|Year : 2014 | Volume
| Issue : 2 | Page : 74-79
In-vitro assessment of effect of a hydroxyapatite containing desensitizing agent on dentinal tubule occlusion
Muniba Mohammad Sultan Ansari1, Nihal Dattatraya Devkar1, Suresh Vasant Lele1, Deepak Jairamdas Nagpal2, Shwetambari Rajiv Navale1, Anupama Satyamurthy Jayamangal1
1 Department of Periodontics, Sinhgad Dental College and Hospital, Pune, Maharashtra, India
2 Department of Oral Pathology, Sinhgad Dental College and Hospital, Pune, Maharashtra, India
|Date of Web Publication||18-Jun-2015|
Dr. Nihal Dattatraya Devkar
Department of Periodontics, Sinhgad Dental College and Hospital, Vadgaon (Budruk), Off. Sinhgad Road, Pune - 411 041, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim and Objectives: Dentinal hypersensitivity (DH) is a common clinical condition usually associated with exposed dentinal surfaces. The present study evaluated the influence of topical application of hydroxyapatite containing desensitizing agent (Remin Pro) on dentinal tubule occlusion. Materials and Methods: A total of 22 dentin discs obtained from 11 anterior teeth were divided into two groups. Test group (Remin Pro) and control group (distilled water). The specimens treated with 17% ethylene diamine tetraacetic acid gel prior to the application of test agents followed by rinsing and application of test agents for 5 min. After rinsing, 5% Evans blue dye was applied on both the specimens. The specimens were observed under light microscope. A quantitative analysis of tubular occlusion was done by counting the number of dentinal tubules/1000 μm 2 , diameter of dentinal tubules (μm), and closed tubular area (μm 2 ). Percentage of tubular occlusion was calculated in both the groups. Results: Mean number of occluded dentinal tubules in Group A was 78.01% whereas in case of Group B it was 4.97%. With a higher percentage of tubular occlusion, the diameter and number of dentinal tubules decreased and closed tubular area of each tubule increased. Conclusion: The desensitizing agent produced significant greater dentinal tubular occlusion as compared to the control group. Clinical implications: Though in-vitro studies provide evidence for immediate dentine hypersensitivity treatment, further well-designed multicenter prospective long-term clinical trials with scanning electron microscopic evaluation are needed to find the gold standard treatment of DH.
Keywords: Dentin disc, dentinal hypersensitivity, Evans blue dye, Remin Pro, tubular occlusion
|How to cite this article:|
Ansari MS, Devkar ND, Lele SV, Nagpal DJ, Navale SR, Jayamangal AS. In-vitro assessment of effect of a hydroxyapatite containing desensitizing agent on dentinal tubule occlusion. J Dent Allied Sci 2014;3:74-9
|How to cite this URL:|
Ansari MS, Devkar ND, Lele SV, Nagpal DJ, Navale SR, Jayamangal AS. In-vitro assessment of effect of a hydroxyapatite containing desensitizing agent on dentinal tubule occlusion. J Dent Allied Sci [serial online] 2014 [cited 2019 Sep 18];3:74-9. Available from: http://www.jdas.in/text.asp?2014/3/2/74/159084
| Introduction|| |
Dentin hypersensitivity is clinically described as a painful response to thermal, chemical, mechanical, evaporative, or osmotic stimuli applied to opened dentinal tubules, which cannot be ascribed to any other form of dental defect or pathology.  It has been demonstrated that hypersensitive dentin has a larger number of wide, open dentinal tubules on the surface than does nonsensitive dentin.  A rational approach to the control of pain arising from exposed dentin would thus be to block the tubules or to reduce their diameter to prevent fluid movements inside the tubules. 
Although the mechanisms of pain transmission across dentin are not fully understood, both dentin permeability and sensitivity are reduced when the dentin tubules are occluded. ,, Most of the treatments for dentin hypersensitivity which range from toothpastes to dentin bonding agents (in-office) attempt to inhibit sensitivity either by sealing the tubules, by altering their contents or by creating insoluble calcium complexes, thus forming mechanical or chemical plugs. However, current therapies provide only temporary effects and require multiple applications to take effect, which explains the large number of in-vitro and in-vivo studies evaluating the reduction of pain in the short-term as well as the lack of long-term effects associated with these materials. ,,
Hydroxyapatite (HAP) is one of the most biocompatible and bioactive materials which has a tendency to cause remineralization of tubules which is more stable. 
In-vitro studies have also suggested that the use of HAP containing dentifrices is better than fluoride dentifrices, as HAP induces a surface remineralization, forming a biomimetic apatite coating on enamel and dentin surface, which quickly occurs due to the chemical-physical characteristics of innovative nano-structured HAP particles, which closely resemble mineral enamel constituents. ,
Studies on relationship between diameter, closed tubular area, number of dentinal tubules, and percentage of dentinal tubules occluded have been done which showed higher percentage of tubular occlusion with decreased diameter and number of dentinal tubules, and increased closed tubular area of the dentin. ,, This study is the continuation for further research in this field.
Furthermore, there has been only few studies on the bioactivity of toothpastes in apatite formation, which may be extremely important in preventing the recurrence of dentine hypersensitivity by remineralization effects. 
Thus, the present study was undertaken to assess the effect of a hydroxyapatite containing desensitizing agent (Remin Pro) on dentinal tubule occlusion in-vitro. The objectives of the study were:
- To compare the percentage of occlusion in test and control groups.
- To find the relation between dentinal tubular diameter, closed tubular area, and number of dentinal tubules and percentage of tubular occlusion.
- The percentage of tubular occlusion before and after application of test agents in both the groups.
| Materials and Methods|| |
Sample preparation for dentin tubular occlusion analysis
The study included 11 human permanent anterior teeth with complete crowns and without decay, cracks or previous reparative therapies that were extracted for orthodontic treatment or treatment of periodontal disease and were collected from Department of Oral Surgery after approval by the Local Ethics Committee. Sample size was determined by GiPA sample size calculator software based on mean difference of 0.750 and standard deviation of 1.000 using the following formula:
S = Standard deviation
d = Expected mean difference
The teeth were cleaned and stored in neutral 10% formaldehyde solution for no longer than a month prior to use. Dentin discs of about 1 mm thickness were obtained by the method described by Kolker et al.  However, in this study graduated scale was used instead of an isomet saw for the measurement and sections obtained using diamond disc under chilled water. The dentin discs were ground to a thickness of about 10 μ on Arkansas silicon carbide combination stone. The sections were polished with 600- and 800-grit sandpaper to form a homogeneous, flat and smooth surface.
The specimens were washed with 20 ml of distilled water and were ultrasonicated for 10 min in an ultrasonic cleaner to remove superficial debris. Dentin discs were etched with 17% ethylene diamine tetraacetic acid (EDTA) gel  applied every 30 s for 3 min to remove the smear layer and open the dentinal tubules for desensitizing agent to act. Later the specimens were rinsed with distilled water. 
The dentin discs were randomly divided into two groups each containing 11 specimens that received the following treatments: Test group in which desensitizing agent was applied and control group had distilled water application for 5 min.
The test agents were applied according to the manufacturer's instructions. After rinsing with distilled water for 2 min, 1 μL of 5% Evans blue More Details dye was applied with a dropping pipette on both the specimens and was left to penetrate for 5 min.  The dentin discs were fixed on glass slides and kept wet with distilled water. 
Dentinal tubular occlusion analysis
A total of 22 dentin discs were used in this study for dentinal tubule occlusion analysis. Photomicrographs of the prepared specimens were taken from selected fields in the central portion of each disc using Leica research microscope DM 1000 at ×40 magnification and images of 8.1 megapixels obtained. The number of dentinal tubules occluded from total number of dentinal tubules seen in the field was counted using image analysis software (Leica Application Suite V 4.1, IBM Corporation) and the data were saved. These stored digitalized images were not enhanced, and no transformation procedures carried out. The image analysis software with Leica research camera attachments was used for measuring the diameter of dentinal tubules (μm), closed tubular area of each tubule (μm 2 ), and number of dentinal tubules per 1000 μm 2 before and after treatment in both the groups. The software can automatically draw the highlighted outline of a dentinal tubule from the difference in gray pixels between the dentinal tubule and the outer area, and then calculate the mean total dentinal tubule area and closed dentinal tubule area.
Percentage of the mean number of occluded dentinal tubules was obtained using formula: 
The data were expressed as the mean and the percentage of total number of the dentinal tubules partially or completely mineralized after the application of the experimental agents. The tubular occlusion was measured at 2 time points: EDTA etched specimens with Evans blue dye applied and later after experimental agents were applied.
Intra-examiner reproducibility was assessed twice within 48 h to check the reproducibility of tubular diameter, area, and number of dentinal tubules measurements. A number of dentinal tubules, diameter, and closed tubule area were calculated using automatic methods (digital computer image analysis). The reproducibility was tested using the Bland and Altman procedure.  SPSS 17.0 software (Chicago, IL, USA) was used for calculation of mean, standard deviation and standard error of number of dentinal tubules per 1000 μm 2 , diameter of dentinal tubules (μm), closed dentinal tubular area (μm 2 ) before and after treatment in both the groups. Student's t-test was used for intergroup comparison with P < 0.05 considered to be statistically significant.
| Results|| |
Reproducibility for tubule diameter (intra-examiner), dentin tubule number, and closed tubular area (comparing manual and automatic methods) were within the limits of agreement.
Tubular occlusion measurements
[Table 1] shows the mean number of total, occluded and nonoccluded dentinal tubules in test and control groups at baseline and after application of test agents. Remin Pro produced significantly high tubular occlusion 23.20 (78.01%) compared to the control group which showed 1.57 (4.97%) of the tubules occluded [Table 1]. The two groups also showed statistically significant difference in the mean number of dentinal tubules that were not occluded (P = 0.000). The percentage of the nonoccluded dentinal tubules in the test group after application of desensitizing agent was 21.99%.
|Table 1: Comparison of mean number of occluded and nonoccluded dentinal tubules in test and control groups |
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The desensitizing agent in the test group produced 78.01% tubular blockage after 5 min of application [Graph 1]. The percentage of mean number of nonoccluded dentinal tubules in the control group was 95.03%, respectively. [Figure 1] shows the microphotographs of specimens from test group after treatment in which dye has penetrated few patent dentinal tubules as Remin Pro produced partial or complete mineralization of majority of the tubules. However, in the control group, the dye was taken up by the majority of dentinal tubules indicating that distilled water has no effect on the degree of tubular blockage [Figure 2].
|Figure 1: Light microscope images (×40) of test specimen treated with the desensitizing agent. (a) Evans blue dye applied (b) software image with contrast|
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|Figure 2: Light microscope images (×40) of control specimen treated with distilled water. (a) Evans blue dye applied (b) software image with contrast|
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After the application of the agents, the total number of dentinal tubules did not differ much in the control group [Table 2]. However, in the test group, the mean number of dentinal tubules reduced to 30.51 (P = 0.976). The two groups showed no significant difference in the total number of dentinal tubules at baseline and after application of test agents.
|Table 2: Comparison of change in the scores from baseline to other time intervals in both the groups |
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The mean diameter after application of desensitizing agent in the test group reduced to 3.89 μm from 4.06 μm whereas in the control group with distilled water application diameter reduced to 3.70 μm from 3.75 μm. The intragroup difference in both the groups was significant. At baseline, the closed tubular area in the test group was 2.62 μm 2 . After the application of Remin Pro, the closed tubular area increased significantly to 3.17 μm 2 . The closed tubular area also increased in the control group. However, no significant differences were reported between both the groups [Table 2].
[Table 3] shows the intra- and inter-group comparison in both test and the control groups for variations in the closed dentinal tubular area, number dentinal tubules, and the mean diameter of dentinal tubules.
| Discussion|| |
The results of this in-vitro study showed that the majority of dentinal tubules were blocked by Remin Pro (78.01%) whereas in the control group, only few dentinal tubules were blocked (4.97%).
The in-vitro assessment of dentinal tubule blockage by dentin disc model has become the gold standard of dentin hypersensitivity. , Previous studies assessing the tubule occluding effects of desensitizing agents provided only descriptive terms, that is, partial or complete blockage of tubules.  Other investigators described their results with symbols, like +, ++, +++, etc.,  or provided indices based on percentages of occluded tubules.  Although these investigations claimed that their methods were quantitative, in reality, they were mainly descriptive (qualitative) studies.
Kolker et al. in 2002 reported that desensitizers may cover the dentin surface without completely occluding the dentinal tubules.  Therefore, it is necessary to evaluate the efficacy of materials that infiltrate the tubules. The present study improved the methodology involved in the assessment of dentin morphology and the surface characteristics of surfaces treated with selected desensitizing agents by means of quantitative image analysis.
Most people with dentin hypersensitivity prefer to use a desensitizing dentifrice because tooth-brushing is one of the easiest methods in a home care system. Clinically, dentifrices containing HAP are used to treat dentine hypersensitivity because of its chemical similarity to enamel. Potassium-based toothpastes do not provide instant relief from dentine hypersensitivity as they require a time period of generally 4 to 8 weeks to demonstrate significant levels of pain relief.  Calcium hydroxide although provides instant relief but requires multiple applications to maintain its effect , and also causes irritation of gingival tissues.  Fluoride iontophoresis has produced an immediate reduction in sensitivity after treatment with, but the symptoms gradually return over the ensuing 6 months. 
Recently desensitizing agents containing arginine 8%, casein phosphopeptide or calcium sodium phosphosilicate that is, NovaMin have shown satisfactory results.  However, they require multiple applications or continuous use by the patients which does not guarantee their effectiveness. , A recent systematic review stated that there is no agent that can provide complete relief from dentinal hypersensitivity (DH).  However, the major feature of hydroxyapatite containing desensitizing agents is that they cause permanent occlusion and remineralization of tubules. Hydroxyapatite is the major constituent of Remin Pro besides fluoride (1450 ppm) and xylitol.
Remin Pro resulted in greater tubular occlusion than reported in an in-vitro study (57.09%).  Despite a reduction in the tubular lumen and complete occlusion of some tubules [Figure 2], control group still has most of the tubular orifices patent.
Remin Pro has been recommended for the management of DH, to prevent enamel demineralization and promote remineralization of enamel subsurface lesions.  A recent study in 2012 reported that dentifrice containing hydroxyapatite (HA) could be effective in occluding the dentinal tubules with a plugging rate >90% with persistent effect.  The results of the current study are in favor of this study. However, an in-vivo study comparing four different desensitizing agents (Remin Pro, Ultra EZ, Fluoritop, Propolis) over a period of 7 days showed Remin Pro to be least effective in reducing hypersensitivity.  Other studies have confirmed occlusion of dentinal tubules predominantly with apatite mineral, not only on the dentin surface, but also deep inside the dentinal tubules to a depth of 10-15 μm from the dentin surface. 
Evans blue dye which is a 1,3-naphthalenedisulfonic acid has been used in previous studies for assessment of dentine permeability. , This is the first study to evaluate tubular occlusion with the help of this dye.
In the current study, an attempt was made to obtain dentin from the same area of the tooth, supra-pulpal mid-coronal dentin and of approximately 1 mm thickness, however, replication of the dentin substrate is impossible due to natural variations in dentin, such as number and diameter of tubules. 
In the present study, Remin Pro produced a highly significant increase in the mean closed tubular area. The hydroxyapatite crystals mineralized the tubules resulting in an increase in closed tubular area. Previous studies have estimated dentinal tubular area that remained open after application of the desensitizing agent. ,,, An in-vitro study showed mean dentinal tubule area in the etched baseline specimens before tooth-brushing to be 5.26 μm 2 which decreased after tooth-brushing using 20% nano-sized carbonate apatite to 1.08 ± 0.42 that is, 79.5% less open tubular area than the baseline.  This is the first study in the literature to measure closed dentinal tubular area after desentizing agent is applied. The closed dentinal tubule area exhibited significant differences among the groups.
In the current study, no standardized force was used for application of the desensitizing agent. However, literature has reported the use of an electric toothbrush to apply uniform force. 
The diameter of dentinal tubules is an important parameter when assessing occluding property of the desensitizing agent. A scanning electron microscopic study on effect of desensitizing toothpastes on dentin showed that the dentin tubular diameter was smallest (0.88-1.36 μm) in teeth that were brushed with desensitizing toothpastes as compared to baseline diameter of 1.26 μm.  The diameter of dentinal tubule was reduced more in this study as compared to previous studies indicating the greater efficacy of this agent. Another study reported that the diameter was smallest in teeth that were brushed with 2% potassium nitrate plus 2% sodium fluoride gel. 
Blockage of the tubules at the dentin surface may also occur by other means, e.g., toothpaste components (abrasive agents), calculus, and oral debris.  Kerns et al. suggested that natural desensitization can occur from the deposition of salivary minerals around tubules, which would result in tubule diameter reduction and subsequent reduction in fluid flow.  The results obtained were probably due to the action of the desensitizing agent in combination with these factors resulting in higher number of partially obliterated tubules.
Specimens in the test group showed lower dentinal tubular counts compared to the control group as desensitizing agent occluded majority of the dentinal tubules. A number of studies have reported that with the application of desensitizing agent the number of dentinal tubules decreases. Another important finding of this study was an increase in closed dentinal tubular area, a reduction in the number and the mean diameter of dentinal tubules with increasing percentage of dentinal tubular occlusion. This is in favor of findings of the previous studies. Absi et al. in 1987 reported that sensitive teeth have significantly increased numbers of tubules per unit area (approximately 8 times), and the diameter was approximately 2 times that of nonsensitive teeth.  In normal conditions, the human dentin has 21,000/mm 2 -48,000/mm 2 dentinal tubules, at a position halfway between the pulp and enamel. 
The present study did not evaluate the dentin permeability. There appears, however, to be no available evidence to suggest that "hypersensitive" dentin is more permeable than "nonsensitive" dentin.  Studies have attempted to analyze dentin permeability using hydraulic conductivity.  However, in the current scenario, there is no way to reproduce satisfactorily the pulpal pressure. ,
Although studies have provided accurate estimation of degree of dentinal tubular occlusion using different parameters but in true clinical situations, there are many factors which affect the results such as the age, condition and source of the dentin specimen, the groove size of the specimen following cutting, the presence or absence of a smear layer, the density, diameter, direction, and orientation (cross-sectional or longitudinal) of the tubules, the presence or absence of highly mineralized peritubular dentin, and the variation of the intricate branching tubule system as well as the types and locations of these branches. 
The period of evaluation for the effect of different desensitizing agents varies from single application to over a period of months. Majority of the studies done in the past evaluated the effect of these agents over a period of 3 months. The different point in this study was the main focus on the short-term effects of the dentifrices. Kerns et al. 1991 have used a 3-min brushing or treatment time; and was considered reasonable for observing any changes produced by the test agents.
Thus, the results of this in-vitro dentinal tubule occlusion analysis test support the concept that desensitizing toothpaste with remineralizing properties may be more effective for long-term home treatment of DH.
Though in-vitro studies provide evidence for immediate dentine hypersensitivity treatment, further well-designed multicenter prospective long-term clinical trials with scanning electron microscopic evaluation are needed to find the gold standard treatment of DH and to compare its bioactivity to common desensitizing toothpastes and prove its effect over time following daily brushing and citric acid challenge.
| Acknowledgments|| |
I would like to thank for great support from Department of Oral surgery, Department of Oral Pathology, Department of Public Health Dentistry, Sinhgad Dental College and Hospital, Pune and Sinhgad College of Pharmacy to conduct this study. I am thankful to Remin Pro, Voco Company, Germany for supply of the product.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]