Knoop microhardness and FT-Raman evaluation of composite resins : influence of opacity and photoactivation source

The aim of this study was to evaluate the degree of conversion by Knoop microhardness (KHN) and FT-Raman spectroscopy (FTIR) of one nanofilled (Filtek Supreme-3M-ESPE [FS]) and one microhybrid composite (Charisma-Heraeus-Kulzer [CH]), each with different opacities, namely enamel, dentin, and translucent, which were photo-activated by a quartz-tungsten-halogen lamp (QTH) and a light-emitting diode (LED). Resin was bulk inserted into a disc-shaped mold that was 2.0 mm thick and 4 mm in diameter, obtaining 10 samples per group. KHN and FTIR values were analyzed by two-way ANOVA and Tukey’s tests (α = 0.05). Nanofilled resin activated by a LED presented higher microhardness values than samples activated by a QTH for dentin opacity (p < 0.05). The microhybrid resin showed no differences in KHN or FTIR values with different activation sources or opacity. The nanofilled dentin and enamel resins showed lower FTIR values than the translucent resin. The KHN values of the translucent resins were not influenced by the light source. Descriptors: Composite Resins; Hardness; Spectrum Analysis; Light. Introduction The clinical performance of composite resin restorations depends on several factors, which may alter the physical-chemical properties of the resin; such properties include the concentration of monomer and filling particles and those related to the clinician’s manipulation.1 Also, the quality of the light emitted by the curing units may affect monomer conversion and improved mechanical properties.2 Curing units activate the camphoroquinone, which is the photo-initiator most commonly present in resin composites that is sensitive to blue light in the wavelengths between 400 and 515 nm.1,3-5 Commonly, curing units are composed of a halogen lamp that provides a white light spectrum filtered to produce a spectral output between 400 and 500  nm.6 However, a wide spectrum of light is still emitted when this energy is spent. The light emitting diode (LED) curing units provide a narrow bandwidth of light with a wavelength of 470-490  nm, which is capable of polymerizing the composite resin with less energy than the halogen lamp because LED light is close to the camphoroquinone excitation wavelength.4 Declaration of Interests The authors have no interest in any of the companies or products mentioned in this article. Knoop microhardness and FT-Raman evaluation of composite resins: influence of opacity and photoactivation source 268 Braz Oral Res. 2011 May-Jun;25(3):267-73 The polymerization efficiency of a composite resin is influenced by the irradiation intensity, exposure time, and the light source.7 Concerns about these variables have led to several studies recently to determine the degree of conversion (DC) of dental composites.7-15 The sensitivity of molecular vibrational methods, such as infrared spectroscopy (FTIR) and Raman spectroscopy (FT-Raman), offers a direct approach to quantify the ratio of monomers’ conversion into polymers; this quantification is accomplished by assessing the specific band positions and by comparing the residual unpolymerized aliphatic C=C stretching band at 1640  cm-1 to the aromatic C=C stretching band at 1610 cm-1.8,9,16 Thus, the ratio of double carbon bonds that are converted into single bonds determines the DC of the resin composite.8,11-13,15 On the other hand, the hardness achieved by the polymerized composite is widely used as an indirect method of determining the quality of the light-initiated polymerization process.17 The light that initiates the polymerization may be absorbed or scattered through the body of the resin, jeopardizing the polymerization process. The lower transmittance of the light results in a low DC18 and consequently low microhardness,19 which is strongly influenced by the resin’s opacity and its filler contents.6,18,20 It has been speculated that darker resins, which are more saturated in croma, need a higher density of light energy to reach an acceptable monomer conversion than do lighter colored resins.20 However, there is little information regarding the opacity and light influence in the conversion because translucent resins are frequently used to reproduce enamel, and opaque resins are frequently used to reproduce dentin with the same croma.21 The aim of this study was to evaluate the degree of conversion by Knoop microhardness (KHN) and FT-Raman spectroscopy (DC) of one nanofilled and one microhybrid composite, each with different opacities (enamel, dentin, and translucent), which were photo-activated by a QTH or a LED. Methodology One nanofilled and one microhybrid commercial composite resin, each with three different opacities related to their translucence, were selected for this study (Table 1). Two light curing units with different light sources – a halogen source with a light intensity of 700 mW/cm2 (Optilux 501 Demetron/Kerr, Danbury, USA) and a LED source with an intensity of 1200 mW/cm2 (Radii SDI Limited, Victoria, Australia) – were used. The power density was measured with a radiometer (Demetron/Kerr, Danbury, USA). Table 2 presents the energy density calculated after multiplying the power density by the exposure time recommended by the manufacturers.22 Specimen preparation A total of 120 specimens were prepared according to the twelve groups (n = 10; Table 2). A two-part white polytetrafluoroethylene mold with a cylindrical window (4 mm in diameter and 2 mm high) was placed on a 10 x 120 x 0.05-mm Mylar strip (Polidental, Cotia, Brazil) on a matte black background and was kept stable with a metal ring. The composite was inserted in a single increment, and a second strip was placed on top of the unpolymerized material. A glass slide was put on top of the Mylar strip, and a 500 g load was applied for 30 s on the top of the mold to provide a smooth surface. The glass slide was removed before exposMaterial Manufacturer Classification Composition Opacity Lot No Filtek Supreme (3M/ESPE, St. Paul, USA) Nanofilled Bis-GMA, Bis-EMA (6), UDMA, TEGDMA, zirconia-silica nanocluster, silica nanoparticles A2D A2E GT 5AM 7EC 7BM Charisma (Heraeus-Kulzer, Hanau, Germany) Microhybrid Bis-GMA, TEGDMA, barium aluminum silicate and colloidal silica OA2 A2 Incisal 010302 010306 010302 Bis-GMA = bisphenol glycidyl methacrylate; Bis-EMA = bisphenol A polyethylene glycol diether dimethacrylate; UDMA = urethane dimethacrylate; TEGMA = triethylene glycol dimethacrylate. Table 1 Composite resin materials evaluated and their classification, composition, opacity and lot number (No). Albino LGB, Rodrigues JA, Kawano Y, Cassoni A 269 Braz Oral Res. 2011 May-Jun;25(3):267-73 ing the composite resin to light, and the light tip of each curing unit was centered on the specimen with no intervening space.8,19 Degree of Conversion Five prepared specimens were evaluated immediately after light activation for each group. As a control, the spectra of fi ve unpolymerized composites were analyzed for each group. The DC of the side opposite to the irradiated surface of the specimens was determined by an RFS 100/S FT-Raman spectrometer (Bruker Optics, Billerica, USA) equipped with a liquid nitrogen-cooled germanium detector. The 1064-nm line of an aircooled Nd:YAG near-infra-red laser was used to excite the samples.8,10 The Raman spectra were obtained using 64 scans at a spectral resolution of 4  cm-1 and were analyzed by selecting a spectral region of 1000-2000  cm-1. The Raman spectrum obtained from the uncured (translucent) FS is represented in Figure 1. The DC of each sample was determined by comparing the aliphatic carbon double bond peak with the aromatic component for the unpolymerized and polymerized states.9 The ratio between the aliphatic (1640  cm-1) and the aromatic (1610  cm-1) carbon double bond peaks was used to calculate the DC according to the following formula:8,11-13 DC = [1 (polymerized/unpolymerized)] × 100 Knoop microhardness profile After polymerization, each specimen was removed from the mold and was stored in deionized water in light-proof containers for 24 h at a constant temperature of 37oC. A Knoop diamond indenter was used to make fi ve indentations in the center of the surface opposite to the light-irradiated surface of the specimen with a 25  g load for 20  s.8,19 The mean of the fi ve microhardness evaluations of each specimen was used as the specimen’s microhardness value. Statistical analysis For each composite, a two-way analysis of variance was carried out, and the means of the Knoop microhardness values (KHN) and the degree of conversion were compared by a Tukey’s interval calculated at a 95% confi dence level. Resin composite Groups Exposure time (s) Halogen Energy density (J/cm2) LED Energy density (J/cm2) Filtek Supreme (FS) D 28 D 48 40


Introduction
The clinical performance of composite resin restorations depends on several factors, which may alter the physical-chemical properties of the resin; such properties include the concentration of monomer and filling particles and those related to the clinician's manipulation. 14][5] Commonly, curing units are composed of a halogen lamp that provides a white light spectrum filtered to produce a spectral output between 400 and 500 nm. 6owever, a wide spectrum of light is still emitted when this energy is spent.The light emitting diode (LED) curing units provide a narrow bandwidth of light with a wavelength of 470-490 nm, which is capable of polymerizing the composite resin with less energy than the halogen lamp because LED light is close to the camphoroquinone excitation wavelength. 4he polymerization efficiency of a composite resin is influenced by the irradiation intensity, exposure time, and the light source. 7][9][10][11][12][13][14][15] The sensitivity of molecular vibrational methods, such as infrared spectroscopy (FTIR) and Raman spectroscopy (FT-Raman), offers a direct approach to quantify the ratio of monomers' conversion into polymers; this quantification is accomplished by assessing the specific band positions and by comparing the residual unpolymerized aliphatic C=C stretching band at 1640 cm -1 to the aromatic C=C stretching band at 1610 cm -1 . 8,9,1613]15 On the other hand, the hardness achieved by the polymerized composite is widely used as an indirect method of determining the quality of the light-initiated polymerization process. 17he light that initiates the polymerization may be absorbed or scattered through the body of the resin, jeopardizing the polymerization process.The lower transmittance of the light results in a low DC 18 and consequently low microhardness, 19 which is strongly influenced by the resin's opacity and its filler contents. 6,18,20It has been speculated that darker resins, which are more saturated in croma, need a higher density of light energy to reach an acceptable monomer conversion than do lighter colored resins. 20However, there is little information regarding the opacity and light influence in the conversion because translucent resins are frequently used to reproduce enamel, and opaque resins are frequently used to reproduce dentin with the same croma. 21he aim of this study was to evaluate the degree of conversion by Knoop microhardness (KHN) and FT-Raman spectroscopy (DC) of one nanofilled and one microhybrid composite, each with different opacities (enamel, dentin, and translucent), which were photo-activated by a QTH or a LED.

Methodology
One nanofilled and one microhybrid commercial composite resin, each with three different opacities related to their translucence, were selected for this study (Table 1).
Two light curing units with different light sources -a halogen source with a light intensity of 700 mW/cm² (Optilux 501 -Demetron/Kerr, Danbury, USA) and a LED source with an intensity of 1200 mW/cm² (Radii -SDI Limited, Victoria, Australia) -were used.The power density was measured with a radiometer (Demetron/Kerr, Danbury, USA).Table 2 presents the energy density calculated after multiplying the power density by the exposure time recommended by the manufacturers. 22

Specimen preparation
A total of 120 specimens were prepared according to the twelve groups (n = 10; Table 2).
A two-part white polytetrafluoroethylene mold with a cylindrical window (4 mm in diameter and 2 mm high) was placed on a 10 x 120 x 0.05-mm Mylar strip (Polidental, Cotia, Brazil) on a matte black background and was kept stable with a metal ring.The composite was inserted in a single increment, and a second strip was placed on top of the unpolymerized material.A glass slide was put on top of the Mylar strip, and a 500 g load was applied for 30 s on the top of the mold to provide a smooth surface.The glass slide was removed before expos-  ing the composite resin to light, and the light tip of each curing unit was centered on the specimen with no intervening space. 8,19

Degree of Conversion
Five prepared specimens were evaluated immediately after light activation for each group.As a control, the spectra of fi ve unpolymerized composites were analyzed for each group.
The DC of the side opposite to the irradiated surface of the specimens was determined by an RFS 100/S FT-Raman spectrometer (Bruker Optics, Billerica, USA) equipped with a liquid nitrogen-cooled germanium detector.The 1064-nm line of an aircooled Nd:YAG near-infra-red laser was used to excite the samples. 8,10The Raman spectra were obtained using 64 scans at a spectral resolution of 4 cm -1 and were analyzed by selecting a spectral region of 1000-2000 cm -1 .The Raman spectrum obtained from the uncured (translucent) FS is represented in Figure 1.
The DC of each sample was determined by comparing the aliphatic carbon double bond peak with the aromatic component for the unpolymerized and polymerized states. 9The ratio between the aliphatic (1640 cm -1 ) and the aromatic (1610 cm -1 ) carbon double bond peaks was used to calculate the DC according to the following formula: 8,[11][12][13] DC = [1 -(polymerized/unpolymerized)] × 100

Knoop microhardness profile
After polymerization, each specimen was re-moved from the mold and was stored in deionized water in light-proof containers for 24 h at a constant temperature of 37ºC.A Knoop diamond indenter was used to make fi ve indentations in the center of the surface opposite to the light-irradiated surface of the specimen with a 25 g load for 20 s. 8,19 The mean of the fi ve microhardness evaluations of each specimen was used as the specimen's microhardness value.

Statistical analysis
For each composite, a two-way analysis of variance was carried out, and the means of the Knoop microhardness values (KHN) and the degree of conversion were compared by a Tukey's interval calculated at a 95% confi dence level.

Knoop microhardness
The data were submitted to a two-way ANOVA and to a Tukey's test (α = 0.05) considering the factors of photoactivation source and resin opacity and their interaction.
There were statistically significant differences for the photoactivation source (p = 0.001; Table 3) for the FS.There were no statistically significant differences for the resin opacity (p = 0.982).Statistically significant differences were found in the interaction of the photoactivation source * resin opacity (p = 0.003, Table 3) with a higher KHN for the dentin resin activated with the LED than that activated with the QTH.
No statistically significant differences were observed for photoactivation source (p = 0.14), resin opacity (p = 0.52), or for the interaction of the photoactivation source * resin opacity (p = 0.51, Table 4) for the microhybrid resin.

Degree of Conversion
Figure 1 shows the representative FT-Raman spectra of the FS (translucent) cured by a halogen light (14 J/cm²).Data were submitted to two-way ANOVA and to Tukey's test (α = 0.05) considering the factors of photoactivation source and resin opacity and their interactions.The data obtained for the DC and for the standard deviation for each experimental group are shown in Tables 5 and 6.
There were statistically significant differences for resin opacity (p < 0.001, Table 5).The dentin and enamel resins showed a lower DC than did the translucent resin (FS).There were no statistically significant differences for photoactivation source (p = 0.65) or for the interaction of the photoactivation source * resin opacity (p = 0.17).
There were no statistical differences for the resin opacity (p = 0.52), the photoactivation source (p = 0.18), or for the interaction of the photoactivation source * resin opacity (p = 0.88) for the CH; the results are presented in Table 6.

Discussion
The advancement in LED technology is comparable with the advancement in computer technology. 4The quality of the polymerization process can influence the performance of composite resins and plays an important role in the success of dental restorations. 7This process may be predicted by the proportion of monomers effectively converted into polymers.This proportion is described as the degree of conversion and is usually evaluated by several methods, including the non-destructive technique Raman spectroscopy. 8,16The most important vibrational bands have been previously reported, 9 and the    Raman spectrum of the uncured FS, with the main vibrational modes assigned, is shown in Figure 1.The nanofilled resin selected for the present study was Filtek Supreme.It is classified as a particulate, reinforced, polymer-based resin. 23Filtek Supreme has been described as having a 46.6% conversion at surface but only 38% DC at a 2-mm thickness with the same nanofilled dentin resin; these data emphasize the decrease in light transmission that occurs throughout the composite resin. 23The DC of the nanofilled resin was influenced by its translucence in the present study.Thus, in this study, the translucent resin showed a higher DC than did the dentin or the enamel opacities.Presumably, there was high light scattering though the translucent resin because a higher transmittance of the light results in a higher DC, 18 which is strongly influenced by the resin opacity and filler contents. 6,18For these reasons, composite resin with the dentin opacity needs to receive a higher exposure time to reach a similar DC of composite resin with enamel opacity (Table 5).
Because the radiant exposure is calculated as the product of irradiance and time of irradiation provided by the light unit, 10 the results obtained using the LED light source are probably due to the generation of a high irradiance of this unit associated with the maximum absorption wavelength of the camphoroquinone.
Moreover, the absence of statistical differences in the degree of conversion in the microhybrid resin in function of opacity and photo-activation factors may be explained by the theory that above a certain energy density threshold, the power density does not influence the polymerization kinetics of the resin polymerization process. 22Moreover, above an irradiance of 10 J/cm², there is a tendency for the DC to stabilize. 24However, higher DCs lead to concerns because they promote higher shrinkage stress during polymerization, and there might be an increased risk of adhesive failures. 13,24he nanofilled resin (FS) showed an interaction of the studied factors.The dentin resin showed a statistically lower KHN when activated by the QTH (39.6 KHN) as compared to the group activated by the LED (56 KHN).An increase in irradiance leads to a higher hardness in the composite resin, 10 and polymerization depends not only on the intensity of the light, but also on the total amount transmitted throughout the polymer. 22Thus, one possible reason for this difference may be related to the energy density of the LED associated with the pigments of the resin composite that were able to scatter the light emitted from the QTH.
It has been reported that the KHN of the A1 shade of a nanofilled composite (FS) was higher (52.3 KHN) than that of the A3 shade (42.7 KHN) after activation by a QTH lamp, showing that the degree of conversion may be impaired by croma saturation. 19In another study, a 37.4 KHN and a 48.6% degree of conversion were reported for the Filtek Supreme-A3 activated with a QTH lamp; these values were considerably lower than the values observed in the present study due to the saturation of the nanofilled resin chosen. 8The translucent resin (shade YT) showed no statistical differences between QTH or LED activation, and these results are similar to those of the present study, which showed similar KHN values independently of the light source. 25igher KHN values at a 2-mm depth have been reported for Charisma samples activated by LED 26 rather than by QTH.Nevertheless, these values are not in agreement with the values achieved in the present study; one probable explanation for this difference is the lower irradiance values in the prior study. 26In the present study, no differences related to opacity or photo-activation factors were observed for the microhybrid resin in the microhardness profiles or in the DC evaluation.One possible reason for this result is the presence of colloidal silica particles in the composite resin, which could cause dispersion of the incident light, thus decreasing the polymerization depth of the composite. 13lthough the results were not statistically comparable, it was observed that the nanofilled resin showed the highest mean microhardness values of those tested.Furthermore, the present study highlights the influence of the amount of energy delivered on the degree of composite polymerization.

Conclusions
The microhardness values of the enamel and the translucent color of the nanofilled composite resin were not influenced by the light activation source.Further, the activation of the dentin shade by the LED resulted in higher microhardness values than did the QTH activation.The dentin and enamel shades of the nanofilled composite showed lower DCs than did the translucent shade.The microhy-brid composite showed no differences in microhardness values or in the degree of conversion for all of the experimental groups.

Figure 1 -
Figure 1 -FT-Raman spectrum of nanofilled composite (translucent) cured by halogen light (14 J/cm²) with the main vibrational modes assigned: C=O stretching mode at 1730 cm -1 ; the methacrylate C=C stretching mode at 1640 cm -1 ; the C=C stretching of the aromatic group at 1610 and 1452 cm -1 .

Table 1 -
Composite resin materials evaluated and their classification, composition, opacity and lot number (No).

Table 2 -
Composite resins' shades and photoactivation sources, showing the calculated irradiation time and the energy density (J/cm²).

Table 4 -
Mean Knoop microhardness values (KHN) and standard deviations for each experimental group of microhybrid composites (CH).

Table 5 -
Mean DC (%) of the nanofilled resin (FS) after activation and standard deviations, and the results of Tukey's test for the opacity factor.

Table 3 -
Mean Knoop microhardness values (KHN) and standard deviations for each experimental group of nanofilled resin (FS) and a Tukey's post-hoc test.