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Year : 2022  |  Volume : 7  |  Issue : 2  |  Page : 31-35

Comparative evaluation of hydroxyapatite fluoride and casein phosphopeptide amorphous calcium phosphate fluoride as remineralizing agents in primary teeth using pH cycling and single-sectioning technique

1 Ex-Post-Graduate, Department of Pediatric and Preventive Dentistry, STES's Sinhgad Dental College and Hospital, Pune, Maharashtra, India
2 Professor and Head, Department of Pediatric and Preventive Dentistry, STES's Sinhgad Dental College and Hospital, Pune, Maharashtra, India

Date of Submission21-Nov-2022
Date of Decision28-Nov-2022
Date of Acceptance05-Dec-2022
Date of Web Publication30-Dec-2022

Correspondence Address:
Dr. Raju Umaji Patil
Department of Pediatric and Preventive Dentistry, STES's Sinhgad Dental College and Hospital, S.no. 44/1 Vadgaon Bk, Off Sinhgad Road, Pune - 411 041, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmo.ijmo_15_22

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Background: Hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate are known to remineralize subsurface carious lesions. Aim: The aim of this study was to evaluate the effect of topical application of hydroxyapatite fluoride (HF) and caseinphosphopeptide-amorphous calcium phosphate fluoride paste (CPP-ACPF) in bringing about remineralization changes in artificially induced carious lesions of primary teeth using pH cycling and single-sectioning technique. Materials and Methods: Seventy extracted deciduous teeth were divided into two groups: Group A: HF (Remin Pro) n = 35 and Group B: CPP-ACPF (Tooth Mousse Plus®) n = 35. The samples were immersed in demineralizing solution for 96 h followed by pH cycling for 10 days which included cycles of demineralization and remineralization of their respective groups. Results: Intergroup comparison of the change in the mean value after demineralization and remineralization showed statistically highly significant difference as the change in Group A HF was 213.52 ± 113.17 (standard deviation [SD]) as compared to that in Group B (CPP-ACPF) 76 ± 57.22 (SD). Conclusion: HF was found to be better when compared to CPP-ACPF for tooth remineralization.

Keywords: Demineralization, fluoride, remineralization

How to cite this article:
Nair MC, Patil RU, Bahutule SR. Comparative evaluation of hydroxyapatite fluoride and casein phosphopeptide amorphous calcium phosphate fluoride as remineralizing agents in primary teeth using pH cycling and single-sectioning technique. Int J Med Oral Res 2022;7:31-5

How to cite this URL:
Nair MC, Patil RU, Bahutule SR. Comparative evaluation of hydroxyapatite fluoride and casein phosphopeptide amorphous calcium phosphate fluoride as remineralizing agents in primary teeth using pH cycling and single-sectioning technique. Int J Med Oral Res [serial online] 2022 [cited 2023 May 28];7:31-5. Available from: http://www.ijmorweb.com/text.asp?2022/7/2/31/366310

  Introduction Top

An ounce of prevention is worth a pound of cure” says an old proverb. Dental caries is the most common disease that affects a large number of people and is a worldwide public health problem, affecting numerous urban and rural communities.[1],[2],[3] In children, dental caries is more common, due to their food habits.[4] The caries process is said to be active when periods of demineralization are more than periods of remineralization.[5] White-spot lesions are the earliest macroscopic evidence of early enamel caries.[6] The enamel surface layer stays intact during subsurface demineralization, but without any treatment, it will eventually collapse into a full cavity.[7] Various calcifying fluids were developed for remineralization of white-spot lesions, by several investigators for the reversal of intact enamel lesions.

The potential for demineralization in children at low oral pH is greater while remineralization at normal oral pH is lower than in adults.[8] The highly effective measure for the prevention of caries is the delivery of topical fluoride.[9],[10] More recently, this has led to the introduction of newer materials containing calcium and phosphate ions.[11] There are studies which have focused on the concentration of calcium and phosphate present in the tooth and since both ions are major components of the tooth and are related to remineralization, most of the efforts have been directed toward their deposition or enhancement in the dental structure.[12] These casein phosphopeptide amorphous calcium phosphate (CPP-ACP) nanocomplexes have been demonstrated to have anticariogenic potential in animal and human in situ experiments.[13],[14],[15],[16],[17],[18] In in vitro studies, CPP-ACP solutions have been shown to remineralize subsurface lesions in human third molar enamel. Casein phosphopeptides containing the cluster sequence -Ser (P)-Ser (P)-Ser (P)-Glu-Glu- bind fluoride as well as calcium and phosphate and thus can also stabilize calcium fluoride phosphate as soluble complexes and are designated as CPP-ACPF.[19] Greater remineralization is produced by caseinphosphopeptide amorphous calcium phosphate fluoride (CPP-ACPF) than CPP-ACP, and the major product formed when remineralization is undertaken with CPP-ACPF is fluorapatite, which is highly resistant to acid dissolution.[20]

Hydroxyapatite fluoride (HF) (Remin Pro) is another type of remineralizing paste which contains calcium and phosphate in the hydroxyapatite form. An added benefit is that fluoride and xylitol have also been included in this product.[21] Therefore, this study was performed to evaluate the possible effect of topical application of HF (Remin Pro) and CPP-ACPF (Tooth Mousse Plus®) [Figure 1] in bringing about remineralization changes in the artificially created carious lesion.[22]
Figure 1: Test materials – Tooth Mousse Plus® and Remin Pro

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  Materials and Methods Top

Seventy deciduous teeth with at least one smooth surface not undermined by caries were collected and immersed in the distilled water at room temperature. A window of 2 mm × 2 mm was created on the sound, intact buccal, or lingual surface of teeth by coating the remaining surface of enamel using acid-resistant nail varnish. Samples were randomly divided into two groups (n = 35) with 35 samples in each group.

Group A = HF (Remin Pro-VOCO, Germany) (n1 = 35)

Group B = CPP-ACPF (GC-Tooth Mousse Plus®) (n2 = 35)

Process of demineralization

Artificial carious, lesions[23],[24],[25],[26] were produced by suspending the samples in the demineralizing solution for 96 h in an incubator at a temperature of 35° ± 2° [Figure 2]. One liter of demineralizing solution was prepared by adding 2.2 mM CaCl2, 2.2 mM KH2PO4, and 0.05M acetic acids at the pH adjusted to 4.4 with 1M KOH (prepared indigenously) by Ten Cate and Duijsters.[27] After induction of enamel lesions, all samples were then assessed under a stereomicroscope to obtain the lesion depth.
Figure 2: Samples immersed in demineralizing solution pH cycle

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Process of remineralization

Longitudinal sectioning of the samples was done. The cut inner part of the section was then painted with nail varnish except for the artificial carious lesion surface. The remineralizing solution was prepared which had 1.5 mM CaCl2, 0.9 mM NaH2PO4, and 0.15M KCL adjusted at a pH of 7.0 by Ten Cate and Duijsters.[27] and the samples were immersed in the remineralizing solution following the pH cycling method along with supernatant solution.

Toothpaste supernatant preparation

Toothpaste supernatants in groups A and B were achieved by suspending 15 g of the respective toothpaste in 45 ml of deionized water to achieve 1:3 (toothpaste: deionized water) ratio.[23]

pH cycling procedure

Each specimen was subjected to different solutions for a specific period in a pH cycle. The pH cycle used, as in accordance with Ten Cate and Duijsters,[23] was as follows:

  • Step 1: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 2: Demineralizing solution (10 ml/section) for 3 h
  • Step 3: Remineralizing solution (10 ml/section) for 2 h
  • Step 4: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 5: Demineralizing solution (10 ml/section) for 3 h
  • Step 6: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 7: Remineralizing solution (10 ml/tooth) overnight
  • Step 8: This pH cycle continues for 10 days.

After 10 days, the remineralized sections were studied under the stereomicroscope and evaluated for lesion depth.

Evaluation of depth of the lesion

The evaluated values for the demineralized sections and remineralized sections were noted in units using “micromotor eyepieces.” The unit's value was then converted into micrometers and millimeters using the following formulas:

Statistical analysis and results

Descriptive statistics such as mean and standard deviation (SD) of demineralization and remineralization of specimens and changes in the values before and after have been expressed. As the data did not follow a normal curve, (as determined by Kolmogorov–Smirnov test P < 0.01: highly skewed data), nonparametric tests were used.

Intergroup comparison of means of demineralization and remineralization of specimens, change has been done using Mann–Whitney test. Intragroup comparison is done using the Wilcoxon signed-rank test. For all the statistical tests, P < 0.05 was considered to be statistically significant, keeping α error at 5% and β error at 20%, thus giving power to the study as 80%. The present in vitro study was conducted to evaluate and compare the remineralization potential of HF and CPP-ACPF; and the recorded values have been presented as mean ± SD, range, and percentage changes. The values were analyzed using the Mann–Whitney test and Wilcoxon signed-rank test.

On comparison, it was seen that the mean of Group A (HF) was 678.72 ± 235.57 (SD) and 465.19 ± 170.25 (SD), and Group B (CPP-ACPF) was 644.52 ± 162.60 (SD) and 568.40 ± 159.35 (SD) after demineralization and remineralization respectively. However, after remineralization with HF (Group A), there was reduction in lesion depth (μm) and the mean of the group was 465.19 ± 170.250 (SD). Furthermore, there was a reduction in lesion depth when specimens were remineralized using CPP-ACPF (Group B) and the mean of this group was 568.40 ± 159.35 (SD) [Table 1], [Table 2], [Table 3] and [Graph 1].
Table 1: Intergroup comparison of demineralization and remineralization values of specimens among both groups

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Table 2: Intragroup comparison for lesion depth before and after remineralization for Group A hydroxyapatite fluoride

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Table 3: Intragroup comparison for lesion depth before and after remineralization for Group B casein phosphopeptide-amorphous calcium phosphate fluoride paste

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On intergroup comparison of lesion depth (μm) after remineralization, there was a statistically highly significant difference seen (P = 0.002). Intergroup comparison of change in the mean value after demineralization and remineralization of the specimens also showed a statistically highly significant difference as the change in Group A (HF) was 213.52 ± 113.17 (SD) as compared to that in Group B (CPP-ACPF) 76 ± 57.22 (SD). As the mean was higher in Group A than in Group B the remineralization efficiency of Group A was found to be better when compared to Group B.

  Discussion Top

Dental caries is a complex multifactorial disease that involves interactions among the tooth structure, oral microbial biofilm formed on the tooth surface, dietary carbohydrates, and salivary and genetic influences.[28],[29],[30] Fluoride dentifrices have a dose-response relationship;[31] and it is important because dentifrices for children offen contain between 250 and 500 ppm fluoride, to reduce the risk of fluorosis. In the present study, CPP-ACPF (Tooth Mousse Plus®) 900 ppm F and HF (Remin Pro) 1450 ppm F were used to remineralize the artificially developed carious lesion on primary teeth.

The concept of in vitro pH cycling was first proposed by Tencate and Duijsters in 1982 in experiments where they exposed artificial carious lesions in enamel to a combination of remineralizing and demineralizing solutions. These experiments were designed to simulate the dynamic variations in mineral saturation and pH associated with the natural carious process.[23] Similar chemical caries model for the production of carious lesion was used in the present study and the solution was kept at a temperature of 37°C.[32]

Casein has the ability to stabilize calcium and phosphate ions by releasing small sequences of peptides (CPPs) through partial enzymatic digestion that led to the development of a remineralization technology based on casein phosphopeptide stabilized amorphous calcium phosphate complexes (CPP-ACP) and casein phosphopeptide stabilized amorphous calcium fluoride phosphate complexes (CPP-ACPF).[33] The caseinphosphopeptide-amorphous calcium phosphate interacts with fluoride ions to produce an amorphous calcium fluoride phosphate stabilized by the CPP on the tooth surface.[34] Tooth creams using CPP-ACP (Recaldent technology) such as MI Paste and Tooth Mousse[35] recognize the importance of the neutral ion species, gaining access to the subsurface lesion through a porous enamel surface. That is why arrested white-spot lesions should have a surface etching treatment before remineralization with recaldent products, unlike fluoride treatments with conventional dentifrices (1000 ppm) that deposit surface minerals but do not eliminate a white-spot lesion.[36] These findings are consistent with a recent in vitro study by Lennon et al. who showed that when casein calcium phosphate was used followed by a toothpaste containing 250 ppm fluoride in an erosive cycle, the enamel loss was less than when using fluoride (250 ppm) and casein / calcium phosphate alone.[37] The above-mentioned studies proved the combined benefits of CPP-ACP and fluoride (Tooth Mousse Plus®) which was used in this study.[24]

Remin Pro is a water-based cream containing hydroxyapatite (calcium and phosphate), fluoride (1450 ppm), and xylitol. The mineral component of enamel is composed mainly of calcium-deficient hydroxyapatite (HAP, Ca10(PO4)6(OH)2), crystals with various impurities, mainly carbonate ions.[38],[39] Fluoride and hydroxyapatite in this material reinforce remineralization and strengthen the enamel surface, as well as help acid neutralizing and balance the oral flora.[40] The high concentration of fluoride which is bound to the tooth surface acts as a fluoride reservoir at the enamel–plaque–saliva interface[41] and aids in the neutralization of acids in plaque, therefore, it balances oral flora.

The present study was designed to test the remineralizing effects of two remineralizing agents on enamel caries initiation and lesion depth progression in in vitro condition. The samples were divided into two groups: Group A (Remin pro) and Group B (Tooth Mousse Plus®) with 35 samples in each group. These samples were subjected to experimental application and subsequent acid challenge for 96 h and were subjected to remineralization for 10 days. Similar studies were done by Advani et al. where they compared the efficacy of remineralization of two pediatric dentifrices and one regular dentifrice on artificial carious lesions in primary teeth by evaluating the depth of the lesion under stereomicroscope.[23] In this study, intergroup comparison of means of demineralization and remineralization of specimens, change (between the groups) has been done using the Mann–Whitney test. Intragroup comparison (before and after values in each group) was done using the Wilcoxson signed-rank test. Intragroup comparison of each group showed statistically significant differences in both groups but after intergroup comparison after demineralization values were not significantly different for Group A (HF) and Group B (CPP-ACPF) [Table 1] and [Table 2]. A statistically nonsignificant difference was seen between Group A and Group B after demineralization. However, after remineralization with HF (Group A), there was a reduction in lesion depth (μm). Furthermore, there was a reduction in lesion depth when specimens were remineralized using CPP-ACPF (Group B). It was observed that the change in Group B was more as compared to Group A. As the mean was higher in Group A (HF) as compared to Group B (CPP-ACPF) it proved that the remineralization efficiency of Group A (HF) is better when compared to Group B (CPP-ACPF). The formation of FHAP could be the most probable reason for it to be resistant to demineralization in an acidic environment and for better remineralization properties. In the present study, HF provided additional protection against caries formation in sound enamel compared with CPP-ACPF. The HF (Remin Pro) provides calcium, phosphate, and fluoride ions for incorporation into clinically and macroscopically undetectable demineralized and hypomineralized enamel before lesion formation. As HF significantly increased remineralization, it is recommended for conservative treatments, after whitening, during orthodontic treatments, and also in high-risk children. In the present study, the remineralizing potential of HF was found to be better than CPP-ACPF.

More in vivo studies need to be carried out with a larger sample size incorporating permanent teeth as well as making the use of different durations for demineralization and remineralization.

  Conclusion Top

There was a significant difference in the lesion depth of specimens following demineralization and after the treatment of specimens with the remineralizing agents

  1. It was observed that both the remineralizing agents used in the study significantly decreased the lesion depth of the specimens after 10 days and when compared both, HF had better remineralizing capacity than CPP-ACPF
  2. Remin Pro can be used as an alternative to fluoride as remineralizing agent as it has shown higher remineralizing potential than Tooth Mousse Plus®.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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