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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 238-246
 

Effect of Aerva sanguinolenta (Lal bishalyakarani) plant extract on biofilm-induced human enamel demineralization: An in vitro study


1 Department of Pedodontics & Preventive Dentistry, Guru Nanak Institute of Dental Sciences and Research, Kolkata, West Bengal, India
2 Department of Oral & Dental Sciences, JIS University, Kolkata, West Bengal, India
3 Department of Pharmacy, Guru Nanak Institute of Pharmaceutical Science and Technology, Kolkata, West Bengal, India
4 Department of Orthodontics, Burdhaman Dental College and Hospital, Bardhaman, West Bengal, India

Date of Submission13-Jul-2020
Date of Acceptance14-Jul-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Dr. Shabnam Zahir
Department of Pedodontics and Preventive Dentistry, Guru Nanak Institute of Dental Sciences and Research, Kolkata - 700 114, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_312_20

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   Abstract 


Background: Controlling cariogenic biofilm formation by plant extracts could add to preventive strategies to dental caries. Objective: To evaluate in vitro the role of Aerva Sanguinolenta ethanolic extract on biofilm-induced microbial human enamel demineralization. Methodology: The prepared enamel sections of study group (SG), positive control group (PCG), and negative control group (NCG) were immersed in 2 ml of 0.2% ethanolic extract of A. sanguinolenta, 0.12% chlorhexidine, and distilled water, respectively, for 2 min before subjecting to closed batch culture technique utilizing mono- or dual-species culture media of Streptococcus mutans and Lactobacillus acidophilus. Quantification of biofilm and demineralization of enamel was performed by crystal violet (CV) assay and scanning electron microscope (SEM) attached to energy-dispersive X-ray analysis, respectively. Statistical Analysis: Two-way ANOVA and Tukey's test were used for analysis. Results: CV assay of biofilm recorded the highest and lowest optical absorbance value in NC3 (2.728660) and PC3 (0.364200), respectively. Thus, biofilm formation is highest in NCG and lowest among PCG. Surface roughness and porosity in enamel are greatest among NCG and lowest among SG as evident by SEM. Wt% of calcium (S3 47.7170) and phosphorus ion (S3 22.7330) was highest in SG, closely resembling that of B enamel (Ca = 41.9530, P = 19.6650). Wt% of oxygen is lowest in SG (S3 28.8920) and resembles baseline O2 (37.4950). Thus, the amount of biofilm formation is moderate and amount of demineralization of enamel is least among SGs. Conclusion: Enamel exposed to 2 ml of 0.2% solution of A. sanguinolenta for 2 min could fairly inhibit formation of biofilm and positively inhibit underlying demineralization in cariogenic environment.


Keywords: Human enamel, microbial demineralization, plant extracts


How to cite this article:
Zahir S, Pal TK, Sengupta A, Biswas S, Bar S. Effect of Aerva sanguinolenta (Lal bishalyakarani) plant extract on biofilm-induced human enamel demineralization: An in vitro study. J Indian Soc Pedod Prev Dent 2020;38:238-46

How to cite this URL:
Zahir S, Pal TK, Sengupta A, Biswas S, Bar S. Effect of Aerva sanguinolenta (Lal bishalyakarani) plant extract on biofilm-induced human enamel demineralization: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2020 Oct 30];38:238-46. Available from: https://www.jisppd.com/text.asp?2020/38/3/238/296633





   Introduction Top


Dental caries is a chronic disease resulting from interaction of microorganisms in the dental biofilm with fermentable carbohydrates from the diet on a susceptible tooth.[1] Among numerous oral bacteria, Streptococcus mutans and Lactobacillus acidophilus have been strongly implicated in the carious process.[2],[3],[4],[5],[6] Reducing the levels of these highly cariogenic bacteria in the oral cavity will provide additional means for the prevention of dental caries.[7]

Dental caries usually begin with demineralization of enamel corresponding to a loss of about 1%–2% mineral (calcium and phosphate), leading to small number of relatively large pores. Qualitative and quantitative measurements of changes in the structure and mineral content of enamel before and after demineralization can be performed by a scanning electron microscope (SEM) with energy-dispersive X-ray analysis (EDAX) attachment.[8]

For research purpose, artificial demineralization is produced byin vitro experimental cariogenic setups such as pH cycling, acid immersion, bacterial slurry, or bacterial biofilm system.[9] Growth of biofilms has been shown to occur via a sequence of colonization events in which initial adhesion to the enamel surface is followed by further bacterial–enamel binding, bacteria–bacteria interaction, and growth.[10] The present authors in an earlierin vitro study produced monospecies (S. mutans) biofilm-induced microscopic human enamel demineralization using the closed batch culture technique as evidenced by SEM of the study group (SG), showing an increase in surface roughness and porosities of enamel surface than the control group. EDAX of SG revealed a statistically significant decrease in calcium (wt%) and phosphorus (wt%) and increase in oxygen (wt%) ions, indicating demineralization of enamel in SG.[11]

Few studies have demonstrated antimicrobial activity against selected oral pathogens from natural plant source.[12],[13] Only 1% of plant source has been evaluated till date for research, thus a lot more to be investigated. Aerva sanguinolenta (L.) Blume of the family Amaranthaceae is a perennial herb with ornamental or wild cultivation status. It is commonly known as Lal bishalyakarani (Bengali), Sufed fulia (Hindi). It is ethnomedicinaly used for the treatment of hematuria, painful menstruation,[14] asthma, and jaundice.[15] Experimentally, it has exhibited antioxidant, antidiabetic, hepatoprotective, diuretic, antileishmania, and anticancer activity.[16]

In vitro antibacterial evaluation of the extract of A. sanguinolenta has shown effective antibacterial activity against Gram-positive bacteria.[17],[18],[19] In a previous study by the present authors the 1000 μg/ml (100%), ethanolic extracts of A. sanguinolenta showed mean inhibition zone (I.Z) of 11.860 mm against S. mutans and a mean I.Z. =9.730 mm against L. acidophilus in the agar disc diffusion assay. It was more than the negative control (NC) (90% dimethyl sulfoxide [DMSO]) with (I.Z)of 6.560mm but less than the positive control (PC) (30% vancomycin)) with (IZ) of 18.200 mm. in the agar disc diffusion assay. A. sanguinolenta exhibited minimum inhibitory concentration (MIC) of 0.02050 μg/ml and 0.02050 μg/ml against S. mutans and L. acidophilus, respectively, in a microbroth dilution method in the same study.

Aim

The study aimed to evaluate the role of whole plant ethanolic extract of A. sanguinolenta on monospecies (S. mutans or L. acidophilus as single species) and multispecies (S. mutans and L. acidophilus) biofilm-induced microbial human enamel demineralization, using the closed batch culture technique. It is also aimed to quantify the biofilm if formed by crystal violet (CV) assay and to quantify the demineralization of human enamel sections structurally and elementally with SEM and EDAX analysis, respectively, in both study and control groups.


   Methodology Top


Requisite ethical clearance (GNIDSR/IEC/18-18) and permission to undertake the study were obtained from the respective committee.

Preparation of tooth sample

Fifty caries-free, human premolar teeth, extracted for the purpose of orthodontic treatment, were collected and analyzed using a stereomicroscope (Olympus SZX7; Olympus Corporation, Tokyo, Japan). Finally, 45 teeth without cracks, pits, and white spots were selected. Teeth were disinfected by autoclaving in 121°C/20 psi for 30 min following the protocol of the Centre for Disease Control, USA.[20] Tooth samples derooted and the crown portion cut longitudinally in a mesio - distal plane using diamond discs, yielding ninety tooth sections. Tooth sections were stored in sterilized and coded containers with double deionized (DI) water.

Preparation of tooth, wire, and test tube assembly

3 mm × 3 mm wax pieces were placed in the middle of the labial surface of the tooth sections, coated with acid-resistant nail varnish and once the paint dried the wax sheets were removed to form an isolated window. The tooth sections were attached to orthodontic wires on the palatal surface, to leave the free enamel window. The test tubes were loosely closed with a cotton plug to allow gas exchange with the environment and this assembly was sterilized in autoclave.

Collection, authentication, pretreatment, and preparation of extract of Aerva sanguinolenta

Whole plant of A. sanguinolenta was collected (from medicinal garden of Ramkrishna Mission Ashram, Narendrapur, West Bengal during the month of December), authenticated (by Botanical Survey of India, Howrah, West Bengal-711103), washed, shade dried, chopped, powdered (mixer grinder - Philips HL), stored, labeled, and refrigerated (at 4°C). Hot continuous extraction of coarse powder was done with 300 ml ethanol (Mercks) by Soxhlet apparatus (Borosil). Wet plant extract (20.5462 g) by weight thus obtained, were concentrated by distillation using a rotary evaporator (RE100PR0MFGD silicogex/USA Takashi) to get dry plant extract (12.5462 g) by weight. Dry extract (1000 μg) was then mixed with 1 ml of DMSO (Mercks) to prepare the 0.2% solution of whole plant ethanolic extract of A. sanguinolenta. It was ten times of MIC of A. sanguinolenta against S. mutans and L. acidophilus which was determined in a previous study by the present authors.

Collection and revival of bacterial sample

The stored bacterial sample of S. mutans (MTCC-890) and Lactobacillus acidophilus (MTCC-10307) were obtained from the Microbial Type Culture Collection and Gene Bank, Chandigarh, inoculated on brain–heart infusion (BHI) broth (Himedia, Mumbai) in separate glass containers (Borosil), and were incubated at 37°C for 24 h. The optical density of the culture was adjusted to obtain a standard amount of cells of approximately 2.15 × 10^8 CFU/ml.

Formation of positive and negative control

0.12% W/V Chlorhexidine gluconate solution (hexidine) was taken as the Positive control (PC) and double distilled water as Negative control (NC).

In vitro biofilm growth

The enamel section with wire assembly was immersed in 2 ml of 0.2% solution of ethanolic whole plant extract of A. sanguinolenta (SG), 2 ml of 0.12% chlorhexidine solution (positive control group [PCG]), and 2 ml of double distill water (negative control group [NCG]) for 2 min before immersing the assembly in sterile BHI broth – BHI (Himedia, Mumbai) with 5% sucrose (Himedia, Mumbai) and 10 ml of fresh 24 h inoculum of bacteria. Subgroup NC1, PC1, S1 inoculated with 10 ml of fresh inoculum of S. mutans (monobacterial culture medium), Subgroup NC2, PC2, S2 with 10 ml of fresh inoculum of L. acidophilus (monobacterial culture medium) and Subgroup NC3, PC3, S3 inoculated with 10 ml of fresh inoculum of both S. Mutans and of L. acidophilus (dual-bacterial culture medium) [Table 1]. All groups had their medium changed at the same time to prevent any kind of contamination and were incubated at 37°C in a CO2 rich atmosphere in a candle jar. The above steps were repeated ten times for ten new sets of test tubes for consecutively 10 days. Contamination at test recipients was verified at each 24 h by inoculation in blood agar media.
Table 1: Distribution of study sample

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Quantification of biofilm formed on tooth sections by crystal violet assay

At the end of 10 days of batch culture technique, enamel sections were separated from the wire, washed with DI water, leaving only attached biofilm and were incubated with 1% solution of CV dye in DI water at room temperature for a period of 30 min. The enamel sections were then washed several times with DI water to remove free dye. 10 ml of decoloring solution (90%–95% of alcohol) was added (to a volume greater than or equal to the original culture media volume) and incubated with the enamel sections for 30 min. Finally, the CV dye infused decoloring solution is assessed for absorbance at 530 nm, with a spectrometer. Higher optical absorption value relates to more biofilm mass.

Structural and elemental analysis of tooth sections with scanning electron microscope attached to energy-dispersive X-ray analysis

Following biofilm quantification, tooth sections dried with a hair dryer, coded as subgroups NC1, NC2, NC3, PC1, PC2, PC3, S1, S2, S3 and B (Baseline). The ten groups of tooth samples were gold and paladium sputtered in SC 7620 Mini Sputter Coater. Photomicrographs of representative areas were taken at ×1500 and ×3000 magnifications with a SEM (Phenom Pro-X) for structural analysis. Although EDAX can assess a wide range of elements, attention was focused on calcium, oxygen, and phosphorus Wt%.

Statistical analysis

The Statistical software IBM SPSS statistics 20.0 (IBM Corporation, Armonk, NY, USA) was used for the analyses of the data and Microsoft word and Excel were used to generate graphs, tables etc., Results from the study were analyzed for statistical significance using two-way ANOVA and Tukey's test for multiple comparison. P < 0.05 was considered statistically significant and P < 0.001 was considered very highly statistically significant.


   Results Top


Result of colorimetric assay of biofilm

Comparison of mean optical absorbance values among all the subgroups of PCG, NCG and SG (PC1, PC2, PC3, NC1, NC2, NC3, S1, S2, S3) using ANOVA test reveals statistically significant difference (F = 208.422) (P < 0.001) [Table 2] and [Graph 1]. Further, using Tukey's post hoc analysis, highly significant difference was observed between all the groups except between PC2 and PC3 (P = 0.719), S1 and S2 (P = 1.000), S2 and S3 (P = 0.996), and S1 and S3 (P = 0.999).
Table 2: Comparison of optical absorbance values (nm) of biofilm following spectrometric analysis in terms of (mean [standard deviation]) among all the subgroups using ANOVA test

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Result of scanning electron microscope micrograph

SEM micrograph of enamel surface exhibits a significant increase in the surface roughness and enamel porosity in a descending order, NC group > PC group > S group. The order of increase in surface roughness and micro porosity among NC subgroups, NC3 > NC1 > NC2 [Figure 1], [Figure 2], [Figure 3], [Figure 4] among PC subgroup, PC3 > PC1 > PC2 [Figure 5], [Figure 6], [Figure 7] among study subgroup, S1 > S3 > S2 [Figure 8], [Figure 9], [Figure 10]. Thus 2 min exposure to 2 ml of 0.2% solution of A. sanguinolenta could inhibit roughness and porosity of enamel more than PC and NC group and it's more effective in monospecies L. acidophilus (S2) than multispecies S. mutans and L. acidophilus (S3) and monospecies S. mutans (S1) biofilm model.
Figure 1: Scanning electron micrograph of baseline (B) enamel

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Figure 2: Scanning electron micrograph of subgroup (NC1) enamel

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Figure 3: Scanning electron micrograph of subgroup (NC2) enamel

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Figure 4: Scanning electron micrograph of subgroup (NC3) enamel

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Figure 5: Scanning electron micrograph of subgroup (PC1) enamel

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Figure 6: Scanning electron micrograph of subgroup (PC2) enamel

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Figure 7: Scanning electron micrograph of subgroup (PC3) enamel

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Figure 8: Scanning electron micrograph of subgroup (S1) enamel

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Figure 9: Scanning electron micrograph of subgroup (S2) enamel

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Figure 10: Scanning electron micrograph of subgroup (S3) and enamel

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Result of energy-dispersive X-ray analysis

On comparing the calcium, phosphorus and oxygen (Wt%) levels in terms of (mean [SD]) among the nine subgroups and baseline enamel samples using Anova test, a statistically significant difference observed between the groups (P <0.001) [Table 3], [Table 4], [Table 5].
Table 3: Comparison of calcium levels (Wt%) of enamel in terms of (mean [standard deviation]) among all the subgroups (NC1, NC2, NC3, PC1, PC2, PC3, S1, S2, S3) and baseline (B) using ANOVA test following energy-dispersive X-ray analysis

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Table 4: Comparison of phosphorus levels (Wt%) of enamel in terms of (mean [standard deviation]) among all the subgroups (NC1, NC2, NC3, PC1, PC2, PC3, S1, S2, S3 ) and Baseline (B) using ANOVA test following energy-dispersive X-ray analysis

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Table 5: Comparison of oxygen levels (Wt%) of enamel in terms of (mean [standard deviation]) among all the subgroups (NC1, NC2, NC3, PC1, PC2, PC3, S1, S2, S3) and baseline (B) using ANOVA test following energy-dispersive X-ray analysis

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The Wt% of calcium and phosphorus ion in the SG (calcium - S3-47.7170, S2-44.4910 and S1-28.0550) and (phosphorus - S3-22.7330, S1-14.9350) resembles the baseline (B) value of (Ca - 41.9530, P - 19.6650) is more than PCG and NCG [Graph 2], [Graph 3] and [Table 3], [Table 4] indicating minimum loss of calcium and phosphorus ion in the SG. Regarding concentration of oxygen ion highest and lowest mean score was recorded in subgroup NC2 (70.0650), S3 (28.8920). The concentration of calcium ion in base line (B) enamel (37.4950) most closely resemble to that of subgroup S3 (22.7330) and S2 (28.8920) [Graph 4] and [Table 5] indicating minimum porosity thereby minimum demineralization of enamel of SG felicitated by 2 min exposure to 2 ml of 0.2% solution of A. sanguinolenta.




   Discussion Top


A. sanguinolenta is the chosen plant as it is widely and easily grown, all season, hardy herbaceous plant of the North Gangetic Plain of West Bengal. In a toxicological study by the present authors, mean LC50 of ethanolic whole plant extract of A. sanguinolenta is found to be 575.42800 μg/ml, at an time interval of 24 h on Zebrafish embryos.[21]

There are a variety of dental caries experimental model systems available, each one presenting advantages and disadvantages.[22],[23] The closed batch biofilm models are used to develop multi-, dual-, or monospecies biofilm, require small amounts of reagents, are convenient, reproducible, and economical to use.[22],[23] In the present study the closed batch culture technique is utilized as it uses bacterial biofilm to induce artificial demineralization of human enamel which simulate more closely toin vivo oral microcosm[24] and it is a modified version of the model used by Oliveira et al. in 2007.[24] In this study, BHI broth is chosen as culture media as it is found to be more effective in biofilm formation than trypticase soy broth.[25]

Among the fermentable carbohydrates, sucrose is the unique substrate necessary for synthesis of insoluble extracellular polysaccharides.[26] It increases the thickness and porosity of biofilms[27] and lead to a deeper pH fall on the tooth-dental biofilm interface.[28] According to Cai et al. (2016) a sucrose concentration of above 2.5% produces optimum biofilm formation, bacterial adhesion and thickness.[29] So for the present study 5% sucrose was added in the culture media. The present batch culture technique is performed in a CO2 rich environment as culturing S. mutans strains under aerobic conditions induces an 80% reduction of bacterial ability to form biofilms.[30] BHI broth with bacterial inoculum is changed every 24 h. As it allows checking for any contamination by Gram staining in blood agar and periods of medium change (6, 12 or 24 h) did not have an effect on biofilm-induced demineralization of enamel.[24] Arthur et al. 2014 in anin vitro study found out that deeper lesions and higher integrated mineral loss were found after 9 days of biofilm formation (P <0.005). In the present study batch culture technique was continued for 10 days.[31]

The present study attempted to grow three types of biofilm setup that is S. mutans monospecies, L. acidophilus monospecies, and combined S. mutans and L. acidophilus dual-species biofilm setup. Simple monobacterial biofilm models have been developed as it is simpler and allows the study of the properties of the individual bacterial species present.[32] Compared to monospecies biofilm, dual-species provide a more complex microbial ecosystems[33] and with increased antimicrobial tolerance.[34] Hence, in the present study, a dual-species biofilm model is also used. However, it does not mimicin vivo multispecies plaque biofilms in both survival and pathogenic potential.

A variety of direct methods i.e., plate counts, microscopic cell counts, coulter cell counting, flow cytometry, and fluorescence microscopy and indirect methods i.e., determination of dry mass, total organic carbon, microtiter plate assays, adenosine triphosphate bioluminescence, total protein, quartz crystal microbalance and CV assay have been used to quantify cells in biofilms.[35] Similar to a study by Barbosa et al. 2016,[36] in the present study CV assay, an indirect method has been used to quantify the biofilm formed as it is relatively easy to perform, relatively inexpensive, reproducible, and allows researchers to rapidly analyze multiple samples simultaneously.

At the end of 10 days of the batch culture technique, amount of biofilm formation is highest among NC and lowest among PC whereas surface roughness and porosity in enamel surface greatest among NC and lowest among SG. Wt% of Calcium and Phosphorus ion is highest in SG closely resembling that of base line enamel. The Wt% of Oxygen ion is highest in the NCG and lowest in the SG. Demineralization is the removal of minerals (mainly calcium, phosphorus) from enamel. Thus the amount of biofilm formation is moderate and amount of demineralization of enamel is least among the SGs. Thus exposing enamel surface for 2 min to 0.2% solution of whole plant ethanolic extract of A. sanguinolenta could inhibit biofilm-induced demineralization of human enamel in an experimental setup partialy simulating the conditions in the oral cavity. Similar to present study Smullen et al. in 2012,[37] John et al. in 2013,[38] Eid et al. in 2015,[39] Ramalingam and Amaechi in 2018[40] showed that the plant extracts inhibited biofilm formation on enamel in artificial mouth model with monospecies, dual-species, or multispeciesinoculum.

In the present study while comparing the subgroups with three types of bacterial model, the amount of biofilm formation, enamel surface roughness, Calcium and Phosphorus Wt% are greatest in the dual bacterial model in SG, PCG and NCG group. Wen in 2010 also reported that the capacity of Lactobacilli to form biofilm was enhanced significantly in the presence of S. mutans. Lactobacilli cannot form plaque on the tooth on their own and depend on extracellular polysaccharide produced mainly by Streptococci, for colonization.[41] On the contrary Ahmed et al. 2014 through anin vitro study revealed Lactobacillus acidophilus reduced the biofilm formation by S. mutans more potently as compared to Lactobacillus rhamnosus. The appropriate mechanism of the inhibition of biofilm was not determined, but it might be due to interference of adherence, fighting for nutrients and antibacterial peptide production by Lactobacillus species. L. acidophilus also reduced the viable count of S. mutans.[42] In the PCG the subgroup P1 forms the greatest amount of biofilm and the highest amount of phosphorus content of enamel may be because the ability of S. mutans to synthesize extracellular glucan-homopolymers from sucrose, play a critical role in initial attachment, colonization and accumulation of biofilms on tooth surfaces (Tanzer, 1992).[43]


   Conclusion Top


Enamel exposed to 2 ml of 0.2% solution of A. sanguinolenta for 2 min could fairly inhibit formation of biofilm and positively inhibit underlying demineralization in cariogenic environment even better than 0.12% of chlorhexidine.

Acknowledgment

We acknowledge West Bengal University of Health Sciences, Bidhannagar, Kolkata-64 and Guru Nanak Institute of Dental Sciences and Research, Kolkata - 114 for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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    Abstract
   Introduction
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    References
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  2005 - Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 
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