Effects of Experimental Nanohydroxyapatite Gel on Enamel Surface After Bleaching Open Access

Dental bleaching is considered safe, efficient, and user-friendly, providing acceptable esthetic outcomes as the most conservative treatment for discolored teeth compared to restorative treatment methods.¹  In recent years, several manufacturers have produced and promoted various tooth-bleaching products with different ingredients. Bleaching agents containing high concentrations (25-40%) of hydrogen peroxide (HP) or carbamide peroxide (CP) can be applied in the office by a dentist. Bleaching agents with lower concentrations of HP (3-7%) or CP (6-20%) can be applied at home by a patient under a dentist's supervision.² 

Dental bleaching involves the release of oxygen and perhydroxyl free radicals by HP. Free radicals diffuse through the tooth structure, breaking down large molecules (chromophores) into smaller molecules, changing the reflection of the wavelength of light and the tooth color.³  HP can demineralize enamel and dentin due to its acidic pH.^4,5  The mineral content of the enamel decreases with the use of high concentrations of peroxide, while the minerals that detach from the surface cause deformation of the prismatic and interprismatic regions, resulting in increased porosity and microcracks, decreased protein content, calcium loss, and altered Ca/P ratios.^6–8  Consequently, there is a decrease in hardness and an increase in surface roughness.⁹  Since bleaching is a chemical reaction, the process and effect of these reactions vary depending on the conditions of the reaction environment and the catalyst.¹⁰  Therefore, the effectiveness of vital tooth bleaching depends on factors such as the concentration of the bleaching agent, pH, application time, and chemical additives.^1,7,11  Other factors include patient-specific characteristics such as the effects of saliva and the accessibility of remineralizing agents.¹¹ 

Several treatments are recommended to follow tooth whitening. Remineralizing agents have been added to bleaching agents to lessen the undesirable effects of the treatment. For this reason, new bleaching products and technologies, such as nano additives and alternative carrier systems, have been developed. Furthermore, these remineralizing agents can be applied to the tooth surface after bleaching to minimize the adverse effects of these treatments.^12–14  These agents are combined with bleaching agents to reduce changes in mineral enamel content.¹⁵  For this purpose, remineralizing agents such as fluoride,¹⁶  potassium nitrate,¹⁷  amorphous calcium phosphate (ACP),¹⁸  chitosan,¹⁹  and hydroxyapatite (HA)²⁰  can be used to minimize the adverse effects of bleaching treatments on the enamel surface.

Human tooth enamel is composed of HA crystals (Ca₁₀(PO₄)₆(OH)₂) containing 17.4 weight percentage (wt%) phosphorus and 37.1 wt% calcium, hydrogen, and oxygen.¹²  Although calcium phosphate exists in many forms in nature, HA is the most stable.²¹  Synthetic HA can mimic the morphology, composition, and size of natural enamel apatite.²²  When reduced to nano-size, it can adhere to the surface of the tooth to cover grooves and pores.²³  Demineralized surfaces have a highly porous enamel surface, thus allowing greater penetration of n-HA. Nano-sized HA particles act as a template with calcium and phosphate ions to promote crystal growth and integrity. Biopolymers with remineralizing or anti-erosion potential are also used for this purpose.²⁴ 

Chitosan is an important biopolymer used in pharmaceutical and biomedical processes such as drug delivery, tissue engineering, and cosmetic product production. This naturally occurring molecule is produced when chitin is deacetylated.^25–27  Chitosan works as a carrier due to its ability to form a thin film.²⁸  It protects the tooth surfaces from demineralization or erosive attacks by forming a stable barrier on the enamel or dentin.²⁹  In a previous study, the addition of 2% chitosan to bleaching gels was reported to reduce surface roughness by binding minerals such as calcium and phosphorus to the enamel surface.¹⁹ 

The purpose of this study was to investigate the effect of chitosan-added experimental n-HA gel on the physical and morphological changes of the enamel surface after the use of two vital bleaching agents. The research hypothesis was that the use of chitosan-added n-HA gel after bleaching would not change bleaching efficiency and would result in an increase in microhardness and a decrease in roughness on the enamel surface.

Nano-sized HA particles were obtained by a high-temperature oxidation method³⁰  using micro-sized HA powder (Sigma Aldrich Co, Madrid, Spain). In the production of n-HA, chitosan (Sigma Aldrich Co.) was used as an additive. Titanium powder (88 wt%), chitosan (2%), and 10% distilled water were added to reach 100 wt%. The solution was thoroughly mixed sonically for 15 minutes and magnetically for one hour until it became homogeneous. It was then allowed to dry in an oven at 80°C for 24 hours. The resultant mass was transferred to an alumina crucible and placed into a muffle furnace (ProTherm, Lebanon, TN, USA), heated to a temperature of 800°C by heating at a rate of 10°C/minute and burned for 30 minutes. The synthesized n-HA particles were characterized by X-ray diffraction (XRD) and SEM.

Chitosan-added n-HA powder was mixed with phosphate-buffered saline (PBS) at a ratio of 1 ml to 1 gram to act as a carrier for application to the tooth surface. PBS is a valuable buffer solution that mimics the ion concentration, osmolarity, and pH of human body fluids. PBS is used to simulate the biodegradation and mineralization processes of HA.³¹  The phosphate ions in PBS combine with the calcium ions of synthetic HA, resulting in the production of hydroxyapatite-like crystals on the tooth surface.^32,33 

For PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ salts were added to 1 liter of distilled water and mixed until a homogeneous mixture was obtained. HCl was used to adjust the pH value. The final pH value of the obtained solution was 7.3.

Fifty-two human maxillary incisors extracted for periodontal reasons without caries, cracks, or hypoplastic defects were used. The materials used in our study and their contents are shown in Table 1. As a result of a G*Power (University of Düsseldorf, Düsseldorf, Germany) analysis, when the effect size was taken as d=0.50 for microhardness, surface roughness, and color change measurements, a minimum sample size of four specimens per treatment group was determined at 95% power and 5% type 1 error rate.

The root surfaces of the teeth were cleaned of debris using a periodontal curette, brush, and fine-grained fluoride-free polishing paste. For each specimen, the crown and root were separated from each other two millimeters below the cementum-enamel junction (CEJ) using a Mecatome T180 (Presi, Eybens, France) device and a diamond disk (Metkon, Bursa, Turkey) under water cooling. The enamel blocks were cleaned in an ultrasonic bath with deionized water for 15 minutes to remove any residues.³⁴ 

The dental crowns were embedded in acrylic resin with their facial surfaces exposed. Each specimen was polished with 800-, 1000-, and 1200-grit silicon carbide abrasive papers at 50 rpm for 10 seconds and finished with a polishing machine (Minitech 233, Presi, Eybens, France). The specimens were placed on a flat surface to obtain standardized enamel surfaces and create a parallel area (7 mm in diameter) on the facial surface.⁷  A new silicon carbide abrasive paper was used for each specimen, and all specimens were sanded for the same duration in the same direction with the same force. The tooth surfaces were then examined for dentin exposure with a loupe at 3.5× magnification.³⁵  All specimens were stored in 37°C distilled water for 24 hours.

The teeth were randomly divided into four groups (n=13) and treated as follows:

Group B — Biowhiten In-office 40% n-HP (BioWhiten, Biodent Ltd., Istanbul, Turkey) was applied for a total of 50 minutes (five applications for 10 minutes each) according to the manufacturer's instructions.

Group O — Opalescence Boost PF 40% HP (Ultradent Products Inc., South Jordan, UT, USA) was applied for a total of 50 minutes (two applications for 10 minutes each) according to the manufacturer's instructions.

Group BN — Biowhiten In-office 40% n-HP (Biodent Ltd.) was applied, similarly to Group B. After bleaching treatments, the gel was rinsed off the enamel surface with running distilled water for 30 seconds. Next, a 1–1.5 mm thick layer of the experimental chitosan-added n-HA gel was applied to cover the buccal surface of the specimens. A microbrush was used with circular movements to agitate the gel on the tooth surface for five minutes before rinsing under distilled water for 30 seconds.

Group ON— Opalescence Boost PF 40% HP (Ultradent) was applied, similarly to Group O. The experimental chitosan-added n-HA gel was applied in the same manner as in Group BN. Afterward, the gel was rinsed off the enamel surface with running distilled water for 30 seconds.

All specimens were kept in distilled water in an oven at 37°C throughout the treatment period. Three specimens from each group were separated for SEM and EDS analyses. Surface roughness, microhardness, and color change were measured on the remaining 10 teeth in each group before, immediately after, and one week after bleaching. Surface roughness was measured first, followed by surface microhardness. Microhardness indentations were made in an area away from the reading path of the profilometric evaluation.

Color measurements were determined using a spectrophotometer (Vita EasyShade Advance 4.0, Vita Zahnfabrik, Germany). A circular area 5 mm in diameter was measured in the middle third of the specimen. In accordance with the manufacturer's instructions, the spectrophotometer was calibrated using a white reflectance standard before each measurement session. Three readings were obtained for each specimen, and the average reading was included in the analysis. The measurements were made before, immediately after, and one week after bleaching, under the same lighting conditions at the same time of day. The measurements were recorded as mean L* (lightness or darkness), a* (redness or greenness), and b* (yellowness or blueness) values in the International Commission on Illumination (CIE) L*a*b* system. The L*a*b* values measured at baseline and immediately after bleaching (ΔE₁) and at baseline and one week after bleaching (ΔE₂) were calculated as ΔE values. Total color changes were calculated using the following formula:³⁶ 

ΔE* is the color difference between two objects. In terms of clinical detectability, the human eye can perceive color differences when ΔE is greater than a certain threshold.³⁷  An inexperienced observer can notice the color difference when ΔE>2.³⁸ 

Surface roughness (Ra) was measured using a contact profilometer (Mahr M300C, Carl-Mahr, Germany). Three measurements were taken at different locations on each specimen, rotating the specimen clockwise by approximately 120° after each measurement. The surface roughness (in μm) was calculated using the arithmetic mean of the measurements. A cut-off length of 0.25 mm, a reading length of 1.25 mm, and a speed of 0.05 mm/second were used.

Surface microhardness was determined using a Vickers microhardness tester (HMV-2, Shimadzu, Kyoto, Japan) with a load of 300 grams for 15 seconds. The final microhardness values (VHNs) were obtained as arithmetic means of three indentations, 100 μm apart, made in the central region of each specimen.

Three specimens from each treatment group, as well as unbleached specimens, were analyzed using SEM/EDS. Specimen surfaces were coated, under vacuum, with a gold-palladium mixture using a sputtering device (Quorum Technologies Ltd, East Sussex, UK) to increase conductivity. Specimens were analyzed by SEM/EDS (Evo LS10, Zeiss, Germany) at 10 kV voltage and 1000× and 5000× magnification. Imaging preferences were concentrated near the center of the specimens. A point analysis was performed to examine the presence of n-HA on the tooth surface. The calcium weight, phosphorus weight, and calcium-phosphorus ratio obtained from each specimen were recorded as percentages.

Statistical analysis was performed using SPSS 23.0 for Windows (IBM Corp, Armonk, NY, USA). The mean (± standard deviation) values for continuous variables were provided for descriptive statistics before and after the bleaching process. For microhardness and Ra results, variance analysis was applied through repeated measurements, and Bonferroni testing was used for pairwise comparisons. A Welch ANOVA (for unequal variances) was used to measure the statistical results of the color change (ΔE). Statistical significance was determined at a confidence level of 0.05 for all analyses.

After characterization by XRD, the experimentally synthesized powder was confirmed to be n-HA. Figure 1 shows the characteristic diffraction peaks of the n-HA sample. The crystal structure in the figure is exactly in accordance with the characteristic index values defined by the Joint Committee on Powder Diffraction Standards (JCPDS) for HA (JCPDS no. 09-0432).³⁹ 

The SEM images of chitosan-added n-HA are shown in Figure 2. It was observed that the HA particles were nanosized, and the size of the particles produced by chitosan-added n-HA was less than 50 nm.

The ΔE values of each group immediately after and one week after bleaching are shown in Table 2. There were no statistically significant differences among the treatment groups at either time period (both p>0.92). In all groups, the color differences at both intervals were noticeable to an inexperienced observer (both ΔE₁ and ΔE₂ >2).³⁸ 

The mean surface roughness values (Ra) and statistical differences among the groups are shown in Table 3.

In the measurements made immediately and one week after bleaching, the lowest and highest Ra values in all groups were determined to be between 0.11 μm and 0.15 μm. In the literature, a clinically acceptable Ra is 0.2 μm, the critical value for plaque retention and accumulation.⁴⁰  The results of this study showed that Ra for all groups was below 0.2 μm.

Before bleaching, all groups exhibited statistically similar surface roughness (p=0.202). After bleaching, a significant increase in Ra value was observed only in Group O (p<0.001). Surface roughness among Groups B, BN, and ON remained unchanged across all time intervals (all p>0.075)

The microhardness results are shown in Table 4. Before bleaching, there were no statistically significant differences among the four treatment groups (p=0.155). Microhardness decreased significantly in all groups immediately after bleaching (p<0.001), but did not decrease further after one week of bleaching (p>0.05). Group O showed the lowest microhardness values both immediately and one week after bleaching (p<0.001).

The unbleached enamel surface is shown in Figures 3A and 3B at 1000× and 5000× magnification. Group B showed a more regular surface structure than Group O (Figure 3C and 3D). Crack-like formations along the surface were observed in Group O (Figure 3E and 3F). Groups BN and ON showed more regular enamel surfaces than the other groups (Figures 3G-3H and 3I-3J).

The chemical compositions of all groups in terms of O, F, Na, P, Cl, and Ca elements in wt% and Ca/P ratio appear in Table 5. The lowest Ca/P ratio was observed for Group O.

At a higher magnification (5000×), single and clustered particles were observed on the enamel surfaces of Groups B, BN, and ON (Figures 3D, 3H, and 3K). The distribution of these particles was not homogeneous. Point analyses were performed on the particles. The SEM images and EDS elemental content (%) of the analyzed particles appear in Figure 4. The chemical elements Ca, P, and O were found in the formula Ca₁₀(PO₄)₆(OH)₂ of the HA structure. In addition, the size of these particles was less than 50 nm. Therefore, the particles observed could belong to the n-HA structure.

Based on the results of this study, the research hypothesis was accepted. The use of the experimental chitosan-added n-HA gel after bleaching treatments increased the microhardness and decreased the roughness of the groups without negatively affecting the color change.

One aim of our study was to evaluate whether hydroxyapatite influences the outcome of tooth bleaching. Nano-hydroxyapatite (n-HA) does not penetrate the dentin layer and does not change the effectiveness of the bleaching agent.⁴¹  Studies have shown that varying concentrations of HA used during or after office bleaching do not affect the bleaching ability of the gel, with the primary bleaching effect attributed to hydrogen peroxide (HP).⁴²  Similar to the results of previous studies,^41,42  all groups tested in our study caused significant discoloration of the enamel surfaces, and the application of chitosan-added n-HA gel after bleaching did not change this effect. In addition, the ΔE value measured one week after bleaching was lower than the ΔE value measured immediately after bleaching, though the difference was not statistically significant. The decrease in ΔE could be attributed to the development of a more opaque tooth surface due to immediate dehydration.

In our study, both vital bleaching agents contained 40% HP and initially had a neutral pH. A significant increase in surface roughness was observed for Opalescence Boost 40% PF treatment likely because of its more acidic pH than that of Biowhiten In-office 40%. Similar to our results, Lugo-Varillaset and others⁴³  reported a significant increase in Ra values after bleaching with 40% HP. It has been reported that the pH level of the bleaching agent is an important feature in avoiding roughness on the enamel surface.⁴⁴  Biowhiten In-office 40% contains n-HA, which may cause ion precipitation onto the tooth surface and consequently reduce Ra and prevent material loss. Experimental chitosan-added n-HA gel application decreased the Ra of the enamel surfaces after the Opalescence Boost 40% PF. Similar to our results, Nishio and others⁴⁵  evaluated the effect of n-HA solution on enamel surfaces bleached with Opalescence Boost 40% PF in vitro and observed the most regular enamel surfaces in the groups immersed in n-HA solution. Similarly, our study determined that the experimental chitosan-added n-HA gel application after both bleaching treatments caused more regular enamel surfaces in SEM images.

In the present study, microhardness decreased significantly for all groups immediately after both bleaching agents were used, with the lowest values found in Group O. Similar to our study, Moradi and others⁴⁶  compared Opalescense Boost 40% PF with other bleaching gels and reported that the lowest microhardness value after bleaching was in the Opalescense Boost 40% PF group. The decrease in enamel surface microhardness in the Opalescence Boost groups might have been due to the low levels of phosphorus and calcium ions and high concentrations of sodium and chlorite. The use of experimental chitosan-added n-HA gel increased the microhardness values of enamel surfaces after both bleaching treatments. Similarly, Coceska and others⁴⁷  reported that enamel substance loss and morphological changes occurred after whitening treatment (the effects were reversible) due to the use of n-HA-containing pastes. Another study, by Scribante and others,⁴⁸  showed that a remineralization treatment with a HA-based prophylaxis paste after bleaching effectively increased the microhardness of the enamel. Similarly, Freiria and others⁴⁹  investigated the effect of n-HA-induced remineralization of artificial white spot lesions after bleaching with 10% CP and reported that n-HA positively affected remineralization by increasing surface microhardness while not changing the esthetic result.

In the literature, the deposition of nanocrystals promoted by n-HA appears to form a replacement layer of calcium and phosphate ions on demineralized surfaces.⁵⁰  In our study, specimens treated with Biowhiten in-office 40% n-HP containing nanohydroxyapatite exhibited higher Ca/P values compared to those treated with Opalescence Boost 40% PF. However, with only three specimens per treatment group, these results must be viewed cautiously. Further study is indicated.

Studies in the literature have revealed that bleaching agents containing high levels of HP cause the appearance of irregular areas on the enamel surface.^47,51  In our SEM analysis, we observed that the group treated only with 40% HP bleaching gel exhibited areas of damaged enamel (Figure 3E and 3F). These results are consistent with the literature.

Nano-hydroxyapatite has been shown to play a critical role in the remineralization of enamel due to its similarity to the mineral structure of teeth, bioactivity, and biocompatibility.⁵²  Notably, n-HA particles can aggregate together, maintaining their crystalline structure to form a stable prismatic structure in the enamel.⁵³  Studies have shown that the application of remineralization agents together with bleaching agents causes mineral precipitation and forms a protective layer on the enamel surface.^47,51  Similarly, in the SEM images examined in this study, single and multiple particles adhered to the enamel surfaces in the groups receiving the experimental chitosan-added n-HA gel. The EDS point analysis obtained from these particles showed that these crystallites, with a size of less than 50 nm, contained Ca, P, and O elements. According to the literature, tooth enamel is primarily composed of HA crystals ranging in size from 20 to 40 nm.⁵⁴  The n-HA crystals produced in our study conform to these established size ranges.

The application time of experimental chitosan-added n-HA gel (five minutes)⁵⁵  was sufficient to obtain satisfactory results on the enamel surface. This finding suggests a suitable indication for the clinical use of these agents. For treatment purposes, these agents can be combined with bleaching treatments without adversely affecting the provider's or patient's time.

This in vitro study does have some limitations. First, the specimens were not stored in artificial saliva and were not subjected to pH cycling, which may reduce the effectiveness of chitosan-added n-Ha gel. Further, the gel was applied only once, rather than repeatedly at different times. In future studies, the effects of experimental chitosan-added n-Ha gel on dentin sensitivity and remineralization on enamel erosion should be investigated.

When used after bleaching, an experimental chitosan-added n-HA gel enhanced the physical and morphological properties of the enamel surface without compromising the effectiveness of two 40% hydrogen peroxide bleaching agents. Therefore, chitosan-added n-HA shows great potential as a post-bleaching treatment material.

This study was funded by Bezmialem Vakif University Research Fund (Project No.: 2021040). This study was presented at the CED/NOF IADR 2023 Oral Health Research Congress.