Effect of strontium fluorophosphate bioactive glass on color,microhardness and surface roughness of bleached enamel

Abstract

BACKGROUND:

Undesirable effects of tooth bleaching can alter the biomechanical properties of enamel.

OBJECTIVE:

To determine the influence of strontium fluorophosphate bioactive glass (Sr-FPG) on color, microhardness and surface roughness of enamel bleached with 35% hydrogen peroxide.

METHODS:

The labial enamel of 36 extracted intact human anterior teeth were divided into 3 groups ( $n=$ 12), group 1 (HP): bleaching with 35% hydrogen peroxide only, group 2 (Sr-HP): bleaching with Sr-FPG incorporated 35% hydrogen peroxide and group 3 (HP-SrFPG): bleaching with 35% hydrogen peroxide followed by remineralization with Sr-FPG. Four consecutive eight-minute applications of the bleaching gel were done twice in all the groups. Color change ( $\mathrm{\Delta }$ E), microhardness and surface roughness were evaluated at baseline, post-bleaching and post-remineralization using spectrophotometer, Vickers hardness tester and profilometric analysis respectively.

RESULTS:

The mean $\mathrm{\Delta }$ E among the groups was statistically similar ( $p>$ 0.05). Bleaching with HP significantly reduced microhardness ( $p<$ 0.05), whereas bleaching with Sr-HP and HP-SrFPG did not ( $p>$ 0.05). Post-bleaching microhardness in Sr-HP was significantly higher than HP-SrFPG ( $p<$ 0.05). An increased surface roughness was seen in Sr-HP bleached samples ( $p<$ 0.05).

CONCLUSION:

The addition of Sr-FPG to hydrogen peroxide significantly improved enamel microhardness than its use post-bleaching. An increase in surface roughness was seen post-bleaching with HP and Sr-HP.

1. Introduction

Favourable and immediate tooth whitening results make bleaching a conservative, cost-effective, easy to perform and popular treatment modality to manage discoloration [1, 2]. A wide array of tooth whitening products ranging from abrasives, antiredeposition agents, colorants, enzymes, peroxides and surfactants are available in the market. Among these, chemical whitening using peroxides has been practised with predictable success [2, 3]. Low concentration of hydrogen peroxide found in over-the-counter whitening products like toothpaste (0.1–1 wt%), mouth wash (1.5%) and chewing gum produces results which are inferior to professional in-office treatment and dentist-supervised at-home bleaching [1, 4]. Bleaching in dental practice involves topical application of an oxidative chemical, such as hydrogen peroxide, in concentrations ranging from 35–45% that results in an oxidation-reduction reaction releasing free radicals. These free radicals infiltrate through enamel and dentin, breaking down complex organic chromophore macromolecules into simple and lighter ones, that alter their light absorption and reflection [3]. Mechanical weakening of the tooth, post-bleaching sensitivity arising from breach in surface integrity of calcium phosphate crystals that make up the inorganic component of tooth and gingival irritation have been reported following the use of high concentration of peroxides ( $>$ 10%) [3, 4]. Several studies reported that minerals are lost from tooth and undesirable changes occur in enamel due to bleaching, leading to a decrease in enamel microhardness and an increase in surface roughness [5, 6, 7]. Pigments might adhere more easily to the rough surface of a bleached enamel, leading to an increased susceptibility to future staining resulting in further discoloration and an increased tendency towards bacterial adhesion [8, 9]. To compensate for the mineral loss and to reduce the ultramorphological defects caused by bleaching, several studies recommend inclusion of a remineralization agent either as part of the bleaching gel or as a pre- or post-bleaching regimen. Demineralized enamel facilitates ionic exchange that results in increased absorption of minerals which was previously lost due to bleaching.

A wide range of remineralizing agents such as fluoride, hydroxyapatite, casein phosphopeptide-amorphous calcium phosphate and bioactive glass (BAG) have been experimented and used as part of the bleaching regimen to prevent such adverse outcomes [10]. BAG consists of oxides of silica, sodium, calcium and phosphorus and has the ability to form a layer of hydroxycarbonate apatite on their surface in an aqueous medium. BAG has been experimented as an additive to improve bioactivity and mechanical properties of dental adhesives, glass ionomer cement and resin composites. They are also researched as an alternative to alumina in air abrasion, to remineralize white spot lesions, as bioactive agents for dentin-pulp complex regeneration and as tubule occluding agents for treating dentin hypersensitivity [11, 12]. Compared to the conventional silica-based BAG that degraded at a low rate and formed less apatite, phosphate-based BAG exhibited complete dissolution in aqueous media and offered the flexibility of incorporation of ions into its structure that enabled extension of its application in dentistry [13]. Among the various modifications of BAG, fluorophosphate bioactive glass (FPG) developed by Rajkumar et al., showed enhanced bioactivity compared to fluoride-free phosphate-based glass. Fluoride inhibits bacterial activity and enamel demineralization by its effective interaction with the tooth surface, thereby enhancing remineralization. Fluorophosphate bioactive glass showed enhanced apatite forming potential and thus holds the future for fluoride releasing biomaterials in dentistry [14].

Recently, Dhivya et al. synthesized strontium fluorophosphate bioactive glass (Sr-FPG) by incorporating 6 mol% of strontium oxide to fluorophosphate bioactive glass and recommended it for therapeutic use in teeth and bone. Strontium has the ability to replace calcium in hydroxyapatite, thereby converting it to a more acid-resistant compound. Strontium and fluoride exhibit synergism in apatite formation and remineralization of enamel [15]. But, the ability of Sr-FPG to remineralize bleached enamel has not yet been explored. Contradictory reports are also found regarding the efficacy of use of a remineralizing agent either in combination with the bleaching agent or after the bleaching procedure [16, 17]. Spectrophotometers are tools to assess discrepancy between tooth color following bleaching in a standardized, reproducible and quantifiable manner, with reduced subjective errors [18]. Microhardness assessments provide indirect evidence of mineral loss or gain by evaluating enamel hardness [19]. The change in surface roughness caused by loss of enamel can be quantified by surface profilometry [20]. Hence, this in vitro study aimed to determine the impact of Sr-FPG on color, microhardness and surface roughness of enamel bleached with 35% hydrogen peroxide using spectrophotometer, Vickers microhardness tester and contact profilometer respectively. The null hypothesis proposed was that using Sr-FPG as a remineralizing agent during and after the bleaching procedures will not affect the color, microhardness and surface roughness of enamel.

2. Materials and methods

The study protocol was duly submitted to the Institutional Review Board of SRM Dental College and approval was obtained (SRMU/M&HS/SRMDC/2022/PG/014). The ethical guidelines outlined in the Declaration of Helsinki (1964) and its ensuing amendments were followed in tooth sample collection. The teeth were extracted after securing patient’s written informed consent.

2.1 Preparation of experimental bleaching and remineralizing agents

The Sr-FPG glass composition 48P2O5-29CaO-3CaF2-14Na2O-6SrO was prepared through melt quenching technique at 1150 ${}^{\circ }$ C using chamber furnace (Kanthal, Hosur, India) as mentioned elsewhere [15]. The obtained sample was powdered in planetary ball mill (PM 100, Retsch, Germany) for 1 h at 300 rpm. 5.1 g of commercially available 35% hydrogen peroxide (Pola office $+$ Advanced tooth whitening system, SDI Ltd., Victoria, Australia) was mixed with 500 mg of Sr-FPG to obtain the experimental bleaching agent, Sr-FPG-modified hydrogen peroxide (Sr-HP). For application post bleaching, Sr-FPG was mixed with glycerol to make a slurry paste.

2.2 Sample preparation

This study is a first of its kind to evaluate the effect of Sr-FPG on bleached enamel as substrate. Since no previous literature is available to calculate effect size, sample size analysis was not done in this study. 36 extracted, intact human anterior teeth stored not more than one month in 0.2% thymol were used. The teeth were decoronated at the level of the cemento-enamel junction and the crown was embedded in a resin block exposing the labial surface. The enamel surfaces were polished with pumice using a prophy rubber cup at slow speed.

2.3 Grouping

The 36 tooth specimens were randomly divided into 3 groups of 12 teeth each based on the bleaching/remineralization regimen. The specimens in group 1 (HP) were bleached with 35% hydrogen peroxide according to the manufacturer’s instructions. The bleaching gel was left in place for a duration of 8 minutes and suctioned off using a surgical aspirator tip. This process was repeated 4 times following which the gel was rinsed off for 15 seconds. For group 2, Sr-HP was used as a bleaching agent, left in place for 8 minutes and suctioned off using a surgical aspirator tip. This process was repeated 4 times following which the gel was rinsed off for 15 seconds. Group 3 (HP-SrFPG) specimens were bleached according to the protocol followed for group 1, following which SrFPG-glycerol slurry was then applied on the enamel surface using an applicator tip, left in place for 5 mins and the paste was rinsed off for 15 seconds. All the samples were maintained at 37 ${}^{\circ }$ C in artificial saliva (Wet mouth, ICPA Health Products Ltd., Mumbai, India). The entire bleaching/remineralizing protocol was repeated after one week again in all the groups before final analysis.

2.4 Parameters analysed

Analysis of color, microhardness and surface roughness was done at baseline and finally at the end of the intervention.

2.4.1 Color analysis

A spectrophotometer (Vita Easyshade Advance 4.0, VITA Zahnfabrik, Bad Säckingen, Germany) with a spectrum range of 400–700 nm analysed color using Commission Internationale De L’Eclairage L*a*b* (CIELAB) system with L* denoting dark-light, a* green-red and b* blue-yellow. Color difference ( $\mathrm{\Delta }$ E) was calculated using the formula, ( $\mathrm{\Delta }$ L* ${}^2+\mathrm{\Delta }$ a* ${}^2+\mathrm{\Delta }$ b* ${}^2){}^{1/2}$ , where $\mathrm{\Delta }$ L*, $\mathrm{\Delta }$ a* and $\mathrm{\Delta }$ b* represent the difference between baseline and final L*, a* and b* values.

2.4.2 Enamel microhardness analysis

A Vickers microhardness tester (Micro HMV-G, Shimadzu, Kyoto, Japan) measured microhardness of samples as Vickers hardness number (VHN) following application of 0.1 N load for 15 s.

2.4.3 Surface roughness analysis

The stylus (tip radius of 2 $\mu$ m) of the contact profilometer (Surftest SJ310, Mitutoyo, Japan) was moved on the enamel surface from mesial to distal at a speed of 1.25 mm/s and pressure of 0.75 mN. Three successive measurements were recorded for each sample at different locations and the average surface roughness (Ra in $\mu$ m) was obtained.

2.5 Statistical analysis

The data was analysed for normality using Shapiro-Wilk test. Since the data was not normally distributed, non-parametric tests were used. Kruskal-Wallis test was used to test significant difference between the groups in all the tested parameters. Wilcoxon signed rank test was used to test significant difference between baseline and final analysis values under microhardness and surface roughness data in each group. Statistical significance was set at $p<$ 0.05.

3. Results

The mean $\mathrm{\Delta }$ E values of groups 1, 2 and 3 were 9.17, 7.57 and 8.11 respectively which were statistically similar ( $p>$ 0.05). The mean microhardness and surface roughness values of all the groups at baseline and final analysis is given in Table 1.

Table 1

Mean pre- and post-intervention microhardness (MH) and surface roughness (SR) values of all the groups

Tested parameters	Group 1 (HP)		Group 2 (Sr-HP)		Group 3 (HP-SrFPG)
	Pre	Post	Pre	Post	Pre	Post
MH
Mean $\pm$ SD	355.29 $\pm$ 22.65	309.91 $\pm$ 23.95	340.37 $\pm$ 54.29	357.37 $\pm$ 69.52 ${}^{\ast }$	342.33 $\pm$ 16.58	338.04 $\pm$ 29.01
Median	348.25	306.00	349.50	386.00	339.25	346.50
IQR	36.38	44.75	76.63	75.63	19.25	37.75
SR
Mean $\pm$ SD	0.55 $\pm$ 0.14	0.80 $\pm$ 0.70	0.57 $\pm$ 0.30	1.00 $\pm$ 0.45 ${}^{\mathrm{\#}}$	0.59 $\pm$ 0.27	0.63 $\pm$ 0.26
Median	0.51	0.54	0.56	1.03	0.55	0.52
IQR	0.25	0.83	0.46	0.67	0.48	0.42

${}^{\ast }$ denotes significant difference between the post-intervention microhardness of the groups ( $p<$ 0.05). ${}^{\mathrm{\#}}$ denotes significant difference between the pre- and post-intervention surface roughness under group 2 ( $p<$ 0.05). SD – Standard deviation, IQR – Interquartile range.

The baseline microhardness values were statistically similar among groups indicating even distribution of samples ( $p>$ 0.05). Bleaching with HP showed a significant reduction in microhardness from baseline ( $p<$ 0.05). Post-bleaching microhardness in Sr-HP and HP-SrFPG were statistically similar to baseline ( $p>$ 0.05). Intergroup comparisons showed that Sr-HP had a significantly higher post-bleaching microhardness compared to groups HP and HP-SrFPG ( $p<$ 0.05).

Baseline Ra values showed no significant difference among the groups indicating uniform distribution of samples ( $p>$ 0.05). An increase in surface roughness was seen following intervention in all the groups, but it was significant only in Sr-HP samples ( $p<$ 0.05). Intergroup comparison revealed statistically similar post-bleaching Ra values among the groups.

4. Discussion

Sr-FPG as a remineralizing agent did not impede the tooth whitening potential of hydrogen peroxide, hence can be safely indicated during the bleaching procedure. Therefore, a part of the null hypothesis stating that use of Sr-FPG as a remineralizing agent during and after the bleaching procedures will not affect the color of enamel is accepted. Acidic pH and higher peroxide concentration causes morphologic and physical changes in enamel [21]. Since the bleaching gel used here has a neutral pH (as mentioned by the manufacturer), the changes in microhardness could be the result of higher peroxide concentration. These results are in accordance with previous studies which reported a decrease in enamel microhardness following bleaching with hydrogen peroxide [5, 6, 22]. The results of the current study showed that Sr-FPG is capable of remineralizing bleached enamel. Earlier research reported comparable results following the use of BAG during bleaching [6, 7, 23, 24]. On the contrary, El-Damanhoury et al. observed that BAG exhibited inferior ability to restore the reduced microhardness of bleached enamel compared to calcium silicate-sodium phosphate-fluoride salts used in the study [5].

The remineralizing ability of Sr-FPG used in the present study could be attributed to its increased mineral deposition and enhanced apatite forming ability [15]. In an aqueous environment, a rapid exchange of sodium ions with H ${}^{+}$ cations (in the form of H ${}_3$ O ${}^{+}$ ) occurs that releases calcium and phosphate ions from the glass. The pH of Sr-FPG, as stated by Dhivya et al. is 6.9, that increases transiently with the release of sodium ions from the glass, which in turn encourages precipitation of additional calcium and phosphate from it [15]. A carbonate enriched hydroxyapatite layer crystallizes on the surface thereby preventing any further demineralization [6, 25]. The pH of Sr-FPG might have also buffered hydrogen peroxide thereby decreasing the deleterious effects of demineralization [15]. Considering that the pH of both the bleaching gel and Sr-FPG are in the neutral range, the pH of the combination (Sr-HP) was not tested in this study.

Complete disappearance of the demineralized enamel morphology was observed with the use of Sr-FPG as a remineralizing agent by Dhivya et al. Compared to commercial BAG, Sr-FPG presented an elevated Ca/P ratio [15]. This property could compensate for the reduction in Ca/P ratio of enamel following bleaching [5]. Studies have shown that strontium and fluoride exhibit synergistic effect in promoting remineralization [26, 27]. Though the use of HP-SrFPG could restore the microhardness values equivalent to baseline, it was not superior to that of Sr-HP. The enhanced microhardness seen with Sr-HP clearly demonstrates that single step bleaching-remineralization was more effective than remineralization after bleaching. This is desirable as it reduces the need for additional dental visits and necessary patient compliance. These results are in accordance with previous reports which showed that combined use of hydrogen peroxide and BAG prevented mineral loss and preserved enamel. Deng et al. proved that using BAG with hydrogen peroxide brought better protective effect than its use pre- or post-bleaching [6]. Khoroushi et al. observed that incorporating biomaterials like BAG in the bleaching agent effectively remineralized and reduced enamel microhardness changes subsequent to bleaching [28]. Yang et al. inferred that combination of hydrogen peroxide and BAG significantly increased enamel microhardness following bleaching and minimized enamel surface damage during bleaching procedure [24]. On the contrary, Saffarpour et al. showed that the use of BAG before bleaching offered greater enamel protection compared to its use during bleaching [29].

The Ra of Sr-HP treated samples was elevated, which could be due to the presence of residual glass particles and the recently formed hydroxycarbonate apatite layer [25]. Marques et al. observed significant increase in surface roughness and altered wettability following BAG coating on Titanium samples [30]. El-Damanhoury et al. observed a threefold increase in surface roughness of enamel following bleaching. They also noted that remineralization with BAG-based NovaMin significantly increased the surface roughness of bleached and non-bleached enamel [5]. The results are in contradiction to previous reports which have shown that BAG reduces the surface roughness of bleached enamel [10, 31]. The compositional and particle size difference between the standard BAG and the Sr-FPG used in the present study could have contributed to the difference in results [15]. This difference could also be due to enamel preparation such as an additional polishing step following bleaching to smoothen the enamel surface [10]. Polishing enamel following bleaching will reduce the surface roughness caused by bleaching. But it will also result in loss of tooth structure. However, polishing enamel bleached with Sr-HP will decrease the surface roughness as well as prevent further tooth structure loss, as the enamel might be protected by a layer of bioactive glass on the surface. Vieira-Junior et al. used Novamin in a similar study and inferred that this group showed the least post-remineralization surface roughness. A notable addition of a fluoride agent in the form of sodium monoflorophosphate to BAG in that study could have influenced the results [22]. Since considerable differences were elicited in the microhardness and surface roughness of enamel between the groups, the null hypothesis is rejected.

An increase in surface roughness could increase the susceptibility to bacterial adhesion [8]. The bacterial enzyme inhibitory action of fluoride component in Sr-FPG along with the antibacterial property of the original glass formulation, will counteract any bacterial adhesion if it occurs [32, 33]. In addition, the presence of strontium ions in Sr-FPG could inhibit bacterial metabolism, growth and reproduction, cell wall synthesis and chromosomal replication [34]. Synergistic anticaries activity of fluoride and strontium has been reported by previous studies [27]. Future studies should assess the bacterial adhesion characteristics of the newly developed Sr-FPG. The morphologic alterations of enamel and dentin following this modified bleaching protocol and their long-term bond durability to resin composite restorations need to be studied. Bleaching-induced change in color and surface texture of resin composite restorations also needs to be investigated. The cytotoxicity of this novel combination of bleaching agent and Sr-FPG has to be assessed. Dentist-supervised in-office and at-home bleaching procedures are widely and successfully practised globally. Caution must be exercised while using higher peroxide concentrations found in over-the-counter commercial products. Remineralization regimen must be recommended as part of bleaching treatment to minimize the risks associated with dental hard tissue damage. The results of the present study show that Sr-FPG with its higher remineralizing ability and enhanced bioactivity could mitigate the deleterious effects of bleaching on enamel microhardness and serve as a potential biomimetic additive to hydrogen peroxide during bleaching procedures. The findings of this study must be interpreted with caution as these results were elucidated using limited sample size. In addition, discretion must be exercised while extrapolating the results of this in vitro study to the clinical scenario.

5. Conclusion

Within the limitations of this in vitro study, it can be concluded that single-step combined use of Sr-FPG and hydrogen peroxide notably improved tooth whiteness as well as protected enamel from the deleterious effects of bleaching.