Evaluation of the Clinical and Antimicrobial Effects of the Er:YAG Laser or Topical Gaseous Ozone as Adjuncts to Initial Periodontal Therapy

Abstract

Objective: The aim of this study was to evaluate the clinical and microbiological results of treatment with the Er:YAG laser and topical gaseous ozone application as adjuncts to initial periodontal therapy in chronic periodontitis (CP) patients. Background data: Although many studies have evaluated the effectiveness of the Er:YAG laser as an adjunct to initial periodontal therapy, few studies have focused on the use of gaseous ozone as an adjunct. Materials and methods: Thirty patients with CP were randomly divided into three parallel groups, each composed of 10 individuals with at least four teeth having at least one approximal site with a probing depth (PD) of ≥5 mm and a sulcus bleeding index (SBI) ≥2 in each quadrant. Groups of patients received: (1) Scaling and root planing (SRP)+Er:YAG laser; (2) SRP+topical gaseous ozone; or (3) SRP alone. The microbiological and clinical parameters were monitored at day 0 and day 90. Results: At the end of the observation period, statistically significant improvements in clinical parameters were observed within each group. Parallel to the clinical changes, all treatments reduced the number of total bacteria and the proportion of obligately anaerobic microorganisms. Although intergroup comparisons of microbiological parameters showed no significant differences, clinical findings, including attachment gain and PD reduction, were found to be statistically significant in favor of the SRP+Er:YAG laser group. Conclusions: Although statistically nonsignificant, the fact that the obligate anaerobic change was mostly observed in the SRP+Er:YAG laser group, and a similar decrease was noted in the SRP+topical gaseous ozone group, shows that ozone has an antimicrobial effect equivalent to that of the Er:YAG laser.

Introduction

P eriodontal disease is characterized by chronic inflammatory processes that are caused by the triggering of the host response by specific pathogens, progressive destruction of alveolar bone, and apical migration of connective tissue and epithelial attachments.^1,2,3 However, mechanical periodontal therapy alone may be insufficient to eradicate the pathogenic bacteria, because of their location within the gingival and dental tissues or in other sites that are not easily accessed by periodontal instruments.^2,3 Therefore, treatment strategies have been proposed that primarily aim to suppress or eliminate periodontal pathogens and control their overgrowth.^4
–6 The adjunctive antimicrobial therapeutic basis depends upon the systemic and local application of antibiotics. Although systemic and local antibiotics are occasionally administered into periodontal pockets to promote disinfection, the risk of developing resistant microbial species increases with the frequent use of antibiotics.⁷ Therefore, some authors have suggested the development of new treatment modalities for the elimination of specific periodontopathogens. Additionally, new tools, such as lasers, ozone (O₃) gas, or photodynamic therapy, have also been proposed.⁸ The Er:YAG laser has specifically drawn attention because of its antibacterial and photomechanical effects. Therefore, Er:YAG laser treatment provides an environment that is favorable for the attachment of periodontal tissue. Another recent suggestion is treatment with O_3, which is a naturally occurring compound consisting of three oxygen atoms. O₃ is one of the most important oxidants present in nature, and it is highly reactive with organic compounds.^9
–11 A survey of the literature shows that O₃ has applications in different areas of dentistry, and specifically garners attention in periodontal treatment because of its antibacterial activity. Currently, several devices, primarily found in the European dental market, produce aqueous and gaseous O₃, and claim to be effective in treating periodontitis.¹² However, there is little information in the current literature regarding the antimicrobial activity of O₃ against periodontal pathogens. Therefore, in this study we aimed to compare the clinical and microbiological results obtained by treatment with the Er:YAG laser and a topical ozonized gas used as adjuncts to mechanical subgingival debridement in chronic periodontitis patients. The null hypothesis was that there would be no significant microbiological difference in terms of the effectiveness of the Er:YAG laser and topical gaseous O₃ as adjuncts to initial periodontal therapy.

Materials and Methods

This study was designed as a randomized controlled clinical trial. The patient population consisted of 30 systemically healthy chronic periodontitis patients between 37 and 67 years of age who were referred to the Department of Periodontology, Yeditepe University Faculty of Dentistry between April 2011 and October 2011. All the details of the study were explained, and an informed written consent to participate was obtained from all patients. The study design and consent were approved by the Yeditepe University Ethical Committee. The patient selection criteria were as follows: (1) no periodontal or antimicrobial treatment within the past 6 months, (2) no pregnancy, (3) the presence of at least four teeth having at least one approximal site with a probing depth (PD) of ≥5 mm and a sulcus bleeding index (SBI) of ≥2 in each quadrant, and (4) no smoking.

Randomization and treatment groups

Patients who were suitable for the study were randomly distributed into three treatment groups according to a computer-assisted randomization table (www.randomizer.org/Copyright ©1997–2011 by Geoffrey C. Urbaniak and Scott Plous). They were treated with scaling and root planing (SRP) plus Er:YAG laser treatment (Group 1 [n=10]), SRP plus topical gaseous O₃ (Group 2 [n=10]), or SRP alone (Group 3 [n=10]) (Fig. 1).

FIG. 1.

Flow chart of the study.

Site selection for microbiological sampling

Four teeth (one in each quadrant) having at least one approximal site with a PD ≥5 mm and SBI ≥2 were selected in each patient, and the selected teeth were sampled as one site/tooth. Therefore, representative periodontal sites were sampled in each patient, and the samples were pooled for the assessment of microbiological parameters. Microbiological samples were collected at day 0 and day 90 and analyzed by an examiner other than the therapist and the clinical examiner.

Clinical examination

Clinical examination was performed on all teeth (by a clinician who was blinded to the type of the treatments) at day 0 and day 90. Clinical parameters, such as plaque index (PI),¹³ SBI,¹⁴ PD, and relative attachment levels (RAL), were measured to the nearest mm with a calibrated periodontal probe (CP 15 UNC, Hu-Friedy, IL) using an individual occlusal stent as a reference point for probe placement.

The SBI was scored as follows:

Score 0 - gingiva of normal texture and color and no bleeding

Score 1 - gingiva apparently normal and bleeding on probing

Score 2 - bleeding on probing, change in color, and no edema

Score 3 - bleeding on probing, change in color, and slight edema

Score 4 - eitherbleeding on probing, change in color, and obvious edema; orbleeding on probing and obvious edema

Score 5 - bleeding on probing and spontaneous bleeding, change in color, and marked edema

Treatment procedures

In all groups, initial periodontal therapy including SRP was performed as full-mouth treatment. In Group 1, an adjunctive disinfection procedure was applied with an Er:YAG laser (Versawave^®, Hoya ConBio, San Francisco, CA; 20 hz, 50 mJ/pulse, 1 min/pocket, and apicocoronal direction in parallel paths with 30 degree angle tips under water irrigation) after SRP according to the manufacturer's instructions.

In Group 2, topical gaseous O₃ (OzonytronX, MYMED-Germany) was applied twice a week for 2 weeks after SRP according to the manufacturer's instructions.

In Group 3, only the initial periodontal therapy was performed, without any adjunctive procedure.

Microbiological procedures

After superficial cleaning of the selected sites with cotton pellets and drying of the supragingival area with a stream of air, samples were taken by sterile paper points inserted to the depth of the pocket and left for 10 sec. Each sample was aseptically transferred to 4.5 mL of phosphate buffered saline (PBS), immediately homogenized using a vortex mixer at the maximal setting for 30 sec, and then serially diluted 10-fold. From each dilution (10⁻¹,10⁻²,…. 10⁻⁵), two 0.1 mL portions were plated separately onto trypticase soy agar (Oxoid Ltd, Basingstoke, Hampshire, England) medium supplemented with 5% defibrinated sheep blood, 0.0005% hemin (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 0.00005% menadione (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

The first trypticase soy agar plate was incubated at 37°C for 7–10 days in Gas Pak Jars (AnaerogenGen kit, Oxoid Ltd, Basingstoke, Hampshire, England), while the other was incubated at 37°C in 10% CO₂ for 4 days. The total viable count (TVC) was determined as the total number of bacterial colonies on anaerobically incubated plates. All the microbiological data were transformed into colony forming units/milliliter (CFU/mL) of transport medium. In addition, the amount of obligate anaerobic bacteria was calculated as the TVC minus the total counts of colonies on plates incubated in 10% CO₂ conditions, and was expressed as a percentage of TVC, as previously described by Noyan et al.¹⁵

Statistical analysis

A software package (SPSS for Windows, Release 15.0, SPSS Ins., USA) was used for the statistical analysis. In the presentation of clinical and microbiological data, descriptive statistics were used, including the arithmetic mean±standard deviation, median, and minimum and maximum values. Intragroup comparisons of pre- and post-treatment values were performed by the Wilcoxon test. Multiple intergroup comparisons for clinical variables were performed using the Kruskall–Wallis test. The multiple comparison Mann–Whitney U test was used when the Kruskall–Wallis test presented a significant difference (p<0.05).

Results

All patients were followed up and none were excluded. Healing was uneventful, and no complications were detected. Table 1 shows the patients' demographic and baseline data. The intragroup statistics of clinical parameters are shown in Table 2. All of the clinical parameters displayed significant improvements from day 0 to day 90.

Table 1.

Demographic Data of the Treatment Groups

	Group 1	Group 2	Group 3	p^a
Number of patients	10	10	10
Gender (male/female)	4/6	5/5	3/7
Age (mean±standard deviation)	43.00±5.01	41.40±8.86	41.40±4.62	0.816

Kruskal-Wallis, p<0.05

Table 2.

Intragroup of the Clinical Parameters at Day 0 and Day 90

	Group 1			Group 2			Group 3
Parameters	Day 0	Day 90	p^a	Day 0	Day 90	p^a	Day 0	Day 90	p^a
PI	1.94±0.30	1.01±0.12	0.005	2.03±0.27	1.01±0.13	0.005	2.06±0.13	1.09±0.21	0.005
SBI	3.35±0.25	0.66±0.26	0.005	3.00±0.42	0.78±0.37	0.005	3.16±0.17	0.75±0.21	0.005
PD	4.02±0.17	2.61±0.28	0.005	3.91±0.24	2.83±0.27	0.005	3.95±0.13	2.91±0.12	0.005
RAL	10.51±0.83	9.38±0.85	0.005	10.16±1.02	9.34±1.04	0.005	10.06±0.81	9.26±0.87	0.005

Wilcoxon test, p<0.05

PI, plaque index; SBI, sulcus bleeding index; PD, probing depth; RAL, relative attachment level.

When the mean differences in PD and RAL values were compared among the groups, statistically significant differences were detected (p=0.001; p=0.001, respectively) (Table 3). It was noted that this significance was in favor of Group 1 in the double comparisons of Group 1–Group 2, Group 1–Group 3 and Group 2–Group 3 for PD (p=0.002; p=0.09; and p=0.365, respectively) and attachment gain values (p=0.001; p=0.001; and p=0.451, respectively) (Table 4).

Table 3.

Intergroup Comparisons of the PI, SBI, and PD Reductions and Attachment Gain of the Groups

	Group 1	Group 2	Group 3	p^a
PI reduction	0.94±0.30	1.02±0.30	0.96±0.23	0.787
SBI reduction	2.69±0.37	2.22±0.44	2.41±0.30	0.025
PD reduction	1.41±0.23	1.08±0.14	1.04±0.08	0.001
Attachment gain (mm)	1.13±0.15	0.82±0.09	0.80±0.14	0.001

Kruskal-Wallis, p<0.05.

PI, plaque index; SBI, sulcus bleeding ındex; PD, probing depth.

Statistically significant values are in bold.

Table 4.

Double Comparison of the Group 1- Group 2, Group 1-Group 3, Group 2-Group 3

Parameters	Group 1- Group 2	Group 1-Group 3	Group 2-Group 3
SBI reduction	0.016	0.041	0.385
PD reduction	0.002	0.009	0.365
Attachment gain (mm)	0.001	0.001	0.451

Mann-Whitney U test, p<0.05.

SBI, sulcus bleeding ındex; PD, probing depth.

Statistically significant values are in bold.

When used as an adjunct to SRP, the Er:YAG laser produced superior PD reduction and attachment gain in comparison with SRP+ozone or SRP alone.

Tables 5 and 6 show the TVC values and the inter- and intragroup comparisons of the proportions of obligately anaerobic microorganisms (expressed as a % of TVC) at day 0 and day 90. Statistically significant reductions were detected in the intragroup comparisons of the groups (p=0.05; p=0.028; and p=0.05), whereas these reductions were not found to be statistically significant in the intergroup comparison of the treatment groups.

Table 5.

Total Viable Counts (TVC) of Subgingival Samples at Day 0 and Day 90

	TVC (×10⁵ CFU/mL)	Group 1	Group 2	Group 3
Day 0	Median	30.55	30.62	27.29
	Range	2.00-46.00	14.04-63.00	11.00-46.00
Day 90	Median	3.43	5.24	3.07
	Range	0.10-6.85	0.010-17.20	0.30-11.00

CFU, colony-forming units.

Table 6.

Inter- and Intragroup Comparison of Proportions of Obligately Anaerobic Bacteria at Day 0 and Day 90

		Group 1	Group 2	Group 3	p^b
Obligate anaerobes (%)	Day 0	47.36±26.38	51.44±31.79	43.68±28.69	0.879
	Day 90	9.96±5.99	17.63±10.67	17.61±11.28	0.996
	Difference	37.40±20.59	33.80±25.01	26.07±19.58	0.456
	p ^a	0.005	0.028	0.005

Wilcoxon test, p<0.05; ^bKruskal Wallis, p<0.05.

Discussion

The results obtained from controlled clinical and microbiological studies and case reports have shown that when used as an adjunctive to initial periodontal therapy, the Er:YAG laser leads to significant gains in attachment levels and reductions in subgingival bacteria.^16

–20 The results of the present study are consistent with those of studies that indicate that there is no difference in terms of the elimination of bacteria when the Er:YAG laser is used as an adjunct to SRP. The finding that a laser application showed no difference in terms of bacterial reduction may be the result of the laser parameters used in our study. The null hypothesis was approved.

In recent years, topical gaseous O₃ has been proposed as a new adjunctive treatment strategy in the management of periodontal disease. However, few studies are available in the literature regarding the evaluation of antimicrobial effectiveness of O₃ against periodontopathogens.

In a study in which O₃ was microbiologically compared with chlorhexidine (CHX), the ratio of A.a, as detected using the PCR method, showed a decrease in the O₃ group, whereas Porphyromonas gingivalis and Tannerella forsythensis showed no reduction with either O₃ or CHX application.¹²

Although Kshitish et al.²¹ showed that O₃ had an antibacterial effect under in vivo conditions, Eick et al.²² detected a decrease in the antibacterial properties of O₃ in gingival crevicular fluid in a similar environment containing saline. This might explain the different results obtained in the present study.

In a clinical and microbiological study conducted on recall patients, Tomasi et al. made a microbiological comparison using DNA-DNA hybridization methodology; however, they determined that there was no statistically significant difference between the groups.²³ The authors attributed this result to the fact that the periodontal pockets were not infected with a high number of bacteria because of recall treatment, as well as to the microbiological technique they used. They indicated that the culturing method is a more reliable means of detecting bacteria, because of its higher accuracy. On the other hand, Eberhard et al.²⁴ reported no difference between the two methods in their in vitro study. The researchers performed SRP with ultrasonics and the Er:YAG laser. They explained their results with the antibacterial effects of both lasers and ultrasonics and further added that the antibacterial effect of ultrasonic devices is related to the cavitation effect. The study presented in this manuscript is the first study that clinically and microbiologically compares the Er:YAG laser and topical gaseous O₃ application as adjuncts to mechanical treatment in periodontal therapy.

Within the limitations of this study, it can be concluded that there was a better resolution of infection in the SRP+Er:YAG laser group. However, although not statistically reflected, the fact that the obligate anaerobic change was mostly observed in the SRP+Er:YAG laser group and that a similar decrease was noted in the SRP+topical gaseous O₃ group shows that O₃ has an antimicrobial effect equivalent to that of the laser.

Conclusions

O₃, in all forms, has recently gained attention as a topic warranting investigation. Long-term follow-up studies in larger groups of patients and in different dosages and durations are necessary, in which O₃ is evaluated along with clinical, microbiological, and immunological parameters.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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