Resin-modified glass ionomer containing calcium glycerophosphate: physico-mechanical properties and enamel demineralization

Abstract Sources of calcium and phosphate have been added to dental restorative materials to improve their anticaries effect. Objective This study evaluated the effect of adding calcium glycerophosphate (CaGP) to resin-modified glass ionomer cement (RMGIC) on the physico-mechanical properties, ion release, and enamel demineralization. Material and Methods: Specimens were fabricated for each experimental group: RMGIC without CaGP (Control), RMGIC with 1, 3 and 9% CaGP. To determine the release of fluoride (F), calcium (Ca) and phosphorus (P), six specimens were immersed in demineralization and remineralization solutions for 15 days. In another experimental trial, the following physico-mechanical properties were evaluated at time intervals of 1 and 7 days after fabrication: compressive strength (n=12), diametral tensile strength (n=12), surface hardness of material (n=6) and the degree of conversion of monomers (n=8). To study enamel demineralization, specimens (n=12) were attached to enamel blocks and submitted to pH-cycling. Subsequently, surface and cross-sectional hardness and the concentration of F, Ca and P in enamel were determined. Results The addition of CaGP to RMGIC led to higher mean release of F, Ca and P when compared with control (p<0.001). Mechanical properties were within the range of those of the ionomer cements after addition of 1% and 3% CaGP. The degree of conversion did not differ between groups at the 1st and the 7th day (p>0.439). The addition of 3% and 9% CaGP reduced mineral loss and increased F, Ca and P in the enamel when compared with control (p<0.05). Conclusion The addition of 3% CaGP in RMGIC increased the release of F, P and Ca, reduced enamel demineralization, and maintained the physico-mechanical properties within the parameters for this material.


Introduction
Dental restorative materials with superior clinical performance regarding the occurrence of secondary carious lesions in primary teeth release fluoride ions (F − : nominated as F) into the oral medium. 1 Among these, the glass ionomer cements (GICs) release large quantities of F and they are the material of choice for use in patients with high caries activity.
Nevertheless, GICs have reduced fracture strength 2 and esthetic appearance 3 compared with composite resin. As the process of demineralization and remineralization depends on the presence of calcium (Ca 2+ : abbreviated as Ca) and phosphate (PO 4 3− : abbreviated as P) ions in the medium, 4 compounds containing amorphous calcium phosphate stabilized by casein phosphopeptides (CPP-ACP) have been added to improve the anticariogenic potential of GIC material. 5 These results were associated with the release of F, Ca and P ions by the GIC. Notwithstanding, the incorporation of CPP-ACP into the GICs decreased their diametral tensile and compressive strength. 5 Another calcium phosphate with anticariogenic action, calcium glycerophosphate (CaGP), is an organic phosphate with affinity for tooth enamel. CaGP provides Ca and P ions increasing their levels into the plaque 6,7 and with a plaque-pH buffering effect.
In some studies, CaGP (50% α-and 50% β-isomer) has been added to low-fluoride toothpastes showing an improvement in their anticaries effect. [8][9][10] This effect was related to its capacity of adsorption onto the enamel surface and increased ionic activity of neutral species, such as CaHPO 4 0 and HF 0 , in dental biofilm. 4,8 The neutral species have a higher diffusion coefficient into enamel than that of charged species. 4 Based on the above studies, the addition of CaGP to the GICs would be another alternative to increase their anticariogenic capacity. The null hypothesis of the study was that the addition of CaGP to the RMGIC would not alter the release of F, Ca and P, its physico-mechanical properties and the effect on enamel demineralization.

Material and methods
Preparation of the RMGIC mixture with CaGP

Surface hardness analysis
Six disc-shaped specimens (5 mm diameter × 2 mm thickness) 11 of each material were manufactured and maintained in relative humidity for 1 day.
Subsequently, five indentations 500 µm equidistant from each other were made on the top surface ( Figure   1), using a microhardness tester (Micromet 5114, Buehler, Lake Bluff, IL, USA), Knoop diamond indenter, 100 g load applied for 10 seconds. 11 After this, the specimens were stored in relative humidity for 7 days, after which the hardness test was repeated.

Degree of conversion (DC) of the monomers of RMGIC
RMGICs were manipulated (n=4/group), as previously described, inserted between two glass In the RMGIC+CaGP spectra, the absorption peak at 1540 cm -1 is not well-defined. In these cases, it was necessary to fit the curves using Gaussian functions to evaluate the Abs(C=C)/Abs(COO − ) ratio ( Figure 2B).

Preparation of enamel blocks
Enamel blocks (4×4×3 mm) were prepared from freshly extracted bovine incisors and the enamel surface was ground flat, resulting in the removal of a depth of approximately 120 mm of the enamel. 15 After polishing, cross-sections were cut at 1 mm from the border of the block to obtain 4×3×3 mm enamel slabs. For selection purposes, the initial surface hardness (SH 1 ) was measured (Knoop) by making five indentations spaced 100 μm from each other, at a distance of 300 μm from the sectioned enamel border using a hardness tester (Micromet 5114, Buehler, Lake Bluff, IL, USA), with 25 g load applied for 10 seconds.   ppm F, pH 7.0 -1.1 mL/mm 2 ) solution, completing one-day cycle. 15 The block/specimen sets were always washed with distilled/deionized water for 30 seconds and dried with absorbent paper between the changes of solutions. After the 5 th day, the remineralization solution was renewed, in which the block/specimen sets remained for 48 hours.

Enamel hardness analysis
After pH-cycling, the final surface hardness on enamel (SH 2 ) was determined as described for SH 1

Analysis of F, Ca and P in enamel
The other halves of the enamel slabs were cut again-transversally-to obtain 2×1×3 mm slabs that were subjected to microabrasion with 400-grit silicon carbide paper in crystal polystyrene flasks. 16  After confirming the homogeneous distribution, the variables were submitted to analysis of variance (1way) followed by the Student-Newman-Keuls test.
The P release values were higher on the first day for 9% CaGP group (p<0.001) when compared with the other groups ( Figure 3E). The highest total value was presented by 9% CaGP group (p<0.003), followed by 1 and 3% CaGP groups. All groups showed a similar release pattern, with rising and falling periods ( Figure   3E). Cumulative mean of P released was higher for 9% CaGP group (p<0.001) ( Figure 3F). When the values of P released by CaGP groups were subtracted from the values of the control group, the quantity of P from CaGP ( Figure 3G) was noted to be higher in the group with 9% (p<0.042). Cumulative release of P from CaGP did not increase over time, but the total mean value was higher for 9% CaGP group ( Figure 3H) (p<0.001). There was a positive correlation between the F and Ca released by materials (Pearson's r=0.890; p<0.001), but there was no correlation of these ions with the P release. values than the other groups (p<0.022) ( Table 2).

Discussion
The experimental design of this in vitro study allowed to verify that the addition of CaGP to RMGIC     In this study, the values obtained (Table 1) for the Control group and RMGIC with 1% and 3% CaGP were close to the variation for this material, but with the addition of 9% CaGP they presented a reduction of 54% (diametral tensile), 54% (compressive strength) and 26% (hardness), after 1 st day. As in previous studies that added the CPP-ACP to the GIC, 21,28 surface hardness tests were shown to be less sensitive to the addition of CaGP than tests of diametral tensile and compressive strength, 5 mainly on 1 st day. It appears that the additions of Ca-P source over 3% produce much alteration in the composition of the powder and the powder:liquid ratio. 5 The better outcomes of diametral tensile and compressive strength and F release obtained with addition of 1.5% CPP-ACP by Mazzaoui, et al. 19 (2003) is probably due to incorporation of low concentration, as observed by the RMGIC with 1% and 3% CaGP in the present study. Between the 1 st and the 7 th day, the mechanical properties of the RMGIC increased, because of the late acid-base reaction that occurred within the material. 29 The addition of CaGP delayed the reaction between the polyacrylic acid and the glass particles, because glycerophosphate would consume H + and increase the amount of Ca 2+ in the acid-base reaction on the 1 st day. Glycerophosphate acted as a filler in the matrix together with the calcium fluorosilicate, since part of the powder was replaced by organic phosphate.
Thereby, the effect on reducing mineral loss was caused by the high values of Ca and F released into the medium, and not due to adsorption of CaGP onto the enamel surface, as observed in previous studies. 9,10 Although 9% CaGP had less ability to reduce mineral loss, the addition of 3% CaGP to the RMGIC produced better results than those of the Control group. This was confirmed by the greater presence of F in enamel, and by the 60% increase in Ca and P in comparison with the Control group. In 9% CaGP group, the presence of higher Ca values together with the increase in F and P produced lower mineral loss because of higher release of Ca and F from the material. However, a higher level of availability of Ca in the medium may lead to less presence of F in the enamel 10 , as observed in this study, when 3% and 9% CaGP groups were compared (Table 2). Thus, an appropriate amount of organic phosphate can be added to the resin-modified glass ionomer powder, thereby improving its effect against demineralization with minimal changes in its physico-mechanical properties.

Conclusion
It was concluded that the incorporation of 3% CaGP into RMGIC increased the release of Ca and F and reduced enamel demineralization, thereby maintaining the physico-mechanical properties within the parameters for this material.