|Year : 2014 | Volume
| Issue : 1 | Page : 4-12
Comparative evaluation of remineralization, fluoride release and physical properties of conventional GIC following incorporation of 1% and 2% zinc acetate: An in vitro study
Prabhakar Attiguppe Ramasetty, Karuna Yarmunja Mahabala Bhat, Mythri Prasanna
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
|Date of Web Publication||18-Feb-2015|
Prabhakar Attiguppe Ramasetty
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka
Source of Support: None, Conflict of Interest: None
Context: Modern concepts of operative dentistry, popularly known as "Minimal intervention dentistry," propose that only the infected dentin should be removed, leaving the affected dentine that has the potential to remineralize. Mineralization of the tooth structure left in the cavity is likely to occur due to the application of fluoride- releasing material such as glass ionomer cement (GIC). Aim: The aim of this study was to evaluate whether the remineralizing potential of GIC can be enhanced with the addition of zinc acetate. Study Design: An experimental in vitro intergroup randomized control trial. Materials and Methods: This study consisted of three groups. Group I was conventional GIC, Group II was GIC with 1% zinc acetate and Group III was GIC with 2% zinc acetate. Thirty human premolars (10 in each group) extracted for therapeutic reasons were used in the first part of this study. Standardized class V cavities were prepared on the buccal and lingual surfaces. Teeth were sectioned longitudinally to get one buccal and one lingual half, in which one half was used as the control and the other half was used as the test specimen. Artificial caries- like lesions were produced and subsequently restorations of experimental specimens were performed. The depth of demineralization and remineralization were photographed under a polarized light microscope and quantified using a computerized imaging system. In the second part of the study, fluoride ions released were measured after 24 h and 7 days using an ion sensitive electrode. In the third part of the study, the setting time was calculated using Vicker's apparatus, as the time elapsed from the beginning of mixing till the time when the needle could no more make a circular indentation on the cement. In the fourth part, specimens prepared using Teflon moulds of standardized dimensions were subjected to shear bond strength analysis using a universal testing machine. Statistical Analysis: The obtained results were subjected to statistical analysis using Student's paired and unpaired 't' test, One way ANOVA, Post hoc Tukey's test. Results: In part one of the study, Group III showed the highest amount of remineralization, followed by Group II. All the groups showed statistically highly significant (P < 0.001) zones of remineralization; the difference between the groups was statistically highly significant (P < 0.001) as well. Part two of the study showed that fluoride release was highest in Group III, the difference being statistically highly significant from Group II and Group I at both the time intervals (P < 0.001). However, the amount of fluoride ions released on the 7 th day were lesser than that at 24 h in all the groups (P < 0.001). Parts three and four of the study showed that there was no statistically significant difference between the groups with respect to setting time and shear bond strength (P = 0.82 and 0.64, respectively). Conclusions: Incorporation of zinc acetate to glass ionomer at 2% w/w was effective in enhancing the remineralization property and fluoride release of the cement without affecting its setting time and shear bond strength.
Keywords: Fluoride release, glass ionomer cement, remineralization, setting time, shear bond strength, zinc acetate
|How to cite this article:|
Ramasetty PA, Bhat KM, Prasanna M. Comparative evaluation of remineralization, fluoride release and physical properties of conventional GIC following incorporation of 1% and 2% zinc acetate: An in vitro study. Int J Oral Health Sci 2014;4:4-12
|How to cite this URL:|
Ramasetty PA, Bhat KM, Prasanna M. Comparative evaluation of remineralization, fluoride release and physical properties of conventional GIC following incorporation of 1% and 2% zinc acetate: An in vitro study. Int J Oral Health Sci [serial online] 2014 [cited 2019 Nov 14];4:4-12. Available from: http://www.ijohsjournal.org/text.asp?2014/4/1/4/151613
| Introduction|| |
Minimal intervention dentistry (MID) focuses on the least-invasive treatment options possible in order to minimize tissue loss and patient discomfort. Concentrating mainly on prevention and early intervention of caries, MID's first basic principle is the remineralization of early carious lesions, advocating a biological or therapeutic approach rather than the traditional surgical approach for early surface lesions. Thus, remineralizing agents are part of a new era of dentistry aimed at controlling the demineralization/remineralization cycle, depending upon the microenvironment around the tooth. 
The practice of "Minimal intervention dentistry" has evolved as a consequence of our increased understanding of the carious process and the development of adhesive restorative materials.  New knowledge of caries progression rates has also led to substantial modification of restorative intervention thresholds and further handling of the disease. 
The scientific knowledge is now available to allow major changes to be introduced into the science and art of restorative dentistry. Thus, the profession should now be encouraged to adopt an entirely new attitude to the repair and restoration of demineralized tooth structure.
Glass ionomer cement (GIC) is one such adhesive restorative material developed by Wilson and Kent in 1972.  The two most important properties of GIC in the context of MID are adhesion to tooth structure and release of fluoride and other ions.  The release of calcium and hydroxide ions facilitate remineralization of the affected dentine and the presence of fluoride ions enables remineralization to occur at a pH of 4.5 instead of the normal pH of 5.5. In vitro, GIC has been found to exert an effect on enamel and dentin remineralization and to attenuate enamel demineralization in the neighborhood. 
It would be very beneficial if, by some means, this restorative material can be made more biologically compatible with the tooth structure by increasing the therapeutic properties.  One such attempt can be made by enhancing the fluoride- induced remineralization property of the GIC.
Salivary macro molecules associated with crystal growth inhibition have an ability to enhance remineralization.  This concept of crystal growth inhibitors potentially enhancing the remineralization has been proven by various studies. ,
Zinc is one such salivary macro molecule. It is an essential trace element. In the mouth, zinc is present naturally in plaque, saliva and enamel. It is implicated in biomineralization, where it influences both bone growth and mineralization. Effects of zinc on teeth are multifold. It is found to have an anticaries effect, antitartar effect, reduction of hydroxy apatite (HA) solubility, modification of calcium phosphate crystal growth, enhancing the effectiveness of fluoride and influence on demineralization and remineralization.
Thus, zinc is formulated into oral health products to control plaque, reduce malodor and inhibit calculus formation.  When zinc acetate was added to the remineralizing solution containing fluoride, findings demonstrated that zinc has the potential to enhance fluoride- induced remineralization of early carious lesions. 
However, while the effects of zinc on calculus and plaque growth have been reviewed extensively, its interaction with the dental hard tissues and possible role in de- and remineralization have received less attention.
Enhancement of remineralization with the incorporation of zinc is desirable. But, meanwhile, it should not have adverse effects on the physical properties of GIC. Thus, the aim of the present study was to evaluate whether incorporation of zinc acetate to universally used adhesive restorative material, GIC, enhances the fluoride-induced remineralization of the affected dentine. This study was also aimed at evaluating the effect of zinc incorporation on fluoride release and physical properties of GIC, viz setting time and shear bond strength.
| Materials and Methods|| |
Ethical clearance was obtained from the Institutional Review Board. The study consisted of three groups:
Preparation of experimental cements
The commercially available GIC, Ketac Molar from 3M ESPE, was used. The physical form of zinc acetate that was commercially available (Gababhai Chemicals, Baroda, India) was the crystal form. It was converted into fine powder by using a mortar and pestle. Two of the experimental cements were prepared by adding 1% and 2% zinc acetate to the powder of GIC [Table 1].
Weight ratios (%) of conventional GIC and zinc acetate particles were obtained by weighing the pre-determined quantity of both the powders and then by thoroughly triturating them incrementally by using a mortar and pestle to get a homogenous mixture.
Part I: Evaluation of remineralization effects
Thirty human premolars extracted for therapeutic reasons were used in this study. Only teeth that were free of caries and restorations and showed no evidence of white spots or cracks on the buccal or lingual surfaces were selected. 
Preparation of extracted teeth
After extraction, the teeth were polished with pumice on a prophylactic brush, autoclaved and immediately stored in cold distilled water at 4°C until use.
Preparation of cavity
Standardized class V cavities (one on the buccal and one on the lingual surface of each tooth) were prepared with a high- speed diamond flat cylinder bur using water as a coolant. The cavity prepared was 3 mm wide, 2 mm high and 1.5 mm deep,  and it was placed parallel to the cement-enamel junction (CEJ), with the preparation extending 1 mm above the CEJ.  The bur was replaced after every 5 th preparation. Each cavity was measured with a periodontal probe to ensure uniform size. 
The teeth were covered with two coats of acid-resistant nail varnish, except for the window, which included the cavity and a 2 mm rim of sound tooth structure surrounding the restoration. 
Preparation of samples for demineralization
Artificial caries- like lesions were created on the exposed cavities by suspending all teeth in an artificial caries system for 2 days (50 mL per sample). The caries solution consisted of 2.2 mM Ca +2 , 2.2 mM PO 4 -3 and 50 mM acetic acid at a pH of 4.4. The solution was kept at a temperature of 37°C under constant circulation. 
After 2 days, the teeth were removed from the artificial caries system. Thirty teeth were randomly divided into three groups. Each tooth was sectioned longitudinally in the occluso gingival direction to get one buccal and one lingual half, in which one half was used as the control and the other half was used as the test specimen randomly. The test specimens were preserved to be used later.
Control specimens were mounted on acrylic blocks for sectioning. A section of 100 μm thickness was obtained by cutting through the center of the cavity using a Silverstone-Taylor hard tissue microtome. The sections were mounted in glass slides to be viewed under polarized light microscopy using an Olympus dual- stage polarized light microscope (model BX-51; Dualmount Corporation, Minneapolis, MN, USA). Photomicrographs were made at 4x magnification. The demineralized areas were quantified with a computerized imaging system, the Image Pro Plus. 
Quantification of the lesions using the Image Pro Plus
The artificial lesion was quantified at three points. Lesion depth was measured from the surface of the lesion to the depth of the lesion, at D1, D2 and D3.  Final lesion depth for each section (in μm) was taken as the average of the three representative measurements from the surface to the depth of the lesion.
Preparation of samples for remineralization
The cavities of teeth segments were restored with respective experimental materials. All the specimens were preserved at room temperature for 24 h. After that, the excess restorative material was removed and polished.  The restored teeth were then stored in artificial saliva at 37°C for 30 days at pH 7 with constant circulation, which was changed every 48 h. The artificial saliva consisted of 20 mM NaHCO 3 , 3 mM NaH 2 PO 4 and 1 mM CaCl2 . 
At the end of 30 days, the specimens were removed from the artificial saliva  and were mounted on acrylic blocks. The 100 μm sections from individual specimens were oriented longitudinally on the glass cover slides for evaluation under polarized light microscope. The remineralized lesions were quantified using an imaging system, the Image Pro Plus, as described for demineralization.
Part II: Evaluation of fluoride release
To evaluate fluoride release, 10 specimens were prepared of each group, using cylindrical brass mould and kept in a 100% humid environment for 1 h. After that, the specimens were removed from the molds, immersed in individual plastic vials containing double-deionized water and incubated at 37°C.  After 24 h, 1 mL of distilled water was extracted from each container and analyzed for fluoride release after 1:1 dilution with TISAB (Total Ionic Strength Adjustment Buffer) using a fluoride ion selective electrode connected to an Orion 940 Ion analyzer.  The pellets were then kept immersed in new double-deionized water for 7 days. The double- deionized water was changed daily and the same procedure of fluoride ion estimation was repeated after 1 week.  Thus, fluoride ions released were measured after 24 h and 7 days.
Part III: Evaluation of setting time
The net setting time is the time measured from the end of mixing until the material sets. The test was undertaken in a climatic condition of 37°C using a Vickers needle (300 g, 1.12 mm) with a flat end that was plane and perpendicular to the long axis of the needle.
Ten specimens per group were prepared in a brass mold, having an inner diameter of 10 mm and 5 mm thickness, and positioned on an aluminum foil. The brass mold was then filled to a level surface with mixed experimental cement as per the manufacturer's recommendation and the upper surface was made flat by pressing down with a glass slide.
The assembly, comprising of mold, foil and cement, was placed in the cabinet, following which the indenter was carefully lowered vertically into the surface of the cement every 15 s. The net setting time was recorded as the time that elapsed between the end of mixing and time when the needle failed to make a complete circular indentation on the cement. 
Part IV: Evaluation of shear bond strength
Fifteen extracted sound human premolars were used, which were bisected in the mesio distal direction with a diamond disc under water cooling. Thus, we obtained 30 segments out of 15 teeth, which were randomly divided into three groups. Each group received 10 segments.
The teeth segments were embedded in the self-cure acrylic so that either the buccal or the lingual/palatal aspect of the tooth is visible. The exposed portion of the tooth along with the acrylic was made flat by grinding in a polishing machine using water as the coolant. It was ensured after grinding that the dentin of the tooth was exposed. This exposed dentinal surface was conditioned using 25% polycarboxylic acid for 25 s. The conditioned surface was rinsed and air dried.
The experimental cements were manipulated according to manufacturer's instructions and were placed on a smoothened dentinal surface by using a Teflon mould of standard dimension (diameter 2 mm and height 3 mm). The samples were stored in deionized water for 24 h and were subjected to the shear bond strength test using a universal testing machine (825, University Ave, Norwood, MA 02062-2643, USA). A cross-head speed of 1 mm/min was applied to each specimen until bonding between the dentin and the GIC failed. The values were calculated as Newtons (N) and were later converted to Mega Pascals (MPa). 
The results obtained were subjected to statistical analysis using Student's paired and unpaired "t" test, one-way ANOVA and post hoc Tukey's test.
| Results|| |
All the groups showed significant zones of remineralization after restorative intervention (P < 0.001) [[Table 2], Graph 1]. When the percentage of remineralization occurring in the three groups were compared, there was a statistically highly significant difference between the groups (P < 0.001). Group III showed the highest amount of remineralization (47.0-63.9%), followed by Group II (39.8-59.8%), the least being in Group I (30.4-40.6%) [Table 3], Graph 2]. Group-wise comparisons showed a highly significant difference between Groups I- II and Groups I-III, also a significant difference between Group II-III [Table 4], [Figure 1], [Figure 2], [Figure 3] and [Figure 4].
|Table 2: Descriptive analysis showing the mean, standard deviation and the significance (P) value of difference in the demineralization and remineralization among the control and experimental groups |
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|Table 3: Descriptive statistics showing the mean and standard deviation and the percentage remineralization among experimental groups |
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|Table 4: Descriptive statistics showing the intergroup comparison of the mean difference in percentage of remineralization and significance P value among the experimental groups |
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Another important finding of this study was that there was a highly significant difference in the amount of fluoride release between the three groups, both after 24 h and after 7 days. At the end of 24 h, Group III showed the highest amount of fluoride release, followed by Group II and Group I (P < 0.001 for both intra- and intergroup comparisons). When fluoride ions released on the 7 th day were compared with that released after 24 h, there was a statistically highly significant decrease. However, the same trend was maintained even after 7 days, viz Group III> Group II > Group I [Table 5] and [Table 6], [Graph 3].
|Table 5: Descriptive statistics showing the intragroup comparison of the mean, t value, level of significance P value and intergroup comparison of F value and P value of difference in fluoride release among the experimental groups after 24 h and on the 7th day |
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|Table 6: Descriptive statistics showing group-wise comparison of the significance P value of differences in fluoride release among the various experimental groups after 24 h and on the 7th day |
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[Table 7] and [Table 8] show the setting time and shear bond strength of the three different groups. There was no statistically significant difference observed between the groups with respect to both the physical parameters [Graphs 4 and 5].
|Table 7: Descriptive statistics showing mean, standard deviation, F value and significance P value differences between groups with respect to setting time |
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|Table 8: Descriptive statistics showing mean, standard deviation, F value and significance P value differences between groups with respect to shear bond strength |
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| Discussion|| |
Globally, dental caries ranks among the most prevalent diseases of humans.  Realization that dental caries is a dynamic biochemical event at a micron level that is reversible in the initial stages has changed the way the profession recognizes the caries process.  Today, the most effective preventive approaches focus on decreasing the tooth's susceptibility to caries by using various fluoride combinations and with the use of dental sealants. Despite their existence, these methods of caries prevention are not used in many areas either because of their unavailability or because of their high cost. As a result, the caries process frequently progresses beyond the reversible stage. In communities with few dental facilities and care providers, alternative measures for treating caries are often used. One such alternative, called the Alternative Restorative Treatment or ART, is to perform minimal cavity preparation using only hand instruments followed by restoration of the cavity with an adhesive filling material, such as GIC. 
Previous studies have shown that fluoride-releasing conventional GICs have a high cariostatic effect.  This is partly attributed to the enhancing effect of fluoride on calcium phosphate precipitation; hence, remineralization.  However, if this remineralizing property of GIC can be enhanced, it would be an added advantage.
In this direction, this study was carried out to evaluate whether incorporation of zinc into conventional GIC makes it a more effective remineralizing adhesive restorative material.
Evaluation of remineralization effects
Because dentin is less mineralized than enamel, diffusion through dentin is more than diffusion through the enamel, owing to the higher porosity, resulting in a larger mineralizing effect.  Also, the current consensus is that caries beyond the dentino-enamel junction (DEJ) should be treated with restorations, where as lesions up to that point should receive preventive care.  Thus, in this study, artificial carious lesions were created on the dentinal surface (class V cavities extending into the dentin).
When test specimens were evaluated for zones of remineralization using an imaging system, the Image Pro Plus, it was observed that GIC with 2% zinc acetate showed the highest amount of remineralization, followed by GIC with 1% zinc acetate.
Enhanced remineralization in the presence of zinc is in accordance with the previous studies conducted by Featherstone et al.,  Tencate et al.,  Lippert  and Lynch et al. 
The most likely explanation for the enhanced remineralization in the presence of combination of zinc and fluoride is the smaller increase in surface zone porosity during remineralization, as zinc retards the crystal growth at the surface thereby facilitating ingress of more ions. Thus, zinc allows preferential deposition of mineral in the deeper parts of the lesions enhancing lesion body remineralization. 
However, plain GIC also showed some amount of remineralization, as supported by previous studies.  It has been reported that fluoride levels in the plaque are elevated substantially for months after a GIC filling has been placed.  Also, silica released from the GIC restoration could have a mineralization promoting effect, as has been reported from crystal growth studies of hydroxyapatite. 
Evaluation of fluoride release
It is well established that during the fluoride elution process, at least two reactions occur. The first is a short-term initial elution occurring rapidly, but ceasing after some time. The second reaction is a long-term, prolonged and more slowly occurring elution process. 
Many authors have confirmed the occurrence of an initial high release from glass ionomers over the first 24 h, probably due to the burst of fluoride released from the glass particles when reacting with the polyalkenoate acid during the setting reaction. Further, according to Momoi and McCabe, the most rapid release of fluoride occurred during the first 7 days for both conventional and light- activated GICs. 
Considering these previous studies, in the present study, it was deemed necessary to evaluate the influence of the addition of zinc to the fluoride-releasing properties of conventional GIC at two time periods, 24 h and 7 days.
An interesting finding in the study was concentration-dependent increase in fluoride release among zinc-containing glass ionomers when compared with conventional glass ionomer alone. This may be attributed to the physical presence of zinc acetate in the matrix of the glass ionomer system, which may have created a pathway for the release of fluoride ions. Or, the presence of zinc acetate may have led to an increase in the solubility of the cement, which might have in turn facilitated a greater fluoride ion release.
This is in accordance with the study conducted by Osinaga,  who added zinc sulfate to glass ionomer-based cements to study its influence on physical and antibacterial properties and zinc and fluoride release of GIC. This study showed that the addition of zinc enhanced fluoride release by GIC. Also, there was an increase in solubility; however, it was maintained below the requirements of the ISO 7489 and, moreover, the flexural strength of the cement was not affected.
Even the study conducted by Ibrahim M Hammouda to assess the interaction of zinc with glass ionomer restorative materials concluded that zinc addition increased the amount of fluoride released from the glass ionomer restorative materials, which was reflected in bacterial growth inhibition. 
Another important observation from this study was the "initial fluoride burst effect," which occurred during the first 24 h, in agreement with Preston and others.  The pattern of fluoride release was found to be similar for all the groups. This initial fluoride "burst" effect is desirable, as it will reduce the viability of bacteria that might have been left in the inner carious dentin. Furthermore, it may be expected that fluoride will facilitate the remineralization of the uninfected inner dentin and demineralized enamel. ,
Evaluation of setting time and shear bond strength
Findings of the present study demonstrated that addition of zinc had no effect on the physical properties of GIC, viz setting time and shear bond strength. This is the first study that has evaluated the effect of incorporation of zinc on setting time and shear bond strength of GIC.
Measurement of the bond strength of GIC to enamel and dentin is complicated by the brittle nature of the GIC. Laboratory bond strength tests invariably result in cohesive failure of the GIC, rather than failure within the ion exchange layer.  Consequently, the true strength of the ion exchange layer is not known.  Thus, shear bond strength is more a measure of the tensile strength of the cement itself as fractures are usually cohesive within the cement, leaving the enriched residue attached to the tooth.
The mean values of shear bond strength observed in this study were well coinciding with the commonly reported values, which are in the range of 3-10 MPa, i.e., approximately the cohesive strength of GIC.
Analyzing all the four parameters tested in this study, it can be stated that the addition of zinc conferred remineralizing properties to the conventional GIC. It may be drawn that fluoride release alone from the conventional glass ionomer may not be sufficient to cause subsurface remineralization. Incorporation of zinc was beneficial, and it acted as an adjuvant in enhancing the fluoride-induced remineralization. Also, the addition of zinc increased the fluoride release from GIC, which might have further enhanced the remineralization property of GIC.
| Conclusions and Points of Clinical Relevance|| |
- Incorporation of 2% zinc acetate to conventional GIC enhanced the remineralization property of GIC
- The incorporation of zinc acetate to GIC also enhanced its fluoride-releasing ability at the end of study periods
- The pattern of fluoride release exhibited by the zinc acetate- incorporated glass ionomer was in tandem with that of conventional GIC during the experimental period
- Setting time and shear bond strength of GIC were not affected by incorporation of zinc acetate to GIC.
Within the limitations of the present study, it may be concluded that the incorporation of zinc acetate to glass ionomer at 2% w/w may be an effective tool in enhancing the remineralization of demineralized dentin. Hence, we recommend zinc acetate-modified GIC as a suitable restorative material for use in minimally invasive dentistry, which is the key phrase of today's preventive dental practice.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]