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Bacterial adhesion on direct and indirect dental restorative composite resins: An in vitro study on a natural biofilm
RESEARCH AND EDUCATION
Bacterial adhesion on direct and indirect dental restorative composite resins: An in vitro study on a natural biofilm Giacomo Derchi, DDS, MSc, PhD,a Michele Vano, DDS, MSc, PhD,b Antonio Barone, DDS, MSc, PhD,c Ugo Covani, MD, MSc,d Alberto Diaspro, MSc, PhD,e and Marco Salerno, MSc, PhDf Composite resins are widely ABSTRACT used in restorative dentistry Statement of problem. Both direct and indirect techniques are used for dental restorations. Which and include products intended technique should be preferred or whether they are equivalent with respect to bacterial adhesion is for use as either direct or inunclear. direct restoratives. However, Purpose. The purpose of this in vitro study was to determine the affinity of bacterial biofilm to one of the major drawbacks to dental restorative composite resins placed directly and indirectly. the use of composite resins is Material and methods. Five direct composite resins for restorations (Venus Diamond, Adonis, their susceptibility to plaque Optifil, Enamel Plus HRi, Clearfil Majesty Esthetic) and 3 indirect composite resins (Gradia, Estenia, 1,2 accumulation. An increase Signum) were selected. The materials were incubated in unstimulated whole saliva for 1 day. The in plaque retention places pabiofilms grown were collected and their bacterial cells counted. In parallel, the composite resin tients at risk for secondary surface morphology was analyzed with atomic force microscopy. Both bacterial cell count and caries adjacent to the comsurface topography parameters were subjected to statistical analysis (a=.05). posite resin margins, and Results. Indirect composite resins showed significantly lower levels than direct composite additionally the formation of resins for bacterial cell adhesion, (P<.001). No significant differences were observed within the biofilm may result in gingival direct composite resins (P.05). However, within the indirect composite resins a significantly inflammation.3 Direct comlower level was found for Gradia than Estenia or Signum (P<.01). A partial correlation was observed between composite resin roughness and bacterial adhesion when the second and posite resin restorations are particularly the third-order statistical moments of the composite resin height distributions were preferred by many clinicians considered. for reasons of minimal interConclusions. Indirect dental restorative composite resins were found to be less prone to biofilm vention.4 Direct restorations adhesion than direct composite resins. A correlation of bacterial adhesion to surface morphology are made in a single treatment exists that is described by kurtosis; thus, advanced data analysis is required to discover possible session at relatively low cost. insights into the biologic effects of morphology. (J Prosthet Dent 2016;-:---) However, when the mass to be polymerized is large (layer processed in the laboratory are more resistant to wear thickness above about 2 mm), the shrinkage stress may than direct composite resins, especially in occlusal concause significant marginal discrepancies and related tact areas,6,7 and they show reduced polymerization defects.5 Additionally, composite resin inlays or onlays
a Research associate, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. b Research associate, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. c Associate Professor, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. d Professor, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. e Director, Nanophysics Department, Italian Institute of Technology, Genova, Italy. f Research Technologist, Nanophysics Department, Italian Institute of Technology, Genova, Italy.
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Clinical Implications When possible, indirect composite resins are to be preferred over direct ones for dental restorations owing to their reduced bacterial adhesion.
shrinkage, which is limited to the thin luting layer.8-10 The enhanced properties of indirect composite resins are associated with a higher degree of conversion obtained by the use of different polymerization procedures that involve activation by heat between 90 C and 140 C and/or vacuum or a nitrogen atmosphere.11 However, the procedure is more expensive and time consuming. Many clinical studies identified secondary caries as one of the main reason for failure of composite resin restorations.1,6-8,12-14 The presence of cariogenic biofilms in the marginal areas of dental restorations is responsible for the development of secondary caries; therefore, bacterial colonization of composite resin surfaces plays a central role in this process. However, little is known about bacterial adherence on the surface of indirect composite resins. Experimental data demonstrate that high surface roughness, and to a lesser extent the high surface free energy of a dental-restorative material, are related to increased biofilm formation on its surface.15-21 Similar considerations also apply to all surfaces of biomedical devices, for example in dental and orthopedic implants.20 Composite resins are complex hybrid materials consisting of an organic resin matrix and inorganic filler particles coated with a bonding agent, usually a silane, to produce a strong interface between the 2 phases. This implies that the surface of a resin-based dental restorative composite resin is not a homogeneous interface because it is the result of the distribution of different physical-chemical phases that present both topographical and chemical differences on a spatial scale of the filler particles, across the microscale to the nanoscale. As a result, commercially available restorative composite resins have different surface roughnesses21-24 and surface chemistry after polishing.3 Surface topography in particular was reported to play an important role in biofilm formation of oral bacteria on composite resins.3,25 The purpose of this in vitro study was to analyze the formation of oral bacterial biofilms on the surface of different direct and indirect composite resin materials. The hypotheses tested were that biofilm formation would be lower on the surface of composite resins used for indirect restorations than those used for direct restorations and that the role of surface topography would be important such that a correlation could be established
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between surface roughness parameters and biofilm formation. MATERIAL AND METHODS Eight different commercially available composite resins were selected, with 5 direct composite resins assessed: Venus Diamond; Heraeus Kultzer (VD), Adonis; Sweden and Martina, Optifil; IDS Dental, Enamel Plus HRi; Micerium (EPH), Clearfil Majesty Esthetic; Kuraray (CME); and 3 indirect composite resins: Gradia; GC Corp, Estenia; Kuraray, and Signum; Heraeus Kultzer (Table 1). A total of 184 disk-shaped test specimens (23 for each material) with a diameter of approximately 4 mm and a height of approximately 2 mm were fabricated. The materials were formed in a calibrated circular steel mold and then covered with a Mylar strip to minimize the formation of an oxygen-inhibited layer. To ensure a high degree of conversion of the direct composite resins, each specimen of this class of materials was then polymerized for 40 seconds in direct contact with the strip with a lightpolymerization unit (Valo; Ultradent Products Inc). The lamp had a nominal power of 1 W, and the lamp tip had a diameter of approximately 5 mm and was kept at a distance of about 1 mm from the surface. These conditions were the same for all the specimens. For the indirect composite resin class of materials, the specimens were polymerized in a laboratory oven (Labolight LV-III; GC Corp) for 2 minutes. All the specimens were then removed from the mold, evaluated for visible surface defects, and polished according to the recommendations of the manufacturers. After polishing, the indirect composite resin specimens were reinserted in the oven for 5 minutes to obtain the final gloss. Finally, all the specimens were stored under lightproof conditions in distilled water at 37 ±1 C. The specimens were kept in these conditions for 5 days to minimize the impact of residual monomer leakage on subsequent tests of cell viability, before which they were cleaned using ethanol (70%) and applicator brush tips (3M ESPE) and thoroughly rinsed in distilled water. After receiving their informed consent to participate in the study, unstimulated whole saliva (UWS) was collected from healthy volunteers who had no acute dental caries or periodontal lesions. UWS collection was routinely performed between 9:00 AM and 11:00 AM to minimize the effects of diurnal variability on salivary composition. Twenty-three different UWS specimens were grown aerobically in braineheart infusion in polystyrene petri dishes for 24 hours at 37 C. The culture medium of each specimen was divided in 8 Eppendorf tubes of 0.5 mL according to the 8 different sets of composite resins to be tested. The respective composite resin specimens were
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Table 1. Composite resin materials investigated Composite Material
Manufacturer
Class of Indication
Class of Filler Size
Filler Type
Gradia
GC Corp
Indirect
Microhybrid (100 nm to 17 mm)
SiO2, fumed SiO2, Sr and lanthanoid F, Al F silicate (prepolymerized)
Resin Base
Estenia
Kuraray
Indirect
Nanohybrid (2 nm to 2 mm)
treated Al2O3
UDMA
Signum
Heraeus Kultzer
Indirect
Microhybrid (w0.6 mm)
SiO2
Bis-GMA, UDMA, TEGMA
Optifil
IDS dental
Direct
Nanohybrid (50 nm to 1.5 mm)
Ba glass
Di- and polyfunctional MA
Enamel Plus HRi (EPH)
Micerium
Direct
Nanohybrid (40 nm to 1 mm)
Nanozirconia, high refractive index glass
Bis-GMA, UDMA, butandiol-MA
Venus Diamond (VD)
Heraeus Kultzer
Direct
Microhybrid (5 nm to 20 mm)
Ba Al F glass (prepolymerized)
TCD-DI-HEA, UDMA
Clearfil Majesty Esthetic (CME)
Kuraray
Direct
Microhybrid (370 nm to 1.5 mm)
Al2O3, glass ceramic (prepolymerized)
4- and 6-functional urethane MA
Adonis
Sweden and Martina
Direct
Nanohybrid (50 nm to 0.9 mm)
Ba glass, pyrogenated Si
Di- and polyfunctional MA
MA
MA, methacrylate.
inserted, one in each tube, with sterile tools. After incubation of the composite resin specimens in the UWS for 1 day, the meniscus of the culture medium was absorbed with sterile tissue paper to prevent modification of the bacterial concentration of the adhesive biofilm. Then the biofilm was removed from the surface of the incubated composite resin specimens and placed into tubes with a known quantity of braineheart infusion (sterile). The tubes were submitted to agitation for 3 minutes (Medicaline) in order to separate the bacterial cells embedded in the biofilm. Each resulting solution was analyzed to calculate the amount of bacterial cells by using the aerobic plate count method.26 The plates with nonselective plate count agar medium were incubated upside-down in the thermostat at 37 C for 48 hours. Then the bacterial colonies were counted, choosing those plates where the colonies were distinct and nonconfluent and thus easily readable.27 Thus, the number of bacteria per unit area (UFC/cm2) of the tested composite resin specimens was determined. The surface morphology of the polished composite resin specimens was measured on 3 different areas with an atomic force microscope (AFM) for 3 specimens per sample. Images with different scan size of 10×10 mm2, 30×30 mm2, and 90×90 mm2 were collected at each specimen position to evaluate for continuity of the morphologic features. The AFM instrument used (MFP3D; Asylum Research) was operated in Tapping mode with silicon probes (NCHR; Nanosensors) of nominal resonance frequency of approximately 330 kHz, a tip length of approximately 15 mm, and a tip apex diameter of approximately 14 nm. Given the large scan size, and in order to keep the Z range within the vertical piezo scanner limit (about 12 mm), in some specimens, the sample base tilt was corrected manually by trial and error with modeling clay (Plasticine; Flair Leisure) as the base holder material for the specimen. All the images were 2562 pixels. After acquisition, the images of height were plane fitted with order 1 for background removal. Where necessary, they were line-by-line flattened with Derchi et al
order 1 after sparse particles assigned to polishing debris or other material not pertaining to the composite resin surface had been masked away. From the height images so treated, the distribution of relative heights was considered. For these not to be affected by artifacts, the flat tails of the distributions were removed by masking the images accordingly at opposite ends where necessary. When the images were not masked, the average values of calculated parameters from both trace and retrace images for the same area were considered to minimize possible artifacts. In addition to qualitative observations, quantitative information of height was extracted from the AFM images. From the corrected height distributions, the 2-dimensional root mean square roughness, Sq, was extracted first, representing the width of the distribution of heights. Additionally, higher moments of the same distribution were considered, namely the third moment (skewness, Sk) and fourth moment (kurtosis, Ku). The quantitative information was extracted from the largest scan size only, as this improved the comparison with the size of the bacterial cells and, we assumed, the correlation with their adhesion. Both the data resulting from the microbiologic experiment and the morphologic measurements of the composite resin surfaces were subjected to statistical analysis by means of 1-way analysis of variance and a post hoc test (Tukey) for multiple pair comparisons (family error rate a=.05) carried out with software (Origin v8.1; OriginLab). RESULTS The mean and standard deviation values of the bacterial cell amount (UFC/cm2) within the biofilms formed on the different composite resins are presented in Figure 1 (n=23). The bacterial counts of all the individual materials are reported independently (Fig. 1A). No statistically significant differences (P.05) were found between any pairs of materials within the class of direct composite resins. However, THE JOURNAL OF PROSTHETIC DENTISTRY
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Bacterial Contents (1023 ufc/cm2)
3.5 c
c
3.0
c
c
2.5
c
2.0 1.5 1.0
b
b
a
0.5
Ad on is
CM E
VD
EP H
l tifi Op
Gr ad ia Es te ni a Si gn um
0.0
Bacterial Contents (1023 ufc/cm2)
A B
2.5 2.0 1.5 A 1.0 0.5 0.0 Indirect
Direct
B Figure 1. Bacterial cell count. A, All individual materials. B, Materials grouped in 2 classes of direct (red bars) and indirect (blue bars) composite resins. Error bars are standard deviations. A, Different lowercase letters identify groups with statistically significant differences (P<.05). B, Different uppercase letters identify groups with statistically significant differences (P<.01).
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along with a few cracks and 1 mm wide holes. These appeared to be prepolymerized fillers. The direct composite resins (Fig. 2D-H) exhibited both common characteristics and differences with respect to the indirect composite resins. For example, the lay (the direction of the predominant surface pattern) due to polishing appeared in different degrees in all the materials, and particularly in EPH (Fig. 2E) and Optifil (Fig. 2D). Additionally, Optifil (Fig. 2D) and Adonis (Fig. 2H) exhibited larger scale grooves that could not be assigned to tool size or finishing defects. VD (Fig. 2F) appeared to have the surface with the greatest adhered contaminants. However, the only composite resin that allowed for clear identification of large filler particles was CME (Fig. 2G). The results of the morphologic analysis of the most relevant images with 90 mm scan size are presented in Figure 3. For the main roughness parameter, Sq, some differences, also statistically significant, emerged among the composite resins. However, this could not be ascribed to their belonging to either of the 2 classes of direct and indirect composite resins. For example, in a comparison among the indirect composite resins, the observation from Figure 2 that Estenia is the roughest of the 3 materials (showing highest Sq) was confirmed, with Signum scoring second and a statistically significant difference (P<.05) appearing between Gradia and Estenia. The origin of the roughness not appearing to be of different type across the 2 classes of composite resins was confirmed by the observation that the 2 materials with the highest Sq were Gradia (indirect) and CME (direct). The same occurred for the 2 materials with the highest Sq, Estenia and Optifil. As a result, in the global data grouping the materials according to their class (Fig. 3B), no statistically significant difference was observed for Sq between direct and indirect composite resins. DISCUSSION
within the class of indirect composite resins, a difference was found between Gradia and both Estenia and Signum, as Gradia had significantly lower (P<.05) bacterial adhesion. Additionally, each indirect material was significantly different from each direct material, with the indirect composite resins showing less bacterial adhesion than the direct composite resins (P<.01, Fig. 1B). Representative AFM images of the composite resin surfaces are displayed in Figure 2. Among the indirect composite resins, especially in Gradia (Fig. 2A), fine parallel grooves were seen, a consequence of the surface treatment. In Estenia (Fig. 2B), the surface features appeared spherical (that is not as stretched along a single direction as in Fig. 2A). In both Estenia and particularly in Signum (Fig. 2B,C), large fillers incompatible with the expected filler size according to Table 1 were visible, THE JOURNAL OF PROSTHETIC DENTISTRY
In this study, different direct and indirect composite resins used in clinical practice were tested in vitro. In order to simulate clinical conditions, a biofilm formed in UWS with an in vitro culture time of 1 day was used to represent the maximum time interval between oral hygiene procedures. A limitation of the present work is that the chemistry of the different composite resins was not addressed, which may affect the resulting surface energy, the evaluation of which would require contact angle and streaming potential measurements. Some monomers released from composite resins have been found to stimulate the growth of the cariesassociated bacteria Streptococcus sobrinus and Lactobacillus acidophilus.28 For example, a higher amount of S sobrinus was found on Tetric Evoceram than compomers and resin-modified glass ionomer cements.29 Derchi et al
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Figure 2. Representative large scan area atomic force microscope images of composite resins tested. A, Gradia. B, Estenia. C, Signum. D, Optifil. Color shade, as described by scale bar at right, corresponds to height of respective position on image.
The amount of accumulated bacteria usually depends on the type of materials tested30 and may also be related to the filler size.31 However, the importance of surface morphology has been pointed out.32,33 The results of the present in vitro study suggest the acceptance of the first research hypothesis, indicating a significantly lower amount of biofilm formation on indirect composite resin restoration, and a partial rejection of the second research hypothesis proposing that the role of surface topography is dominant for adhesion such that a straightforward correlation can be established between surface roughness parameters and biofilm formation. For the surface morphology data, the protruding features identified as deposited debris were removed before the analysis, as toothbrushing would remove them easily. Nevertheless, by extending the surface roughness analysis from Sq
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(which would be equivalent to Sa, and not considered here) to Sk and Ku, more detailed observations could be made. For a better understanding of the Sk and Ku roughness parameters, it is worth first recalling the respective physical meaning. Sk qualifies the symmetry of the distribution of heights in the considered surface area: negative Sk indicates a predominance of valleytype regions, while positive Sk appears on surfaces with predominant peak-type regions; Ku qualifies instead the surface flatness: spiky surfaces have Ku>3, whereas bumpy surfaces have Ku<3, while 3 indicates a surface with neither predominant character as to the type of protrusions (either spiky or bumpy). Thus, it appears that all the composite resins after polishing/ finishing present a dominating valley-type roughness, the indirect composite resins more so than the direct ones.
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Figure 2. (continued). Representative large scan area atomic force microscope images of composite resins tested. E, EPH. F, VD. G, CME. H, Adonis. Color shade, as described by scale bar at right, corresponds to height of respective position on image.
Overall, the material with the largest negative Sk was Adonis, and second was Optifil, which should correspond to large/more relevant valleys/holes. Ku was instead very low for Estenia, Optifil, and EPH, in all cases <3, which means bumpy (that is, rounded) type of asperities may be valleys or mountains, whereas for Signum and partly VD, Ku was much higher than 3, meaning a spiky surface. After grouping the materials into direct and indirect composite resins, it appeared that although Sq is equivalent for both classes of materials, what is most different is rather the Ku (Fig. 3B). Obviously, not only the width of the distribution of heights is important (the lower the better) but also, if not just Gaussian (bell) shaped, the shape of the distribution; for example, the extension of the tails on the low- or the high-height side contains useful information.
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When the morphologic data in Figure 3 is compared with the microbiologic data in Figure 1, one can see that despite Estenia having a similar Sq to the direct composite resins, it performed better (that is, it exhibited lower bacterial cell count) than any of them. The effects of surface roughness and topography on biofilm formation could be explained by the fact that deeper (higher Sq) depressions on the composite resin surface can provide more favorable places for bacterial colonization and biofilm formation because of the difficulty of dislodging bacterial colonies, for example by toothbrushing. Polishing the composite resins is intended to minimize the critical aspect of surface roughness. However, when the first-order parameter Sq is equivalent, additional parameters describing the type of roughness (Sk, Ku) may also become important.
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400 300
b
6
a
a
b
a
–4 Sk
Ku
a
A
b a
120
2.4
1.6
a
40
0.8 a
a
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Direct
Sq
Sk
Sk and Ku (nm)
80
Sq (nm)
nis
E
–2
Sq
–40
0
Ad o
CM
VD
H
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a a
a EP
m
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a
a
a a
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Sq (nm)
b
a
Sk and Ku (nm)
b
200
kurtosis of the height distributions of the composite resin surfaces.
8
b
0.0
–0.8 Ku
B
Figure 3. Morphologic parameters extracted from atomic force microscope images of composite resin surfaces height. Error bars are standard errors. A, All individual materials independently. B, Materials grouped in 2 classes of direct and indirect restorative composite resins. Different letters identify groups with statistically significant differences (P<.05).
If the second most important parameter for bacterial adhesion after Sq is Ku instead of Sk, the interpretation may be that less spiky surfaces such as those of indirect composite resins provide fewer adhesion points for planktonic bacteria. CONCLUSIONS Within the limits of the present in vitro investigation, the following conclusions were drawn: 1. A significantly lower bacterial accumulation was found in the indirect composite resins group than in the direct composite resins group, regardless of the material tested. 2. No statistically significant correlation could be established between major surface roughness parameter root mean square and biofilm formation. 3. The type of roughness in addition to its absolute value seemed to influence bacterial adhesion on dental materials, especially as described by the Derchi et al
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nanostructured surfaces on bacterial adhesion and biofilm formation. PLoS One 2011;6:e25029. 33. Riedewald F. Bacterial adhesion to surfaces: the influence of surface roughness. PDA J Pharm Sci Technol 2006;60:164-71. Corresponding author: Dr Marco Salerno Nanophysics Department Italian Institute of Technology Genova ITALY Email: [email protected] Copyright © 2016 by the Editorial Council for The Journal of Prosthetic Dentistry.
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Bacterial adhesion on direct and indirect dental restorative composite resins: An in vitro study on a natural biofilm Giacomo Derchi, DDS, MSc, PhD,a Michele Vano, DDS, MSc, PhD,b Antonio Barone, DDS, MSc, PhD,c Ugo Covani, MD, MSc,d Alberto Diaspro, MSc, PhD,e and Marco Salerno, MSc, PhDf Composite resins are widely ABSTRACT used in restorative dentistry Statement of problem. Both direct and indirect techniques are used for dental restorations. Which and include products intended technique should be preferred or whether they are equivalent with respect to bacterial adhesion is for use as either direct or inunclear. direct restoratives. However, Purpose. The purpose of this in vitro study was to determine the affinity of bacterial biofilm to one of the major drawbacks to dental restorative composite resins placed directly and indirectly. the use of composite resins is Material and methods. Five direct composite resins for restorations (Venus Diamond, Adonis, their susceptibility to plaque Optifil, Enamel Plus HRi, Clearfil Majesty Esthetic) and 3 indirect composite resins (Gradia, Estenia, 1,2 accumulation. An increase Signum) were selected. The materials were incubated in unstimulated whole saliva for 1 day. The in plaque retention places pabiofilms grown were collected and their bacterial cells counted. In parallel, the composite resin tients at risk for secondary surface morphology was analyzed with atomic force microscopy. Both bacterial cell count and caries adjacent to the comsurface topography parameters were subjected to statistical analysis (a=.05). posite resin margins, and Results. Indirect composite resins showed significantly lower levels than direct composite additionally the formation of resins for bacterial cell adhesion, (P<.001). No significant differences were observed within the biofilm may result in gingival direct composite resins (P.05). However, within the indirect composite resins a significantly inflammation.3 Direct comlower level was found for Gradia than Estenia or Signum (P<.01). A partial correlation was observed between composite resin roughness and bacterial adhesion when the second and posite resin restorations are particularly the third-order statistical moments of the composite resin height distributions were preferred by many clinicians considered. for reasons of minimal interConclusions. Indirect dental restorative composite resins were found to be less prone to biofilm vention.4 Direct restorations adhesion than direct composite resins. A correlation of bacterial adhesion to surface morphology are made in a single treatment exists that is described by kurtosis; thus, advanced data analysis is required to discover possible session at relatively low cost. insights into the biologic effects of morphology. (J Prosthet Dent 2016;-:---) However, when the mass to be polymerized is large (layer processed in the laboratory are more resistant to wear thickness above about 2 mm), the shrinkage stress may than direct composite resins, especially in occlusal concause significant marginal discrepancies and related tact areas,6,7 and they show reduced polymerization defects.5 Additionally, composite resin inlays or onlays
a Research associate, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. b Research associate, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. c Associate Professor, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. d Professor, Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Italy; and Tuscan Dental Institute, Lido di Camaiore, Pisa, Italy. e Director, Nanophysics Department, Italian Institute of Technology, Genova, Italy. f Research Technologist, Nanophysics Department, Italian Institute of Technology, Genova, Italy.
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Clinical Implications When possible, indirect composite resins are to be preferred over direct ones for dental restorations owing to their reduced bacterial adhesion.
shrinkage, which is limited to the thin luting layer.8-10 The enhanced properties of indirect composite resins are associated with a higher degree of conversion obtained by the use of different polymerization procedures that involve activation by heat between 90 C and 140 C and/or vacuum or a nitrogen atmosphere.11 However, the procedure is more expensive and time consuming. Many clinical studies identified secondary caries as one of the main reason for failure of composite resin restorations.1,6-8,12-14 The presence of cariogenic biofilms in the marginal areas of dental restorations is responsible for the development of secondary caries; therefore, bacterial colonization of composite resin surfaces plays a central role in this process. However, little is known about bacterial adherence on the surface of indirect composite resins. Experimental data demonstrate that high surface roughness, and to a lesser extent the high surface free energy of a dental-restorative material, are related to increased biofilm formation on its surface.15-21 Similar considerations also apply to all surfaces of biomedical devices, for example in dental and orthopedic implants.20 Composite resins are complex hybrid materials consisting of an organic resin matrix and inorganic filler particles coated with a bonding agent, usually a silane, to produce a strong interface between the 2 phases. This implies that the surface of a resin-based dental restorative composite resin is not a homogeneous interface because it is the result of the distribution of different physical-chemical phases that present both topographical and chemical differences on a spatial scale of the filler particles, across the microscale to the nanoscale. As a result, commercially available restorative composite resins have different surface roughnesses21-24 and surface chemistry after polishing.3 Surface topography in particular was reported to play an important role in biofilm formation of oral bacteria on composite resins.3,25 The purpose of this in vitro study was to analyze the formation of oral bacterial biofilms on the surface of different direct and indirect composite resin materials. The hypotheses tested were that biofilm formation would be lower on the surface of composite resins used for indirect restorations than those used for direct restorations and that the role of surface topography would be important such that a correlation could be established
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between surface roughness parameters and biofilm formation. MATERIAL AND METHODS Eight different commercially available composite resins were selected, with 5 direct composite resins assessed: Venus Diamond; Heraeus Kultzer (VD), Adonis; Sweden and Martina, Optifil; IDS Dental, Enamel Plus HRi; Micerium (EPH), Clearfil Majesty Esthetic; Kuraray (CME); and 3 indirect composite resins: Gradia; GC Corp, Estenia; Kuraray, and Signum; Heraeus Kultzer (Table 1). A total of 184 disk-shaped test specimens (23 for each material) with a diameter of approximately 4 mm and a height of approximately 2 mm were fabricated. The materials were formed in a calibrated circular steel mold and then covered with a Mylar strip to minimize the formation of an oxygen-inhibited layer. To ensure a high degree of conversion of the direct composite resins, each specimen of this class of materials was then polymerized for 40 seconds in direct contact with the strip with a lightpolymerization unit (Valo; Ultradent Products Inc). The lamp had a nominal power of 1 W, and the lamp tip had a diameter of approximately 5 mm and was kept at a distance of about 1 mm from the surface. These conditions were the same for all the specimens. For the indirect composite resin class of materials, the specimens were polymerized in a laboratory oven (Labolight LV-III; GC Corp) for 2 minutes. All the specimens were then removed from the mold, evaluated for visible surface defects, and polished according to the recommendations of the manufacturers. After polishing, the indirect composite resin specimens were reinserted in the oven for 5 minutes to obtain the final gloss. Finally, all the specimens were stored under lightproof conditions in distilled water at 37 ±1 C. The specimens were kept in these conditions for 5 days to minimize the impact of residual monomer leakage on subsequent tests of cell viability, before which they were cleaned using ethanol (70%) and applicator brush tips (3M ESPE) and thoroughly rinsed in distilled water. After receiving their informed consent to participate in the study, unstimulated whole saliva (UWS) was collected from healthy volunteers who had no acute dental caries or periodontal lesions. UWS collection was routinely performed between 9:00 AM and 11:00 AM to minimize the effects of diurnal variability on salivary composition. Twenty-three different UWS specimens were grown aerobically in braineheart infusion in polystyrene petri dishes for 24 hours at 37 C. The culture medium of each specimen was divided in 8 Eppendorf tubes of 0.5 mL according to the 8 different sets of composite resins to be tested. The respective composite resin specimens were
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Table 1. Composite resin materials investigated Composite Material
Manufacturer
Class of Indication
Class of Filler Size
Filler Type
Gradia
GC Corp
Indirect
Microhybrid (100 nm to 17 mm)
SiO2, fumed SiO2, Sr and lanthanoid F, Al F silicate (prepolymerized)
Resin Base
Estenia
Kuraray
Indirect
Nanohybrid (2 nm to 2 mm)
treated Al2O3
UDMA
Signum
Heraeus Kultzer
Indirect
Microhybrid (w0.6 mm)
SiO2
Bis-GMA, UDMA, TEGMA
Optifil
IDS dental
Direct
Nanohybrid (50 nm to 1.5 mm)
Ba glass
Di- and polyfunctional MA
Enamel Plus HRi (EPH)
Micerium
Direct
Nanohybrid (40 nm to 1 mm)
Nanozirconia, high refractive index glass
Bis-GMA, UDMA, butandiol-MA
Venus Diamond (VD)
Heraeus Kultzer
Direct
Microhybrid (5 nm to 20 mm)
Ba Al F glass (prepolymerized)
TCD-DI-HEA, UDMA
Clearfil Majesty Esthetic (CME)
Kuraray
Direct
Microhybrid (370 nm to 1.5 mm)
Al2O3, glass ceramic (prepolymerized)
4- and 6-functional urethane MA
Adonis
Sweden and Martina
Direct
Nanohybrid (50 nm to 0.9 mm)
Ba glass, pyrogenated Si
Di- and polyfunctional MA
MA
MA, methacrylate.
inserted, one in each tube, with sterile tools. After incubation of the composite resin specimens in the UWS for 1 day, the meniscus of the culture medium was absorbed with sterile tissue paper to prevent modification of the bacterial concentration of the adhesive biofilm. Then the biofilm was removed from the surface of the incubated composite resin specimens and placed into tubes with a known quantity of braineheart infusion (sterile). The tubes were submitted to agitation for 3 minutes (Medicaline) in order to separate the bacterial cells embedded in the biofilm. Each resulting solution was analyzed to calculate the amount of bacterial cells by using the aerobic plate count method.26 The plates with nonselective plate count agar medium were incubated upside-down in the thermostat at 37 C for 48 hours. Then the bacterial colonies were counted, choosing those plates where the colonies were distinct and nonconfluent and thus easily readable.27 Thus, the number of bacteria per unit area (UFC/cm2) of the tested composite resin specimens was determined. The surface morphology of the polished composite resin specimens was measured on 3 different areas with an atomic force microscope (AFM) for 3 specimens per sample. Images with different scan size of 10×10 mm2, 30×30 mm2, and 90×90 mm2 were collected at each specimen position to evaluate for continuity of the morphologic features. The AFM instrument used (MFP3D; Asylum Research) was operated in Tapping mode with silicon probes (NCHR; Nanosensors) of nominal resonance frequency of approximately 330 kHz, a tip length of approximately 15 mm, and a tip apex diameter of approximately 14 nm. Given the large scan size, and in order to keep the Z range within the vertical piezo scanner limit (about 12 mm), in some specimens, the sample base tilt was corrected manually by trial and error with modeling clay (Plasticine; Flair Leisure) as the base holder material for the specimen. All the images were 2562 pixels. After acquisition, the images of height were plane fitted with order 1 for background removal. Where necessary, they were line-by-line flattened with Derchi et al
order 1 after sparse particles assigned to polishing debris or other material not pertaining to the composite resin surface had been masked away. From the height images so treated, the distribution of relative heights was considered. For these not to be affected by artifacts, the flat tails of the distributions were removed by masking the images accordingly at opposite ends where necessary. When the images were not masked, the average values of calculated parameters from both trace and retrace images for the same area were considered to minimize possible artifacts. In addition to qualitative observations, quantitative information of height was extracted from the AFM images. From the corrected height distributions, the 2-dimensional root mean square roughness, Sq, was extracted first, representing the width of the distribution of heights. Additionally, higher moments of the same distribution were considered, namely the third moment (skewness, Sk) and fourth moment (kurtosis, Ku). The quantitative information was extracted from the largest scan size only, as this improved the comparison with the size of the bacterial cells and, we assumed, the correlation with their adhesion. Both the data resulting from the microbiologic experiment and the morphologic measurements of the composite resin surfaces were subjected to statistical analysis by means of 1-way analysis of variance and a post hoc test (Tukey) for multiple pair comparisons (family error rate a=.05) carried out with software (Origin v8.1; OriginLab). RESULTS The mean and standard deviation values of the bacterial cell amount (UFC/cm2) within the biofilms formed on the different composite resins are presented in Figure 1 (n=23). The bacterial counts of all the individual materials are reported independently (Fig. 1A). No statistically significant differences (P.05) were found between any pairs of materials within the class of direct composite resins. However, THE JOURNAL OF PROSTHETIC DENTISTRY
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Bacterial Contents (1023 ufc/cm2)
3.5 c
c
3.0
c
c
2.5
c
2.0 1.5 1.0
b
b
a
0.5
Ad on is
CM E
VD
EP H
l tifi Op
Gr ad ia Es te ni a Si gn um
0.0
Bacterial Contents (1023 ufc/cm2)
A B
2.5 2.0 1.5 A 1.0 0.5 0.0 Indirect
Direct
B Figure 1. Bacterial cell count. A, All individual materials. B, Materials grouped in 2 classes of direct (red bars) and indirect (blue bars) composite resins. Error bars are standard deviations. A, Different lowercase letters identify groups with statistically significant differences (P<.05). B, Different uppercase letters identify groups with statistically significant differences (P<.01).
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along with a few cracks and 1 mm wide holes. These appeared to be prepolymerized fillers. The direct composite resins (Fig. 2D-H) exhibited both common characteristics and differences with respect to the indirect composite resins. For example, the lay (the direction of the predominant surface pattern) due to polishing appeared in different degrees in all the materials, and particularly in EPH (Fig. 2E) and Optifil (Fig. 2D). Additionally, Optifil (Fig. 2D) and Adonis (Fig. 2H) exhibited larger scale grooves that could not be assigned to tool size or finishing defects. VD (Fig. 2F) appeared to have the surface with the greatest adhered contaminants. However, the only composite resin that allowed for clear identification of large filler particles was CME (Fig. 2G). The results of the morphologic analysis of the most relevant images with 90 mm scan size are presented in Figure 3. For the main roughness parameter, Sq, some differences, also statistically significant, emerged among the composite resins. However, this could not be ascribed to their belonging to either of the 2 classes of direct and indirect composite resins. For example, in a comparison among the indirect composite resins, the observation from Figure 2 that Estenia is the roughest of the 3 materials (showing highest Sq) was confirmed, with Signum scoring second and a statistically significant difference (P<.05) appearing between Gradia and Estenia. The origin of the roughness not appearing to be of different type across the 2 classes of composite resins was confirmed by the observation that the 2 materials with the highest Sq were Gradia (indirect) and CME (direct). The same occurred for the 2 materials with the highest Sq, Estenia and Optifil. As a result, in the global data grouping the materials according to their class (Fig. 3B), no statistically significant difference was observed for Sq between direct and indirect composite resins. DISCUSSION
within the class of indirect composite resins, a difference was found between Gradia and both Estenia and Signum, as Gradia had significantly lower (P<.05) bacterial adhesion. Additionally, each indirect material was significantly different from each direct material, with the indirect composite resins showing less bacterial adhesion than the direct composite resins (P<.01, Fig. 1B). Representative AFM images of the composite resin surfaces are displayed in Figure 2. Among the indirect composite resins, especially in Gradia (Fig. 2A), fine parallel grooves were seen, a consequence of the surface treatment. In Estenia (Fig. 2B), the surface features appeared spherical (that is not as stretched along a single direction as in Fig. 2A). In both Estenia and particularly in Signum (Fig. 2B,C), large fillers incompatible with the expected filler size according to Table 1 were visible, THE JOURNAL OF PROSTHETIC DENTISTRY
In this study, different direct and indirect composite resins used in clinical practice were tested in vitro. In order to simulate clinical conditions, a biofilm formed in UWS with an in vitro culture time of 1 day was used to represent the maximum time interval between oral hygiene procedures. A limitation of the present work is that the chemistry of the different composite resins was not addressed, which may affect the resulting surface energy, the evaluation of which would require contact angle and streaming potential measurements. Some monomers released from composite resins have been found to stimulate the growth of the cariesassociated bacteria Streptococcus sobrinus and Lactobacillus acidophilus.28 For example, a higher amount of S sobrinus was found on Tetric Evoceram than compomers and resin-modified glass ionomer cements.29 Derchi et al
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Figure 2. Representative large scan area atomic force microscope images of composite resins tested. A, Gradia. B, Estenia. C, Signum. D, Optifil. Color shade, as described by scale bar at right, corresponds to height of respective position on image.
The amount of accumulated bacteria usually depends on the type of materials tested30 and may also be related to the filler size.31 However, the importance of surface morphology has been pointed out.32,33 The results of the present in vitro study suggest the acceptance of the first research hypothesis, indicating a significantly lower amount of biofilm formation on indirect composite resin restoration, and a partial rejection of the second research hypothesis proposing that the role of surface topography is dominant for adhesion such that a straightforward correlation can be established between surface roughness parameters and biofilm formation. For the surface morphology data, the protruding features identified as deposited debris were removed before the analysis, as toothbrushing would remove them easily. Nevertheless, by extending the surface roughness analysis from Sq
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(which would be equivalent to Sa, and not considered here) to Sk and Ku, more detailed observations could be made. For a better understanding of the Sk and Ku roughness parameters, it is worth first recalling the respective physical meaning. Sk qualifies the symmetry of the distribution of heights in the considered surface area: negative Sk indicates a predominance of valleytype regions, while positive Sk appears on surfaces with predominant peak-type regions; Ku qualifies instead the surface flatness: spiky surfaces have Ku>3, whereas bumpy surfaces have Ku<3, while 3 indicates a surface with neither predominant character as to the type of protrusions (either spiky or bumpy). Thus, it appears that all the composite resins after polishing/ finishing present a dominating valley-type roughness, the indirect composite resins more so than the direct ones.
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Figure 2. (continued). Representative large scan area atomic force microscope images of composite resins tested. E, EPH. F, VD. G, CME. H, Adonis. Color shade, as described by scale bar at right, corresponds to height of respective position on image.
Overall, the material with the largest negative Sk was Adonis, and second was Optifil, which should correspond to large/more relevant valleys/holes. Ku was instead very low for Estenia, Optifil, and EPH, in all cases <3, which means bumpy (that is, rounded) type of asperities may be valleys or mountains, whereas for Signum and partly VD, Ku was much higher than 3, meaning a spiky surface. After grouping the materials into direct and indirect composite resins, it appeared that although Sq is equivalent for both classes of materials, what is most different is rather the Ku (Fig. 3B). Obviously, not only the width of the distribution of heights is important (the lower the better) but also, if not just Gaussian (bell) shaped, the shape of the distribution; for example, the extension of the tails on the low- or the high-height side contains useful information.
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When the morphologic data in Figure 3 is compared with the microbiologic data in Figure 1, one can see that despite Estenia having a similar Sq to the direct composite resins, it performed better (that is, it exhibited lower bacterial cell count) than any of them. The effects of surface roughness and topography on biofilm formation could be explained by the fact that deeper (higher Sq) depressions on the composite resin surface can provide more favorable places for bacterial colonization and biofilm formation because of the difficulty of dislodging bacterial colonies, for example by toothbrushing. Polishing the composite resins is intended to minimize the critical aspect of surface roughness. However, when the first-order parameter Sq is equivalent, additional parameters describing the type of roughness (Sk, Ku) may also become important.
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400 300
b
6
a
a
b
a
–4 Sk
Ku
a
A
b a
120
2.4
1.6
a
40
0.8 a
a
Indirect
Direct
Sq
Sk
Sk and Ku (nm)
80
Sq (nm)
nis
E
–2
Sq
–40
0
Ad o
CM
VD
H
b
–200
0
2
a a
a EP
m
Op tifi l
Sig nu
Est en ia
–100
a
a
aa
a
a
a a
0
4
b a
a
100
Gra dia
Sq (nm)
b
a
Sk and Ku (nm)
b
200
kurtosis of the height distributions of the composite resin surfaces.
8
b
0.0
–0.8 Ku
B
Figure 3. Morphologic parameters extracted from atomic force microscope images of composite resin surfaces height. Error bars are standard errors. A, All individual materials independently. B, Materials grouped in 2 classes of direct and indirect restorative composite resins. Different letters identify groups with statistically significant differences (P<.05).
If the second most important parameter for bacterial adhesion after Sq is Ku instead of Sk, the interpretation may be that less spiky surfaces such as those of indirect composite resins provide fewer adhesion points for planktonic bacteria. CONCLUSIONS Within the limits of the present in vitro investigation, the following conclusions were drawn: 1. A significantly lower bacterial accumulation was found in the indirect composite resins group than in the direct composite resins group, regardless of the material tested. 2. No statistically significant correlation could be established between major surface roughness parameter root mean square and biofilm formation. 3. The type of roughness in addition to its absolute value seemed to influence bacterial adhesion on dental materials, especially as described by the Derchi et al
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27. Zipkes MR, Gilchrist JE, Peeler JT. Comparison of yeast and mold counts by spiral, pour, and streak plate methods. J Assoc Off Anal Chem 1981;64: 1465-9. 28. Konishi N, Torii Y, Kurosaki A, Takatsuka T, Itota T, Yoshiyama M. Confocal laser scanning microscopic analysis of early plaque formed on resin composite and human enamel 2003;790-5. 29. Kawai K, Takaoka T. Inhibition of bacterial and glucan adherence to various light-cured fluoride-releasing restorative materials. J Dent 2001;29:119-22. 30. Hansel C, Leyhausen G, Mai UE, Geurtsen W. Effects of various resin composite (co)monomers and extracts on two caries-associated microorganisms in vitro. J Dent Res 1998;77:60-7. 31. Takahashi Y, Imazato S, Russell RRB, Noiri Y, Ebisu S. Influence of resin monomers on growth of oral streptococci. J Dent Res 2004;83:302-6. 32. Singh AV, Vyas V, Patil R, Sharma V, Scopelliti PE, Bongiorno G, et al. Quantitative characterization of the influence of the nanoscale morphology of
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nanostructured surfaces on bacterial adhesion and biofilm formation. PLoS One 2011;6:e25029. 33. Riedewald F. Bacterial adhesion to surfaces: the influence of surface roughness. PDA J Pharm Sci Technol 2006;60:164-71. Corresponding author: Dr Marco Salerno Nanophysics Department Italian Institute of Technology Genova ITALY Email: [email protected] Copyright © 2016 by the Editorial Council for The Journal of Prosthetic Dentistry.
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