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ORIGINAL ARTICLE
Year : 2013  |  Volume : 31  |  Issue : 1  |  Page : 29-35
 

In vitro remineralization of enamel subsurface lesions and assessment of dentine tubule occlusion from NaF dentifrices with and without calcium


1 Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
2 Department of Pedodontics and Preventive Dentistry, PGIMER, Chandigarh, India

Date of Web Publication27-May-2013

Correspondence Address:
A R Prabhakar
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
India
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DOI: 10.4103/0970-4388.112403

PMID: 23727740

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   Abstract 

Currently, fluoride is the most effective preventive treatment for remineralization of incipient carious lesions and dentinal hypersensitivity due to wasting disorders. The products containing fluoride, calcium and phosphate are also claim to remineralize early, non-cavitated enamel demineralization. The aim of this study was to investigate and compare the efficacy of two such products, Tooth Mousse and Clinpro tooth crème on remineralization and tubule occluding ability with 5000ppm fluoride-containing toothpaste. Materials and Methods :Thirty third molar teeth were placed in demineralizing solution for 5 days such that only a window of 1mm x 5mm was exposed to the environment to produce artificial caries-like lesions and randomly assigned to three groups: Group I, 5000ppm sodium fluoride; Group II, GC MI paste plus and Group III, Clinpro tooth crème. Axial longitudinal sections of 140-160 μm of each tooth which included the artificial carious lesion taken and were photographed under polarized light microscope. The demineralized areas were then quantified with a computerized imaging system. The experimental materials were applied onto the tooth sections as a topical coating and subjected to pH-cycling for 28 days. To evaluate tubule occlusion ability, thirty dentin specimens of 2mm thickness were obtained from cervical third of sound third molars. Specimens were ultrasonicated and etched with 6% citric acid for 2 minutes to simulate the hypersensitive dentin. Specimens were randomly divided into above mentioned three groups (n=10). The test agents were brushed over the specimens with an electric toothbrush, prepared and observed under Scanning Electron Microscope for calculation of the percentage of occluded tubules. Results: Group I showed a significantly greater percentage of remineralization than Group III and Group II. Comparison of the remineralization potential between group II and group III were not significant.In case of dentine hypersensitivity, Group I and group III showed greater percentage of tubule occlusion ability than Group II. Intergroup comparison of the tubule occlusion potential of group I and group III were not significant. Interpretation and Conclusion: Within the limitations of our study, sodium fluoride showed relatively greatest remineralizing and dentinal tubule occlusion property when compared with GC MI paste plus and Clinpro tooth crème.


Keywords: Demineralization, Remineralization, Dentinal hypersensitivity, CPP-ACPF, Tricalcium phosphate, Sodium fluoride


How to cite this article:
Prabhakar A R, Manojkumar A J, Basappa N. In vitro remineralization of enamel subsurface lesions and assessment of dentine tubule occlusion from NaF dentifrices with and without calcium. J Indian Soc Pedod Prev Dent 2013;31:29-35

How to cite this URL:
Prabhakar A R, Manojkumar A J, Basappa N. In vitro remineralization of enamel subsurface lesions and assessment of dentine tubule occlusion from NaF dentifrices with and without calcium. J Indian Soc Pedod Prev Dent [serial online] 2013 [cited 2014 Nov 23];31:29-35. Available from: http://www.jisppd.com/text.asp?2013/31/1/29/112403



   Introduction Top


The concept of tooth demineralization is undergoing a paradigm shift where thetreatment is now directed toward reverting the process to near normalcy. There has been an explosion of interest in technologies that may have value for remineralization of enamel or for desensitization of exposed dentin. [1] Recently studies have focused on the concentration of calcium and phosphate present in the tooth. Since both the ions are major components of the tooth and are ultimately related to remineralization, most of the efforts have been directed toward their deposition or enhancement in the dental structure. [2] This study is an attempt to evaluate two such products Clinpro tooth crème and GC MI paste plus for their remineralization and desensitization properties as compared with sodium fluoride 5000 ppm.


   Materials and Methods Top


Sixty-third molars extracted for therapeutic reasons were included in this study. All soft tissue deposits and calculus were removed from the teeth with a periodontal scaler. The teeth were cleaned using slurry of pumice and autoclaved. All teeth were then stored in distilled water containing 0.2% thymol at 4°C to inhibit the microbial growth. [3]

The teeth were then randomly assigned to each of following three groups. Group I - Sodium fluoride (NaF) 5000ppm; Group II - GC MI paste plus; Group III - Clinpro tooth creme.

The study was carried out in two parts; first to evaluate the remineralization potential of the above-mentioned materials and second to assess their desensitization ability by examining the dentinal tubule occlusion.

Evaluation of remineralization potential

Among the sixty 3 rd molars selected, thirty were allotted to evaluate the remineralization potential of the experimental groups (n=10). A rectangular piece of plaster adhesive tape measuring 1 mm length and 5 mm width (mesio-distal) was fixed to the cervical one-third of the buccal surface of each of thethirty 3 rd molars and the remaining portion of the teeth was painted with acid resistant nail varnish. Once the varnish dried, the masking tape was removed and the window dimensions were reconfirmed with a Williams's graduated periodontal probe to ensure the uniformity of the enamel window, in all the samples. Artificial caries like lesions were created on the exposed enamel by suspending all teeth in an artificial caries system for 5 days (50 mL per sample). The caries solution consisted of 2.2 mM CaCl 2 .2H 2 O, 2.2 mM KH 2 PO 4 , and 0.05 M acetic acid. The pH was adjusted to 4.4 by the addition of 10 M KOH. [2] The solution was kept at a temperature of 37°C under constant circulation.

After 5 days the teeth were removed from the artificial caries system and mounted on acrylic blocks for sectioning. Each tooth was sectioned longitudinally to a thickness of 140-160 μm by a Silverstone-Taylor hard tissue microtome. These sections were wetted and photographed using the polarized light microscope (Olympus, SZX12, Japan). The demineralized areas were then quantified with imaging system software, Image Pro-Plus at 3 points, D1, D2, and D3 from the surface to the depth of the lesion (μ). The artificial carious lesion was quantified at three points [Figure 1],[Figure 3]. Lesion depth was measured from the surface to the depth of the lesion at D1, D2, and D3. [4] Lesion depth for each section was taken as the average of the three representative measurements from the surface of the lesion to the depth of the lesion.
Figure 1: Polarized microscope showing demineralized area in groupI

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The experimental dentifrices were then applied onto the sections in slurry formed by one part experimental product and three parts deionized water for 4 min twice a day for 28 days. All surfaces of the sections were covered with an acid-resistant varnish using a brush under the microscope except for a window the width of the artificial lesion on natural outer surface of the section. The sections were then subjected to 28 days pH cycling consisting of a four minute treatment application, followed by a demineralization solution for 3 h, artificial saliva for 2 h, demineralizing solution for 3 h, and second treatment application for 4 min. The sections were stored overnight in artificial saliva. In-between all the steps of pH - cycling, each group were rinsed with distilled water.

The artificial saliva prepared for this study consisted of 20 mM NaHCO 3 and 3 mM NaH 2 PO 4 and 1 mM CaCl 2 . The pH was adjusted to 6.8 by the addition of 0.1 M sodium hydroxide. The demineralization and remineralization solutions were changed on the fourth day of pH cycling so that concentration of calcium and phosphate ions in solution would not affect the results. Once the pH cycling was finished the acid resistant varnish was removed using acetone 99.6% with a brush. The sections were then kept in plastic container containing deionized water for less than 24 h until they were viewed and photographed using polarized light microscopy for evaluation of remineralization. The difference in area between the initial and the final lesion was calculated [Figure 2],[Figure 3].
Figure 2: Polarized light microscope showing remineralized area in Group I

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Figure 3: Measurement of carious lesion at 3sites (D1,D2,D3)

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The second part of the study was the assessment of the desensitization ability of the dentifrices.

The remaining (thirty 3 rd )molars were used to prepare dentin discs of 2 mm thickness from the coronal portion of the tooth just below the level of the cementoenamel junction using double-sided diamond disk operated in a micromotor hand piece with water coolant. These dentin discs were then polished with sequential grades of silicon carbide paper, namely 400, 600, 800, and 1000. Then the specimens were ultrasonicated in distilled water for 12 min to remove the residual smear layer and then etched by immersing the specimens in a tray containing 6% citric acid for 2 min to simulate dentin hypersensitivity in vivo situations. [5],[6]

The 30 resulting dentin specimens were divided into the three above-mentioned groups.

The test agents were brushed over the specimens using an electric toothbrush (Colgate 360° sonicpower® ) at 20 000 strokes per minute for 2 min twice a day for 7 days. [5]

The specimens were rinsed in distilled water after each brushing session and stored in a closed container containing artificial saliva. After the last brushing session, specimens were washed with distilled water and sputter coated (MED 010- Jeol, Japan) with a thin gold layer following which the specimens were analyzed in a scanning electron microscope (DSM 840-A-Geol.Japan) operating at 20 kv in 1000× magnification. The area in the center of each specimen was scanned so as to obtain tubules in a circular cross section.

Photomicrographs were taken to analyze the following study parameters [Figure 4],[Figure 5] and [Figure 6].

  1. Number of tubules occluded per unit area.
  2. Number of tubules patent per unit area
    Figure 4: Dentinal tubule occlusion seen in Group I

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    Figure 5: Dentinal tubule occlusion seen in Group II

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    Figure 6: Dentinal tubule occlusion seen in Group III

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To facilitate the manual counting, grids were applied on the photomicrograph using ADOBE PHOTOSHOP CS3 software. Tubules at the right and upper sides of the grid margins were counted and included in the total.


   Results Top


One way ANOVA showed that the sections treated with sodium fluoride 5000 ppm displayed the highest change in lesion depth that was statistically highly significant compared to the other experimental groups. Tukey's posthoc test revealed that the comparison of remineralization potential between group II and III was found to be statistically nonsignificant (P>0.05) [Table 1].
Table 1: Comparison of the mean and standard deviation and the significance (P) value of difference in de and remineralization for the various experimental and control groups.

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The paired t test showed that Groups I, II and III had a highly significant decrease in lesion depth after the specified treatment [Table 2].
Table 2: Showing intergroup comparison of change in lesion depth between the experimental and control groups.

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Kruskall-Wallis test revealed that intergroup comparison of the mean percentage of tubule occlusion was statistically highly significant. Mann-Whitney U test showed that the tubule occlusion potential was statistically significant among all groups except for Group I and Group III where it was not significant [Table 3].
Table 3: Comparison of the difference in the tubule occlusion ability of the experimental and control groups.

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   Discussion Top


The use of fluoride toothpaste is generally recognized as the main reason for the decline in caries in industrialized countries over the last four decades. [7] It is now realized that the primary caries preventive mechanisms of the action of fluoride are posteruptive through topical effects that can interfere with the dynamic equilibrium at the interface between mineral surface and oral fluid. The frequent delivery of fluoride to the tooth surface is currently the most efficient measure leading to caries arrest and reversal. [8]

The current trend has been to increase the concentration of fluoride in dentrifices; 2800-5000 ppm fluoride containing dentrifices have recently been introduced in several countries for patients with a high caries risk. [9] Caries rate was shown to decrease by approximately 70% when compared to regular toothpaste, that is, there seemed to exist a dose-dependent linear relationship between the levels of fluoride in toothpaste and level of anticaries effect (r=0.85). [10]

Although fluoride is the cornerstone of the noninvasive management of noncavitated caries lesions, the ability of the ion to promote net remineralization is limited by the availability of calcium and phosphate ions. The availability of calcium and phosphate ions can be the limiting factor for fluoride retention and for net enamel subsurface lesion remineralization to occur, upon the application of topical fluoride ions. However, combining calcium phosphate and fluoride ions in oral care products is problematic and can lead to loss of bioavailable fluoride ion due to a reaction between the calcium phosphate phase and the fluoride ion. In an approach to overcome this incompatibility of calcium phosphates and fluoride ions, two calcium phosphate technologies have been developed: the first of these calcium phosphate technologies is casein phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP) delivered in a product called Tooth Mousse or MI Paste (TM) containing CPP-ACP or Tooth Mousse Plus or MI Paste plus (TMP) containing CPP-ACP plus 900 ppm F. [11] It is proposed that the presence of the casein phosphopeptides (CPP) stabilizes the amorphous calcium phosphate phase to deliver bioavailable calcium, phosphate and fluoride ions to the tooth surface to promote remineralization of enamel subsurface lesions. Due to the size and electroneutrality of the nanocomplexes, it is expected that they would enter the porosities of the enamel subsurface lesion and diffuse down the concentration gradient into the body of the subsurface lesion thus promoting remineralization. When CPP-ACP is provided with a low background of fluoride, electron microprobe analysis has shown that the mineral formed is consistent with fluorapatite or fluorhydroxyapatite. [12],[13]

The second technology is functionalized tricalcium phosphate (fTCP) where tricalcium phosphate particles have been ball milled with sodium lauryl sulfate. This technology has been included in a tooth cre`me with sodium fluoride marketed as Clinpro tooth cre`me (3 M ESPE). [11]

One other area in dentistry that is fast gaining importance is the concern over dentin hypersensitivity. Dentin hypersensitivity can be a potential threat to the individual's oral health because such pain may interfere with the maintenance to good oral hygiene. However, it still remains a poorly understood area and consequently there appears to be no permanent treatment for this clinical condition. [14]

Dentin hypersensitivity is closely related to the exposure and patency of dentinal tubules. Many factors may contribute to the exposure of dentinal tubules such as occlusal wear, caries, abrasion due to brushing, dietary erosion, parafunctional habit, aging, gingival recession, chronic periodontal disease, abnormal tooth positioning, periodontal surgery, root preparation, and abfractive lesions. [3] The condition has been treated by a number of agents, which have been claimed to reduce pain by occluding dentinal tubules. Various chemical agents have been used for dentinal tubule occlusion and for treatment of dentin hypersensitivity. Desensitizing toothpastes contain some active agents, such as fluorides, strontium salts, and potassium. Further, tooth brushing is generally performed with dentifrices containing abrasives that can increase tubule occlusion. This additional blockage may occur by directly occluding the tubules with abrasive or indirectly by the formation of a smear layer during tooth brushing. In vivo studies have shown considerable decrease in hypersensitivity when teeth were brushed with desensitizing dentifrices. [5]

In view of the previous concepts, this study was intended to evaluate the remineralization and desensitizing potential of CPP-ACP containing fluoride and TCP containing fluoride in comparison with sodium fluoride (NaF).

In this study, teeth free of any anomalies were included as previous studies have revealed that any pre-existing alteration of surface morphology of the tooth can directly influence the caries progression. [15],[16]

The tooth samples were then subjected to a chemical caries model for the production of artificial caries lesion. Such a model with a closed agitated, acidic, aqueous environment will facilitate the production of artificial caries like lesions. Such techniques that involve the direct use of acid to produce caries like lesions have lately replaced the ones involving the microbial production of acid. These methods have improved our understanding of the mechanism of de and remineralization processes. Further they provide information about the effects of caries preventive agents on the de/remineralization dynamics at the surface and in the subsurface of the teeth. [17],[18]

Following the creation of artificial carious lesion, the teeth were sectioned longitudinally to a thickness of 140-160 μm. The tooth sections were then observed under the polarized light microscope.

Polarized light microscopic (PLM) analysis was chosen in the current study since it is a technique extremely sensitive to changes in hard tissues. With respect to de- and remineralization, it can qualitatively show the areas of mineral loss and mineral gain represented by the visualization of areas with different porosities and birefringence.

The depth of demineralization was measured at three different points (D1, D2, and D3) in order to avoid errors. The demineralized depth was quantified with the Image Pro-Plus imaging system. [4] The mean value of the depth was taken into consideration for statistical analysis.

In the current study, the pH cycling was performed to simulate the frequency of tooth brushing and acid challenges. Therefore, immediately after the first treatment regimen, the sections were immersed in a demineralization solution as recommended by Reynolds and Walsh. [2]

At the end of the 28th day cycling period, the specimens were removed and were then quantified for remineralization using imaging system, Image Pro-Plus as described for demineralization.

For the determination of the dentinal tubule occlusion ability of each of the dentifrices, dentin specimens were obtained from molars. To simulate the location of the clinical hypersensitive dentin, dentin samples were obtained at the level of the cementoenamel junction of cervical third of molars. Further polishing and ultrasonication of the samples removed the smear layer occluding the dentinal tubules. All the samples were brushed for 2 min with motorized tooth brush to standardize the brushing pressure and strokes for each sample, since the motorized brush (Colgate 360° sonicpower) oscillates at a constant speed. After each brushing session the samples were kept in artificial saliva to simulate the oral condition.

The results of this study revealed that on quantifying the depth of remineralization it was evident that all the three groups showed significant remineralization, measured as a decrease in lesion depth. NaF 5000 ppm showed the highest degree of remineralization while the remineralization observed from GC MI paste plus and Clinpro tooth crème were found to be comparable.

The high magnitude of remineralization seen with the NaF 5000 ppm group was in agreement with Pulido MT, AL-Mulla et al. Nordstrom A. [2],[9],[7],[19] This observed response can be explained by assuming that F-deposition during treatment depends on lesion depth. With elevated external F-levels, the F-gradient will be higher, driving the fluoride deeper into the lesion, in spite of the F-diffusion being slowed by adsorption onto and reaction with hydroxyapatite crystallites in the pore walls. [19]

In the present study, although the GC MI paste plus and Clinpro tooth crème differed in their basic formulation, the remineralization effect observed was found to be comparable.

The evaluation of desensitization revealed that NaF showed a mean tubule occlusion of 51.52%. This is probably because fluoride decreases the dentinal permeability by precipitation of calcium fluoride crystals inside the dentinal tubules. These crystals are partially insoluble in saliva. [7],[9] This precipitated fluoride compounds might block the dentinal tubules mechanically which will prevent the hydraulic fluid transmission of the pain reducing stimuli. [20],[21]

On the other hand, some toxicological concern has been raised using high F toothpaste at concentration 5000 ppm. This can be explained by the fact that approximately only 5-10% of the dentifrice is swallowed during tooth brushing with toothpaste using a minimum amount of water. The amount of swallowed fluoride is thereby 0.25-0.50 mg, when using 1 g of a toothpaste containing 5 mg F. This quantity corresponds to the amount of F in 1-2 fluoride tablets containing 0.25 mg F and is considered to have no toxic effect in teenagers and adults. [7],[9]

Clinpro tooth crème showed a mean tubule occlusion of 45.74%. Similar findings were noted in the previous study conducted by Mackey AC. [22] Further, the presence of fluoride in concentration of 950 ppm may also contribute to the occlusion of the tubules. The tubule occluding ability of Clinpro Tooth Crème was found to be comparable with NaF 5000 ppm.

In our study, GC MI Paste plus showed a mean tubule occlusion of 29.51%. It showed a weak ability to occlude the dentinal tubules when compared to other experimental groups which was in accordance with the study conducted by Kowalczyk A, Giovanna GM. [23],[24]


   Conclusion Top


It can be concluded that NaF 5000 ppm have found to be relatively superior remineralizing and desensitizing agent when compared with GC MI paste plus and Clinpro tooth crème.

 
   References Top

1.Reynolds EC. Calcium phosphate-based remineralization systems: Scientific evidence? Aust Dent J 2008;53:268-73.  Back to cited text no. 1
    
2.Pulido MT, Wefel JS, Hernandez MM, Denehy GE, Chalmers JM, Qian F, et al. The Inhibitory Effect of MI Paste, Fluoride and a Combination of Both on the Progression of Artificial caries-like lesions in Enamel. Oper Dent 2008;33:550-5.  Back to cited text no. 2
    
3.Lee BS, Chang CW, Chen WP, Lan WH, Lin CP. In vitro study of dentine hypersensitivity treated by Nd:YAP laser and bioglass. Dent Mater 2005;21:511-9.  Back to cited text no. 3
    
4.Todd AM, Staley NR, K anellis JM, Donly JK, Wefel SJ. Effect of a fluoride varnish on demineralization adjacent to orthodontic brackets. Am J Orthod Dentofacial Orthop 1999;116:159-67.  Back to cited text no. 4
    
5.Arrais GC, Micheloni CD, Giannini M, Chan DC. Occluding effect of dentifrices on dentinal tubules. J Dent 2003;31:577-84.  Back to cited text no. 5
    
6.Gillam DG, Khan N, Mordan NJ, Barber PM. Scanning electron microscopy (SEM) investigation of selected desensitizing agents in the dentine disc model. Endod Dent Traumatol 1999;15:198- 204.  Back to cited text no. 6
    
7.AL-Mulla A, Karlsson L, Kharsa S, Kjellberg H, Birkhed D. Combination of high-fluoride toothpaste and no post-brushing water rinsing on enamel demineralization using an in-situ caries model with orthodontic bands. Acta Odontol Scand 2010;68:323- 8.  Back to cited text no. 7
    
8.AobaT. Solubility properties of human tooth mineral and pathogenesis of dental caries. Oral Diseases 2004;10:249-57.  Back to cited text no. 8
    
9.Nordstrom A, Birkhed D. Preventive effect of high-fluoride dentifrices (5,000ppm) in caries active adolescents: A 2-year clinical trial. Caries Res 2010;44:323-31.  Back to cited text no. 9
    
10.Tavss EA, Mellberg JR, Joziak M. Relationship between dentifrice fluoride concentration and clinical caries reduction. Am J Dent 2003;16:369-74.  Back to cited text no. 10
    
11.Shen P, Manton DJ, Cochrane NJ, Walker GD, Yuan Y, Reynolds C, et al. Effect of added calcium phosphate on enamel remineralization by fluoride in a randomized controlled in situ trial. J Dent 2011;39:518-25.  Back to cited text no. 11
    
12.Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC. Enamel Subsurface Lesion Remineralisationwith Casein Phosphopeptide Stabilised Solutions of Calcium, Phosphate and Fluoride. Caries Res 2008;42:88-97.  Back to cited text no. 12
    
13.Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV, et al. Fluoride and Casein Phosphopeptide-Amorphous Calcium Phosphate. J Dent Res 2008;87:344-8.  Back to cited text no. 13
    
14.Burman LH. Dentinal sensation and hypersensitivity- A review of mechanisms and treatment alternatives. J Periodontol 1984;45:216-21.  Back to cited text no. 14
    
15.Montero MJ, Doughlass JM, Mathew GM. prevalence of dental caries and enamel defects in Connecticut head start children. Pediatr Dent 2003;25:235-9.  Back to cited text no. 15
    
16.Ellwood RP, O′Mullane D. The association between developmental enamel defects and caries in populations with and without fluoride in their drinking water. J Public Health Dent 1996;56:76-80.  Back to cited text no. 16
    
17.Ten cate JM, Mundorff-Shrestha SA. Working group report 1: Laboratory models for caries (in vitro and animal models). Adv Dent Res 1995;9:332-4.  Back to cited text no. 17
    
18.Pearce EI. A microradiographic and chemical composition of in vitro systems for the stimulation of incipient caries in abraded bovine enamel. J Dent Res 1983;62:969-74.  Back to cited text no. 18
    
19.TenCate JM, Buijs MJ, Miller CC, Exterkate MA. Elevated Fluoride Products Enhance Remineralization of Advanced Enamel Lesions J Dent Res 2008;87:943-7.  Back to cited text no. 19
    
20.Maglani, Aggarwal V, Ahuja B. Dentine hypersensitivity: Recent trend in management. J Conserv Dent 2010;13:218-24.  Back to cited text no. 20
    
21.Gedalia TM, Emlich J. X-ray defraction and scanning electron microscopic investigations of the fluoride treated dentin in man. Arch Oral Biol 1976;21:285-8.  Back to cited text no. 21
    
22.Mackey AC. In Vitro Assessment of Dentin Tubule Occlusion by Hypersensitivity Dentifrices. 2009 April 1-4; Available from: http://iadr.confex.com/iadr/2009miami/webprogram/ Paper 120922.Html.   Back to cited text no. 22
    
23.Kowalczyk A, Botuliñski B, Jaworska M, Kierklo A, Pawiñska M, Dabrowska E. Evaluation of the product based on Recaldent technology in the treatment of dentin hypersensitivity. Adv Med Sci. 2006;51 Suppl 1:40-2.  Back to cited text no. 23
    
24.Giovannaa GM, Silvia F, Pashley DH, Gasparotto G, Carlo P. Calcium silicate coating derived from Portland cement as treatment for hypersensitive dentine. J Dent 2008;36:565-78.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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