The goal of this research was to experimentally evaluate the surface morphology and adhesion capacity of Streptococcus mutans (U159) on brackets with thin films of titanium nitride (TN) and of titanium nitride doped with calcium phosphate (TNCP).
Twenty-four metallic brackets were equally allocated to 3 groups (n = 8), according to the type of covering (no covering, TNCP, and TN). The coatings were deposited by cathodic cage (TNCP and TN groups) and were evaluated by scanning electron microscopy and energy dispersive x-ray spectrometry. The biofilm formation of S. mutans on the surface of brackets was determined by crystal violet assay and subsequent optical density quantification.
There was homogeneity on the surface morphology of the tie wing area in all groups, whereas the TNCP group has presented particles in the slot. After 24 hours, a biofilm of S. mutans was formed in all the observed groups. The optical density obtained in all 3 groups was similar (no covering, 0.347 ± 0.042; TNCP, 0.238 ± 0.055; TN, 0.226 ± 0.057), with no statistically relevant difference ( P = 0.06).
The thin film of TNCP has altered the surface of the bracket’s slot, whereas the coatings of TN and TNCP have not altered the superficial morphology of the tie wings. The presence of coatings have not influenced the formation of the S. mutans biofilm on the surface of metallic brackets.
The method for depositing the thin films has darken the brackets.
The film of titanium nitride doped with calcium phosphate has altered the bracket’s slot.
The brackets tie wings surfaces have not suffered any alterations with the thin films.
No antimicrobial characteristic has been observed in the evaluated films.
A biofilm of Streptococcus mutans was formed in all groups.
White spot lesions affect the majority of orthodontic patients. As brackets work as retention sites, areas of demineralized enamel may occur when binomial biofilm-sugar is present. The antimicrobial activity of some substances has been widely studied for the purpose of reducing biofilm formation and, therefore, the development of white spot lesions and caries. For orthodontic patients, preventive actions are limited to oral hygiene instructions, fluoridation, and application of varnishes and fluorides.
Among all orthodontic materials, brackets play a significant role in biofilm retention and, as a consequence, in enamel demineralization, for they remain attached to the teeth during orthodontic treatment. In addition, their complex design results in difficult cleansing. The incorporation of antimicrobial agents in the bracket is an option to prevent caries regardless of patient compliance.
Titanium nitride (TN) coating has been used in dentistry since the 1980s, but it was only by the year 2000 that its antimicrobial potential began to be studied, especially in implants. Several authors have reported smaller bacteria attachment to surfaces covered with TN in comparison with control groups.
Amorphous calcium phosphate is a biomaterial used for dental regeneration. Its addition to orthodontic bonding materials means an alternative for secondary prevention of caries, especially in noncooperative patients who have a high cariogenic potential. In addition, its known positive effect on the remineralization of areas peripheral to brackets, , an inhibitory action on microorganisms’ growth was recently observed in amorphous calcium phosphate-coated titanium.
The bactericidal action reported in the literature and the excellent biocompatibility of calcium phosphate and TN led us to select these experimental thin films for this research. Thus, the objective of this study was an in vitro evaluation of the surface morphology and the adhesion capacity of Streptococcus mutans on brackets with thin films of titanium nitride doped with calcium phosphate (TNCP) and of TN on its own.
Material and methods
Twenty-four stainless steel brackets for mandibular incisors (Edgewise, 0.022-in slot, area size 10.72 mm 2 ; Morelli, Sorocaba, Brazil) were equally and randomly divided into 3 groups according to coating types. Brackets belonging to the no covering (NC) group did not receive any coating. In the TNCP and TN groups brackets were coated with TNCP and TN alone, respectively, through cathodic cage deposition, an innovative technique based on multiple hollow cathode effects. This technique presents some advantages compared with the conventional methods, including the simplicity of the apparatus involved and formation of homogenous thin films even in surfaces of complex geometry, such as orthodontic brackets.
The depositions were made in a plasma chamber with a perforated cathodic cage made of commercially pure titanium (Ti) grade II (100 mm × 64 mm × 1 mm), in a plasma atmosphere of 20% nitrogen and 80% hydrogen, for 330 minutes with constant pressure and temperature (106.66 Pa and 240 ± 5°C). The brackets were positioned within the cage, on an insulating surface. The cathodic cage acted as a cathode and plasma was not formed on the surface of the samples.
Two brackets from each group were used for sample characterization, whereas the remaining ones were used in the microbiological evaluation. Surface morphology of the bracket slot and tie wing areas of each group were examined using a Carl Zeiss EVO 40 series scanning electron microscope (SEM), at 1,000 X magnification (Carl Zeiss, Jena, Germany). Energy dispersive x-ray spectroscopy (EDS) analysis was performed, allowing the identification of chemical elements in the brackets through the interaction between the primary electron beam and the samples.
Streptococcus mutans U159 was inoculated in a brain heart infusion (BHI) medium (Becton Dickinson, Franklin Lakes, New Jersey) and incubated for 24 hours at 100 rpm, 37°C and 5% CO 2 partial pressure. From this inoculum, standardized suspensions were prepared at 10 8 Colony Forming Unit (CFU)/mL (optical density [OD] 620 = 1.0). OD is a common method for estimating the concentration of bacteria in a liquid medium, being related to the number of actual bacterial cells through a standard curve relating these 2 parameters. CFUs were verified by plating serial dilutions of each inoculum onto a BHI medium (Becton Dickinson).
The remaining brackets of each group (NC, TNCP, and TN) were sterilized at 121°C, 1 atm for 30 minutes (Stericlan 12; Sandersmedical, Santa Rita, Brazil) and evaluated after 24 hours of incubation. Each experiment was conducted in independent triplicates, in 6 independent repetitions with a total of 18 tests. Blanks consisted of brackets put under the same experimental conditions, but with no microorganisms.
On 96-well microtiter plates, the brackets were submerged in aliquots of 100 μL of S. mutans suspension in the standardized BHI (or only BHI for blank groups) for 24 hours at 37°C and 5% carbon dioxide partial pressure. The growth medium did not contain any added sugar or sucrose.
After the incubation period, the brackets were gently washed with distilled water and adhered cells were fixed with 99% methanol (Isofar, Rio de Janeiro, Brazil) for 15 minutes. The brackets were dried and immersed in a 0.1% crystal violet solution (Isofar, Rio de Janeiro, Brazil) for 20 minutes. The excess of crystal violet was removed and each well washed 5 times with distilled water. Then, the crystal violet impregnated in the biofilm was dissolved with the addition of 100 μL of acetic acid 33% (Isofar) and the OD of the cells measured at 590 nm (BioPhotometer Plus, Eppendorf, Hamburg, Germany), to quantify the total biomass of the adhered biofilm. Absorption values from the control group were subtracted from those obtained from the experimental groups to eliminate medium interference.
Intraclass correlation coefficient was applied to test the repeatability of assessments and normality was assessed through the Shapiro-Wilk test. A comparative analysis of S. mutans biofilm formation and the type of coating among the groups was analyzed by 1-way analysis of variance. The level of significance adopted was α = 5%. The software Bioestat statistical (version 5.3; Mamirauá, Belém, Brazil) was used for statistical analysis.
Deposition of thin films resulted in darkened brackets in the TNCP and TN groups, when compared with the NC group ( Fig 1 ).
Figure 2 depicts scanning electron micrographs of superficial aspects of the brackets in NC, TNCP, and TN groups, respectively. A homogeneous surface morphology could be observed on the bracket tie wings of all groups. In contrast, Figure 3 shows the presence of particles of different sizes on the bracket’s slot area with a thin film of TNCP, what does not occur in NC and TN groups.
Analysis through EDS confirmed the presence of Ti, calcium, and phosphorus on the surface of brackets from the TNCP group and Ti on those from the TN group. Shadows of the elements iron, chrome, manganese, nickel, and silicon are present in all evaluations, as these are chemical constituents of the brackets ( Table I ).