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Year : 2019  |  Volume : 22  |  Issue : 3  |  Page : 305-309
Remineralization potential of fluoride, amorphous calcium phosphate-casein phosphopeptide, and combination of hydroxylapatite and fluoride on enamel lesions: An in vitro comparative evaluation

1 Department of Conservative Dentistry and Endodontics, Yogita Dental College and Hospital, Ratnagiri, Maharashtra, India
2 Department of Conservative Dentistry and Endodontics, Vasantdada Patil Dental College and Hospital, Sangli, Maharashtra, India
3 Department of Conservative Dentistry and Endodontics, D.Y. Patil Dental School, Pune, Maharashtra, India

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Date of Submission05-Feb-2019
Date of Decision18-Mar-2019
Date of Acceptance03-Apr-2019
Date of Web Publication03-Jul-2019


Aim: The purpose of this study was to evaluate and compare remineralization potential of fluoride, amorphous calcium phosphate-casein phosphopeptide (ACP-CPP), and combination of hydroxyapatite (HAP) and fluoride on enamel lesions.
Materials and Methodology: Ten intact caries-free human premolars were selected. The coronal portion of each tooth was sectioned into four parts to make four enamel blocks. The baseline surface microhardness (SMH) was measured for all the enamel specimens using Vickers microhardness testing machine. The artificial carious lesion was created by immersing the specimens in demineralizing solution for 3 consecutive days at 35° The SMH of each specimen was evaluated. All the four enamel sections of each tooth were subjected to various surface treatments, i.e., Group A – Fluoride varnish, Group B – ACP-CPP, Group C – Combination of HAP and fluoride (Clinpro), and Group D – Control group; no surface treatment. A carious progress test (pH cycle) was carried out which consisted of alternative demineralization (3 h) and remineralization using artificial saliva (21 h.) for 5 consecutive days. After pH cycling, SMH readings of each specimen were again assessed to evaluate remineralization potential of each surface treatment agent.
Results: Data obtained were statistically analyzed using one-way ANOVA followed by Tukey–Kramer multiple comparison test which was applied to detect significant differences between different surface treatments at different phases of studies.
Conclusion: Fluoride varnish shows higher remineralization potential of early carious lesion compare to ACP-CPP and Clinpro.

Keywords: Amorphous calcium phosphate-casein phosphopeptide; Clinpro; demineralization-remineralization; fluoride

How to cite this article:
Bandekar S, Patil S, Dudulwar D, Moogi PP, Ghosh S, Kshirsagar S. Remineralization potential of fluoride, amorphous calcium phosphate-casein phosphopeptide, and combination of hydroxylapatite and fluoride on enamel lesions: An in vitro comparative evaluation. J Conserv Dent 2019;22:305-9

How to cite this URL:
Bandekar S, Patil S, Dudulwar D, Moogi PP, Ghosh S, Kshirsagar S. Remineralization potential of fluoride, amorphous calcium phosphate-casein phosphopeptide, and combination of hydroxylapatite and fluoride on enamel lesions: An in vitro comparative evaluation. J Conserv Dent [serial online] 2019 [cited 2023 Mar 20];22:305-9. Available from:

   Introduction Top

Tooth surfaces within the oral cavity are colonized by a variety of bacterial species, capable of producing organic acids through the metabolism of dietary carbohydrates. These oral bacteria are the primary constituents of dental plaque. The exchange of mineral (i.e., loss or gain) continues at the surface of enamel as long as the biofilm at the surface creates an environment favoring demineralization or remineralization. The pH of biofilm, levels of calcium, phosphate, and hydroxyl ions in the saliva determine the shift of the balance toward demineralization or remineralization.[1]

Demineralization starts only after the pH drops below the critical pH of 5.5. Below this pH, there is an acidic environment, which results in loss of hydroxyl and phosphate ions by reacting with excess of hydrogen ions.[2] White-spot lesions are the earliest macroscopic evidence of enamel caries. White spots are evident when the amount of subsurface minerals lost (demineralization) exceeds the amount of minerals gained (remineralization) for a long period of time.[3] The majority of demineralization in white-spot lesions occurs in the subsurface region of enamel. This subsurface demineralization increases porosity and changes the optical properties of the enamel. Typically, the enamel surface layer stays intact during subsurface demineralization, but without treatment, the subsurface loss will continue and eventually the surface layer will collapse and lead to a cavity formation.[4]

Remineralization is nothing but the net gain in minerals at the surface of enamel, which were lost due to demineralization. Remineralization is dependent on the bioavailability of calcium, phosphate, and hydroxyl ions. It is however enhanced by the presence of sublevels of fluoride.[5],[6]

In the past decade, various remineralizing agents containing fluoride, calcium, and phosphate ions in varied forms and concentrations were introduced. These agents tend to remineralize the subsurface caries lesion by providing calcium phosphate with or without fluoride and control the surrounding microenvironment.

Fluor Protector (Intro Pack; Ivoclar Vivadent) is a fluoride varnish containing 1000 ppm of fluoride. Fluoride is the most commonly used remineralizing agent. When the acid attacks the enamel surface, the pH begins to rise and fluoride present in microenvironment cause enamel dissolution to stop.[7] As pH rises, newer and larger crystals that contain more fluoride (fluorhydroxyapatite) are formed, therefore reducing demineralization by forming fluorhydroxyapatite crystals and enhancing remineralization.[8]

Tooth mousse cream (GC International, Itabashi-Ku, Tokyo, Japan) contains nanocomplexes of milk protein, casein phosphopeptide (CPP) with amorphous calcium phosphate (ACP). It has been suggested that CPP has the ability to stabilize calcium phosphate in solution by binding ACP with their multiple phosphoserine residues, thereby allowing the formation of small CPP-ACP clusters.[9] It has been claimed that it promotes remineralization of the carious lesions by maintaining a supersaturated state of enamel minerals, at the same time, it also hinders colonization of dental surfaces by cariogenic bacteria.[8]

Clinpro™ Tooth Creame (3M ESPE Dental Products, St. Paul, MN, USA) contains 950 ppm fluoride and a functionalized tri-calcium phosphate ingredient (fTCP) also known as hydroxyapatite (HAP). One main feature of this calcium phosphate system is that, it is stable in an aqueous environment and does not affect the fluoride activity added in the dentifrice.[10]

All the three remineralizing agents mentioned above, differ in their composition and mechanism of action yet, each one has a promising ability to remineralize the enamel. Thus, the present study was undertaken to determine and compare the remineralizing potential of fluoride varnish, ACP-CPP, and combination of HAP and fluoride on early enamel carious lesion.

   Materials and Methodology Top

Ten freshly extracted premolars from patients ranging in the age group of 14–20 years, for orthodontic purpose, were collected, and the radicular part of each tooth was removed. The coronal part of each tooth was then longitudinally sectioned buccolingually and mesiodistally into four sections using a high-speed diamond tipped disc. Four enamel specimens were prepared. Custom made plastic cylindrical molds were made and self-cured acrylic resin was poured on it; then, each enamel block was embedded in, on top of partially set, and allowed to set. An acid-resistant nail varnish was applied around the exposed enamel surface leaving a window of 3 mm × 3 mm of enamel exposed at the center.

Leica Japan, Tokyo, Vickers micro hardness tester was used to evaluate microhardness. A load of 25 g was applied, for 5 s, for all the specimens. The Vickers hardness numbers (VHNs) of five indentations at spacing of 100 μ were taken and the average value was considered the mean baseline surface microhardness (SMH) of the corresponding specimen. The objective of baseline SMH determination is to compare and calculate the changes that occur after induction of enamel lesions and after pH cycling.

Carious lesions representing preliminary stage of subsurface enamel demineralization were produced by suspending four sections of each tooth into glass tubes containing 20 mL of demineralization solution, for 72 h, in an incubator at a temperature of 35°.[9] After induction of enamel lesions, all the specimens were evaluated for SMH measurements under 25 g loads for 5 s duration. As mentioned earlier, the four sections of each tooth were distributed in the following groups:

Group A – A thin layer of fluoride varnish was applied, allowed to be absorbed for 20 s and then air dried. Group B – A generous layer of ACP-CPP cream was applied by an applicator brush and left undisturbed for a minimum of 3 min. Group C – A thin layer of Clinpro was applied and allowed to be absorbed for 3 min. Group D – This served as the control group where no surface treatment was performed.

A pH cycling regimen included alternative demineralization (2 h) and remineralization (21 h) for 5 consecutive days. For the demineralization phase, the demineralization solution for the induction of enamel lesions was used; and for the remineralization phase, synthetic saliva preparation was carried out.[11] The inorganic composition of synthetic saliva is similar to that of natural saliva. After pH cycling, again the SMH was assessed for all the specimens under 25 g load for 5 s.

   Results Top

The scores obtained were statistically analyzed using one-way ANOVA followed by Tukey–Kramer multiple comparison test was applied to detect significant differences between various surface treatments at different phases of studies are summarized in [Table 1]. By applying Tukey–Kramer multiple comparison test variation among column means is significantly higher/greater than expected by chance [Table 2].
Table 1: Distribution of mean and standard deviation of microhardness in hardness numbers (25 g load) at initial hardness, after demineralization, and after pH cycling in Group I, II, III, and IV (n=10)

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Table 2: Intragroup and intergroup comparison after surface treatment

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   Discussion Top

In spite of growing awareness toward oral health care, the prevalence of dental caries still remains high. Clinically, the early enamel lesion appears white because the normal translucency of the enamel is lost. The surface becomes fragile and is susceptible to damage from probing. The most important feature of white-spot lesion is the presence of relatively intact surface layer overlying subsurface demineralization (40%–70%). Even though initial enamel lesions have intact surfaces, they have a low mineral content at the surface layer when compared to sound enamel; thus showing a lower hardness value at the surface than for sound enamel tissue.[12],[13]

Organic acids are produced by the metabolic activity of microorganisms in the bacterial plaque. These acids diffuse through the pellicle into the surface enamel. These acids attack the apatite crystals, particularly at the vulnerable lattice points where carbonate ions are present. This causes Ca 2+, OH , PO42−, F , CO 3−, Na +, and Mg 2+ to be removed from the crystal lattice and to diffuse into the solution phase between the crystals. The dissolving calcium ions and phosphate ions form various calcium phosphate salts that either diffuse to the exterior or provide an environment that facilitates the repair of the faulty crystallites beneath the surface of enamel facilitating remineralization.[14] Mineral loss or demineralization proceeds as long as sufficient acid is available. As more enamel dissolves, concentration of the Ca 2+ ion and PO42− ions increase. As calcium and phosphate ions diffuse outward, remineralization at the surface becomes more and more likely. This leads to the formation of an apparently intact enamel surface layer about 20–40 μ where the mineral content is higher than the body of the lesion.[14]

In the present study, the specimens kept in the demineralization solution (CaCl2, NaH2 PO4, lactic acid, and fluoride) for 72 h at 37°C created a subsurface demineralization of approximately 150 μ width with an intact surface simulating an early enamel lesion.[15] The concentration of both calcium and phosphates, in the demineralization solution, was at 50% of saturation level, causing dissolution of only enamel subsurface. The addition of fluoride prevented surface demineralization by forming fluorapatite at the surface, which simulated the naturally occurring early enamel lesions having an intact surface layer.

Considering the importance of the surface layer in caries progression, the evaluation of changes in this region is relevant. SMH measurement is a suitable technique for this purpose. Microhardness measurement is appropriate for a material having fine microstructure, nonhomogeneous, or prone to cracking like enamel. SMH indentation provides a relatively simple, nondestructive, and rapid method in demineralization and remineralization studies. Therefore, in the present study, the microhardness values for each specimen were measured in three steps; the baseline microhardness, after induction of carious lesion (demineralization), and after pH cycling.[16]

The values (VHN) obtained during the initial baseline microhardness measurements in the present study were in the range of VHN 246–372, which satisfies the VHN range of normal enamel tissue.[16] The SMH values for each group of the enamel specimens were decreased to 165–193 at the end of 72 h of demineralization which is in accordance with the study conducted by Lata et al.[7] The period for demineralization in the pH cycling phase is for 3 h, which was to simulate the duration of demineralization (low cariogenic challenge) that occurs in the oral cavity.[17] The test material was applied on enamel blocks twice a day to simulate the normal recommended daily oral prophylaxis.

In the present study, after the pH cycling phase, the mean SMH (VHN) for Group A was 219, for Group B group was 225, for Group C group was 219, and for the control group, it was 164, respectively. It indicates that topical fluoride varnish application produced maximum remineralization compare to ACP-CPP and combination of HAP and fluoride.

Fluoride has been recognized as a valuable therapeutic agent to provide partial protection against dental caries for approximately one-half century. Recent evidence suggests that the primary mechanism of action of fluoride may be its ability to facilitate the remineralization of white-spot lesions. Enamel is primarily made of calcium phosphate-based crystalline mineral called HAP. The HAP crystals are packed tightly together to form millions of microscopic prisms and lattices. Fluoride is incorporated into solid crystalline lattice by iso-ionic exchange to form fluorohydroxyapatite. This form of enamel is harder than naturally occurring enamel.[18]

The new phosphopeptide isolated from the milk protein casein, combined with calcium phosphate (CPP-ACP nanocomplexes), has recently been shown to be efficacious in both the prevention and reversal of enamel subsurface lesions in caries.[19],[20],[21]

In the present study, demineralized enamel specimens were subjected to a remineralizing cycle of 5 days and treated with the remineralizing agent twice daily. It was found that in Group A microhardness increased significantly to 219.0 ± 20.33 (mean increase of 31.9 VHN, [P = 0.001]). The mean increase in microhardness of enamel is in accordance with the study conducted by Lata et al.[7] This indicated that there is a significant increase in microhardness, therefore ACP-CPP can also aid in remineralization, but not as effective as fluoride.

The fTCP contains TCP, especially milled with a simple organic ingredient known as “Sodium Lauryl Sulfate.” During the milling process, they functionalize the TCP resulting in an organic-calcium phosphate hybrid being formed. This importantly ensures that the calcium oxides are protected from the undesirable interactions with fluoride, which could render both calcium and fluoride inactive preventing calcium phosphate reaction with fluoride and formation of calcium fluoride. As a result, fluoride, calcium, and phosphate are available in an aqueous form for the remineralization process.[22]

In a study conducted by Karlinsey et al., 2010,[23] compared 5000 ppm containing dentifrice with Clinpro 5000 (5000 ppm fluoride + fTCP). Clinpro 5000 showed a mean increase of 105.6 ± 5.6 VHN, in microhardness of enamel after 10 days. In the present study, the microhardness of enamel specimens treated with fTCP + NaF showed a mean increase of 48.2 + 13.8 VHN. Thus, in the present study, a lesser increase in microhardness was seen for the specimens were subjected to a 5-day cycle compared to 10 days in the study done by Karlinsey et al.

   Conclusion Top

Within the limits of the present in vitro study, we conclude that: fluoride varnish is effective in remineralization of early enamel carious lesion. ACP-CPP and combination of HAP and fluoride are effective too, but to a lesser extent compared to fluoride group, no statistical difference was found between ACP-CPP and combination of HAP and fluoride groups.

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Conflicts of interest

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   References Top

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Correspondence Address:
Dr. Siddhesh Bandekar
Department of Conservative Dentistry and Endodontics, Yogita Dental College and Hospital, Khed, Ratnagiri - 415 709, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCD.JCD_13_19

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