| Abstract|| |
Background: Periodontitis is a multifactorial disease, and the causative periodontal pathogens have been detected in patient samples like plaque, gingival crevicular fluid (GCF), saliva, crevicular blood, gingival tissues, etc. Laser disinfection is an advanced treatment that has been shown to alter the bacterial flora and have greater efficacy in reducing the progression of the disease. Aim: This study is aimed to evaluate the immediate effects of a diode laser (970 ± 15 nm) on the microbial load in GCF, crevicular blood, and saliva samples of patients suffering from chronic periodontitis. Materials and Methods: A total of 45 subjects were recruited for the study. Each patient's mouth was divided equally into two halves, which were allotted randomly into two groups. In group I (test group), diode laser disinfection was performed, and in group II (control group), saline irrigation was performed. The GCF, saliva, and crevicular blood samples were collected before and immediately after disinfection for microbial analysis. Results: Clinical observations showed a reduction in microbial load in both groups, which were statistically significant in the test group. GCF and saliva samples showed greater reduction when compared with crevicular blood samples in both groups. Conclusion: This study concludes that the application of diode laser (970 ± 15 nm) has immediate reducing effects on the microbial load in GCF, crevicular blood, and saliva samples in patients with chronic periodontitis.
Keywords: Chronic periodontitis, diode laser, gingival crevicular fluid, microbial colony count, saliva
|How to cite this article:|
Chandrasekaran K, D Chavan SK, Kripal K, Anuroopa P. Immediate effects of diode laser (970±15 nm) on microbial load in patients with chronic periodontitis: A split-mouth study. Indian J Dent Res 2021;32:453-8
|How to cite this URL:|
Chandrasekaran K, D Chavan SK, Kripal K, Anuroopa P. Immediate effects of diode laser (970±15 nm) on microbial load in patients with chronic periodontitis: A split-mouth study. Indian J Dent Res [serial online] 2021 [cited 2022 Dec 7];32:453-8. Available from: https://www.ijdr.in/text.asp?2021/32/4/453/345417
| Introduction|| |
Periodontitis is a multifactorial inflammatory disease that destroys the supporting periodontal tissues. It is proved beyond doubt that dental biofilm acts as the initiator of periodontal disease. In contrast, the clinical manifestation and progression of the disease are affected by a wide variety of determinants and risk factors. The subgingival microbial complexes are persistently found near the gingival sulcus and play a key role in the pathogenesis of periodontitis. The bacteria may affect the periodontal attachment apparatus by activating the inflammatory and immune processes., Larry Wolff et al., in 1994, concluded from their research that bacteria and their endotoxins might serve as pronounced risk indicators for periodontitis.
Many scientific studies suggest a close association among specific bacteria, their gingival crevicular fluid (GCF) levels and products, and their effect on clinical measures of periodontitis., These microorganisms may be seen attached to the tooth, epithelial surfaces of the gingival crevice or periodontal pocket, and also to other bacteria which are attached to these surfaces. The increased flow of GCF, as a serum transudate or inflammatory exudate, washes periodontal pockets and thereby provides host-derived substances that shape subgingival bacterial populations. The GCF, enriched with bacterial material from subgingival space, finally enters the oral cavity at the periodontal pocket orifice. Only a few studies to date have been able to throw light on the host-associated constituents of GCF and its bacterial composition.,
In periodontal therapy, lasers are beneficial for control of bacteremia, better removal of the pocket epithelium in the pockets,, reduction of bacteria,,,, and improvement of periodontal regeneration in animals and humans without damaging the surrounding bone and pulp tissues., Among the various types of lasers currently available, soft tissue lasers like diode laser (980 nm) are considered a good choice for bacterial reduction. A study conducted by Fontana et al. showed that diode lasers could reduce the bacterial load even without scaling and root planing.
Thus, this study was carried out with the hypothesis that diode lasers reduce the microbial load when performed before oral prophylactic measures. This aimed to assess the immediate effects of diode laser (970 ± 15 nm) on microbial load in GCF, crevicular blood, and saliva samples in patients from the South Indian population suffering from chronic periodontitis.
| Materials and Methods|| |
A randomised, double-blind split-mouth clinical trial was carried out at the Department of Periodontology, Rajarajeswari Dental College & Hospital, Bangalore, India. The ethical committee approved the study protocol of the institution (Ref no: RRDC&H/PG-258/2017-2018). The study has been registered with Clinical Trials Registry India (CTRI) (Reg no: CTRI/2017/12/011030). The patients were explained about the study, and written consent was obtained from all the patients before the study.
A total of 90 samples from 45 patients, aged 35 to 65 years, diagnosed with chronic periodontitis were collected for the study after the initial screening, which included oral hygiene index (1964) (score = 3.1–6.0, Fair) and Russell's periodontal index (1956) (score = 2.0–4.9, beginning destructive periodontal disease). The inclusion criteria for the study were as follows: (1) all the patients who were willing to participate in the study; (2) age range = 35–65 years; (3) patients having at least 20 teeth with probing depth of ≥5 mm, clinical attachment level ≥3mm, and radiographic evidence of alveolar bone loss on at least two teeth per quadrant excluding the third molars; and (4) systemically healthy patients who have not received periodontal treatment for at least 6 months before the clinical examination and sampling.
Patients not willing to participate in the study, smokers, alcoholics, pregnant and lactating women, and patients on any medication taken within the last 6 months were excluded. Each patient's mouth was divided bilaterally, and each half was allotted to a group based on the coin toss method. The two groups were group I (test group) and group II (control group). The quadrants in group I was subjected to diode laser disinfection, and quadrants in group II were subjected to saline irrigation. Scaling and root planing were performed in both groups only after a week of sample collection. The CONSORT flowchart is shown in [Figure 1].
In group I (test group), a diode laser of 970 ± 15 nm wavelength and 1.2 W power were used for sulcular or pocket disinfection. The fiber tip was placed subgingivally and used in a noncontact mode for 60 s at each site, as shown in [Figure 2] (i). In group II (control group), each site was irrigated with saline for 60 s. Following this, the GCF, crevicular blood, and saliva samples were collected again.
|Figure 2: (i) Sulcular disinfection using diode Laser; (ii) Collection of various samples - (a) GCF collection using micropipettes, (b) Crevicular blood collection using micropipettes, (c) Saliva collection using a disposable syringe; (iii) Microbiological analysis - growth seen on sheep blood agar culture plates|
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Post sample collection
A reassessment of the gingival and periodontal status of the patient was carried out after 1 week of sample collection. Following this, scaling and root planing were performed in all the patients, and oral hygiene maintenance was advised.
GCF, crevicular blood, and saliva samples were collected from different sites from 45 patients before and immediately after laser disinfection and saline irrigation. The saliva samples were collected first, followed by GCF samples, with a gap of 15 min between the two procedures. GCF samples were collected by drying the gingival surface with sterile cotton, after which the area was isolated to prevent contamination with saliva. A graduated micropipette was inserted subgingivally to collect 3 μL of GCF. The collected GCF was flushed into an Eppendorf tube with Phospate-buffered saline (PBS) solution. The GCF samples were examined for any blood contamination. In the case of visible blood contamination, the sample was discarded. The crevicular blood samples were also collected using graduated micropipettes from the sulcus and stored in Eppendorf tubes to GCF. Pooled saliva samples from the surrounding vestibular region were collected by aspiration in a 2-mL syringe and stored in PBS in Eppendorf tubes. All the samples were subjected to microbiological analysis. [Figure 2] (ii) (a), (b) and (c) shows the method of collection of the samples from the patients.
The GCF, crevicular blood, and saliva samples collected were subjected to microbiological analysis. Separate agar plates were used for GCF, crevicular blood, and saliva samples. The colony-forming units (CFUs) of bacteria were counted by growing the colonies by aerobic culture on sheep blood agar plates, as shown in [Figure 2] (iii). The counts were made at the end of the second day after inoculation on the culture plates.
Statistical Software Package SPSS for Windows Version 22.0 Released 2013, Armonk, NY: IBM Corp., was used to perform statistical analyses. Shapiro–Wilk test indicated that the data were not following a normal distribution. Henceforth, relevant nonparametric tests were used for the analysis of data. The level of significance was set at P < 0.05.
| Results|| |
A total of 45 patients amounting to 90 samples (GCF, saliva, and crevicular blood for test and control groups) were analyzed to reduce microbial load following disinfection with a diode laser. The microbial analysis showed a similar microbial load between the test and control groups; the difference was not statistically significant. Post disinfection, the intragroup and intergroup comparison showed a statistically significant reduction in the CFU count in samples of the test group. The reduction was more significant in the GCF and saliva samples as compared with crevicular blood samples.
[Table 1] shows the comparison of the CFU count in all the samples of the test and control group before and after disinfection. The results revealed that there was no statistically significant difference demonstrated among the samples before disinfection. The post disinfection data revealed a statistically significant reduction in the CFU count of test group samples compared with control group samples, with a greater difference in crevicular blood and saliva samples than GCF samples.
|Table 1: Comparison of mean CFUs between test and control group for different types of samples during Pre- & Post- treatment period using Mann Whitney U test|
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[Table 2] and [Figure 3] show the comparison of CFU count in all the samples in the test and control group before and after disinfection. The results revealed a statistically significant reduction in the CFU count in all the samples in test group, whereas, there was no statistically significant reduction in the CFU count in the samples of control group.
|Table 2: Comparison of mean CFUs between Pre and Post treatment period in Test and Control groups for different types of samples using Wilcoxon Signed Rank test|
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|Figure 3: (i) Comparison of mean CFUs between pre and post treatment period in test group for different types of samples; (ii) Comparison of mean CFUs between pre and post treatment period in control group for different types of samples|
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[Table 3] shows Spearman Correlation carried out to assess the relationship between CFU values in all the samples in the test and control group. The results revealed a very weak positive correlation between the CFU of GCF and crevicular blood in pretreatment samples of both groups. The CFU of GCF and saliva showed a weak negative and a weak negative correlation in the test and control groups, respectively. After disinfection, a very weak positive correlation was seen between CFU of GCF and crevicular blood and a weak negative correlation between CFU of GCF and saliva samples in the test group.
|Table 3: Spearman's Correlation test for assessing the relationship between the CFUs from GCF with those from Blood & Saliva|
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| Discussion|| |
In-depth knowledge of the great diversity of microbial species involved in the periodontal disease progression is of utmost importance in understanding the subgingival site in health and disease. It also involves the interactions between subgingival species and the epithelial cells which line the gingival crevice and periodontal pocket. A passive and active detachment of bacterial cells from biofilms occurs as part of their biofilm life cycle., These detached (planktonic) bacteria play an important role by downregulating genes required for biofilm formation and upregulating the expression of virulence properties for acute infection. Identifying unattached bacteria may provide an insight into the activity of subgingival biofilm in vivo.
In this study, three types of samples, that is, GCF, crevicular blood, and saliva, were analyzed to study the effect of diode laser therapy on microbial load. GCF is considered an accurate and important diagnostic factor as it helps detect various biomarkers in health and disease. In a study conducted by Asikainen et al. in 2010, it was proposed that bacterial species found in GCF reflect the bacterial populations detached from subgingival biofilms.
To the best of our knowledge, this is the first study to analyze and compare the microbial load after sulcular disinfection in GCF, crevicular blood, and saliva samples in patients suffering from chronic periodontitis. It is also supposedly the first study to assess the immediate effects of diode laser on microbial load in GCF, crevicular blood, and saliva samples in patients. Thus, this study results could not be directly compared with the results of other similar studies. However, the effect of diode laser on the microbial load is similar, as shown in studies conducted by Fontana et al. (2004), Assaf et al. (2007), and Kamma et al. (2009).,,
This study's results have shown that there was a reduction in microbial load in both the groups following sulcular disinfection with a significant reduction in the test group. In the test group, a significant reduction was observed in GCF samples (from 6.44 × 103 to 6.84 × 102) and saliva (from 2.38 × 104 to 2.28 × 103) when compared with the crevicular blood samples (from 5.6 × 104 to 2.02 × 104). The results in the test group can be attributed to the bactericidal effects of the diode laser. The bacterial load reduction occurs due to the high absorption of a specific wavelength of the laser beam by bacterial chromophores.
The diode laser of 970 ± 15 nm wavelength is used in the study for sulcular disinfection of the sites in the test group. Using a diode laser for disinfection has several advantages. It is beneficial for the control of bacteremia and bacterial reduction apart from other applications in periodontics. The diode laser has a coagulation effect due to which there is reduced bleeding post irradiation. A study conducted by Fontana et al. in 2004 showed that diode lasers have bactericidal and fungicidal effects, with the highest reductions observed for Prevotella sp. and Fusobacterium sp., even without scaling and root planing before laser irradiation. The use of a diode laser for disinfection also reduces the need for anesthesia during subgingival scaling.
A study conducted by Assaf et al. in 2007 showed that diode lasers effectively reduce bacteremia and bacterial load and recommended it for immunocompromised patients. Another study conducted by Kamma et al. in 2009 confirmed that it was possible to reduce the total microbial load in the pockets in aggressive periodontitis patients without using systemic antimicrobial therapy. Thus, the use of lasers may eliminate systemic antimicrobials in periodontal therapy and reduce the chances of adverse drug reactions in patients on other systemic therapies.
There are a few shortcomings of this study. First, the microbial analysis needs to be strengthened with long-term studies utilising more reliable and accurate methods like ELISA and polymerase Chain Reaction. Second, a specific assessment of periodontopathogens needs to be carried out to highlight the efficacy of lasers in disinfection against particular bacterial species. Third, a larger sample size would play a role in bringing about better results. Fourth, bias may have occurred due to a collection of pooled saliva exposed to the disinfecting agent of either group as the samples are collected from two halves of the same patient.
Future perspectives and clinical implications
On the basis of this study's results, the immediate microbial reducing effect with diode lasers can be clinically applied for reducing the tendency to bleed when laser irradiation is performed before scaling and root planing or any other nonsurgical treatment procedures. It may help reduce anxiety in patients and increase acceptance of treatment procedures. It may even reduce microbial dissemination to other sites, thereby preventing infections like bacteremia. Studies may also be performed to assess the similar effect in systemically compromised patients at a susceptible edge of contracting the bacterial infection by periodontal pathogens.
| Conclusions|| |
This study concludes that applying diode laser (970 ± 15 nm) for sulcular disinfection has immediate reducing effects on the microbial load in GCF, crevicular blood, and saliva samples in patients with chronic periodontitis with the greater reduction seen in GCF and saliva samples compared with crevicular blood samples.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Sunil Kumar D Chavan
Assistant Professor, Department of Microbiology, All India Institute of Medical Sciences (AIIMS), Bibinagar, Hyderabad Metropolitan Region - 508126, Telangana
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]