|Home | About | Journals | Submit | Contact Us | Français|
The mainstay of periodontal therapy is mechanical removal of subgingival plaque. There is considerable interest in supplementing it with the use of antibiotics and antiseptics. Many drawbacks are associated with these adjunctive pharmacological regimens such as development of resistance to antibiotics and disruption of microflora of the gastrointestinal tract. Hence, alternate means of killing subgingival bacteria are clearly desirable. One such method is the use of laser.
This study aimed to investigate antibacterial capabilities of gallium-arsenic (Ga-As) laser on photosensitized periodontopathic organisms. The three bacteria selected for the study were Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia.
The subjects for the study were selected from the patients visiting the Department of Periodontics, Karnataka Lingayat Education Society's Institute of Dental Sciences, Belgaum.
In vitro study design.
Subgingival plaque samples collected from chronic periodontitis patients were cultured anaerobically for 72 h. Predetermined number of colonies of each bacterium was taken and was then divided into cases and control groups. Both groups were photosensitized using toluidine blue O (TBO) dye and the case groups were irradiated with Ga-As laser. Bacterial colonies were then serially diluted and were incubated for subculture. After incubation period, the number of viable bacterial count was performed.
Wilcoxon-signed rank test was carried out to determine significance of reduction on subsequent dilution within the bacterial group. Mann–Whitney U-test was performed to determine the significance of reduction between cases and control of particular bacterial group.
The results revealed substantial reduction in the viable bacterial count. F. nucleatum was found to be most sensitive to killing by laser irradiation followed by P. intermedia and then P. gingivalis. Further, the TBO dye per se did not have any significant bactericidal effect.
Photodynamic Therapy may prove to be a promising method for eradicating periodontopathic bacteria in near future.
Light has been used as a therapeutic agent for many centuries. In ancient Greece, the Sun was used in heliotherapy or the exposure of the body to the Sun for the restoration of health. Chinese used the Sun to treat conditions such as rickets, skin cancer, and even psychosis. This use of light for treatment of various pathologies is referred as phototherapy.
As long as 1900, it was recognized that the microbes could be killed by visible light by treating with a photosensitizing compound. More recently, the idea of sensitizing bacterial cells to killing by otherwise harmless doses of visible light has been used as the basis of new therapeutic modality known as photodynamic therapy (PDT). This involves the administration of a photosensitizer which exhibits some predilections for bacterial cells and then irradiating with light. Singlet oxygen and free radicals generated by irradiated photosensitizer bring about the destruction of bacteria.
Periodontal diseases are chronic infectious condition resulting from accumulation of bacterial biofilm on the tooth surface below the level of gingival margin. The mainstay of periodontal therapy remains the physical removal of subgingival plaque. However, mechanical therapy alone may fail to eliminate the pathogenic bacteria because of their location within gingival tissue or in other areas inaccessible to periodontal instrumentation. These limitations and the improved biological understanding of periodontal diseases have led to a move in emphasis from a pure mechanical approach to other methods which include the use of antiseptics and antibiotics. Besides the development of resistance, other problems associated with these adjunctive pharmacological regimes are disruption of microflora of oral cavity and gastrointestinal tract. Moreover, it is difficult to achieve bactericidal concentration of antibiotics in periodontium required for complete eradication of periodontopathic organisms. Alternate means of killing subgingival bacteria which could surpass these problems are clearly desirable. One such method is the use of laser along with photosensitizer.
Main advantages of using laser for killing subgingival bacteria are that bactericidal activity is achieved in seconds, which minimizes the need for maintaining high concentration of chemical within the lesion for long time. In addition, killing is mediated by free radicals singlet oxygen, so resistance development would be unlikely and killing would be confined to diseased lesion, so disturbances of microflora at other sites would not occur.
Periodontitis appears to be disease well suited to treatment by PDT since such diseases (localized aggressive periodontitis and chronic periodontitis) are essentially localized infections and it is easy to access the periodontal pocket. Periodontitis would be very amenable to treatment by PDT, wherein the photosensitizer could be placed directly into the pocket which could then be irradiated either through the thin gingival tissue or through optical fiber placed directly into the pocket.
Although PDT is more widely known for its application in the treatment of neoplasms, there is also an interest in antimicrobial PDT because a large number of microorganisms (oral species) have been reported to be killed in vitro by this approach.[2,3] Furthermore, the potential of some key virulence factors (lipopolysaccharide and proteases) has also been shown to be reduced by photosensitization. Hence, an attempt has been made in this study to investigate the bactericidal effect of gallium-arsenic (Ga-As) laser (λ =904 nm) on photosensitized predominant periodontopathogenic organisms.
The subjects for the study were selected from the patients visiting the Department of Periodontics, Karnataka Lingayat Education Society's (KLES's) Institute of Dental Sciences, Belgaum. Ethical clearance was taken before the study by the Ethical Committee, KLES's Institute of Dental Sciences, Belgaum. A special pro forma was designed to obtain written consent of patients for their voluntary participation in the study and explaining the purpose of study in their vernacular language.
Patients diagnosed with chronic periodontitis (according to the American Academy of Periodontology, 1999) with:
A total of thirty subgingival plaque samples were collected from chronic periodontitis patients.
After supragingival scaling, subgingival plaque was collected from pockets >5 mm depth using sterile curettes under aseptic conditions. Samples were then immediately transported to microbiology laboratory using transport media, thioglycollate broth with Vitamin K and hemin. Samples were cultured anaerobically for 72 h at 37°C on blood agar, Brewer's agar culture plates.
After 72 h, growth of bacterial colonies was seen on culture plates.
Bacteria were identified based on colony characteristics, hemolysis, pigmentation, and fluorescence. Three bacteria selected for the study were Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia.
Colony count for the above three mentioned bacteria was performed and expressed as colony-forming unit (CFU)/ml. Predetermined number of colonies (CFU/ml) of each bacteria was taken equally as control and case groups (for P. intermedia 500 colonies, P. gingivalis 400 colonies, F. nucleatum 300 colonies taken initially).
To both groups, 30 µl of 100µg/ml of toluidine blue O (TBO) dye was added and only the case groups were exposed to Ga-As diode laser for 1 min in noncontact mode. Bacterial colonies of cases and control groups were then serially diluted (in thioglycollate broth) in 1:10, 1:50, and 1:100 ratio and subcultured under the same culture conditions and incubated for 72 h. After incubation period, again, the number of viable bacterial count was performed using magnified glass and expressed as CFU/ml to determine the bactericidal effect of Ga-As laser. The above-mentioned procedure was done in an identical manner for each of the bacterial group selected for the study.
The study was statistically analyzed using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). Mean values and standard deviation were calculated. Wilcoxon-signed rank test was performed to determine significance of reduction on subsequent dilution within the bacterial group. Mann–Whitney U-test was performed to determine the significance of reduction between cases and control of particular bacterial group.
A different bactericidal effect of the three bacterial groups investigated was obtained in the present study. Mean reduction values for P. gingivalis: Control = 395.0 ± 11.37 CFU/ml, cases = 299.67 ± 12.45 CFU/ml [Table 1, Figure 1]. Mean reduction values for F. nucleatum: Control = 293.67 ± 12.17 CFU/ml, cases = 211.67 ± 18.21 CFU/ml [Table 2, Figure 2]. Mean reduction values for P. intermedia: control = 494.67 ± 13.58 CFU/ml, cases = 369.0 ± 36.99 CFU/ml [Table 3, Figure 3]. Within the framework of this study, the reductions obtained in F. nucleatum (29.44%) were markedly greater than that of P. gingivalis (25.08%) and P. intermedia (26.20%) [Table 4].
The term “PDT” was established as early as 1900 by Raab, who realized that interaction between acridine dye and visible light in the presence of oxygen, killed paramecia.
PDT is based on the principle that a photoactivable substance, the photosensitizer binds to the target cell and can be activated by light of suitable wavelength. During this process, free radicals are formed (singlet oxygen) which then produce an effect that is toxic to the cell.[4,5]
PDT involves the use of low-power laser light with appropriate wavelength to kill cells or microbes previously treated with photosensitizer. The photosensitizer interacts with the outer wall at the surface of several types of bacteria to increase their permeability and allows a significant amount of photosensitizer to accumulate at the level of cytoplasmic membrane.
PDT has been used as a means of eradicating periodontopathic bacteria and photosensitizer have been tested in vitro and in vivo in combination with low-power laser to determine their bactericidal effect. A number of studies have shown that not only can this approach be used to kill bacteria but it can also be used to reduce the impact of bacterial virulence factors.
The present study aimed to investigate the bactericidal effect of Ga-As laser (λ =904 nm) on predominant anaerobic periodontopathic bacteria. The results of the present investigation have shown that short-term (60 s) exposure to light from Ga-As laser (λ =904 nm, power output = 11 mW) has bactericidal effect on bacteria present in subgingival plaque samples once they have been sensitized using TBO dye. Significant reduction in bacterial count has also been seen on subsequent dilution (P < 0.0001).
The results of the present study were in agreement with previous reports of bactericidal effect of lasers.[7,8,9,10,11,12,13,14] Sarkar and Wilson have shown that 7.3 mW helium-neon (He-Ne) laser irradiation for 30 s significantly reduce the viable count of aerobic and anaerobic bacteria with 100% reduction in black-pigmented anaerobes such as P. gingivalis and F. nucleatum. Furthermore, Wilson and Dobson observed significant bactericidal effects of 7.3 mW He-Ne laser irradiation on pure culture of periodontopathic bacteria photosensitized with TBO dye and methylene blue.
Selection of an effective photosensitizer is essential for the success of the technique. Wilson et al. 1993 have screened a number of chemical compounds for their ability to sensitize oral bacteria to killing by low-power laser from He-Ne laser. Sixteen of the compounds tested were able to act as lethal photosensitizer of Streptococcus sanguis. Some of the most effective photosensitizer were TBO and methylene blue which rendered periodontopathogenic species of P. gingivalis, F. nucleatum, and Aggregatibacter actinomycetemcomitans susceptible to killing by He-Ne laser light following exposure for 30 s. However, neither TBO nor methylene blue affected the viability of organism in the absence of laser at the concentration used (50 mg/ml). Thus, the bactericidal effect seen in their study is mainly because of laser.
In the present study, focus was laid on the bactericidal effect of Ga-As laser (λ = 904 mm) on three predominant periodontopathic bacteria namely P. gingivalis, F. nucleatum, and P. intermedia. In the present study, the mean reduction in bacterial count obtained in control group when compared to cases was not statistically significant. This suggests that dye itself at the concentration (100 µg/ml) used has no significant effect on viability of bacteria. However, it sensitizes the bacteria to killing by laser irradiation.
Overall, the results reveal that periodontopathogenic bacteria display a varying degree of sensitivity to PDT. One can only speculate as to the possible reasons contributing to this observation. Probably, different sensitiveness for the action of Ga-As laser radiation for the investigated strains of bacteria can be assumed which may be determined by cell morphology of various bacterial species.
It is also proposed that the kind of pigmentation of cell wall especially determines the susceptibility of various bacterial species to laser radiation.
Since lethal photosensitization requires binding of sensitizer to the cytoplasmic membrane of the target cell, such variation in the susceptibility may be attributed at least in part to differences in membrane binding and/or cell wall permeability.
Furthermore, surface components such as capsule, fimbriae, and fibrils may protect the species by binding the sensitizer. Quantitative and qualitative differences in the surface components of the three bacteria (P. gingivalis, F. nucleatum, and P. intermedia) therefore may also have contributed to their ranging susceptibility to lethal photosensitization.
The nature of laser light also contributes to nonequal killing rate of bacteria; the most important characteristics are its wavelength, power output, exposure time, whether it is delivered continuously or intermittently, and the diameter of beam. The low level of killing achieved by Ga-As laser radiation–TBO dye combination may be partially attributed to the mismatch between the wavelength of light emitted by laser (λ =904 mm) and the absorption maximum (632 mm) of the dye (range 620–660 mm). Environmental factors of importance include pH, color, thermal conductivity of water and organic matter, and density of cell population which might have overshadowed any accompanying photochemical-induced change. Nitzan et al. in 1989 reported that culture media can reduce photodynamic effect by binding porphyrins to the substrate it contains before these can reach bacterial cells.
In addition, the relatively reduced sensitivity of P. gingivalis and P. intermedia might have been because of their ability to counter the harmful effects of oxygen radicals generated. Nonequal killing rates of bacterial group by laser have been previously reported for neodymium-doped yttrium aluminum garnet, He-Ne, CO2, Ar dye laser.[7,9,16]
In the present investigation, we have used CFU/ml as a method of quantification for bacterial colonies. This method of CFU quantification has been proved superior to the bacterial viability test as well as to the inhibition zone analysis.
Within the limitation of the present study, following conclusions are drawn. Substantially, greater killing is achieved with laser-dye combination when compared to dye alone, and quantitative reduction in the viable count of photosensitized bacteria can be achieved using Ga-As laser (λ = 904nm) at power output 11 mW for 60 s regardless of the type of bacterial species. The results of the present investigation are encouraging and hold promise for future implementation of PDT in anti-infectious treatment of periodontal disease.
There are no conflicts of interest.
We would like to acknowledge Dr. Kishore Bhat M.D., Professor and Head, Department of Microbiology, Maratha Mandal Dental College, Belgaum, for carrying out bacterial culture work. and Oralia Co., Laser Research Laboratory, Germany for laser parameters.