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Reduction of bacterial adhesion on dental composite resins by silicon–oxygen thin film coatings
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015017 (http://iopscience.iop.org/1748-605X/10/1/015017) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 149.150.51.237 This content was downloaded on 04/04/2015 at 05:54
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 015017
doi:10.1088/1748-6041/10/1/015017
Paper
received
9 September 2014
Reduction of bacterial adhesion on dental composite resins by silicon–oxygen thin film coatings
re vised
21 December 2014 accep ted for publication
5 January 2015 published
29 January 2015
Pietro Mandracci1, Federico Mussano2, Paola Ceruti2, Candido F Pirri1,3 and Stefano Carossa2 1
Politecnico di Torino, Department of Applied Science and Technology – Materials and Microsystems Laboratory (ChiLab), corso Duca degli Abruzzi 24, I-10129, Torino, Italy 2 Università di Torino, Dipartimento di Scienze Chirurgiche, CIR Dental School, via Nizza 230, 10127, Torino, Italy 3 Istituto Italiano di Tecnologia, Center for Space Human Robotics, Corso Trento 21, 10129 Torino, Italy E-mail: [email protected] Keywords: bacterial adhesion, oral streptococci, thin film coatings, silicon oxide
Abstract Adhesion of bacteria on dental materials can be reduced by modifying the physical and chemical characteristics of their surfaces, either through the application of specific surface treatments or by the deposition of thin film coatings. Since this approach does not rely on the use of drugs or antimicrobial agents embedded in the materials, its duration is not limited by their possible depletion. Moreover it avoids the risks related to possible cytotoxic effects elicited by antibacterial substances released from the surface and diffused in the surrounding tissues. In this work, the adhesion of Streptococcus mutans and Streptococcus mitis was studied on four composite resins, commonly used for manufacturing dental prostheses. The surfaces of dental materials were modified through the deposition of a-SiOx thin films by plasma enhanced chemical vapor deposition. The chemical bonding structure of the coatings was analyzed by Fourier-transform infrared spectroscopy. The morphology of the dental materials before and after the coating deposition was assessed by means of optical microscopy and high-resolution mechanical profilometry, while their wettability was investigated by contact angle measurements. The sample roughness was not altered after coating deposition, while a noticeable increase of wettability was detected for all the samples. Also, the adhesion of S. mitis decreased in a statistically significant way on the coated samples, when compared to the uncoated ones, which did not occur for S. mutans. Within the limitations of this study, a-SiOx coatings may affect the adhesion of bacteria such as S. mitis, possibly by changing the wettability of the composite resins investigated.
1. Introduction The adhesion of bacteria on the surface of biomaterials, and more specifically on dental materials, is a common issue [1, 2] that is frequently faced whatever the type of medical device studied. A promising strategy, which is increasingly implemented to reduce the number of bacteria living on artificial surfaces, relies on growing onto them films enriched in antimicrobial agents, particularly silver ions [3–5] or nanoparticles [6]. Alternatively, these agents are directly embedded within these materials during their preparation [7, 8]. However, incorporating Ag within either the bulk material or the coating cannot prevent the diffusion of metal ions or nanoparticles toward the material or coating surface and hence, possibly, within the recipient tissues, where a potentially harmful action has recently been described in rats [9]. © 2015 IOP Publishing Ltd
A different approach aiming at the reduction of bacterial colonization of the biomaterials is based on achieving physical and chemical properties unfavorable for bacteria adhesion. Thus, the biomaterial features are modulated either by means of surface treatments able to change the material itself (such as wet or dry etching, ion implantation, surface nitruration) [10–12] or by the deposition of a suitable thin film coating [13, 14]. The latter method has the advantage that further useful characteristics can be imbued in the coating other than the antibacterial ones, such as barrier properties against the diffusion of potentially harmful elements from the bulk materials [15]. Although this prospective is promising, it is still challenging to design surfaces endowed with physico-chemical characteristics able to reduce the bacterial contamination, since the mechanisms involved in the adhesion process are not completely understood. Indeed, the interaction at the
IOP Publishing
Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Table 1. Composition of dental resins and protocols used for their production. Resin
Composition monomer and fillers
Manufacturer
Polymerization apparatus and protocol Alogen light: Coltolux II, Coltene/ Whaledent Inc., USA
Gradia
UDMA EDMA, microfine ceramic / prepolymer, 75% wt
GC Europe, NV Leuven, Belgium
Fluorescent light: GC labolight LV-III, GC Dental Products First cycle: alogen light-curing unit (400–420 mw cm−2 for 40 s Second cycle: 12 fluorescent lamps in laboratory light source for 5 min light
Signum
Bis-GMA and TEGDMA–SiO2, Ba-Al-Si (1.0 µm)—70 wt%
Heraeus Kulzer, GmbH, Hanau, Germany
180 s at 1100 W using xenon strobe light
Adoro
UDMA + silicon dioxide
Ivoclar Vivadent, Schaan Liechtenstein
Heat-Light (Lumamat 100)
50% HEMA and 10% to 30%(octahydro-4,7methano-1H-indenediyl) bis(methylene) diacrylate), strontium-aluminum borosilicate glass, silicon oxide, silane and photoinitiators
3M ESPE, Seefeld, Germany
Light-Vacuum Visio Alfa (ESPE) Visio Beta Vario (ESPE)
Aliphatic and cycloaliphatic monomers— Fillers: Al-B-Si and B-SiO2 (0,6 µm)—50 wt%
OR ESPE, St Paul, MN
Sinfony
25 min heat/light
First cycle: Polymerization for 15 s in the Visio Alpha unit (400 mW cm−2) Second cycle: Polymerization for 15 min in the Visio Beta unit (470 mW cm−2) under vacuum at 40 C
Notes. UDMA: Urethane dimethacrylate, EDMA: Ethyleneglycol dimethacrylate, HEMA: hydroxyethyl methacrylate.
interface between bacteria and materials is a complex phenomenon, where morphology of the material is paramount along with roughness and wettability [2]. In this context, the authors focused on the influence that silicon-based inorganic coatings have on the wettability of some representative dental composite resins and consequently on the adhesion of two main oral bacteria species chosen among the Streptococcus genus. Among the wide range of available coating materials, the authors have selected to study silicon–oxygen amorphous thin film alloys (a-SiOx), since they are endowed with very good chemical inertness and biocompatibility [14, 15]. Also, if the oxygen content is sufficiently high, these materials show a very high transparency in the optical region [16]. This is a very important factor when considering the application of a coating on dental prostheses, since the aesthetic features of dental materials are developed carefully to mimic as accurately as possible the color of natural teeth, and should not be modified. A-SiOx thin films can be grown by plasma enhanced chemical vapor deposition (PECVD) directly on the surface of a wide range of materials, including composite resins. Moreover, the growth process can be carried out at low temperatures, allowing for the deposition of coatings on materials unable to stand high temperatures, such as resins or other polymers. It is also possible to apply a plasma-assisted pre-deposition treatment on the surface of the dental material before the growth of the coating in the same reactor. This adhesion treatment relies on the exposure of the material surface to a plasma of O2, Ar, CO2 or other gases, and often shows the added advantage of exerting an antimicrobial action on the treated surface [17–19]. Some preliminary results concerning the effect of a-SiOx coatings on the S. mutans adhesion on composite resins, which were previously reported [13] by 2
the same authors, suggested that the wettability could have a role in reducing the bacterial adhesion on some of these materials. However, that work was limited to the S. mutans bacteria. Moreover, the results on bacterial adhesion were qualitative and related only to a single type of dental resin. In this paper, instead, a more complete study is presented, considering a quantitative comparison between the adhesion of two important oral bacterial species, S. mitis and S. mutans, on four different composite resins, and investigating whether the adhesion process is influenced by an a-SiOx layer grown on these materials by PECVD. More specifically, the null hypothesis that there is no difference between bacterial adhesion on uncoated and coated materials was assessed by the unpaired two-tailed Student’s t-test.
2. Materials and methods 2.1. Substrate preparation Four types of composite resins (Gradia by GC, Japan, Signum by Heraeus-Kulzer, Germany, Adoro by Ivoclar Vivadent, Liechtenstein and Sinfony by 3M, USA) for dental laboratory were shaped as parallelepiped samples of approximately 10 mm × 10 mm × 2 mm. The composite resins were polymerized, following the manufacturers’ protocols, as reported in table 1. Afterwards, the samples were polished by a two component, rubber-based, polishing system (mediumand fine-grained rotating tips). A cleaning process was performed, only on the samples to be coated, before the film deposition, in order to remove from the resins surface any organic contaminant that could reduce film adhesion. The process consisted in the immersion of the samples in an acetone ultrasonic bath for 5 min. No damage to the samples was observed after this treatment, consistently
IOP Publishing
Biomed. Mater. 10 (2015) 015017
P Mandracci et al
with [20], where a similar process was described for the extraction of unpolymerized monomers from dental resins, reporting the absence of any damage to resins samples even after 7 d of acetone immersion. One of the samples was masked in the center before deposition, obtaining a step between the film and the uncoated region, which was used for the film thickness estimation by mechanical profilometry. In order to allow FTIR characterization of the a-SiOx films, also silicon substrates cut from wafer of [1 1 1] orientation were added to each deposition run. 2.2. Film deposition A-SiOx films of about 2 µm thickness were grown on the dental materials by a radio frequency-PECVD (RFPECVD) reactor, using silane (SiH4) and nitrous oxide (N2O) as silicon and oxygen precursors, respectively. The reactor and the growth conditions were already described elsewhere [13]. The uncertainty of film thickness was about 5% as estimated in previous works [13, 14]. The composite resin samples were located in contact with the rf electrode during the deposition processes and both sides of the samples were coated during two separate deposition runs, using the same process parameters. All the deposition processes were run at the same process conditions. 2.3. Surface characterization A Tencor P-10 (KLA-Tencor Corp., USA, California, Milpitas) mechanical profiler was used to estimate the film thickness, on the sample that had been masked before deposition, by measuring the step between the film top surface and uncoated area of the substrate. The deposition rate, which was obtained as the ratio between film thickness and deposition time, resulted 0.4 nm s−1. The same profiler was used to measure the roughness of both the uncoated and coated surfaces. Surface profiles were measured along five different paths of length 5 mm over an area of 25 mm2 in the central part of each sample surface. They were filtered to separate the long-wave component (waviness) from the short-wave one (roughness) by a Gaussian filter, provided by the data acquisition software of the profiler, using a cutoff wavelength of 800 µm. Photos of the samples surfaces before and after the coating deposition were taken using an optical reflected light microscope Nikon ME 600 (Nikon Corp., Japan, Tokio) equipped with a PC-interfaced digital video camera Sony ExwaveHAD SSC-DC50AP (Sony Corp., Japan, Tokio) in order to analyze the morphology of dental materials and deposited films. All photos were taken at the same magnification and the bar shown on each of them marks a length of 100 µm. In some of the images, some parts of the surface are out of focus, since the sample surface is not perfectly planar, but is characterized by a certain waviness. As a consequence, it is not possible to focus all the points of the sample surface at the same time, due to the low depth of field of the microscope objective lenses. 3
2.4. FTIR characterization The a-SiO x films grown on c-Si substrates were characterized as for their chemical bonding structure by means of Fourier-transform infrared (FTIR) spectroscopy, to evaluate the composition of the material. The transmission spectrum in the IR spectral region of the films deposited on c-Si substrates was measured by a Perkin Elmer System 2000 (Perkin_Elmer Corp., USA, Massachusetts, Waltham) FTIR spectrophotometer. The acquisition performed on the uncoated silicon substrate was repeated on the coated sample, after the film deposition. A total number of 64 scans was acquired (range: 400–1700 cm−1, resolution: 1 cm−1). The film spectrum was then obtained by the acquisition software, as the ratio between the transmitted spectrum of the film + substrate structure and the one previously measured on the uncoated Si substrate. 2.5. Contact angle characterization The samples wettability was estimated by the measurement of water contact angle, which was carried out by a dynamic contact angle tester Data Physics OCAH 200 (Data Physics Corp., USA, California, San Jose). The test consisted in pouring a drop of water with a volume of 1.5 µl and acquiring an image of the drop by the high resolution camera of the contact angle tester immediately after. The contact angle was then determined by the data analysis software provided by the instrumentation by fitting the sessile drop profile. For each sample, the contact angle measurement was repeated seven times. 2.6. Bacterial adhesion tests Bacterial adhesion tests were performed on uncoated and coated samples of each dental material. The samples were washed in 70% ethanol and subsequently into sterile water. Finally the specimens were dried under a sterile hood. S. mutans and S. mitis were purchased by American Type Culture Collection (ATCC) and suspended following these indications: 100 µl were seeded on defibrined blood Columbia agar + 5% and incubated for 24 h (at 37 °C, 5% CO2). Three colonies were transferred onto Todd-Hewitt terrain, incubated for 24 h (at 37 °C, 5% CO2) and then 100 µl of bacterial suspension were refreshed in new broth for 4–6 h. Bacteria were then centrifuged at 4000 rpm at 4 °C for 15 min, the pellet was gently washed twice, resuspended in 0.15 M Phosphate-buffered saline (PBS) (pH 7) and sonicated at 70 W for 10 s. Some dilutions of the suspension were plated on blood agar to quantify the bacteria. Meanwhile, the samples were placed into 120 ml of bacterial suspension under constant stirring (650–700 rpm), at 37 °C for 2 h. The samples were (a) washed in PBS 0.15 M at RT for 10 min, (b) fixed in glutaraldehyde 2.5% for 30 min at 4 °C, (c) washed with pure water and (d) colored with acridine orange 1% for 30 min. At least 10 different fields were acquired for each sample. Three different samples were used for each condition tested and the experiments were
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Figure 1. Images taken at the confocal microscope of the surface of Gradia samples uncoated (a) and coated (b) during bacterial adhesion test with S. mitis.
Figure 2. FTIR spectroscopy transmission spectrum of an a-SiOx thin film.
replicated three times (n = 3). Sample observation was carried out using a confocal microscope Radiance 2100, Biorad at 1000 × magnification. Sample images taken from uncoated and coated Gradia samples are shown in figure 1.
which was 10°, for the test it was considered equal to 10°. Also in this case, a p-value lower than 0.05 was considered significant, and a p-value lower than 0.01 was considered highly significant.
3. Results 2.7. Data analysis The statistical analysis of the data was carried out using the PSPP software (version 0.7.9). The significance of the difference between the roughness measured on uncoated and coated samples was analyzed by the unpaired two-tailed Student’s t-test. The same test was also used to analyze results of S. mitis and S. mutans bacterial adhesion tests. A p-value lower than 0.05 was considered significant, and a p-value lower than 0.01 was considered highly significant. The unpaired two-tailed Student’s t-test was also used to analyze the significance of the wettability test results. Since the contact angle measured on coated samples was found always lower than the sensitivity of the instrumentation, 4
3.1. FTIR characterization The results of FTIR characterization are shown in figure 2, where the peaks appear as regions of lower intensity in the transmission spectrum: the more intense peak at 1065 cm−1 is assigned to stretching vibration of Si–O bonds in Si-based amorphous thin films [21]. At the high-wavenumber side of this peak, there is evidence of a convolution structure with another one at about 1150 cm−1, which can be related to bending vibrations of N–H bonds [21]. Moreover, the peak at 810 cm−1 is usually related to bending vibrations of Si–O bonds, while the one at 460 cm−1 can be related to the out of plane rocking vibration of Si–O bonds [21].
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Figure 3. Images taken at the optical microscope of the surface of dental resins: Gradia uncoated (a) and coated (b); Signum uncoated (c) and coated (d); Adoro uncoated (e) and coated (f); Sinfony uncoated (g) and coated (h).
3.2. Optical characterization Images of the dental material surfaces, taken at the optical microscope before and after the coating deposition, are shown in figures 3(a)–(h). Figures 3(a) and (b) show the surface of a Gradia sample, before and after the film deposition respectively. It is possible to observe the presence of some microstructures with an approximately polyhedral shape, embedded in the material bulk. Figures 3(c) and (d) show the surface of a Signum sample, before and after it has been coated. Both images show the presence of scratches, which are due to the mechanical polishing procedure applied to the samples before coating. In this case, the surface waviness is much greater, as can be deduced by the fact that a consistent part of the image is out of focus. Figures 3(e) and (f) show respectively the surfaces of uncoated and coated samples of Adoro dental resin. Also in this case scratches are visible on the surface, which are due to 5
the mechanical polishing procedure. Figures 3(g) and (h) show the uncoated and coated surfaces of Sinfony samples. In this case a greater amount of the sample areas is not focused, due to the greater waviness of these samples, compared to the other ones. For all samples, the color and appearance of the surface was not considerably altered after the coating deposition. 3.3. Roughness analysis The results of roughness analysis are reported in table 2: the Student’s t-test could not find out any difference between the roughnesses of coated and uncoated samples for any of the dental materials, at a significance level of 5% (p > 0.05). 3.4. Contact angle analysis The results of contact angle measurements are reported in table 3. The contact angle on all coated samples
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Table 2. Roughness of dental materials before and after coating deposition. Gradia
Signum
Adoro
Sinfony
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
618 (327)
421 (130)
561 (131)
456 (134)
701 (198)
704 (272)
715 (197)
545 (57)
p = 0.26
p = 0.24
p = 0.99
p = 0.13
π = 0.31
π = 0.31
π = 0.1
π = 0.52
Notes RMS: root mean square roughness. Table 3. Contact angle of dental materials before and after coating deposition. Gradia
Signum
Adoro
Sinfony
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
110.58 (0.15)
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Reduction of bacterial adhesion on dental composite resins by silicon–oxygen thin film coatings
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015017 (http://iopscience.iop.org/1748-605X/10/1/015017) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 149.150.51.237 This content was downloaded on 04/04/2015 at 05:54
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 015017
doi:10.1088/1748-6041/10/1/015017
Paper
received
9 September 2014
Reduction of bacterial adhesion on dental composite resins by silicon–oxygen thin film coatings
re vised
21 December 2014 accep ted for publication
5 January 2015 published
29 January 2015
Pietro Mandracci1, Federico Mussano2, Paola Ceruti2, Candido F Pirri1,3 and Stefano Carossa2 1
Politecnico di Torino, Department of Applied Science and Technology – Materials and Microsystems Laboratory (ChiLab), corso Duca degli Abruzzi 24, I-10129, Torino, Italy 2 Università di Torino, Dipartimento di Scienze Chirurgiche, CIR Dental School, via Nizza 230, 10127, Torino, Italy 3 Istituto Italiano di Tecnologia, Center for Space Human Robotics, Corso Trento 21, 10129 Torino, Italy E-mail: [email protected] Keywords: bacterial adhesion, oral streptococci, thin film coatings, silicon oxide
Abstract Adhesion of bacteria on dental materials can be reduced by modifying the physical and chemical characteristics of their surfaces, either through the application of specific surface treatments or by the deposition of thin film coatings. Since this approach does not rely on the use of drugs or antimicrobial agents embedded in the materials, its duration is not limited by their possible depletion. Moreover it avoids the risks related to possible cytotoxic effects elicited by antibacterial substances released from the surface and diffused in the surrounding tissues. In this work, the adhesion of Streptococcus mutans and Streptococcus mitis was studied on four composite resins, commonly used for manufacturing dental prostheses. The surfaces of dental materials were modified through the deposition of a-SiOx thin films by plasma enhanced chemical vapor deposition. The chemical bonding structure of the coatings was analyzed by Fourier-transform infrared spectroscopy. The morphology of the dental materials before and after the coating deposition was assessed by means of optical microscopy and high-resolution mechanical profilometry, while their wettability was investigated by contact angle measurements. The sample roughness was not altered after coating deposition, while a noticeable increase of wettability was detected for all the samples. Also, the adhesion of S. mitis decreased in a statistically significant way on the coated samples, when compared to the uncoated ones, which did not occur for S. mutans. Within the limitations of this study, a-SiOx coatings may affect the adhesion of bacteria such as S. mitis, possibly by changing the wettability of the composite resins investigated.
1. Introduction The adhesion of bacteria on the surface of biomaterials, and more specifically on dental materials, is a common issue [1, 2] that is frequently faced whatever the type of medical device studied. A promising strategy, which is increasingly implemented to reduce the number of bacteria living on artificial surfaces, relies on growing onto them films enriched in antimicrobial agents, particularly silver ions [3–5] or nanoparticles [6]. Alternatively, these agents are directly embedded within these materials during their preparation [7, 8]. However, incorporating Ag within either the bulk material or the coating cannot prevent the diffusion of metal ions or nanoparticles toward the material or coating surface and hence, possibly, within the recipient tissues, where a potentially harmful action has recently been described in rats [9]. © 2015 IOP Publishing Ltd
A different approach aiming at the reduction of bacterial colonization of the biomaterials is based on achieving physical and chemical properties unfavorable for bacteria adhesion. Thus, the biomaterial features are modulated either by means of surface treatments able to change the material itself (such as wet or dry etching, ion implantation, surface nitruration) [10–12] or by the deposition of a suitable thin film coating [13, 14]. The latter method has the advantage that further useful characteristics can be imbued in the coating other than the antibacterial ones, such as barrier properties against the diffusion of potentially harmful elements from the bulk materials [15]. Although this prospective is promising, it is still challenging to design surfaces endowed with physico-chemical characteristics able to reduce the bacterial contamination, since the mechanisms involved in the adhesion process are not completely understood. Indeed, the interaction at the
IOP Publishing
Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Table 1. Composition of dental resins and protocols used for their production. Resin
Composition monomer and fillers
Manufacturer
Polymerization apparatus and protocol Alogen light: Coltolux II, Coltene/ Whaledent Inc., USA
Gradia
UDMA EDMA, microfine ceramic / prepolymer, 75% wt
GC Europe, NV Leuven, Belgium
Fluorescent light: GC labolight LV-III, GC Dental Products First cycle: alogen light-curing unit (400–420 mw cm−2 for 40 s Second cycle: 12 fluorescent lamps in laboratory light source for 5 min light
Signum
Bis-GMA and TEGDMA–SiO2, Ba-Al-Si (1.0 µm)—70 wt%
Heraeus Kulzer, GmbH, Hanau, Germany
180 s at 1100 W using xenon strobe light
Adoro
UDMA + silicon dioxide
Ivoclar Vivadent, Schaan Liechtenstein
Heat-Light (Lumamat 100)
50% HEMA and 10% to 30%(octahydro-4,7methano-1H-indenediyl) bis(methylene) diacrylate), strontium-aluminum borosilicate glass, silicon oxide, silane and photoinitiators
3M ESPE, Seefeld, Germany
Light-Vacuum Visio Alfa (ESPE) Visio Beta Vario (ESPE)
Aliphatic and cycloaliphatic monomers— Fillers: Al-B-Si and B-SiO2 (0,6 µm)—50 wt%
OR ESPE, St Paul, MN
Sinfony
25 min heat/light
First cycle: Polymerization for 15 s in the Visio Alpha unit (400 mW cm−2) Second cycle: Polymerization for 15 min in the Visio Beta unit (470 mW cm−2) under vacuum at 40 C
Notes. UDMA: Urethane dimethacrylate, EDMA: Ethyleneglycol dimethacrylate, HEMA: hydroxyethyl methacrylate.
interface between bacteria and materials is a complex phenomenon, where morphology of the material is paramount along with roughness and wettability [2]. In this context, the authors focused on the influence that silicon-based inorganic coatings have on the wettability of some representative dental composite resins and consequently on the adhesion of two main oral bacteria species chosen among the Streptococcus genus. Among the wide range of available coating materials, the authors have selected to study silicon–oxygen amorphous thin film alloys (a-SiOx), since they are endowed with very good chemical inertness and biocompatibility [14, 15]. Also, if the oxygen content is sufficiently high, these materials show a very high transparency in the optical region [16]. This is a very important factor when considering the application of a coating on dental prostheses, since the aesthetic features of dental materials are developed carefully to mimic as accurately as possible the color of natural teeth, and should not be modified. A-SiOx thin films can be grown by plasma enhanced chemical vapor deposition (PECVD) directly on the surface of a wide range of materials, including composite resins. Moreover, the growth process can be carried out at low temperatures, allowing for the deposition of coatings on materials unable to stand high temperatures, such as resins or other polymers. It is also possible to apply a plasma-assisted pre-deposition treatment on the surface of the dental material before the growth of the coating in the same reactor. This adhesion treatment relies on the exposure of the material surface to a plasma of O2, Ar, CO2 or other gases, and often shows the added advantage of exerting an antimicrobial action on the treated surface [17–19]. Some preliminary results concerning the effect of a-SiOx coatings on the S. mutans adhesion on composite resins, which were previously reported [13] by 2
the same authors, suggested that the wettability could have a role in reducing the bacterial adhesion on some of these materials. However, that work was limited to the S. mutans bacteria. Moreover, the results on bacterial adhesion were qualitative and related only to a single type of dental resin. In this paper, instead, a more complete study is presented, considering a quantitative comparison between the adhesion of two important oral bacterial species, S. mitis and S. mutans, on four different composite resins, and investigating whether the adhesion process is influenced by an a-SiOx layer grown on these materials by PECVD. More specifically, the null hypothesis that there is no difference between bacterial adhesion on uncoated and coated materials was assessed by the unpaired two-tailed Student’s t-test.
2. Materials and methods 2.1. Substrate preparation Four types of composite resins (Gradia by GC, Japan, Signum by Heraeus-Kulzer, Germany, Adoro by Ivoclar Vivadent, Liechtenstein and Sinfony by 3M, USA) for dental laboratory were shaped as parallelepiped samples of approximately 10 mm × 10 mm × 2 mm. The composite resins were polymerized, following the manufacturers’ protocols, as reported in table 1. Afterwards, the samples were polished by a two component, rubber-based, polishing system (mediumand fine-grained rotating tips). A cleaning process was performed, only on the samples to be coated, before the film deposition, in order to remove from the resins surface any organic contaminant that could reduce film adhesion. The process consisted in the immersion of the samples in an acetone ultrasonic bath for 5 min. No damage to the samples was observed after this treatment, consistently
IOP Publishing
Biomed. Mater. 10 (2015) 015017
P Mandracci et al
with [20], where a similar process was described for the extraction of unpolymerized monomers from dental resins, reporting the absence of any damage to resins samples even after 7 d of acetone immersion. One of the samples was masked in the center before deposition, obtaining a step between the film and the uncoated region, which was used for the film thickness estimation by mechanical profilometry. In order to allow FTIR characterization of the a-SiOx films, also silicon substrates cut from wafer of [1 1 1] orientation were added to each deposition run. 2.2. Film deposition A-SiOx films of about 2 µm thickness were grown on the dental materials by a radio frequency-PECVD (RFPECVD) reactor, using silane (SiH4) and nitrous oxide (N2O) as silicon and oxygen precursors, respectively. The reactor and the growth conditions were already described elsewhere [13]. The uncertainty of film thickness was about 5% as estimated in previous works [13, 14]. The composite resin samples were located in contact with the rf electrode during the deposition processes and both sides of the samples were coated during two separate deposition runs, using the same process parameters. All the deposition processes were run at the same process conditions. 2.3. Surface characterization A Tencor P-10 (KLA-Tencor Corp., USA, California, Milpitas) mechanical profiler was used to estimate the film thickness, on the sample that had been masked before deposition, by measuring the step between the film top surface and uncoated area of the substrate. The deposition rate, which was obtained as the ratio between film thickness and deposition time, resulted 0.4 nm s−1. The same profiler was used to measure the roughness of both the uncoated and coated surfaces. Surface profiles were measured along five different paths of length 5 mm over an area of 25 mm2 in the central part of each sample surface. They were filtered to separate the long-wave component (waviness) from the short-wave one (roughness) by a Gaussian filter, provided by the data acquisition software of the profiler, using a cutoff wavelength of 800 µm. Photos of the samples surfaces before and after the coating deposition were taken using an optical reflected light microscope Nikon ME 600 (Nikon Corp., Japan, Tokio) equipped with a PC-interfaced digital video camera Sony ExwaveHAD SSC-DC50AP (Sony Corp., Japan, Tokio) in order to analyze the morphology of dental materials and deposited films. All photos were taken at the same magnification and the bar shown on each of them marks a length of 100 µm. In some of the images, some parts of the surface are out of focus, since the sample surface is not perfectly planar, but is characterized by a certain waviness. As a consequence, it is not possible to focus all the points of the sample surface at the same time, due to the low depth of field of the microscope objective lenses. 3
2.4. FTIR characterization The a-SiO x films grown on c-Si substrates were characterized as for their chemical bonding structure by means of Fourier-transform infrared (FTIR) spectroscopy, to evaluate the composition of the material. The transmission spectrum in the IR spectral region of the films deposited on c-Si substrates was measured by a Perkin Elmer System 2000 (Perkin_Elmer Corp., USA, Massachusetts, Waltham) FTIR spectrophotometer. The acquisition performed on the uncoated silicon substrate was repeated on the coated sample, after the film deposition. A total number of 64 scans was acquired (range: 400–1700 cm−1, resolution: 1 cm−1). The film spectrum was then obtained by the acquisition software, as the ratio between the transmitted spectrum of the film + substrate structure and the one previously measured on the uncoated Si substrate. 2.5. Contact angle characterization The samples wettability was estimated by the measurement of water contact angle, which was carried out by a dynamic contact angle tester Data Physics OCAH 200 (Data Physics Corp., USA, California, San Jose). The test consisted in pouring a drop of water with a volume of 1.5 µl and acquiring an image of the drop by the high resolution camera of the contact angle tester immediately after. The contact angle was then determined by the data analysis software provided by the instrumentation by fitting the sessile drop profile. For each sample, the contact angle measurement was repeated seven times. 2.6. Bacterial adhesion tests Bacterial adhesion tests were performed on uncoated and coated samples of each dental material. The samples were washed in 70% ethanol and subsequently into sterile water. Finally the specimens were dried under a sterile hood. S. mutans and S. mitis were purchased by American Type Culture Collection (ATCC) and suspended following these indications: 100 µl were seeded on defibrined blood Columbia agar + 5% and incubated for 24 h (at 37 °C, 5% CO2). Three colonies were transferred onto Todd-Hewitt terrain, incubated for 24 h (at 37 °C, 5% CO2) and then 100 µl of bacterial suspension were refreshed in new broth for 4–6 h. Bacteria were then centrifuged at 4000 rpm at 4 °C for 15 min, the pellet was gently washed twice, resuspended in 0.15 M Phosphate-buffered saline (PBS) (pH 7) and sonicated at 70 W for 10 s. Some dilutions of the suspension were plated on blood agar to quantify the bacteria. Meanwhile, the samples were placed into 120 ml of bacterial suspension under constant stirring (650–700 rpm), at 37 °C for 2 h. The samples were (a) washed in PBS 0.15 M at RT for 10 min, (b) fixed in glutaraldehyde 2.5% for 30 min at 4 °C, (c) washed with pure water and (d) colored with acridine orange 1% for 30 min. At least 10 different fields were acquired for each sample. Three different samples were used for each condition tested and the experiments were
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Figure 1. Images taken at the confocal microscope of the surface of Gradia samples uncoated (a) and coated (b) during bacterial adhesion test with S. mitis.
Figure 2. FTIR spectroscopy transmission spectrum of an a-SiOx thin film.
replicated three times (n = 3). Sample observation was carried out using a confocal microscope Radiance 2100, Biorad at 1000 × magnification. Sample images taken from uncoated and coated Gradia samples are shown in figure 1.
which was 10°, for the test it was considered equal to 10°. Also in this case, a p-value lower than 0.05 was considered significant, and a p-value lower than 0.01 was considered highly significant.
3. Results 2.7. Data analysis The statistical analysis of the data was carried out using the PSPP software (version 0.7.9). The significance of the difference between the roughness measured on uncoated and coated samples was analyzed by the unpaired two-tailed Student’s t-test. The same test was also used to analyze results of S. mitis and S. mutans bacterial adhesion tests. A p-value lower than 0.05 was considered significant, and a p-value lower than 0.01 was considered highly significant. The unpaired two-tailed Student’s t-test was also used to analyze the significance of the wettability test results. Since the contact angle measured on coated samples was found always lower than the sensitivity of the instrumentation, 4
3.1. FTIR characterization The results of FTIR characterization are shown in figure 2, where the peaks appear as regions of lower intensity in the transmission spectrum: the more intense peak at 1065 cm−1 is assigned to stretching vibration of Si–O bonds in Si-based amorphous thin films [21]. At the high-wavenumber side of this peak, there is evidence of a convolution structure with another one at about 1150 cm−1, which can be related to bending vibrations of N–H bonds [21]. Moreover, the peak at 810 cm−1 is usually related to bending vibrations of Si–O bonds, while the one at 460 cm−1 can be related to the out of plane rocking vibration of Si–O bonds [21].
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Figure 3. Images taken at the optical microscope of the surface of dental resins: Gradia uncoated (a) and coated (b); Signum uncoated (c) and coated (d); Adoro uncoated (e) and coated (f); Sinfony uncoated (g) and coated (h).
3.2. Optical characterization Images of the dental material surfaces, taken at the optical microscope before and after the coating deposition, are shown in figures 3(a)–(h). Figures 3(a) and (b) show the surface of a Gradia sample, before and after the film deposition respectively. It is possible to observe the presence of some microstructures with an approximately polyhedral shape, embedded in the material bulk. Figures 3(c) and (d) show the surface of a Signum sample, before and after it has been coated. Both images show the presence of scratches, which are due to the mechanical polishing procedure applied to the samples before coating. In this case, the surface waviness is much greater, as can be deduced by the fact that a consistent part of the image is out of focus. Figures 3(e) and (f) show respectively the surfaces of uncoated and coated samples of Adoro dental resin. Also in this case scratches are visible on the surface, which are due to 5
the mechanical polishing procedure. Figures 3(g) and (h) show the uncoated and coated surfaces of Sinfony samples. In this case a greater amount of the sample areas is not focused, due to the greater waviness of these samples, compared to the other ones. For all samples, the color and appearance of the surface was not considerably altered after the coating deposition. 3.3. Roughness analysis The results of roughness analysis are reported in table 2: the Student’s t-test could not find out any difference between the roughnesses of coated and uncoated samples for any of the dental materials, at a significance level of 5% (p > 0.05). 3.4. Contact angle analysis The results of contact angle measurements are reported in table 3. The contact angle on all coated samples
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Biomed. Mater. 10 (2015) 015017
P Mandracci et al
Table 2. Roughness of dental materials before and after coating deposition. Gradia
Signum
Adoro
Sinfony
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
RMS (nm)
618 (327)
421 (130)
561 (131)
456 (134)
701 (198)
704 (272)
715 (197)
545 (57)
p = 0.26
p = 0.24
p = 0.99
p = 0.13
π = 0.31
π = 0.31
π = 0.1
π = 0.52
Notes RMS: root mean square roughness. Table 3. Contact angle of dental materials before and after coating deposition. Gradia
Signum
Adoro
Sinfony
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Uncoated
Coated
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
Theta (°)
110.58 (0.15)
