Adjunctive Nd:YAG laser irradiation in the treatment of stage III/IV periodontitis: a 12-month, randomized, controlled trial

The aim of this study was to evaluate the clinical efficacy of the adjunctive use of Nd:YAG laser (1064 nm) to full-mouth scaling and root planning (FMS), in stage III/IV periodontitis patients. Sixty stage III/IV periodontitis patients were randomly assigned to three groups. The control group received FMS, laser 1 group received combined FMS/single Nd:YAG laser irradiation (3 W, 150 mJ, 20 Hz,100 μs), and laser 2 group received combined FMS/double Nd:YAG laser irradiation with 1-week interval (2.0 W, 200 mJ, 10 Hz, 100 μs). PD, CAL, FMPS, GI, FMBS, and GR were evaluated at baseline, 6 weeks, 3, 6, and 12 months after treatment. Patient-reported outcomes were evaluated 1 week after treatment. A significant improvement (p < 0.001) for all clinical parameters was observed during the entire study period, with the exception of mean CAL gain for the laser 2 group at 12 months. The percentage of pockets ≤ 4 mm was significantly higher compared to baseline for all groups throughout the study, with no inter-group differences at any time point. Patient-reported analgesic consumption was higher for laser 1 group. The adjunctive use of Nd:YAG laser irradiation was similarly effective to FMS alone, during the entire study period. A slightly higher, though not statistically significant improvement was reported for PD at 6 and 12 months after a single post-FMS application of Nd:YAG laser for pocket epithelium removal and coagulation. Additional Nd:YAG laser application for sulcular epithelium removal and coagulation may provide minor long-term improvements compared to FMS or laser irradiation for pocket disinfection and detoxification. ISRCTN26692900. Registration date: 09/06/2022


Introduction
There is profound evidence to warrant the efficacy of traditional root instrumentation in clinical and microbiological parameters of non-surgical periodontal therapy [1][2][3]; however, it cannot be regarded as the sole mode of treatment in deep (≥ 7 mm) pockets [4], furcation areas [5], and generally where access to areas, such as grooves and concavities, is restricted [6]. Instrumentation of infected root surfaces can either be performed via the conventional per quadrant approach (SRP), or alternatively with the implementation of the full-mouth scaling and root planning (FMS) with or without antiseptics [7][8][9]. FMS has been proved to be equally effective and acceptable [10] with SRP, although minor differences in favor of FMS protocol in moderate pockets of single-rooted teeth [11] do not appear to yield clinical significance [12].
The application of neodymium-doped yttrium aluminium garnet (Nd:YAG) laser irradiation as a monotherapy or as a complementary tool in non-surgical periodontal therapy lies on the purported anti-inflammatory and antimicrobial irradiation properties [13,14], which might enhance the effectiveness of traditional scaling and root planning.
Participants were recruited from a pool of patients initially presented to the Periodontal Department, 401 Athens Military Hospital, Athens, Greece, seeking periodontal treatment. Every patient diagnosed with severe chronic generalized periodontitis according to the previous classification system at the time [26], corresponding to periodontitis state III or IV [27], was enrolled in the study. All patients received a panoramic x-ray before treatment initiation. Inclusion criteria were (i) subjects of 35-65 years old, (ii) presence of at least 16 teeth, and (iii) periodontitis with PD ≥ 5 mm with bleeding on probing (BOP) in at least 30% of the teeth. Exclusion criteria were (i) poorly controlled diabetes, (ii) autoimmune diseases, (iii) genetic disorders, (iv) bone metabolic diseases, (v) bisphosphonate drugs, (vi) drug-induced gingival overgrowth, (vii) tumors or other oral pathology, (viii) pregnant or lactating women, and (ix) antibiotic use for any purpose within 3 months before entering the study.

Sample-size calculation
The sample-size calculation determined that 18 subjects per treatment group would provide 95% power to detect a true difference of 1 mm between test and control groups using PD reduction as the primary outcome variable, assuming that the common standard deviation was 0.8 mm. Accordingly, a sample of 20 subjects per group (60 in total) was recruited to compensate for possible dropout during the study period. Sample size was determined by using Graph-Pad StatMate version 2.0 for Windows, GraphPad Software, San Diego California USA, ww. graph pad. com.

Blinding of participants and personnel
All clinical measurements were performed by the same examiner (A.P.), who was masked to the treatment and was not involved in patients' therapy. Access to the data of former assessments was not allowed during the course of the study. Treatment procedures including non-surgical periodontal therapy and laser application were accomplished by the same experienced periodontist (N.M.). Until the completion of FMS and subsequent randomization, the operator (N.M.) was masked to the treatment group and unaware of the measurements performed throughout the study by the examiner (A.P.).

Examiner calibration
Prior to the initiation of the study, the clinical examiner (A.P.) was calibrated to the operator (N.M.). Concordance between the two examiners regarding their measurements of PD, GR, and CAL was assessed on data from 10 periodontitis patients not included in the study (contributing a total of 752, 786, and 786 measurements, respectively), after a similar pilot study with comparable data from 8 patients. Results of this analysis showed a satisfactory degree between examiners' agreement with Lin's concordance coefficient [29] being 0.672, 0.729, and 0.714, respectively. For PD, there was a perfect agreement for 59.8% of the measurements with only 4.8% being outside the Bland & Altman limits of agreement [30]. The corresponding percentages were 63.2% and 6.6% for measurements of GR and 46.3% and 5.3% for measurements of CAL. Percentages where difference in the measurements of the two examiners were ≥ 2 mm in absolute values and were 4.8%, 6.6%, and 9.4% for the three types of measurements, respectively.

Questionnaire
Immediately after treatment completion, a questionnaire form was provided to each subject. Each subject was asked to fill out the questionnaire at the end of the first week after the completion of all treatment interventions. A visual analogue scale was used to assess patients' perception of pain, sensitivity, swelling, bleeding, acceptance of the protocol during and after therapy, and treatment recommendation to a friend. Subjects marked a point on a 10-cm-long uncalibrated line with the negative extreme response (0) on the left end and the positive extreme response (10) at the right end. Additionally, the number of analgesic tablets taken were recorded.

Non-surgical therapy
The study included two test and one control group with a parallel design. All patients received oral hygiene instructions in Bass technique and interdental brushes and were reinforced whenever indicated during the course of the trial. The 3 patient groups consisted of 60 patients, who initially received FMS in two consecutive sessions, under local anesthesia. During each session, FMS was initially performed with a piezoelectric ultrasonic unit 2 complemented by Gracey curettes. 3

Randomization and allocation concealment
After completion of FMS therapy, the 60 patients were consecutively randomized into 3 treatment groups (laser 1, laser 2, control). Randomization was performed using a blocked randomization list stratified by sex and smoking status with a block size of 6. The list was created using the Sealed Envelope online tool. 4 The study coordinator (I.K.) was responsible for concealing the allocation. Sealed envelopes stated which patients would receive additional laser treatment. These envelopes were opened only after FMS was accomplished. Allocation to the treatment group was not revealed to the clinical examiner (A.P.) or the statistician (D.N.).

Laser treatment
For both test groups, an Nd:YAG Laser (1064 nm) 5 was utilized. All laser procedures were performed 1 week after FMS, in order to benefit from the short-term reduction of inflammation expected after scaling and root planning. Laser application was accomplished with protective eyewear delivered to the patient, dentist, and assistant. To avoid pain and sensitivity, all sites treated with laser were anesthetized. The 320-μm diameter optic fiber was calibrated in accordance to the periodontal probe charting before starting the procedure and was adjusted for every patient. The sites with initial PD ≥ 4 mm were treated with the Nd:YAG laser device. For laser 1 group, laser therapy was performed once, 1 week after FMS. The optical fiber tip of the laser moved slowly in a sweeping motion parallel to the tooth axis, during laser emission. Effort was made to achieve the biggest possible angle toward the soft wall of the pocket. Depending on the morphology of the roots and the depth of the pockets, irradiation angles varied between 20° and 30°. The procedure started from the top of the sulcus, maintaining contact of the fiber tip with the pocket wall, eventually reaching close to the base of the pocket, removing its epithelial lining and superficial part of lamina propria. The laser settings for this pass were set to 3 W, 150 mJ, 20 Hz with a pulse duration of 100 μs. The fiber tip was inspected frequently, and any accumulated tissue and debris were wiped off to avoid inefficiency [31]. For laser 2 group, laser therapy was performed 1 week after FMS, by inserting the optical fiber, after calibration of its length according to the pocket depth. Optical fiber tip was inserted into the pocket close to the pocket base and then was slowly moved in a sweeping motion parallel with the soft-tissue wall, using 2.0 W, 200 mJ, 10 Hz with a pulse duration of 100 μs. The purpose of this procedure was to eliminate pathogenic microbiota from the pocket environment without any soft-tissue removal. The laser was applied for approximately 30 s per tooth surface. The procedure was repeated 1 week later, with identical laser settings.

Statistical analysis
The statistical analysis was performed using commercially available software. 6 A subject-level analysis was performed for each of the parameters. All teeth were included in the statistical evaluation. Primary clinical outcome variables were changes in CAL and PD. Secondary clinical outcome variables were changes in GR, FMBS, GI, and FMPS. Mean and standard deviation (SD) for the clinical variables were calculated for each treatment group. The method of Kolmogorov and Smirnov was used to confirm that the data were sampled from a Gaussian distribution. One-way ANOVA with Tukey post-test was used for comparison among groups regarding continuous variables after confirming normality of the data distribution. Likewise, the significance of the difference within each group before and after treatment was evaluated with the paired samples t test. Nonparametric data was analyzed with the Kruskal-Wallis test, whereas the χ 2 test was used for categorical data. Differences were considered statistically significant when the P value was < 0.05.

Results
The outline of the study is depicted in the flowchart of Fig. 1. A total of 60 patients were enrolled in the study and none of them was excluded during the follow-up period. The postoperative healing was uneventful in all cases. At baseline, all treatment groups were balanced for age, sex, smoking status, and clinical indices (Table 1).
Primary clinical outcome variables were changes in CAL and PD. Secondary clinical outcome variables were changes in GR, FMBS, GI, and FMPS.
For all groups, treatment led to statistically significant improvement in FMPS, GI, FMBS (Table 2), PD (Table 3, Fig. 2), % of sites with PD ≥ 7 mm, and % of sites with PD ≤ 4 mm (Table 4), as compared to baseline. There was a statistically significantly intra-group difference for CAL at all time points after treatment, except for the 12-month CAL for laser 2 ( Table 3, Fig. 3). In all groups, a statistically significant increase was reported for GR throughout the study period (Table 3, Fig. 4).
Inter-group comparison did not show any significant difference for FMPS, GI, FMBS, PD, CAL, GR, PD difference (PDdiff), % of sites with PD ≥ 7 mm, and % of sites with PD ≤ 4 mm at any time point between the treatment groups (Tables 2, 3, and 4).
In a subgroup analysis including smokers, a statistically significant post-treatment PD reduction was maintained for all groups compared to baseline (Table 4). However, no statistical significant difference was observed for PD at any time point between groups (Table 4).
A similar pattern was observed for the post treatment patients' perception, as evaluated via analogue scale, except for the reported number of analgesics which was statistically significantly higher for laser 1 group, as compared to laser 2 and control groups (Table 5).

Discussion
The present triple-arm parallel prospective, doublemasked, randomized controlled trial evaluated the possible additional clinical benefit of Nd:YAG laser irradiation to FMS, when applied with two distinct laser modalities (single irradiation by using a 320-μm fiber tip, 3 W, 150 mJ, 20 Hz with a pulse duration of 100 μs, double irradiation with 1-week interval by using a 320-μm fiber tip, 2.0 W, 200 mJ, 10 Hz with a pulse duration of 100 μs).
In the present study, FMS alone or in combination with Nd:YAG laser irradiation led to significant clinical improvement, which is in accordance with previous reports [2,3,7,[11][12][13][14] and showed that Nd:YAG irradiation in conjunction with FMS is a viable treatment option.
The results of this study did not demonstrate clinical superiority of the adjunctive use of Nd:YAG laser irradiation to scaling and root planning (SRP), as compared to SRP alone, which is in accordance with the outcomes of systematic reviews [22,24,25]. The absence of additional clinical benefit of Nd:YAG laser over SRP was previously reported in class II furcation defects at 6 weeks [32] with laser settings (100 mJ/pulse, 15 Hz, 1.5 W) similar to those for laser 2 group (2W, 200 mJ/pulse, 10 Hz).

3
A meta-analysis on the adjunctive use of Nd:YAG laser to SRP [22], where three split-mouth studies [33][34][35] with follow-up range 1-9 months were included, showed that SRP could potentially benefit a mean 0.55 mm PD reduction from the adjunctive Nd:YAG laser application. Significant superiority in PD reduction was recently found with the combined Nd:YAG laser/SRP over SRP at 6 months by Dortaj et al. [36].
In our study, a non-statistically significantly higher PD reduction was found for laser 1 group compared to  control group, 6 and 12 months after treatment. Other studies reported a clinical improvement in terms of CAL gain after the adjunctive Nd:YAG laser irradiation, immediately after treatment [37] and at 6-12 months [14], especially in pockets ≥ 7 mm [25,38]. Recent experimental data [39] support combined therapies, such as Nd:YAG laser irradiation plus 0.5% NaOCl or 0.5% H 2 O 2 , for their bactericidal effects.
The similar GR among groups reveals the absence of significant negative effect of laser irradiation on GR for a 12-month period. This is in agreement with previous reports [32,40] and in disagreement with Dortaj et al.'s [36] findings on significant GR aggravation with laser at 6 months.
The present findings do not support the significance of the double irradiation in the attempt to further improve the clinical outcome. Though laser beam traits slightly differed between protocols, the double laser irradiation, aiming to enhance its bactericidal effect, did not significantly improve the clinical outcome achieved with the single irradiation. On the contrary, the non-statistically significantly higher PD reduction at all time points for laser 1 group than laser 2 group implies that laser 1 group was slightly more effective in PD reduction than laser 2 group, which sustained for 12 months. To our knowledge, the removal of the lining of the pocket soft tissue wall, through gingival curettage, has not been proved as mandatory.
However, the 1064 nm wavelength is capable to ablate inflamed soft tissues, such as the ulcerated pocket wall [41]. This was claimed to enhance connective tissue attachment formation [20].
Numerous irradiation settings have been suggested to achieve an optimal effect [14,24,[41][42][43]. The present study evaluated laser settings that had been arbitrarily used in clinical applications, in order to prevent possible detrimental effects at the surrounding tissues [24,44]. Since the Nd:YAG laser might penetrate deeper into the tissue before being absorbed, the local morphology of the periodontal tissues and the level of inflammation (i.e., Amount of hemoglobin in the treated tissue) must also be taken into account. Tissue necrosis is related to laser exposure time, type of laser delivery tip (fiber), direction of the beam, and applied laser energy. An inhibitory effect on cell viability and proliferation has been reported as a result of an increase of pulse energy, pulse repetition rate, and power output [45]. Most studies reported the use of Nd:YAG 1064 nm with power output range 0.5-4 W, whereas substantially higher power settings up to 9 W are not recommended for clinical application [44]. The use of 3 W power output for laser 1 group is within the range suggested for sulcular epithelium removal and coagulation [42].
The non-significantly higher percentage of deep pockets (≥ 7 mm) at 6 weeks for laser 1 group, as compared to the other groups, might reflect delayed healing due to the thermal effect of the laser beam.
The combined use of Er:YAG and Nd:YAG laser, as compared to SRP alone, demonstrated significantly greater improvement in PD and CAL for deep pockets at 1 and 3 months [46] and for moderately deep pockets (4-6 mm) at 6 months [47]. Unlike Nd:YAG laser, erbium lasers are capable of ablating subgingival calculus effectively without causing irreversible thermal damage on the root surface and the bone [44], whereas they are not capable of pocket epithelium ablation resulting in a concomitant microvessel rupture and bleeding [18].
One of the most significant endpoints after active periodontal therapy is the presence of shallow periodontal pockets (≤ 4 mm) that do not bleed on probing in patients with FMBS < 30% [48]. All groups achieved significant high percentages of PD ≤ 4 mm for the entire follow-up period, without inter-group differences, implicating that in patients with effective plaque control, there is a high chance of clinical stability up to 12 months after SRP alone or in combination with laser treatment.
When a sub-analysis was performed regarding smoking, the additional application of Nd:YAG laser irradiation did not show further benefit, compared to FMS, as previously reported [34,35].
With respect to patient-reported outcomes as analyzed from the questionnaires, the patients in laser 1 group received a significantly elevated number of analgesics, as compared to those in laser 2 and FMS groups. A possible explanation lies behind the increased power output compared to laser 2 group or to thermal damage at the surrounding tissues [44]. This observation corresponds with previous findings [39,49] referring to a more pronounced postoperative pain in a group of patients that received combined SRP/Nd:YAG laser treatment, which according to the authors [49] was directly associated with receiving more analgesics, on day one after laser irradiation.
In the present study, laser treatment was not performed prior to FMS, in order to minimize the operator bias, since he had to be masked to the treatment groups. One week after FMS, it is rational to expect reduction of inflammation and of profound bleeding during laser treatment.
It seems that the present clinical trial has several strong characteristics as follows. Final evaluation was performed at 12 months. Mean plaque scores were < 10% for all groups and for the entire 12-month period, which supports previous findings on the correlation between optimal oral hygiene and clinical stability over time [50], irrespective of the treatment modality [46]. In order to avoid any impact of smoking which is a confounding factor that may affect the clinical outcome of the treatment [51], smokers in this study were equally distributed among groups (n = 10 per group).
Considering the limitations of the study, patients in laser 1 and laser 2 groups were not masked to laser irradiation application, which could have influenced plaque control throughout the entire study period. Though, no statistically significant differences in FMPS were reported at any time point among groups.
In the present study, a manual probe was utilized for pocket depth measurements, which cannot identify minor differences (like 0.5 mm). Clinical practice requires modification of the settings, occasionally several times during the laser treatment of a patient. Pulse duration, frequency, pulse energy, and duration of irradiation may differ depending on the depth of the pocket, the degree of inflammation, and the gingival phenotype. However, in a research protocol, these settings should be standardized in order to ensure valid comparisons.

Conclusions
Within the limitations of the present study, the following conclusions can be drawn: (1) Both FMS and the combined FMS/Nd:YAG laser application achieved significant clinical improvement, which sustained for 12 months. (2) FMS and the combined FMS/Nd:YAG laser application led to similar clinical improvement for a 12-month period. (3) The combination of FMS and Nd:YAG laser (single irradiation by using a 320-μm fiber tip, 3 W, 150 mJ, 20 Hz, with a pulse duration of 100 μs) might be nonstatistically significantly superior in PD reduction for a 12-month period, as compared to FMS.
Author contributions P.M., I.K. and N.M. have made substantial contributions to conception and design of the study. A.P. has been involved in data collection. N.M. has been involved in the patients' therapies. D.N. has been involved in data collection, interpretation and statistical analysis. P.M., E.P., I.K. and N.M. have been involved in drafting the article. All authors have been involved in revising it critically and have given final approval of the version to be published. The authors report no conflict of interest to this study.
Funding No funding was obtained for this study.

Declarations
Ethics approval and consent to participate A written informed consent was obtained from all study participants.

Conflict of interest
The authors declare no competing interests.