Effect of carbamide peroxide and bromelain bleaching agents by the activation of diode laser on the tooth at different time intervals: An in vitro study

Abstract

Objective:

To evaluate and compare the peroxide and nonperoxide organic bleaching agents with and without 940 nm diode laser activation on the tooth at different time intervals.

Materials and Methods:

An in vitro study was conducted on 56 extracted human teeth. Following polishing, specimens were standardized by immersion in black tea solution for 1 week to achieve uniform discoloration. Initial and subsequent shade assessments were performed using a spectrophotometer based on the Commission Internationale de l’Eclairage Lab* system. Teeth were randomly divided into two groups: 10% carbamide peroxide (CP) and bromelain. Each group was further subdivided into four subgroups: Control, gel-only, gel + laser for 2.5 min, and gel + laser for 5 min. Bleaching procedures were performed in two sessions, 1 week apart, with repeated gel applications per session. Color changes were calculated and statistically analysed using the one-way ANOVA with Tukey’s post hoc test (P < 0.05).

Results:

Both bleaching agents produced perceptible shade improvements, influenced by diode laser activation and exposure time. Progressive shade enhancement was observed across evaluation points, with some groups showing more stable and pronounced outcomes. Shade relapse varied among protocols, indicating treatment-dependent differences in color stability.

Conclusion:

Diode laser activation improved the bleaching efficacy of both CP and bromelain systems. Shorter laser exposure proved safer and more effective than prolonged activation. Bromelain demonstrated potential as a biologically friendly alternative to peroxide agents. These findings support incorporating diode lasers into bleaching protocols and encourage further clinical research to validate and standardize treatment strategies.

INTRODUCTION

Esthetic dentistry plays a key role in improving patient confidence and satisfaction. Tooth discoloration, arising from intrinsic or extrinsic factors such as dietary habits, aging, fluorosis, or trauma, is a prevalent esthetic concern in clinical practice.[1] Tooth bleaching, defined by Sturdevant as the lightening of the tooth’s color through chemical oxidation of organic pigments, is a widely accepted cosmetic solution.[2]

Vital bleaching can be performed through in-office treatments, dentist-supervised at-home protocols, or over-the-counter products.[3] In-office bleaching uses high concentrations of agents and adjunctive technologies such as lasers or lights to accelerate the process.[4] Hydrogen peroxide and carbamide peroxide (CP) remain the benchmark bleaching agents, given their consistently high effectiveness in treating intrinsic and extrinsic discoloration.[4,5] Their action involves the release of reactive oxygen species (ROS), which penetrate enamel and dentin to oxidize and break down stain-causing chromogens.[6]

Hydrogen peroxide acts directly, while CP decomposes into hydrogen peroxide and urea, offering a slower, sustained release.[7,8] However, concerns remain over the adverse effects of peroxides, such as enamel demineralization, reduced microhardness, increased tooth sensitivity, and potential pulp damage.[9,10] High concentrations (30%–37%) of CP, although effective for short sessions (8–15 min), increase dentin permeability and risk soft-tissue irritation.[7,8]

Due to these drawbacks, nonperoxide, enzyme-based alternatives such as bromelain – an extract from pineapple stem – are gaining popularity. These agents act by proteolytic breakdown of proteinaceous stain matrices without harming enamel integrity.[9] Bromelain gels have demonstrated effective removal of extrinsic stains from the substances such as coffee and fruit juice, while maintaining enamel smoothness and biocompatibility.[10] Their safety and natural origin make them particularly suitable for patients with sensitivity or those preferring eco-friendly treatments.[11,12]

Enhancement of bleaching efficacy is commonly achieved through activation techniques such as heat, halogen light, LED, and lasers.[13,14] Diode lasers, especially at wavelengths such as 810, 940, and 980 nm, offer efficient stain removal by accelerating the release of ROS and improving bleaching outcomes with minimal side effects.[14] Laser activation reduces overall treatment time, enhances chromophore penetration, and aids in minimizing sensitivity by promoting dentinal tubule occlusion.[15] However, care must be taken to avoid thermal damage to pulp and enamel with overexposure.[16]

Laser activation has also shown promise in amplifying the action of nonperoxide agents such as bromelain. The heat and light energy from lasers may enhance the proteolytic stain breakdown capability of bromelain, providing a balanced, safer, and effective whitening outcome.[17] Recent studies by Saluja et al. support short-duration diode laser activation (2.5–5 min) as effective for peroxide bleaching without compromising pulp health.[3]

This study aims to compare the bleaching efficacy of peroxide-based (CP) and nonperoxide-based (bromelain) agents when activated using a 940 nm diode laser. It will evaluate whitening outcomes, enamel surface changes, and safety at various time intervals. The results are expected to guide future protocols that merge effectiveness with biocompatibility, contributing to the advancement of safer, patient-centered approaches in esthetic dentistry.

MATERIALS AND METHODS

This in vitro study was conducted to evaluate and compare the bleaching efficacy of two agents – 35% CP and bromelain-based gel – activated with a 940 nm diode laser, on artificially stained human teeth. A total of 56 freshly extracted, sound human permanent anterior teeth (central incisors, lateral incisors, and canines) were selected [Figure 1]. The specimens were cleaned using rubber cups and polishing paste, rinsed, and stored in distilled water at 8°C to maintain hydration until the study commenced [Figure 2a].

Figure 1.

Stained 56 extracted teeth specimen

Figure 2.

(a) Scaling of the samples. (b) Bromelain mixture. (c) 35% Carbamide peroxide application on the samples (d) bromelain gel application on the samples. (e) Activation by the laser on the samples. (f) Shade selection of the samples using spectrophotometer

Baseline tooth shade measurements were recorded using a spectrophotometer. Enamel surfaces were etched using 35% phosphoric acid for 60 s to facilitate stain uptake, followed by rinsing. The specimens underwent artificial staining by being immersed in a standardized black tea solution, prepared using four tea bags in 200 mL of water, for 7 days. The solution was stirred once daily to maintain uniform staining across all the samples.

To assess shade changes, the Commission Internationale de l’Eclairage Lab* colour system was employed. L* indicates brightness, a* represents the red-green axis, and b* the yellow-blue axis. The total shade change (ΔE*) was calculated using the formula: ΔE = √([ΔL] ²+ [Δa*]²+ [Δb*]²)**, before and after bleaching.

For bromelain bleaching gel preparation, 1 g of xanthan gum (Purix) was mixed with 15 mL of glycerine (Gas Lab) as a first vial. Next, in a second vial, 0.5 mL of the sodium bicarbonate (Gas Lab) and xylitol (Research labs) were mixed with 1 mL of the polyvinyl pyrrolidone (Gas Lab) and 0.5 mL of polyethylene glycol 400 (Purenso select). Subsequently, 0.1 g of bromelain enzyme powder (Bioven) and dissolved it in 9.9 mL of distilled water to make a 1% w/v solution, was added to the second vial, along with 50 μL of essential peppermint oil (Gas Lab); the vials were then mixed together, resulting in the bromelain-based gel. The completed gel was stored under refrigeration at 4°C–8°C [Figure 2b].

After staining, tooth shade was recorded and teeth were randomly divided into two groups (n = 28 each) based on the bleaching agent:

• Group A: 35% CP

• Group B: Bromelain gel.

Each group was subdivided into four subgroups (n = 7) based on treatment modality:

• Group 1 (control): Stored in distilled water for 2 weeks

• Group 2: Bleaching gel applied for 10 min

• Group 3: Bleaching gel applied + laser activation for 2.5 min

• Group 4: Bleaching gel applied + laser activation for 5 min.

The bleaching procedure was done in two sessions, 1 week apart [Figure 2c and d]. Each session consisted of three applications of the bleaching gel resulting in a total gel contact time of 10 min for each session. After each application, the gel was removed with sterile gauze and the tooth was rinsed. Teeth were stored in distilled water at 37°C between sessions.

Laser activation was carried out using a 940 nm diode laser set at 1.5 W in continuous-wave mode. The laser tip was maintained 2–3 mm above the gel surface and moved in a sweeping pattern to provide uniform irradiation [Figure 2e]. Postirradiation, the gel remained on the teeth until the 10-min contact time was completed.

Shade measurements were taken at five intervals:

• T0 – Baseline

• T1 – After staining

• T2-1 week after first bleaching

• T3-1 week after second bleaching

• T4-2 weeks post-bleaching.

To ensure consistent spectrophotometer readings, the same point on each tooth was marked and used for all measurements [Figure 2f].

RESULTS

At the baseline evaluation (T0), spectrophotometric analysis demonstrated no statistically significant differences among any of the subgroups, confirming uniform initial shade values 7.3–7.5 across all samples. Following artificial staining (T1), shade measurements remained comparable between groups, validating the consistency of the staining protocol. This uniformity ensured that any subsequent color changes observed could be attributed exclusively to the applied bleaching interventions. By the first posttreatment evaluation (T2), all active treatment groups (A2–A4 in Group A and B2–B4 in Group B) demonstrated significant improvement in shade compared to their respective controls (A1 and B1), indicating the effectiveness of both bleaching agents. Notably, the subgroups subjected to laser activation (A3, A4, B3, and B4) exhibited more pronounced color changes, suggesting that diode laser irradiation enhanced the bleaching potential of both CP and bromelain [Tables 1 and 2].

Table 1.

Descriptive statistics of colour change in the group A (Carbamide Peroxide) at different time intervals between the different subgroups

Group	Mean	SD	SE	Minimum	Maximum
T1
A1	9.54	2.42	0.91	6.98	14.07
A2	9.69	4.41	1.67	4.23	17.07
A3	10.47	3.34	1.26	6.02	13.58
A4	10.34	3.06	1.16	4.73	13.44
T2
A1	9.54	2.42	0.91	6.98	14.07
A2	2.77	1.56	0.59	0.62	4.37
A3	3.88	1.92	0.73	1.28	5.63
A4	7.03	2.14	0.81	3.97	8.64
T3
A1	9.03	2.44	0.92	6.87	13.90
A2	6.18	1.26	0.48	3.95	7.67
A3	5.99	0.76	0.29	5.00	7.16
A4	7.69	2.25	0.85	4.15	10.74
T4
A1	9.06	2.78	1.05	6.88	14.79
A2	7.59	1.52	0.58	5.06	9.33
A3	6.87	0.82	0.31	5.67	7.74
A4	7.90	2.32	0.88	4.25	11.05

This table describes the mean, SD and the minimum and maximum values of colour change of all the sub-groups. SD: Standard deviation, SE: Standard error

Table 2.

Descriptive statistics of colour change in the group B (Bromelain) at different time intervals between the different subgroups

Group	Mean	SD	SE	Minimum	Maximum
T1
B1	3.738	1.033	0.391	2.830	5.890
B2	8.624	3.013	1.139	4.450	12.050
B3	11.573	3.612	1.365	7.530	19.110
B4	10.013	1.432	0.541	8.960	12.520
T2
B1	3.673	1.041	0.394	2.830	5.790
B2	7.266	3.038	1.148	2.320	10.620
B3	4.412	1.601	0.605	2.700	5.840
B4	4.561	1.404	0.531	2.360	6.310
T3
B1	3.806	4.256	1.609	1.670	13.400
B2	8.292	2.517	0.951	4.250	10.850
B3	6.930	1.258	0.476	5.140	8.030
B4	6.210	2.513	0.950	3.020	10.990
T4
B1	3.661	4.278	1.617	1.440	13.290
B2	9.069	4.163	1.574	3.430	15.310
B3	8.379	2.258	0.853	5.140	11.820
B4	8.210	2.630	0.994	5.520	13.350

This table describes the mean, SD and the minimum and maximum values of colour change of all the sub-groups. SD: Standard deviation, SE: Standard error

Within Group A, which utilized 35% CP, Subgroups A3 (35% CP with 2.5 min of laser activation) and A4 (35% CP with 5 min of laser activation) showed significantly greater bleaching than A2 (35% CP without laser), confirming the benefit of laser-enhanced activation. Subgroup A4 demonstrated a more immediate whitening effect by T2, though a plateau in improvement was observed thereafter. In contrast, A3 showed a more gradual yet consistent shade enhancement up to T3, indicating a sustained bleaching response with shorter laser exposure. By the final evaluation at T4, differences among subgroups in Group A had reduced, reflecting stabilization in the bleaching effect across all treated teeth, regardless of the laser exposure duration [Figure 3].

Figure 3.

Descriptive statistics of colour change in the group A (carbamide peroxide) and B (Bromelain) at different time intervals between the different subgroups

In Group B, which was treated with the bromelain-based bleaching agent, laser activation also played a critical role. Subgroup B3 (bromelain with 2.5 min of laser) achieved the highest degree of bleaching among all bromelain groups at earlier time points, particularly at T2. This suggests that lower-duration laser exposure was more effective in rapidly activating the enzymatic bleaching potential of bromelain. Subgroup B4 (bromelain with 5 min of laser) also showed considerable improvement but was accompanied by a slight relapse in color between T3 and T4, possibly indicating a threshold beyond which prolonged laser exposure may diminish bleaching stability. Subgroup B2 (bromelain without laser) presented moderate whitening effects, although some rebound in staining was noted between T2 and T4, highlighting the limited efficacy of enzymatic bleaching without external activation [Figure 2].

An intergroup comparison revealed that, at T2, the bromelain-laser subgroup B3 produced a faster and more immediate whitening response than its peroxide counterpart. However, as time progressed (T3 and T4), both CP and bromelain groups, when activated with diode laser, achieved comparable outcomes in terms of total color change (ΔE*). This points toward the potential of bromelain as a viable natural alternative to traditional peroxide agents when used in conjunction with light or laser activation. In contrast, the control groups A1 and B1, which received no bleaching intervention, exhibited minimal to no change in shade throughout the entire duration of the study, further reinforcing the effectiveness of the applied bleaching protocols.

DISCUSSION

Tooth discoloration remains a prevalent aesthetic concern, driving continuous innovation in bleaching agents and techniques. This in vitro study explored the comparative efficacy of 35% CP and bromelain-based bleaching agents, both with and without 940 nm diode laser activation. The findings demonstrated that while both agents effectively improved tooth shade, their performance was significantly enhanced when combined with laser activation, aligning with previous studies supporting the role of lasers in improving bleaching outcomes.[1,2]

CP is a widely used bleaching agent in clinical practice, breaking down into hydrogen peroxide and urea during its decomposition.[18] Hydrogen peroxide subsequently forms hydroxyl radicals, which oxidize chromogenic molecules within enamel and dentin, resulting in color lightening.[4] The current study confirmed the superior bleaching capacity of 35% CP, especially when activated with diode laser. Subgroup A3 (CP + 2.5 min laser) consistently exhibited the most consistent and sustained shade improvement through the T3 time point. This supports prior research suggesting that shorter laser activation durations effectively accelerate peroxide breakdown without inducing excessive enamel damage.[5]

Interestingly, the whitening effect plateaued in subgroup A4 (CP + 5 min laser), suggesting a threshold for peroxide activity beyond which further exposure does not translate into enhanced results. This may be attributed to oxidative saturation or enamel fatigue, phenomena noted in previous investigations involving prolonged bleaching exposure.[6] In addition, concerns related to the aggressive oxidative potential of high-concentration CP were evident, as prolonged exposure did not yield proportional improvements in whitening efficacy, indicating a saturation effect. This observation is consistent with earlier reports by Efeoglu et al. and Faraoni-Romano et al., which describe diminishing returns with increased peroxide concentration and exposure time. Although diode laser activation enhanced the bleaching response, careful optimization of peroxide concentration and activation duration remains essential.[7,8]

The introduction of bromelain – a proteolytic enzyme from pineapple – as a bleaching agent offers a novel, biologically compatible alternative.[19] Bromelain cleaves peptide bonds in protein-based stains rather than oxidizing chromogens, thereby presenting a less aggressive mechanism.[9] While its stand-alone application (subgroup B3) achieved moderate whitening, laser-activated bromelain showed a faster early whitening response, becoming comparable to peroxide-treated groups at later intervals. These findings suggest that thermal activation by diode laser may enhance bromelain’s enzymatic diffusion and interaction with intrinsic stains, consistent with hypotheses proposed by Cuc et al. and Vejai Vekaash et al.[10,11]

However, subgroup B4 (bromelain + 5 min laser) showed slight color rebound by T4, highlighting that extended laser exposure does not linearly correlate with increased efficacy. This trend mirrors observations in the CP group, where longer durations resulted in either effect plateauing or minor relapse, potentially due to dehydration or thermal-induced enamel changes that affect light scattering.[12,13] These outcomes emphasize the importance of optimizing laser exposure time.[20] Notably, 2.5 min of diode laser activation yielded the most favorable results across both bleaching agents, balancing efficacy and enamel safety.

Comparative analysis of both agents revealed that CP demonstrated a slightly stronger initial response, while laser-activated bromelain achieved comparable long-term color change. Despite its superior bleaching efficacy, CP may pose risks of enamel alteration and cytotoxicity. Bromelain, by comparison, provides a biologically safer and less aggressive approach, especially beneficial for patients with tooth sensitivity or compromised enamel.[14,15] Nevertheless, its long-term stability without laser enhancement remains limited, indicating the necessity for adjunctive activation or repeated applications.[21,22,23]

The biocompatibility profile of bromelain was further validated by its negligible impact on enamel microhardness, aligning with previous studies by Münchow et al. and Ribeiro et al.[18,19] The formulation used in this study – comprising xanthan gum, PEG 400, sodium bicarbonate, and other stabilizers – ensured appropriate gel consistency, enzymatic activity, and safety, providing a feasible foundation for clinical use.[24,25]

While the results of this study reinforce the benefits of laser-assisted bleaching, several limitations must be acknowledged.[26,27] Being an in vitro investigation, the findings may not fully replicate clinical conditions, where variables such as salivary enzymes, pH fluctuations, and oral hygiene influence treatment outcomes. In addition, color perception in a clinical setting involves complex interactions with lighting, translucency, and surrounding tissues, which spectrophotometric readings alone cannot entirely capture. Hence, in vivo clinical trials with a long-term follow-up are essential to validate and translate these findings into patient-centered care.

From a clinical perspective, diode laser-enhanced bromelain bleaching offers a promising alternative for patients contraindicated for peroxide-based agents. In-office protocols using 2.5-minute laser exposure appear optimal for achieving efficient and safe whitening outcomes. For home-use systems, stabilized bromelain formulations could be developed for milder cumulative results, although this requires further research into enzyme stability and delivery systems.

CONCLUSIONS

In summary, this study highlights the growing potential of integrating natural bleaching agents with adjunctive technologies such as diode lasers. While CP remains the gold standard in tooth whitening,[28] bromelain – especially when laser-activated – emerges as a viable, patient-friendly alternative. Treatment plans should be personalized based on patient age, enamel condition, sensitivity, and esthetic expectations, ensuring optimal outcomes while preserving tooth structure. Future studies should continue exploring enzyme-based agents and refining photoactivation protocols to expand safe and effective whitening solutions in esthetic dentistry.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.