University Journal of Dental Sciences. Original Research Paper

Transcription

1 A COMPARATIVE EVALUATION OF THE REMINERALIZING POTENTIAL OF FLUORIDE VARNISH, ACP-CPP- F & TCP-F ON ARTIFICIALLY DEMINERALIZED ENAMEL LESIONS : AN IN-VITRO STUDY Shilpa S Magar, Shaliputra Magar, Aparna Palekar, Siddharth Mosby 1 Senior Lecturer, Department of Conservative Dentistry and Endontics, Sri Aurobindo College of 2 3 Dentistry, Indore, Reader, Department of Oral Medicine & Radiology, Professor & Head, Department of Conservative Dentistry and Endontics, Modern Dental College And Research 4 Center Indore, Professor, & Head, Department of Oral Pathology and Microbiology, Maharanatapa Dental College Gwalior University Journal of Dental Sciences Original Research Paper ABSTRACT: This in vitro study was conducted on enamel blocks of human premolars with the aim of evaluating the remineralization potential of fluoride varnish, ACP-CPP-F, TCP-F on early enamel lesions. Methods: Thirty extracted premolars were collected for the study. The final sample size consisted of 60 derived from half section of 30 teeth. 60 enamel specimens were equally divided into four study groups: Group I: Fluoride varnish (fluoroprotectors Vivadent) treatment group, Group II: CPP-ACP-F paste treatment group Group III: TCP-F paste treatment group & Group IV: Control group (No surface treatment). Before and after ph cycling surface microhardness by Vickers hardness tester was assessed for all the specimens under 25 grams load for 5 seconds. Statistically analysis using oneway ANOVA followed by multiple comparisons Test (multiple Duncan test) was applied to detect significant differences at the level of p _ 0.05, between various surface treatments at different phases of study. Results: The mean hardness of TCP-F group has the highest hardness at the mean of VHN followed by ACP-CPP-F group has hardness at the mean of VHN. Fluoride varnish group has the higher mean hardness of VHN than the control group, whereas the hardness of control group has VHN which is the lowest among all the groups. Conclusion: The hardness of enamel carious lesion increases when the agents are applied in remineralization phase. Thus, it can be said that although the fluoridated toothpaste offered some protective potential, samples treated with CPP-ACP-F and TCP-F paste were best able to resist the acidic challenge. Key words : CPP, ACP, Fluoride, Demineralization, Enamel, Dentin, Remineralization Source of support : Nil Conflict of Interest : None INTRODUCTION : Prevention is one of the main concepts and forms the cornerstone of health care. Prevention has been recognized as one of the important characteristics of public health1. Dental caries is an acknowledged global health problem. Dental caries has been defined as the localized destruction of susceptible dental hard tissues by acidic byproducts produced from the bacterial fermentation of dietary carbohydrates2. There exists a delicate balance between the demineralization and remineralization process at the tooth micro-environment3. Prolonged multiple exposures to cariogenic food substrates results in an imbalance of this process leading to formation of an early enamel lesion, which may progress into caries or may remain stable and remineralize with time in plaque with a suitable composition4. Reports on remineralization of enamel and dentin in vivo can be found in early literature in the form of clinical observations of naturally occurring arrestment of carious lesion. Other in vitro studies have shown that artificially formed lesions in enamel can be partially remineralized as evidenced by increasing hardness or mineral content. Consequently, remineralization is accepted as a viable non-invasive approach for restoring carious teeth at least at the earlier stages of the disease. Currently with the increase in preventive dental care & awareness, the progression of caries is slow in the majority of individuals.5 Fluoride is a preventive agent that has mesmerized the dental research. It is one of the elements categorized by Navia as strongly cariostatic. The current concepts on the theories of the mechanism of action of fluoride indicate that fluoride works primarily via topical mechanisms by inhibition of demineralization and enhancement of remineralization. According to the World Health Organization expert committee the decline in dental caries prevalence observed in many countries can be attributed to the widespread use of toothpastes that contain fluoride6. Fluoride act by inhibiting demineralization through deposition of fluorapatite& promotes remineralization in the presence of adequate amounts of calcium and phosphate7,8 and also inhibits the growth of plaque micro-organism9. Thus several new agents have been proposed and recommended for personal and professional applications10. Casein Phosphopeptides (CPP) with the sequence Ser (P)-Ser (P)-Ser (P)-Glu-Glu- that

2 stabilizes amorphous calcium phosphate in solution is one such non fluoridated agent.11 Phosphopeptides of casein are produced from a tryptic digest of milk protein casein by aggregation with calcium and phosphate. They form nanocomplexes at the tooth surface, thus providing a reservoir of non-structurally bound calcium and phosphate ions which favor remineralization during a cariogenic attack12. TCP, or functionalized TCP, is a unique technology involving mechanochemical ball-milling of tri-calcium phosphate with simple organic ingredients that results in a functionalized or bioactive tri-calcium phosphate. Tri-calcium phosphate was selected over other calcium phosphate systems because it appears as a transitional phase in hydroxyapatite conversion; is biocompatible and bioactive in the oral cavity; and contains sites within its structure that can be activated with, for instance, simple organic molecules. Unlike other calciumbased additives, and by meticulous design, only low levels of functionalized TCP are needed to produce strong, acidresistant mineral nucleation without negatively affecting fluoride's proven benefits. Additionally, tri-calcium phosphate can be custom-tailored for a variety of oral care products.13 This study was done to evaluate the ability of topically applied Fluoride varnish, CPP-ACP-F and TCP-F on enamel mocrohardness after remineralization of artificial caries. MATERIALS AND METHOD: An in vitro study was designed and conducted in the Department of Conservative Dentistry and Endodontics, Modern Dental College & Research Centre. Ethical clearance for the study was taken from ethical committee for the use of human extracted teeth. Thirty premolars extracted from patients ranging in the age group of years, for orthodontic purpose, were collected for the study. The final sample size consisted of 60 derived from half section of 30 teeth. Extracted teeth washed in running tap water and placed in an antifungal solution containing 0.1% Thymol. The collected teeth obtained and screened for any macroscopically visible caries or surface defects. The selected teeth were cleaned using ultrasonic scaling tips. Freshly extracted premolars for orthodontic purpose and macroscopically caries free are selected for this study. Teeth having intrinsic stains, dental caries, gross surface defects like pits and cracks were be excluded. Rest of the teeth fulfilling all the selection criteria was stored in antifungal solution containing 0.1% Thymol until the experimental procedure is initiated. This procedure also helped to prevent dehydration. Radicular part of each tooth was removed. The coronal part of each tooth was then longitudinally sectioned mesio-distally into two sections using a high speed diamond tipped disc. Two enamel specimens were prepared. Rounded shaped impression of putty were made and self cured acrylic resin was poured on it; then each enamel block was embedded in on top of partially set, and allowed to set. An acid resistant nail varnish was applied around the exposed enamel surface leaving a window of 3 x 3 mm of enamel exposed at the centre. Different colour nail varnish was used with all groups to indicate its corresponding code. Sixty enamel specimens equally divided among four groups. All specimens were assigned to one of the following study groups: Group I: Fluoride varnish (fluoroprotectors Vivadent) treatment group, Group II: CPP-ACP-F paste treatment group, Group III: TCP-F paste treatment group &Group IV: Control group (No surface treatment). Acidic medium was prepared to have 0.05 M acetic acid, 2.2mM of calcium chloride and 2.2mM of sodium dihydrogen orthophosphate. To prepare one liter of acidic medium, 244mg calcium chloride and 344mg sodium dihydrogen orthophosphate were dissolved in one liter of distilled water. To this solution 2.78ml of acetic acid added. Potassium hydroxide pellets were used to adjust the ph of the solution to 4.5. All the enamel blocks were placed for ten days in bottles containing 20 ml of acidic medium solution. The acidic medium solution was freshly prepared and was changed in every 24 hours. After five days of acid demineralization of the exposed part of tooth, all the sections were result into the artificial enamel lesions and they were ready for the measurement of microhardness. Lesion microhardness measurement at first phase (lesion initiation phase): Immediately after acid dissolution, all the tooth sections were washed in distilled water and placed in a plastic cover. The tags with identification codes served as identification for each tooth section. The acid resistant nail varnish was removed using acetone. Vickers microhardness tester was used to evaluate micro hardness. A load of 25 grams was applied, for five seconds, for all the specimens. The microhardness numbers (VHN) of five indentations at spacing of 100 microns were taken and the average value was considered the mean base line micro hardness (SMH) of the corresponding specimen. The objective of base line surface micro hardness determination was to compare and calculate the changes that occurred after induction of enamel lesions and after ph cycling.

3 Second Phase (Paste application): After the lesion microhardness was be measured at end of first phase, the tooth sections were reapplied with nail varnish leaving the 3X3 mm window open and the caries like lesions in each test group. All specimens were subjected to the following surface treatment. Group 1 - A thin layer of fluoride varnish was applied, allowed to absorbed for 20 second and then air dried. Group 2 A generous layer of ACP-CPP Plus cream was applied by an applicators brush and left undisturbed for a a minimum of three minutes. Group 3 A layer of TCP- F cream was applied by and left undisturbed for a minimum of three minutes. The test group was treated for 5 days with respective tests agents twice a day for 3 minutes. Preparation of remineralizing solution: Synthetic solution was prepared by g/l gastric mucin g/l sodium chliride (NaCl) g.l calcium chloride (CaCl2. 2H2O) G/L potassium hydrogen phosphate (K2HPO4. 3H2O) g/l potassium chloride (KCL) 85% Lactic acid to adjust ph 7.00 at 37 C. PH CYCLING PHASE: A ph cycling regimen included alternative demineralization (three hours) and remineralization (21 hours) for five consecutive days. For the demineralization phase, demineralization solution used for the induction of enamel lesions was used and for the remineralization phase a synthetic saliva preparation was carried out. Lesion microhardness measurement at second phase: After ph cycling again surface microhardness was assessed for all the specimens under 25 grams load for 5 seconds. Statistically analysis: Statistically analysis using one-way ANOVA followed by multiple comparisons Test (multiple Duncan test) was applied to detect significant differences at the level of p _ 0.05, between various surface treatments at different phases of study. RESULTS : Results showed that the mean hardness of TCP- F group has the highest hardness at the mean of VHN followed by ACP-CPP-F group has hardness at the mean of VHN. Fluoride varnish group has the higher mean hardness of VHN than the control group, whereas the hardness of control group has VHN which is the lowest among all the groups(table no. 1). Group I compared with other groups shown that the mean difference compared with control group and and mean difference with ACP-CPP-F and TCP-F respectively. Group II shown the mean difference compared with control group, with group I and with Group III. Group III shown the mean difference compared with control group, and compared with group I and group II respectively. Control group has shown the highly mean difference with remaining three groups at the 0.05 level of significance( Table no.2) DISCUSSION : Minimally invasive dentistry adopts a philosophy that integrates prevention, remineralization and minimal intervention for the placement and replacement of restorations. Minimally invasive dentistry reaches the treatment objective using the least invasive surgical approach, with the removal of the minimal amount of healthy tissues. The onset of dental caries requires the establishment of necessary physicochemical conditions for mineral dissolution. Chemical agents can modify this progressive mineral loss caused by organic acids. Demineralization begins at the atomic level on the crystal surface inside the enamel or dentin and can continue unless halted with the end point being cavitation. Remineralization is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. Fluoride is known to promote remineralization, but is dependent on calcium and phosphate ions from saliva to accomplish this. Recent investigations have primarily focused on various calcium phosphate based technologies which are designed to supplement and enhance fluoride's ability to restore tooth mineral. The present study utilized an in vitro model to test hardness of artificial enamel lesions before and after remineralization on application of different fluoride containing agents named as ACP-CPPF, TCP-F and Fluoride varnish. Thirty premolars extracted for orthodontic purpose were included in the study. The purpose of choosing the premolar in patients from the age group of years is that the teeth in this age group have sound enamel and no ageing process has taken place. Also they are extracted for orthodontic purpose. The study consisted of two phases. The first phase represented lesion initiation (demineralization) and the second phase represented remineralization phase. The mean hardness of artificial lesions in each of the test and control groups was compared before and after the application of the remineralizing agents. In the present study, the specimens were kept in the demineralization solution for 72 hours at 37 C creating a subsurface demineralization of

4 approximately 150 microns width with an intact surface simulating an early enamel lesion. Artificial enamel carious lesions were produced by using solutions similar to those proposed by Tencate and Duijsters (1982)14. Artificial carious lesions are considered to be more reproducible than natural carious lesions and thus make the experimental model more reliable (Silverstone, 1983). They facilitate the testing of multiple areas in any lesion at different time intervals, in order to assess the remineralizing phenomena (Arends and Christoffersen, 1986)15. Prior to the start of the ph cycle, hardness of samples were studied and found to be statistically insignificant. This precluded the possibility of a sampling bias at baseline. In the interest of standardization, all of the specimens in the present study were subjected to 5 days of ph cycling which involved three hours of demineralization twice a day, with two hours of remineralization in between. During the ph cycle, the specimens were treated thrice daily; before the first demineralization, and before and after the second demineralization. This mimicked to a greater extent the daily eating patterns that occur in vivo. The present study used single section technique as used by Donly KJ and colleagues 16 that allows measurements of exactly the same tissue before and after treatment, which helps investigators to observe and quantitatively measure changes in lesion characteristics. The agents used for remineralization in the present study were Fluoride varnish, ACP-CPPF & TCP-F. The cariostatic efficacy of fluorides has been convincingly demonstrated and the recent decline in caries prevalence is primarily attributed to the increased use of fluoride agents. The mechanism by which fluoride increases caries resistance may arise from both systemic and topical applications of fluoride and can be broadly be grouped as follows: 1] Increased enamel resistance 2] Increased rate of maturation 3] Remineralization of incipient lesions 4] Interference with microorganisms 5] Improved tooth morphology. Fluoride enhances the rate of remineralization from calcium phosphate solutions. Remineralization of etched surfaces is accelerated by as much as four to five times by only 1 ppm F, and remineralization of white spot may be increased twofold. Perhaps the higher fluoride concentrations favor calcium fluoride formation, which prevents hydroxyapatite crystal growth, or rapid mineral deposition blocks diffusion pathways. The presence of plaque and pellicle is very important in remineralization. It can act as a reservoir for remineralizing agents, concentrating them near the lesion surface. As a reservoir plaque usually contains much higher concentrations of fluoride, calcium, and phosphate than saliva. The remineralizing property of fluoride has been demonstrated in various studies.1, 2 4, 8 12, 17 Various fluoride agents are available such as sodium fluoride, stannous fluoride. It also available in gel form which is used for topical application and tablet form used for systemic application. In present study fluoride varnish, a topical application agent has used. Fluoride varnish (Fluoroprotectors, Ivoclar Vivadent) is a protective varnish, containing 0.1% fluoride in a homogeneous solution; after the varnish has dried the concentration is approx. 10x higher. It has property of optimum flow and wetting properties, low viscosity Clear, colourless and has excellent adhesion on tooth surface because of its fast drying property. It is available in VivAmpoule. However, there are various reasons to seek alternatives to fluorides. These are as follows18 : 1. Fluoride is highly effective on smooth-surface caries; its effect would seem to be more limited on pit and fissure caries. 2. A high-fluoride strategy cannot be followed to avoid the potential for adverse effects (e.g. fluorosis) due to overexposure to fluoride. 3. Although fluoride presents no problems when used properly, among certain parts of the world, there has been the suggestion that fluoride exposure should be limited. Fluoride is known to promote remineralization, but is dependent on calcium and phosphate ions from saliva to accomplish this. Recent investigations have primarily focused on various calcium phosphate based technologies which are designed to supplement and enhance fluoride's ability to restore tooth mineral. This new calcium phosphate based technology is the alternative to fluorides and introduced as Non fluoridated remineralizing agent. These are Complexes of casein phosphopeptides-amorphous calcium phosphate, Amorphous calcium phosphate, Sodium calciumphosphosilicate (bioactive glass), Nanohydroxyapatite, Calcium carbonate carrier-sensistat, The trimetaphosphate ion Alpha-tricalcium phosphate, Dicalcium phosphate dihydrate, Xylitol carrier. 18,19 CPP-ACP is the acronym for a complex of casein phosphopeptides (CPPs) and amorphous calcium phosphate (ACP). Caseins are a heterogeneous family of proteins predominated by alpha 1 and 2 and beta- caseins. CPPs are phosphorylated casein-derived peptides produced by tryptic digestion of casein. CPP contains the Ser (P) Ser (P) Ser (P)

5 -Glu-Glu active cluster sequence which has a remarkable ability to stabilize calcium and phosphate in a metastable solution. In neutral and alkaline supersaturated calcium phosphate solutions, amorphous calcium phosphate (ACP) nuclei form spontaneously (Reynolds 1998b). The above phosphopeptide through the -Ser-sequence is able to bind to the forming ACP nanoclusters in metastable solutions. These CPP-ACP nanocomplexes which are of around 1.5nm radius prevent growth of the nanoclusters to the critical size required for nucleation and phase transformation (Cochrane et al 2008). Hence, the calcium and phosphate are maintained in high concentration forms that are readily available at the tooth surface without allowing their precipitation into calculus.19,20 For mineral deposition (remineralization) to occur within the body of a subsurface lesion, calcium and phosphate ions must first penetrate the surface layer of the enamel. The highly mineralized and charged nature of the surface layer poses a challenge for ion penetration. In the presence of CPP-ACP, the acid results in dissociation of the compound with the release of calcium, phosphate and formation of a neutral ion pair CaHPO4. Reynolds (1997) in an in vitro study analyzed the remineralizing solutions and process of remineralization using densitometry. It was revealed that the neutral ion pair along with the associated calcium and phosphate ions is able to actively diffuse through the surface layer and penetrate the protein/water filled pores of the subsurface carious enamel into the body of the lesion. Once within, the CaHPO4 dissociates, increasing the concentration of calcium and phosphate thereby increasing the degree of saturation with respect to hydroxyapatite. Formation of hydroxyapatite within the lesion produces acid and phosphate which diffuse out of the lesion. The efficacy of the CPP-ACP solution may be attributed to its ability to take up the released acid to dissociate and allow further remineralization through generation of CaHPO4. The bound ACP component acts as a reservoir for free calcium and phosphates and maintains the high concentration required to allow their diffusion into the lesion.21 Apart from the above described remineralization mechanism, CPP also plays a role in prevention of demineralization. This is affected by the ability of the CPP-ACP to buffer plaque ph during the acidogenic challenge. During the process, the compound CPP-ACP dissociates into calcium, phosphate and CaHPO4 and formation of these products offset any fall in ph and initiates remineralization. Hence the beneficial actions of CPP-ACP are twofold, by preventing enamel demineralization as well as promoting remineralization.20 Fluoride, when added to CPP-ACP, gives a synergistic effect on remineralization of early carious lesion. Elsayad reported that addition of fluoride to CPP-ACP could give a synergistic effect on enamel remineralization. Karlinsey found CPP- ACP with fluoride is found to be effective in remineralizing bovine enamel specimens. In present study CPP-ACP-F (GC mousse) with fluoride was used to test the remineralization. The major disadvantages of CPP-ACP includes that the CPP- ACP containing products over fluoride containing products, which pose a risk if ingested in a significant quantity (Hawkins et al, 2003). However, the potential side effects from the consumption of casein derived proteins in people with immunoglobulin E allergies to milk proteins should be taken into consideration. However, CPP-ACP is digestible even by individuals with lactose intolerance.21 Many in vitro studies have proved that CPP-ACP-F have a remarkable ability to remineralize caries While in one study authors have concluded that although cpp-acp can remineralize surface lesion, it is not effective in remineralizing the early enamel caries at the subsurface level.21 In early 2009, 3M ESPE introduced two dentifrice formulations, one with 5,000 ppm fluoride (Clinpro 5000 Anti-Cavity Toothpaste) and one with 950 ppm fluoride (Clinpro Tooth Crème), that contain a calcium phosphate ingredient called TCP. TCP, or functionalized TCP, is a unique technology involving mechanochemical ball-milling of tri-calcium phosphate with simple organic ingredients that results in a functionalized or bioactive tri-calcium phosphate. Tri-calcium phosphate was selected over other calcium phosphate systems because it appears as a transitional phase in hydroxyapatite conversion; is biocompatible and bioactive in the oral cavity; and contains sites within its structure that can be activated with, for instance, simple organic molecules. Unlike other calcium-based additives, and by meticulous design, only low levels of functionalized TCP are needed to produce strong, acid-resistant mineral nucleation without negatively affecting fluoride's proven benefits. Additionally, tri-calcium phosphate can be custom-tailored for a variety of oral care products. In late 2010, company introduced several patented, white fluoride varnish formulations containing TCP. In this case, TCP was custom-tailored for varnishes by milling it with a different organic ingredient. This resulted in greater mineral deposition onto dentin surfaces, providing

6 greater antihypersensitivity potential.26,27 Tri-calcium phosphate (TCP), present in some 3M ESPE products, is an intelligent material that provides bioavailable calcium and phosphate ions to the teeth, generating significant remineralization even when used in small amounts. It functions synergistically with fluoride to enable high fluoride bioavailability at normal and dry mouth conditions. Our exclusive milling process protects the TCP so that the calcium does not degrade the fluoride during storage. TCP functions in neutral or slightly basic ph environments. Different organic materials can be used to tailor the TCP system to a variety of topically applied oral care preparations, such as toothpaste, oral rinses and varnishes.28 TCP containing chewing gums are considered to be potential anticaries agents, because, as a consequence of their effect on salivary flow, they can induce increases in plaque and salivary ph. This higher ph can increase tooth mineral saturation during a challenge and thus decrease demineralization. Increased calcium and phosphate concentrations in the oral environment can also increase tooth mineral saturation in oral fluids. Recently, it was demonstrated that, when an acidic gum was used to increase the solubility of a calcium phosphate additive (alpha-tricalcium phosphate), a substantial increase in plaque fluid and saliva calcium and phosphate could be attained in subjects who chewed the gum following a sucrose rinse.57 The value (VHN) obtained during the initial base line micro hardness measurement in the present study were in the range of VHN , which satisfies the VHN range of normal enamel tissue. The surface micro hardness values for each group of the enamel specimens were decreased to at the end of 72 hours of demineralization (table no. 1) which is in accordance with the study conducted by Lata S. et al30 and Maupome et al31 The period for demineralization in the ph cycling phase is for three hours, which was to simulate the duration of demineralization (low cariogenic challenge) that occurs in the oral cavity. The test material was applied in enamel blocks twice a day to simulate the normal recommended daily oral prophylaxis. In the present study, after the ph cycling phase the mean SMH (VHN) was , for ACP-CPP-F group, for TCP-F group, and for the control group respectively. It indicates that combination of fluoride with ACP-CPP and TCP group provide additive remineralization potential when compared to fluoride varnish alone. In the present research, group III (TCP-F) has shown 27 % recovery of surface micro hardness, Group II (CPP-ACP F) showed 24% recovery, group I (Fluoride varnish) showed 18% recovery in 5 days whereas control group showed least recovery of surface micro hardness after remineralization. TCP-F was found to be effective than both CPP-ACP-F and fluoride varnish. Higher concentration of calcium ion in TCP- F in group III might have led to better remineralization capacity than CPP-ACP-F. Also both fluoride containing groups showed better performance than fluoride varnish. These results are in accordance with the studies conducted by Lata S. et al30 Inevitably, certain limitations are associated with in vitro studies such as the present study. These include the lack of saliva, plaque and the salivary pellicle which would be present in the oral cavity. These variations in the characteristics and quantities of these factors, which vary between individuals, need equalization in in vivo studies. Nevertheless, the lack of these factors could have influenced the results of the present study, because the main mechanism of action of CPP-ACP is for it to bind to the dental plaque and provide a reservoir of calcium and phosphate ions, thus inhibiting demineralization and enhancing the remineralization process (Rose, 2000; Reynolds, 2003).32 Another limitation may be that although the samples were randomly divided into the six treatment groups, to prevent a sampling bias, we cannot exclude the possibility that one group was dominated by multiple sections from the same tooth. Furthermore, some teeth can be expected to have greater susceptibility than others to demineralization due to the age of the donor and exposure to environmental factors such as fluoride. Also, the specimens in the ph cycle were subjected to repeated cycles of remineralization and demineralization (Ten Cate and Duijsters, 1982) which is more aggressive than the acid attacks that a tooth is exposed to, on a daily basis, in the oral cavity. Although there are limitations in the methodology in respect of this ph cycling model, and the use of the test reagents which were not used as recommended by the respective manufacturers', the conditions were at least standardized and repeatable.33 The results of this study, in conjunction with the results of the above-mentioned studies, suggest that CPP stabilizes and localizes ACP at the tooth surface. It maintains a super saturation level of the calcium and phosphate ions in close proximity to the tooth surface. It thereby buffers plaque ph, depresses enamel demineralization and enhances remineralization.

7 Table 1: One way ANOVA for Hardness after ph cyclin Table 2: Comparison of different surface treatment groups at different phase of the study CONCLUSION: Within the limitations, and based on the findings of this in vitro study, the following conclusions can be drawn: 1] Statistically significant differences were evident when comparing the hardness before demineralization and after remineralization between the Fluoride varnish, ACP- CPP-F, TCP-F and control group. 2] ACP-CPP-F and TCP-F both group are highly significant than fluoride varnish in their remineralizing potential. 3] TCP-F group is highly significant than ACP-CPP-F group in remineralizing potential. 4] The hardness of enamel carious lesion increases when the agents are applied in remineralization phase. 5] Thus, it can be said that although the fluoridated toothpaste offered some protective potential, samples treated with CPP-ACP-F and TCP-F paste were best able to resist the acidic challenge. These are valuable new products to complement the usual toothpaste. It is a highly effective means of practicing active caries prevention in patients susceptible to dental caries. However, one must bear in mind that remineralization in vitro may be quite different when compared to dynamic complex biological system which usually occurs in oral cavity in vivo. Thus direct extrapolations to clinical conditions must be exercised with caution because of obvious limitations of in vitro studies. REFERENCES: 1. Brudevold F, Tehrani A, Attarzadeh F, Goulet D, van Houte J. Effect of some salts of calcium, sodium, potassium, and strontium on intra-oral enamel demineralization. J Dent Res Jan; 64(l): Margolis HC, Moreno EC, Murphy BJ. Effect of low levels of fluoride in solution on enamel demineralization in vitro. J Dent Res Jan; 65(1): Silva MF, Burgess RC, Sandham HJ, Jenkins GN. Effects of water-soluble components of cheese on experimental caries in humans. J Dent Res Jan; 66(1): Featherstone JD, Glena R, Shariati M, Shields CP. Dependence of in vitro demineralization of apatite and remineralization of dental enamel on fluoride concentration. J Dent Res Feb; 69: Herkstroter FM, Witjes M, Arends J. Demineralization of human dentine compared with enamel in a ph-cycling apparatus with a constant composition during' de- and remineralization periods. Caries Res. 1991; 25(5): Duckworth RM, Morgan SN. Oral fluoride retention after use of fluoride dentifrices. Caries Res. 1991; 25(2): Nelson DG, Coote GE, Shariati M, Featherstone JD. High resolution fluoride profiles of artificial in vitro lesions treated with fluoride dentifrices and mouthrinses during ph cycling conditions. Caries Res. 1992; 26(4): Featherstone JD, Zero DT. An in situ model for simultaneoui assessment of inhibition of demineralization and enhancemen of remineralization. J Dent Res Apr; 71: Kong K. G. Role of fluoride toothpastes in a caries preventive strategy. Caries Res. 1993: (Suppl 1): Roberts AJ. Role of models in assessing new agents for caries prevention - non-fluoride systems. Adv Dent Res 1995 Nov;9(3): Schupbach P, Neeser JR, Golliard M, Rouvet M, G u g g e n h e i m B. I n c o r p o r a t i o n o f caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci. J Dent Res Oct; 75(10): Itthagarun A, Wei SH. Analysis of fluoride ion concentrations and in. vitro fluoride uptake from different commercial dentifrices. Int Dent J Aug; 46(4): Klein U,: Kanellis MJ, Drake D. Effects of four anticaries agents on lesion depth progression in an in vitro caries model. PediatrDent May-Jun; 21(3):

8 14. Tencate and Duijsters (1982) Alternative demineralization and remineralization o artificial enamel lesions.caries Res 1982;16: Arends and Christoffersen, The nature of early caries lesions in enamel. J Dent Res Jan;65(1): Cai F, Shen P, Morgan MV, Reynolds EC. Remineralization of enamel subsurface lesions in situ by sugar-free lozenges containing Casein Phosphopeptide- Amorphous Calcium phosphate. Aust Dent J Dec; 48(4): Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF. Johnson IH, Perich JW. Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat. J Dent Res Jun; 74(6): Goswami M Saha S. Chaitra TR (2012) Latest developments in non-fluoridated remineralizing technologies. Journal of ISPPD Jan-Mar 2012/Issue 1 Vol Amir Azarpazhooh and Hardy LimebackClinical Efficacy of Casein Derivatives : ASystematic Review of the Literature JADA 2008;139(7): El-Sayad I.I.1, Sakr A.K. 2, Badr Y.A Combining CPP- ACP with fluoride. A synergistic remineralization potential of artificially demineralised enamel or not? Proc. of SPIE Vol A Lata S. N O Varghese. Jolly Mary Varughese.(2010) Remineralization potential of fluoride and amorphous calcium phosphate-casein phospho peptide on enamel lesions: An in vitro comparative evaluation. J Conserv Dent/Jan-Mar 2010 Vol 13 Issue1 22. Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res Sep; 76(9): Hicks MJ, Flaitz CM. Enamel caries formation and lesion progression with a fluoride dentifrice and a calciumphosphate containing fluoride dentifrice: a polarized light microscopic study. ASDC J Dent Child Jan- Feb; 67(1) 24. Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC Remineralization of enamel subsurface lesions by sugarfree chewing gum containing Casein Phosphopeptide- Amorphous Calcium Phosphate.J Dent Res Dec; 80(12): Ramalingam L, Messer LB, Reynolds EC Adding casein phosphopeptide-amorphous calcium phosphate to sports drinks to eliminate in vitro erosion. Pediatr Dent Jan-Feb; 27(1): Bhat SS et al (2012) Incipient enamel lesions remineralization using casein phosphopeptide amorphous calcium phosphate cream with and without fluoride: a laser fluorescence study. J. Clinical pediatric dentistry 2012; 36(4): R.L. Karlinsey, A.C. Mackey et al (2010) Preparation, Characterization and In Vitro Efficacy of an Acid- Modified ß-TCP Material for Dental Hard-Tissue R e m i n e r a l i z a t i o n. A c t a B i o m a t e r Mar;6(3): Carolina Simonetti Lodi, Kikue Takebayashi Sassaki Evaluation of some properties of fermented milk beverages that affect the demineralization of dental enamel Braz Oral Res Jan-Mar;24(1): Goswami M Saha S. Chaitra TR (2012) Latest developments in non-fluoridated remineralizing technologies. Journal of ISPPD Jan-Mar 2012/Issue 1 Vol Lata S. N O Varghese. Jolly Mary Varughese.(2010) Remineralization potential of fluoride and amorphous calcium phosphate-casein phospho peptide on enamel lesions: An in vitro comparative evaluation. J Conserv Dent/Jan-Mar 2010 Vol 13 Issue Maupome G, Aguilar-Avila M, Medrano-Ugaldo H, Borges-Yanez A. In vitro quantitative micro hardness assessment of enamel with early salivary pelllicles after exposure to an eroding cola drink. Caries Res 1999;33: El-Sayad I.I.1, Sakr A.K. 2, Badr Y.A Combining CPP- ACP with fluoride. A synergistic remineralization potential of artificially demineralised enamel or not? Proc. of SPIE Vol A Pradeep k., 2. Prasanna kumar rao(2011)remineralizing agents in the non-invasive treatment of early carious lesions. Int J Dent Case Reports 2011; 1(2): CORRESPONDENCE AUTHOR : Dr. Shilpa S Magar Senior Lecturer, Department of Conservative Dentistry and Endontics, Sri Aurobindo College of Dentistry, Indore