Introduction gallic acid and polymeric and oligomeric procyanidins.


                     Dental caries is an
infectious disease and its initiation and progression depends on several
factors. Teeth are constantly going through cycles of demineralization and
remineralization. The ultimate goal of clinical intervention is the
preservation of tooth structure and prevention of lesion progression to the
point where restoration is required.

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                     While fluoride is an
established agent in promoting remineralization; there are other remineralizing
agents available such as synthetic nanohydroxyapatite, casein phosphopeptide
amorphous calcium phosphate with fluoride(CPP-ACPF) and grape seed

                        Hydroxyapatite is an important
biomaterial and a major component of the mineralized structure of the teeth and
bones . It is also an important bioceramic for medical and dental applications
(including dental implants, orthopedics, alveolar reconstruction and drug
delivery systems) due to its biocompatibility and biological and chemical
similarity to the bone structure. HA can be used for bone, cementum and
artificial root formation and can induce tooth remineralization1.

                       It has been observed that CPP-ACPF has the
ability to localize ACP at the tooth surface, which brings about buffering of
calcium and phosphate free ion activities, thereby helping to maintain a state
of super saturation with respect to tooth enamel negating demineralization and
enhancing remineralization with added fluoride effects. 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 monomeric
catechin and epicatechin, gallic acid and polymeric and oligomeric
procyanidins. Proanthocyanidins has been reported to strengthen collagen-based
tissues by increasing collagen crosslinks. There is evidence that PA increases
collagen synthesis and accelerates the conversion of soluble collagen to
insoluble collagen. PA has proved safe in various clinical applications and has
been used as dietary supplements. It has been shown that GSE positively affects the
remineralization process of root caries. Studies claim that collagen can serve
as a substrate for apatite formation3.

                        Therefore, our study
aims at quantitatively evaluating the enamel remineralizing potential of Nanohydroxyapatite,
CPP-ACPF, GSE using surface microhardness analysis(Vickers hardness test).


and Methods:


criteria for teeth under study were:-

Non carious teeth.

Teeth free of restorations.

Teeth with no cracks
on the crown as a result of pressure from extraction forceps.

Exclusion criteria
for teeth under study were:-

Teeth with abnormal
morphology such as hypoplastic defects.

Grossly carious teeth.

Damaged teeth


Infection control protocol:

were cleansed of visible blood and gross debris and were maintained in a
hydrated state during storage and placed in 3% sodium hypochlorite solution
diluted with saline in a ratio of 1:10 in the container. Microbial growth was eliminated by using an autoclave cycle for
40 min.


Sample preparation:

samples (2 mm thickness) were prepared from the buccal or lingual surfaces of
the teeth selected, using a double faced diamond disc mounted on a contra-angle
handpiece. After sample preparation windows were created (dimension of 5 mm × 5
mm) using adhesive tape and the sample was made completely resistant to acid
attack by coating nail varnish (nail varnish, Maybelline). After drying, the
adhesive tape was removed from the enamel using a sharp tipped instrument
exhibiting a rectangular area on the enamel surface.


Lesion preparation

of the enamel samples were then immersed in 40 ml of demineralizing solution
for a period of 4 days at a constant temperature of 37°C, in an incubator to
induce artificial caries formation, simulating an active area of
demineralization. Fresh preparation of demineralized solution was done.4The
demineralizing solution used was:

calcium 2.0 mMol/L

phosphate 2.0 mMol/L

acetic acid 75.0
mMol/L, pH 4.4

preparation of remineralized solution was done.4The remineralizing
solution used was:

1.5 mM/L calcium

0.9 mM/L phosphate

130 mM/L KCl in
20mM/L, Tris buffer, pH 7


Slurry preparation of CPP-ACPF, Nano-hydroxyapatite:

Slurry of CPP-ACPF
and Nano-hydroxyapatite were prepared by agitating the preparations in
deionized water in the ratio of 1:3 (Stookey et al. 2011). To achieve this, 17 gm of dentifrice was dispensed using high
precision balance weighing machine from the respective tube and then
transferred into two tubes to which 51 ml of deionized water was added and
stirred 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. The
Proanthocyanidins 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:

enamel slabs were randomly divided into five groups of 10 samples in each based
on the type of remineralizing agent to be used

I:  Sound enamel (no treatment)

               Control group consisted of tooth
sections immersed in normal saline.

II:  Demineralized and treated with
slurry of nano-hydroxyapatite 

                 (APAGARD m-plus standard

III: Demineralized and treated with slurry of CPP-ACPF (GC Tooth 

                Mousse Plus, 0.2% fluoride
(900ppm), 10% RECALDENT)

IV: Demineralized and treated with solution of grape seed extract.  

                (95%  proanthocyanidins, Brazil, Nutrija

Group V:  Demineralized and not treated with any


pH cycling model:

cycling model was adopted to simulate the dynamic process of demineralization
and remineralization that occurs in the oral cavity. Each of the enamel samples
were treated with the respective remineralizing agents for a period of 2 min,
following which the samples were immersed in 20 ml of 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. This was followed-up with treatment of the
samples again with slurries of the respective remineralizing agents for 2 min.
All the enamel samples were immersed in 30 ml of remineralizing solution (1.5
mM/L Ca(NO3)2•4H2O,
0.9 mM KH2PO4, 20 mM/L Tris buffer
pH 7.0 and 130 mM/L KCl) for a period of 17 h. The remineralizing solution was
replaced every 48 h and the demineralizing agent replaced every 5 days.

pH cycling was carried out for a period of 28 days. After the completion of the
process of pH cycling, all the groups of enamel samples were assessed for
surface microhardness using Vickers hardness test (Micro Vickers Hardness
tester, Matsuzawa Co., Ltd, Toshima, Japan). All the samples were embedded in
acrylic blocks.


Surface microhardness

surface microhardness of specimens was determined using digital microhardness
tester (MATSUZAWA Co., Ltd. Model – MMT X7, Japan) with a Vickers elongated
diamond pyramid indenter and a ×40 objective lens. A load of 100 g was applied
to the surface for 10 s.7Five indentations were placed on the
surface and the average value was considered. Precision microscopes of
magnification of ×400 were used to measure the indentations. The diagonal
length of the indentation was measured by built in scaled microscope and
Vickers values were converted to microhardness values.


Statistical analysis

results were analyzed by one-way analysis of variance (ANOVA). Multiple
comparisons between groups were performed by posthoc Tukey test.










Std. Deviation

Std. Error

95% Confidence Interval for Mean



Lower Bound

Upper Bound
























































Table 1 – Mean values
of microhardness (HV)
in different groups using oneway ANOVA



Sum of Squares


Mean Square



Between Groups





<.001** Within Groups 9253.665 45 205.637 Total 64725.160 49   Table 2- Presenting the statistically significant difference between the mean values (presented in table 2) of different 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 mean values of microhardness of one group with the other four groups using Post hoc test           Discussion: The casein phosphopeptides (CPPs) are produced from the tryptic digest of casein, aggregated with calcium phosphate and purified through ultrafiltration.Casein has the ability to stabilize calcium and phosphate ions. This technology was developed 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 the precise ratio of 144 calcium ions plus 96 phosphate ions and six peptides of CPP. The nanocomplexes form over a pH range of 5.0-9.0. A 1% CPP solution at pH 7.0 can stabilize 60 mM calcium and 36 mM phosphate.9, 10 Calcium interacts with CPP through the negatively charged residues of the peptides.11  The size and electroneutrality of the CPP nanocomplexes allows them to diffuse down the concentration gradient into the body of the sub-surface lesion.12, 13 Once 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 for apatit15; thus, on entering the lesion, the CPPs binds to the more thermodynamically favored surface of an apatite crystal face. In this study, CPP-ACPF was used. High remineralization potential of CPP-ACPF can be attributed to the synergistic anticariogenic effects of CPP-ACP and fluoride. The fluoride ions are adsorbed onto the surface of enamel crystals, inhibiting dissolution and increasing remineralization. With the use of low fluoride concentration as is present in CPP-ACPF (0.2% or 900 ppm of NaF), there is a complex localization of free calcium phosphate and fluoride ion activities, which helps in maintaining a state of supersaturation by suppressing demineralization.16 Thus CPP-ACPF (Tooth Mousse Plus™) is an excellent local slow-delivery system to treat the white spot lesion.  Nanoparticle Hydroxyapatite containing toothpastes were first introduced and tested in Japan in the 1980s (e.g. Apadent, Apagard, and others by Sangi Co., Ltd., Tokyo). They were used as anticaries agents by Japanese Government in 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.17 nano-hydroxyapatite in dentrifrice is reported to function by directly filing up micropores on demineralized tooth surfaces. When it penetrates the pores of tooth tissues, it acts as a template in the remineralization process by continuously attracting large amounts of calcium and phosphate ions from the remineralization solution to the tooth tissue, thus promoting crystal integrity and growth.18 GSE contribute to mineral deposition on the superficial layer of the lesion by formation of insoluble complexes when mixed with bufferic phosphate solution. GSE interact with proteins to induce cross-links by four different mechanisms: covalent interaction, ionic interaction, hydrogen bonding interaction and hydrophobic interaction. In the present study GSE containing 95 percent proanthocyanidin (as advocated by manufacturer) was used. In the present study 2mm thick enamel samples were prepared form crown portion of buccal or lingual / palatal surface of selected maxillary and mandibular molars. The middle region of cut sample was chosen for window preparation in order to avoid both the enhanced fluoride surfaces of cervical enamel and the reduced fluoride surfaces coronally, the latter being due to the loss of original surface through wear. Acid resistant nail varnishes of different colors were used to cover the enamel surface leaving a window of enamel.19 In this study demineralization similar to enamel subsurface lesion was created. An intermediate pH 5 was therefore employed for 4 days and composition similar to the one employed by Featherstone in 1992 was used (Featherstone & Zero 1992). Standardized demineralization and remineralization solutions were prepared according to the compositions shown in Table 4 below. Solutions were stored in sealed containers at 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 with 50% NaOH after all ingredients were dissolved completely. +Standard volume prepared in 4L glass beaker. Adjusted to pH 7.0 with concentrated HCl. Shelf life - no more than 7 days. According to Zero, (Surface Micro Hardness) SMH measurement is a highly sensitive and reproducible method used to evaluate in situ studies.21 Using SMH it is possible to study early stages of enamel-dentine demineralization22 or enamel remineralization.23 In addition, White (1987) found a high correlation (r2= 0.94; p < 0.01) between the remineralization of early carious lesions measured by SMH. Thus, a pH-cycling model that promotes early enamel carious lesion, which may be evaluated by SMH measurement using Vickers hardness was employed in this study. The values 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 which makes it more capable to remineralize the enamel as advocated by Reynolds et al.12 According to a study done by Shetty, Hegde & Bopanna addition of Fluoride to CPP-ACP shows improved remineralization of initial enamel caries when compared with CPP-ACP.24 Effect of remineralization by CPP-ACPF and nano-hydroxyapaptite was observed better than grape seed extract on enamel subsurface lesion. According to a study done by Pavan et al. GSE significantly decreased the mineral loss and lesion depth of in vitro demineralized dentin. This is not in accordance with this study. It may be due to difference in samples used in this study i.e. enamel samples instead of dentin samples.25                              Multiple group post hoc Tukey test showed a significant difference between demineralized group (groups V) and all other groups.  According to the results obtained in the present study, remineralizing agents i.e. CPP-ACPF containing Tooth mousse plus and nano-hydroxyapatite containing Apagard paste significantly increased microhardness compared to demineralized group (groups V). On further analysis by comparing individual groups in pairs it was observed that the comparison between (Group III) CPP-ACPF and (Group II) nano-hydroxyapaptite was insignificant statistically. This is in accordance with the study done by Attia and Kamel.26 Although there is slight increase in microhardness with grape seed extract but comparison between group V and group IV was insignificant statistically. Traditionally mature dental enamel is considered to be free of collagen, Acil et 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 as compared to that in dentin.27 Furthermore, Felszeghy et al. found that type X collagen is one of the candidate molecules present in the enamel matrix, which might be involved in mineralization of enamel.28 Considering these findings, it is not surprising to find small amount of exogenous collagen cross-links produced by the positive effects of remineralization of enamel defects by GSE in the present study. Based on data obtained in our in vitro study, it may be proposed that GSE promotes the remineralization process of artificial carious lesions in the enamel. This is in accordance with another study done by Mirkarimi et al.29 Due to limitations of invitro study effect of remineralizing agents on enamel lesion still need further investigation. Conclusion Within the limitations of this study we can conclude that all of the three materials used CPP-ACPF, nano-hydroxyapatite, Grape Seed Extract substantially increased hardness of the enamel lesion. But, CPP-ACPF can be considered as a better remineralizing material of white spot or initial enamel caries. Nano-hydroxyapatite and Grape Seed Extract also increased microhardness but lesser than CPP-ACPF.