INTRODUCTION
Teeth go through a natural, continuous process of demineralization and remineralization . Demineralization occurs due to bacterial acids, foods, and physiological processes. Mineralization occurs from the calcium and phosphorus (normally present in saliva) precipitating into a crystalline form of calcium phosphate called hydroxycarbonate apatite, which is the mineral component of all teeth and bones. Since natural remineralization is inadequate to maintain strong enamel, the natural remineralization process needs to be augmented.[ 1 ] Caries is just not a disease, but instead a disease process.[ 2 ] The understanding of this basic fact has opened up newer avenues of interception of this disease process through remineralization . The current concept considers caries as a dynamic and reversible process and is the result of the interplay of a number of etiological factors.[ 3 , 4 ] Some of these factors cause demineralization, whereas others promote remineralization of the tooth. When the demineralization process continues, cavitation will eventually result. Caries can be arrested or even reversed at the precavitated stage, provided a balance toward remineralization can be established.[ 3 ] The progressive increase in processed sugars and acidic foods and beverages in the human diet provides oral bacteria greater opportunity to produce acids that dissolve tooth mineral. Tooth mineral is composed mainly of calcium and phosphorous. Methods for providing these constituents of minerals to facilitate the remineralization of teeth have been the backbone for newer remineralization strategies.[ 2 ] Demineralization and remineralization are balanced processes that normally occur in the oral cavity. Sometimes, diet variations, oral hygiene or microbial activity can lead to the predominance of demineralization. Remineralization is facilitated by the buffering action of saliva, permitting calcium, and phosphate ions to precipitate onto the tooth and form the new mineral. Therefore, modulation of the demineralization–remineralization balance is the key to prevention of dental caries.[ 4 , 5 ]
Shelcal 500 mg is used for the growth of strong teeth and to prevent dental cavities. The composition of the shelcal includes (1) Calcium carbonate from an organic source (power oyster shell) – 1250 mg and (2) Vitamin D3 – 259 Iu.[ 6 ]
Vitamin C is water-soluble vitamin synthesized from glucose. As it cannot be synthesized by the body, it must be consumed in fruits, vegetables, and supplements. It is recommended to take 75 mg/day (women) and 90 mg/day (men). Its effects on the body are: antioxidant as it protects cells against damage caused by free radicals present in cigarette smoke, environmental pollution, and ultraviolet rays, production of collagen necessary for wound healing; improves absorption of the iron present in foods of vegetal origin, contributes to the system immune to protect the body against diseases, and can delay the macular disease progression and age-related cataracts.[ 7 , 8 ] Calcium sandoz contains calcium carbonate as the main ingredient. However, calcium sandoz –250 contains Khatika Churna, and it is claimed to have 250 mg calcium equivalent in each chewable tablet.[ 9 ] The aim of the study is to evaluate the remineralization efficacy of shelcal , calcium sandoz and shelcal with Vitamin C on artificially induced caries lesions in permanent teeth and to assess the Surface roughness in microhardness testing in tooth specimens. There is no difference between the remineralization efficacy of shelcal , calcium sandoz, and shelcal with Vitamin C on artificially induced caries lesions in permanent teeth.
MATERIALS AND METHODS
Study design
An in vitro study.
Number of samples
The number of samples included in the study was 45 sound human permanent teeth (15 in each group).
Materials used
Permanent teeth
Demineralizing solution 2.2 mM CaCl2 .2H2 O (calcium chloride), 2.2 mM NaH2 PO4 .7H2 O (monosodium phosphate), and 0.05 M (lactic acid)
Artificial saliva – 2.200 g/L (gastric mucin), 0.381 g/L sodium chloride (NaCl), 0.213 g/L CaCl2 .2H2 O (calcium chloride), 0.738 g/L K2 HPO4 .3H2 O (potassium hydrogen phosphate), and 1.114 g/L KCl (potassium chloride)
Shelcal (calcium and Vitamin D3)Vitamin C
Calcium sandoz tablet.
Inclusion criteria
Freshly extracted human permanent premolars for orthodontic purpose were included
Exclusion criteria
Teeth with any visible
Detectable caries
Hypoplastic/white spot lesion
Enamel cracks /fracture
Developmental defects
Deciduous teeth
Process
Freshly extracted 45 sound human permanent teeth with at least one sound tooth surface, without any visible caries, hypoplastic lesions, stains, or white spot lesions were included in the study. After extraction, the teeth were cleaned, washed with water, and stored in isotonic saline. The root of the teeth was separated from the crown portion at the cementoenamel junction using a diamond-coated disc [Figure 2 ]. The labial surface of all samples was progressively ground flat and hand polished with the aqueous slurry of progressively finer grades of silicon carbide, up to 4000 grit to obtain flat surface and about 150 mm thickness of the enamel tissue was removed from the original tooth surface. To prevent dehydration, the specimens were stored in normal saline. Each sample was embedded in the self-cured acrylic resin. To create the artificial carious lesions, acid-resistant nail varnish was applied around the exposed tooth surfaces, leaving a 4 mm × 4 mm window of the enamel exposed in the center.[ 10 ]
Figure 1: Flowchart representing the methodology
Figure 2: Sectioned tooth samples
Then, the baseline enamel was measured.
Demineralizing solution was prepared using 2.2 mM CaCl2 .2H2 O (calcium chloride), 2.2 mM NaH2 PO4 .7H2 O (monosodium phosphate), and 0.05 M (lactic acid). Each ingredient was added separately to deionized water under continuous stirring and was allowed to dissolve completely before the next ingredient was added. The solution was maintained at 37°C and the pH was adjusted to 4.5 using 50% NaOH solution.[ 11 ]
Artificial saliva was prepared by mixing 2.200 g/L (gastric mucin), 0.381 g/L NaCl (sodium chloride), 0.213 g/L CaCl2 .2H2 O (calcium chloride), 0.738 g/L K2 HPO4 .3H2 O (potassium hydrogen phosphate), and 1.114 g/L KCl (potassium chloride). Each ingredient was added separately to deionized water under continuous stirring and was allowed to dissolve completely before the next ingredient was added. The solution was maintained at 37°C and the pH was adjusted to 7.00 using 85% lactic acid.[ 11 ] The schematic representation depicted the methodology in [Figure 1 ].
Remineralization /demineralization study protocolThe samples were randomly divided into three groups (15 each):
Group I – Shelcal
Group II – Shelcal with Vitamin C
Group III – Calcium sandoz tablets.
The specimens of the enamel blocks were immersed in the demineralized solution. The solution was stirred and the demineralization was performed at 37°C for 48 h and the enamel surface microhardness (SMH) was measured after demineralization.
The specimens underwent were subjected to the remineralization process twice a day for 10 days.[ 12 ]
The demineralization solution and artificial saliva were changed daily. After the 10 days of remineralization procedure, the groups were evaluated on the basis of surface micro hardness (SMH) appearance of the enamel surface [Figure 3 ].[ 13 ]
Figure 3: Picture showing hardness using vickers hardness tester
Hardness testing
Vicker’s hardness test to check the microhardness of the enamel surface was done. The testing was done with a FIE microhardness tester in India. Forty-five (n = 15) samples were placed on the tester after leveling the dental stone block so that a plane is achieved [Figure 4 ].
Figure 4: HMV G micro hardness tester (Shimadzu’s)
The diamond tip was used to create a nanoindenter. Under a ×100 microscope, the sample positioning was done so that the indent falls on the enamel portion of the section. A load of 100 gf for 15 s was applied, and the rhomboid indent is measured for length, and depth and the hardness values were calculated.[ 13 ]
Intragroup comparisons
Table 1 is Intragroup comparison.
Table 1: Post hoc comparison within groups with Bonferroni correction
Intergroup comparisons
Tables 2 and 3 is Intergroup comparison
Table 2: Posttest (remineralization ) comparison between shelcal , shelcal with Vitamin C, and calcium sandoz using one-way analysis of variance*
Table 3: Post hoc comparison between shelcal , shelcal with vitamin-c and calcium sandoz with bonferroni correction
Statistical analysis
The mean and standard deviation of the microhardness of tooth samples were obtained for each group and compared using one-way ANOVA. The pair-wise comparison was carried out using a post hoc Bonferroni test. The statistical analysis was performed using statistical packages for social sciences SPSS (Version 20.0; SPSS Inc.,Chicago, IL, USA).
RESULTS
The results showed no significant difference between groups at baseline and demineralization with P < 0.05.
Intergroup comparison between groups showed statistical significant difference with P < 0.05 [Table 2 ]. Post hoc Bonferroni test proved that there is a significant difference between shecal and shelcal with Vitamin C, and calcium sandoz [Table 3 ].
Intragroup comparison within groups showed significant differences at all levels (baseline, demineralization, and remineralization ). The post hoc test also showed signification change at all levels in the three groups [Table 1 ]. Graph 1 represents the diagrammatic representation of mean values of three different groupsusing calcium sandoz,shelcal & Vitamin C and shelcal at baseline, demineralization and remineralization .
Graph 1: Diagrammatic representation of mean value of the three different groups
DISCUSSION
Mineralization occurs as a lifelong process. Demineralization is the process of removing mineral ions from hydroxyapatite (HA) crystals of hard tissues such as enamel, dentin, cementum, and bone, whereas restoring these mineral ions again to the HA crystals is called remineralization . It occurs as a continuous process in the oral cavity until there is an imbalance that sets in due to numerous known etiological factors. Demineralization is a reversible process; hence, the partially demineralized HA crystals in teeth can grow to their original size if they are exposed to oral environments that favor remineralization .[ 14 ] Calcium, phosphate, and fluoride ions play an important role in the remineralization process which modify the susceptibility of the tooth to caries progression.[ 15 ] During demineralization, calcium release precedes phosphate release from enamel and dentin. Therefore, using calcium to suppress the demineralization process would be effective.[ 16 ]
Decades of research have led to the advancement of technologies that can promote enamel remineralization or downscale demineralization, thereby reinforcing and aiding oral health.[ 17 ] Considerable efforts have been made to limit the progression of carious lesions while clinical studies remain the gold standard. Standardized in vitro models are the most conventional techniques in cariology research and can serve as a valuable tool for assessing the anticaries efficacy of remineralizing agents.[ 18 ]
Many remineralizing substitutes such as shelcal , calcium sandoz, and shelcal with Vitamin-C aid in strengthening teeth through mineralization and are being recommended. Shelcal and calcium sandoz are recommended based on their remineralizing potential, observed in research related to bones and dentoalveolar bone.[ 19 ] Recent epidemiological studies have provided convincing evidence for the increased risk of osteoporosis and fractures caused by the decreased bone formation in patients with Vitamin C deficiency, where there is an improvement with Vitamin C supplements that aids in the synthesis of collagen.[ 20 ]
There are lacunae in literature with regard to the remineralization potential of these calcium substitutes with respect to the tooth surface along with the estimation of hardness values. Hence, the present study was carried out to compare the efficacy of these products such as shelcal , shelcal with Vitamin C, and calcium sandoz for the remineralization potential on teeth.
Calcium substitutes such as shelcal , calcium sandoz, and shelcal with Vitamin-C can be used for strengthening teeth through mineralization.
The present study revealed a significant increase in SMH after remineralization with shelcal compared to shelcal with Vitamin C and calcium sandoz. This study shows that plain calcium ions in shelcal are more effective compared with the synergistic form of calcium ions with Vitamin C (shelcal with Vitamin C) and gelatin form substitutes (calcium sandoz). This study is the first of its kind; hence, findings from the study could not be compared.
The intragroup comparison showed that the remineralization potential of all three groups increased after demineralization but not up to the mark of baseline this could be a restraint factor with calcium ion substitutes. Further, SEM-based studies can be recommended for analysis at HA crystal levels to analyze the actual remineralization potential using these three substitutes.
CONCLUSION
Shelcal was observed to aids in remineralization and turns have a higher SMH compared to shelcal with Vitamin C and calcium sandoz.
Strengths
To the best of our knowledge this is the first invitro study aiding in assessing the remineralizing potential using calcium substitutes
Recommendations
Further Invivo studies can be carried out with larger samples
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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