Enamel is the hardest tissue in the human body due to its 95–97% mineral content, which protects teeth from physical and chemical damage [6]. Enamel that is interior to the outermost area of the tooth has a microstructure consisting of geometric prism structures, known as rods, and inter-prismatic structures, known as inter-rods. At the nanoscopic level, both the rods and inter-rods are highly organized structures of hydroxyapatite crystals [7]. These crystals are hexagonal in shape with a diameter of approximately 30 nm, and they are arranged with all their c-axes parallel [8]. In contrast, enamel located in the outermost area of tooth has no prisms. This enamel is normally found in the occlusal, fissural, and cervical regions of all deciduous teeth and 70% of permanent teeth. This layer is 15–30 µm thick [9]. The structure of this region has two components: one with a scale-like appearance, and the other with a laminated appearance. The crystallites of the scale-like prismless enamel are arranged parallel to each other and their c-axis is perpendicular to the striae of Retzius, while the crystals of the laminated prismless enamel are perpendicular to the enamel surface [10–11]. The prismless enamel has more negative birefringence [12] and stronger resistance to demineralization than the prismatic enamel. This layer functions as a barrier against the acid dissolution that can result in dental caries [13].
In all previous studies encountered by the current authors, a portion of the tooth sample was flattened by grinding in preparation for the microhardness test, in the belief that a flat surface is necessary for an accurate test result. Unfortunately this grinding unavoidably removes the prismless layer that plays an important role resisting acid challenges. In the current study, the demineralization procedure was designed and performed with the goal of mimicking the natural in vivo demineralization process. Therefore the tooth samples were not flattened, by grinding or any other means, and the prismless enamel was thus preserved, keeping the laboratory procedures of the study closer to actual clinical circumstances. Regarding concern that the natural contour of the tooth will prevent an accurate microharness test, the current authors make the following observations. The diameter of the Vicker hardness test indenter is 0.5 µm, which is quite small. On that tiny scale, the natural contour of the tooth can be considered negligible and inconsequential, just as a person standing on Earth will generally perceive the Earth as more or less flat, with the Earth’s natural curve becoming negligible and inconsequential for purposes in the person’s immediate area. When performing the hardness test, the tiny indenter is placed somewhere near the apex of the most convex surface of the sample, and the point of contact at that scale will for all practical purposes be flat. This can be confirmed by the fidelity of the indentations to the shape of the indenter itself. Therefore testing the enamel surface without preparatory flattening is considered to have no influence on the outcome of the hardness measurement.
The particular chemical composition as well as the physical and mechanical properties of enamel vary from one person to the next, from one tooth to the next, and even from one area of a tooth to the next. Since the tooth specimens in this study were collected from different patients, their exact chemical composition as well as physical and mechanical properties are expected to be different. Since the purpose of this study is to observe the series of changes that occur when tooth samples undergo the demineralization and remineralization procedures, using and following the same enamel pieces all the way through the study is vital in order to be able to compare before and after figures within the same sample at each experiment stage.
The most familiar properties of color are hue, chroma, value and translucency [14]. Value, also called brightness, is the amount of light returned from an object [15], and this can also be referred to as the lightness (L*) of a color [16]. Lightness can be measured independently of hue as grayscale. The relationship between lightness and chroma is a negative correlation. Lightness is a dominant color dimension which determines 75% of shade selection [14]. White index (W) is quantified measurement of the common idea of “whiteness”. When comparing two values of W, the higher value is “whiter”. White index represents the amount of visible light reflection compared to the complete reflection of all light spectra and is a function of lightness, hue, and chroma, each of which contribute to the total perception of whiteness [17]. Thus the value of W can be calculated from the three fundamental dimensions of color: L*, a*, and b*. The current study utilizes L*, a*, and b*, along with W as a concomitant perception of color.
In the current study, the whiteness index of the demineralized enamel significantly increased. The effect was that the demineralized teeth became excessively white in subjective appearance and thus lost their natural look. Use of any of the three remineralizing agents (but not the water control) succeeded in improving the subjective appearance of the demineralized teeth, not by lowering whiteness but by increasing lightness. However none of the remineralizing agents completely recovered the natural appearance of the initial teeth. The three-day period that the remineralizing agents were in contact with the teeth allowed all three agents to ameliorate the appearance of the demineralized enamel, and a treatment period of additional length might yield even more pronounced results in future studies.
The ΔE values that were calculated in this study are used to evaluate the perceptibility of color differences. Given any two objects of different color, the difference between the two colors can be calculated as a ΔE value. Importantly, if the value of ΔE is greater than 1, this means that the color difference in question can be perceived by the human eye as long as the two colors are displayed side by side [18]. If ΔE is higher than 3.3, this means that the human eye can detect the color difference even when the two colors are not seen side by side [19]. When ΔE is greater than 3.7, this means that the two colors are quite dissimilar and of little relation. In this case, the color difference between the observed subjects is obvious to the human eye [20]. In the current study, the ΔE values calculated between all three stages of all three remineralizing agents were all over 3.7. Therefore both the demineralization and remineralization processes alter the enamel color to an extent that people can obviously see.
Regarding the SEM pictures, the demineralized enamel revealed tiny porosities all over its surface, which together appear similar to the subsurface lesion seen in the naturally demineralized reference tooth. The area of this lesion has lower mineral composition compared to the intact ename [21]. It is believed that the porosities on the enamel surface may provide access for the demineralizing solution to get into the enamel and dissolve some amount of minerals, thus creating the subsurface lesion. This in turn affects the refractive index of the enamel surface, leading to the tooth color alteration. In 2019, the scattering coefficient was observed at wavelength 543–1060 nm in both intact and demineralized enamels. The latter had two times the scattering coefficient (8.46 mm1−) compared to the intact enamel (4.60 mm1−) when 37% phosphoric acid was applied for 120 seconds, and as the application time increased, so did the scattering coefficient [22]. The color alteration in demineralized enamel is likely the result of this increase in the porous enamel’s scattering coefficient.
Energy Dispersive X-ray Spectroscopy (EDX or EDS) is used in conjunction with a Scanning Electron Microscope (SEM) for element analysis. Electrons from the x-ray photons hit atoms in the observed material, causing the material’s electrons to be ejected, and the electron vacancy is then filled by electrons from the higher shell. The resulting energy difference causes x-ray energy to be emitted. That x-ray energy is used to characterize the elements from which it is emitted [23]. The depth of EDS analysis depends on the strength of the primary x-ray beam energy sent to stimulate the material. The current study employed a beam energy of 20 kV. At this strength, the electrons can reach a depth of approximately 1 µm [24]. Thus the element analysis using EDX in this study was investigated within that depth of the enamel surface.
In previous studies, the amount of calcium and phosphate significantly decreased in demineralized enamel in both the surface area [25] and the subsurface area [21], reflecting the same results found here. No previous study is known to have observed the amount of fluoride in enamel after undergoing demineralization. As mentioned earlier, application of the fluoride-containing remineralizing agents in the current study caused the percentage of fluoride in the enamel to increase, and particularly so in the case of the fluoride varnish. The fluoride percentage increase from fluoride varnish was significantly higher than any of the other remineralizing agents. This higher percent increase may be a result of the fact that the fluoride varnish contains more fluoride (22,500 ppm fluoride ions) than CPP-ACPF (900 ppm fluoride ions). The hardness test in this study showed that the fluoride varnish also produced the highest hardness result, followed by the CPP-ACPF, and then the CPP-ACP. This might be because the first two reagents contain fluoride, so they can form fluorapatite or hydroxyfluorapatite, whereas CPP-ACP contains no fluoride and so can only form hydroxyapatite, which is not as hard as fluorapatite or hydroxyfluorapatite.
It is noteworthy that both CPP-ACP and CPP-ACPF possess an advantageous ability that fluoride varnish does not. CPP-ACP and CPP-ACPF contain peptide (CPP) in an alkaline supersaturated calcium phosphate solution (ACP). One molecule of this peptide can bind as many as 21 calcium ions or 14 phosphorous ions. The purpose of the peptide is to prevent the calcium and phosphate ions in the solution from precipitating into calciumphosphate, because the precipitate form is of no clinical use (Reynolds 1997). In contrast, the peptide-separated calcium and phosphate ions in the form of CPP-ACP or CPP-ACPF are able to enter carious lesions in the enamel to form hydroxyapatite or hydroxyfluorapatite. In this way, CPP-ACP or CPP-ACPF can deliver calcium and phosphate ions to the enamel due to the different concentration gradient [26–27]. In the acid condition, the peptide could maintain high concentrations of calcium and phosphate in the lesions [26–29].
The roughness of the demineralized enamel in this study increased insignificantly compared to the natural enamel, which corresponds to the results of previous studies [22, 30–31]. The AFM micrographs show many tiny peaks for both the demineralized and remineralized teeth. Each peak represents a porosity that the scanning probe investigated. The higher the peak, the deeper the porosity. The AFM probe is able to characterize the porosities because the probe diameter is smaller than 20 nm, while the diameter of the enamel porosities ranges approximately 21.27–191.50 nm. The enamel specimens in this study were submerged in pH4.4 demineralizing solution for 3 days, and the AFM probe determined that the resulting depth of the porosities was in the range of 100–200 nm (data not shown). By contrast, Yu et al. (2017) submerged enamel pieces in a pH5.0 acetate buffer demineralizing solution for 21 days and found that the resulting average depth of the porosities in the demineralized enamel was approximately 134 µm [32]. This difference in porosity depth seems to indicate that the type of acid used and the duration of acid exposure influence the degree of demineralization. After applying the remineralizing agents for 3 days in the current study, the roughness of the remineralized enamel was still similar to that of the demineralized enamel, and there was no significant difference in roughness between the different remineralizing agents. Thus all three remineralizing agents were able to increase the hardness of the demineralized enamel at the molecular level through formation of fluorapatite or hydroxyfluorapatite, although surface texture was not improved.