
이번 연구의 목적은 정적인 방법과 동적인 방법으로 형성된 지르코니아 표면에 부착된 바이오필름에 대한 LED 칫솔의 항균 효과를 평가하고자 하였다.
구강 바이오필름을 형성하기 위해 직경 12 mm, 두께 2.5 mm의 지르코니아 디스크를 24-well plate(정적 방법)와 Center for Disease Control and Prevention (CDC) biofilm reactor (동적 방법)에 디스크를 넣어 바이오필름을 형성하였다. 디스크는 아무 처치도 하지 않은 대조군, 상용화된 광역학(PDT) 키트, 치솔질(brushing) 단독, LED 치솔군, LED 치솔과 에리스로신을 같이 적용한 군, 이렇게 5개 그룹으로 구성하였다. 각 군별 처치 후, 개별 디스크를 시험관에 넣고 60 초 동안 vortexing하여 세균을 분리한 후, 분리된 세균 용액을 선택 배지를 이용하여 살아있는 세균 수를 확인한 후 실험 방법에 따른 항균 효과를 계측하였고, 주사전자현미경(SEM)을 통하여 세균의 형태 변화를 관찰하였다.
바이오필름의 형성과 구성비는 동적인 방법과 정적인 방법에 따른 차이는 관찰되지 않았다. 대조군과 실험군간에 세균의 생존률에 유의한 차이가 있었다(
이번 연구 결과 지르코니아 표면에 부착된 바이오필름을 효과적으로 제거하는 방법으로 LED 치솔과 에리스로신을 같이 적용하는 것이 추천된다.
The purpose of this study was to evaluate the antimicrobial effects of a toothbrush with light-emitting diodes (LEDs) on periodontitis-associated dental biofilm attached to a zirconia surface by static and dynamic methods.
Zirconia disks (12 mm diameter, 2.5 mm thickness) were inserted into a 24-well plate (static method) or inside a Center for Disease Control and Prevention (CDC) biofilm reactor (dynamic method) to form dental biofilms using
No significant difference in biofilm formation was observed between dynamic and static methods. A significant difference was observed in the number of viable bacteria between the control and all experimental groups (
The findings suggest that an LED toothbrush with erythrosine is more effective than other treatments in reducing the viability of periodontitis-associated bacteria attached to zirconia in vitro.
Peri-implantitis is an infectious disease caused by bacteria from dental biofilms.1 A history of periodontitis, cigarette smoking, poor oral hygiene, and lack of periodic supportive periodontal therapy are considered risk factors for peri-implantitis.2-4 The composition of biofilm microorganisms around the implant is similar to that observed in periodontitis, which may increase the risk of peri-implantitis in patients with active periodontal disease.5 Dental biofilm formation begins with the pellicle from saliva that covers the surface of the tooth within a few minutes of mechanical cleansing. Early colonizers including
Periodontal disease begins with plaque deposition in the gingival crevice; thus, peri-implant disease progression is associated with plaque deposition on the implant abutment surface and surrounding crevice of the peri-implant area. Therefore, the properties and surface modification of the implant abutment material may affect peri-implant conditions. Zirconium oxide (zirconia) has been recently used as an implant abutment for aesthetic purposes, as its color is similar to that of natural teeth.11,12 In addition, zirconia has a high-loading capacity, and excellent corrosion resistance and biocompatibility, indicative of its suitability as an implant abutment material.13,14 Nascimento et al.15 revealed no significant difference between the bacterial species attached to zirconia and titanium disks but observed a significant difference in the degree of colonization; titanium disks presented higher counts of bacteria than zirconia disks. Another study observed no significant differences between the species and the amount of bacteria present on zirconia and titanium surfaces.16 These results indicate that zirconia, like titanium, is vulnerable to periodontal bacteria in the mouth, and that surface cleaning of zirconia abutments may be significant in the prevention of peri-implant disease.
Treatment studies of peri-implant disease have focused on the cleansing of the implant and abutment material surfaces. Limited accessibility around the implant and abutment may complicate the removal of the bacterial load with mere mechanical debridement.17 Also, mechanical devices are known to damage the surfaces of implants and abutments. Although local and systemic antibiotics have been used to manage this problem, these bacteria have shown resistance to antibiotics. Several methods have been proposed for cleaning the surface of implants, but none of these methods have demonstrated superiority over other methods.18
Photodynamic therapy (PDT) is a newly proposed method for treating periodontitis and peri-implant diseases that applies a combination of light, photosensitizers, and oxygen.19 Irradiation with light of a specific wavelength results in the transition of the photosensitizer from a low energy state to a singlet state. Clinical plaque disclosing agent erythrosine is a potential photosensitizer for the PDT of oral plaque biofilms.20 This process produces reactive oxygen species such as free radicals and singlet oxygen, which are extremely toxic to bacteria.21 Bassetti et al.22 compared the effects of PDT and local drug delivery on the treatment of peri-implant disease and found that PDT could essentially replace local drug delivery. Recently, adjunctive PDT with mechanical debridement in the management of peri-implantitis has been suggested to improve periodontal conditions.23,24 We recently evaluated the antibacterial effect of a newly devised toothbrush with light-emitting diodes (LEDs) on
Researchers have used an in vitro periodontitis-associated dental biofilm model for evaluating the antimicrobial effects of various treatment options for peri-implant disease because in vivo periodontitis-associated biofilm can be altered by host problems and many ethical considerations.26 Frankline et al.27 suggested that an in vitro dental biofilm model using a Center for Disease Control and Prevention (CDC) biofilm reactor (dynamic method) can create an environment similar to the saliva and gingival crevicular fluid in the oral cavity. However, few studies have reported the efficacy of an LED toothbrush on dental biofilm attached to a zirconia surface by CDC biofilm reactor. Therefore, the present study aimed to evaluate the antimicrobial effects of the LED toothbrush on periodontitis-associated dental biofilm attached to a zirconia surface prepared by static and dynamic methods.
Zirconia disks (HASS Corporation, Gangneung, Korea) measuring 12 mm in diameter and 2.5 mm in thickness were manufactured. One side of the zirconia disk was covered with a putty-type hydrophilic vinyl polysiloxane material (Eli-dent Group S.P.A., Fiorentino, Italy) so that bacteria attach to only one side of the disks. The disks were soaked in 70% ethanol for 60 s and sterilized in an autoclave. The disks were then placed in the wells of a 24-well polystyrene cell culture plate (SPL Life Sciences Co., Ltd., Pocheon, Korea) with 2 mL of artificial saliva (Kolmar Korea Co., Ltd., Sejong, Korea), and the plate was incubated at 37°C with gentle shaking for 4 h to form acquired pellicles.
The two strains of periodontitis-associated bacteria used in this study were
For biofilm formation using the static method, 25 μL of
For biofilm formation using the dynamic method, disks were mounted on polypropylene coupon holders and placed in a CDC biofilm reactor (BioSurface Technologies Corporation, Bozeman, USA). The reactor was filled with 350 mL of
After 5 days of incubation, the zirconia disks with multi-species biofilms formed using the dynamic and static methods were divided into five groups, comprising eight disks each (Table 1): negative control; commercial photodynamic therapy (PDT; Periowave system, Ondine Biomedical Inc., Vancouver, Canada) as positive control; brushing with toothbrush (B; Smart E-care, AinA Co., Ltd., Daegu, South Korea) only; brushing with an LED light (BL): and brushing with an LED light and erythrosine (BLE). The LED light comprised one red LED (630 nm, 44 mW), two blue LEDs (465 nm, 64 mW), and one white LED (550 nm, 64 mW). The disks from the PDT group were placed in methylene blue (1 mL, 100 μg/mL) for 60 s, followed by irradiation (670 nm, 160 mW) with the diode laser for 60 s. The disks from the B group were only brushed for 60 s. A brush attached electric toothbrush was applied by a constant speed and direction for desirable experimental results. The disks from the BL group were brushed with an LED light for 60 s. The disks from the BLE group were placed in erythrosine (1 mL, 22 μM) for 60 s before being brushed with an LED light for 60 s. After treatment, each disk was placed in a test tube and vortexed with 3 mL phosphate-buffered saline (PBS) and glass beads (0.15 mm diameter, DAIHAN Scientific, Wonju, Korea) for 60 s to detach the bacteria. The solution containing detached bacteria was spread directly onto trypticase soy agar plates containing 1 mg/mL yeast extract, 1 μg/mL menadione, 5 μg/mL hemin, 5% sheep blood (Hanil-Komed Co., Ltd., Seongnam, Korea), and 1.5% Bacto agar (Becton, Dickinson and Company) using a spiral plate system (IUL, Barcelona, Spain). The plates were incubated under anaerobic conditions for 96 h at 37°C. An automatic counter (IUL) was used to determine the number of colony-forming units (CFUs). The percentage of surviving bacteria was determined by counting the CFUs after incubation by dividing the number of CFUs on the treatment group disks with the number on the control group disks.
Treatment protocol
Group | Treatment | N |
---|---|---|
Control | No treatment | 8 |
PDT | Methylene blue (60 s), diode laser (60 s) | 8 |
B | Brush (60 s) | 8 |
BL | Brush with LED (60 s) | 8 |
BLE | Erythrosine (60 s), brush with LED (60 s) | 8 |
PDT, photodynamic therapy; B, brush alone; BL, brushing with a LED light; BLE, brushing with a LED light and erythrosine.
Scanning electron microscopy (SEM) was used to visualize the changes in bacterial cell walls and observe the number of attached cells. The disks with attached bacteria were fixed in 2.5% glutaraldehyde for 2 h at room temperature. The fixed samples were washed 3× with PBS for 10 min each and dehydrated for 30 min in graded ethanol solutions (30%, 50%, 70%, 90%, and 100%). After critical point drying, samples were mounted on a stub, coated with gold, and observed with SEM. The surface of the disk was observed using variable pressure field emission SEM (SUPRA55VP, Carl Zeiss, Oberkochen, Germany).
The data were analyzed with a statistical program (SPSSTM 22.0, IBM Inc., Armonk, USA). Paired t-test and one-way analysis of variance with the Duncan correction were applied to assess differences in application. The level of significance was set at
Table 2 presents the mean values of log CFU/mL and proportion of surviving bacteria according to static and dynamic culture methods. The mean values of log CFU/mL of biofilms in the dynamic method group were lower than those in the static group, but no significant difference was observed in the level of cell growth between the two methods. Regardless of culture method, the proportion of
Quantitative analysis of biofilm formation with each method
Log CFU/mL (mean± SD) | ||
---|---|---|
Static method (%) | Dynamic method | |
4.69± 0.14 (49.3) | 4.55± 0.12 (48.6) | |
4.83± 0.31 (50.7) | 4.83± 0.09 (51.4) | |
Total cell count | 9.51± 0.22 (100) | 9.37± 0.20 (100) |
CFU, colony forming unit; SD, standard deviation.
Scanning electron microscopy images of attachment on zirconia surface according to culture method. (A, C Biofilm in well plate (static method), (B, D) Biofilm in CDC biofilm reactor (dynamic method). The arrowheads indicate
Table 3 shows the mean counts of viable bacteria and percentages of bacterial reduction according to static and dynamic culture methods and treatments. Regardless of culture method, the control group showed significantly higher mean counts of viable bacteria than all of the experimental groups (
Counts of viable bacteria and percentages of bacterial reduction on the zirconia surface in different treatment groups
Group | Log CFU/mL (mean± SD) | Bacterial reduction (%) | ||
---|---|---|---|---|
Static method | Dynamic method | Static method | Dynamic method | |
Control | 5.10± 0.08a | 5.01± 0.09a | - | - |
B | 4.66± 0.33b | 4.68± 0.28b | 54.3 | 47.5 |
PDT | 4.64± 0.29b | 4.38± 0.22c | 59.4 | 74.6 |
BL | 3.88± 0.24c | 3.72± 0.33d | 93.3 | 93.5 |
BLE | 3.35± 0.25d | 3.44± 0.22d | 97.8 | 97.1 |
CFU, colony-forming unit; SD, standard deviation; B, brush alone; PDT, photodynamic therapy; BL, brushing with a LED light; BLE, brushing with a LED light and erythrosine. Different superscript letters (a, b, c, d) indicate significant differences (
Fig. 2 shows SEM images of
Scanning electron microscopy images of
CDC, Center for Disease Control and Prevention; LED, light-emitting diodes.
This study was designed to evaluate the antimicrobial effects of LED toothbrushes on periodontitis-associated dental biofilm attached to zirconia surfaces by CDC biofilm reactor in vitro. We used
The incomplete removal of plaque around an implant may result in bacterial settlement, leading to peri-implant mucositis. The consequences give rise to peri-implantitis, leading to supportive marginal bone loss. Thus, cleaning the initial bacterial deposits plays an important role in the prevention of peri-implant disease. Several methods have been proposed for the initial treatment of peri-implant disease in clinical settings.29 A few conventional methods, including adjunctive antiseptic rinse, powered toothbrush, and irrigation, allow patients to control plaque; however, their efficacies are unproven,30 leading to the development of new instruments such as the LED toothbrush. The LED toothbrush may have dual action for dental biofilm; brushing is essential for oral hygiene to break the biofilm and LED light may be as effective as photodynamic therapy (PDT). Park et al.31 applied brushing with dentifrice to resorbable blasting media titanium disks incubated with
A limitation of this study is that only two different bacterial strains were used for biofilm formation. Roder et al.36 suggested the possibility of including as many strains or natural environmental samples as possible for the development of biofilms with characteristics similar to those under natural conditions. Hence, further studies should consider the cultivation of several species of bacteria for biofilm formation.
Within the limitations of this study, the use of a blue LED toothbrush with erythrosine was shown to be more effective than conventional PDT for the removal of bacteria attached to zirconia surfaces. This method induced cell wall destruction of
This study was supported by the Scientific Research of Gangneung-Wonju National University Dental Hospital (SR1704). The LED toothbrush used in the experiment was provided by AinA (Daegu, South Korea). Zirconia discs were supplied by HASS Corporation (Gangneung, Korea). No potential conflict of interest relevant to this article was reported.
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