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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 33  |  Issue : 1  |  Page : 94-99
Comparative evaluation of remineralisation potential of bioactive glass, casein phosphopeptide-amorphous calcium phosphate and novel strontium-doped nanohydroxyapatite paste: An In-Vitro study


1 Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Saveetha University, Chennai, Tamil Nadu, India
2 Department of Oral and Maxillofacial Surgery, Noorul Islam College of Dental Science, Thiruvananthapuram, Kerala, India
3 Department of Oral Pathology and Microbiology, Noorul Islam College of Dental Science, Neyyatinkkara, Thiruvananthapuram, Kerala, India
4 Department of Oral Pathology and Microbiology, Azeezia College of Dental Sciences and Research, Kollam, Kerala, India
5 Department of Orthodontics and Dentofacial Orthopedics, PMS College of Dental Sciences, Thiruvananthapuram, Kerala, India

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Date of Submission17-Jan-2022
Date of Decision09-May-2022
Date of Acceptance17-May-2022
Date of Web Publication09-Aug-2022
 

   Abstract 


Background: Many studies explained the importance of remineralisation of early carious lesions with various remineralising agents. In the present study, we incorporated the remineralising agents in a dentifrice, applied that in artificial enamel caries and evaluated their remineralising potential and compared the efficacy among the three. Aim: To evaluate and compare the remineralisation potential of a dentifrice containing bioactive glass, casein phosphopeptide-amorphous calcium phosphate and novel laboratory synthesised strontiumdoped nanohydroxyapatite paste in artificial enamel caries. Methods and Materials: 120 enamel specimens were divided into 4 groups of 30 specimens each, based on the type of dentifrice applied: GI - conventional toothpaste (control group), GII - calcium sodium phosphosilicate (Novamin), GIII - casein phosphopeptide-amorphous calcium phosphate (GC tooth mousse) and GIV- Novel strontiumdoped nanohydroxyapatite paste (SrnHAp paste). Specimens in all the groups were subjected to demineralisation, and calcium/phosphorous ratio was analysed followed by remineralisation and the mean calcium–phosphorus ratio was assessed using a scanning electron microscope and energy dispersing X-ray analysis. Statistical Analysis: Data were analysed using IBM SPSS Statistics for Windows Software, version 22 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to calculate the mean and standard deviation. Kruskal-Wallis, ANOVA and Mann–Whitney tests were used. The level of significance was set at 5%. Results and Conclusion: All except the control group showed a net increase in calcium and phosphorous values after application of the respective remineralising agents in respective groups. Inter-group comparison revealed that Group IV - SrnHAp paste yields higher net calcium and phosphorous values than other groups. Hence, novel SrnHAp can be considered as the material of choice in remineralising early enamel carious lesions.

Keywords: Calcium phosphate, dental caries, fluorides, strontium

How to cite this article:
Rajendran R, Hussain M S, Sandhya R, Thomas AJ, Ameena M, Saleem S. Comparative evaluation of remineralisation potential of bioactive glass, casein phosphopeptide-amorphous calcium phosphate and novel strontium-doped nanohydroxyapatite paste: An In-Vitro study. Indian J Dent Res 2022;33:94-9

How to cite this URL:
Rajendran R, Hussain M S, Sandhya R, Thomas AJ, Ameena M, Saleem S. Comparative evaluation of remineralisation potential of bioactive glass, casein phosphopeptide-amorphous calcium phosphate and novel strontium-doped nanohydroxyapatite paste: An In-Vitro study. Indian J Dent Res [serial online] 2022 [cited 2022 Oct 4];33:94-9. Available from: https://www.ijdr.in/text.asp?2022/33/1/94/353537



   Introduction Top


According to Sturdevant, dental caries is an infectious microbiologic disease of the teeth that results in localised dissolution and destruction of calcified tissues.[1] Dental caries, though easily detectable and reversible at an early stage, remains the most common cause of enamel loss in a clinical situation as the incipient lesion proceeds to cavitation, a condition which is irreversible.[2] Hence, the focus of caries management has to be shifted to early detection and non-invasive management like remineralising agents containing fluoride, calcium, phosphate ions in varied forms and concentrations.[3] Remineralisation helps in regaining the lost calcium, phosphate, and fluoride ions of the tooth structure and is replaced in the form of fluorapatite crystals.[4] Enamel is subjected to numerous cycles of demineralisation and reminerlisation throughout lifetime controlling progression or reversal of caries through controlling the demineralisation or reminerlisation cycle, depending upon the microenvironment around the tooth.[3] Non-cavitated lesions as well as caries extending up to the dentino-enamel junction can be arrested by adequately controlling the cariogenic challenges of the specific microenvironment or/and applying therapeutic agents for tissue healing.[2]

Bioactive glass (Novamin)

Various remineralising agents incorporate casein phosphopeptide-amorphous calcium phosphate and bioactive glass (Novamin). Both are widely used for remineralisation.[5] Novamin (calcium sodium phosphosilicate) is a bioactive glass, originally developed as bone regenerative material.[6] It turns reactive when exposed to body fluids and deposits hydroxycarbonate apatite.[7] On tooth surface, it acts as a biomimetic mineraliser forming a strong and acid-resistant layer of natural crystalline hydroxyl carbonate.[2],[8] Novamin contains calcium, sodium, phosphorous and silica ions. In the aqueous oral environment, the glass particles rapidly release calcium and phosphate ions leading to localised, transient increase in pH leading to precipitation of extra calcium and phosphate ions forming a calcium phosphate layer which further crystallises into hydroxycarbonate apatite (HCA), resulting in remineralisation and prevention of further demineralisation.

Casein phosphopeptide-amorphous calcium phosphate

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) contains nanocomplexes of milk protein casein with ACP. It promotes remineralisation by incorporating nanocomplexes into plaque and tooth surfaces, thereby maintaining a supersaturated state of essential minerals, hindering the colonisation of cariogenic bacteria.[9]

Novel strontium-doped nanohydroxyapatite paste

Recently, nanoparticles of hydroxyapatite (nHAp) with higher Ca2+ ion release rates and superior functional properties due to uniform grain size were introduced.[8] However, nHAp has limitations such as lack of strength, brittleness, high degree of crystallinity and low solubility at neutral pH.[10] Researchers have tried replacing calcium (Ca2+) ion with strontium ion (Sr2+) in varied ratios to increase the acid reactivity of apatite, improve solubility and increase fluoride release successfully.[11],[12] Hence, strontium-doped nHAp (SrnHAp) may be beneficial for inducing enamel repair and remineralisation.


   Methods Top


Preparation of enamel specimens

One twenty intact premolars extracted for orthodontic reasons were cleaned and used for the study after approval from the Institutional Ethical Committee (Saveetha dental college and hospitals, IHEC Ref No SDC/Ph.D/07/18/46). The specimens were 4 × 4 × 1 in size and were prepared from buccal surfaces of teeth by sectioning. Teeth were sectioned horizontally using a diamond diac with a slow speed straight handpiece at 1500 rpm at the level of CEJ separating the crown part of tooth.[13] Specimens were divided into four groups of thirty specimens each. Group I was specimens treated with conventional toothpaste (control group), Group II was treated with calcium sodium phosphosilicate (Novamin) while Group III treated with casein phosphopeptide-amorphous calcium phosphate (GC tooth mousse), and Group IV was treated with novel strontiumdoped nanohydroxyapatite paste (SrnHAp paste). The surface topography of all sound enamel specimens was evaluated by a scanning electron microscope; the mean calcium and phosphorus values were obtained using energy dispersing X-ray analysis (JEOL, JSM-840A, TOKYO, JAPAN).

Demineralisation of enamel specimens

McInne's demineralising solution was used for the demineralisation of specimens. Previous studies have proved the efficacy of McInne's in causing enamel demineralisation. Darshan and Shashikiran[14] concluded the study by stating that 'McInnes agent does decrease the microhardness of enamel by causing enamel demineralisation'. The specimens in four groups were subjected to demineralisation by applying freshly prepared McInne's demineralising solution (1 mL of 36% hydrochloric acid, 1 mL of 30% hydrogen peroxide and 0.2 mL of anaesthetic ether in the ratio of 5:5:1) for 5 min, then washed under running tap water, damped dry with absorbent paper, and stored in artificial saliva for 24 h to prevent dehydration. After 24 h, the demineralisation cycle was repeated, specimens were washed, damped dry and the mean calcium and phosphorus content was recorded via SEM-EDX analysis.

Remineralisation of the specimens

The demineralised specimens were subjected to remineralisation with conventional toothpaste in Group I (control group), calcium sodium phosphosilicate (Novamin) in Group II, casein phosphopeptide-amorphous calcium phosphate (GC tooth mousse) in Group III and novel strontiumdoped nanohydroxyapatite paste (SrnHAp paste) in Group IV. Toothpaste for elemental analysis we used 1 mm of toothpaste sample. All the specimens were brushed using a motorised toothbrush for 3 min twice daily (12 h interval), rinsed with running tap water and stored in artificial saliva for 28 consecutive days. The mean calcium and phosphorus content after remineralisation was then evaluated.


   Results Top


Group I (control group) showed a mean calcium value of 65.27 ± 0.58 for sound enamel, while demineralised specimens showed a mean calcium value of 55.09 ± 0.54. After the application of conventional toothpaste, the mean calcium value obtained was 55.24 ± 0.49. No change in the calcium level was noted after the application of conventional toothpaste. Group II (Novamin group) showed a mean calcium value of 65.27 ± 0.58 for sound enamel, while demineralised specimens showed a mean calcium value of 55.09 ± 0.54. After remineralisation with Novamin containing toothpaste, the mean calcium value obtained was 61.29 ± 0.62. There was a considerable amount of rise in calcium level noted after remineralisation. Group III (GC tooth mousse group) showed a mean calcium value of 65.32 ± 0.66 for sound enamel, while demineralised specimens showed a mean calcium value of 55.22 ± 0.47. After remineralisation with GC tooth mousse containing toothpaste, the mean calcium value obtained was 63.05 ± 0.37. The rise in calcium level was noted after remineralisation, which is higher than Group II. Group IV (SrnHAp group) showed a mean calcium value of 65.25 ± 0.59 for sound enamel, while demineralised specimens showed a mean calcium value of 55.03 ± 0.59. After remineralisation with SrnHAp containing toothpaste, the mean calcium value obtained was 65.4 ± 0.86. The remineralised values of the specimens were higher than the demineralised specimens and it was statistically significant (P < 0.001) [Table 1].
Table 1: Mean calcium (SD) of Groups I, II, III, IV

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The mean phosphorus values of sound enamel, demineralised and remineralised specimens in Group I were 20.79 ± 0.78, 15.71 ± 0.57 and 15.76 ± 0.49, respectively. The mean phosphorus values were found to be decreased than all other groups after remineralisation using a conventional toothpaste since no remineralising agents were used. The mean phosphorus values of sound enamel, demineralised and remineralised specimens in Group II were 20.79 ± 0.78, 15.71 ± 0.57 and 17.56 ± 0.43, respectively. The mean phosphorus values were found to be decreased after remineralisation using a Novamin toothpaste. The mean phosphorus values of sound enamel, demineralised and remineralised specimens in Group III were 20.89 ± 0.76, 15.69 ± 0.53 and 18.54 ± 0.39, respectively. The mean phosphorus values were found to be decreased after remineralisation using GC tooth mousse but the values were high when compared to those of Novamin toothpaste. The mean phosphorus values of sound enamel, demineralised and remineralised specimens in Group IV were 20.76 ± 0.01, 15.59 ± 0.48 and 20.5 ± 0.76, respectively. The mean phosphorus values were almost the same as those of sound enamel after remineralisation using a SrnHAp paste. The values of Group IV were higher than the other groups and were statistically significant (P = 0.000) [Table 2].
Table 2: Mean phosphorus (SD) of Groups I, II, III, IV

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Inter-group comparison showed statistically significant differences in the mean calcium and phosphorus levels, with Group IV yielding higher mean calcium and phosphorus levels similar to sound enamel and Group I showing the least values [Table 3].
Table 3: Comparison of mean calcium and phosphorous of four groups after remineralisation

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The topographic changes of the enamel layer in sound enamel [Figure 1], demineralised [Figure 2] and remineralised specimens were assessed using an SEM. The SEM images of Groups I [Figure 3], II [Figure 4], III [Figure 5] specimens show mineral deposition, pores covered partially with crystals.The SEM images of Group IV specimens [Figure 6] treated with SrnHAp paste showed a smoother surface with almost complete obliteration of surface pores.
Figure 1: Scanning microscopic image and energy dispersing xray analysis of sound enamel specimen

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Figure 2: Scanning microscopic image and energy dispersing xray analysis of demineralized enamel specimen

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Figure 3: Scanning microscopic image and energy dispersing xray analysis of group 1 (conventional tooth paste without specific remineralising agent)

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Figure 4: Scanning microscopic image and energy dispersing xray analysis of group 2 ( tooth paste containing calcium sodiumphosphosilicate -novamin)

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Figure 5: Scanning microscopic image and energy dispersing xray analysis of group 3 (topical cream containing casein phosphopeptide amorphous calcium phosphate-gc tooth mousse)

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Figure 6: Scanning microscopic image and energy dispersing xray analysis of group 4 ( tooth paste containing strontium doped nano hydroxy apatite-SrnHAp)

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   Discussion Top


White spot lesions are the earliest clinically detectable sign of the caries process and are reversible with therapeutic measures unless the lesion progresses into the enamel and dentin.[10] The mean calcium and phosphorus values were determined using EDX analysis which is considered as the “gold standard” while determining loss or gain of minerals in experimentally induced early carious lesions by precisely quantifying the mineral content.[15] Strategies aiming at controlling, arresting, or reversing the disease process can reduce the pain and suffering of placing and replacing restorations thereby, economic burden. Acidogenic bacteria produce organic acids which diffuse through the enamel and dentin organic matrix leading to demineralisation. The minerals dissolve into the surrounding aqueous phase at a critical pH of about 5.5. If the calcium and phosphate supersaturation levels are restored, minerals will diffuse into the tooth and deposit a new, more acid-resistant layer.[16] It may be explained by two mechanisms: one is the deposition of fluoride and other ions from saliva and the other is the outward diffusion of minerals and ions from the subsurface lesion.[17]

In this study, McInne's solution was used and demineralisation of enamel specimens was done in two cycles in order to simulate an incipient caries process. A previous study revealed a significant reduction in the microhardness of enamel only after two cycles of demineralisation.[18] Various remineralising agents are available such as tricalciumphosphate, calcium sodiumphosphosilicate (Novamin) and casein phosphopeptide-amorphous calcium phosphate which are incorporated either into toothpaste or a topical cream. Tricalcium phosphate is a new hybrid material composed of beta-tricalcium phosphate (β-TCP) and sodium lauryl sulfate or fumaric acid. Novamin, a bioactive glass, acts as a biomimetic mineraliser. It binds to the tooth surface and continuously deposits a natural crystalline hydroxyl carbonate. The efficacy of CPP-ACP nanocomplexes in the prevention and reversal of early enamel lesions has been proven in clinical in-situ trials and in-vivo randomised control trial.[15]

In the present study, Group I showed mean calcium and phosphorous levels of 61.23 ± 0.65 and 17.52 ± 0.42, respectively, after remineralisation with Novamin. The study results are in accordance with the previous studies, showing that bioactive glass containing remineralisation toothpaste showed significant remineralising potential.[5] In Group II, the remineralising agent used was CPP-ACP and it resulted in mean calcium and phosphorous levels of 63.05 ± 0.37 and 18.54 ± 0.39, respectively, after remineralisation. Thus, CPP-ACP has better remineralising potential than calcium sodium phosphosilicate (Novamin). A similar study evaluating remineralising potential of Novamin and CPP-ACP revealed that CPP-ACP has better remineralising potential than the former.[5]

The mean calcium and phosphorous levels after remineralisation with SrnHAp paste in Group III were 65.4 ± 0.86 and 20.5 ± 0.76, respectively. After remineralisation with the novel SrnHAp paste, the calcium and phosphorus values were almost similar to those of natural teeth. The study by Krishnan et al.[19] attributed this ability to the reduced particle size, easing penetration and increasing solubility.

The net calcium and phosphorous content increased after remineralisation in all the three groups concluding that Novamin, CPP-ACP and SrnHAP are capable of remineralising the demineralised artificial caries lesion. These changes are found to be statistically significant (P < 0.001). The inter-group comparison showed significantly higher mean calcium and phosphorus levels in Group IV (SrnHAP) than Groups I, II and III. Similar results were observed in a study by Krishnan et al.[19] SrnHAp in solution was found to be superior to CPP-ACP cream and nHAp toothpaste as strontium improves solubility and retention on tooth surface, making it a better choice than CPP-ACP and Novamin.[8] Additionally, the quantitative results of the present study are supported by SEM images, which clearly show that Group IV specimens [Figure 6] treated with SrnHAp paste showed a smoother surface almost similar to sound enamel in contrast to Groups I [Figure 3], II [Figure 4] and III [Figure 5].

Thus, the improved remineralisation potential and reduced cytotoxicity make this novel SrnHAp paste a suitable remineralising agent for incipient caries and white-spot lesions. However, further evaluation with different concentrations of SrnHAp and assessment of other characteristics is required before the application of the paste clinically.

Within the limitations of this study, SrnHAp paste showed better remineralisation potential and favourable surface changes in enamel compared to calcium sodium phosphosilicate and casein phosphopeptide-amorphous calcium phosphate. Casein phosphopeptide-amorphous calcium phosphate showed better remineralising potential than calcium sodium phosphosilicate (Novamin). Thus, the novel SrnHAp paste with improved cell viability and reduced cytotoxicity can be considered for enamel repair and remineralisation in incipient caries and white-spot lesions.

Certain factors within the oral cavity such as salivary pH, intake of acidic beverages and foodstuffs were not considered though they might have an effect on remineralisation. The duration of the application of paste in the present study was limited to 3 min. But studies have shown proportionality between application time and remineralisation potential of the tooth.[17]


   Conclusion Top


Within the limitations of the present study, it can be concluded that Novamin, GC tooth mousse as well as SrnHAP showed a significant remineralisation potential compared to regular dentifrice. SEMEDAX evaluation revealed favourable surface changes in enamel with Novamin, GC tooth mousse as well as SrnHAP. The novel laboratory synthesised strontium-doped nanohydroxyapatite (SrnHAP) showed better remineralisation than Novamin and GC tooth mousse and can be considered for enamel repair in incipient carious lesions. Further evaluation of the remineralisation potential of SrnHAP will provide a basis for the support of the outcome obtained in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Rajendran R, Kunjusankaran RN, Sandhya R, Anilkumar A, Santhosh R, Patil SR. Comparative evaluation of remineralizing potential of a paste containing bioactive glass and a topical cream containing casein phosphopeptide-amorphous calcium phosphate: An in vitro study. Pesqui Bras Odontopediatria Clin Integr 2019;19:e4668.  Back to cited text no. 5
    
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Hench LL, Andersson O. Bioactive glasses. In: Hench LL, Wilson J, editors. An Introduction to Bioceramics. Singapore: World Scientific; 1993. p. 41-62.  Back to cited text no. 6
    
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Andersson OH, Knagasniemi I. Calcium phosphate formation at the surface of bioactive glass in vitro. J Biomed Mater Res 1991;25:1019-30.  Back to cited text no. 7
    
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Li L, Pan H, Tao J, Xu X, Mao C, Gu X, et al. Repair of enamel by using hydroxyapatite nanoparticles as the building blocks. J Mater Chem 2008;18:4079-84.  Back to cited text no. 8
    
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Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Spec Care Dentist 1998;18:8-16.  Back to cited text no. 9
    
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Nan K, Wu T, Chen J, Jiang S, Huang Y, Pei G. Strontium doped hydroxyapatite film formed by micro-arc oxidation. Mater Sci Eng C 2009;29:1554-8.  Back to cited text no. 10
    
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O'Donnell MD, Fredholm Y, de Rouffignac A, Hill RG. Structural analysis of a series of strontium-substituted apatites. Acta Biomater 2008;4:1455-64.  Back to cited text no. 11
    
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Thuy TT, Nakagaki H, Kato K, Hung PA, Inukai J, Tsuboi S, et al. Effect of strontium in combination with fluoride on enamel remineralization in vitro. Arch Oral Biol 2008;53:1017-22.  Back to cited text no. 12
    
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Featherstone JD. The science and practice of caries prevention. J Am Dent Assoc 2000;131:887-99.  Back to cited text no. 13
    
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Darshan HE, Shashikiran ND. The effect of McInnes solution on enamel and the effect of tooth mousse on bleached enamel: An in vitro study. J Conserv Dent 2008;11:86-91.  Back to cited text no. 14
[PUBMED]  [Full text]  
15.
Oliveira GM, Ritter AV, Heymann HO, Swift E Jr., Donovan T, Brock G, et al. Remineralization effect of CPP-ACP and fluoride for white spot lesions in vitro. J Dent 2014;42:1592-602.  Back to cited text no. 15
    
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Featherstone JD. The continuum of dental caries--evidence for a dynamic disease process. J Dent Res 2004;83(Spec No C):C39-42.  Back to cited text no. 16
    
17.
Kim MY, Kwon HK, Choi CH, Kim BI. Combined effects of nano-hydroxyapatite and NaF on remineralization of early caries lesion. Key Eng Mater 2007;330:1347-50.  Back to cited text no. 17
    
18.
Hora BS, Kumar A, Bansal R, Bansal M, Khosla T, Garg A. Influence of Mcinnes bleaching agent on hardness of enamel and the effect of remineralizing gel GC tooth mousse on bleached enamel-An in vitro study. Ind J Dent Res 2012;4:4:13-6.  Back to cited text no. 18
    
19.
Krishnan V, Bhatia A, Varma H. Development, characterization and comparison of two strontium doped nano hydroxyapatite molecules for enamel repair/regeneration: An in vitro study. J Dent Mater 2016;646-59.  Back to cited text no. 19
    

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Correspondence Address:
Dr. Ratheesh Rajendran
Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Science, Saveetha University, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_45_22

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