Dental caries is an infectiousdisease and its initiation and progression depends on several factors. Teethare constantly going through cycles of demineralization and remineralization.The ultimate goal of clinical intervention is the preservation of tooth structureand prevention of lesion progression to the point where restoration isrequired.                     While fluoride is anestablished agent in promoting remineralization; there are other remineralizingagents available such as synthetic nanohydroxyapatite, casein phosphopeptideamorphous calcium phosphate with fluoride(CPP-ACPF) and grape seedextract.

                                      Hydroxyapatite is animportant biomaterial and a major component of the mineralized structure of theteeth and bones . It is also an important bioceramic for medical and dentalapplications (including dental implants, orthopedics, alveolar reconstructionand drug delivery systems) due to its biocompatibility and biological andchemical similarity to the bone structure. HA can be used for bone, cementumand artificial root formation and can induce tooth remineralization1.                       It has been observedthat CPP-ACPF has the ability to localize ACP at the tooth surface, whichbrings about buffering of calcium and phosphate free ion activities, therebyhelping to maintain a state of super saturation with respect to tooth enamelnegating demineralization and enhancing remineralization with added fluorideeffects. Casein phosphopeptide amorphous calcium phosphate nanocomplexes  have shown to localize at the tooth surface2.                      Grape seed extract (GSE)is a rich source of proanthocyanidin (PA), mainly composed of monomericcatechin and epicatechin, gallic acid and polymeric and oligomericprocyanidins. Proanthocyanidins has been reported to strengthen collagen-basedtissues by increasing collagen crosslinks.

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There is evidence that PA increasescollagen synthesis and accelerates the conversion of soluble collagen toinsoluble collagen. PA has proved safe in various clinical applications and hasbeen used as dietary supplements. It has been shown that GSE positively affects theremineralization process of root caries.

Studies claim that collagen can serveas a substrate for apatite formation3.                        Therefore, our study aims at quantitativelyevaluating the enamel remineralizing potential of Nanohydroxyapatite, CPP-ACPF,GSE using surface microhardness analysis(Vickers hardness test). Materialsand Methods: Inclusioncriteria for teeth were:-·        Non carious teeth.·        Teeth free ofrestorations.·        Teeth with no crackson the crown.Exclusion criteriafor teeth were:-·        Teeth with defectivemorphology like hypoplastic defects.

·        Grossly carious teeth.·        Damaged teeth Infection control protocol:Teethwere cleansed of visible blood and gross debris and were maintained in ahydrated state during storage and placed in 3% sodium hypochlorite solutiondiluted with saline in a ratio of 1:10 in the container. Microbial growth was eliminated by using an autoclave cycle for40 min.  Sample preparation:Enamelsamples (2 mm thickness) were prepared from the buccal or lingual surfaces ofthe teeth selected, using a double faced diamond disc. After sample preparationwindows were created (dimension of 5 mm × 5 mm) using adhesive tape and thesample was made completely resistant to acid attack by coating nail varnish(Maybelline). Afterwards, adhesive tape was removed using a sharp instrumentexhibiting a rectangular area on the enamel. Lesion preparationEachof the enamel samples were then immersed in 40 ml of demineralizing solutionfor a period of 4 days at a constant temperature of 37°C, in an incubator toinduce artificial caries formation, simulating an active area ofdemineralization.

Fresh preparation of demineralized solution was done.4Thedemineralizing solution used was:·        calcium 2.0 mMol/L·        phosphate 2.0 mMol/L·        acetic acid 75.

0mMol/L, pH 4.4Freshpreparation of remineralized solution was done.4The remineralizingsolution used was:·        1.5 mM/L calcium·        0.9 mM/L phosphate·        130 mM/L KCl in20mM/L, Tris buffer, pH 7 Slurry preparation of CPP-ACPF, Nano-hydroxyapatite:Slurry of CPP-ACPFand Nano-hydroxyapatite were prepared by agitating the preparations indeionized water in the ratio of 1:3 (Stookey et al. 2011). To achieve this, 17 gm of dentifrice was dispensed using highprecision balance weighing machine from the respective tube and thentransferred into two tubes to which 51 ml of deionized water was added andstirred with stirring rod until it was well mixed. Grape seed extract preparation: The grape seed extract consistes of 95%Proanthocyanidins according to data provided by manufacturer.

TheProanthocyanidins in the Grape seed extract is composed mainly of monomers(catechins). A 6.5% (W/V) solution in phosphate buffer (0.025M K2HPO4,0.025 M KH2PO4, ph 7.4) was used in the study.

6 Grouping of samples:50enamel slabs were randomly divided into five groups of 10 samples in each basedon the type of remineralizing agent to be usedGroupI:  Sound enamel (no treatment)               Control group consisted of toothsections immersed in normal saline.GroupII:  Demineralized and treated withslurry of nano-hydroxyapatite                   (APAGARD m-plus standardpaste)GroupIII: Demineralized and treated with slurry of CPP-ACPF (GC Tooth                  Mousse Plus, 0.2% fluoride(900ppm), 10% RECALDENT)GroupIV: Demineralized and treated with solution of grape seed extract.                   (95%  proanthocyanidins, Brazil, NutrijaLifescienses)Group V:  Demineralized and not treated with anysolution pH cycling model:Tosimulate the dynamic process of demineralization and remineralization in oralcavity pH cycling model was used.

Enamel samples were treated with therespective remineralizing agents for 3 min than samples were immersed in 20 mlof demineralizing solution (2.0 mM/L Ca(NO3)2•4H2O,2.0 mM/L KH2PO4, 75 mM acetic acid,pH 4.8), for a period of 3 h. Samples were again treated with slurries of therespective remineralizing agents for 3 min.

All the enamel samples wereimmersed in 30 ml of remineralizing solution (1.5 mM/L Ca(NO3)2•4H2O,0.9 mM KH2PO4, 20 mM/L Tris bufferpH 7.0 and 130 mM/L KCl) for a period of 17 h. The remineralizing solution wasreplaced every 48 h and the demineralizing agent replaced every 5 days. ThepH cycling was done for a period of 28 days. After this, all the samples wereembedded in acrylic blocks and assessed for surface microhardness using Vickershardness test.

 Surface microhardnessThesurface microhardness of specimens was determined using digital microhardnesstesting machine (MATSUZAWA Co., Ltd. Model) with a pyramid indenter and a ×40objective lens. A load of 100 g was applied to the surface for 10 s.

7Fiveindentations were placed on the surface and the average value was considered.Precision microscopes of magnification of ×400 were used to measure theindentations. The diagonal length of the indentation was measured by built inscaled microscope and Vickers values were evaluated to determine microhardnessvalues. Statistical analysisTheresults were analyzed by one-way analysis of variance (ANOVA). Multiplecomparisons between groups were performed by posthoc Tukey test.      N     Mean   Std. Deviation   Std. Error 95% Confidence Interval for Mean   Minimum     Maximum Lower Bound Upper Bound I 10 278.

4416 9.13575 2.8889 271.9062 284.9769 262.452 290.056 II 10 220.9484 14.

92140 4.7185 210.2742 231.6225 197.

600 247.600 III 10 232.9654 14.08529 4.4541 222.8893 243.0414 207.

334 247.600 IV 10 196.4546 7.258504 2.2953 191.

2621 201.6470 178.800 205.576 V 10 182.1532 21.70238 6.

8628 166.6282 197.6781 165.000 215.800 Total 50 222.1926 36.34448 5.1398 211.

8636 232.5216 165.000 290.056  Table 1 – Mean valuesof microhardness (HV)in different groups using oneway ANOVA   Sum of Squares Df Mean Square F Sig. Between Groups 55471.495 4 13867.874 67.439 <.

001** Within Groups 9253.665 45 205.637 Total 64725.

160 49  Table 2-Presenting the statistically significant difference between the mean values ofdifferent groups using ANOVA test   (I) Group (J) group   Mean Difference (I-J)   Std. Error   Sig. 95% Confidence Interval Lower Bound Upper Bound I II 45.476200(*) 6.

413065 <.001** 27.25380 63.69860 III 57.493200(*) 6.

413065 <.001** 39.27080 75.

71560 IV 81.987000(*) 6.413065 <.001** 63.76460 100.20940 V 96.288400(*) 6.

413065 <.001** 78.06600 114.51080 II I -57.493200(*) 6.413065 <.

001** -75.71560 -39.27080 III -12.

017000 6.413065 .346 -30.23940 6.

20540 IV 24.493800(*) 6.413065 .004** 6.27140 42.

71620 V 38.795200(*) 6.413065 <.001** 20.57280 57.01760 III I -45.

476200(*) 6.413065 <.001** -63.69860 -27.25380 II 12.017000 6.

413065 .346 -6.20540 30.23940 IV 36.510800(*) 6.413065 <.

001** 18.28840 54.73320 V 50.812200(*) 6.413065 <.001** 32.58980 69.03460 IV I -81.

987000(*) 6.413065 <.001** -100.2094 -63.76460 II -36.510800(*) 6.

413065 <.001** -54.73320 -18.28840 III -24.493800(*) 6.

413065 .004** -42.71620 -6.27140 V 14.

301400 6.413065 .187 -3.

92100 32.52380 V I -96.288400(*) 6.413065 <.001** -114.

5108 -78.06600 II -50.812200(*) 6.413065 <.001** -69.03460 -32.58980 III -38.795200(*) 6.

413065 <.001** -57.01760 -20.57280 IV -14.301400 6.413065 .187 -32.52380 3.

92100 * The mean difference is significant at the .05 level. Table 3- Comparison of meanvalues of microhardness of one group with the other four groups using Post hoc test Discussion: Thecasein phosphopeptides (CPPs) are produced from the tryptic digest of casein,aggregated with calcium phosphate and purified through ultrafiltration.Caseinhas the ability to stabilize calcium and phosphate ions. This technology wasdeveloped by Eric Reynolds, Australia. CPPs contain the cluster sequence of-Ser (P)-Ser (P)-Ser (P)-Glu-Glu from casein.8 This protein nanotechnology combines theprecise ratio of 144 calcium ions plus 96 phosphate ions and six peptides ofCPP. The nanocomplexes form over a pH range of 5.

0-9.0. A 1% CPP solution at pH7.0 can stabilize 60 mM calcium and 36 mM phosphate.9, 10 Calciuminteracts with CPP through the negatively charged residues of the peptides.

11  The size and electroneutrality of the CPPnanocomplexes allows them to diffuse down the concentration gradient into thebody of the sub-surface lesion.12, 13Once present in the enamel sub-surface lesion,the CPP-ACP releases the weakly bound calcium and phosphate ions14,depositing them into crystal voids. The CPPs have a high binding affinity forapatit15; thus, on entering the lesion, the CPPs binds to themore thermodynamically favored surface of an apatite crystal face.

In this study, CPP-ACPF was used. Highremineralization potential of CPP-ACPF can be attributed to the synergisticanticariogenic effects of CPP-ACP and fluoride. The fluoride ions are adsorbedonto the surface of enamel crystals, inhibiting dissolution and increasingremineralization. With the use of low fluoride concentration as is present inCPP-ACPF (0.2% or 900 ppm of NaF), there is a complex localization of freecalcium phosphate and fluoride ion activities, which helps in maintaining astate of supersaturation by suppressing demineralization.16 ThusCPP-ACPF (Tooth Mousse Plus™) is an excellent local slow-deliverysystem to treat the white spot lesion.  Nanoparticle Hydroxyapatite containing toothpastes were first introducedand tested in Japan in the 1980s (e.

g. Apadent, Apagard, and others by SangiCo., Ltd., Tokyo). They were used as anticaries agents by Japanese Governmentin 1993.  In our study we used nano-hydroxyapatite containing paste by Sangi Co., Ltd., Japan.

It contains 10%Hydroxyapatite with Hydroxyapatite (HA) particle size of < 100 nm.17nano-hydroxyapatite in dentrifrice is reported to function by directlyfiling up micropores on demineralized tooth surfaces. When it penetrates thepores of tooth tissues, it acts as a template in the remineralization processby continuously attracting large amounts of calcium and phosphate ions from theremineralization solution to the tooth tissue, thus promoting crystal integrityand growth.18 GSE contribute to mineral deposition on the superficial layer of thelesion by formation of insoluble complexes when mixed with bufferic phosphatesolution. GSE interact with proteins to induce cross-links by four differentmechanisms: covalent interaction, ionic interaction, hydrogen bondinginteraction and hydrophobic interaction.

In the present study GSE containing 95percent proanthocyanidin (as advocated by manufacturer) was used.In the present study 2mm thickenamel samples were prepared form crown portion of buccal or lingual / palatalsurface of selected maxillary and mandibular molars. The middle region of cutsample was chosen for window preparation in order to avoid both the enhancedfluoride surfaces of cervical enamel and the reduced fluoride surfaces coronally,the latter being due to the loss of original surface through wear. Acidresistant nail varnishes of different colors were used to cover the enamel surfaceleaving a window of enamel.19Inthis study demineralization similar to enamel subsurface lesion was created.

Anintermediate pH 5 was therefore employed for 4 days and composition similar tothe one employed by Featherstone in 1992 was used (Featherstone & Zero 1992). Standardizeddemineralization and remineralization solutions were prepared according to thecompositions shown in Table 4 below. Solutions were stored in sealed containersat room temperature throughout each of the experiments.  Table 4.Composition of demine ralizing and remineralizing solution. Demineralizing solution* Calcium 2.0 mmol/L Ca(NO3)2•4H2O mwt = 236.16 0.

4723 g/L Phosphate 2.0 mmol/L KH2PO4 mwt = 136.09 0.2722 g/L Acetic acid 75.

0 mmol/L CH3COOH mwt = 60.05 4.5083 g/L Remineralizing solution+ Calcium 1.5 mmol/L Ca(NO3)2•4H2O mwt = 236.16 0.

3542 g/L Phosphate 0.9 mmol/L KH2PO4 mwt = 136.09 0.1225 g/L KCl 130.0 mmol/L KCl mwt = 74.55 9.

6915 g/L Tris buffer 20.0 mmol/L (HOCH2)3CNH2 mwt = 121.14 2.4228 g/L *Adjusted to appropriate pH with50% NaOH after all ingredients were dissolved completely. +Standard volume prepared in 4L glassbeaker. Adjusted to pH 7.0 with concentrated HCl. Shelflife – no more than 7 days.

According to Zero,(Surface Micro Hardness) SMH measurement is a highly sensitive and reproduciblemethod used to evaluate in situ studies.21Using SMH it is possible to study early stages of enamel-dentinedemineralization22 or enamel remineralization.23 In addition,White (1987) found a high correlation (r2= 0.94; p < 0.01)between the remineralization of early carious lesionsmeasured by SMH. Thus, a pH-cycling model that promotes early enamel cariouslesion, which may be evaluated by SMH measurement using Vickers hardness wasemployed in this study. Thevalues of surface microhardness was more in samples of group III (CPP-ACPF)followed by group II (nano-hydroxyapatite) and group IV (Grape seed Extract).This may be because of the additional effect of Fluoride in CPP-ACPF whichmakes it more capable to remineralize the enamel as advocated by Reynolds et al.

12 Accordingto a study done by Shetty,Hegde & Bopanna addition of Fluoride to CPP-ACP shows improvedremineralization of initial enamel caries when compared with CPP-ACP.24Effectof remineralization by CPP-ACPF and nano-hydroxyapaptite was observed betterthan grape seed extract on enamel subsurface lesion. According to a study doneby Pavan et al. GSE significantly decreased the mineral loss and lesion depthof in vitro demineralized dentin.

This is not in accordance with this study. Itmay be due to difference in samples used in this study i.e. enamel samplesinstead of dentin samples.25                             Multiplegroup post hoc Tukey testshowed a significant difference between demineralized group (groups V) and allother groups.

  According to the resultsobtained in the present study, remineralizing agents i.e. CPP-ACPF containingTooth mousse plus and nano-hydroxyapatite containing Apagard pastesignificantly increased microhardness compared to demineralized group (groupsV). On further analysis by comparing individual groups in pairsit was observed thatthe comparison between (Group III) CPP-ACPF and (Group II) nano-hydroxyapaptitewas insignificant statistically. This is in accordance with the study done byAttia and Kamel.26Althoughthere is slight increase in microhardness with grape seed extract butcomparison between group V and group IV was insignificant statistically.Traditionally mature dental enamel is considered to be free of collagen, Acilet al.

showed that this is not the case and type I collagen is found in enamel;however, the concentration of collagen in enamel was considerably lower ascompared to that in dentin.27 Furthermore, Felszeghy et al. foundthat type X collagen is one of the candidate molecules present in the enamelmatrix, which might be involved in mineralization of enamel.28Considering these findings, it is not surprising to find small amount ofexogenous collagen cross-links produced by the positive effects ofremineralization of enamel defects by GSE in the present study. Based on dataobtained in our in vitro study, it may be proposed that GSE promotes theremineralization process of artificial carious lesions in the enamel. This isin accordance with another study done by Mirkarimi et al.29 Due tolimitations of invitro study effect of remineralizing agents on enamel lesionstill need further investigation.

ConclusionWithinthe limitations of this study we can conclude that all of the three materialsused CPP-ACPF, nano-hydroxyapatite, Grape Seed Extract substantially increasedhardness of the enamel lesion. But, CPP-ACPF can be considered as a betterremineralizing material of white spot or initial enamel caries.Nano-hydroxyapatite and Grape Seed Extract also increased microhardness butlesser than CPP-ACPF.  

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