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Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 2  |  Page : 157-163
Evaluation of the remineralization potential of amorphous calcium phosphate and fluoride containing pit and fissure sealants using scanning electron microscopy


1 Department of Pedodontics and Preventive Dentistry, College of Dental Science and Research Centre, Lucknow, India
2 Department of Pedodontics and Preventive Dentistry, BBDCODS, Lucknow, India
3 Department of Pedodontics and Preventive Dentistry, Ahmedabad Dental College and Hospital, Ahmedabad, India
4 Department of Oral Medicine and Radiology, BBDCODS, Lucknow, India

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Date of Submission09-Mar-2011
Date of Decision30-Jun-2011
Date of Acceptance05-Mar-2012
Date of Web Publication3-Sep-2012
 

   Abstract 

Aim: To evaluate the remineralization potential of Amorphous Calcium Phosphate (ACP) and Fluoride containing pit and Fissure Sealants using Scanning Electron Microscopy.
Materials and Methods: Thirty maxillary first premolars were divided into three groups of ten each and were randomly selected for ACP containing (Aegis- Opaque White, Bosworth Co. Ltd.), Fluoride containing (Teethmate F1 Natural Clear, Kuraray Co. Ltd.), resin based (Concise- Opaque White, 3M ESPE Co. Ltd.) pit and fissure sealant applications. The Concise group served as a control. The teeth weresubjected to the pH-cycling regimen for a period of two weeks. After two weeks, the teeth were sectioned bucco-lingually into 4mm sections and were observed under Scanning Electron Microscope at 50X, 250X, 500X, 1000X and 1500X magnifications. The qualitative changes at the tooth surface and sealant interface were examined and presence of white zone at the interface was considered positive for remineralization.
Results: Both ACP containing (Aegis) and Fluoride containing (Teethmate F1) group showed white zone at the tooth surface-sealant interface. The resin based group (Concise) showed regular interface between the sealant and the tooth structure, but no clear cut white zone was observed.
Conclusion: Both, Aegis and Teethmate F1 have the potential to remineralize. Release of Amorphous Calcium Phosphate molecules in Aegis group and formation of Fluoroapetite in Teethmate F1 group, were probably responsible for the remineralization.

Keywords: Amorphous calcium phosphate, fluoride, pit and fissure sealants, remineralization

How to cite this article:
Choudhary P, Tandon S, Ganesh M, Mehra A. Evaluation of the remineralization potential of amorphous calcium phosphate and fluoride containing pit and fissure sealants using scanning electron microscopy. Indian J Dent Res 2012;23:157-63

How to cite this URL:
Choudhary P, Tandon S, Ganesh M, Mehra A. Evaluation of the remineralization potential of amorphous calcium phosphate and fluoride containing pit and fissure sealants using scanning electron microscopy. Indian J Dent Res [serial online] 2012 [cited 2020 Jul 6];23:157-63. Available from: http://www.ijdr.in/text.asp?2012/23/2/157/100419

   Introduction Top


Deep pits and fissures (Deep, narrow I-shaped and K shaped) which are not accessible for cleaning, have the highest caries susceptibility and have always remained an area of concern for the dentists. [1] Pits and fissure sealants are effective methods of sealing. These caries susceptible sites and undergone a major evolution since the initial studies were performed by Buonocore in 1955. [2],[3] .They are defined as the materials, which are placed in the pits and fissures of teeth in order to prevent or arrest the development of dental caries. [4] Several types of resin, both filled and unfilled, have been employed as a pit and fissure sealants. These resin systems includes Cyanoacrylates, Polyurethanes, and Bis-GMA. The main component of the fissure sealant is Bis-GMA resin. The success of the sealant technique is highly dependent on obtaining and maintaining an intimate adaptation of the sealant to the tooth surface. [5] However, microleakage between the sealant and tooth surface causing bacterial invasion and secondary caries is one of the biggest deterrent of the success of the pit and fissure sealants. Over the years, research have been conducted on sealant materials and methods to improve their properties like retention, marginal integrity etc. but today there is a need for materials with thequintessential potential of the remineralization. Calcium and Phosphate ions from saliva or other sources (dentifrices, chewing gums, beverages, remineralizing solutions and restorative materials), and Fluoride from topical or systemic sources, have the potential to remineralize the carious lesions or minimize caries development.

The anticariogenic property of the Fluoride is well documented and involves various mechanisms including the reduction of the demineralization, the enhancement of the remineralization, the interference of pellicle and plaque formation and the inhibition of microbial growth and metabolism. [6],[7],[8],[9],[10],[11],[12] The beneficial effects of the Fluoride arise from its incorporation in tooth mineral as fluoroapatite leading to the decreased solubility of the tooth enamel. [13] Fluoride-releasing materials may act as a Fluoride reservoir and may increase the Fluoride level in saliva, plaque and the dental hard tissues. However, clinical studies exhibited conflicting data as to whether or not these materials significantly prevent or inhibit secondary caries and promote remineralization. [6]

Amorphous Calcium Phosphate (ACP), thought to be a precursor in the formation of Hydroxyapatite, has also shown anti-cariogenic properties with remineralization potential [14],[15],[16],[17] ACP containing bioactive materials stimulates mineral growth by increasing the Calcium and Phosphate concentrations within the lesion, especially in the acidic oral environment, to levels that exceed those existing in ambient oral fluids thereby shifting the solution thermodynamic driving forces toward the formation of apatite. ACP can sustain these super saturation conditions over extended periods of time. [18],[19],[20],[21] ACP and Fluoride have been incorporated in restorative composites, glass ionomer cements, orthodontic adhesives, crown and bridge adhesives, pit and fissure sealants or as colloidal suspensions in toothpaste, chewing gum or mouthwash. [6],[5],[17],[18],[22],[23],[24],[25],[26]

The aim of the present study was to evaluate the remineralization potential of Amorphous Calcium Phosphate (ACP) containingand Fluoride containing pit and fissure sealants using Scanning Electron Microscope.


   Materials and Methods Top


Thirty healthy, non-hypoplastic, non-carious maxillary first premolars extracted for orthodontic reasons, were collected from the Department of Oral and Maxillofacial Surgery, MCODS, Manipal and were divided into the three groups of ten premolars each. Two groups were randomly selected for ACP containing (Aegis- Opaque White, Bosworth Co. Ltd.) and Fluoride containing (Teethmate F1- Natural Clear, Kuraray Co. Ltd.) pit and fissure sealants applications. In the third group, a plain resin based pit and fissure sealant (Concise- Opaque White, 3M ESPE Co. Ltd.) was applied which served as a control because it does not have any remineralizing potential. All the teeth were then polished with the pumice to remove any residual plaque or stain, especially from the occlusal surfaces, and were stored in artificial saliva at room temperature (22°C-25°C). Artificial saliva was prepared at Manipal College of Pharmaceutical Sciences, Manipal, and the composition was as follows: Sodium Carboxymethylcellulose 10.00 g, Potassium Chloride 0.62 g, Sodium Chloride 0.87 g, Magnesium Chloride 0.06 g, Calcium Chloride 0.17 g, Di- Potassium Hydrogen Orthophosphate 0.80 g, Potassium Di-Hydrogen Orthophosphate 0.30 g, Sorbitol 29.95 g, Compound Tartrazine solution 0.1 ml, Methyl P-Hydroxybenzoate 1.00 g and Water to 1 litre. [27] Prior to the placement of sealant, an acid resistant varnish coating was applied on all the surfaces of the teeth except on the occlusal surface wheresealant placement was to be done. Sealant placement in each group was then carried out according to the respective manufacturer's instructions.

Dynamic model (pH-Cycling Regimen)

Following sealant application, teeth in all the groups were subjected to the pH-cycling regimen (dynamic model) for a period of two weeks. pH- cyling was carried out at the Department of Oral Pathology, Manipal. The purpose of pH- cycling was to simulate the drop of pH occurring in the oral environment every day. All the groups were kept in artificial saliva for 11.5 hours and then in demineralizing solution for 0.5 hour. The composition of the demineralizing solution was: 3.0 m mol/liter CaC l2 , 1.8 mmol/liter K 2 HPO 4 , 0.1 mol/liter Lactic Acid and a mass fraction of 1% Carboxymethyl Cellulose. The pH of solution was adjusted to 4.0 by addition of KOH solution. [17],[28] After 0.5 hours, the teeth were removed and cleaned with double de-ionized distilled water and were kept back in artificial saliva till another cycle. So a total of two cycles were completed in 24 hours. The temperature was kept at 37°C, by keeping all the groups with solutions in the incubator.

Preparation of sample for scanning electron microscopic (SEM) evaluation

After 2 weeks, the teeth were removed and were prepared for SEM evaluation (JEOL JSM 840ASM, Japan). SEM evaluation was carried out at the Department of Metallurgy, National Institute of Technology (N.I.T), Surathkal. The root portions were cut using diamond disc. The crowns were sectioned buccolingually to allow studying of the marginal topography of the fissure sealant and crystal changes along the depth of the fissure. The thicknesses of the samples were kept at 4 mm as per requirements of theScanning Electron Microscope. Sectioning was done using Arkansas stone and a diamond disc, and care was taken to avoid damage to the filled sealants. The specimens were allowed to dry for 24 hours before subjecting them to the Platinum ion sputtering. This was done to prevent any moisture contamination during SEM study. The specimens were then mounted on brass specimen stubs using silver paste and a vacuum resistant adhesive. The specimens were mounted in such a way that the area to be studied faced upwards. Each stub contained three to four specimens. The mounted surfaces were then coated with a thin layer (30 μm thickness) of pure Platinum using an ion sputtering unit of 1000 volts for a period of 30 seconds. This ensured proper conducting surface to the non-conducting specimens. This tray containing the specimen stubs were later placed in the vacuum chamber of the Scanning Electron Microscope. The accelerating voltage was kept at 50kV and the angle of tilt and the aperture was adjusted according to the specimen to optimize the quality of micrograph. The surface was scanned and observed on the screen under 50X, 250X, 500X, 1000X and 1500X magnifications. The qualitative changes at the tooth surface and the sealant interface were observed, and the presence of white zone at the interface was considered positive for remineralization. [29]


   Results and Observations Top


Microscopic examination of each section was done at 50X, 250X, 500X, 1000X and 1500X magnifications. The qualitative changes at the tooth surface and the sealant interface were observed in each group.

All the tooth samples in Aegis and Teethmate F1 groups showed white zone at the tooth surface-sealant interface. The zone increased in width in the regions of fissure slopes with increased irregularities and it appeared to form from the sealant towards the tooth

surface. With the increase in the resolution, the clarity of the white border increased and a white irregular granular or globular zone was evident in few areas in the Aegis group [Figure 1], [Figure 2], [Figure 3] and [Figure 4]. However, these granular or globular zones were not evident in the Teethmate F1 group. In the Concise group, a smooth border between the sealant and tooth interface was observed, and complete merging of the sealant with the tooth surface present. However, there was no white zone present at the tooth surface-sealant interface in any of these samples under a higher magnification as well. [Figure 5] and [Figure 6].
Figure 1: Buccolingual section (Aegis) under SEM at 250X showing white zone at the tooth surface- sealant interface.

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Figure 2: Buccolingual section (Aegis) under SEM at 1500X showing white zone at the tooth surface- sealant interface. A white irregular granular or globular zone can also be seen.

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Figure 3: Buccolingual section (Teethmate F1) under SEM at 250X showing white zone at the tooth surface- sealant interface.

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Figure 4: Buccolingual section (Teethmate F1) under SEM at 1000X showing white zone at the tooth surface- sealant interface.

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Figure 5: Buccolingual section (Concise) under 250X showing smooth border at the tooth surface- sealant interface. White zone at the tooth surface-sealant interface was not evident.

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Figure 6: Buccolingual section (Concise) under 1000X showing smooth border at the tooth surface- sealant interface. White zone at the tooth surface-sealant interface was not evident.

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   Discussion Top


Pits and fissures are the most susceptible sites for caries. Pit and fissure sealants prevent these pits and fissures from becoming carious, and reduce the formation of major cavitated lesions. However, the major disadvantage of these sealants is microleakage between the sealant and the tooth, which leads to bacterial invasion and secondary dental caries. [30] Therefore, there is a need for a material with the quintessential potential of remineralization, so that the incidence of secondary caries due to microleakage can be reduced. The bioactive materials, which increases Calcium and Phosphate concentrations or release Fluoride within the lesion to levels, consistently above those that exist in ambient oral fluids, have the potential to cause the remineralization. In the present study, the remineralization potential of ACP containingand Fluoride containing pit and fissure sealants were evaluated using the Scanning Electron Microscopy.

The teeth were divided into 3 groups, (10 teeth in each group) and were randomly selected for A.C.P containing (Aegis), Fluoride containing (Teethmate F1), resin based (Concise) pit and fissure sealants applications. Aegis was chosen because, it is the only ACP containing sealant available. Teethmate F1 was chosen because, it was found to release the highest amount of Fluoride over a period of 30 days. [31] Concise group served as a control because it does not have any remineralizing potential. Following the sealant application, all the groups were subjected to pH-cycling regimen, [17] sectioned bucco-lingually and examined under the Scanning Electron Microscope at 50X, 250X, 500X, 1000X and 1500X magnifications. [32] The qualitative changes at the tooth surface and sealant interface were examined, and presence of white zone at the interface was considered positive for remineralization. [29]

Aegis group showed white zone at the tooth surface-sealant interface, with irregular granular or globular zone evident in few areas [Figure 2]. The width and irregularities of the white zone increased in the regions of fissure slopes, and it appeared to form from the sealant towards the tooth surface. It was considered as remineralization zone formed by the precipitation of Hydroxyapatite due to the release of Amorphous Calcium Phosphate at the sealant tooth interface.

A similar white granular pattern was observed under the Scanning Electron Microscope by Park et al., [29] They found that, as the amount of Hydroxyapatite was increased, the white granular zone was more clearly seen. They concluded that this zone could be the remineralization zone because there was no sign of remineralization in the control group. Ramalingam, Messer and Reynolds, [33] in their study observed adherent granules or globules under the SEM when they used CPP- ACP on enamel after erosion from the sports drinks and concluded that, they might represent the redeposit mineral phases following mobilization of Calcium and Phosphate from CPP- ACP.

Skrtic et al [17] reported A.C.P, to be an effective agent for remineralizing in vitro, caries- like enamel lesions artificially induced in extracted bovine incisors.

Langhorst et al, [19] evaluated the efficacy of an experimental orthodontic ACP composite to remineralize in vitro subsurface enamel lesions micro-radiographically and concluded that, ACP composite efficiently established mineral ion transfer throughout the body of the lesions, and restored the mineral lost due to acid attack. Recently, Silva et al, [14] evaluated the remineralizing potential of pit and fissure sealants containing Amorphous Calcium Phosphate (ACP) and/or Fluoride in artificially induced carious lesions on smooth enamel surfaces and concluded that, the sealants containing ACP provided either more efficient or similar remineralization, than the other sealants containing Fluorideand ACP containing pit and fissure sealants were able to promote remineralization of artificially induced carious lesions on the smooth enamel surfaces.

In the Teethmate F1 group also, similar white zone was observed at the tooth surface- sealant interface, and was considered as remineralization zone formed by the Fluorapetite [Figure 4]. Recently Silva et al, [14] in their study, also concluded that the sealants containing Fluoride were also able to promote remineralization of artificially induced carious lesions. However, various studies have shown conflicting results regarding the cariostatic and remineralization potential of the Fluoride releasing materials as compared to non-fluoridated restorative materials. [6] Some studies concluded that, the Fluoride releasing sealants reduced the enamel demineralization and increased the acid resistance of the enamel by the formation of Fluorapetite [34],[35],[36],[37],[38],[39] whereas, the other studies have shown contradictory results. [40],[41]

In the Concise group, it was observed under low resolution that, a smooth regular border existed between the sealant and the tooth without any evidence of the white zone. Under higher resolution, it showed a complete merging of the sealant with the tooth, without any appreciable gap at the interface. Similar observations have been made by Irinoda et al, [42] Simmonsen [43] and Ganesh and Tandon. [44] .

The Calcium Phosphate and Fluoride release from the sealants, occurs in two stages: (1) Penetration of water in the sealant and (2) Leaching of Calcium Phosphate and Fluoride from the sealant due to the hydrolysis. The rate of hydrolysis is inversely proportional to the pH of the solution i.e., at acidic pH, the rate of hydrolysis is greater and at neutral or alkaline pH, it is less. Also, these sealants are advantageous because the rate of release of Ca 2+ , PO 4 and F - ions is almost constant for a prolonged period of time, and they functions as both reservoir and sealer. [45],[46],[47],[48],[49]

Amorphous Calcium Phosphate (ACP) formation precedes the Crystalline Hydroxyapatite (HAP) formation, which is considered to be the final, stable product in the precipitation of the Calcium and Phosphate ions from neutral or basic solutions. The relatively high solubility of ACP and its conversion to HAP in aqueous media makes it a potential remineralizing agent in dental applications. It releases supersaturating levels of Calcium and Phosphate ions intralesionally and shifts the solution thermodynamic driving forces toward the formation of apatite. Also, when it is stabilized by ions that retard its conversion to HAP (primarily Pyrophosphate P 2 O7 4- , and to the lesser extent Mg 2+ ), a substantial release of Ca 2+ and PO 4 ions for sufficient periods of time can be achieved, which is an important pre-requisite for the remineralization. [20],[21],[48]

In the presence of low concentrations of Fluoride in solution (such as saliva or plaque fluid), the released Calcium and Phosphate ions are reprecipitated as Fluorapatite. The Fluoride ions leached into saliva from the fluoride-containing sealant protects the teeth against caries, by increasing the Fluoride content of the enamel and altering the metabolic activity of plaque in the vicinity of the sealants. Also, Fluorapatite formation is a sluggish reaction and requires low Fluoride concentrations because at high fluoride concentrations, Calcium Fluoride is formed in enamel instead of the Fluorapatite. Therefore, a long-term constant fluoride release can be achieved with these materials and thus, are more effective in the formation of the Fluorapatite. [6],[45]

Even though in the present study, both the ACP containing and Fluoride containing pit and fissure sealants showed potential for the remineralization. ACP has certain advantages over fluoride releasing materials: (1) Remineralization by Fluoride is a self-limiting surface phenomenon that prevents penetration of ions into the depth of the lesion. These materials had shown to deposit more mineral only in the initial 30% of the lesion. Thereafter, it occludes the surface pores and limits the extent to which the rest of the lesion may be repaired. ACP containing bio-active materials delivers mineral deeper into the enamel and deposit significantly more mineral overall than their fluoridated counterpart. Therefore, the depth of penetration of mineral by fluoridated bio-active materials puts it at a distinct disadvantage as compared to the ACP. [19] (2) The effect of the Fuoride ion may be limited by the availability of the Calcium and Phosphate in the mouth (plaque /saliva) whereas, ACP dissolves as Calcium and Phosphate ions thereby supersaturating the area locally and promoting hydroxyapetite formation. [48],[50],[51] (3) ACP containing materials are active "smart material" i.e., they release Calcium and Phosphate ions in the surrounding only when the pH drops below 5.5, and ceases when the pH rises above 5.5. It neutralizes the acid and buffers the pH, has a long life, does not wash out and is non-reliant on patient compliance whereas fluoride releasing materials are "passive smart materials" i.e., they release Fluoride when the pH of the oral fluid drops below 5.5 but, the release continues even when the pH rises above 5.5. Therefore, the release of Fluoride is not long lasting as compared to that of the ACP. [52],[53]


   Conclusion Top


In summary, both A.C.P containing (Aegis) and Fluoride containing (Teethmate F1) sealant groups showed signs of remineralization at the tooth surface-sealant interface. Aegis group showed white zone at the tooth surface-sealant interface with irregular, granular or globular zone evident in few areas. This was considered as remineralization zone formed by the precipitation of Hydroxyapatite due to the release of Amorphous Calcium Phosphate at the sealant tooth interface. In the Teethmate F1 group also, similar white zone was observed at the tooth surface-sealant interface and was considered as remineralization zone formed by the Fluorapetite. However, granular or globular zones were not evident in this group. The resin based group (Concise) showed regular interface between the sealant and the tooth structure but no clear cut white zone was observed.

 
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Correspondence Address:
Prashant Choudhary
Department of Pedodontics and Preventive Dentistry, College of Dental Science and Research Centre, Lucknow
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.100419

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