| Abstract|| |
Context: Remineralization is defined as the process whereby calcium and phosphate ions are supplied from a source external to tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. The remineralization produced by saliva is less and also a slow process, therefore remineralizing agents are required.
Aims: The study was planned to evaluate the effectiveness of homeopathic Calcarea Fluorica (calc-f) tablets as remineralizing agents on artificial carious lesions using scanning electron microscope (SEM) and surface microhardness (SMH) testing.
Subjects and Methods: A total of 24 patients needing removable orthodontic treatment were included in the study. They were divided into two groups of 12 patients each. The Group I consisted of patients in whom no tablets were given while Group II consisted of patients in whom calc-f tablets were given in a dosage of 4 tablets twice a day. Four enamel samples with the artificial carious lesions were then embedded in the removable appliance for a period of 6 weeks. After 6 weeks, the enamel samples were retrieved and evaluated by SEM and SMH.
Statistical Analysis Used: One-sample Kolmogorov-Smirnov test and Student's t-test were applied to analyze the difference in the Vickers microhardness number (VHN) values of remineralized enamel obtained from control and experimental group.
Results: The signs of remineralization such as reduction in depth prismatic holes or decrease in porosity, variable sized uneven distribution of deposits and amorphous deposits were seen in enamel samples of both the groups. The mean SMH of remineralized enamel sample of Group I and Group II were 270.48 and 302.06, respectively, and the difference was statistically significant.
Conclusions: (1) Remineralization occurred in both the groups as indicated by SEM and the increase in surface hardness values in both the groups. (2) Remineralization of enamel samples in the control group as indicated by SEM and also by increase in VHN values indicated that the saliva has a tendency of remineralizing the early carious lesions. Conclusions drawn from the study are that the calc-f tablets can be used as safe and cost effective remineralizing agent.
Keywords: Artificial carious lesions, Calcarea Fluorica tablets, remineralization, scanning electron microscope and surface microhardness testing
|How to cite this article:|
Bansal K, Balhara N, Marwaha M. Remineralizing efficacy of Calcarea Fluorica tablets on the artificial carious enamel lesions using scanning electron microscope and surface microhardness testing: In vivostudy. Indian J Dent Res 2014;25:777-82
Caries is a biofilm (plaque)-induced acid demineralization of enamel or dentin, mediated by saliva. Early childhood caries, is the presence of 1 or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a child 71 months of age or younger.  It is the most common oral disease of children. It can rapidly develop and causes several health problems in children.  White spot lesion, the earliest clinical sign of dental caries, is generally characterized by enamel demineralization of the subsurface, with increasing porosity due to removal of minerals into the outer surface.  The acid-producing bacteria in the biofilm metabolize dietary carbohydrates to produce weak acids which cause the local pH to fall below a critical value. This fall in pH causes the mineral component of the tooth (hydroxyapatite) to dissolve releasing calcium and phosphate ions. This process is known as demineralization. When the pH rises, a certain amount of mineral can be re-precipitated by diffusion of calcium and phosphate ions from the saliva back into the porous surface created by mineral loss. This is known as remineralization. 
|How to cite this URL:|
Bansal K, Balhara N, Marwaha M. Remineralizing efficacy of Calcarea Fluorica tablets on the artificial carious enamel lesions using scanning electron microscope and surface microhardness testing: In vivostudy. Indian J Dent Res [serial online] 2014 [cited 2019 Jun 24];25:777-82. Available from: http://www.ijdr.in/text.asp?2014/25/6/777/152204
The prevention of dental caries is uniquely associated with fluorides. They act to impede demineralization and to enhance the remineralization of enamel, both of which prevent caries.
Fluoride has a profound effect on the level of caries prevalence, but it is far from a complete cure.  However, its real mechanism of action and its indiscriminate use is questionable.  Furthermore, fluoride can cause fluorosis through overexposure, especially in young children. The analysis of these issues, especially fluorosis, shows that new therapeutic approaches must be tested. Therefore, an appropriate nonfluoride anticaries agent is the demand of time. 
Other new technology for remineralization or alternatives to fluoride have been developed and investigated over period of time such as sugar substitute xylitol, casein phosphopeptides with amorphous calcium phosphate (CPP-ACP), calcium sodium phosphosilicate, nano-hydroxyapatite,  ozone,  grape seed extract,  galla chinensis  available in various ways like dentifrices,  chewing gums,  mouth rinses,  tablets,  lozenges,  restorative material,  pit and fissure sealants,  and dental floss. 
Various homeopathic medicines have been also used systemically such as Calcarea Phosphorica (calc-p) or phosphate of lime, Calcarea Fluorica (calc-f) or fluoride of lime (CaF 2 ) for the treatment of various dental problems as they contain mineral salts that they play role in the mineralization of teeth and bone.  Various medical uses of calc-f are chronic suppuration of middle ear, vomiting, diarrhea, hemorrhoids, hydrocele, varicose veins, etc.  Till date, only one study has been conducted on the animal model (rats) investigating various benefits of homeopathic medicines on caries. Hence, the present study was planned with the following aims and objectives:
- To evaluate the remineralizing efficacy of homeopathic medicine calc-f on artificial carious lesions on enamel specimens using a scanning electron microscope (SEM)
- To evaluate the strength of the in vivo remineralized enamel samples using surface microhardness test (SMH).
| Subjects and methods|| |
The study was approved by the institutional ethical committee. Subjects, requiring removable orthodontic appliance treatment, were explained about the purpose of the study. Only the subjects who agreed to comply with the instructions to be followed during the study were selected. Informed consent was taken from the parents of all the subjects who participated in the study.
Twenty-four subjects (13 girls and 11 males, aged ranged 10-16 years) requiring removable orthodontic treatment were included in the study. Inclusion criteria for the subjects were (i) 22 natural teeth present with no current caries activity, periodontal disease or other oral pathology. (ii) None of the subjects would be using antibiotics or medication and (iii) the patients who were willing to comply with the instructions regarding usage of test regime. The subjects were then randomly divided into two groups of 12 patient each; Group I (Not receiving any tablets) and Group II (the experimental group receiving the tablets of calc-f [×6], Dr. Reckeweg German).
Freshly extracted caries-free/sound premolars (extracted for orthodontic purpose), and third molars were collected and cleaned. Any soft tissue attachments were removed. Then the extracted teeth were sterilized with 10% formalin for 1 week and washed three times with distilled water and stored in 70% (v/v) ethanol till further use. From each extracted tooth, four enamel samples of approximately 3 mm × 2 mm × 2 mm dimensions were prepared by sectioning with the help of carborundum discs attached to micromotor straight hand piece. The artificial caries were produced in enamel samples using demineralizing solution containing: 3 mM calcium, 1.8 mM phosphate, 0.1 mM lactic acid, and 1% carboxymethyl cellulose sodium salt, 0.1M HCl was added to adjust the pH of the solution to 4.0 using electronic pH meter.  The prepared enamel samples were then placed in the 30 ml of demineralizing solution for a 16 h time period for the production of artificial caries. The specimens were tested for artificial caries under SEM at magnifications ×1000, ×3000, ×5000.
Incorporation of demineralized enamel samples into intraoral appliances
Four demineralized enamel samples were placed in each of the intra-oral appliances during acrylization of the orthodontic appliance. The two samples were placed in the canine region, and other two were placed in molar region. Care was taken while acrylization that no cold cure acrylic was present on the samples [Figure 1].
|Figure 1: Removable intraoral appliance with artificial carious enamel samples|
Click here to view
The subjects of the group I were instructed to follow the routine oral hygiene regime. Subjects in this group were not provided with any tablets. While the subjects of the Group II were given calc-f (×6) by the investigator. The frequency of administration prescribed to subjects was four tablets 2 times a day for home use for 6 weeks period. Patients were advised not to chew the tablets and keep the tablets in the mouth until it dissolves on its own. Patients were instructed to take the tablets half an hour before any meal and not to drink water or rinse or eat for half an hour after the taking the tablets. Subjects of both the group were advised to brush their teeth 2 times a day with nonfluoridated toothpaste (Meswak, Dabur) during the study. The enamel samples were subjected to in vivo remineralization for a period of 6 weeks during which the subjects of both the groups were followed up on a weekly basis. During each follow-up visit, the embedded enamel samples were checked for their retention. Subjects were also reinforced to follow the instructions as per the group allocation. In the experiment group, the subjects were provided with calc-f tablets for home use. After 6 weeks of in vivo experimental period, the enamel samples were retrieved from the acrylic plates and stored in distilled water till they were evaluated.
For scanning electron microscope evaluation
For the purpose of SEM examination, the samples retrieved from the right side of the intraoral appliance were air-dried and sputter coated with gold (Polaron SC 7640, UK) and were observed under SEM (Carl Zeiss EVO 40 at 20 kV, Germany). A representative image of all the samples was obtained at ×1000, ×3000 and ×5000 magnification. The SEM images were also obtained for the corresponding demineralized samples which were subjected to demineralization medium only and not subjected to the in-vivo treatment, to compare with the corresponding remineralized samples.
For surface microhardness
The enamel samples on the left side of appliances were subject to SMH test. Custom made cylindrical molds were prepared using self-cured acrylic resin, and then each enamel block was embedded in, on top of partially set, and allowed to set in order to have a stable base for testing. Vickers microhardness tester (VH1000B, BCS/E/04) was used to evaluate microhardness. The microhardness values were obtained of the sound enamel that is, baseline values, after demineralization and after the experimental period of 6 weeks of the samples from the left side of the intraoral appliance. A load of 100 g was applied, for fifteen seconds, for all the specimens. The Vickers microhardness number (VHN) of three indentations was taken, and the average value was considered the mean micro hardness (SMH) of the corresponding specimen.
In order to determine the difference in VHN values of control, experimental group, one-sample Kolmogorov-Smirnov test and student t-test was applied.
| Results|| |
0Scanning electron microscope findings
The SEM images at ×1000 showed homogenously arranged enamel rods and surface appeared smooth. Overlapping of incremental layers of perikymata [Figure 2]a]. At ×3000, the sample showed prism pits in the form of depressions [Figure 2]b]. At ×5000, Tome's process pits were seen as shallow depressions [Figure 2]c].
|Figure 2: Scanning electron microscope pictures of sound enamel (a-c) and artificial caries (d-f) at ×1000, ×3000, ×5000|
Click here to view
Artificial carious enamel lesions
The surface of enamel specimens was intact with increased porosity at ×1000. Deepened Tome's processes due to dissolution of the process pits were observed. Irregular pattern of surface destruction was located in the vicinity of prismatic pattern of destruction [Figure 2]d]. At ×3000, prismatic pattern of destruction is evident, that is, destruction of the prism cores but intact interprismatic sheaths [Figure 2]e]. At ×5000, focal holes were found to be distributed non-uniformly, and smaller focal holes coalesced together to form a larger ones [Figure 2]f].
In vivo remineralized samples
After in-vivo test period for 6 weeks, the enamel samples from the intraoral appliances were retrieved and subjected to SEM A total of 48 samples were obtained from the appliances in the Group I and II and then 24 samples were analyzed under SEM at ×1000, ×3000 and ×5000 and were compared with their corresponding demineralized samples. Other 24 samples were analyzed using SMH test.
In Group I, on analyzing the samples, 14 out of 24, showed the partial filling of the prism pits with focal holes at ×1000, ×3000, ×5000 [Figure 3]a-c]. Whereas, eight samples showed a reduction in the depth of prism pits and irregular deposits. In addition, two samples also showed globules of deposits scattered on the surface.
|Figure 3: Scanning electron microscope pictures of in vivo remineralized enamel samples of Group I and II at ×1000, ×3000, ×5000|
Click here to view
In Group II, all the samples showed signs of remineralization after the test period of 6 weeks. However, in the Group II, ten samples out of 24 showed generalized reduction in the depth of prism pits resulting in a smooth flat, homogenous surface with slightly discernible prism outlines after 6 weeks while eight samples showed deposits of irregular particles with uneven distribution. Only two samples showed irregularly arranged large globule like deposits scattered on the surface of the lesion giving a rough appearance at higher magnification [Figure 3]d-f]. Whereas, four samples showed partial filling of the prism pits and also the presence of thin slits which is a feature of the demineralized sample. The deposits were present with still existing porosities indicating partial remineralization of the samples.
Surface hardness testing
The enamel hardness value of sound, demineralized, and remineralized enamel samples for both Group I and Group II is presented in [Table 1]. A statistical significant difference was observed between the VHN values of the remineralized enamel obtained from control and experimental group with P < 0.00. While, there is no statistically significant difference in the VHN values of the sound and demineralized enamel samples of Group I and II as P > 0.05.
|Table 1 :Enamel hardness values (VHN) of sound, demineralized and remineralized enamel samples in|
Group I and Group II
Click here to view
One-sample Kolmogorov-Smirnov test and Student's t-test were applied, and statistical analysis showed statistical significant difference between the VHN values of the remineralized enamel obtained from control and experimental group with P < 0.00.
Following equation was used for calculating the percentage of SMH recovery  (SMH R ):
VHN R - Remineralized value, VHN D - Demineralized, VHN S - Sound enamel
The SMH R percentage of the Group I is 60.17% and for Group II is 85.66%, respectively.
| Discussion|| |
The present study clearly proved the natural remineralizing capacity of the saliva. However, in the presence of the calc-f tablets, the hardness of remineralized samples increased significantly in comparison to the enamel hardness in the no tablet group. The mineral loss or gain in the enamel because of demineralization or remineralization can be measured by changes in the enamel SMH.  The indentation hardness test with Vickers indenter has been used for measuring of initial/sound enamel hardness of artificial caries and the remineralized enamel specimens. This could be further substantiated using SEM technique to depict the surface changes following demineralization and remineralization.
The surface hardness value of the sound enamel obtained in the present study is in accordance with the VHN value of sound enamel obtained by Gutéιrrez-Salazar and Reyes-Gasga (VHN 268-375).  The decrease in VHN values obtained after the production of the artificial carious lesion in the enamel samples in both group I and group II were in accordance to the studies reported in the literature. ,, After the in vivo experimental period of 6 weeks, the difference between the VHN values of Group I and Group II was statistically significant. The increase in the VHN values was more in Group II as compared to Group I.
In the present study, the percentage of SMH R was calculated and found to be 60.17% in the control group and 85.66% in the experimental group. Corpron et al. found that the percentage recovery of the NaF dentifrice and APF containing mouthrinse was 69.05% and 66.01% respectively.  While Damle et al. studied the effect of the dentifrice containing sodium monofluorophosphate in vivo, and the percentage of surface hardness recovery obtained was 64.6%.  However, Wang et al. in their in vivo study have obtained the percentage SMH R by fluoride gum and fluoride-releasing devices to be 44.4% and 43.3%.  In in vitro study on primary molars using various fluoride dentifrices containing NaF observed, the percentage SMH R to be ranging from 28.6% to 45.4%.  The percentage surface hardness recovery obtained for CPP-ACP as test agent in in vitro study was found to be 13-53.37% in various studies. ,,
An increase in the surface hardness values and the percentage of the surface hardness recovery ranges have been observed in several studies in which no test regimen is given and this percentage has been found to be ranging from 2.53% to 38.3%. ,,,, This increase in surface hardness can be attributed to the remineralizing efficacy of saliva itself.
The features of demineralized lesion in our study were found to be to be well-correlating with the observation of natural caries and of artificial carious lesion that is, the increased porosity, varying amount of openings, relatively large focal holes and deepened Tome's processes on SEM examination. ,,,
The surface characteristics before and after the remineralization of artificial carious lesions could be appreciated on SEM examination after the experimental period of 6 weeks. The signs of remineralization such as reduction in the depth of the prism pits resulting in a smooth flat, homogenous surface with slightly discernible prism outlines, deposits of irregular particles with uneven distribution that is, concentrated at some places while scarce at other places could be seen. Also at places of the specimen, few focal holes were still evident. Gupta et al.  found the extremely variable morphological features on the enamel surface, ranging from an unevenly distributed superficial deposit to a smooth surface with tightly packed crystals.
Jayarajan et al.  also observed the calcified deposits to be more evident that were seen concentrated along the porous defects at ×1000 magnification and at higher magnification, mineralized deposits were discernible and were seen profusely scattered along the porous defects after remineralization by CPP-ACPF groups. In CPP-ACP group, the interprismatic substance was evident with porosities and areas of remineralization.
In the present study, at ×1000 irregularly arranged large globule like deposits were seen scattered on the surface of two samples and at higher magnification that is, ×3000 and ×5000 the surface appeared rough due to the presence of the globule like deposits. These findings were in accordance to those observed by Vashisht et al. 
Only two specimens of our experiment group showed partial filling of the prism pits. The presence of thin slits was seen in two samples of the experimental group indicating still existing porosities indicating partial remineralization of the samples. Similar findings have been reported by Gupta et al. where they observed partial filling of prism pits in fluoridated dentifrice group and amorphous precipitate like deposit scattered unevenly on the surface or globular particles deposited uniformly on the enamel surface in the urea-based mouthrinse group. 
In the present study, in vivo removable appliance model has been used to assess the remineralization of enamel caries. Removable appliances with enamel slabs having artificially created demineralized lesions were inserted into the oral cavities of the children undergoing orthodontic treatment for the experimental period of 6 weeks. There was no loss of enamel samples during the experimental period, and subjects did not complain of any discomfort. Moreover, no specimens were destroyed when applied and removed from the appliance. The same observation has been found by Sjögren et al., 2002. 
The duration of in situ studies ranges from as short as 2 days to as long 6 months used to study the remineralization. Only a few studies having experimental period of more than 6 weeks have been reported in the literature while most of the studies have short experimental period. ,,, The experimental period in the present study is 6 weeks which can be considered adequate to study the effect of these experimental tablets. During the experimental period, the patients were advised not to chew the tablets and keep them in the mouth until it dissolves on its own so as to have more intraoral time and maximum topical effects while intraoral appliance was in mouth.
The present concepts of the cariostatic mechanism of action of topical fluoride application developed during the last few years, established that the calcium fluoride particles that form on dental enamel on application of high concentrations of fluoride are not lost during the subsequent 24 h, as previously thought, but are retained for extensive periods of time and provide free fluoride during pH cycles in the dental plaque. Calcium fluoride thus constitutes a reservoir of fluoride that releases fluoride when the pH drops to 6 or below in dental plaque. This is caused by surface adsorption of secondary phosphate onto the calcium fluoride crystals. Calcium Fluoride thus provides a pH controlled reservoir of fluoride, which is mobilized on a carious challenge and helps in remineralization reactions in the enamel.  Similarly, the main component of calc-f tablets is calcium fluoride. Thus, it can also act as a reservoir of fluoride and remineralize the demineralized enamel.
Almeida et al. evaluated the effects of homeopathic medicines on teeth of rats that were fed on cariogenic diet. They proposed the hypothesis for the presence of deposits on the surface of the teeth of rats that were treated with these medicines viz.; calc-p, nat-f, kreos calc-f, and 0.05% NaF. They also stated that the deposits are due to the fact that these medicinal substances are more related to the physiopathology of caries (pathogenesis). The exaggerated medicinal stimulation that is, daily administration of medicine for 35 days resulted in a form of aggravation represented by the deposit. 
The results of SEM and SMH in our study confirm the effectiveness of calc-f tablets as a remineralizing agent. Thus, it can be concluded that calc-f tablets can also be used as a remineralizing agent. The calc-f tablets have several advantages such as these tablets are of low cost, easily available, no side effects, therefore, can be used for low socioeconomic population. However, the present study is only study till date to access the effectiveness of the calc-f tablets as a remineralization agent, therefore there is need for more studies with different analytical techniques to study the remineralization potential of the homeopathic tablets and compare them with the other remineralizing agents such as fluoride and CPP-ACP.
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Department of Pedodontics and Preventive Dentistry, SGT Dental College, Hospital and Research Institute, Village Budhera, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]