|Year : 2014 | Volume
| Issue : 1 | Page : 19-25
Remineralization efficiency of bioactive glass on artificially induced carious lesion an in-vitro study
Sai Sathya Narayana1, Vinoth Kumar Deepa2, Shafie Ahamed1, Emmanuel Solomon Sathish1, R Meyappan1, KS Satheesh Kumar2
1 Department of Conservative Dentistry, Rajah Muthiah Dental College and Hospital, Annamalai University, Annamalai Nagar, Tamil Nadu, India
2 Department of Endodontics, Rajah Muthiah Dental College and Hospital, Annamalai University, Annamalai Nagar, Tamil Nadu, India
|Date of Web Publication||15-Feb-2014|
Vinoth Kumar Deepa
Department of Conservative Dentistry and Endodontics, Rajah Muthiah Dental College, Annamalai University, Annamalai Nagar, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The objective of this study is to investigate the efficacy of bioactive glass containing product on remineralization of artificial induced carious enamel lesion and to compare its efficiency with other remineralization products using an in-vitro pH cycling method. The null hypothesis tested was bioactive glass has no effect on enamel remineralization. Materials and Methods: A total of 20 enamel samples of human molar teeth were subjected to artificial caries lesion formation using pH cycling method and was verified using high resolution scanning electron microscope (HRSEM). Each demineralized sample was then divided into five test groups each containing twenty. Group A - Bioactive glass (SHY-NM), Group B - Fluoride tooth paste (Amflor), Group C - CPP-ACP (Tooth mousse), Group D - CPP-ACPF (Tooth mousse plus), Group E - control. All the test groups were exposed to the pH cycling regime, the remineralizing agents were applied for 10 min except control. After 10 days period, the entire test groups were evaluated with HRSEM and quantitative assessment by energy dispersive X-ray spectroscopy. The obtained data was analyzed statistically using one-way ANOVA, Student's t-test and Tukey's multiple comparison tests. P ≤ 0.05 was considered to be significant. Results: Rejection of the null hypothesis and highlights the concept of biomimetic bioactive glass as an effective remineralizing agent. Clinical Relevance: To focus on the importance of minimal invasive treatment on incipient carious lesion by remineralization.
Keywords: Biomimetics, casein phosphopeptide-amorphous calcium phosphate, demineralization, fluoride, remineralization
|How to cite this article:|
Narayana SS, Deepa VK, Ahamed S, Sathish ES, Meyappan R, Satheesh Kumar K S. Remineralization efficiency of bioactive glass on artificially induced carious lesion an in-vitro study. J Indian Soc Pedod Prev Dent 2014;32:19-25
|How to cite this URL:|
Narayana SS, Deepa VK, Ahamed S, Sathish ES, Meyappan R, Satheesh Kumar K S. Remineralization efficiency of bioactive glass on artificially induced carious lesion an in-vitro study. J Indian Soc Pedod Prev Dent [serial online] 2014 [cited 2018 Dec 10];32:19-25. Available from: http://www.jisppd.com/text.asp?2014/32/1/19/127047
| Introduction|| |
In the past, dental caries was considered as an infectious bacterial disease that was irreversible. Recent concept in caries states that caries is a complex multifactorial disease caused by an imbalance in physiologic equilibrium between tooth mineral and biofilm. The caries process is said to be active when demineralization periods are more than periods of remineralization. This new insight into caries provided the scope for remineralizing incipient carious lesion.  The demineralization process can be stopped by creating an environment conducive for remineralization by various remineralizing agents.  The process of restoring lost mineral ions to the tooth structure and strengthening the lattice work is known as remineralization.
The remineralized enamel crystallites are generally more resistant to decalcification and also have the same orientation as the original enamel crystallites.  The early enamel lesions have a potential for remineralization with an increased resistance to further acid challenge, particularly with the use of enhanced remineralization treatments.  Thus invasive treatments of precavitated lesions are not required. Various remineralizing agents like fluoride,  Casein phosphopeptide stabilized amorphous calcium phosphate (CPP-ACP; Recaldent), , unstabilized ACP (ACP, Enamelon), CPP stabilized amorphous calcium phosphate with fluoride (CPP-ACPF; Recaldent) , has been studied in both in-vitro,, and in-vivo studies. ,
Recently, bioactive glass materials have been introduced in many fields of dentistry. This unique material has numerous novel features,  most important of which are its ability to act as a biomimetic mineralizer matching the body's own mineralizing traits while also affecting cell signals in a way that benefits the restoration of tissue structure and function. Bioactive glass is considered to be a breakthrough in re-mineralization technology,  it is a multi-component inorganic compound made up of elements such as silicon, calcium, sodium and phosphorus. The active ingredient is amorphous calcium sodium phosphosilicate. This compound in an aqueous environment release bioavailable calcium, sodium and phosphate ions contributing to the remineralization process. 
However, the remineralization potential of bioactive glass has so far not been evaluated and compared with fluoride, CPP-ACP and CPP-ACP containing fluoride using an in-virto pH cycling method. The pH cycling system has been shown to work successfully to evaluate lesion progression and mineral changes in enamel lesions.  Artificial early caries like lesions of enamel show all the principal histological features of natural caries and are a useful analog for natural lesions when studying demineralization and remineralization of enamel in-vitro. Hence the aim of this in-vitro study was to investigate the efficacy of bioactive glass containing toothpaste (Novamin) on remineralization of artificial incipient enamel lesion using pH cycling method and to compare its efficacy with fluoride containing toothpaste (Amflor), CPP-ACP containing tooth cream (Tooth mousse) and CPP-ACPF containing tooth cream (Tooth mousse plus) [Table 1]. The null hypothesis tested was bioactive glass has no effect on enamel remineralization.
| Materials and Methods|| |
A total of 20 human molar teeth extracted due to periodontal problems were taken. The teeth were cleaned of soft-tissue debris and inspected for intact surfaces free from caries, hypoplasia and white spot lesions. The teeth were stored in 0.1% Thymol until further processing. The radicular part of each tooth was removed. The coronal part of each tooth was sectioned mesiodistally into two halves using high speed diamond tipped disc. Pumice prophylaxis was done on the enamel surface of one half of the tooth to remove debris and fluoride rich layer. Nail varnish was applied to the rest of the tooth surface.
The sectioned enamel samples were subjected to artificial caries lesion formation. In pH cycling method, samples were subjected to pH cycling for 7 days.  During 5 days, the samples were immersed in demineralization solution (2 mM Ca(NO 3 ) 2 4H 2 O, 2 mM NaH 2 PO 4 2H 2 O, 0.075 mM acetate buffer, 0.02 ppm F pH 4.7) for 6 h and the remineralization solution (1.5 mM Ca(NO 3 ) 2 4H 2 O, 0.9 mM NaH 2 PO 4 2H 2 O, 150 mM KCl, 0.1 mol/l Tris buffer, 0.03 ppm F pH 7) for 18 h. In the last 2 days, the samples were maintained only in remineralization solution. Demineralization of the test sample was assessed by high resolution scanning electron microscope (HRSEM) and mineral loss quantified with energy dispersive X-ray spectroscopy (EDS). Each demineralized test sample was then divided into 5 quadrants one for each test groups (n = 20) for five groups.
- Test Group A - Bioactive glass (Novamin)
- Test Group B - Fluoride tooth paste (Amflor)
- Test Group C - CPP-ACP (Tooth mousse)
- Test Group D - CPP-ACPF (Tooth mousse plus)
- Test Group E (control group) - Demineralized sample with no treatment.
The pH cycling regime
The teeth samples were dipped in 50 ml of the demineralization solution (1.5 mM CaCl 2, 0.9 mM KH 2 PO 4, 50 mM for acetic acid, pH 4.8) for 4 min. After the demineralization challenge, the enamel specimens were rinsed in distilled water for 1 min and then placed in 50 ml of artificial saliva. This process was repeated until the teeth samples had been subjected to 5 demineralization challenges.  After the last demineralization challenge, the samples were rinsed in distilled water for 1 min and then each test group was added for 10 min. After that it was rinsed in distilled water for 1 min and stored in artificial saliva in an incubator at 37°C. Group E (control) received no intervention throughout the cycling regime. The demineralization solution and artificial saliva were changed daily. After the 10-day period of pH cycling regime the entire test groups were evaluated quantitatively and qualitatively.
Evaluation criteria and statistical analysis
Qualitative topographical assessment was performed by HRSEM. The specimens were viewed under ×500 and ×2000 magnification. The quantitative assessment of the changes in mineral profile was studied by EDS. The obtained data's were subjected to mean and standard deviation. The values were tabulated and statistically analyzed using one-way ANOVA. Comparison of each group with the control group was analyzed using "Student's t-test". The comparison of means between groups was conducted using Tukey's multiple comparison tests. P ≤ 0.05 was considered to be significant.
| Results|| |
The following were the results obtained by mineral profile analysis for elements calcium, phosphorus and fluorine [Table 2] and Graphs 1 and 2]. One-way ANOVA analysis showed a statistically significant difference in the mean value between the groups.
|Table 2: Depicts the comparison of individual|
group with the control for calcium, phosphorus,
fluorine elements in weight and atomic
Click here to view
Group A showed statistically significant results when compared with Group E for elements Ca and P. When F was considered there was no statistically significant difference. Group B showed statistically significant results when compared with Group E for elements Ca, P and F. Group C showed statistically significant results when compared with Group E for elements Ca and P. When F was considered there was no statistically significant difference. Group D showed statistically significant results when compared with Group E for elements Ca, P and F [Table 2]. The HRSEM pictures and energy dispersive X-ray analysis (EDAX) graph revealed mineral deposits on the surface when each test group was compared with the control group [Figure 1] and [Figure 2] and [Graph 1].
|Figure 1: High resolution scanning electron microscope pictures of various test groups at ×2000|
Click here to view
|Figure 2: High resolution scanning electron microscope pictures of various test groups at ×5000|
Click here to view
| Discussion|| |
For studying remineralization, pH cycling regime was followed in this in-vitro study as it exactly simulated the changes in the oral cavity. , Modern prospective caries studies require the measurement of small changes in tooth mineral content.  Quantitative measurements of changes in mineral content in a single caries lesion are desirable. One of the most recent techniques is scanning electron microscope (SEM) with EDAX attachment. It is a microanalytical technique that is employed to estimate quantitatively the amounts of mineral in a given tooth sample.  HRSEM gives the topographical pictures and is used to assess the surface changes seen on enamel, , EDAX gives quantification of various elements such as Ca, P, F, O, Mg, Na etc.,  in both atomic and weight percentage. The atomic and weight percentage of Ca, P and F are evaluated in the study groups as these ions take part in remineralization process. Bioactive glass is made of synthetic mineral containing sodium, calcium, phosphorous and silica (sodium calcium phosphosilicate) which is all elements naturally found in the body. Unlike other calcium phosphate technologies, the ions that bioactive glass releases form hydroxycarbonate apatite (HCA) directly, without the intermediate ACP phase. These particles also attach to the tooth surface and continue to release ions and re-mineralize the tooth surface after the initial application. These particles have been shown, in in-vitro studies  to release ions and transform into HCA for up to 2 weeks. , Ultimately these particles will completely transform into hydroxyapatite which is the mineral our teeth and bones are made. HCA layer is believed to contribute to the re-mineralization process of the tooth surface. Bioactive glass in the aqueous environment immediately begins surface reaction in three phases, leaching and exchange of cations, network dissolution of SiO 2 and precipitation of calcium and phosphate to form an apatite layer.  Alaudin and Fontana studied the remineralization efficiency of Novamin and fluoride using confocal laser scanning microscopy and stated that Novamin dentifrice produced significantly more remineralization than did the fluoride dentifrice.  In this study when Group A was compared with Group E, the results were statistically significant implying greater degree of remineralization. There was a marked increase of calcium in terms of atomic and weight percentage when compared with the control [Table 2] and Graph 1]. The HRSEM picture revealed uniformly dispersed mineral deposits along the porous defects [Figure 1] and [Figure 2]. Thus the null hypothesis was rejected and bioactive glass is considered as a remineralizing agent. When compared with Group B and Group D there was a decrease of fluoride in both atomic and weight percentage [Graph 2]. This can be substantiated as Novamin (SHY-NM) is non-fluoridated toothpaste.
For the caries preventive effect the bioavailability of fluoride is of importance. Bioavailability of fluoride is dependent from the solubility of the fluoride containing compound and from the adhesion of the fluoride compound to the surface. In dentifrices different fluoride formulations are used as the carrier for fluoride ions of which the most frequent are sodium fluoride, sodium monofluorophosphate and amine fluoride. The effect of amine fluoride on enamel remineralization has been studied in experimental and clinical investigations. , The different solubility of NaF, sodium monofluorophosphate and amine fluoride may result in different amounts of CaF 2 formation and hence influence the bioavailability of fluoride in saliva and the demineralization and remineralization potential of enamel. For amine fluoride, being a cationic tenside, the surface tension has also been discussed being responsible for the caries protective effect.  Studies with amine fluoride and sodium monofluorophosphate revealed complete remineralization of the lesion.  This leads to the conclusion, that amine fluoride not only enhances enamel remineralization but also results in a more stable, less soluble superficial enamel layer. 
In this study, the results showed a significant difference between Group B (Amflor) and Group E [Table 2] and Graph 1] by the increase in atomic and weight percentage of calcium, phosphorus and fluoride and favored the above studies. The EDAX analysis showed a greater percentage of fluoride both in terms of atomic and volume percentage when compared between the groups suggesting that the increase of fluoride is attributed to the use of organic fluoride [Graph 2]. HRSEM pictures showed mineral deposits which are evenly scattered on the enamel surface [Figure 1] and [Figure 2].
The CPPs have an important role as an ACP carrier localizing the highly soluble calcium phosphate phase at the tooth surface. , CPP-ACP have been found to increase the levels of calcium and phosphate in plaque up to five folds in in-situ studies.  The proposed mechanism of their anticariogenicity is that they act as a calcium-phosphate reservoir, buffering the activities of free calcium and phosphate ions in the plaque fluid helping to maintain a state of supersaturation with respect to enamel, thereby depressing enamel demineralization and enhancing remineralization.  The remineralizing potential of CPP-ACP has been shown in various in-vitro studies ,, and in-vivo studies. 
The present study showed a significant difference between Group C (CPP-ACP) with Group E in terms of both atomic and weight percentage of calcium and phosphorus [Table 2] and Graph 1]. Considering the element fluorine there was not much significant difference existing as CPP-ACP do not contain fluoride. The HRSEM pictures revealed redeposited mineral phases as adherent granules or globules when compared with the control group [Figure 1] and [Figure 2]. Jayarajan et al. in his study investigated the efficacy of CPP-ACP and CPP-ACPF using Diagnodent (KaVo) and SEM. He concluded that there was significant demineralization of both the groups when compared to control and because of the added benefit of fluoride (NaF 0.2%), CPP-ACPF (Tooth Mousse-Plus) showed marginally more amount of remineralization than CPP-ACP (Tooth Mousse).  A dentifrice containing CPP-ACPF provides remineralization which is superior to both CPP-ACP alone and to conventional and high fluoride dentifrices. ,
Since additive effects were obtained when CPP-ACP is used in conjunction with fluoride, it can be recommended that CPP-ACP should be used as a self-applied topical coating after the teeth have been brushed with fluoridated toothpaste by children who have a high caries risk.  Fluoride enhances the rate of remineralization from calcium phosphate solutions. Comparison between Group D (CPP-ACPF) and Group E revealed statistically significant results with Ca, P and F in terms of both atomic and weight percentage [Table 2] and Graph 1]. HRSEM pictures revealed mineral deposits evenly dispersed throughout the surface [Figure 1] and [Figure 2]. Comparing Group D with Group A and Group C revealed an increase of F in terms of atomic and weight percentage in Group D and this can be attributed to the presence of F in CPP-ACPF [Graph 2].
| Conclusion|| |
Within the limitations of this in-vitro study, the following inferences were drawn
- Each test group when compared with the control group showed a significant difference existing for element Ca and P.
- Group A (SHY-NM) showed an increase of calcium suggesting that bioactive glass can be considered as an effective remineralizing agent.
- F increase was seen in Group B (Amflor) followed by Group D (CPP-ACPF) as both contained fluoride.
- Group A and Group C showed less of F as both are non-fluoridated.
Further studies on enamel crystal formation and chemical structure using advanced quantification technique and the resistance of acid solubility of these remineralized crystallites have to be investigated in order to achieve more conclusive results.
| Acknowledgments|| |
The authors would like to thank the Biochemistry Department, Annamalai University for manufacturing the solutions necessary for the research. We extend our thanks to the staff of Sophisticated Analytical Instrumental Facility (SAIF), Indian Institute of Technology, Chennai, for his skillful assistance with the high resolution scanning electron microscope (HRSEM) and energy dispersive X-ray analysis (EDAX).
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[Figure 1], [Figure 2]
[Table 1], [Table 2]