BIP - A new method: measurement of microleakage volume using human, dog and bovine permanent teeth

This study investigates the feasibility of a different new approach to determining the microleakage volume associated with dental restorations and the relative marginal adaptation deficiency of dog, bovine and human permanent teeth in in vitro conditions. Also researched is the appropriateness of using dog and bovine teeth in in vitro studies rather than human teeth.

This preliminary investigation showed that the new microleakage volume measurement method may be a valuable new technique for the in vitro study of microleakage dynamics around dental restorations.

BIP Article

One of the most important problems of restorative dentistry today is the failure of restorative materials to completely bond to enamel and dentin, causing microleakage. (Tjan and Tan, 1991; Taylor and Lynch, 1992; Yavuz and Aydin, 2005). The aim of some researchers is to develop an in vitro model to replicate microleakage at a tooth/restoration interface (Iwami et al. 2000; Matharu et al. 2001; Yavuz et al. 2003; Yavuz and Aydin, 2005). The significant differences between these models and materials suggest that an ideal method for the determination of microleakage has not yet been established.

The aim of this study was to develop an in vitro model to determine the microleakage volume and ability to use dog and bovine teeth instead of human teeth in in vitro studies.

Materials and Methods

Sixty teeth were selected by Binocular Stereo Microscope (Olympus Co., Japan) for this study; 20 human, 20 bovine and 20 dog permanent canines were used (Figure 1).

The teeth in all groups were restored in the following way: The type 2 light-hardening glass ionomer cement (Variglass VLC, Dentsply, USA), following polymerization, all preparations were filled with a high copper amalgam (Cavex Avalloy, Cavex Co., Holland).

The specimens were subjected to thermo cycling between 5ºC ± 4ºC and 60ºC ± 4ºC for 500 cycles. After thermo cycling, was coated with a layer of nail varnish, melted utility wax (Derhami et al. 1995; Iwami et al. 2000; Gungor et al. 2003; Yavuz et al. 2003; Olmez et al. 2004; Yavuz and Aydin, 2005).

The methylene blue(MB) solution was prepared to a concentration of MB 4.75 g/l. A stock solution was prepared with a pH of 6.98 and 24 hrs did storage the specimens in the MB solution. Section made at the middle of the restorations to examine dye penetration.

In our study, the first stage was to evaluate the marginal leakage. The results were evaluated using the microleakage score; 0, 1, 2, 3, 4 and 5 (Figure 2, Table 1) (Yavuz and Atakul, 2000; Yavuz and Atakul, 2001a; Yavuz and Aydin, 2005).

The second stage was the measurement of the volume of the marginal gaps. Theoretically, the volume measurement method was created and applied as described below.

Absorption is the accumulation of dissolved molecules over the surface of a solid matter, the dissolved molecules could be atoms or ions of matter present in any solution of a gas, vapour or liquid phase. The phase, which allows the accumulation to occur on its surface, is known as the absorber (the teeth), the matter, which accumulates, is known as the absorbed (MB) (Davies, 1952; Nelsen et al. 1957; Aydin and Tez, 1996).

Absorption, in the liquid form, is usually measured using an indirect method. After the experiment, the teeth were dissolved in a 50% solution of nitric acid, the MB that filled the microleakage gaps dissolves into the solution and it’s the MB concentration is determined.

To draw the calibration graph, a part of the MB solution was taken and determined to have a wavelength of 664 nm in a spectrophotometer l_max (maximum absorption wavelength).

These measurements were then used to construct the calibration graph (Figure 3).

A=E C (A: absorption, E: molar absorption coefficient, C: concentration)

The molar absorption coefficient was determined to be 170.57 dm³.cm^-1.g^-1. After the calibration graph was drawn, the concentrations were calculated using the absorption values. In the absorption experiments of our study, the teeth were placed in MB solution, dye penetration at 37ºC for 24 hrs, (Yavuz and Atakul 2000; Yavuz et al. 2003; Yavuz and Aydin, 2005).

Afterwards, the tooth tissue around the restoration surfaces was removed in a block by making cuts 1.5 mm around the restored area (Figure 4 and Figure 5). These blocks were then dissolved separately in 50% nitric acid (Figure 6) and the amount of MB absorption was calculated using the previously created calibration graph. These values were converted to volume values (V=m/d) and the individual tooth volume values are reported in Table 2.

Findings

In our study, the first stage was dye penetration. We used Kruskal-Wallis non-parametric test. The differences were not statistically significant between all groups (p > 0.05).

In the second stage of this research, another three groups were evaluated statistically for microleakage volume at the restorations/cavity wall interface. The measurements in all groups were compared using One Way ANOVA Test and groups had no statistically significant differences (p > 0.05).

Discussion

Variety of methods have been used to research microleakage (Yavuz and Atakul, 2001b; Kelsey et al. 2004; Turgut et al. 2004; Ersin and Eronat, 2005). Some of these methods have been successfully used to in order to determine microleakage, but they are not quantitative methods. But as of today, there are no valuable quantitative methods applicable and valuable for the microleakage determination; we have above indicated the amount of microleakage through quantification.

In the stereo microscopic studies, the method is a semi-quantitative approach where the leakage is calculated solely at the surface where the section is made (Yavuz and Atakul, 2001b; Yavuz and Aydin, 2005). In our method, the researcher’s interpretation do not come into play in the determination of microleakage volume quantity and all surfaces where a leakage occurs between tooth/restoration material is quantitatively measured by a chemist.

When the three groups were compared using the One Way Anowa Test, there weren’t statistically significant difference (p > 0.05), also the dye penetration test (control groups), evaluated using the Kruskal-Wallis non-parametric test confirmed our method and the results had no significant difference (p > 0.05).

As a result of this study, the relative microleakage volume of dog and bovine permanent teeth to human permanent teeth in in vitro conditions was found to be similar. Within the limitations of these experiments, the following can be concluded; the ability to use dog and bovine teeth instead of human teeth in in vitro studies was confirmed.

It was also concluded in this preliminary investigation that the method of measuring the microleakage volume can be best a valuable tool for the in vitro study of microleakage dynamics around dental restorations, and this method can be use as a new technique for the determination of microleakage volume. Further work to establish the true scope of the model remains to be undertaken, but this preliminary investigation shows promise.

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