Ó 2014 Eur J Oral Sci
Eur J Oral Sci 2014; 122: 154–160 DOI: 10.1111/eos.12120 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Intensity and duration of in-vitro antibacterial activity of different adhesives used in orthodontics
Claudio Passariello1, Gianpaolo Sannino2, Stefano Petti1, Pierangelo Gigola3 1
Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, Rome; 2Department of Prosthodontics, University of Rome “Tor Vergata”, Rome; 3 Department of Surgical Specialities, Radiologic and Medico-Forensic Sciences University of Brescia, Brescia, Italy
Passariello C, Sannino G, Petti S, Gigola P. Intensity and duration of in-vitro antibacterial activity of different adhesives used in orthodontics. Eur J Oral Sci 2014; 122: 154–160. © 2014 Eur J Oral Sci This work investigated the antibacterial activity of 14 bonding agents to predict their ability to inhibit white-spot development during orthodontic treatment. Standardized, sterilized disks of each material were continuously rinsed (for up to 180 d) in a flow of sterile saline. At predetermined time points, the residual ability of each material to inhibit bacterial growth (determined by measuring the size of inhibition halos around disks placed onto appropriate culture media seeded with Streptococcus gordonii DSM6777, Streptococcus sanguinis DSM20567, Streptococcus mutans DSM20523, or Lactobacillus acidophilus DSM20079) and biofilm formation (determined by measuring the numbers of bacteria adherent to disks following incubation in appropriate broths) was tested in triplicate and compared with the baseline activities of freshly prepared materials. Overall antibacterial and anti-biofilm activities, adjusted for exposure time and strain of bacteria, were assessed. The decrease of antibacterial activity was faster (30–60 d) and complete for fluoride-enriched materials, but slower (90 d) and partial for antimicrobial-containing materials (benzalkonium chloride, zinc oxide, chlorexidine, or MDPB). Materials enriched with benzalkonium chloride, chlorexidine, or MDPB showed the highest antibacterial activities. Anti-biofilm assays yielded similar results. These data could be helpful for clinicians in the choice of the best performing bonding agent also in light of duration of the clinical application.
During the last few decades orthodontic treatment has undergone significant evolution and at present the majority of individuals undergoing such treatment have strong requirements also to improve their dental and facial esthetics (1, 2). The possible incidence of secondary effects of orthodontic treatment on dental esthetics has consequently become a major concern, needing diagnostic and technical considerations. Complications that occur during and following treatment with fixed orthodontic appliances are mainly caused by the excessive accumulation of dental plaque (3), and include dental and gingival complications. In fact, the placement of fixed orthodontic appliances causes significant ecological changes in the dental biofilm that affect the composition, metabolic activity, and pathogenicity of the oral microbiota, favoring an increase in the incidence of periodontal inflammation and incipient carious lesions (4–8). Ecological alterations and periodontal inflammation are mainly the consequences of mechanical hindrance caused by the appliance, and these are largely reversible in children and adolescents (9). In contrast, esthetic and functional damage that is caused by the creation of persistent white spots and incipient caries contrast greatly with the esthetic
Dr Claudio Passariello, Dipartimento di Sanita “La Pubblica e Malattie Infettive, Universita Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy E-mail: [email protected] Key words: adhesives; antibacterial activity; cariogenic bacteria; white-spotting Accepted for publication January 2014
requirements of patients and their parents, and must be regarded as permanent lesions (10, 11). Several studies have demonstrated that enamel decalcification (persistent white spotting) and the formation of incipient caries in these patients could be the consequence of an increased prevalence of Streptococcus mutans and Lactobacillus species in the dental biofilm surrounding brackets and other fixed orthodontic appliances (12, 13). Recently we demonstrated that, in vitro, biofilms formed by cariogenic bacteria at the surface of brackets develop more rapidly than those formed by non cariogenic streptococci and that this occurs in a material dependent manner (14), which suggests that fixed orthodontic appliances could selectively promote the local development of pathologic biofilms. The possibility that incipient caries and gingival inflammation are induced during treatment with fixed orthodontic appliances has stimulated researchers and commercial organizations to develop orthodontic bonding agents with antibacterial activity (15–19). Most of these modified orthodontic adhesives possess significant antibacterial activity and are expected to reduce enamel demineralization and gingival inflammation in subjects undergoing treatment with fixed orthodontic appliances
Antibacterial activity of adhesives
(20–22). However, only limited information is available on the durability of this antibacterial activity and no study has compared the various aspects of this activity in different orthodontic bonding agents. Consequently, in the study reported herein, we compared the antibacterial activity of different orthodontic bonding agents containing, or not containing, antimicrobial substances, immediately after preparation and after rinsing for different periods of time with physiological saline to simulate the action of saliva.
Material and methods This study was designed to provide a comparative evaluation, in vitro, of the strength and duration of the antibacterial activity of a representative selection of materials available for fixation of orthodontic appliances. The purpose of the study was to generate experimental observations that were easier to interpret than those obtained from in-vivo experimental models, in which the complexity of the dental microbiota, the variability of physico-chemical conditions, mechanical disturbance, and a multitude of other subjective factors would require analysis of extremely large population to obtain statistically meaningful results. Observations were performed on pure cultures of reference strains of two species of cariogenic bacteria and on two species of bacteria present in the normal plaque of humans. These bacteria are discussed in more detail below. Separate investigations were performed on the inhibition, by the selected materials, of planktonic growth and sessile growth of the selected species to investigate, in more detail, the mechanisms potentially implicated in the activity exerted by these materials.
155
Streptococcus spp. strains were maintained during the experiments by weekly passage on Todd Hewitt agar plates. Lactobacillus acidophilus DSM 20079 was maintained by weekly passage on MRS agar plates. Processing of disks Following gas sterilization, the 192 disks of each test material were placed in a sterile glass bottle (capacity 100 ml), containing 15 ml of sterile physiological saline (time = T0), and subjected to a constant flow of sterile physiological saline at 7.5 ml h 1 using the pumps and controller of a BioSys 2000 FPLC system (Beckman Coulter, Milan, Italy) at 35°C for up to 180 d. At predetermined time points (1, 7, 30, 60, 90, 120, 150, and 180 d from T0), 24 disks of each material were removed from the bottle with sterile forceps, placed inside a sterile Petri dish, and allowed to dry inside a biohazard II cabinet. Twelve disks (three for each strain tested) were used for residual antibacterial activity assays and 12 disks (three for each strain tested) were used for biofilm-formation assays (Fig. 1). Antibacterial activity A modification of the antimicrobial disk-diffusion assay was used to assess the antimicrobial activity of the tested orthodontic bonding agents. Standardized cultures of each strain to be tested were prepared by plating 1.0 9 106 colony-forming units (CFUs) of each strain onto the surface of a 50-mm Petri dish that contained the appropriate culture medium. Then, three disks of each test material were placed on the surface of the inoculated plates, which were incubated at 35°C for 24 h (Fig. 1). Following incubation, the diameters of the resultant inhibition zones were measured using a digital caliper.
Bonding agents and sample preparation Fourteen orthodontic bonding agents, with or without antibacterial agents, were tested for antibacterial activity (Table 1). Seven are commercially available, fluorideenriched products (one of which, material G, is a product that is intended for a different use but has already been tested elsewhere as a bonding agent for orthodontic appliances); one (material J) is a commercially available, fluoride-free product (22); and the remaining six are commercial materials that were modified for the present experiments by the addition of antibacterial agents, according to previously published methods (15–19, 21). For agar-diffusion tests and biofilm-formation assays, 192 disks (of 6 mm diameter and 2 mm height) of each test material were prepared using custom-made molds. The discs were allowed to polymerize appropriately, sterilized by exposure to ethylene oxide gas for 5 h, and then degassed for 48 h. Bacterial strains and culture As mentioned earlier, four reference strains of bacteria were used: Streptococcus gordonii DSM6777, Streptococcus sanguinis DSM20567, S. mutans DSM20523, and Lactobacillus acidophilus DSM 20079. All strains were maintained in stock cultures, containing 20% [volume by volume (v/v)] glycerol, at 80°C.
Biofilm formation Twelve disks of each test material were placed inside the wells of a 24-well plate (three disks per well) that contained 1 ml of culture medium (Todd Hewitt broth for streptococci and MRS broth for L. acidophilus). Each well was then inoculated with 1.0 9 105 CFUs of each test strain and incubated for a further 72 h at 35°C (Fig. 1). Following incubation, the culture medium was carefully removed, the disks were washed five times with 1 ml of PBS (pH 7.2), and the amount of adherent bacteria on each disk was determined using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (23, 24). Briefly, each well that contained a disk was emptied carefully of any residual liquid, the disks were transferred individually, using sterile forceps, to 1.5-ml microcentrifuge tubes, and 0.15 ml of PBS was added, followed by 0.05 ml of MTT [0.3% (vol/vol) in PBS; Sigma Chemical, St Louis, MO, USA]. Samples were then incubated for 2 h at 37°C. The MTT was replaced with 0.15 ml of dimethylsulfoxide and 0.025 ml of glycine buffer (0.1 M, pH 10.2) and the samples were incubated for 15 min at room temperature. In the MTT reduction assay, bacteria with an active electron transport system reduce the pale yellow tetrazolium salt to water-soluble purple formazan. The amount of formazan produced by each reaction was determined by measuring
156
Passariello et al. Table 1 List of orthodontic cements used during the study
Material
Manufacturer
Base and antibacterial compound
Reference
Identification
GC BJM Laboratories Pulpdent Kuraray Reliance Orthodontic Reliance Orthodontic Kuraray
Glass ionomer + fluoride Light-cured adhesive + fluoride
Commercial Commercial
A B
Light-cured adhesive + fluoride Light-cured adhesive + fluoride Autopolymerizable monocomponent adhesive + fluoride Autopolymerizable bicomponent adhesive + fluoride
Commercial Commercial Commercial
C D E
Commercial
F G
Superbond C&B+BAC-5 Superbond C&B+BAC-2.5 Superbond C&B Fuji Ortho LC Fuji Ortho LC + ZnO
Generique
SAITO et al. (18)
I
Commercial Commercial SPENCER et al. (17)
J K L
Fuji Ortho GC + CHL10 Fuji Ortho GC + CHL18
GC
Autopolymerizable bicomponent adhesive + benzalkonium chloride 5% Autopolymerizable bicomponent adhesive + benzalkonium chloride 2.5% Autopolymerizable bicomponent adhesive Light-cured resin-modified glass ionomer + fluoride Light-cured resin-modified glass ionomer + 23% zinc oxide Glass ionomer + fluoride + 10% chlorhexidine
IMAZATO et al. (15) UYSAL et al. (22) SAITO et al. (18)
FARRET et al. (19)
M
GC
Glass ionomer + fluoride + 18% chlorhexidine
FARRET et al. (19)
N
Fuji Ortho GC High Q Bond Bracket Cement Ortho Choice OBA Kurasper-F Rely-A-Bond Phase-II Clearfil Protect Bond
Generique Generique GC GC
MDPB
Fig. 1. Flow chart of samples used in the study. The chart shows the flow of the 192 gas-sterilized disks, prepared for each of the 14 test materials, through the 180-d time course of rinsing in sterile saline (to simulate exposure to the oral environment) and testing of residual antibacterial activity and residual biofilm-inhibiting activity.
the absorbance at 550 nm using a BioRad model 680 microplate reader (BioRad Laboratories, Segrate, Milan, Italy). Quantitative analysis was performed following construction of species-specific standard curves for each test strain.
H
For each material, bacterial strain, and time point, significant differences from values obtained at baseline were assessed by applying the Student’s t-test for paired samples. The level of significance was set at 95%. The presence of significant differences between materials in their overall antibacterial activity on planktonic and sessile growth, adjusted for strain and time of exposure, were assessed using the two-way analysis of covariance (ANCOVA), with bacterial strains and materials as categories and time as covariate. Two ANCOVAs were performed, with the log number of adherent bacteria and the size of inhibition halos as dependent variables, for activity on sessile and planktonic bacteria, respectively. The ANCOVA provided the overall differences among strains and materials and the potential interactions between time, strains, and materials. In the event that the ANCOVA provided statistically significant results at P < 0.05, post-hoc tests were performed to make pairwise comparisons between strains and between materials. The Bonferroni–Dunn test was chosen. According to the Bonferroni correction, the levels of significance of this test were 0.0083 for bacterial strains (i.e. 0.05 divided by 6, the number of pairwise comparisons between strains) and 0.00055 for materials (i.e. 0.05 divided by 91, the number of pairwise comparisons between materials). This post-hoc test allowed bacterial strains and materials to be ranked according to different levels of resistance and of sessile/planktonic activity, respectively. The statistical software STATVIEW 5.0.1 (SAS Institute, Cary, NC, USA) was used for the ANCOVA, and MICROSOFT EXCEL 2010 was used for the paired t-test.
Statistical analysis Counts of adherent bacteria were normalized by log transformation; undetected values were changed into 1.752 log, corresponding to one half of the distance between zero and the lowest detectable value. The size (in mm) of the inhibition halos was already normalized.
Results The disk-diffusion assays demonstrated that all bonding materials tested, except material J (the fluoride-free
Antibacterial activity of adhesives
control of materials H and I), had significant antibacterial activity toward all test bacteria during the early part of the 180-d experimental period. Although L. acidophilus DSM 20079 yielded significantly larger inhibition halos than the three test streptococci at day 1 (P < 0.05), no statistically significant differences were observed at subsequent time points (data not shown). The antimicrobial activity of the commercial fluorideenriched materials (materials A–F and K) decreased significantly after 30–60 d of continuous rinsing with sterile saline (Fig. 2). Between 150 and 180 d, all fluoride-enriched materials, except for material A, had completely lost their activity, so that bacterial growth extended under the disks (Fig. 2). In contrast, the other materials (materials G–I, M, and N) retained significant antimicrobial activity against all test strains after 180 d of rinsing with saline, although from day 90 onwards, significant differences were observed in the diameters of the inhibition halos compared with those obtained at T0 (Fig. 2). Material L showed a different behavior: in fact, it completely lost its activity against S. sanguinis DSM 20567 and retained minimal activity against the other test strains, with the inhibition halos showing diameters of
Eur J Oral Sci 2014; 122: 154–160 DOI: 10.1111/eos.12120 Printed in Singapore. All rights reserved
European Journal of Oral Sciences
Intensity and duration of in-vitro antibacterial activity of different adhesives used in orthodontics
Claudio Passariello1, Gianpaolo Sannino2, Stefano Petti1, Pierangelo Gigola3 1
Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, Rome; 2Department of Prosthodontics, University of Rome “Tor Vergata”, Rome; 3 Department of Surgical Specialities, Radiologic and Medico-Forensic Sciences University of Brescia, Brescia, Italy
Passariello C, Sannino G, Petti S, Gigola P. Intensity and duration of in-vitro antibacterial activity of different adhesives used in orthodontics. Eur J Oral Sci 2014; 122: 154–160. © 2014 Eur J Oral Sci This work investigated the antibacterial activity of 14 bonding agents to predict their ability to inhibit white-spot development during orthodontic treatment. Standardized, sterilized disks of each material were continuously rinsed (for up to 180 d) in a flow of sterile saline. At predetermined time points, the residual ability of each material to inhibit bacterial growth (determined by measuring the size of inhibition halos around disks placed onto appropriate culture media seeded with Streptococcus gordonii DSM6777, Streptococcus sanguinis DSM20567, Streptococcus mutans DSM20523, or Lactobacillus acidophilus DSM20079) and biofilm formation (determined by measuring the numbers of bacteria adherent to disks following incubation in appropriate broths) was tested in triplicate and compared with the baseline activities of freshly prepared materials. Overall antibacterial and anti-biofilm activities, adjusted for exposure time and strain of bacteria, were assessed. The decrease of antibacterial activity was faster (30–60 d) and complete for fluoride-enriched materials, but slower (90 d) and partial for antimicrobial-containing materials (benzalkonium chloride, zinc oxide, chlorexidine, or MDPB). Materials enriched with benzalkonium chloride, chlorexidine, or MDPB showed the highest antibacterial activities. Anti-biofilm assays yielded similar results. These data could be helpful for clinicians in the choice of the best performing bonding agent also in light of duration of the clinical application.
During the last few decades orthodontic treatment has undergone significant evolution and at present the majority of individuals undergoing such treatment have strong requirements also to improve their dental and facial esthetics (1, 2). The possible incidence of secondary effects of orthodontic treatment on dental esthetics has consequently become a major concern, needing diagnostic and technical considerations. Complications that occur during and following treatment with fixed orthodontic appliances are mainly caused by the excessive accumulation of dental plaque (3), and include dental and gingival complications. In fact, the placement of fixed orthodontic appliances causes significant ecological changes in the dental biofilm that affect the composition, metabolic activity, and pathogenicity of the oral microbiota, favoring an increase in the incidence of periodontal inflammation and incipient carious lesions (4–8). Ecological alterations and periodontal inflammation are mainly the consequences of mechanical hindrance caused by the appliance, and these are largely reversible in children and adolescents (9). In contrast, esthetic and functional damage that is caused by the creation of persistent white spots and incipient caries contrast greatly with the esthetic
Dr Claudio Passariello, Dipartimento di Sanita “La Pubblica e Malattie Infettive, Universita Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy E-mail: [email protected] Key words: adhesives; antibacterial activity; cariogenic bacteria; white-spotting Accepted for publication January 2014
requirements of patients and their parents, and must be regarded as permanent lesions (10, 11). Several studies have demonstrated that enamel decalcification (persistent white spotting) and the formation of incipient caries in these patients could be the consequence of an increased prevalence of Streptococcus mutans and Lactobacillus species in the dental biofilm surrounding brackets and other fixed orthodontic appliances (12, 13). Recently we demonstrated that, in vitro, biofilms formed by cariogenic bacteria at the surface of brackets develop more rapidly than those formed by non cariogenic streptococci and that this occurs in a material dependent manner (14), which suggests that fixed orthodontic appliances could selectively promote the local development of pathologic biofilms. The possibility that incipient caries and gingival inflammation are induced during treatment with fixed orthodontic appliances has stimulated researchers and commercial organizations to develop orthodontic bonding agents with antibacterial activity (15–19). Most of these modified orthodontic adhesives possess significant antibacterial activity and are expected to reduce enamel demineralization and gingival inflammation in subjects undergoing treatment with fixed orthodontic appliances
Antibacterial activity of adhesives
(20–22). However, only limited information is available on the durability of this antibacterial activity and no study has compared the various aspects of this activity in different orthodontic bonding agents. Consequently, in the study reported herein, we compared the antibacterial activity of different orthodontic bonding agents containing, or not containing, antimicrobial substances, immediately after preparation and after rinsing for different periods of time with physiological saline to simulate the action of saliva.
Material and methods This study was designed to provide a comparative evaluation, in vitro, of the strength and duration of the antibacterial activity of a representative selection of materials available for fixation of orthodontic appliances. The purpose of the study was to generate experimental observations that were easier to interpret than those obtained from in-vivo experimental models, in which the complexity of the dental microbiota, the variability of physico-chemical conditions, mechanical disturbance, and a multitude of other subjective factors would require analysis of extremely large population to obtain statistically meaningful results. Observations were performed on pure cultures of reference strains of two species of cariogenic bacteria and on two species of bacteria present in the normal plaque of humans. These bacteria are discussed in more detail below. Separate investigations were performed on the inhibition, by the selected materials, of planktonic growth and sessile growth of the selected species to investigate, in more detail, the mechanisms potentially implicated in the activity exerted by these materials.
155
Streptococcus spp. strains were maintained during the experiments by weekly passage on Todd Hewitt agar plates. Lactobacillus acidophilus DSM 20079 was maintained by weekly passage on MRS agar plates. Processing of disks Following gas sterilization, the 192 disks of each test material were placed in a sterile glass bottle (capacity 100 ml), containing 15 ml of sterile physiological saline (time = T0), and subjected to a constant flow of sterile physiological saline at 7.5 ml h 1 using the pumps and controller of a BioSys 2000 FPLC system (Beckman Coulter, Milan, Italy) at 35°C for up to 180 d. At predetermined time points (1, 7, 30, 60, 90, 120, 150, and 180 d from T0), 24 disks of each material were removed from the bottle with sterile forceps, placed inside a sterile Petri dish, and allowed to dry inside a biohazard II cabinet. Twelve disks (three for each strain tested) were used for residual antibacterial activity assays and 12 disks (three for each strain tested) were used for biofilm-formation assays (Fig. 1). Antibacterial activity A modification of the antimicrobial disk-diffusion assay was used to assess the antimicrobial activity of the tested orthodontic bonding agents. Standardized cultures of each strain to be tested were prepared by plating 1.0 9 106 colony-forming units (CFUs) of each strain onto the surface of a 50-mm Petri dish that contained the appropriate culture medium. Then, three disks of each test material were placed on the surface of the inoculated plates, which were incubated at 35°C for 24 h (Fig. 1). Following incubation, the diameters of the resultant inhibition zones were measured using a digital caliper.
Bonding agents and sample preparation Fourteen orthodontic bonding agents, with or without antibacterial agents, were tested for antibacterial activity (Table 1). Seven are commercially available, fluorideenriched products (one of which, material G, is a product that is intended for a different use but has already been tested elsewhere as a bonding agent for orthodontic appliances); one (material J) is a commercially available, fluoride-free product (22); and the remaining six are commercial materials that were modified for the present experiments by the addition of antibacterial agents, according to previously published methods (15–19, 21). For agar-diffusion tests and biofilm-formation assays, 192 disks (of 6 mm diameter and 2 mm height) of each test material were prepared using custom-made molds. The discs were allowed to polymerize appropriately, sterilized by exposure to ethylene oxide gas for 5 h, and then degassed for 48 h. Bacterial strains and culture As mentioned earlier, four reference strains of bacteria were used: Streptococcus gordonii DSM6777, Streptococcus sanguinis DSM20567, S. mutans DSM20523, and Lactobacillus acidophilus DSM 20079. All strains were maintained in stock cultures, containing 20% [volume by volume (v/v)] glycerol, at 80°C.
Biofilm formation Twelve disks of each test material were placed inside the wells of a 24-well plate (three disks per well) that contained 1 ml of culture medium (Todd Hewitt broth for streptococci and MRS broth for L. acidophilus). Each well was then inoculated with 1.0 9 105 CFUs of each test strain and incubated for a further 72 h at 35°C (Fig. 1). Following incubation, the culture medium was carefully removed, the disks were washed five times with 1 ml of PBS (pH 7.2), and the amount of adherent bacteria on each disk was determined using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (23, 24). Briefly, each well that contained a disk was emptied carefully of any residual liquid, the disks were transferred individually, using sterile forceps, to 1.5-ml microcentrifuge tubes, and 0.15 ml of PBS was added, followed by 0.05 ml of MTT [0.3% (vol/vol) in PBS; Sigma Chemical, St Louis, MO, USA]. Samples were then incubated for 2 h at 37°C. The MTT was replaced with 0.15 ml of dimethylsulfoxide and 0.025 ml of glycine buffer (0.1 M, pH 10.2) and the samples were incubated for 15 min at room temperature. In the MTT reduction assay, bacteria with an active electron transport system reduce the pale yellow tetrazolium salt to water-soluble purple formazan. The amount of formazan produced by each reaction was determined by measuring
156
Passariello et al. Table 1 List of orthodontic cements used during the study
Material
Manufacturer
Base and antibacterial compound
Reference
Identification
GC BJM Laboratories Pulpdent Kuraray Reliance Orthodontic Reliance Orthodontic Kuraray
Glass ionomer + fluoride Light-cured adhesive + fluoride
Commercial Commercial
A B
Light-cured adhesive + fluoride Light-cured adhesive + fluoride Autopolymerizable monocomponent adhesive + fluoride Autopolymerizable bicomponent adhesive + fluoride
Commercial Commercial Commercial
C D E
Commercial
F G
Superbond C&B+BAC-5 Superbond C&B+BAC-2.5 Superbond C&B Fuji Ortho LC Fuji Ortho LC + ZnO
Generique
SAITO et al. (18)
I
Commercial Commercial SPENCER et al. (17)
J K L
Fuji Ortho GC + CHL10 Fuji Ortho GC + CHL18
GC
Autopolymerizable bicomponent adhesive + benzalkonium chloride 5% Autopolymerizable bicomponent adhesive + benzalkonium chloride 2.5% Autopolymerizable bicomponent adhesive Light-cured resin-modified glass ionomer + fluoride Light-cured resin-modified glass ionomer + 23% zinc oxide Glass ionomer + fluoride + 10% chlorhexidine
IMAZATO et al. (15) UYSAL et al. (22) SAITO et al. (18)
FARRET et al. (19)
M
GC
Glass ionomer + fluoride + 18% chlorhexidine
FARRET et al. (19)
N
Fuji Ortho GC High Q Bond Bracket Cement Ortho Choice OBA Kurasper-F Rely-A-Bond Phase-II Clearfil Protect Bond
Generique Generique GC GC
MDPB
Fig. 1. Flow chart of samples used in the study. The chart shows the flow of the 192 gas-sterilized disks, prepared for each of the 14 test materials, through the 180-d time course of rinsing in sterile saline (to simulate exposure to the oral environment) and testing of residual antibacterial activity and residual biofilm-inhibiting activity.
the absorbance at 550 nm using a BioRad model 680 microplate reader (BioRad Laboratories, Segrate, Milan, Italy). Quantitative analysis was performed following construction of species-specific standard curves for each test strain.
H
For each material, bacterial strain, and time point, significant differences from values obtained at baseline were assessed by applying the Student’s t-test for paired samples. The level of significance was set at 95%. The presence of significant differences between materials in their overall antibacterial activity on planktonic and sessile growth, adjusted for strain and time of exposure, were assessed using the two-way analysis of covariance (ANCOVA), with bacterial strains and materials as categories and time as covariate. Two ANCOVAs were performed, with the log number of adherent bacteria and the size of inhibition halos as dependent variables, for activity on sessile and planktonic bacteria, respectively. The ANCOVA provided the overall differences among strains and materials and the potential interactions between time, strains, and materials. In the event that the ANCOVA provided statistically significant results at P < 0.05, post-hoc tests were performed to make pairwise comparisons between strains and between materials. The Bonferroni–Dunn test was chosen. According to the Bonferroni correction, the levels of significance of this test were 0.0083 for bacterial strains (i.e. 0.05 divided by 6, the number of pairwise comparisons between strains) and 0.00055 for materials (i.e. 0.05 divided by 91, the number of pairwise comparisons between materials). This post-hoc test allowed bacterial strains and materials to be ranked according to different levels of resistance and of sessile/planktonic activity, respectively. The statistical software STATVIEW 5.0.1 (SAS Institute, Cary, NC, USA) was used for the ANCOVA, and MICROSOFT EXCEL 2010 was used for the paired t-test.
Statistical analysis Counts of adherent bacteria were normalized by log transformation; undetected values were changed into 1.752 log, corresponding to one half of the distance between zero and the lowest detectable value. The size (in mm) of the inhibition halos was already normalized.
Results The disk-diffusion assays demonstrated that all bonding materials tested, except material J (the fluoride-free
Antibacterial activity of adhesives
control of materials H and I), had significant antibacterial activity toward all test bacteria during the early part of the 180-d experimental period. Although L. acidophilus DSM 20079 yielded significantly larger inhibition halos than the three test streptococci at day 1 (P < 0.05), no statistically significant differences were observed at subsequent time points (data not shown). The antimicrobial activity of the commercial fluorideenriched materials (materials A–F and K) decreased significantly after 30–60 d of continuous rinsing with sterile saline (Fig. 2). Between 150 and 180 d, all fluoride-enriched materials, except for material A, had completely lost their activity, so that bacterial growth extended under the disks (Fig. 2). In contrast, the other materials (materials G–I, M, and N) retained significant antimicrobial activity against all test strains after 180 d of rinsing with saline, although from day 90 onwards, significant differences were observed in the diameters of the inhibition halos compared with those obtained at T0 (Fig. 2). Material L showed a different behavior: in fact, it completely lost its activity against S. sanguinis DSM 20567 and retained minimal activity against the other test strains, with the inhibition halos showing diameters of
