|Year : 2020 | Volume
| Issue : 4 | Page : 355-360
Evaluation of mineral loss in primary and permanent human enamel samples subjected to chemical demineralization by international caries detection and assessment system II and quantitative light-induced fluorescence™: An in vitro study
Ragavee Veeramani, Raghavendra Shanbhog, Nandlal Bhojraj, Shefali Kaul, NK Anoop
Department of Pediatric and Preventive Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Research, Mysuru, Karnataka, India
|Date of Submission||15-May-2020|
|Date of Acceptance||03-Dec-2020|
|Date of Web Publication||5-Jan-2021|
Dr. Ragavee Veeramani
Department of Pediatric and Preventive Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Research, Mysuru - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose: The purpose of the study was to understand the mineral loss in primary and permanent enamel samples and an attempt is made to standardize the process of chemical demineralization to generate more meaningful results in research studies involving the remineralization of demineralized samples. Materials and Methods: Due to variability among enamel samples theoretically, it is impossible to standardize demineralization by running time-based chemical demineralization cycle without frequent monitoring. Instead of carrying out demineralization cycles for a fixed duration of time, we quantified the mineral loss 24 hourly using the International Caries Detection and Assessment System (ICDAS) and Quantitative Light-induced Fluorescence System (QLF™). Twenty primary and permanent enamel samples were subjected to demineralization, and ICDAS and QLF™ evaluation were done at 0, 24, 48, 72, 96, 120, 144, and 168 h of demineralization. Results: The first visual change in permanent enamel is appreciated at 24 h (ICDAS II code1, QLF™ code1 −16.353 – ΔF) of demineralization, at 48 h (ICDAS II code2, QLF™ code2, −24.515 – ΔF), there was localized white spot lesion in permanent enamel and remained until 96 h (ICDAS II code 2, QLF™ code 2, −25.739 – ΔF) of demineralization. In primary samples, distinct visual change was seen at 24 h (ICDAS II code2, QLF™ code2, −19.431 – ΔF), and at 48 h clinically, there was a distinct visual change, but optically mild enamel breakdown was appreciated (ICADSII code 2 QLF™ code3, −27.201 – ΔF), which remained constant till 120 h of demineralization (ICDAS II code2 QLF™ code3 −37.645 – ΔF). Conclusion: Different samples demineralize at different rates. The demineralization in primary samples was 1.25 times higher than permanent samples. Recommendation: due to inherent variability in the samples continuous monitoring of the demineralization process on a 24 hourly basis is required to standardize the process.
Keywords: Artificial caries, International Caries Detection and Assessment System II, permanent enamel, primary enamel, Quantitative Light-Induced Fluorescence
|How to cite this article:|
Veeramani R, Shanbhog R, Bhojraj N, Kaul S, Anoop N K. Evaluation of mineral loss in primary and permanent human enamel samples subjected to chemical demineralization by international caries detection and assessment system II and quantitative light-induced fluorescence™: An in vitro study. J Indian Soc Pedod Prev Dent 2020;38:355-60
|How to cite this URL:|
Veeramani R, Shanbhog R, Bhojraj N, Kaul S, Anoop N K. Evaluation of mineral loss in primary and permanent human enamel samples subjected to chemical demineralization by international caries detection and assessment system II and quantitative light-induced fluorescence™: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2022 May 22];38:355-60. Available from: https://www.jisppd.com/text.asp?2020/38/4/355/306214
| Introduction|| |
Incipient caries lesion of enamel plays an important role in cariology research. They are created and researched in an attempt to understand the pattern, formation, and prevention of such lesions. Artificial lesions are preferred over natural lesion in research so as to reduce the variability in lesion formation and progression. A wide variety of demineralization systems have been practiced for the preparation of artificial caries lesion in dental enamel and are correlated to type and degree of lesion progression. Among the systems in use, in vitro chemical models are frequently utilized owing to its advantages such as simplicity, economy, efficiency, reproducibility, and stability. Mild organic acid buffers (pH from 4.4 to 5.0) are used to generate demineralized lesions in the chemical models by arresting the process of re-mineralization. Such demineralization cycle lasts for 72–96 h to produce a lesion about 80–130 μm in depth.,, Standardization of this demineralization process is a prime concern to generate valid and meaningful results in research studies involving remineralization of demineralized samples. However, due to the inherent variability in human enamel samples, theoretically, it is impossible to produce standardized demineralization in human enamel by running time-based chemical demineralization cycle without frequent monitoring and quantification of mineral loss. Instead of carrying out demineralization cycles for fixed duration of time as suggested in literature, quantifying the mineral loss on daily basis using a reliable and sensitive tool during demineralization cycle will help us to generate the standardized demineralized samples.
The International Caries Detection and Assessment System (ICDAS) is one of the systems which can classify the level of demineralization visually and is validated as one of the reliable methods to assess demineralization. Furthermore, several optical and nondestructive techniques enable the assessment of enamel demineralization and remineralization over time. One such optical technology is quantitative light-induced fluorescence system (QLF™), which has been highly validated for its sensitivity and reliability., The QLF™-D Biluminator™ 2 which was used in this study consists of a Biluminator™ mounted on a Single Lens Reflex camera fitted with a 60 mm macro lens. The Biluminator™ provides the light sources and filters for making white-light and QLF™-images and a connection to a computer that runs the necessary software for archiving and analysis.
The aim of present study was to understand mineral loss behavior among primary and permanent human enamel samples when subjected to chemical model of demineralization, classified by ICDAS II and quantified by QLF™ for 7 days in an attempt to develop standardized demineralization lesions in human enamel samples for in vitro research.
| Materials and Methods|| |
Study design and location
This was an in vitro experimental study. The sample preparation and intervention were conducted in the Special Interest Group, Cariology Research, Department of Pedodontics and Preventive Dentistry. The demineralizing solution was prepared in the Pharmaceutics laboratory, College of Pharmacy.
Sample size calculation
Using 80% power and 95% confidence interval, the sample size was found to be 18. To overcome errors during the procedure, it was rounded off to 20 in each group [n Master sample size calculation software].
Sample collection and preparation
Twenty primary and twenty permanent teeth enamel samples were collected from disease and restoration free ten primary molars and ten premolars, with no visible enamel infarctions, wasting diseases, stains, and developmental anomalies. All the collected teeth were therapeutically extracted or exfoliated primary molars from age of 7 to 11 years and premolars from volunteers aged 15 to 25 years which were extracted for orthodontic purposes from individuals brushing with fluoridated toothpaste twice daily. The collected samples were thoroughly cleaned for debris and calculus, were washed with 0.1M phosphate buffer solution (pH 7.4), rinsed with deionized water, and were stored in distilled water at 4°C till further use, in a refrigerator. The collected teeth were decoronated at the cemento-enamel junction, and the crown using diamond disc (Diatech CH9435, Swiss Dental Instruments, Lot: 9605) mounted on a slow speed Micromotor Straight Handpiece (SH-E, NSK, Nakanishi Inc., Shimohinata, Kanuma, Japan) under continuous irrigation with deionized water, to obtain a total of twenty primary and twenty permanent samples. Each sample was mounted uniformly in an acrylic block with the help of a customized mounting jig. Following this, a polyvinyl adhesive tape of 2 mm × 2 mm dimension was placed on the surface of samples and they were coated with an acid resistance transparent nail varnish (Lakmé™, Lakmé Cosmetics, Hindustan Unilever Limited, Mumbai, Maharashtra, India). After setting of the varnish, the adhesive tape was removed leaving a window of 2 mm × 2 mm on the enamel surface (area of interest [AOI]). The AOI was cleaned with a micro brush under low magnification microscope.
Preparation of demineralizing solution
The demineralizing solution (2.2 mM calcium chloride, 2.2 mM Sodium dihydrogen orthophosphate dehydrate, and 0.05 M Acetic acid) was prepared according to J. M ten Cates protocol, 1982. pH of the solution was adjusted to 4.4 using 50% Sodium hydroxide. All the chemicals used were industrial grade procured from Sigma-Aldrich (Sigma-Aldrich Corporation, St. Louis, Missouri, United States)
All the prepared samples were individually stored in 30 ml of freshly prepared demineralizing solution. The samples were placed in an Orbital Shaking Incubator (REMI, Mumbai, Maharashtra, India) at 37°C with 50 RPM to provide gentle agitation. The demineralizing solution was prepared and replaced for all the samples every 24 h.
Evaluation and quantification of mineral loss
The samples were evaluated at baseline (0), 24, 48, 72, 96, 120, 144, and 168 h of demineralization cycle using ICDAS II and QLF™ performed by two previously calibrated examiners. The AOI was dried with compressed air for 5 s, visual examination was done, QLF™, and White light photographs were taken with QLF™ (QLF™-D Biluminator™, Inspektor Research Systems BV, Amsterdam, The Netherlands) under standardized lighting and camera settings. White spot lesion analysis was done for all the images using QA2 v 1.26.0 (Inspecktor Reasearch Systems BV, Amsterdam) by two trained examiners and the mean of % loss of Fluroscence (ΔF) was taken for the results. The obtained data were transferred to a datasheet and subjected to appropriate statistical analysis using SPSS® (v 23 2015, IBM, Armonk, New York, USA) software. Mean and standard deviations were used for descriptive statistics, and intergroup comparison was carried by Mann–Whitney U-test.
| Results|| |
Primary and Permanent human enamel samples subjected to chemical demineralization for 168 h, evaluated for ICDAS II with clinical examination, showed a gradual increase from code 0 (baseline) to code 3 (168 h) in permanent enamel and code 0 (baseline) to code 3 (168 h) in primary enamel. Similarly, the mean % loss of florescence (ΔF) evaluated longitudinally with QLF™ showed an increase from −10.753 (baseline) to − 34.163 (168 h) in permanent enamel and −8.316 (baseline) to −42.808 (168 h) in primary enamel [Table 1].
|Table 1: Means and standard deviation of ΔF values for both deciduous and permanent teeth as compared to the quantitative light.induced fluorescence index|
Click here to view
During demineralization cycle, the first visual change in permanent enamel samples (ICDASII code1) were seen at 24 h demineralization which shifted to distinct visual change (ICDASII code 2) at 48 and remained constant till 96 h of demineralization. At 120, 144, and 168 h of demineralization, the samples displayed localized enamel breakdown (ICDASII code 3). Corresponding to this observation first observed change in the green fluorescence (QLF™ code 1) was appreciated at 24 h demineralization (−16.353 –ΔF) which increased to distinct visual change in green fluorescence (QLF™ code 2) at 48 h (−24.515 –ΔF) and remained constant till 96 h of demineralization (QLF™ code 2, −25.739 –ΔF). At 120 h of demineralization, the samples showed distinct dark spots and the appearance of orange-red florescence (QLF™ code 3, −32.642 –ΔF) and remained constant till 168 h of demineralization (QLF™ code 3, −34.163 –ΔF).
In primary samples, distinct visual change (ICDASII code 2) was seen at 24 of demineralization, at 48 h and at 72 h. From 96 to 168 h, the samples displayed localized enamel breakdown (ICDASII code 3). Corresponding to this observation, a distinct change in the green fluorescence (QLF™ code 2) was observed at 24 h demineralization (−19.431 –ΔF) which shifted to the appearance of distinct dark spots and appearance of orange-red fluorescence (QLF™ code 3) at 48 h (−27.201 –ΔF) and remained constant till 120 h of demineralization (QLF™ code 3) (−37.645 –ΔF). At 144 h (−39.963 –ΔF) and 168 h (−42.808 –ΔF) of demineralization, the samples showed extensive fluorescence loss and dark spots with orange red fluorescence (QLF™ code 4).
ICDAS II distinct and corresponding QLF™ index code 1 and 2 are indicated for preventive care and are the desired stages of artificial caries lesion generated for remineralization based in vitro studies. Our results show that the QLF™ index by Alammari in 2010 of 2 (ΔF = −18.5 to −26) which equates to ICDAS II score 2 representing white distinct change in enamel is achieved in the primary enamel samples by the end of 24 h of demineralization cycle, and in next 24 h, it showed a minimal increase in ΔF value of −27.20 ± 2.79 making the QLF™ index to 3 (ΔF = −26.5 to −32). The results on the permanent enamel samples showed that QLF™ score of 2 was achieved at 48 h of demineralization (ΔF = −24.51) and is steady over a period of 96 h (ΔF = −25.73). Observed variation among standard deviation of ΔF within primary or permanent samples suggested existence of variation in rate of demineralization among similar enamel samples during chemical demineralization process.
Intergroup comparison of ΔF values for primary and permanent enamel samples, the variation observed of ΔF mean difference values at 0, 72, 96, 144, and 168 h were found statistically significant at P< 0.001 and at 24 and 120 h found significant at P< 0.05 [Table 2].
|Table 2: Intergroup comparison of ΔF values, after exposure to demineralizing solution|
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| Discussion|| |
In the beginning of the study based on known fact, teeth demineralize at different rates and to different extents; theoretically, we hypothesized that it is difficult to produce standardized demineralization in human enamel samples by running time-based chemical demineralization cycle without frequent monitoring and quantification of mineral loss. To understand mineral loss behavior among primary and permanent human enamel samples when subjected to chemical model of demineralization, the present study was carried out. The results of the study clearly demonstrated the existence of variation in the rate of demineralization among similar enamel samples during chemical demineralization process in line with our hypothesis. Inherent biological variability exists in the structure of enamel. Such biological variability in structure and composition of enamel affects the demineralization rate and pattern. Gängler et al. noted changes in pattern of demineralization with increase in age of the tooth samples, which can be explained by posteruptive maturation of enamel. The susceptibility to demineralization also depends on the fluoride content of enamel and conditions in the oral environment. Hence, to achieve approximately same amount of demineralization among all samples, frequent monitoring of samples during demineralization cycle is essential.
The results of the present study also showed that primary enamel is 1.25 times more susceptible to demineralization as compared to succedaneous enamel sample. These results are in agreement with the previous studies. Feartherstone and Mellberg compared the rates of in vitro caries progression by dye uptake method between primary and permanent enamel and concluded that lesion progression is approximately 1½ times more rapid in deciduous than in permanent enamel. Shellis found that, on average, lesion depth was 75% greater in deciduous than in permanent enamel and attributed this to the greater mean prism-junction density and mean volume fraction of interprismatic enamel in primary teeth, as measured by planimetry of scanning electron micrographs. As compared to the permanent enamel, deciduous enamel is much thinner, and the prism themselves are about 2 μm narrower in dimension than the permanent counterpart, this difference might account for a more rapid spread of caries in deciduous enamel, considering that caries attack spreads interprismatically, since there would be twice the number of pathways in any given volume of deciduous enamel compared with permanent enamel. Other than the morphological differences, the overall mineral content of the primary enamel is significantly less as compared to the successor teeth. Permanent enamel is composed of about 92% inorganic compounds, and primary enamel is about 86%–88%. These factors give a plausible explanation for intra- and intergroup variability in the rate of demineralization.
A number of investigations are available for both detection and quantification of early carious lesions. The ICDAS was developed in the year 2002 and revised in the year 2005 to provide an international system for caries detection that would allow for the comparison of data collected in different locations as well as at different points of time and to provide a real-time detection of the lesion, followed by an assessment of the severity of the lesion, which again is followed by an assessment of the activity of the lesion. ICDAS II has been validated as an acceptable caries detection system in several in vitro and in vivo studies, in both permanent and primary teeth.,,
Previous studies have well established that QLF™ can be used as a tool for detection and quantification of early WSL without the disintegration of the structure of sample both in primary and permanent teeth, despite their inherent differences.,,, At the 2011 International Conference on QLF™ (ICQ 2011), study relating ranges of values of ΔF and ΔR to ICDAS II and histology scores by Manal Allammari et al. was presented and they created an index, for the quantitative QLF™ values as compared to the ICDAS II scores.
Comparing this QLF™ index, 2010 by Allammari et al. with our study results, we are able to infer that the QLF™ score of 2, which can be considered a threshold value for enamel remineralization beyond which enamel breakdown and surface loss occurs, hence remineralization of the enamel, is questionable. This score of 2, in the QLF™ index, is achieved at 24 h of demineralization in the primary samples, whereas is achieved at 48 h and is consistent till 96 h. The nature of demineralization in permanent enamel from 48 to 96 h could be attributed again to the higher mineral content, arrangement of crystal structure, and individual sample variation according to age and fluoride history. It is possible that the higher mineral content of permanent enamel causes more supersaturation of the demineralizing solution and hence slower surface layer breakdown of the enamel. QLF™ evaluation along with ICDAS II scoring of demineralization gives us a real-time evaluation of the lesion, without loss of the sample, and hence, it can be used for determining the adequate loss of fluorescence that is required for each sample. The limitation of the present study is we monitored samples only on a 24-h basis for a period of 7 days. Since demineralization was appreciated both clinically, and in QLF™ at 24 h, frequent monitoring of samples within 24 h is required to understand the rate and pattern of demineralization. Chemical model of demineralization can produce direct enamel erosion, instead of white spot lesion. The present study used only clinical and optical method of evaluation, to understand the pattern and depth of in-vitro demineralization on a longitudinal basis we require a histological comparison of the results.
| Conclusion|| |
Within the limitations of our study we conclude that,
- Artificial caries progression in primary enamel is 1.25 times faster as compared to that of permanent enamel
- Within first 24 h localized white spot lesion (ICADS II) is appreciated in primary enamel, whereas this lesion remains over a period of 48–96 h of demineralization in case of permanent enamel
- After 48 h of demineralization, localized enamel breakdown (ICDAS II) occurs in primary teeth, and hence remineralization is questionable in primary sample after 48 h of chemical induction of artificial WSL
- The amount of fluorescence loss is statistically significant between primary and permanent enamel over all time intervals.
Since, there is a lot of individual sample variations due to developmental factors, fluoride history and age, we recommend frequent evaluation of the chemical demineralizing process in the formation of artificial carious lesion at regular intervals, both in primary and permanent teeth. QLF™ being a sensitive device, guides in the detection and quantification of these lesions longitudinally, without the disintegration of the structure of sample along with clinical evaluation with ICDAS II scores. Further studies are required to validate and correlate our results clinically, optically, and histologically with more sample size and using the various chemical demineralizing solution.
The authors acknowledge Dr Elbert de Josselin de Jong, Founder and CSO, Inspektor research Systems, Amsterdam, The Netherlands for sharing his knowledge on QLF™ technology and helping us with this study, and Mr. Sathish, JSS College of Pharmacy, Mysore for helping out with the preparation of solutions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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