The significance of cosmetic dental procedures has grown in recent years as a direct result of the modern concept of smile aesthetics. This concept places an emphasis on having teeth that are white, well-aligned, and framed by the gums and lips (1). Discoloration of the teeth that is visible to the naked eye can have a detrimental outcome on a person's life. As a result, tooth bleaching is consistently ranked as one of the highest priorities for patients who prioritize having an attractive smile (2, 3). Bleaching toothpastes and professional oral prophylaxis that involve scaling and polishing, as well as in-office bleaching, are the various ways that tooth color can be improved (4–6). Current advancements in technology have made it feasible for new aesthetic materials to exist as well as for old methods, such as tooth bleaching, to become more frequently employed (2, 7). One of the most prevalent treatments performed today in the field of cosmetic dentistry is tooth bleaching, which is a process that is widely accepted as being effective in the management of discoloured and stained teeth (6, 8). The active component of the bleaching agents that are now in use are variants of peroxide, and the amounts of each component that constitutes the bleaching agent's active component might vary. They cause the chromogens in the enamel and dentine to become more transparent, which lightens the color of the tooth as a whole (4). It is common knowledge that the application of bleaching treatment can alter the morphological appearance of mineralized deposits (9).

Because enamel is the surface stratum of the tooth, it is more vulnerable to the plethora of variables and circumstances that may be found in the oral cavity. These elements can include bacteria, acids, plaque, and food particles. Hydroxyapatite, a crystalline form of calcium phosphate, makes up the vast bulk of enamel. Hydroxyapatite is a mineral that may be found in teeth. Enamel is the substance that gives teeth their characteristic white color. In spite of the fact that it is the most resistant to demineralization of any tissue in the human body, the bone is constantly losing its minerals (10). The rate of demineralization is accelerated in acidic surroundings, and one of the most common ways this might happen is through the consumption of meals that contain both acid and sugar (11). Alternatively, saliva, antibacterial agents, and ions all contribute to the process of remineralization (such as fluoride, calcium, and phosphate). Due to the extreme fragility of the equilibrium that occurs between the processes of continual demineralization and remineralization, early carious lesions are often the outcome when this balance is disrupted for a lengthy period of time (12, 13). Changes in surface microhardness, color, and lowering of the concentration of carbonate, phosphate, or calcium in enamel may occur. These changes in tooth layers' micromorphology may be associated with the constituents of the bleach, the concentrations of those agents, the pH levels at which they are used, and the various application methods (12–17).

A number of different devices, such as “Scanning Electron and Polarized Light Microscopy”, “Micro Energy-Dispersive x-Ray Fluorescence Spectrometry”, “Fourier Transform-Raman (FT-Raman) Spectroscopy”, & “Atomic Absorption Spectroscopy (AAS)”, are commonly used to study surface changes (6).

When enamel microbiopsy is performed, the use of the method of colorimetric spectrophotometry is beneficial in quantifying the minerals that are extracted from the dental substrate without causing a significant amount of damage to the tooth structure (15). Alterations in the surface's morphology can also be seen through the use of SEM.

Recent in vitro and in situ studies have demonstrated that remineralization agents like nano-hydroxyapatite and casein phosphopeptide can effectively restore mineral content post-bleaching without compromising whitening efficacy (18, 19).

The purpose of this research was to apply the colorimetric spectrophotometry to investigate the reduction in mineral content that occurred after bleaching with three distinct types of bleaching chemicals and scanning electron microscopy to investigate the alterations that occurred on the surface of the material.

In this in vitro laboratory study, ninety (90) human extracted premolar teeth were used. These teeth were obtained from the Department of Oral and Maxillofacial Surgery of Rajarajeshwari Dental College and Hospital, Bengaluru, India. The teeth were extracted for orthodontic or periodontal purposes, were non-carious, and had no enamel or dentin defects. Following extraction, the teeth were thoroughly cleaned and disinfected before being stored in a normal saline solution until further use. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Ethical Committee of Rajarajeshwari Dental College and Hospital, Bengaluru, India.

The practice of bleaching, which dates back more than a century, has been around for quite some time (18–20). As a result of the recent surge in popularity of cosmetic dentistry, many individuals are now opting for bleaching procedures to enhance their dental aesthetics. When vital teeth are bleached, the enamel surface is exposed for a prolonged duration to gels rich in oxidizing agents. The exposure duration depends on the formulation used, which raises concern about potential enamel damage (21–25).

The structural integrity of organic enamel components, including collagen and proteins, can be compromised by oxidizing agents used in bleaching (22, 26, 27). Studies have reported decreased micro-tensile strength, fracture resistance, increased porosity, surface roughness, fluoride loss, mineral loss, altered Ca/P ratio, and organic matrix disruption (28, 29). These effects support the hypothesis that the chemical agents in bleaching formulations may weaken the mineralized structure of teeth (23, 30–34).

Surface microhardness assessment via the Vickers test has proven effective in evaluating enamel changes post-bleaching (23, 24, 31). Mechanical changes vary with testing parameters such as load, dwell time, and indent location (35–37).

Since demineralization was found to be localized primarily to surface enamel, spectrophotometric chemical analysis was selected as a minimally destructive tool for assessing mineral changes. Enamel structure and composition are known to vary regionally depending on its anatomical position, leading to localized differences in mineral response (24, 25, 37–39).

In this study, the enamel was segmented into three parts to independently assess calcium, phosphorus, and micro-morphological changes via SEM. The bleaching agents compared were the McInnes solution, modified McInnes solution, and Pola Office bleach. Previous studies confirm that peroxide-based bleaching agents alter the inorganic content of enamel, reducing Ca and P concentrations and disrupting enamel crystals (26).

Only a limited number of studies have specifically quantified mineral loss using the same bleaching agents as used here. Spectrophotometric analysis provides a novel, reproducible technique for enamel microbiopsy, offering a practical way to evaluate mineral composition without compromising structural integrity (18, 21).

The analysis revealed that all groups experienced a measurable reduction in mineral content post-bleaching, regardless of the agent used. Hydrogen peroxide, a common component in all three agents, acts by generating reactive oxygen species (ROS) that degrade pigmented molecules (chromophores) (30). Exposure of enamel to acidic conditions leads to dissolution of hydroxyapatite by hydrogen ions (31).

Group-wise comparisons indicated that Group 1 (McInnes solution) exhibited the greatest mineral loss, which is attributed to its content of 30% hydrochloric acid. This is consistent with findings by Tezel et al., who demonstrated enamel decalcification with HCl concentrations between 18% and 30% (31). Choi et al. similarly reported notable enamel etching within two minutes of HCl application in vitro (32).

To minimize HCl's erosive effect, Chen Hua Ji et al. substituted 20% sodium hydroxide (NaOH) in the modified McInnes solution (26). The modified version used a 1:1 ratio of 30% H₂O₂ and 20% NaOH, which increased the oxidative efficiency of hydrogen peroxide in alkaline media while reducing enamel damage. Their findings — that modified McInnes produces less enamel damage — align with those of the present study.

Mineral loss observed in the Pola Office group was comparable to the other two groups. Its high 35% H₂O₂ concentration results in a low pH of ∼4.5, contributing to its aggressiveness. Tezel et al. also noted that higher peroxide concentrations extract more Ca²⁺ ions, increasing enamel susceptibility (33).

After treatment, teeth were stored in artificial saliva to simulate oral conditions. Remineralization was observed over 7 and 14 days, supporting prior findings by Haleh Heshmat et al., which attribute this to phosphate and calcium ion saturation (40). This super saturation promotes both resistance to demineralization and enhances natural remineralization potential (37). However, in vivo, saliva alone may be insufficient, necessitating additional remineralizing agents after bleaching.

SEM analysis showed that Group 1 had the most severe surface alterations, corroborating the results of Rajesh A. G. et al., who linked extensive morphological damage to HCl (37). The findings of Ji Chen et al. on the milder effects of modified McInnes were also validated (26). In the Pola Office group, variable surface roughness and cracking were noted. These outcomes are consistent with Souza RO et al., who linked high H₂O₂ concentrations to enamel chemistry and morphology changes (30).

Both SEM and spectrophotometric findings confirm that enamel damage post-bleaching is influenced by agent type, pH, and application duration (39, 41, 42). These results align with prior enamel studies (43–45).

This study has certain limitations that must be considered when interpreting the findings. First, it was conducted exclusively under in vitro conditions. Although artificial saliva was used to simulate the oral environment, it does not fully replicate the complex biological reality of the oral cavity, including dynamic salivary flow, enzymatic activity, biofilm interactions, and mechanical forces such as mastication. These factors may substantially alter enamel demineralization and remineralization in vivo.

Second, the sample size within each subgroup was relatively small (n = 10 per time point). A formal power analysis was not performed, and the limited sample size may have reduced statistical power and increased variability. This is reflected in some groups, particularly in phosphorus concentration measurements, where standard deviations were high. Consequently, there remains a risk of Type II error and overestimation of certain effects.

Third, although standardized bleaching protocols were employed, subtle operator-dependent variations and baseline differences in enamel composition between teeth cannot be fully excluded as potential confounders.

Fourth, the study assessed only short-term changes (up to 14 days) in enamel mineral content and surface morphology. Long-term effects, including the potential for progressive demineralization, were not addressed. Likewise, no patient-centered outcomes, such as hypersensitivity, esthetic improvement, or durability of bleaching, were evaluated.

Finally, the study did not investigate the protective role of post-bleaching remineralizing agents, which are clinically relevant in contemporary bleaching protocols and may mitigate enamel alterations. Future studies should include these comparisons to provide more translational evidence.

All three bleaching protocols tested in this study—McInnes solution, modified McInnes solution, and Pola Office bleach—caused varying degrees of enamel mineral loss and surface alteration.

Among them, the McInnes solution induced the most pronounced mineral depletion and surface roughness, likely due to its high hydrochloric acid content.

The modified McInnes solution demonstrated relatively milder effects on enamel, confirming its potential as a safer alternative.

Pola Office bleach also resulted in mineral loss, though at a level comparable to modified McInnes.

Spectrophotometric analysis and SEM imaging both proved useful in detecting these changes.

Post-bleaching remineralization in artificial saliva showed partial recovery of calcium and phosphorus levels, highlighting the importance of remineralizing protocols in clinical practice.