MICROSCOPY RESEARCH AND TECHNIQUE 77:986–988 (2014)
Influence of Fluorescent Dye on Physical–Mechanical Properties of Luting Cements for Confocal Microscopy Analysis 1,2 DAYANE OLIVEIRA,1* LUCIA PRIETO,1 CINTIA ARAUJO, ERICK COPPINI,1 GISELE PEREIRA,3 AND LUIS PAULILLO1 1
Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas -UNICAMP, Sao Paulo, Brazil Department of Dentistry, Faculty of Sciences of Health, Federal University of Jequitinhonha and Mucuri Valley – UFVJM, Diamantina, Brazil 3 School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 2
rhodamine B; Knoop hardness; flexural strength; young modulus
ABSTRACT Aims: To evaluate the influence of a fluorescent dye (rhodamine B) on the physical and mechanical properties of three different luting cements: a conventional adhesive luting cement (RelyX ARC, 3M/ESPE), a self-adhesive luting cement (RelyX U-200, 3M/ESPE), and a self-etching and self-adhesive luting cement (SeT PP, SDI). Materials and Methods: The cements were mixed with 0.03 wt% rhodamine B, formed into bar-shaped specimens (n 5 10), and light cured using an LED curing unit (Radii, SDI) with a radiant exposure of 32 J/cm2. The Knoop hardness (KHN), flexural strength (FS), and Young’s modulus (YM) analyses were evaluated after storage for 24 h. Results: Outcomes were subjected to two-way ANOVA and Tukey’s test (P 5 0.05) for multiple comparisons. No significant differences in FS or YM were observed among the tested groups (P 0.05); the addition of rhodamine B increased the hardness of the luting cements tested. Conclusion: The addition of a fluorescent agent at 0.03 wt% concentration does not negatively affect the physical–mechanical properties of the luting cement polymerization behavior. Microsc. Res. Tech. 77:986–988, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION Microleakage is widely used to evaluate the interfacial integrity of adhesive restorations; dye penetration has been not correlated to marginal gaps (Dietrich et al., 2000). More sophisticated methods for microleakage analysis have been developed, including confocal laser-scanning microscopy (CLSM), which provides accurate visualization of resin tags and the hybrid layer (Arrais et al., 2009). CLSM is a valuable technique for adhesion studies in dentistry, permitting nondestructive analyses to be performed under humid conditions and producing high-resolution three-dimensional images (Watson, 1989, 1991). Fluorescent dyes and CLSM have been applied to analyze interfacial integrity (Griffiths and Watson, 1995; Watson, 1989) and nanoleakage (D’Alpino et al., 2006a; Pioch et al., 1997). CLSM yields more detailed information concerning the penetration and distribution of resin cements and adhesives than scanning electron microscopy (Bitter et al., 2009). Micromorphological analyses of the bonding interfaces and visualization of interfacial integrity using CLSM may be considerably enhanced by the incorporation of fluorescent dyes into the bonding systems (Watson, 1997). Fluorochromes used for this purpose should be well mixed in advance of placement to ensure total incorporation and reduce any effects on the physical and mechanical properties of the composite material (Schupbach et al., 1997; Watson and Boyde, 1991). Labeling adhesive materials with fluorescent agents may affect the polymerization process, which could then modify the physical and mechanical properties C V
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and the bonding performance (e.g., self-adhesive luting cements) (D’Alpino et al., 2006b; Dewaele et al., 2009), resulting in changes to the hybrid layer properties (Takahashia et al., 2002). Depending on the concentration, fluorochromes have the potential to reduce monomer conversion of the bonding agent by absorbing light. This absorption might inhibit sufficient light from reaching the photoinitiators to induce polymer growth (D’Alpino et al., 2006c). Since CLSM is used to assess the micromorphology of bonded interfaces, it is extremely important to evaluate the effects of adding fluorescent dyes on the physical and mechanical properties of luting cements as was done in previous studies examining adhesive bond systems (Ara ujo et al., 2013). Thus, the objective of the present study was to evaluate whether addition of small amounts (0.03 wt%) of the rhodamine B fluorescent dye affected the physical and mechanical properties of conventional, self-etching, and self-adhesive luting cements. The research hypothesis tested was that rhodamine B would not negatively influence the properties of the cements. *Correspondence to: Dayane Oliveira, Restorative Dentistry Department, Piracicaba Dental School, State University of Campinas, 901 Limeira Av. Arei~ ao, 13414-903 Piracicaba, SP, Brazil. E-mail: [email protected]
Received 27 February 2014; accepted in revised form 31 July 2014 REVIEW EDITOR: Dr. Chuanbin Mao This article was published online on 16 August 2014. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 15 September 2014. Pesquisa do Estado de S~ Contract grant sponsor: Fundac¸~ ao de Amparo a ao Paulo – FAPESP; Contract grant number: 2011/12180-8. DOI 10.1002/jemt.22426 Published online 19 August 2014 in Wiley Online Library (wileyonlinelibrary.com).
RHODAMINE B INFLUENCE ON LUTING CEMENT PHYSICAL PROPERTIES
MATERIAL AND METHODS Luting Cements A conventional luting cement (RelyX ARC, 3M ESPE, St. Paul, MN), a self-adhesive luting cement (RelyX U200, 3M ESPE, St. Paul, MN), and a selfetching and self-adhesive luting cement (SeT PP, SDI, Bayswater, Victoria, Australia) were investigated. Specimen Preparation Light curing was performed in a fixture coupled to the light-curing unit (Radii, SDI, Bayswater, Victoria, Australia). The exposure distance was fixed at 4 mm to simulate the typical distances encountered in indirect restorations. The distance was established using an electronic micrometer (Digimatic, IP-65 Mitutoyo, Tokyo, Japan). To examine the influence of the fluorescent dye on the physical–mechanical properties, 0.03 wt % (Ara ujo et al., 2013; D’Alpino et al., 2006a) of rhodamine B (Sigma, St. Louis, MO, USA) was mixed with the base paste of the luting cement systems tested. The mixture was kept in a continuous stirring device (AP22 Phoenix Luferco, Phoenix Ind and Com Equips Scientific LTDA; Araraquara, SP, Brazil) for at least 2 h to ensure complete dissolution (Arrais et al., 2009). For each luting cement system, two sets of specimens (with and without fluorescent dye) were prepared. Bar-shaped specimens with dimensions of 7 mm 3 2 mm 3 1 mm were prepared to test Knoop hardness (KHN), flexural strength (FS), and Young’s modulus (YM) (ISO 4049/2000) (Diaspro et al., 2001) (n 5 10). Each luting cement was manipulated according to the manufacturer’s instructions and placed into a standardized polydimethylsiloxane (Virtual, Ivoclar Vivadent, Liechtenstein) mold in a single layer using a micropipette. The mixtures were constantly agitated to prevent the formation of air bubbles (Loguercio et al., 2009). Before light activation, a Mylar strip was placed over the mold to produce a flat sample surface. All samples were light cured with an output of 32 J/cm2, after which they were removed from the silicone mold and stored dry in darkness for 24 h at 37 C. Physical–Mechanical Properties The KHN of the samples was determined using a microhardness tester (Shimadzu HMV-2) in which a 50 g load was applied for 10 s to three equally spaced points on each sample. The hardness of each sample was reported as the arithmetic mean of the three readings. FS and YM tests were performed in a universal testing machine (model 4411, Instron, Canton, MA) using a three-point bending fixture (span between supports 6.0 mm; crosshead speed 0.5 mm/s) until failure. Young’s modulus (E—GPa) and flexural strength (FS—MPa) were calculated using the following equations: FS 5 3 3 L 3 D / 2 3 w 3 h2 and E 5 L 3 D3/4 3 w 3 h3 3 d, in which L (N) is the load recorded in the elastic regime, D (mm) is the distance between supports, w (mm) is the width of the sample, d (mm) is the displacement of the beam corresponding to L, and h (mm) is the thickness of the sample. Microscopy Research and Technique
Fig. 1. For each of the luting cements tested, the mean KHN, FS, and YM values 6 standard error for control (blue) and rhodamine (red) is provided. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Statistical Analysis The results were tabulated and analyzed using twoway analysis of variance (ANOVA) and Tukey’s test (P 5 0.05) for multiple comparisons. RESULTS Figure 1 illustrates the results of the physical– mechanical properties evaluated according to the different luting cements tested. There were no significant differences in FS and YM among the luting cements regardless of the addition of rhodamine B dye. KHN increased with addition of rhodamine B in all of the cements tested. DISCUSSION CLSM is a potential visualization technique that is nondestructive, unlike conventional light microscopy and transmission/scanning electron microscopy (Pioch et al., 1997). However, the use of fluorescent dyes, even as tracers, may significantly impact specimen analysis and data interpretation (D’Alpino et al., 2006b). In the present study, the addition of 0.03% of rhodamine B did not statistically affect the mechanical properties of the luting cements. Given the crucial relationship between the light-curing process and the physical– mechanical properties of dental resin-based materials (Bae et al., 2005), the results imply that rhodamine B at the concentration used did not interfere with the process of polymerization. Insufficient monomer conversion may degrade the mechanical properties of the hybrid layer and increase the amount of residual uncured monomer (D’Alpino et al., 2006b). Rhodamine B interferes with the behavior of adhesives by physically scattering light and reducing activation of the photoinitiators. The dyes do not bind chemically to the monomers, and the incorporation of the fluorochrome is based only on a mixing process. Thus, after the curing of the luting cement, the fluorochrome must remain entrapped in the polymer network (Schupbach et al., 1997; Watson, 1997). Although there are no covalent bonds between the fluorescent
D. OLIVEIRA ET AL.
molecules and the adhesive, arbitrary use of the dye should be avoided because it is more visible at increasing concentrations, and at greater concentrations it may dramatically reduce resin polymerization, compromising the mechanical properties of the adhesive. No significant differences in FS or YM were detected between any of the cements, and the mechanical properties of the luting systems with and without dye were similar regardless of the composition. The presence of rhodamine B in the luting systems probably did not affect their performance. However, it is important to consider that the bar specimens for the mechanical measurements were irradiated with 32 J of energy to achieve adequate polymerization. It has been reported that the degree of polymerization of adhesive resins does not change when the resin is irradiated at levels greater than 32 J/cm2 (Yanagawa and Finger, 1994). The increase in KHN values with rhodamine B addition is not a negative effect, since it did not interfere with the mechanical properties of the luting cements. It is likely that the rhodamine B works as a filler to increase the hardness of the luting cements. This study demonstrated that it is possible to incorporate a fluorescent dye (rhodamine B) in luting cements to facilitate optical detection of interfacial leakage, with no deleterious effects on the physical or mechanical properties of these agents, in agreement with the original study of rhodamine B incorporation into adhesive systems (Ara ujo et al., 2013). Rhodamine was selected because it is the most commonly used fluorochrome in fluorescence microscopy (Diaspro et al., 2001; Pioch et al., 1997) and is used in dentistry to analyze bonding to dental hard tissues and interactions between materials used in clinical practice (Arrais et al., 2009; Diaspro et al., 2001). Rhodamine B is stable over a range of pH conditions, is water soluble, and is highly soluble in organic solutions (Pioch et al., 1997). However, other types of fluorochromes and other commercially available luting cements should be evaluated using a similar methodology to assess the effects of the dyes on the luting systems. CONCLUSION Within the limitations imposed by the experimental design used in the current study, the following conclusion can be drawn: the addition of rhodamine B fluorescent agent at 0.03 wt% concentration does not negatively affect luting cement polymerization behavior.
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