Effects of diode laser application on inflammation and mpo in periodontal tissues in a rat model

Abstract Objective In this study, we aimed to histologically and immunologically evaluate the effect of diode laser treatment when applied adjunctive to scaling and root planing (SRP) in an experimental periodontitis model. Materials and methods We used Wistar-Albino rats (n=60) with average weight of 230 g. Experimental periodontitis was induced by ligature at the right and left first mandibular molar teeth in all rats. After 11 days, the ligature was removed and rats were divided into two groups. The control group (n=30) received only SRP treatment, while the laser group (n=30) received a diode laser (GaAlAs, 810 nm, 1 W, 10 J, 20 s) treatment adjunctive to SRP. Ten rats in each group were sacrificed after 7, 15, and 30 days. Histopathological examination was performed in the left mandible of rats. Myeloperoxidase (MPO) was evaluated by western blot in the gingival specimens from the right mandible. Results MPO levels in the laser group were statistically significantly lower compared with the control group (p≤0.05). There was no statistically significance at any time between MPO levels in the control group (p>0.05). MPO levels in the laser group at the 7th day were statistically significantly higher compared to the 15th (p≤0.05) and the 30th day (p≤0.05). Inflammatory cell infiltration decreased over time in both groups and was statistically significantly lower in the laser group than in the control group at all times (p≤0.01). Conclusions Within the limits of this study, we suggest that diode laser application is an adjunctive treatment because it reduced inflammation and MPO when applied in addition to SRP. On the other hand, more studies are needed for the assessment of the effects of diode laser application to periodontal tissues.

found that gingival crevicular fluid MMP-8 and MPO levels in the patients with chronic periodontitis were significantly higher than those with gingivitis and healthy individuals. Wei, et al. 6 (2004), on the other hand, found that MPO levels in the group with periodontitis were significantly high in compliance with clinical periodontal parameters, and they also reported that ROS played a role on periodontal tissue destruction.
The main target of periodontal treatment is to cease the inflammatory process by reducing the microorganisms through surgical and non-surgical treatments. Nowadays, authors think that the application of laser practices in addition to the non-surgical periodontal treatment could be effective in controlling sub-gingival microorganisms because of their antibacterial efficiency 7 .
Nowadays diode lasers are thought to increase the connective tissue attachment by de-epithelizing the pocket epithelium besides the elimination of the bacteria in the periodontal pockets 8,9 . Thus, it was emphasized that the application of diode laser could be used in addition to SRP.
Despite the fact that the use of diode laser in periodontal treatment is on the rise, the results regarding this subject are still contradictory, and further studies need to be conducted [10][11][12][13][14]  Sixty Wistar-Albino rats with an average weight of 230 g were used during the study period. They were randomly placed in the cages within the same center in the way that there would be five animals in each cage; they were kept at an air-conditioned room with a constant temperature of 20-22ºC. Rats were fed with standard rat feed and water. program, the estimated average for the bone loss parameter was determined as 0,4; the standard deviation as 0,2; the power when received was identified as 0,80, whereas the number of samplings determined for α:0,05 was identified as n:5 at miminum for each time period. For each time period, 10 animals were kept, considering the possibility of having drop outs. Sixty rats in total were divided into two groups by being randomly selected using a computer-generated table, in the way that there would be 30 animals in each group. The first animal was the first choice, followed by animals 2 and 3 respectively for better standardization.   the presence of periodontal pockets. The rats that had periodontitis were randomly divided into two groups.

Experimental design
One group received only SRP and the other received diode laser treatment adjunctive to SRP at the same day.

SRP treatment
The rats were anesthetized with ketamine (30 mg/ kg) and xylazine (5 mg/kg) by intraperitoneal injection.

MPO analysis
Gingival tissues around the right mandibular first molar tooth were collected, pooled together and homogenized. The MPO levels in the tissues were evaluated by using the Western Blot method.
Prior to the blotting process, the proteins in the Effects of diode laser application on inflammation and mpo in periodontal tissues in a rat model  After drying them up, the relative densities of the bands were taken to be analyzed the through Image Analyses System program (Image J, National Institute of Health, Bethesda, USA).

Histological analyses
The left mandibular teeth, gum and bone tissues to be used in histological examinations were taken into 10%-neutral formaldehyde, and we made their

Results
The MPO levels of the laser group at T1, T2, and T3 time periods were found to be statistically lower on a significant level when compared with the control group (p≤0.05).
There was no significant difference between the MPO levels in the control group at T1, T2, and T3 time periods (p>0.05). In the laser group, however, there was a statistically significant difference between MPO levels at T1, T2, and T3 time periods. The MPO level at T1 was statistically higher on a significant level than USLU MÖ, ELTAS A, MARAKOĞLU İ, DÜNDAR S, ŞAHIN K, ÖZERCAN İH the MPO levels at T2 (p≤0.05) and T3 (p≤0.05). There was no statistically significant difference between the MPO levels at T2 and T3 (p>0.05) (

Histopathological findings
The ICI levels pertaining to the laser group at the T1, T2, and T3 time periods were found to be statistically lower on a significant level when compared with the control group (p≤0.01).
There was a statistically significant difference between ICI levels at T1, T2 and T3 time periods in both groups. The ICI level at T1 was statistically higher on a significant level than the ICI levels at T2 Effects of diode laser application on inflammation and mpo in periodontal tissues in a rat model     and T3. There was no statistically significant difference between the ICI levels at T2 and T3 (Table 2) ( Figure   10). Normally, periodontitis can be initiated in the rats resistant to periodontal diseases through the use of methods such as bacterial vaccination (bacterin), creating a surgical defect, feeding rats with a diet rich in carbohydrates or receiving ligature around the teeth.

Discussion
Since the experimental periodontitis model created by receiving ligature to molar teeth is the model which is closest to the natural plaque formation, it has been used frequently in several studies 16,25,26,[29][30][31] . There are studies suggesting that the destruction rate of the alveolar bone was mostly seen on 7 th -11 th days after the ligature had been connected, and that this rate The main purpose of these treatments is to achieve a smooth root surface by removing tartar and dental plaque to regain the biological connection.
The SRP process is accepted as the golden standard in the non-surgical periodontal treatment and it has successfully found an area of use up until today.
However, SRP fails to be effective alone in the presence of furcation areas, concavities and deep pockets. Reactive oxygen metabolites were seen to have decreased on a significant level both in the group to which SRP was applied and in the group to which additional laser was applied, whereas no significant difference was determined between the two groups.
In our study, on the other hand, we seen that the MPO level is lower in the laser group and that its tendency to decrease continues up to T3, while it tends to increase in the control group. These results indicate the fact that the laser has positive effects on the oxidative stress, reducing the inflammation. Separately, neutrophils play a major role both in MPO production and in the ICI process. The fact that the MPO level and ICI proved to be reduced in the laser group suggests that the laser may cause a decrease in neutrophil production.
Neutrophils are the major protectors of host defence against microorganisms. Therefore, the findings of our study promote the fact that the laser could reduce the oxidative stress with its antibacterial activity.
In the treatment of periodontitis, the effect of diode laser application on inflammatory changes was determined by evaluating the change in the bacterial load as well as the clinical parameters in the experimental and clinical studies. It was stated that the application of diode laser in addition to SRP alleviated inflammation by reducing the pathogenic bacteria, such as A. actinomycetemcomitans, P. intermedia, and P. gingivalis, ensuring significant recoveries in periodontal tissues 8,9,43 . One study demonstrated that diode laser was effective on inflammation in the treatment of ligature-induced periodontitis in rats both when used alone and when used as adjunctive therapy to SRP 44 . In our study, we aimed to describe the local inflammatory changes in periodontitis through ICI.
While ICI decreased in both groups as time went by, we determined that this decrease was more pronounced in the laser group.
These findings are in compliance with the study This study has a couple of limitations: only the results of diode laser treatment along with SRP were investigated. There is no group in which periodontitis was generated but no treatment was applied. Moreover, there is no group in which periodontitis was generated and treated only with diode laser application. Thus, the pre-and post-treatment levels of the parameters we studied could not be compared. Additionally, only one dose of diode laser application in ligature-induced periodontitis may not be sufficient to assess the actual effectiveness of the diode laser therapy. Thus, the effectiveness of diode laser therapy applied at more frequent and repeated doses should be investigated.

Conclusion
The results of this study provide significant evidence suggesting that the application of diode laser along with SRP could make additional contribution to the alleviation of inflammation and oxidative stress in periodontal tissues. The fact that there is no standard procedure as to the mode, energy level or the application period of diode lasers used in periodontal treatment requires further studies to be conducted on this type of laser. We are of the opinion that the clinical effect of the diode laser in periodontal treatment will gain much more importance along with the developments in this subject.