Comparative evaluation of the remineralising effects and surface microhardness of glass ionomer cement containing grape seed extract and casein phosphopeptide--amorphous calcium phosphate: an in vitro study.
Abstract: AIM: To evaluate and compare in vitro the remineralising effects and surface microhardness of glass ionomer cement (GIC), GIC containing grape seed extract and GIC containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). METHODS: An in vitro study with 45 mandibular premolars with standardised class V cavities prepared on the buccal and lingual surfaces. Artificial caries-like lesions were created and teeth were randomly divided into 3 groups of 15 teeth each. Each tooth was sectioned longitudinally into buccal and lingual halves; lingual half was used as control and the other as the test specimen. Axial longitudinal sections of the control specimens were analysed under polarised light microscopy. The experimental halves were restored with 3 materials. Group I: glass ionomer cement (GIC), Group II: 10% (w/w) grape seed extract and GIC (GSGIC), Group III: 10% (w/w) casein phosphopeptide-amorphous calcium phosphate (Recaldent) and GIC (CPPGIC). The restored tooth specimens were exposed to pH cycling for 28 days and analysed again for remineralisation under polarised microscopy. For the second part of the study, 60 cylindrical specimens (20 each) were made using standardised brass moulds and the three experimental materials. The specimens were immersed individually in de-ionised water. After 7 days of immersion, 10 specimens from each group were subjected to microhardness measurements and repeated on the remaining specimens at the end of 30 days. STATISTICS: One way ANOVA and post-hoc Tukey's test were used for statistical analysis. RESULTS: CPPGIC showed the greatest remineralisation followed by GSGIC and least for GIC. Comparisons of microhardness between the three groups were not statistically significant. CONCLUSION: Grape seed extract and CPPACP can be used for remineralisation of damaged dentine and their incorporation into GIC does not compromise the microhardness properties significantly.

Key words: Glass ionomer cement, grape seed extract, casein phosphopeptide-amorphous calcium phosphate, remineralisation, surface microhardness.
Article Type: Report
Subject: Ionomers (Health aspects)
Dental adhesives (Composition)
Dental glass ionomer cements (Composition)
Casein (Health aspects)
Calcium phosphate (Health aspects)
Authors: Prabhakar, A.R.
Sharma, D.
Sugandhan, S.
Pub Date: 06/01/2012
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: June, 2012 Source Volume: 13 Source Issue: 3
Product: Product Code: 3843075 Dental Adhesives; 2023147 Casein; 2819826 Calcium Phosphates NAICS Code: 339114 Dental Equipment and Supplies Manufacturing; 311514 Dry, Condensed, and Evaporated Dairy Product Manufacturing; 325188 All Other Basic Inorganic Chemical Manufacturing SIC Code: 3843 Dental equipment and supplies; 2821 Plastics materials and resins; 2824 Organic fibers, noncellulosic; 2874 Phosphatic fertilizer industry
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 292992924
Full Text: Introduction

Dental caries is a dynamic process of alternating de- and remineralisation [Featherstone, 2000]. The dentinal lesion presents different levels of demineralisation, having the most advanced areas closer to the biofilm growing on the surface of the cavity [Gonzalez-Cabezas, 2010]. The preservation and stability of dentine collagen may be essential during the remineralising process as it acts as a scaffold for mineral deposition [Qian et al., 2008].

Mineralisation of the dentine left in the cavity after excavation is likely to occur due to the application of chemically adhesive materials, such as GIC's [ten Cate et al, 1995]. The term internal remineralisation was introduced to describe the interaction between GIC and carious dentine, and to suggest that GIC is an essential tool in the management of deep carious lesions as it supplies apatite-forming ions to the partially demineralised dentine at the base of the cavity [Ngo, 2010]. While fluoride is the well-established method to prevent and inhibit caries progression, novel therapies have been introduced that affect the organic dentine matrix component to promote remineralisation.

A majority of studies for natural products in the field of oral health have focused on their antibacterial properties. Very few have reported on effects of natural products on the remineralisation process of dental hard tissues [Wu, 2009]. Proanthocyanidin (PA) is a naturally occurring plant metabolite widely available in fruits, vegetables, nuts, seeds, flowers, and bark. Grape seed extract (GSE) is a rich source of PA, which has been reported to strengthen collagen-based tissues by increasing collagen cross-links [Qian et al., 2008]. Illustrating the scope of responses that may be facilitated by dietary phytochemicals, Wu reviewed the potential of grape products to modulate oral and dental health. GSE, high in PA, positively affected the in vitro demineralisation and/or remineralisation processes of artificial root caries lesions.

The concept of CPP-ACP as a remineralising agent was first postulated in 1998, in which ACP is stabilised by CPP, and these nanocomplexes act as a calcium and phosphate reservoir [Shen et al., 2001]. These nanocomplexes have been shown to prevent demineralisation and promote remineralisation of enamel subsurface lesions in animal and in situ caries models [Reynolds et al., 1995].

As reported in the literature, restorative filling materials used in dentistry are required to have long term durability in the oral cavity along with remineralisation potential. One of the most important physical properties of restorative filling material is surface hardness. Moreover, hardness has been used to predict the wear resistance of a material and its ability to abrade or being abraded by opposing dental structures and materials [Ateyah, 2002].

Therefore the aim of this study was to obtain further insight into the nature of in vitro remineralising effects and surface microhardness after incorporating grape seed extract and CPP-ACP into a conventional GIC.

Materials and methods

Experimental materials used. Reinforced Glass Ionomer Cement (high strength) (GC Fuji II, GC corporation, Tokyo, Japan), Grape seed extract (Falcon international- Bangalore, India) and GC Tooth Mousse -Recaldent (GC corporation, Tokyo, Japan), were used as the test materials.

Part I: Evaluation of remineralisation effects. Forty-five mandibular premolars extracted for orthodontic reasons, were selected for this study. After extraction, the teeth were polished with pumice on a prophylactic brush; steam autoclaved and immediately stored in cold distilled water at 4[degrees]C for 1-2 months before testing [Titley et al.,1998]. Standardised class V cavities (one on the buccal and one on the lingual surface of each tooth) were prepared (3mm wide, 2mm high and 1.5mm deep) with the preparation extending 1mm above the CEJ [Yazici et al., 2003].

The teeth were then randomly divided into 3 experimental groups of 15 teeth each (Table 1). They were covered with two coats of acid-resistant nail varnish except for a window which included the cavity and a 2mm rim of sound tooth structure surrounding the cavity [Creanor, 1998]. Artificial caries-like lesions were created on the exposed cavities by suspending them in an artificial caries system for two days. The caries solution consisted of 2.2mM[Ca.sup.2+], 2.2mMP[O.sub.4.sup.3-], 50mM acetic acid at a pH of 4.4.The solution was kept at a temperature of 37[degrees]C, under constant circulation [ten Cate, 2004].

After two days, the teeth were removed from the artificial caries system. Each tooth was sectioned longitudinally to provide one buccal and one lingual half in which the lingual half was used as the control and the buccal half as a test specimen, when the test specimens were preserved in distilled water to be used later. Control specimens were mounted in acrylic blocks for sectioning. A section of 100pm thickness was obtained by cutting through the centre of each cavity using a Silverstone-Taylor hard tissue microtome. Sections were washed with de-ionised water and mounted on glass cover slides and evaluated under polarised light microscopy (model BX-51, Dualmont Corporation, Minneapolis, USA). Photomicrographs were made at 10x magnification and the demineralised areas were quantified with a computerised imaging system Image Pro-Plus [ten Cate, 2004]. Lesion depth was measured from the surface of the tooth to the depth of the cavity, at D1, D2 and D3 (in u) and an average of the three representative measurements was taken [Todd et al., 1999].

The cavities of the tooth segments (buccal half) were restored with the experimental materials at room temperature (21 [+ or -] 1[degrees]C), in 55% relative humidity. The restored tooth segments were stored in a humid environment at 37 [+ or -] 1[degrees]C for 24 hours. After that, the excess restorative material was removed and polished [Hara et al., 2002].

These restored tooth specimens were exposed to a daily cyclic treatment regime which involved exposing the specimen to de- and remineralising solutions. The remineralising solution used contained 2mM calcium chloride and 2mM sodium dihydrogen orthophosphate. The pH was adjusted to 6.8 by the addition of 0.1M sodium hydroxide. The demineralising solution was the same as that used for lesion creation. Each specimen was immersed in 10ml of remineralising solution for 20 hours at 37[degrees]C, removed and washed with de-ionised water and then immersed in 10ml of demineralising solution within another vial for 4 hours at 37[degrees]C. The cycling program was carried out for 28 days [Creanor et al., 1998].

At the end of the 28th cycling period, the specimens were removed from the pH cycling regime and were mounted on acrylic blocks. The 100pm sections from individual specimens were oriented longitudinally on glass cover slides for evaluation under polarised microscopy. The remineralised lesions were again quantified using the imaging system, Image Pro-Plus as described for demineralisation.

Part II: Evaluation of surface microhardness. A total of 60 cylindrical specimens (20 each in a group) were made from the 3 experimental materials. By placing the mixed materials into standardised cylindrical brass molds (diameter, 10mm; height, 1.5mm), slightly overfilling them and gently compressing them between two glass plates [Yap, 1997]. All the specimens were prepared at room temperature (21 [+ or -] 1[degrees]C), in 55% relative humidity.

The specimens were gently removed from the moulds, stored at 37[degrees]C for 1 h, and immersed individually in test tubes in 20ml of de-ionised water in an incubator at 37[degrees]C. Immersion times were 7 days and 30 days [Kanchanavasita et al., 1998]. The specimens were then mounted on acrylic blocks. Hardness measurements were taken in the central area of each specimen and readings were recorded immediately after removal of the indentor to minimise the effects of elastic recovery of polymers on the results. The same specimens were evaluated after different time periods and the means and standard deviations were calculated and tabulated.

After 7 days of immersion, 10 specimens from each group were randomly selected and subjected to microhardness measurements. The same procedure was repeated on the remaining specimens at the end of 30 days. The microhardness measurements were carried out using a microhardness tester (MITUTOYO). The indentations were made with a 50g load applied for 5s. Under these conditions, sharply defined indentation marks were obtained allowing the determination of the surface hardness of all the test materials with sufficient accuracy. The application of higher loads or a longer contact time invariably initiated cracks on the surface of the materials [De Moor and Veerbeeck, 1998].

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

For each test specimen, the values were read referring to the size of the greater diagonal. The values were transformed into Vickers hardness numbers. Surface microhardness was calculated using the following formula:

VH = 1.854 x F x [10.sup.3]/[d.sup.2]

where F is the applied test load (N) and d is the average of the indentation diagonals (mm) [De Moor and Veerbeeck, 1998].

The results from observations of both the experiments were tabulated and statistically analysed using one-way ANOVA for multiple group comparison followed by post-hoc Tukey's test for pairwise comparisons. For all the tests, a p-value of 0.05 or less was considered for statistical significance.

Results

Remineralisation study. Data representing the mean depth of de- and remineralisation of the various experimental groups are shown in Table 2 and Figures 1-2. The differences in de- and remineralisation were statistically highly significant for the GIC group (p<0.001), significant for CPPGIC (p= 0.02) and not significant for GSGIC (p=0.11).

Inter-group comparison of remineralisation among GSGIC and GIC showed a mean difference of 17.38 which was statistically highly significant (p<0.001), between CPPGIC and GIC a mean difference of 22.23 was observed which was statistically highly significant (p<0.001). On comparison of GSGIC and CPPGIC a mean difference of 5.36 was observed, which was not statistically significant (Table 3 and Figure 3). The remineralisation potential was statistically significant for all the groups. GSGIC and CPPGIC showed higher remineralisation than the conventional GIC.

Surface microhardness study. Comparison of differences between surface microhardness (SMH) among the GIC, GSGIC and CPPGIC groups on the 7th and 30th days are shown in Table 4. GIC showed a mean difference of 6.77 between the two SMH observations, which were not statistically significant (p=0.15). GSGIC showed a mean difference of 5.03 between the two SMH observations were also not statistically significant (p=0.11). CPPGIC showed a mean difference of 9.36 between the two SMH observations and the differences were statistically significant (p=0.02).

Comparisons of GIC, GSGIC and CPPGIC for SMH values were not statistically significant (p=0.14) at either the 7th or 30th days (p=0.47) as shown in Table 3 and Figure 4.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Discussion

There is a constant research for materials and efficient dentistry techniques targeting underprivileged communities [Raggio et al., 2010]. A topic that has received limited attention is whether there is a point beyond which remineralisation can or does no longer occur. Koulourides et al. [1961] stated that if caries has weakened the tooth structure to below a hardness of 150 (KHN), remineralisation could no longer be achieved. The current consensus is that caries beyond the dentino-enamel junction (DEJ) should be treated with restorations, and lesions up to that point should receive extra preventive care. However, it has never studied whether deep lesions, extending into dentine, can be remineralised if such lesions are subjected to a continuous remineralisation scheme.

A major reason for the use of GICs in a variety of clinical applications is their capacity to bond chemically to different surfaces such as enamel, dentine, and composite resin. Previous studies have shown that fluoride releasing conventional GICs have high cariostatic effect [Fross, 1993]. This is partly attributed to the enhancing effect of fluoride on calcium phosphate precipitation, hence remineralisation [Marinho et al., 2003]. However, for net remineralisation to occur, adequate levels of calcium and phosphate ions must be available [Featherstone, 2008].

In our study the greatest degree of depth of remineralisation was found in the GIC containing CPP-ACP followed by GIC containing grape seed extract but the difference between them was not statistically significant, so both were more effective in remineralisation as compared to GIC. Based on the data obtained GSE may positively affect the remineralisation process possibly through two distinct mechanisms. First GSE may contribute to mineral deposition on the superficial layer of the lesion. GSE may form insoluble complexes when mixed with the remineralising solution at pH 7.4. These complexes remained visually insoluble at pH range of 2-7. Thus it is likely that after treatment with GSE, this will combine with calcium from the remineralising solution, thereby enhancing remineralisation. Secondly, GSE may interact with the organic portion of the root dentine through Proanthocyanidin (PA) collagen interaction, thereby stabilising the exposed collagen matrix [Qian et al., 2008].

According to Al Zraikat et al. [2011] GIC containing CPP-ACP inhibited enamel demineralisation, when subjected to an acid challenge using lactic acid buffer at pH 4.8, which is similar to the caries process. The area of demineralised enamel adjacent to the GIC with 3 or 5% CPP-ACP was significantly smaller than the area adjacent the control GIC. The ability of GIC to inhibit demineralisation of enamel and dentine has been well established in previous studies. The anti-cariogenic mechanism of fluoride is the localisation of the fluoride ion at the tooth surface. This localisation promotes remineralisation of enamel with fluorapatite (FA). It is clear that, for the formation of FA [[Ca.sub.10][(P[O.sub.4]).sub.6][F.sub.2]], calcium and phosphate ions must also be present with the fluoride ions. The reported additive anti-cariogenic effect of CPP-ACP and fluoride may therefore be attributable to the localisation of the novel calcium, fluoride, and phosphate ion nanoclusters at the tooth surface by the CPP, which co-localises calcium, phosphate, and fluoride as bioavailable ions in the correct molar ratio to form fluorapatite.

One of the important physical properties of any restorative material is surface hardness, which correlates well to compressive strength and abrasion resistance. Surface hardness is related to the amount of water taken up with a greater uptake resulting in a weaker swollen hydrogel. Estimation of hardness may indicate deteriorating effects on the restorative material when it is placed in the oral cavity. Therefore investigation of the effects of storage media on surface hardness are warranted to enhance better understanding of clinical behaviour [Silva et al., 2007]. In the current study, no significant difference was found between GIC, GSGIC and CPPGIC after 30 days.

There are different schools of thoughts regarding the hardness of GIC over a period of time. One study reported that surface hardness will increase because of the prolonged setting reaction [De Gee et al., 1996]. Few researchers on the other hand, have reported a softening of the surface with time, when GIC is stored in an aqueous solution [De Moor and Veerbreeck, 1998]. Matsuya et al. [1996] showed that ageing time had no significant effect on the surface hardness of conventional GICs. In our study also, there was no significant effect on the microhardness of the GIC at 7 or 30 days.

The incorporation of CPP-ACP into Fuji VII had variable effects on the working and physical properties according to a previous study [Mazzaoui et al., 2003]. The setting time had increased as more CPP-ACP was incorporated, while compressive and diametrical tensile strength values of the material had decreased slightly.

There is a scarcity of data in the literature regarding the effect of grape seed extract on the microhardness of dentine and this study is the first attempt to evaluate the same. In this study, remineralisation was checked using a pH cycling method. Further evidence about the use of these novel materials along with GIC as a remineralising agent can be proved with the amount of fluoride release. It should be noted that in a clinical situation, the behaviour of a material is strongly dependent on the local environment such as the amount and pH of saliva in the close vicinity of the restoration. The surface characteristics of the restorations, e.g. surface roughness also affect their ability to precipitate calcium and phosphorus ions. Wear of the restoration will also naturally affect the material's surface characteristics. However, these clinical variables could not be controlled in this study. We therefore recommend further controlled studies on in vivo models to confirm our observations and ascertain the true clinical efficacy of these materials.

Conclusion

Grape seed extract and CPP-ACP had a significant potential to remineralise demineralised dentine in permanent teeth. The addition of grape seed extract and CPP-ACP to GIC did not compromise the mechanical properties of the materials. Thus, they can be used where their bioactivity can be beneficial, such as in minimal invasive dentistry.

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A.R. Prabhakar, D. Sharma, S. Sugandhan

Department of Paedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, India.

Postal address: Dr. A.R. Prabhakar, Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere 577004, Karnataka, India.

Email: attiguppeprabhakar@yahoo.com
Table 1. Description of the different experimental
materials used in the study

Groups      Colour     Experimental     Abbreviation
             code        Material

Group 1      Red     Reinforced glass       GIC
                      ionomer cement

Group II    Purple   GIC + 10 % grape      GSGIC
                       seed extract

Group III   Golden      GIC + 10 %         CPPGIC
                         CPP-ACP

Table 2. Descriptive analysis showing the mean and standard
deviation for de- and remineralisation among the various
experimental groups

Groups             Demineralisation   Remineralisation

GIC         Mean        106.72             63.13
Group I     SD          13.15               9.29
GSGIC       Mean        122.79             80.51
Group II    SD           5.12              18.80
CPPGIC      Mean        115.97             85.87
Group III   SD          10.76              11.89

Groups                  Mean            p * Value,
                      difference            sig

GIC         Mean        43.59            p<0.001 HS
Group I     SD
GSGIC       Mean        42.28             0.11 NS
Group II    SD
CPPGIC      Mean        30.10              0.02 S
Group III   SD

* Student's paired t test. HS- highly significant.
NS--Not significant. S--Significant

Table 3. Descriptive statistics showing the mean and standard
deviation for remineralisation among the various experimental
groups.

            Remineralisation

                            p * Value,      Significant
Groups      Mean     SD        sig           pairs **

GIC         63.13   9.29
Group I

GSGIC       80.51   18.80   p<0.001 HS   1 and 2 (17.38),
Group II                                 1 and 3 (22.23)

CPPGIC      85.87   11.89
Group III

* One-way ANOVA test. ** post hoc Tukey's test.
HS--Highly significant

Table 4. Descriptive statistics showing the intra-group and
inter-group comparison of the mean and standard deviation
for microhardness among the various experimental materials
at 7 and 30 days.

Groups                7 Days        30 Days         Mean
                                                  difference

GIC         Mean      46.60          39.83           6.77
Group I     SD        10.97           5.01

GSGIC       Mean      42.63          37.60           5.03
Group II    SD         7.91           5.38

CPPGIC      Mean      50.53          41.17           9.36
Group III   SD         6.11           8.59

                   Intra-qroup       Inter-qroup comparison
                    comparison
                                    7th day        30th day

Groups             p * Value,     p * Value,      p * Value,
                   significance   significance   significance

GIC         Mean     0.15 NS
Group I     SD

GSGIC       Mean     0.11 NS        0.14 NS        0.47 NS
Group II    SD

CPPGIC      Mean      0.02 S
Group III   SD

* Student's paired t test. ** Oneway ANOVA test.
*** post hoc Tukey's test. HS- Highly significant.
HS--highly significant. NS--Not significant.
S--Significant
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