there is evidence based on the remineralization potential of agents such as fluoride , casein phosphopeptide amorphous calcium phosphate ( cpp - acp ) , casein phosphopeptide amorphous calcium phosphate with fluoride ( cpp - acpf ) .
it has been observed that cpp - acp remineralized initial enamel lesions and has shown increased remineralization potential when used along with fluoridated toothpaste .
apf gel forms more fluoride in enamel than neutral gel and it is more efficient in reducing enamel demineralization of enamel blocks submitted to cariogenic challenge than the neutral one .
cpp - acpf is proven to produce significantly greater mean percentage of remineralization in comparison with cpp - acp .
mineral deposition throughout the lesion has been proven in relation to use of both cpp - acp and cpp - acpf .
it has been observed that gums comprising of cpp - acp nano complexes have a marked remineralization potential .
the cpp - acp has also been shown to remineralize enamel subsurface lesions in situ when delivered through oral care products .
the probable anticariogenic mechanism of cpp - acp is its 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 .
cpp - acpf due to its added fluoride content has shown improved ability to remineralize initial caries . in incipient enamel caries low concentrations of fluoride are able to penetrate deeper into the body of the lesion and bring about remineralization , hence sodium fluoride ( naf ) ( 200 ppm/0.044% ) has been opted for use in this study . the study aimed at quantitatively evaluating the enamel remineralizing potential of cpp - acp , cpp - acpf and naf using surface microhardness analysis ( vickers hardness test ) .
the teeth were cleansed of visible blood and gross debris and were maintained in a hydrated state during storage .
extracted teeth were placed in sodium hypochlorite solution diluted with saline in a ratio of 1:10 in the container with a secure lid to prevent leaking and labeled with the biohazard symbol .
teeth that do not contain amalgam restorations were preferred because they can be safely autoclaved . however , extracted teeth containing amalgam restorations , were immersed in 10% formalin solution for 2 weeks .
enamel samples ( 2 mm thickness ) were prepared from the buccal and lingual surfaces of the teeth selected , using a double faced diamond disc mounted on a contra - angle hand piece . following 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 .
the adhesive tape was removed from the enamel using a sharp tipped instrument exhibiting a rectangular area on the enamel surface .
a total of 50 enamel slabs were randomly divided into five groups of 10 samples in each based on the type of remineralizing agent to be used [ table 1 ] .
the division of groups each of the enamel samples were then immersed in 40 ml of demineralizing solution
( acetate 0.1 mol / l , calcium 0.1 mol / l , phosphate 0.1 mol / l , fluoride 0.1 mg / l , ph 5.0 ) for a period of 4 days at a constant temperature of 37c , in an incubator to induce artificial caries formation , simulating an active area of demineralization .
slurry of cpp - acp and cpp - acp along with fluoride were prepared by agitating the above mentioned preparations in deionized water in the ratio of 1:3 .
ph 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 individually immersed in 20 ml of demineralizing solution ( calcium 2.0 mmol / l , phosphate 2.0 mmol / l , acetic acid 75.0 mmol / l , ph 4.4 ) 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 individually immersed in 30 ml of remineralizing solution ( 1.5 mm calcium , 0.9 mm phosphate , 0.15 m kcl in 0.1 m tris buffer , ph 7 ) for a period of 17 h. the remineralizing solution was replaced every 48 h and the demineralizing agent replaced every 5 days . the 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 ) .
the surface microhardness of the 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. five indentations were placed on the surface and the average value was considered .
the diagonal length of the indentation was measured by built in scaled microscope and vickers values were converted to microhardness values .
multiple comparisons between groups were performed by posthoc tukey test [ tables 2 and 3 ] .
the teeth were cleansed of visible blood and gross debris and were maintained in a hydrated state during storage .
extracted teeth were placed in sodium hypochlorite solution diluted with saline in a ratio of 1:10 in the container with a secure lid to prevent leaking and labeled with the biohazard symbol .
. however , extracted teeth containing amalgam restorations , were immersed in 10% formalin solution for 2 weeks .
enamel samples ( 2 mm thickness ) were prepared from the buccal and lingual surfaces of the teeth selected , using a double faced diamond disc mounted on a contra - angle hand piece . following 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 .
the adhesive tape was removed from the enamel using a sharp tipped instrument exhibiting a rectangular area on the enamel surface .
a total of 50 enamel slabs were randomly divided into five groups of 10 samples in each based on the type of remineralizing agent to be used [ table 1 ] .
each of the enamel samples were then immersed in 40 ml of demineralizing solution ( acetate 0.1 mol / l , calcium 0.1 mol / l , phosphate 0.1 mol / l , fluoride 0.1 mg / l , ph 5.0 ) for a period of 4 days at a constant temperature of 37c , in an incubator to induce artificial caries formation , simulating an active area of demineralization .
slurry of cpp - acp and cpp - acp along with fluoride were prepared by agitating the above mentioned preparations in deionized water in the ratio of 1:3 .
ph 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 individually immersed in 20 ml of demineralizing solution ( calcium 2.0 mmol / l , phosphate 2.0 mmol / l , acetic acid 75.0 mmol / l , ph 4.4 ) 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 individually immersed in 30 ml of remineralizing solution ( 1.5 mm calcium , 0.9 mm phosphate , 0.15 m kcl in 0.1 m tris buffer , ph 7 ) for a period of 17 h. the remineralizing solution was replaced every 48 h and the demineralizing agent replaced every 5 days . the 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 ) .
the surface microhardness of the 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. five indentations were placed on the surface and the average value was considered .
the diagonal length of the indentation was measured by built in scaled microscope and vickers values were converted to microhardness values .
multiple comparisons between groups were performed by posthoc tukey test [ tables 2 and 3 ] .
in spite of the enamel remineralizing potential of saliva , by itself it fails to initiate the process of increasing the levels of calcium and phosphate . calcium and phosphate ions must first penetrate the surface layer of enamel , to bring about deposition of minerals through the body of the lesion , which confirms the reason for the cpp supported metastable calcium phosphate solutions of being such efficient remineralizing solutions .
majority of them are composed of calcium and phosphate with acetic acid or lactate . the main variation lies in concentrations of each component , which influences the final ph and the sample exposure time .
the ph employed varies from 3.5 to 5 and the time differs from 2 h to 21 days . in this study demineralization
various studies have used different methods to assess the process of enamel remineralization . the commonly used microhardness tests for evaluating enamel remineralization are vickers microhardness test and knoop microhardness test .
as this study put the focus on evaluation of surface microhardness of enamel , vickers surface microhardness test was used .
the values of surface microhardness indicate that remineralization of enamel is more in samples of group iii ( cpp - acpf ) followed by group iv ( naf ) and group ii ( cpp - acp ) . this may be because of the presence of fluoride in cpp - acp makes it more capable to remineralize the enamel .
however , group iv ( f ) showed a marginal increase in remineralization compared with group ii ( cpp - acp ) .
one - way anova shows that there is a significant difference between all the five groups .
however , multiple group posthoc tukey test showed a significant difference only between groups v with the other groups .
no other group showed significance when they were inter - compared . adopting the usage of low fluoride concentration as present in cpp - acpf ( 0.2% ) , there is a complex localization of free calcium phosphate and fluoride ion activities , which aid in maintaining a state of super saturation by suppressing demineralization . even though fluoride is present in tooth pastes manufactured in india and china ,
research has also proven that apf forms more fluoride and is more efficient in reducing enamel demineralization when compared with neutral gel .
sugar free chewing gums containing cpp - acp and citric acid result in higher remineralisation when compared with chewing gums without cpp - acp and citric acid and gums with citric acid alone .
research suggest that fluoride dentifrices in combination with daily applications of acidulated phosphate fluoride ( apf ) topical thrice and four times a day were the only treatments able to increase the surface microhardness of enamel .
the use of fluoride and the cpp - acp in recent years have been the best possible method in prevention of enamel caries and also in halting the progress of the existent enamel lesions .
cpp - acp containing remineralising paste has shown remineralization effect within the first three month evaluation period .
conversely , research also suggest that acp - cpp cream is nt as effective as fluoride in remineralizing early enamel caries at surface level .
combination of fluoride and acp - cpp does not provide any additive remineralization potential compared to fluoride alone .
however , it 's important to note that compliance of patient in oral hygiene maintenance and in home fluoride use is of utmost importance in prevention of enamel caries .
the results of this study showed that cpp - acpf remineralized enamel subsurface lesion better in comparison with naf and cpp - acp .
naf shown to be better than cpp - acp in enamel remineralization although the comparison between these two agents was insignificant statistically . with this basis
we can confirm that cpp - acpf is an excellent material for remineralization of white spot or initial enamel caries .
cpp - acp effectively remineralizes initial enamel caries , but to a lesser extent in comparison to cpp - acpf and naf.the addition of fluoride to cpp - acp shows improved remineralization of initial enamel caries when compared with cpp - acp and naf . 
 
cpp - acp effectively remineralizes initial enamel caries , but to a lesser extent in comparison to cpp - acpf and naf .
the addition of fluoride to cpp - acp shows improved remineralization of initial enamel caries when compared with cpp - acp and naf .
 aim : the aim of this study is to evaluate the enamel remineralization after treatment with three different remineralizing agents using surface microhardness assessment.materials and methods : this in vitro study involves 50 enamel samples divided into five groups of 10 samples each . 
 the positive control group consisted of intact enamel and a negative control group consisted of demineralized enamel samples . 
 all groups excluding the positive control group were subjected to demineralization following which three of these groups were remineralized using remineralizing agents ( casein phosphopeptide amorphous calcium phosphate [ cpp - acp ] [ gc tooth mousse ] , casein phosphopeptide amorphous calcium phosphate with fluoride [ cpp - acpf ] [ gc tooth mousse plus ] , sodium fluoride [ phos - flur ] ) . 
 the groups treated with remineralizing agents were subjected to ph cycling over a period of 28 days . 
 this was followed with assessment of surface microhardness ( micro vickers hardness tester , matsuzawa co. , ltd , toshima , japan).statistical analysis : one - way analysis of variance test and posthoc tukey test were conducted for multiple group comparison.results:there was an improved enamel remineralization in the group , remineralized using cpp - acpf in comparison with the other groups.conclusion:casein phosphopeptide with fluoride is a promising material for remineralization of enamel subsurface lesions . 
INTRODUCTION
MATERIALS AND METHODS
Infection control protocol
Sample preparation
Groups
Lesion preparation
Slurry preparation of CPP-ACP, CPP-ACPF and NaF
pH cycling model
Surface microhardness
Statistical analysis
DISCUSSION
CONCLUSION