| Abstract|| |
Background: Erosion, a dynamic process with periods of demineralisation and remineralisation, has become a common problem in modern societies, owing to changes in life style and dietary habits. Although fluorides have been included in toothpastes that claim to prevent demineralisation and aid remineralisation, their ability to remineralise is limited by low concentration of calcium and phosphate ions available in saliva. Hence, a new paste based on casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF), nanohydroxyapatite and bioactive glass (BAG) were introduced. Aim: To evaluate and compare the effects of BAG, nanohydroxyapatite and CPP-ACPF pastes on surface microhardness of demineralised enamel. Materials and Methods: 48 enamel specimens were randomly divided into five groups: Group I positive control - intact specimens and Group II - demineralised specimens. The test groups, Group III, IV and V, comprised CPP-ACPF, nanohydroxyapatite and BAG, respectively. The test specimens were demineralised with 0.1% citric acid followed by remineralisation using either of the three prepared slurries. The specimens were subjected to pH cycling regime for 15 times. The remineralisation potential of the specimens was studied by evaluating the surface microhardness. One specimen from each group was analysed under SEM. Data was tabulated and analysis performed by one way ANOVA and post hoc Scheffe test. Results: Statistically significant difference was found between the negative control and three test groups based on microhardness evaluation. Nanohydroxyapatite had the least remineralising potential as compared to CPP-ACPF and BAG. Conclusion: Comparatively, BAG and CCP-ACPF paste showed better remineralising potential.
Keywords: Enamel remineralisation, pH cycling, SEM analysis, surface micro hardness
|How to cite this article:|
Suryani H, Gehlot PM, Manjunath MK. Evaluation of the remineralisation potential of bioactive glass, nanohydroxyapatite and casein phosphopeptide-amorphous calcium phosphate fluoride-based toothpastes on enamel erosion lesion –An Ex Vivo study. Indian J Dent Res 2020;31:670-7
|How to cite this URL:|
Suryani H, Gehlot PM, Manjunath MK. Evaluation of the remineralisation potential of bioactive glass, nanohydroxyapatite and casein phosphopeptide-amorphous calcium phosphate fluoride-based toothpastes on enamel erosion lesion –An Ex Vivo study. Indian J Dent Res [serial online] 2020 [cited 2021 Feb 26];31:670-7. Available from: https://www.ijdr.in/text.asp?2020/31/5/670/306459
| Introduction|| |
Dental erosion develops from the chronic exposure to non-bacterial acids resulting in bulk mineral loss with a partly demineralised surface of reduced microhardness.
In the incipient phase, the enamel is dissolved without clinically detectable softening and dentin being affected only at a later point of time. According to Barbour et al., dental erosion is a common problem in modern societies, owing to the increased consumption of acid drinks such as soft drinks, sport drinks, fruit juices, which in turn have a high potential to provoke dental demineralisation. The development of erosion involves a chemical process in which the inorganic phase of the tooth is demineralised, thereby reducing the hardness of the tooth substrates. Thus, preventive measures against erosion are required.
Enamel has no spontaneous capability to repair when affected by specific dental pathologies such as caries, abrasions or fractures because it contains no cells. Therefore, when enamel is exposed to oral environment, the only possibility to be reconstructed depends on the application of alloplastic materials, which provide a sort of prosthetic restoration.
With improved oral hygiene and fluoridated pastes being commonly used, there has been a decline in incidence of tooth demineralisation, resulting in increased fluoride content in saliva. Various fluoridated formulations have been tested but the major shortcoming of these is that their ability to remineralise enamel is limited by the low concentration of calcium and phosphate ions available in saliva. Hence, current conventional fluoride–containing toothpastes do not appear to protect efficiently against tooth erosion.
The casein-phosphopeptide-stabilised amorphous calcium phosphate complex dentifrices (CPP-ACP) significantly prevented and reversed demineralisation in various in vitro and in vivo studies. Newer materials like hydroxyapatite nanoparticles are synthesised and tailored biomimetically and are very similar to hydroxyapatite in morphology. Even after a single treatment with the nanohydroxyapatite dentifrice calcium concentration were elevated in the remineralising solution. Bioactive glass, a breakthrough in remineralisation technology, is now being used in various aspects of dentistry. When in an aqueous environment, BAG undergoes a solution-mediated dissolution resulting in change in composition solution which in turn changes the pH and releases bioavailable calcium, sodium and phosphate ions contributing to the remineralisation process.
As per the author's knowledge, the remineralisation potential of BAG in a fluoridated tooth paste has not been evaluated so far. The current study has been undertaken to evaluate and compare the remineralisation potentials of BAG-containing fluoridated tooth paste (Regenerate™ Enamel Science- Unilever Dept ER Wirral, UK) with CPP-ACPF tooth cream (GC Tooth Mousse Plus - GC Corporation. Tokyo. Japan) and nanohydroxyapatite-containing fluoride toothpaste (SHY XT- Group Pharmaceuticals Limited, Bangalore, India) using surface microhardness.
| Materials and Methods|| |
The study was approved by the Institutional ethical committee for research on human subjects or specimens (JSS/DCH/IEC/MD-14/2015-16). This ex vivo study design consisted of a cyclic demineralisation–remineralisation model in which human enamel specimens were exposed to an erosive challenge, remineralisation toothpaste treatment and storage in artificial saliva. Twenty four human premolar teeth freshly extracted due to orthodontic reason were collected, cleaned of debris and calculus, and were disinfected in 5% sodium hypochlorite solution (Nice Chemicals, Kochi, India) for 1 hour and stored in 1% Thymol prior to specimen preparation. Teeth with caries, cracks or defects, fluorosis, fractures, restorations or root canal treatment were excluded.
Each tooth was decoronated at the level of cemento–enamel junction (CEJ) and the crown was sectioned mesiodistally into two halves using a diamond disc (NTI Diamond Disc. Kavo Kerr, USA) under constant water coolant, resulting in total 48 specimens. The specimens were then evaluated under a dental operating microscope (OPMI Pico, Carl Zeiss and Oberkochen, Germany) at ×21 magnification for any enamel defects, carious lesions or cracks. The buccal and lingual halves of the teeth specimens were then embedded 2 mm into autopolymerising polymethyl methacrylate acrylic resin (Dental Products of India, Mumbai, India) using moulds (4 mm × 4 mm). The buccal/lingual surfaces of the enamel were ground with 400, 600 and 1200-grit silicone carbide abrasive paper (Wol cut abrasives, Delhi, India) under water irrigation to obtain flat surfaces. The specimens were polished with felt paper (3M, USA) moistened with 0.5 μm diamond polishing paste (Ultradent Products, South Jordan, USA) and stored in distilled water until use.
Preparation of remineralising paste slurry
The slurry was prepared by suspending 12 g of the respective remineralising tooth paste [Table 1] in 36 ml bi-distilled water (by weight) to create 1:3 w/v dilutions. The solutions were thoroughly stirred using a magnetic stirrer (Remi Elektrotechnik Ltd. Thane, India) for 1 min at room temperature before starting the pH-cycling. The pH values of all solutions were monitored with pH-sensitive electrode (Mettler-Toledo India Pvt Ltd. Mumbai, India).
The specimens were submitted to a cyclic demineralisation–remineralisation regimen 15 times which mimics 7 days. One complete cycle comprised of following steps:
- Demineralisation by immersion in 5 ml of 0.1% w/w citric acid (pH 3.8) for 2 minutes under constant movement using a platform rocker (Thermo Fisher Scientific India Ltd. Bangalore, India).
- 1-minute rinse in distilled water.
- For remineralisation, the specimens were immersed for 3 minutes in 5 ml of respective remineralising slurry under constant movement using a platform rocker (Thermo Fisher Scientific India Ltd. Bangalore, India). The specimens were incubated (REMI Lab Instruments, Mumbai, India) in artificial saliva for 2 hours at 37°C after every remineralising cycle and before every demineralising cycle.
During the pH cycling for every specimen (Groups II, III, IV and V), fresh solutions were used between the cycles. The pH of citric acid, remineralising paste and artificial saliva were recorded using Bench-top digital pH meter (Mettler-Toledo India Ltd., Mumbai, India) and the values were recorded. Following the completion of all cycles, the samples were stored in distilled water until microhardness and surface evaluation.
Additional specimens were prepared as described earlier and assigned to corresponding group to be used for SEM analysis.
Surface microhardness assessment (VHN)
Vickers hardness number (VHN) was determined by making three indentations in different regions (100 μm apart) of each specimen using a square based Vickers diamond pyramid indenter (HWMMP-XT.Highwood Hardness Tester) under a load of 100 g applied to the enamel surface for 10 seconds. The average of three indentations were collected and tabulated.
Scanning Electron Microscopy (SEM)
One specimen from each group was used for SEM examination (EVO® LS15, Carl Zeiss Microscopy GmbH, Goettingen, Germany). The central beam of the SEM was directed to the surface of each enamel specimen under x10 magnification in order to observe the whole sample surface. Then, 10 areas of each specimen projected onto the screen were randomly selected and magnified × 1000 to × 5000 times.
The data obtained for microhardness was analysed using mean and standard deviation for descriptive statistics. ANOVA (Analysis of Variance) was used for analysing differences between groups followed by Scheffe's post hoc test. The significance was set at the 5% probability level. The analysis was performed with Statistical Package for the Social Sciences (SPSS 22.0, SPSS Inc, Chicago, IL, USA).
| Results|| |
The surface microhardness (Vickers Hardness Number–Kgf/mm2) analysis of all the groups is shown descriptively in [Table 2].
|Table 2: Mean, SD and SE of surface microhardness results of human enamel specimens according to the different groups|
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One-way ANOVA and Scheffe's post hoc analysis showed a statistically significant difference in mean of enamel microhardness between the groups (P < 0.001). Enamel specimens of Group IV (SHY NM) were not significantly different (P = 0.592) to Group II but were statistically different to Groups I (positive control), III (SHY XT) and V (Regenerate Enamel) (P < 0.05). The Groups I, III and V were not statistically different ([Table 1]; P > 0.05).
Scanning electron microscopy (SEM) analysis
In Group A (Positive control; artificial saliva), the sound enamel had homogeneous smooth appearance [[Figure 1]a; 1500×] with the enamel rods barely appreciable. In [Figure 1]b, at a higher magnification of 5000×, the typical key hole pattern of enamel prisms are appreciable. Faint lines of mineralisation can be seen around the interprismatic structures. In Group II, after demineralisation with citric acid, the surface appeared more irregular with voids and numerous micropores [Figure 1]c and [Figure 1]d. In Group III, the surface revealed homogeneous coating with the rods and prismatic substance not discernible [Figure 1]e and [Figure 1]f. In Group IV, at 2500 × magnification, the surfaces of the specimen treated with nanohydroxyapatite showed irregular etched pattern of enamel with the porous interprismatic and prismatic enamel structures appreciable similar to the Group II and at 5000 × the pits and irregular enamel margin were seen along with sparsely distributed apatite crystals [Figure 1]g and [Figure 1]h and in Group V, enamel specimens treated with bioglass showed near-complete coverage of the enamel surface with dense large crystalline structures [Figure 1]i and [Figure 1]j.
|Figure 1: SEM of enamel specimens in (a and b) intact enamel at 1500 × and 5000×. Arrow shows mineralisation of the interprismatic substance; (c and d) Demineralised specimens in Group II at 1500 × and 2500×. Arrow shows the demineralised enamel (Etched appearance) with fish scale appearance; (e and f) Group III at 2500 ×, with homogeneous coating and 5000 × shows the presence of mineralised deposits over the etched enamel surface (Arrow); (g and h) Group IV at 1500 ×, with etched appearance of enamel and 5000 × shows the sparse presence of mineralised deposits and presence of numerous pits over the enamel surface (Arrow); (i and j) Group V at 2000 ×, presence of mineral deposits with few pits and 5000 × shows the dense presence of mineralised deposits over the enamel (Arrow)|
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| Discussion|| |
Over the past few years, the prevalence of erosion has increased predominantly among young adults due to their change in life style and occupation. The aetiology for erosion can be intrinsic or extrinsic in origin with diet being a major causative factor. Dietary acids diffuse into the tooth surface resulting in dissolution of superficial enamel only, which is usually considered reversible. However, initial erosive lesions tend to go undiagnosed clinically until there is extensive loss of dental tissue. In a clinical setting, progression of early erosive lesions is prevented by the remineralising property of agents introduced into the oral cavity like dentifrices, varnishes, gels, foams personal or professional saliva, tooth paste, mouthrinses, varnishes, gels, sugar substitutes, foams, floss, fluoride releasing restorative materials and xylitol containing chewing gums. In an attempt to enhance enamel resistance to erosive attacks, tooth pastes were found to be the most effective, and accessible agents among all of them.
While fluoridated toothpastes strengthen teeth against erosive acid damage, frequent applications are considered potentially effective approaches in preventing dental erosion. The mechanism of action of fluoride against erosion is by inducing the formation of a layer composed of CaF2 or metal-rich precipitates which act like a physical barrier, preventing the contact of acid with the underlying enamel, or as a mineral reservoir that is attacked by the erosive challenge, thus buffering the acids or promoting mineral precipitation. However, studies have shown a limited benefit of conventional 1100 ppm F dentifrices (NaF) compared with non-fluoridated dentifrices. According to Larsen and Richards (2002), fluoride was unlikely to provide a preventive effect against erosion because an acidic drink will rapidly dissolve accessible calcium fluoride and remove the remaining traces of a previous topical fluoride treatment. Hence, it seems important to develop new toothpastes with good protection against erosion irrespective of the presence of fluoride.
In the present ex vivo study, three commercially available fluoridated toothpaste formulations with remineralising agents were evaluated [Table 1]. Human teeth were selected because of ease of availability and the study would closely mimic the clinical situation. Many studies have used bovine teeth. The advantages of bovine specimens include similar mineral distribution as human teeth, up to 4–5 specimens gained out of a single bovine incisor and allocation of samples from the same tooth to different experimental groups. The drawback, however, includes the artificial lesions formed in bovine tooth enamel after cyclic erosion/abrasion were twice as deep as those formed in human teeth.
The enamel surface was ground with 400, 600 and 1200-Grit silicone carbide abrasive paper to obtain flat surfaces and remove the aprismatic enamel layer which is less permeable than the underlying enamel.
Methods to study enamel demineralisation and remineralisation have relied on semiquantitative or qualitative techniques including hardness measurement, microradiographic measurements or electron-microscopic observation. All methods rely on the fact that the demineralised enamel is physically changed or in simplistic terms 'weakened' or 'softened'. The reduction in hardness or abrasion resistance of demineralised enamel clearly indicates an increased susceptibility to physical insult. The present study is quantitative analysis since the surface hardness was evaluated.
The remineralisation potential was evaluated by measuring the surface microhardness of enamel. The methodology followed in the current study is similar to a study by Hegde et al. In order to study remineralising potential, pH cycling regime similar to that of Hornby et al., was followed which simulated an initial acid attack and the changes occurring in the oral cavity.,
The present study used citric acid for demineralising the specimens, since it is commonly found in packed foods and soft drinks/fruit juices. It has been observed to cause far more erosion over the pH range employed than phosphoric acid for enamel and dentine in an in vitro study. Hence, specimens were subjected to 2 minutes of demineralisation with 0.1% w/w citric acid and titrable acidity of 4.2.
In literature, the duration of erosion varied between 15 seconds and 40 min (in vitro) and 40 seconds and 20 min (in situ) per cycle, while mostly an immersion time between 1 and 5 min/cycle. The time span the pH remains low in the oral cavity is not longer than 2 minutes usually. So, to better simulate oral conditions, the pH cycle included a 2-minutes erosive acid challenge.
The 3-minute immersion of specimens in remineralising slurry was done to have a dose dependent effect and the solutions were constantly kept in motion using a gel rocker to simulate swishing of the solution in the mouth and also the tooth surface to solution contact observed in the oral cavity.
After every demineralisation and remineralisation cycle, the specimens were incubated in artificial Saliva, at 37°C for 2 hours, in order to mimic the oral environment and maintain the in vitro specimens at a temperature similar to that seen in human oral cavity. Studies have shown that acid-softened enamel can reharden after exposure to saliva. During an erosive challenge, saliva played a protective role undergoing dilution and clearance, neutralisation and buffering of acids, remineralising of enamel and formation of the acquired pellicle. However, few studies did not find a significant rehardening effect of saliva.
The initial stages of demineralisation and remineralisation in enamel results in change in mineral density which can be monitored by the variation in surface microhardness. Surface microhardness test is considered the most long-established method for evaluation of remineralisation potential. Changes in surface hardness of enamel can be observed even few minutes after exposure to an erosive agent; hence, it is suitable to test initial erosive lesions and remineralisation effect. Another advantage is that the indentations placed on enamel in surface hardness test are not vulnerable to time-dependent changes in their morphology.,
In the present study, the (Group I) intact enamel specimens resulted in mean VHN value of 284.42 kg/mm2 and those in (Group II) demineralised with citric acid, resulted in VHN value of 263.6 kg/mm2. This was similar to the range of 260 to 279 kg/mm2 that was seen in a study by Meredith et al. but lower than the value reported by Maupome et al. The difference in value could be due to the difference in methodology and the force used for VHN and the duration of indentation. As discussed previously, the process of enamel demineralisation depends on the pH and calcium, phosphate, and fluoride contents of teeth. These factors determine the level of mineral saturation. Thus, in condition where the environment is subsaturated, it will result in the dissolution of hydroxyapatite (HA) crystals in the tooth with diffusion of calcium and phosphate ions toward the enamel surface, resulting in decrease in VHN values that are indicative of demineralisation.
As per the pH cycling, remineralisation for 3 minutes in the present study showed an increase in the surface microhardness of all the enamel specimens that were treated with remineralising formulations and statistical significant differences were observed between them. However, hypersaturation of ions on the enamel surface can lead to redeposition of HA crystals, known as remineralisation, and consequently the formation of an intact superficial layer on the enamel surface. An increased VHN indicates remineralisation.
In the current study, the results showed that the VHN values were higher in the teeth treated with Regenerate Enamel (BAG) paste than in the teeth treated with SHY XT paste (nanohydroxyapatite), and there was statistically significant difference between them. The presence of fluoride in Regenerate Enamel paste may be the reason for this difference. Hence, on comparison of Regenerate Enamel paste with GC Tooth Mousse Plus, no significant difference was found between them due to presence of fluoride.
However, comparing the pH of toothpaste slurries, SHY-XT was neutral (pH 7.2) compared to other test toothpastes and would be expected to provide more fluoride release. Brighenti et al. found a small decrease in the percentage of surface microhardness in pH cycling models using acidified toothpastes compared to neutral ones. In our study, the overall performance of the slightly neutral paste (SHY-XT) showed a reduction in VHN when compared to the more basic toothpastes, GC tooth mousse plus and Regenerate Enamel, which showed better rehardening, which was statistically significant. This was in contrast with a study done by Lussi and Megert et al., in which the authors found the role of pH not to be very significant for test products evaluated.
The reasons for reduced VHN values of nanohydroxyapatite are varied. Huang et al. found that an optimal concentration (10%) of nanohydroxyapatite resulted in remineralisation of initial enamel lesions. In the present study the SHY–XT paste contains 1% nanohydroxyapapite, which may be less to obtain the remineralisation of enamel specimens. Moreover, some studies have suggested that the presence of desensitising agents like KNO3 could reduce the bioavailability of fluoride resulting in reduced remineralisation; hence the less VHN values in the present study.
The reason for enhanced VHN for Regenerate Enamel paste could be due to various reasons: paste is based on Bio-active glass (BAG) and is a ceramic material consisting of amorphous sodium-calcium-phosphosilicate. In the presence of saliva in the oral cavity, sodium ions from the BAG particles rapidly exchange with hydrogen cations (in the form of H3O+) and release calcium and phosphate (PO4−) ions from the glass. A localised, transient increase in pH occurs during the initial exposure of the material to water due to the release of sodium. This increase in pH helps to precipitate the extra calcium and phosphate ions provided by the BAG to form a calcium phosphate layer. As these reactions continue, this layer crystallises into hydroxycarbonate apatite (HCA)., Thus, the formulation results in regeneration of structure similar to hydroxyapatite. The VHN values of Regenerate Enamel were similar to intact human enamel which supports the manufacturers' claim to regenerate enamel.
The present study also revealed that CPP-ACP remineralised human enamel in vitro lesion. In a study by Srinivasan et al., CPP-ACP and CPP-ACPF remineralising pastes resulted in a significant increase in post-erosion microhardness and it has been proposed that the remineralisation mechanism of CPP–ACP involves localisation of ACP at the tooth surface, which buffers free calcium and phosphate ions. By maintaining a state of supersaturation with respect to the hydroxyapatite, these ions depress demineralisation and promote remineralisation.
According to Jayarajan et al., after application of CPP-ACPF, the enamel rods and prismatic substance are not discernable but the areas of calcified deposits are more evident and are seen concentrated along the porous defects.
A study by Neto et al. found that there was no difference in the surface microhardness of enamel following treatment with CPP-ACP and CPP-ACP + F pastes during cariogenic challenges. According to them, the reason may be the presence of the fluoride ion in CPP-ACP + F that could interact with the ACP component of the casein complex, rendering both inorganic components ineffective and a lower level of fluoride available when compared to the other fluoride systems.
Thus, the presence of dental plaque seems to be crucial for the synergistic effect of CPP-ACP with fluoride. This enhancement of remineralisation by CPP-ACPF products can be attributed to the level of bioavailable calcium and phosphate ions released by these products. In the oral environment, the CPP-ACPF product increased the concentration of calcium, phosphate and fluoride ions in saliva which prevents spontaneous precipitation and allows penetration of the ions deep into the subsurface lesions.
Scanning electron microscope (SEM) is a time-tested technique to quantitatively assess demineralisation and remineralisation in in vitro studies., In various studies, the specimens have been coated with metals such as gold or palladium to improve the image quality of SEM analysis. However, in specimens that use SEM without metal sputtering, the samples could be reobserved again. This version was considered in our study as there would not be loss of samples and re-evaluation of the same samples after treatment regimen would be possible. Thus, five specimens were evaluated for structural analysis of remineralised enamel using SEM. The SEM analysis of Group I (Intact enamel) revealed a homogeneous smooth appearance and at a higher magnification of 5000×, the typical key hole pattern of enamel prisms are appreciable. Faint lines of mineralisation can be seen in and around the interprismatic structures. In Group II, (Demineralisation only) after demineralisation with citric acid, the surface appeared more irregular with voids and numerous micropores [Figure 1]c and [Figure 1]d. The irregular etched pattern of enamel with the porous interprismatic and prismatic enamel (Fish scale) structures was appreciable. In Group III (Remineralisation with CPP-ACPF/GC Tooth mousse plus), the enamel surface revealed homogeneous coating [Figure 1]e and at higher magnification of 5000X [Figure 1]f, areas of mineralised deposits are appreciable and scattered over the etched surface. In Group IV (Remineralisation with Nanohydroxyapatite/SHY XT paste), at 2500X magnification, the surfaces of the specimen treated with nanohydroxyapatite showed irregular etched pattern of enamel with the porous interprismatic and prismatic enamel structures appreciable similar to Group II [Figure 1]g. At 5000X, the pits and irregular enamel margins were seen along with sparsely distributed apatite crystals [Figure 1]h. In Group V (Remineralisation with BAG/Regenerate Enamel science), specimens treated with bioglass showed near-complete coverage of enamel surface with densely packed crystalline structures [Figure 1]i and [Figure 1]j.
The major limitation of the present study is that it is an ex vivo study. In addition, the study evaluated the remineralisation potential by using slurry and without abrasion using a tooth brush. Hence, the results obtained cannot be directly correlated to the clinical situations.
| Conclusion|| |
Within the limitations of this ex vivo study, Bioactive glass - based paste, Regenerate enamel science had better potential to remineralise enamel and would be beneficial in the management of enamel erosion. The nanohydroxyapatite-containing toothpaste could not significantly inhibit demineralisation. The CPP-ACPF–based toothpaste was as good as Bioglass-based toothpaste. Further, the results of the present in vitro study may be different to in vivo conditions in the oral cavity with dynamic biological systems.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Paras Mull Gehlot
Department of Conservative Dentistry and Endodontics, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, JSS Medical Institutions Campus, Sri Shivarathreeshwara Nagar, Mysuru - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]