|Year : 2020 | Volume
| Issue : 2 | Page : 67-73
The comparative evaluation of the effects of antioxidants pretreatment on remineralization of demineralized dentin - In vitro study
Sadashiv G Daokar, Kalyani S Kagne, Kalpana S Pawar, Kapil D Wahane, Trupti V Thorat, Chaitali R Mahakale, Sweety Kumari, Suraj V Rathi
Department of Conservative Dentistry and Endodontics, CSMSS Dental College, Aurangabad, Maharashtra, India
|Date of Submission||02-Mar-2020|
|Date of Acceptance||19-Jun-2020|
|Date of Web Publication||21-Aug-2020|
Dr. Kalyani S Kagne
Department of Conservative Dentistry and Endodontics, CSMSS Dental College, Aurangabad, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Mechanical performance of dentin is of significant significance for the overall function of the teeth. Remineralization of carious dentin is the ultimate goal in re-establishing the functionality of the affected tissue to regain and maintain the mechanical properties of dentin. Functional remineralization of the affected dentin involves stabilization of both inorganic and organic component, but remineralizing agents stabilize only inorganic content. Hence, to stabilize organic content and to bring in functional remineralization, the use of anti-collagenolytic and anti-elastastic agents was considered for this study. Aims: The aim is to assess and compare the effect of antioxidants pretreatment on remineralization of demineralized dentin-in vitro study. Settings and Design: Experimental randomized analytical- in vitro study. Subjects and Methods: Ninety specimens were subjected to artificial caries lesions and were randomly divided into six groups based on the pretreatment with white tea, and grape seed extract (GSE) followed by casein phosphopeptide amorphous calcium phosphate and casein phosphopeptide amorphous calcium phosphate with fluoride. All the specimens were subjected to pH cycling regimen. The specimens were subjected to Vickers micro-hardness test to obtain the micro-hardness values. Statistical Analysis Used: The values were statistically analyzed using one-way analysis of variance. Results: After pH cycling, GSE showed a significant increase in micro-hardness values (46.57 ± 3.51 VHN), followed by white tea (42.56 ± 5.20) with the level of significance <0.05. Conclusions: Results of this study showed that naturally occurring antioxidants, i.e., 10% White tea and 6.5% GSE can be used as pretreatment regime before remineralization treatment.
Keywords: Bioflavonoid, catechins, polyphenols
|How to cite this article:|
Daokar SG, Kagne KS, Pawar KS, Wahane KD, Thorat TV, Mahakale CR, Kumari S, Rathi SV. The comparative evaluation of the effects of antioxidants pretreatment on remineralization of demineralized dentin - In vitro study. J Interdiscip Dentistry 2020;10:67-73
|How to cite this URL:|
Daokar SG, Kagne KS, Pawar KS, Wahane KD, Thorat TV, Mahakale CR, Kumari S, Rathi SV. The comparative evaluation of the effects of antioxidants pretreatment on remineralization of demineralized dentin - In vitro study. J Interdiscip Dentistry [serial online] 2020 [cited 2020 Sep 22];10:67-73. Available from: http://www.jidonline.com/text.asp?2020/10/2/67/292915
| Clinical Relevance to Interdisciplinary Dentistry|| |
Microhardness is a significant property of the tooth substance that correlates with strength. Remineralizing agents can remineralize inorganic content of tooth substance but it can not stabilize organic component. Antioxidant enhances the mechanical stability of dentin as well prevents collagen degradation, further improves mechanical properties of dentin.
| Introduction|| |
“Dental caries is described as a cyclic process, the metabolism of a fermentable substrate by plaque flora results in periods of demineralization, followed by periods of remineralization.”
In the entire life of an individual demineralization and remineralization processes co-exist in teeth. In pathological conditions, demineralization becomes dominant over remineralization; carious lesions affect the mineral phase of dentin and expose the collagen fibers, results in fast destruction of the entire dentin network, leading to degradation of collagen fibers and decrease in the mechanical properties of dentin.
Fluoride is known as a remineralizing agent, which interacts with oral fluids on the enamel surface and subsurface and bonds chemically to calcium and phosphate ions forming fluorapatite.
Tea is known to contain catechins (natural polyphenols) like epigallocatechin gallate (EGCG), epicatechin gallate, epigallocatechin, epicatechin. White tea contains these catechins in large volumes. White tea exhibits significantly higher anti elastase and anti-collagenase activities.
Grapeseed or Viti's vinifera extract (GSE) contains flavonoids and proanthocyanidin (PA), flavan-3-ols, and catechin. Studies have shown that glutaraldehyde and extracts rich in PA can be efficient for improving the mechanical stability of dentin and preventing collagen degradation.,
PA also increases the collagen synthesis, decreases the rate of enzymatic degradation of the collagen matrix, and promotes the conversion of insoluble collagen to soluble collagen during development.
Casein phosphopeptide amorphous calcium phosphate (CPP-ACP) is derived from milk protein, and it was introduced as a supplemental source of calcium and phosphate ions.
The anti-cariogenic activity of CPP-ACP has introduced its incorporation into food products and dental products to fight against dental caries. It has been claimed that the multifactorial anti-cariogenic mechanism of CPP-ACP has a threefold mode of action:
- It promotes the remineralization of enamel lesions by maintaining a supersaturated state of the enamel minerals calcium and phosphate in the dental plaque
- It delays the formation of biofilm and inhibits bacterial adhesion to the tooth surface
- It acts as a buffering agent, preventing a reduction of pH in the oral micro-environment.
Casein phosphopeptide amorphous calcium phosphate with fluoride (CPP-ACFP) has the same potential with additional benefits of added fluoride.
Currently, modern dentistry is in an era of minimal intervention for maximum preservation of tooth structure and function. Caries management has shifted its focus more toward prevention and control of the disease because of a better understanding of the basic disease process and advances in dental material science.
Hence, this study was undertaken to assess and compare the effect of white tea (antioxidant and anti-collagenolytic agent) and grape seed extract (GSE) (antioxidant and natural cross-linking agent) solution pretreatment on the remineralizing effect of CPP-ACP and CCP-ACFP by micro-hardness test of remineralized dentin.
Aim and objective
- To evaluate and compare the effect of white tea pretreatment on the remineralization capacity of CPP-ACP and CPP-ACFP on demineralized dentin
- To evaluate and compare the influence of GSE pretreatment on the remineralization capacity of CPP-ACP and CPP-ACFP on demineralized dentin
- To evaluate and compare the remineralization capacity of CPP-ACP and CPP-ACFP on demineralized dentin.
| Subjects and Methods|| |
Preparation of samples
Forty-five extracted permanent mandibular molar teeth free of caries and defects were cleaned by ultrasonic scaler to get free from debris and soft tissue. Radicular part of each tooth was removed, and the coronal part was then longitudinally sectioned in the mesiodistal direction into two sections using a diamond disc to prepare ninety specimens.
Having color as criteria to differentiate enamel and dentin, the enamel was removed to expose the dentin, using a coarse grit diamond points. The dentin specimens were horizontally embedded in self-cure acrylic resin and allowed to set to mount dentin blocks.
A small piece of adhesive tape was taken, a window of 5 mm × 5 mm was marked on the adhesive tape with marker and ruler. The marked window was cut with the help of scissors, and it was placed in the center of the dentin specimen. An acid-resistant transparent nail varnish was applied around the exposed dentin surface, leaving a window of 5 mm × 5 mm of dentin at the center. Later on, adhesive tape was removed from the dentin specimens leaving 5 mm × 5 mm dentin experimental area exposed.
Baseline micro-hardness value for all the dentin specimens was obtained using Vickers micro-hardness tester (Mitutoyo HM100, Japan) at a load of 25 g for 5 s at room temperature. The average value was considered the mean baseline micro-hardness for each specimen.
Preparation of demineralizing solution
CaCl2(2.2 mM), NaH2 PO4(2.2 mM), lactic acid (0.05 M), fluoride (0.2 ppm), adjusted with 50% NaOH to a pH 4.5.
All the specimens were immersed in the demineralizing solution for 72 h, at room temperature to induce artificial carious lesion (demineralized lesions). After induction of carious lesions, all the specimens were subjected again for micro-hardness test, and obtained values were recorded.
Preparation of antioxidant solutions
Two antioxidant solutions were prepared.
White tea extract solution (10%)
10% White tea [Figure 1] extracts solution was prepared by weighing 50 g of the white tea powder and dissolved it in 500 ml of distilled water.
Grape seed extract solution (6.5%)
6.5% GSE solution was prepared by weighing 32.5 g of powdered GSE [Figure 1] and dissolved it in 500 ml deionized water.
Based on remineralization protocol, three groups were formed;
- Group AI: White tea pretreatment followed by CPP-ACP application
- Group AII: White tea pretreatment followed by CPP-ACFP application
- Group BI: GSE pretreatment followed by CPP-ACP application
- Group BII: GSE pretreatment followed by CPP-ACFP application
- Group CI: CPP-ACP application without pretreatment
- Group CII: CPP-ACFP application without pretreatment.
The following protocol was followed for all the groups:
Each group was subjected to pH cycling regimen. pH cycling regimen consisted of experimental application protocol and storage in artificial saliva for 21 h. This was done as follows:
Group A: pH cycling regimen with 10% white tea extract
Group A specimens (n = 30) were immersed in white tea solution for 21 h and then immersed in artificial saliva for the remaining 3 h. This was repeated again for another day, after which Group A1 and A2 (n= 15 each) were subjected to CPP-ACP and CPP-ACFP [Figure 2] application respectively and they were left for three minutes. Then they were immersed in artificial saliva for 21 h. This was repeated for a period of 2 days followed by surface micro-hardness evaluation, and values were recorded.
Group B: pH cycling regimen with 6.5% grape seed extract solution
Group B specimens (n = 30) were immersed in GSE solution for 21 h and then immersed in artificial saliva for the remaining 3 h. This was repeated again for another day, after which Group B1 and B2 specimens (n = 15 each) were subjected to CPP-ACP and CPP-ACFP [Figure 2] application respectively and they were left for three minutes. Then they were immersed in artificial saliva for 21 h. This was repeated for a period of 2 days followed by surface micro-hardness evaluation, and values were recorded.
Group C: pH cycling regimen (control group)
Group C specimens were left untreated with antioxidant. Group C1 and C2 specimens (n = 15 each) were subjected to CPP-ACP and CPP-ACFP [Figure 2] application, respectively, and they were left for 3 min. Then they were immersed in artificial saliva for 21 h. This was repeated for a period of 2 days followed by surface micro-hardness evaluation, and values were recorded.
Micro-hardness values were obtained using digital Vickers micro-hardness tester (Mitutoyo HM100, Japan) [Figure 3] at a load of 25 g that was applied for 5 s at room temperature and same were recorded.
All the values obtained from the study were tabulated and subjected to the statistical analysis using analysis of variance (ANOVA) test using IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp, at the significance level of 0.05 (P < 0.05 = significant).
| Results|| |
For statistical analysis of our study, the difference between Vickers micro-hardness number of demineralized dentin and remineralized dentin was taken into consideration, and the average value was mentioned as mean difference in Vickers micro-hardness number.
Baseline micro-hardness values of normal dentin (55.78 ± 2.88 VHN) significantly dropped after demineralization (15.90 ± 3.04 VHN) [Table 1] and [Graph 1]. The mean difference micro-hardness values of all the groups are given in [Table 2] and [Graph 2]. One-way ANOVA is given in [Table 3].
|Table 1: The mean, standard deviation, standard error values of normal and demineralized dentin (vickers hardness number)|
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|Table 2: The mean, standard deviation, standard error values of all groups (vickers hardness number)|
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Mean difference in the Vickers micro-hardness number was highest for Group B2 (46.57 ± 3.51) followed by Group A2 (42.56 ± 5.20), Group B1 (38.36 ± 4.41), Group A1 (35.97 ± 4.35), Group C2 (32.29 ± 3.79) and Group C1 (29.61 ± 4.25). Statistical analysis indicated that Group B2 had the highest difference in the Vickers micro-hardness number, which was significantly different from all other groups. Among both the antioxidants, GSE showed significantly higher values than white tea.
| Discussion|| |
Reversing the radical trend of total excavation, now currently, the primary aim is to eradicate only the highly infected, irreversibly demineralized, and devitalized biomass followed by remineralization. The management of deep dentinal caries and noncarious lesions still poses a major challenge to the restorative dentist because of the fact that there is no infallible way to predict the future development of the lesion.
Recent researches on remineralization are on biomimetic remineralization materials, which were initially proposed by Moradian-Oldak et al. in 2001.
Dentin remineralization is more complex and less effective than enamel remineralization because there are residual mineral crystals on enamel, which are inattentive in dentin lesions. Various strategies have been considered to study the remineralization of dentin, such as fluoride and ACP releasing resin.,
It is well recognized that the collagen matrix functions as a scaffold for crystal deposition but does not provide a mechanism for nucleation of hydroxyapatite. The bio-mineralization process is typically modulated by a series of noncollagenous proteins, although they only comprise approximately 10% of the organic components.,
In the 20th century, the world is witnessing many uprisings in medical management. There is increased attentiveness to the value of good health. Good health cannot be achieved without good oral hygiene. It is believed that mouth is the mirror of the body and the gateway to good health. The field of dentistry plays a key role in the maintenance and improvement of oral hygiene.
In earlier times, herbs were widely used in dentistry for the prevention and cure of dental caries. The major advantages of using herbal preparations are easy availability, cost-effectiveness, increased shelf life, low toxicity, and lack of microbial resistance. Apart from the advantages of a naturally available cross-linking agent over a synthetic one, it shows massive biological activities with good biocompatibility and a faster reaction rate. Hence, natural antioxidants were used.
GSE is a rich source of PA, principally composed of monomeric catechin and epicatechin, gallic acid and polymeric, and oligomeric procyanidins. It is verified that GSE, composed predominantly of PA, can positively affect the tooth structure, thus offering a new remedy for carious lesions.
PA which is a bioflavonoid comprising benzene-pyran-phenolic acid molecular nucleus. PA, a naturally available plant metabolite is an antioxidant and free radical scavenger. The PA enhances the transformation of soluble collagen to insoluble collagen during development and increases collagen synthesis.
Camellia sinensis (L.) Kuntze is a highly branched tree belonging to the Theaceae family (Duarte and Menarim, 2006). Even though it is originated in China, Tibet, and Northern India, today is broadly cultivated all over the world. The white tea has a specific technique of postharvest processing, containing a greater proportion of sprouts, which are covered with a thin layer of silvered hair characterizing the tea coloring (Karori et al., 2007). Unlike black and green tea, white tea is not rolled or crushed but is fermented to some extent, cooked quickly and its leaves are dried naturally in the air to preserve the most polyphenols (Cheng, 2006).
There are various chemical constituents in the C. sinensis tea, such as polyphenols, methylxanthines (caffeine, theophylline and theobromine), vitamins, amino acids, carbohydrates, proteins, chlorophyll, volatile compounds, fluoride, minerals, trace elements, and other undefined compounds (Cabrera et al., 2003). Polyphenols are compounds of great interest because they exhibit potent antioxidant activity bothin vitro andin vivo due to its reducing properties.
Recent studies have evaluated that EGCG exhibits profound inhibitory activity on collagenases that degrade the organic matrix. Kato et al., attributed the inhibitory activity of tea against MMPs to EGCG, which exhibits a hydrogen bonding and hydrophobic interaction with collagenases, which may be responsible for the change in secondary structure of collagenases and consequently their inhibition.
In the late 1950s, milk and dairy products were presented to be effective in the prevention of caries without any side effects on the teeth. Casein is the major phosphoprotein in bovine milk and accounts for around 80% of its total protein, primarily as calcium phosphate stabilized micellular complexes that can be released as small peptides (CPPs) by partial enzymatic digestion. This has led to the development of a remineralization tool based on CPP-stabilized ACP complexes (CPP-ACP) (RecaldentR CASRN691364-49-5), and CPP stabilized ACFP complexes (CPP-ACFP).
The additive anti-cariogenic effect of CPP-ACP and fluoride may be owing to the localization of ACFP at the tooth surface, which, would co-localize calcium, phosphate, and fluoride ions. One of the advantages of CPP-ACP products over fluoride products is that they do not cause fluorosis of the enamel and are ingestible as compared to the topical fluoride products that pose a threat if the patient ingests a significant amount of fluoride.
Hardness is a surface property of a material that indicates its resistance against permanent deformation. Micro-hardness is a significant property of the tooth substance that correlates with strength, proportionality limit, wear-resistance, and surface roughness.
According to the results of our study, baseline micro-hardness values of normal dentin (55.78 ± 2.88 VHN) and the micro-hardness value significantly dropped after artificial demineralization (15.90 ± 3.04 VHN). These Vickers micro-hardness values are in accordance with other micro-hardness studies done by Fuentes et al., and Mollica et al.,
After pH cycling, there was an increase in the hardness values compared to demineralized specimens in all the groups. Mean difference in the Vickers micro-hardness number was highest for Group B2 (46.57 ± 3.51) followed by Group A2 (42.56 ± 5.20), Group B1 (38.36 ± 4.41), Group A1 (35.97 ± 4.35), Group C2 (32.29 ± 3.79), and Group C1 (29.61 ± 4.25).
This shows that GSE as pretreatment regime increased more micro-hardness value as compared to white tea. Furthermore, CPP-ACFP shows a significant increase in micro-hardness than CPP-ACP.
Jose et al. conducted a similar study with 10% white tea; they concluded that both the green tea and white tea extracts increased the micro-hardness values when used as pretreatment regime for remineralization. However, 10% white tea showed better results as compared to green tea indicating stabilization of collagen in dentin resulting in functional remineralization.
The result of our study are in agreement with the previous study conducted by Khamverdi et al. concluded that 6.5% GSE solution pretreatment followed by CPP-ACP application followed by fluoride varnish application showed the highest remineralizing potential.
According to the results of the study, the use of white tea and GSE increased micro-hardness of dentin, indicating functional remineralization. This could be due to the cross-linking of the collagen network which may have stabilized the collagen and maintained the collagen network in an expanded state so that the intra-fibrillar spaces are left open for remineralization. Hence, the results of this study rejects the hypothesis tested.
| Conclusions|| |
Based on the employed methodology, limitations of the study and obtained results in the presentin vitro study, it can be concluded that naturally occurring antioxidants, i.e., 10% white tea and 6.5% GSE can be used as pretreatment regime before remineralization treatment. Further studies should be performed to clinically establish the effect of different antioxidant pretreatment on remineralization with variable concentration and application time of the antioxidant.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
White DJ. The application ofin vitro
models to research on demineralization and remineralization of the teeth. Adv Dent Res 1995;9:175-93.
Bertassoni LE, Habelitz S, Kinney JH, Marshall SJ, Marshall GW Jr. Biomechanical perspective on the remineralization of dentin. Caries Res 2009;43:70-7.
Chokshi K, Chokshi A, Konde S, Shetty SR, Chandra KN, Jana S, et al
. Anin vitro
comparative evaluation of three remineralizing agents using confocal microscopy. J Clin Diagn Res 2016;10:ZC39-42.
Jose P, Sanjeev K, Sekar M. Effect of green and white tea pretreatment on remineralization of demineralized dentin by CPP-ACFP- Anin vitro
microhardness analysis. J Clin Diagn Res. 2016;10:85-9.
Khamverdi Z, Kordestani M, Soltanian AR. effect of proanthocynidin (PA), fluoride and casein phosphopeptide amorphous calcium phosphate remineralizing agents on microhardness of demineralized dentin. J Dent 2017;14:76-83.
Bedran-Russo AK, Pashley DH, Agee K, Drummond JL, Miescke KJ. Changes in stiffness of demineralized dentin following application of collagen crosslinkers. J Biomed Mater Res B Appl Biomater 2008;86:330-4.
Castellan CS, Pereira PN, Grande RH, Bedran-Russo AK. Mechanical characterization of proanthocyanidin-dentin matrix interaction. Dent Mater 2010;26:968-73.
Walter R, Miguez PA, Arnold RR, Pereira PN, Duarte WR, Yamauchi M. Effects of natural crosslinkers on the stability of dentin collagen and the inhibition of root caries in vitro
. Caries Res 2008;42:263-8.
Elsayad I, Sakr A, Badr Y. Combining casein phosphopeptide-amorphous calcium phosphate with fluoride: Synergistic remineralization potential of artificially demineralized enamel or not? J Biomed Opt 2009;14:044039.
Groisman S, Borzino L, Olival A, Borzino T, Corvino M, et al
. Effects of casein phosphopeptide amorphous calcium fluoride paste on white spots lesions during orthodontic treatment: One year follow up- tooth mousse GC in white spot during orthodontic treatment. J Dent Health Oral Disord Ther 2015;2:43.
Padmaja M, Raghu R. An ultraconservative method for the treatment of deep carious lesions-step wise excavation. Adv Biol Res 2010;4:42-4.
Moradian-Oldak J. Amelogenins: Assembly, processing and control of crystal morphology. Matrix Biol 2001;20:293-305.
Shen C, Zhang NZ, Anusavice KJ. Fluoride and chlorhexidine release from filled resins. J Dent Res 2010;89:1002-6.
Xu HH, Moreau JL, Sun L, Chow LC. Nanocomposite containing amorphous calcium phosphate nanoparticles for caries inhibition. Dent Mater 2011;27:762-9.
Gajjeraman S, Narayanan K, Hao J, Qin C, George A. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. J Biol Chem 2007;282:1193-204.
Cao Y, Mei ML, Xu J, Lo EC, Li Q, Chu CH. Biomimetic mineralisation of phosphorylated dentine by CPP-ACP. J Dent 2013;41:818-25.
Boukpessi T, Menashi S, Camoin L, Tencate JM, Goldberg M, Chaussain-Miller C. The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins. Biomaterials 2008;29:4367-73.
Ahamed ST. Awareness of oral hygiene among children in Chennai. Res J Pharm Tech 2016;9:1055-8.
Jain P, Ranjan M. Role of herbs in intracanal medicaments. Int J Pharm Bio Sci 2014;5:126-31.
Lussi A, Linde A. Mineral inductionin vivo
by dentine proteins. Caries Res 1993;27:241-8.
Kao TT, Tu HC, Chang WN, Chen BH, Shi YY, Chang TC, et al
. Grape seed extract inhibits the growth and pathogenicity of Staphylococcus aureus
by interfering with dihydrofolate reductase activity and folate-mediated one-carbon metabolism. Int J Food Microbiol 2010;141:17-27.
Pereira VP, Knor FJ, Vellosa JC, Beltrame FL. Determination of phenolic compounds and antioxidant activity of green, black and white teas of Camellia sinensis
(L.) Kuntze, Theaceae
. Rev Brasil Plantas Med 2014;16:490-8.
Chhabra N, Chhabra A. Enhanced remineralisation of tooth enamel using casein phosphopeptide-amorphous calcium phosphate complex: A review. Int J Clin Prev Dentistry 2018;14:1-10.
Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 1997;76:1587-95.
Hawkins R, Locker D, Noble J, Kay EJ. Prevention. Part 7: Professionally applied topical fluorides for caries prevention. Br Dent J 2003;195:313-7.
Fuentes V, Toledano M, Osorio R, Carvalho RM. Microhardness of superficial and deep sound human dentin. J Biomed Mater Res A 2003;66:850-3.
Mollica FB, Rocha Gomes Torres C, Gonçalves SE, Mancini MN. Dentine microhardness after different methods for detection and removal of carious dentine tissue. J Appl Oral Sci 2012;20:449-54.
Bertassoni LE, Habelitz S, Marshall SJ, Marshall GW. Mechanical recovery of dentin following remineralizationin vitro
– An indentation study. J Biomech 2011;44:176-81.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]