|Year : 2017 | Volume
| Issue : 3 | Page : 229-237
A comparative evaluation of the remineralization potential of three commercially available remineralizing agents on white spot lesions in primary teeth: An in vitro study
Pujan Kamath1, Rashmi Nayak1, Shobha U Kamath2, Deepika Pai1
1 Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences Manipal, Manipal University, Manipal, Karnataka, India
2 Department of Biochemistry, KMC Manipal, Manipal University, Manipal, Karnataka, India
|Date of Web Publication||31-Jul-2017|
Department of Pedodontics and Preventive Dentistry, Room No. 6, Manipal College of Dental Sciences Manipal, Manipal University, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The focus in caries management has shifted to early detection of caries lesions and targeted noninvasive management of incipient lesions using novel remineralizing agents. Aim: This study aimed to compare and evaluate the remineralization potential of commercially available agents containing nano-hydroxyapatite (nano-HA), casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), and Tricalcium phosphate (TCP) on artificially induced white spot lesions in primary teeth. Settings and Design: This is an in vitro double-blind randomized study. Materials and Methods: Forty extracted or exfoliated primary teeth were selected and randomized as follows: Group I: FTCP, Group II: fluoridated dentifrice, Group III: CPP-ACPF, and Group IV: Nano-HA. DIAGNOdent readings and scanning electron microscope (SEM) energy dispersive X-ray (EDX) analysis were carried out at baseline. The samples were subjected to the test agents after inducing white spot lesions. The readings were repeated postdemineralization and postremineralization. Statistical Analysis: The data were analyzed using IBM SPSS version 20 software with one-way ANOVA, post hoc Tukey's HSD, and paired t-test. Results: SEM evaluation showed favorable surface changes in all the four study groups after remineralization therapy. Intragroup comparison of DIAGNOdent and EDX readings showed a highly significant difference between baseline, postdemineralization, and postremineralization values. However, the intergroup comparison was statistically nonsignificant. Conclusion: All test agents were comparable in their remineralization potential.
Keywords: Casein phosphopeptide-amorphous calcium phosphate, DIAGNOdent, hydroxyapatite, primary teeth, remineralizing agents, tricalcium phosphate, white spot lesions
|How to cite this article:|
Kamath P, Nayak R, Kamath SU, Pai D. A comparative evaluation of the remineralization potential of three commercially available remineralizing agents on white spot lesions in primary teeth: An in vitro study. J Indian Soc Pedod Prev Dent 2017;35:229-37
|How to cite this URL:|
Kamath P, Nayak R, Kamath SU, Pai D. A comparative evaluation of the remineralization potential of three commercially available remineralizing agents on white spot lesions in primary teeth: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2017 [cited 2019 Jul 23];35:229-37. Available from: http://www.jisppd.com/text.asp?2017/35/3/229/211848
| Introduction|| |
The current consensus in caries management is that caries should be detected and managed in its earliest stages when nonsurgical reversal can be achieved. While fluoride is a time-tested, effective, caries-preventive agent, the search for compounds with additional and synergistic benefits has yielded a plethora of novel agents for targeted remineralization therapy.
Enamel of primary teeth is less mineralized, exhibits a greater diffusion coefficient, and consequently more susceptible to acid dissolution compared to enamel of permanent teeth. Early childhood caries which affects the primary dentition frequently manifests as white spot lesions, and aggressive preventive therapy for remineralization of these lesions is essential for their reversal. Thus, the aim of the present study was to compare and evaluate the remineralization potential of commercially available agents containing hydroxyapatite (HA), casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), and tricalcium phosphate on artificially induced white spot lesions in primary teeth. A null hypothesis was proposed that there will be no difference in the remineralization potential of the test agents.
| Materials and Methods|| |
The present study was conducted in the Department of Pedodontics and Preventive Dentistry, in collaboration with the Department of Biochemistry with Institutional Ethical Committee's approval (IEC 454/2012).
Test agents used
- Nano-HA – Reminpro (VoCo, Germany; Lot No: 1106330)
- It is a water-based cream containing HA and fluoride (1450 ppm sodium fluoride)
- CPP-ACPF – GC Tooth Mousse Plus (GC Corporation, Tokyo, Japan; Lot No: 1108171)
- It is a water-based cream containing CPP-ACPF with incorporated fluoride (0.2% w/w, 900 ppm)
- Tricalcium Phosphate – Clinpro ™ (3M ESPE, USA; Lot No: 10021)
- Tooth cream containing tricalcium phosphate and 0.21% w/w sodium fluoride
- Fluoridated dentifrice – Colgate (Colgate-Palmolive, India)
- Sodium monofluorophosphate with 1000 ppm fluoride.
Sample size of forty was estimated using the power calculation α = 0.05 and β = 0.20 with 80% being the power of the study. Based on previous findings reported by Hegde and Moany, a total of 65 primary teeth indicated for extraction were obtained from patients in the age group of 3–14 years. A written informed consent was obtained from the parents at the outset. Owing to the difficulty in obtaining sound, caries-free primary teeth, sound tooth surfaces obtained from carious primary teeth were considered in this study. Teeth with at least one sound tooth surface, without any visible caries, hypoplastic lesions, stains, or white spot lesions were included in the study. After extraction, the teeth were cleaned, washed with water, and stored in isotonic saline.
The coronal portion of the teeth was separated from the roots using a diamond disc (Diatech CH-9435, Swiss Dental Instruments, Lot: 9605) mounted on a Micro Motor Straight Hand Piece (NSK, Japan) in the presence of a water coolant. The samples were then embedded into the partially set acrylic resin in cylindrical plastic molds.
A 3 mm × 3 mm sticking plaster was placed on the exposed tooth surface and acid-resistant nail varnish was applied around it to delineate the area to be studied.
The baseline DIAGNOdent (Kaltenbach and Voigt GmbH and Co. KG, Biberach, Germany) readings were recorded for all the samples. The present study used the newly formulated DIAGNOdent cutoff limits given by Goel et al. for detection of early carious lesions in primary teeth. Of the 65 teeth initially collected, 25 samples showing a value >6 were discarded, establishing a final sample size of 40.
The samples were randomly divided into four groups (I–IV) comprising ten samples each using simple randomized sampling (n = 10). Individual sample within each group was assigned an alphabetical denotation (A–J).
The freshly prepared demineralization solution comprised 2.2 mM CaCl2, 2.2 mM NaH2 PO4, 0.05 M lactic acid, and 0.2 ppm fluoride. The pH was adjusted to 4.5 with 50% NaOH. The solution was maintained at 37°C. Each sample was immersed in an individual sterile container containing 12 mL of demineralization solution ensuring that the section was completely immersed in the solution. The samples were maintained at 37°C in an incubator (Autoflow N-5510, NuAire, USA) for 72 h. This demineralization procedure was intended to produce a consistent subsurface lesion.
Recording of readings
After demineralization, the samples were thoroughly washed with deionized water and allowed to dry. The samples were evaluated for demineralization using DIAGNOdent.
Following DIAGNOdent readings, six out of the ten samples from each group (A–F) were subjected to scanning electron microscopic (SEM) evaluation (EVO MA18 with Oxford EDS [X-act]) to evaluate surface morphology. Estimation of calcium (Ca), phosphate, and fluoride (F) (weight percentage) content was done using energy dispersive X-ray (EDX) analysis for which the samples were mounted firmly using a graphite tape on a disc comprising nine specimen holders and maintained under vacuum. The samples were analyzed at 15 kV and × 500, ×1500, ×2000, and × 5000 magnifications.
A double-blind investigation was planned since all the test agents had the same color (white) and similar consistency. However, since the packaging was not similar, in order to confirm the double-blind design of the study, each of the test agents was coded as I, II, III, and IV and entrusted to the chief supervisor of the study. Half pea-sized quantity of test agent was dispensed half an hour prior to the initiation of treatment in a palette with four wells. The sanctity of the double-blind study was strictly maintained and decoding was done after statistical evaluation of the data.
Application of test agents
The samples in each group were treated with the allocated remineralizing agent once every 24 h for 14 days. The test agents were continuously applied onto the tooth surface within the window area with the help of a disposable cotton tip applicator for 3 min. Samples were then washed with deionized water and placed in artificial saliva, maintained at ambient temperature. Artificial saliva was changed every 24 h, just before the immersion of freshly treated samples.
Following is the composition of artificial saliva used:
- Na3 PO4-3.90 mM, NaCl2-4.29 mM, KCl - 17.98 mM
- CaCl2-1.10 mM, MgCl2-0.08 mM, H2 SO4-0.50 mM
- NaHCO3-3.27 mM, distilled water, and the pH was set at 7.2.
Re-evaluation of test parameters
After the 14-day remineralization regimen, the surface was reassessed using DIAGNOdent. The samples (A–F) were also assessed using SEM-EDX to study the change in surface characteristics and estimate the mineral content (Ca, phosphate [P], and F).
The data were analyzed by IBM Statistical Package for Social Sciences version 20 (IBM corp., Armonk, New York) software using one-way ANOVA, post hoc Tukey's HSD, and paired t-test. P < 0.005 was considered statistically significant.
| Results|| |
On comparing the DIAGNOdent readings Paired t-test showed a statistically significant increase in the readings from baseline to post-demineralization [Table 1].
Paired t-test showed a statistically significant increase in the readings from baseline to post-demineralization (P < 0.001) and a statistically significant decrease postremineralization (P < 0.001) and approaching the baseline values for each of the experimental groups [Table 2].
|Table 2: Intragroup comparison of mean fluoride levels postdemineralization and postremineralization|
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However, post hoc Tukey's HSD test showed statistically nonsignificant difference among the four groups at the different experimental stages.
Scanning electron microscopy
SEM images [Figure 1], [Figure 2], [Figure 3], [Figure 4] taken after 72 h of demineralization revealed loss of surface integrity. Loss of aprismatic enamel was evident and it was noticeable that the enamel comprised indistinct, completely destructed enamel rods with loss of prism cores, wide inter-rod spaces, and leftovers of fractured enamel prism bases suggestive of demineralization.
|Figure 1: Group I - Postdemineralization (scanning electron microscopic image)|
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|Figure 2: Group I - Postremineralization (scanning electron microscopic image)|
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|Figure 3: Group II - Postdemineralization (scanning electron microscopic image)|
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|Figure 4: Group II - Postremineralization (scanning electron microscopic image)|
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Postremineralization [Figure 5], [Figure 6], [Figure 7], [Figure 8]
|Figure 5: Group III - Postdemineralization (scanning electron microscopic image)|
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|Figure 6: Group III - Postremineralization (scanning electron microscopic image)|
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|Figure 7: Group IV - Postdemineralization (scanning electron microscopic image)|
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|Figure 8: Group IV - Postremineralization (scanning electron microscopic image)|
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On observation, the samples demonstrated plugging of the porous defects with resultant decrease in the cavities and micropores and re-establishment of surface integrity in all the four groups. Distinct surface coatings deposited by different agents were evident. However, in comparison to other groups, samples of nano-HA group showed a superior and uniform re-establishment of surface integrity.
Energy dispersive X-ray analysis
Calcium and phosphate levels
The Ca/P content (weight %) of demineralized and remineralized enamel was converted into Ca/P ratios for each group.
The percentage gain of Ca/P was calculated using the following formula:
An intergroup comparison of the mean percentage gain of the four study groups showed statistically nonsignificant gain posttreatment (P = 0.159) [Graph 1].
Estimation of fluoride content
One-way ANOVA test showed statistically nonsignificant difference between the mean fluoride content of the four experimental groups postdemineralization (P = 0.275) and postremineralization (P = 0.494) [Graph 2].
Paired t-test demonstrated a statistically significant difference (P < 0.05) in the mean fluoride content from postdemineralization to postremineralization for each of the experimental groups.
Thus, the proposed null hypothesis was accepted.
| Discussion|| |
The oral cavity is a battlefield of activities of demineralization and remineralization, the balance between them being the crucial determining factor of the caries process.
Demineralization of enamel leads to dissolution of HA and diffusion of Ca/P ions toward the enamel surface. Hypersaturation of Ca/P ions on the surface results in a re-precipitation of HA forming the intact superficial layer on the enamel surface. Remineralization of enamel is enhanced by the presence of fluoride ions.
The noninvasive treatment of early lesions by remineralization bridges the traditional gap between prevention and surgical procedures. Central to this vision is the ability to detect caries lesions at an early stage and correctly quantify the degree of mineral loss, ensuring that the correct intervention is instituted.
Assessment of in vitro demineralization and remineralization can be done using different methods. The methods used are SEM/SEM-EDX,,, DIAGNOdent, surface microhardness, and polarized light microscopy., DIAGNOdent was used in the present study with three main objectives. To aid in the selection of caries-free, sound enamel surfaces at the outset of the study, to ensure decalcification prior to subjecting the samples to SEM-EDX, and to detect and compare the changes before and after application of the remineralizing agents. DIAGNOdent (KAVO, Biberach, Germany), a noninvasive method, uses laser fluorescence to measure early demineralization. Various literature reports have shown the value of DIAGNOdent as a reliable noninvasive caries-detecting device.,,
DIAGNOdent has demonstrated promising results for the detection of incipient enamel lesions in studies undertaken in vivo and in vitro in permanent teeth, but literature regarding its use in deciduous teeth is scanty. Results from permanent teeth cannot be extrapolated to primary teeth because these have different morphological, anatomical, and physiological characteristics. In view of this, newly formulated cutoff limits with histological validation developed by Goel et al. were considered in the present study as against the manufacturer's cutoff limits.
Prior to recording DIAGNOdent readings at baseline, postdemineralization, and postremineralization, each sample was air-dried for 10 s. This was in accordance with Pinelli et al. who emphasized that reproducibility of the device increased under dry condition, thereby advising drying to detect caries-like lesions on free smooth surfaces.
In the present study, low frequency (LF) values increased postdemineralization and subsequently decreased postremineralization. Similar results were demonstrated in studies conducted by Al-Khateeb et al.,, Eggertsson et al., Shi et al., Mendes et al., Pai et al., Bahrololoomi et al., and Patil et al. who also used DIAGNOdent to assess remineralization.
Mendes et al. attributed the increase in LF values to enhanced porosity in enamel surface due to decrease in the overall mineral content. Furthermore, decrease in values postremineralization can be due to decrease in porosity of the enamel surface.
Artificially produced caries-like lesions are of considerable interest as they can be compared to the earliest detectable ultrastructural change in the caries process.
To produce and study in vitro enamel demineralization models, many demineralizing agents have been used such as acetic acid, lactic acid, or acidified hydroxyethylcellulose system  for different time periods. In the present study, demineralization was carried out as described by Lata et al. The specimens were kept in the demineralization solution for 72 h at 37°C, creating a subsurface demineralization of approximately 150 μ width with an intact surface simulating an early enamel lesion. The concentration of both Ca and P, in the demineralization solution, was at 50% of saturation level, causing dissolution of only enamel subsurface. Addition of fluoride prevented surface demineralization by forming fluorapatite at the surface, which simulated the naturally occurring early enamel lesions having intact surface layer.
The present study used paste-type formulation of the test agents, applied with disposable cotton tip applicators. This was done in view of replication of patient convenience in using the tooth creams like a toothpaste with toothbrushes or along with cotton tip applicators. The remineralization regimen comprised 3 min daily application for 14 days. This is in accordance with the manufacturer's prescription. Pai et al. followed a similar regimen of 3 min daily application for 14 days. In contrast, in a study by Shirahatti et al., the treatment pastes were applied twice daily for 3 min for 14 days. Hegde and Moany  in their study performed twice daily application of CPP-ACP for 3 min for 7, 14, 21, 28, and 35 days. The difference in remineralization regimen could be the possible reason for variation in outcomes in our study.
In the present study, artificial saliva was changed every 24 h during the 14-day remineralization regimen to ensure ionic balance and maintenance of pH. In contrast, Shirahatti et al. changed the artificial saliva solution every 72 h in their study.
SEM is one of the most sensitive, time-tested techniques to assess the demineralization and remineralization of the carious lesions in vitro as reported in earlier studies,,, while in most studies, using SEM, samples are coated with metals such as gold or palladium to improve image quality., Harding et al. and Nicolae et al. studied the samples using SEM without metal sputtering so that they could be observed again, if necessary, once the study ended. Consequently, this version was considered in our study as there would not be loss of samples and re-evaluation of the same samples after treatment regimen would be possible. A graphite tape was used to aid in conduction.
The demineralized enamel specimens were observed at different magnifications. At ×5000 magnification, numerous depressions in a honeycomb pattern were revealed which corresponded to the observations made by other investigators. Correlation of SEM observations made it possible to conclude that DIAGNOdent is effective in detecting demineralization of enamel. Postremineralization, all the test specimens showed re-establishment of surface integrity. The potential to induce remineralization of the test agents was demonstrated as seen by the increase in crystal size and occlusion of porous defects. However, there were minor differences in the surface morphology, which may be due to their different mechanisms for promoting remineralization. The SEM results were in accordance with Huang et al., Roveri et al., and Swarup and Rao.
A superior surface texture of enamel was noted in the present study on SEM evaluation postremineralization with nano-HA compared to other groups. This could be attributed to similarity of the nano-sized particles to the apatitic crystals of tooth enamel in morphology, crystal structure, and crystallinity.
A correlation of SEM observations and LF readings postremineralization indicated that DIAGNOdent was effective in detecting enamel remineralization.
EDX analysis was used to determine the mineral content postdemineralization and postremineralization, thereby facilitating estimation of alteration in mineral content pre- and post-treatment. Eggerath et al., (2011), used EDX elemental analysis to estimate Ca, P, and F content of enamel after exposure to fl uoride-containing tablets in vitro. used EDX elemental analysis to estimate Ca, P, and F content of enamel after exposure to fluoride-containing tablets in vitro.
In the present study, EDX analysis showed that the percentage gain of Ca and P postremineralization was greatest in the fTCP group followed by nano-HA, CPP-ACPF, and fluoride groups, the difference between the groups being statistically nonsignificant. This is in contrast to the results of the study by Hedge et al. and Hegde and Moany.
The fall in the surface Ca/P ratio following demineralization did not appreciably increase after remineralizing with fluoridated dentifrice. This finding suggests that fluorides alone bring about only structural modification of apatite crystal, restricted to partial hydroxyl group replacement by fluoride ions without influencing the Ca and P content. Furthermore, this reinforces the fact that fluoride in the presence of Ca and P is more effective in driving the mineral for remineralization. The specimens treated with nano-HA exhibited surface Ca/P ratio close to that of the biological enamel and synthetic nano-HA, indicating an apatitic coating deposition on the demineralized enamel surface.
A study done by Roveri et al. demonstrated that biomimetic nonsized HA crystals produce an apatitic coating deposition on the enamel surface, which is much less crystalline than native enamel apatite, but consists of a new apatitic mineral deposition which progressively fills the surface defects as against the mere structural modification induced by fluoride, restricted to a partial hydroxyl group replacement by fluoride ions without affecting the Ca and P structural network appreciably.
The present study is in agreement with previous studies that demonstrated the capability of the chosen test agents to induce remineralization of early enamel lesions.,,,
Although the remineralization potential of fluoride in the present study was found to be lower compared to the other test agents, its utility cannot be underestimated by virtue of the nonsignificant difference in remineralization potential among the test agents. Judicious use at the target site could facilitate remineralization of the early enamel lesions. Sjögren et al. observed that the retention of fluoride in the mouth is site specific and there is minimal transport of fluoride ions between left and right sides of the mouth and between arches. This explains the development of localized lesions in patients using fluoride dentifrice routinely. Hence, judicious use of fluoride at the appropriate localized site could provide an economical alternative to facilitate remineralization of incipient enamel lesions in vivo.
A limitation of the present study is that SEM-EDX was done only for postdemineralization and postremineralization samples. SEM-EDX at baseline could have facilitated a better comparison of the ability of the test agents to induce remineralization and their potential to bring the mineral content closer to baseline levels.
Thus, although the mechanisms of action of the agents were different, the results obtained indicate that each of the tooth creams enhanced enamel remineralization. To the best of our knowledge, this is the first in vitro study comparing the remineralization potential of the four agents in primary teeth. However, one must bear in mind that remineralization in vitro may be quite different when compared with dynamic complex biological system, which usually occurs in the oral cavity in vivo. Thus, direct extrapolations to clinical conditions must be exercised with caution because of obvious limitations of in vitro studies. Long-term clinical trials are required to establish the utility of remineralizing agents in remineralization of white spot lesion in primary teeth in in vivo conditions and to single out the superiority of any one agent.
| Conclusion|| |
Based on the findings of the present study, the following conclusions were drawn:
- All the test agents were comparable in their remineralization potential
- Role of fluoridated dentifrice as a remineralizing agent cannot be underestimated and judicious use of fluoridated dentifrice could serve as a valuable therapeutic aid
- DIAGNOdent has the potential to be used as a noninvasive diagnostic as well as monitoring tool during remineralization therapies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2]