In-Vitro Evaluation of the Whitening Efficacy of GuruNanda Whitening Strips with and without Hydroxyapatite

Objective

 The objective of this in-vitro study was to evaluate and compare the whitening efficacy of a hydrogen peroxide–based GuruNanda® whitening strip with and without hydroxyapatite. The study aimed to determine whether incorporation of hydroxyapatite enhances whitening performance compared with hydrogen peroxide alone and a placebo strip when applied to intensively stained tooth surfaces.

 

Process 

An in-vitro experimental study design was employed using extracted human incisor teeth. A total of 60 teeth free from structural defects or anomalies were selected. The roots were removed, and each tooth was embedded in polymethyl methacrylate resin with the facial enamel surface exposed. All specimens were stored in distilled water prior to testing.

Baseline tooth color was measured digitally using a calibrated VITA EasyShade Advance 4.0 spectrophotometer under standardized color-corrected lighting conditions (5000 K). Tooth shade was recorded using the VITA® Classical shade guide system, which assigns numerical values corresponding to shade intensity from the lightest to the darkest tones.

Following baseline tooth color measurement, enamel surfaces were lightly etched with 32% phosphoric acid for 10 seconds to facilitate stain uptake. The specimens were then subjected to a cyclic staining protocol involving acquired salivary pellicle formation and stain immersion. Pellicle formation was achieved by incubating the enamel blocks in pasteurized human whole saliva at 37 °C for one hour under continuous agitation. This was followed by staining through immersion in a solution containing common dietary chromogens, including tea, coffee, red wine, turmeric, and tobacco, under identical conditions. The pellicle formation and staining cycles were repeated over a 12-hour period, with overnight storage in the staining solution to complete the 24-hour staining regimen. Following staining, tooth shade measurements were repeated to document post-staining discoloration prior to initiation of the whitening protocol.

The 60 stained tooth blocks were randomly allocated into three treatment groups (n = 20 per group):

1. Hydrogen Peroxide + Hydroxyapatite Whitening Strip (HP–HAP)
2. Hydrogen Peroxide–Only Whitening Strip (HP)
3. Placebo Whitening Strip

Randomization was performed to ensure that post-staining shade scores were comparable across the three groups. Each group underwent a daily whitening regimen for 14 consecutive days. On each day, the assigned whitening strip was applied for 30 minutes, followed by gentle brushing with a toothbrush and deionized distilled water to remove residual gel. Specimens were stored in artificial saliva at 37°C between treatments to simulate oral conditions.

Tooth shade measurements were obtained at three time points: original tooth shade, after staining, and after 14 days of whitening treatment.

 

Results 

Comparison of original tooth shade across groups showed no meaningful differences, indicating comparable baseline enamel color. Similarly, post-staining measurements demonstrated uniform stain intensity among the three groups prior to whitening treatment.

Paired analysis demonstrated a significant reduction in stain intensity from post-staining to 14 days of whitening for the hydrogen peroxide + hydroxyapatite strip, while Wilcoxon signed-rank testing confirmed similarly significant reductions for the hydrogen peroxide strip (z = −2.94, p = 0.003) and the placebo strip (z = −2.89, p = 0.004).

 

One-way analysis of variance revealed a significant difference in percentage whitening among groups (F(2,27)=50.48, p<0.001), with both hydrogen peroxide + hydroxyapatite and hydrogen peroxide strips producing significantly greater whitening compared with placebo, and no significant difference between the two active formulations.

 

Table 1. Statistical Comparison of Stain Intensity Before and After 14 Days of Whitening

Variable		M	SD	Mean Difference		t	df	p	Sig?
Hydrogen Peroxide + Hydroxyapatite
	After Staining	7.8	4.26	5.0		5.79	10	<.001	Yes
	After 14 days of Whitening	2.8	1.54
Hydrogen Peroxide
		Mean/Median
After Staining		6.5/4	4.3	4.1		4.05	10	.001	Yes
After 14 days of Whitening		2.4/2	1.4
Placebo
After Staining		5.1/5	1.8	0.8		6.63	10	<.001	Yes
After 14 days of Whitening		4.3/4	1.5

Abbreviations: M = Mean; SD = Standard Deviation; df = Degrees of Freedom; p = Probability value; Sig = Statistical significance.

Figure 1: Comparative trends in stain intensity demonstrating greater stain reduction after 14 days of whitening with hydrogen peroxide–based strips compared with placebo.

Figure 2: Mean percentage whitening demonstrating greater whitening efficacy of hydrogen peroxide–based strips compared with placebo

 

Observation

The whitening strips containing hydrogen peroxide consistently demonstrated effective removal of extrinsic stains from heavily stained enamel surfaces. Incorporation of hydroxyapatite appeared to enhance whitening performance, producing greater overall stain reduction compared with hydrogen peroxide alone. The placebo strip showed limited whitening efficacy, confirming the active role of peroxide-based formulations in stain removal.

 

Conclusion

This in-vitro study demonstrated that hydrogen peroxide–based whitening strips effectively whiten intensively stained tooth surfaces. The addition of hydroxyapatite to the whitening strip formulation enhanced whitening efficacy, resulting in greater stain reduction compared with hydrogen peroxide alone and placebo. These findings support the use of hydroxyapatite as a functional additive in whitening strip formulations to improve overall whitening performance under controlled laboratory conditions.