were fixed to a glass slide using an inert adhesive (double sided tape 3M 950, supplied by Uline) to facilitate handling. It should be noted that the samples are polished flat to remove natural variations in the surface of tooth sites that may result in different responses to acid attack .
Powdered synthetic hydroxyapatite (HAP: Ca 5 (PO 4 ) 3 (OH); molecular weight of 502.31 g/mol) was sourced from Acros Organics™ (CAS 1306-06-5). For dissolution analysis, powder particles with median diameter d 50 = 6.5 μm were fixed on a glass slide using inert double sided tape (3M 950, supplied by Uline) prior to further analysis.
2.2
De-/re-mineralizating treatment protocol
VSI was used to monitor the enamel surfaces over three steps: (Step 1) demineralization, (Step 2) remineralization, and (Step 3) demineralization of the newly remineralized surface ( Fig. 2 ).
Step 1 : Demineralization is carried out at room temperature (25 ± 3 °C) using a pH 4 buffer solution (Fisher Scientific CAS 50-00-0: water 98.91%, 1,2-benzenedicarboxylic acid, monopotassium salt 1.0%, formaldehyde 0.05%, methyl alcohol 0.02%, fluorescein, 2′,4′,5′,7′-tetraiodo, disodium salt 0.02%, all percentages written as mass %). A few drops of buffer solution (50–75 μL) were applied on the sample surface using a micropipette to obtain a liquid-to-solid ratio ( l / s , by mass) between 50,000–75,000 to approximate the dilute limit. This elevated ratio hindered solution saturation with ions and prevents precipitation, if any. After 20 min of solvent contact, the sample is rinsed using distilled water and gently dried with compressed air. The dry sample is analyzed by VSI over fifteen demineralization cycles resulting in a solution contact time of 5 h per sample. For comparison, the dissolution rate of synthetic hydroxyapatite (HAP) was also determined under the same experimental conditions.
Step 2 : Enamel remineralization was carried out at room temperature using 2 commercial agents: (i) PreviDent5000 (Colgate-Palmolive, New York, USA) containing 1.1% NaF, and (ii) MI Paste (GC Corporation, Tokyo, Japan) whose active ingredient is Recaldent™ (CPP–ACP: casein phosphopeptide, CPP, and amorphous calcium phosphate, ACP). Each toothpaste was applied on the enamel surface for a contact time of 60 min. This procedure was repeated 9 times (i.e., for a total application duration of 9 h). Between each remineralization treatments, the samples were rinsed thoroughly and then dried prior to VSI analysis.
Besides comparing the toothpastes’ remineralization efficacy against each other, it would be more beneficial to compare them to standard benchmark remineralization agents. Simulated body fluid (SBF) was selected as a benchmark agent because of its two advantages over artificial saliva:
- (i)
SBF is widely used and its composition (and ionic strength) can be closely controlled, and,
- (ii)
for the purposes of this study, i.e., to demonstrate the ability to detect varying degrees of mineralization, it is important to select an easily reproducible protocol that can be replicated by other investigators around the world.
SBF is a well-established agent (ISO 23317) that is suitable for use in international standardized protocols to evaluate biomineralization . In contrast, it is difficult to replicate the unique properties of human saliva because this complex fluid is a mixture of fluids secreted by several salivary glands. Each of these individual saliva producers make highly variable saliva in terms of viscosity, ionic strength, etc. according to the time of day, diet, and many physiologic factors. For example, Gal et al. investigated several different artificial saliva formulations and found that several were arbitrary formulations which yielded highly variable properties. While some of the more recent artificial saliva formulations contain similar ions as SBF, they do not allow simple additions of calcium and phosphates species to alter ionic strength, without producing insoluble precipitants. On the other hand, SBF is a well-designed system that allows easy manipulation of ionic strength without precipitation in most hands.
Therefore, three SBF concentrations (1×, 2× and 5×) were prepared, and their remineralization rates were used to provide data against which the commercial toothpastes could be compared. After demineralization at pH 4 (Step 1), remineralization is carried out by contacting the enamel surface with 50–75 μL of SBF for 30 min. After each SBF application, the sample is rinsed with distilled water and dried with compressed air prior to VSI analysis. The remineralization step was repeated 14 times for a total contact time of 7 h.
Step 3 : Similar to Step 1, acid exposure (pH 4) was carried out to ascertain the demineralization rates of the “deposited or remineralized” tissue surfaces, as noted in Step 2.
2.3
Vertical scanning interferometry (VSI)
A Zygo NV 8200 vertical scanning interferometer fitted with a 50× Mirau objective (N.A. = 0.55) was used in the analyses. This setup permitted visualization of a scanning area of 152.27 μm × 141.86 μm per image field. Since images are recorded in stitched mode, using a P × Q grid ( P rows × Q columns) composed of PQ tiles, the full-image field is on the order of (152.27 × P ) μm × (141.86 × Q ) μm wherein P and Q values ranged between 2–5. A back-scan length of 145 μm was used, which allows peak-to-valley height differences up to 290 μm to be sampled. The acquisition time of the tiled image field, depending on grid size, ranged between 300-to-1200 s.
For any given sample, a scanning area was selected to monitor the topography of the surface (i.e., enamel or HAP), while establishing the embedment resin (for the enamel) or the polymer substrate (for the HAP), as a reference plane. Both the resin and the polymer used are inert to the solutions applied (see Section 3.1 ). After the images are captured, a data processing procedure consisting of several steps is applied:
- (i)
filtering is carried out to include or exclude specific characteristics of the surface. For the data presented herein, a median filter is chosen to reduce noise.
- (ii)
Form removal is carried out, if needed, to minimize alignment errors and to remove any background tilt that could mask details of the surface. To perform such leveling, mathematical functions are chosen to describe a geometrical form (e.g., a plane) that can be fit to, and then subtracted from the acquired data.
- (iii)
Masking is used to select and analyze a specific region in detail, while excluding (masking) the rest of the image field from the analyses. With the use of multiple masks placed over different regions of a given image-field, topographical parameters spanning a large area can be acquired, and, as such, a statistically significant analysis can be performed.
- (iv)
Topographical parameters are calculated using the Mx software (Ver. 6.1.0.4, distributed by Zygo Corporation) to quantify the changes in surface height. The parameter used in this study is the area averaged height ( h ) that is computed by averaging the height profile over M × N pixels as follows :
h = 1 M N ∑ x = 0 M − 1 ∑ y = 0 N − 1 Z ( x , y ) − Z ¯
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