Remineralization of artificial caries lesions using casein phosphopeptide–amorphous calcium phosphate containing probiotic lozenge: an in vitro study

Abstract

Background

Concerns regarding enamel demineralization due to the acidic nature of probiotic tablets have led to investigations into potential solutions. This study aimed to investigate the remineralization potential of probiotic tablets containing Casein phosphopeptide–amorphous calcium phosphate (CPP-ACP).

Methods

Forty-eight enamel blocks extracted from human teeth were prepared and randomly assigned to four groups (n = 12 per group). Group 1 was treated with a 20 mg CPP-ACP-containing probiotic lozenge dissolved in 5 mL of Phosphate Buffered Saline (PBS) and applied to the enamel surface for 10 min, while Group 2 received the same lozenge dissolved in 10 mL of PBS under identical conditions. Group 3 (Positive Control) was immersed in a remineralization solution containing 1.5 mM calcium, 0.9 mM phosphate, and 0.15 M KCl in 0.1 M Tris buffer, with pH adjusted to 7.0 for 18 h, whereas Group 4 (Negative Control) was exposed only to PBS. Prior to treatment protocols, all specimens were demineralized by immersion in a solution (pH 4.5) for 72 h to induce artificial enamel lesions. Surface microhardness was measured at baseline, after demineralization, and after treatment using a digital Micro-Vickers Hardness Tester. The percentage of surface microhardness recovery (%SMHR) was calculated. Statistical analysis was performed using two-way repeated measures analysis of variance (ANOVA), one-way ANOVA, and the Least Significant Difference (LSD) test.

Results

Both CPP-ACP-containing probiotic lozenge groups (Groups 1 and 2) exhibited significant remineralization, demonstrated by a marked increase in surface microhardness values (p = 0.001). Group 1 demonstrated greater remineralization than Group 2 (p = 0.011). The positive control group (Group 3) exhibited the highest microhardness values after 18 h (p < 0.001). The %SMHR values confirmed a dose-dependent remineralization effect of CPP-ACP-containing probiotic lozenges, with higher concentrations yielding greater remineralization.

Conclusions

CPP-ACP-containing probiotic lozenges significantly enhanced enamel microhardness in a dose-dependent manner, suggesting their potential for enamel remineralization.

Background

Dental caries is a common multifactorial disease influenced by genetic, dietary, environmental and lifestyle factors, arising from an imbalance between demineralization and remineralization [1, 2]. Preventive strategies and minimally invasive dental approaches promote remineralization technologies to prevent caries onset and manage early-stage lesions [3, 4]. Fluoride-containing agents are widely recommended to prevent demineralization of dental tissues and have proven effective in controlling and preventing caries [5]. Another promising therapeutic agent, casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) is a bioactive agent derived from milk protein that has demonstrated potential in enhancing the remineralization of superficial enamel lesions in various studies [6]. CPP-ACP-based agents have shown significant potential in preventing carious lesions and addressing erosive processes [7, 8]. Clinical trials have confirmed the efficacy of CPP-ACP in promoting tooth remineralization, establishing its role as an effective anti-cariogenic agent [9–12]. Moreover, in vitro studies have demonstrated that CPP-ACP improves surface microhardness, thereby enhances remineralization [13–15].

Probiotics are defined by the World Health Organization and the Food and Agriculture Organization of the United Nations (2001) as “live microorganisms that provide a health benefit when administered in sufficient quantities” [16]. Probiotic microorganisms are commercially available in various market forms, including dairy products (cheese, yogurt, ice cream), fruit juices, gum, and dietary supplements [17]. The introduction of oral probiotics in products such as lozenges, tablets, gum, capsules, mouthwash, and toothpaste has driven research into their effects on oral and dental health [18]. Commonly used probiotic microorganisms include Lactobacillus, Bifidobacterium, Streptococcus, and Bacillus [19]. Lactic acid-producing bacteria generate organic acids, which may benefit systemic health, particularly in the gastrointestinal, vaginal, urogenital, and oropharyngeal areas [20]. However, their impact on the balance of the oral microbiome remains a concern [21]. While probiotic bacteria compete with cariogenic species and may reduce the risk of dental caries, their acidic nature can contribute to enamel demineralization [22, 23]. Studies suggest that acids produced by probiotic bacteriamay lower salivary pH, leading to mineral loss from enamel and initiating the demineralization process [24–26]. Incorporating remineralizing agents into probiotics may enhance their ability to reduce reduce dental caries risk and supportremineralization [27].

An experimental sugar-free probiotic lozenge containing CPP-ACP (Stellar Biome, London, ON, Canada), was formulated with Lactobacillus plantarum Stellar™ Lp-199 and Lactobacillus helveticus Stellar™ Lh-170 at a total concentration of 1 × 10⁹ CFU (colony-forming units) and 20 mg of CPP-ACP. Saha et al. [28], evaluated the probiotic lozenge in vitro and reported a decrease in enamel surface microhardness, indicating demineralization. Building on these findings, this study aimed to evaluate the in vitro remineralization potential of the experimental CPP-ACP-containing probiotic lozenge. To the best of our knowledge, this is the first study to assess the remineralization effect of a CPP-ACP-containing probiotic lozenge on permanent teeth using surface microhardness testing. This study aimed to investigate the remineralization potential of probiotic tablets containing casein phosphopeptide–amorphous calcium phosphate (CPP-ACP).Two null hypotheses were proposed: (1) The use of CPP-ACP-containing probiotic lozenge would not significantly differ in its remineralization effect from the positive control (remineralization solution). (2) Different concentrations of CPP-ACP-containing probiotic lozenges would not significantly impact enamel remineralization.

Methods

Study design

This study was approved by the Marmara University Health Sciences Institute Clinical Research Ethics Committee (Protocol number: 22/22022016). The research was conducted at the Faculty of Dentistry, Marmara University and the Research Center of Yeditepe University, Istanbul, Türkiye between May 2016 and January 2017. The minimum sample size required was determined using to G*Power version 3.1.9.6, based on a study by Shetty et al. [29]. The calculation was performed with a significance level (α) of 0.05, a power (1 − β) of 0.95, and an effect size of 1.921, resulting in a required minimum of nine samples per group.

Preparation of samples

This in vitro study was carried out on fifty-four freshly extracted human permanent molars for periodontal or orthodontic reasons were collected from the Department of Oral Surgery, Faculty of Dentistry, Marmara University, İstanbul, Türkiye. Prior to the collection of extracted teeth, informed consent form was obtained from patients, and patients were informed that their extracted teeth would be used in a planned study. The selection criteria required teeth to be free of decay, restorations, or root canal treatments, as well as free of mineralization defects, cracks, or fractures on the enamel surface.

Teeth were ultrasonically cleaned to remove deposits and stains, following the Occupational Safety and Health Administration (OSHA) recommendations [30]. Infrared light transillumination (DIAGNOcam 2170U, Kavo, Biberach, Germany) was utilized to eliminate the possibility of enamel cracks, decalcification, white spot lesions, and extraction damage. Subsequently, all tooth crowns were excised from the roots at the cementoenamel junction and bisected mesiodistally using an ISOMET Low-Speed Saw cutting machine (Buehler, Lake Bluff, IL, USA).

The resulting 48 specimens, each measuring 3 × 2 × 2 mm, were prepared from the labial surfaces and distributed randomly into four groups using random generator software (www.random.org). Group distribution details are presented in Table 1. The methodology was illustrated using a flowchart (Fig. 1).

Table 1.

Distribution of groups with respective active ingredients

Groups	Agents	Active ingredients
1	CPP-ACP-Containing Experimental Probiotic Lozenge (dissolved in 5 mL of Phosphate Buffered Saline)	20 mg of CPP-ACP + Lactobacillus plantarum Stellar™ Lp-199 & Lactobacillus helveticus Stellar™ Lh-170 Stellar Biome, London, ON, Canada
2	CPP-ACP-Containing Experimental Probiotic Lozenge (dissolved in 10 mL of Phosphate Buffered Saline)	20 mg of CPP-ACP + Lactobacillus plantarum Stellar™ Lp-199 & Lactobacillus helveticus Stellar™ Lh-170 Stellar Biome, London, ON, Canada
3	Positive Control (Remineralization Solution)	1.5 mM calcium, 0.9 mM phosphate, 0.15 M KCl in 0.1 m Tris buffer, pH 7
4	Negative Control	Phosphate Buffered Saline, pH 7.4

CPP-ACP: Casein phosphopeptide–amorphous calcium phosphate, CFU: Colony forming unit

Fig. 1.

Flow chart of the study

Specimens were stored in 0.1% thymol solution until the experimental procedure commenced. Following embedding in epoxy resin, the superficial enamel surface was polished using water-cooled carborundum discs and 1200-grit waterproof silicon carbide paper (Amico), removing approximately 200 μm of enamel [31].

Demineralization protocol

To induce enamel lesions, a demineralization solution was prepared by combining 100 mmol/L sodium hydroxide with 100 mmol/L lactic acid, adjusting the pH to 4.5. Specimens were then immersed in this demineralization solution for three days [32]. Following demineralization protocol, the enamel specimens were thoroughly rinsed with sterile water to remove any remaining acid. The specimens were then stored in filtered distilled water for the duration of the study.

Treatment protocol

The CPP-ACP-containing probiotic lozenge was applied to the enamel samples in Groups 1 and 2 as follows:

1. One probiotic lozenge was dissolved in 5 mL of PBS (Group 1) or 10 mL of PBS (Group 2) within 5 min.

2. The resulting solutions were applied to the enamel surface, using an applicator brush, ensuring complete coverage, and left for 10 min.

3. After the 10-minute exposure, enamel samples from Group 1 and Group 2 were rinsed with deionized water to remove residual probiotic solution. The microhardness measurements were then recorded immediately.

For the positive control group (Group 3), specimens were immersed for 18 h in a remineralization solution containing 1.5 mM calcium, 0.9 mM phosphate, and 0.15 M KCl in 0.1 M Tris buffer, with pH adjusted to 7.0 [33]. The negative control group (Group 4) was exposed only to PBS. Treatment protocol was illustrated using a schematic diagram (Fig. 2).

Fig. 2.

Schematic illustration of the study design

Surface microhardness measurements

Surface microhardness analysis was conducted using a digital Micro-Vickers Hardness Tester (Wilson Wolpert Europe BV, 401 MVD, Netherlands) equipped with a Vickers diamond indenter. A 200 N load was applied to create indentations on the enamel surface, with the indenter held in position for 15 s at three distinct points, each 1 mm apart. The average of these measurements was recorded as the Vickers Hardness Number (VHN) after baseline, after demineralization and after treatment, followed by a comparative analysis.

Statistical analysis

The Shapiro-Wilk test was used to assess the normality of VHN baseline, demineralization, and treatment variables. The effect of all groups on VHN at different stages was analyzed using a two-way repeated measures analysis of variance (ANOVA). The percentage of surface microhardness recovery (%SMHR) was calculated using the following formula and analyzed with one-way ANOVA and Least Significant Difference Test [34]. SPSS 22.0 Windows version package program was used in the analyses. P < 0.05 was considered significant.

Results

Table 2 presents the surface microhardness within each group. Baseline VHN values did not differ significantly between groups (p > 0.05). After 72 h immersion in the demineralizing solution, a significant reduction of VHN values was observed in all groups (p < 0.001) (Table 2). Two-way repeated measures ANOVA revaled statistically significant differences between groups, time points, and group-treatment interactions (Table 3). A comparison of VHN after demineralization and VHN after treatment indicated that Groups 1, 2, and 3 were significantly increased enamel microhardness (p_1,2,3= 0.001). Conversely, Group 4 showed no significant difference between the after demineralization and after treatment VHN values (p = 0.718) (Table 3; Fig. 3).

Table 2.

Mean values and standard deviations for changes in surface microhardness

		VHN Baseline Mean ± SD	VHN Demineralization Mean ± SD	VHN Treatment Mean ± SD	P
Group 1	CPP-ACP-Containing Probiotic Lozenge (dissolved in 5 mL of Phosphate Buffered Saline)	336.95 ± 3.73	262.65 ± 10.29	286.9 ± 3.06	0.001*†
Group 2	CPP-ACP-Containing Probiotic Lozenge (dissolved in 10 mL of Phosphate Buffered Saline)	333.48 ± 5.83	265.09 ± 6.61	281.03 ± 4.69
Group 3	Positive Control (Remineralization Solution)	333.08 ± 5.66	265.6 ± 5.36	308.87 ± 5.21
Group 4	Negative Control (Phosphate Buffered Saline)	334.16 ± 4.7	267.04 ± 4.37	267.88 ± 4.3
	P	0.001*‡			0.001*ǂ

*meaning at p < 0.05 level. Two-way repeated measures ANOVA (analysis of variance)

†Group. ‡measurement. ǂgroup*measurement interaction

CPP-ACP: Casein phosphopeptide–amorphous calcium phosphate, PBS: Phosphate Buffered Saline, VHN: Vickers Hardness Number,

Table 3.

Multiple comparison of baseline, demineralization and treatment VHN among groups

		Group 1			Group 2			Group 3			Group 4
		B	D	T	B	D	T	B	D	T	B	D	T
Group 1	B		0.001	0.001	0.131	0.001	0.001	0.093	0.001	0.001	0.224	0.001	0.001
	D			0.001	0.001	0.288	0.001	0.001	0.199	0.001	0.001	0.057	0.024
	T				0.001	0.001	0.011	0.001	0.001	0.001	0.001	0.001	0.001
Group 2	B					0.001	0.001	0.862	0.001	0.001	0.768	0.001	0.001
	D						0.001	0.001	0.822	0.001	0.001	0.394	0.225
	T							0.001	0.001	0.001	0.001	0.001	0.001
Group 3	B								0.001	0.001	0.639	0.001	0.001
	D									0.001	0.001	0.530	0.322
	T										0.001	0.001	0.001
Group 4	B											0.001	0.001
	D												0.718
	T

Bold font: p < 0.05 statistically significant, VHN: Vickers Hardness Number, B:Baseline, D:Demineralization, T: Treatment

Fig. 3.

Vickers hardness number changes for all groups

The mean %SMHR was significantly different among groups (p < 0.001) (Table 4). Pairwise comparison of %SMHR across treatment groups were presented in Table 5. Multiple comparison showed that %SMHR (Table 5) was significantly higher in the group treated with the CPP-ACP-containing probiotic lozenge dissolved in 5 mL of PBS compared to the 10 mL PBS group (p = 0.006).

Table 4.

Mean and standard deviations (SD) of percentage surface microhardness recovery (%SMHR) for all groups

Groups		SMHR% Mean (SD)	p
Group 1	CPP-ACP-Containing Probiotic Lozenge (dissolved in 5 mL of PBS)	23.47 ± 9.52	0.001*
Group 2	CPP-ACP-Containing Probiotic Lozenge (dissolved in 10 mL of PBS)	15.15 ± 6.5
Group 3	Positive Control (Remineralization Solution)	42.47 ± 6.06
Group 4	Negative Control (Phosphate Buffered Saline)	0.03 ± 5.37

*p < 0.05, one-way ANOVA (analysis of variance)

CPP-ACP: Casein phosphopeptide–amorphous calcium phosphate, PBS: Phosphate Buffered Saline, VHN: Vickers Hardness Number, SMHR%: Surface Microhardness Recovery, SD: Standart deviation

Table 5.

The pairwise comparison of percentage surface microhardness recovery (%SMHR) of treatment groups

	Group 2 CPP-ACP-Containing Probiotic Lozenge (dissolved in 10 mL of PBS)	Group 3 Positive Control (Remineralization Solution)	Group 4 Negative Control (Phosphate Buffered Saline)
Group 1 CPP-ACP-Containing Probiotic Lozenge (dissolved in 5 mL of PBS)	0.006*	0.001*	0.001*
Group 2 CPP-ACP-Containing Probiotic Lozenge (dissolved in 10 mL of PBS)		0.001*	0.001*
Group 3 Positive Control (Remineralization Solution)			0.001*

*p < 0.05, Least Significant Difference Test

CPP-ACP: Casein phosphopeptide–amorphous calcium phosphate, PBS: Phosphate Buffered Saline, VHN: Vickers Hardness Number

Discussion

In this study, statistically significant improvements in microhardness values were observed in enamel specimens treated with CPP-ACP-containing probiotic lozenges, leading to the rejection of the initial null hypothesis. These findings aligned with previous in vitro studies, which demonstrated the remineralization potential of CPP-ACP formulations [13, 15,35–40]. This effect was attributed to the mineralizing properties of CPP-ACP, which facilitated the localization of amorphous calcium phosphate (ACP) on the enamel surface, creating a reservoir of free calcium and phosphate ions [41]. These ions acted as a buffer against demineralization and promoted remineralization by maintaining a state of supersaturation, thereby preventing mineral loss [42–44].

To simulate varying salivary flow conditions, 5 mL and 10 mL dilution volumes were selected based on physiological unstimulated and stimulated saliva flow rates [45, 46]. The 5 mL dilution produced a more concentrated CPP-ACP solution, potentially enhancing remineralization, whereas the 10 mL dilution simulated higher saliva flow, leading to increased clearance of active ingredients and reduced remineralization efficacy. Consistent with this, surface microhardness values were significantly higher in the more concentrated solution group, resulting in the rejection of the second null hypothesis. The observed dose-dependent effect of CPP-ACP-containing probiotic lozenges was consistent with previous research, which demonstrated greater enamel remineralization with higher CPP-ACP concentrations [27, 47–49]. Additionally, it was suggested that the protective effect of CPP-ACP depended on application frequency, with multiple exposures producing superior remineralization outcomes compared to a single application [14].

The positive control group exhibited the highest surface microhardness recovery, which was attributed to its optimized remineralizing solution composition and extended application duration, allowing for prolonged enamel interaction. In contrast, the lower microhardness values observed in the probiotic lozenge groups may have resulted from the shorter exposure duration used in this study. Unlike the remineralizing solution, which remained in contact with the enamel surface for an extended period, the probiotic lozenges were applied for a limited time, potentially reducing their remineralization efficacy. However, this approach was intentional, as the study aimed to evaluate the potential of an experimental CPP-ACP-containing probiotic lozenge, marking the first investigation of its kind. Furthermore, there is no established consensus regarding the optimal frequency and dosage of oral probiotic tablets for dental applications, highlighting the need for further research.

Another possible explanation for the differences in remineralization efficacy was the demineralizing effect of probiotic tablets, which was counteracted and reversed by CPP-ACP. Some studies suggested that probiotic solutions may contribute to calcium and phosphorus loss from enamel, exhibiting a demineralizing effect similar to lactate solutions [50, 51]. Consistent with this, in vitro studies on probiotic products reported a decrease in microhardness values [28, 52, 53]. Karataş et al. [52], found that microhardness values decreased following exposure to a probiotic mouthwash solution, while Saha et al. [28], reported a reduction in enamel microhardness in the probiotic solution group. Similarly, Maden et al. [53], observed a statistically significant decrease in Vickers hardness values after the tooth brushing process in groups using probiotic toothpaste.

Surface microhardness testing is a widely used method for assessing enamel remineralization, as it provides quantitative data on mineral alterations in dental hard tissues. Microhardness tests could be performed using either a Vickers or Knoop indenter, is commonly employed to evaluate the efficacy of remineralization agents [54, 55]. Several studies have demonstrated the remineralization potential of CPP-ACP using Vickers surface microhardness testing [13, 15, 29, 36, 40, 56], while others have assessed similar effects using Knoop microhardness testing [57, 58]. Additionally, studies investigating probiotic products have used Vickers hardness testing to assess their effects on enamel microhardness, reporting variable results [28, 52, 53]. Given that our study is the first to evaluate the remineralization efficacy of a CPP-ACP-containing probiotic lozenge, we selected Vickers surface microhardness testing as a reliable and widely used technique to measure enamel surface alterations. To minimize variability, measurements were conducted on the midline surface of each enamel specimen at three time points. No statistically significant differences were observed between the groups at baseline or after demineralization.

CPP-ACP has been incorporated into various materials, including chewing gums, milk, mouth rinses, lozenges, and dental creams, and is widely recommended in minimally invasive dentistry due to its remineralization potential [59–62]. Probiotics have also gained attention for their potential role in maintaining oral health, though their impact on enamel integrity remains controversial [24–26, 63]. The combination of probiotics and CPP-ACP represents a promising approach for enamel remineralization, as probiotics may help modulate the oral microbiome, while CPP-ACP stabilizes calcium and phosphate ions to promote mineral deposition [27]. To the best of our knowledge, this is the first in vitro study to evaluate the enamel remineralization potential of CPP-ACP-containing probiotic lozenges using Vickers surface microhardness testing.

While previous studies have highlighted concerns regarding the potential demineralizing effects of probiotics, the findings of this study provided the first evidence that a CPP-ACP-containing probiotic lozenge enhanced enamel remineralization. This underscores the dynamic interplay between demineralization and remineralization, which is influenced by factors such as application method, exposure duration, and formulation. Future studies should further investigate these variables to optimize the clinical application of probiotic lozenges in enamel remineralization.

This study provided preliminary insights into the effects of CPP-ACP-containing probiotic lozenges on enamel microhardness. However, certain limitations should be considered. First, the in vitro design did not fully replicate the oral environment, including salivary flow, dietary influences, and microbial interactions. Second, the relatively small sample size may have limited the generalizability of the findings. Additionally, only two probiotic strains (Lactobacillus plantarum and Lactobacillus helveticus) were tested, and no control group was included to assess probiotic lozenges without CPP-ACP. Future research incorporating advanced technologies should focus on optimizing the formulation and application of CPP-ACP-containing probiotic lozenges to maximize their remineralization potential. Studies should also investigate different concentrations, application frequencies, and long-term effects. Incorporating lesion depth analysis could provide further insights into subsurface mineralization, while comparative studies with other remineralization agents would help establish clinical applicability. Expanding research on these lozenges will be crucial in defining their optimal role in preventive and restorative dentistry.

Conclusion

CPP-ACP-containing probiotic lozenges significantly enhanced enamel microhardness, confirming their remineralization potential. The dose-dependent effect suggests that higher concentrations improve remineralization efficacy. These findings indicate that incorporating CPP-ACP into probiotic lozenges may enhance their effectiveness as a remineralization agent.

Abbreviations

CPP-ACP

Casein phosphopeptide–amorphous calcium phosphate

CFU

colony forming unit

OSHA

Occupational Safety and Health Administration

PBS

Phosphate Buffered Saline

VHN

Vickers Hardness Number

SMHR%

Surface Microhardness Recovery

SD

Standart deviation

Author contributions

E.A.S. and B.K. contributed to the conception and design of the study. E.A.S performed the experiment. E.A.S. drafted the manuscript. B.K. critically revised the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of this study.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author, [EAS], upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the of Marmara University Health Sciences Institute Clinical Research Ethics Committee (Protocol number: 22/22022016). Written informed consent was obtained from patients involved in the study.

Consent for publication

No publication consent required.

Competing interests

The authors declare no competing interests.