Can Photobiomodulation Therapy Using an 810-nm Diode Laser Increase the Secondary Stability of Orthodontic Mini-Screws? A Split-Mouth Double-Blind Randomized Clinical Trial

: Background: In orthodontic treatment, mini-screws can provide maximum anchorage conditions. If the miniscrew stability is compromised, this could jeopardize the outcomes of the orthodontic treatment or biomechanics. Photobiomodulation therapy (PBMt) is beneﬁcial for biological tissues since it promotes wound healing with its anti-inﬂammatory and osteo-stimulatory properties. Objectives: The purpose of this study was to evaluate the effects of 810-nm Diode PBMt on the stability of orthodontic mini-screws for three months. Trial Design: Parallel, allocation ratio 1:1. Methods: Using a split-mouth technique, a total of 40 mini-screws were randomly allocated (1:1) to either a placebo laser application (P group) or a laser PBMt (L group). An 810-nm diode laser irradiation of 1 W output power, operating in continuous wave mode for 50 s, was applied in a sweeping movement at a 22 mm distance from the mini-screws. The allocated mini-screws ﬁrst received PBMt at baseline, then every other day for ﬁve days, and then at each orthodontic visit (every three weeks) for a period of three months. At each visit, the stability of the mini-screws was measured by the Osstell Implant Stability Quotient (Osstell ISQ) at three points: lower, upper, and distal to the mini-screw. Results: The lower, upper, and distal ISQ values of irradiated and non-irradiated mini-screws signiﬁcantly decreased at each point over time ( p < 0.001). No signiﬁcant difference was found in the global ISQ values between the laser and the placebo group ( p > 0.05). Conclusion: PBM did not signiﬁcantly increase the stability of orthodontic mini-screws over a period of three months.


Introduction
Biomechanics is an important variable in the orthodontic equation. It includes both a biological element involving the tooth and its periodontium, and a mechanical element defining the force system. In fact, Newton's third law of motion, which defines the orthodontic movement, states that for every action (force) in nature there is an equal and opposite reaction. How does one counteract these reactive forces? It took until 1923 for the term "anchorage" to be defined by Louis Ottofy as "the base against which orthodontic force or reaction of orthodontic force is applied" [1]. Many anchorage systems have been developed since, but it was not until the introduction of skeletal anchorage as a source of fixed Our double-blinded split-mouth randomized controlled trial (RCT) purp evaluate the effect of PBMt on the stability of orthodontic mini-screws during fixe ance orthodontic treatment followed the CONSORT guidelines ( Figure 1). The was approved by the Research Ethics Committee of the Faculty of Dental Med Saint Joseph University, Beirut, Lebanon, (#USJ/2019/161). The steps and purpo thoroughly explained to all subjects and written consent was signed by each of t fore the procedure. The study involved the insertion of a total of 40 mini-screws in the maxilla a to the split-mouth technique. Twenty Caucasian patients (8 males and 12 female skeletal and dental Class II relationship and a mean age of 22.9 years ± 4.78 were re After agreeing on a treatment plan, teeth alignment was achieved using Nitinol a (014, 016, and 16 × 22 NiTi), followed by canine distalization on 17 × 25 stainl archwires.
Mini-screws were inserted after the stainless steel arch wire was placed. Elig tients were those needing orthodontics using two orthodontic mini-screws in the non-smokers or light smokers (less than 10 cigarettes/day) with acceptable oral and the presence of an attached gingiva for proper mini-screw insertion. Patie excluded when failing to fulfil one of these criteria.

Randomization and Masking
Mini-screws were randomly allocated (1:1) by either a placebo laser applic group) or by laser PBMt (L group). Randomization was based on a random-seque gram (www.random.org/sequences), accessed on 22 August 2021, where laser app was performed on the right side of the patients who were assigned an odd num on the left side of patients who were assigned an even number. On the contralate the placebo application was performed for each patient.
The laser placebo consisted of photobiomodulation therapy irradiation with vice switched on 0 W [18]. A 632-nm visible red-light guide (negligible power) laser device switched to silent mode was used during all the irradiation in order The study involved the insertion of a total of 40 mini-screws in the maxilla according to the split-mouth technique. Twenty Caucasian patients (8 males and 12 females) with a skeletal and dental Class II relationship and a mean age of 22.9 years ± 4.78 were recruited. After agreeing on a treatment plan, teeth alignment was achieved using Nitinol archwires (014, 016, and 16 × 22 NiTi), followed by canine distalization on 17 × 25 stainless steel archwires.
Mini-screws were inserted after the stainless steel arch wire was placed. Eligible patients were those needing orthodontics using two orthodontic mini-screws in the maxilla, non-smokers or light smokers (less than 10 cigarettes/day) with acceptable oral hygiene, and the presence of an attached gingiva for proper mini-screw insertion. Patients were excluded when failing to fulfil one of these criteria.

Randomization and Masking
Mini-screws were randomly allocated (1:1) by either a placebo laser application (P group) or by laser PBMt (L group). Randomization was based on a random-sequence program (www.random.org/sequences), accessed on 22 April 2021, where laser application was performed on the right side of the patients who were assigned an odd number, and on the left side of patients who were assigned an even number. On the contralateral side, the placebo application was performed for each patient.
The laser placebo consisted of photobiomodulation therapy irradiation with the device switched on 0 W [18]. A 632-nm visible red-light guide (negligible power) with the laser device switched to silent mode was used during all the irradiation in order to keep the therapies blinded [19]. Therefore, patients and study team members responsible for the administration of the treatment were masked in treatment allocation. The investigator responsible for statistical data analysis was also masked to treatment assignment.

Procedure
Regarding mini-screw insertion and stability measurements: 40 mini-screws M.O.S.A.S self-drilling 25M-16208 (Dewimed Medizintechnik GmbH, Tuttlingen, Germany) were inserted with an 8 mm length and a diameter of 1.6 mm using an implant motor with a 40 N torque. All of the mini-screws were positioned at a 90-degree angle in the attached gingiva, 1 mm below the mucogingival junction of the right and the left maxilla between the first and second premolar or the second premolar and first molar (Figure 2). the therapies blinded [19]. Therefore, patients and study team members respon the administration of the treatment were masked in treatment allocation. The inv responsible for statistical data analysis was also masked to treatment assignmen

Procedure
Regarding mini-screw insertion and stability measurements: 40 min M.O.S.A.S self-drilling 25M-16208 (Dewimed Medizintechnik GmbH, Tuttling many) were inserted with an 8 mm length and a diameter of 1.6 mm using an motor with a 40 N torque. All of the mini-screws were positioned at a 90-degree the attached gingiva, 1 mm below the mucogingival junction of the right and the illa between the first and second premolar or the second premolar and first mola 2). The implant stability measurements were taken using the Resonance F Analysis technique of the Osstell ISQ (W&H Dentalwerk GmBH, Bürmoos, Osstellʹs patented technology uses Resonance Frequency Analysis (RFA) meas frequency with which a device vibrates. By comparing resonance frequencies, the of a dental implant can be determined as the resonance frequency changes with stabilities. The ISQ has a value between 1 and 100 and gives the surgeon an ins the stability of the implant after placement. With the ISQ value, the surgeon can the osseointegration. To use the Osstell ISQ on mini-screws, a special device wa cally designed and tested for this study. It had two sides: one side where the S could be screwed, and another side where the head of the mini-screw was fixed 3 and 4). The implant stability measurements were taken using the Resonance Frequency Analysis technique of the Osstell ISQ (W&H Dentalwerk GmBH, Bürmoos, Austria). Osstell's patented technology uses Resonance Frequency Analysis (RFA) measuring the frequency with which a device vibrates. By comparing resonance frequencies, the stability of a dental implant can be determined as the resonance frequency changes with different stabilities. The ISQ has a value between 1 and 100 and gives the surgeon an insight into the stability of the implant after placement. With the ISQ value, the surgeon can monitor the osseointegration. To use the Osstell ISQ on mini-screws, a special device was specifically designed and tested for this study. It had two sides: one side where the SmartPeg could be screwed, and another side where the head of the mini-screw was fixed (Figures 3 and 4).
Three ISQ measures were taken at baseline and at each appointment: lower, upper, and distal to the mini-screw ( Figure 5).
To reduce the risk of bias, the mini-screw insertion and the stability measurements were performed by a well-qualified blinded periodontist (S.G). Appl. Sci. 2023, 13, x FOR PEER REVIEW 5 of 15      Three ISQ measures were taken at baseline and at each appointment: lower, upper, and distal to the mini-screw ( Figure 5). To reduce the risk of bias, the mini-screw insertion and the stability measurements were performed by a well-qualified blinded periodontist (S.G).

Laser Application
Each patient in this study underwent a laser application on one of the mini-screws and a placebo irradiation on the other in the following manner: first at the day of the implantation (T0) followed by every other day for five days at T0 + 2 days, T0 + 4 days, and at each orthodontic visit (every three weeks) for a period of three months at T0 + 3 weeks, T0 + 6 weeks, T0 + 9 weeks, and T0 + 12 weeks.
For the L group, the laser device employed in the treatment was an 810-nm diode laser (Picasso Lite; AMD lasers, Indianapolis IN, USA). The laser energy was applied in continuous wave mode with an output power of 1 W for 50 s and was used in a sweeping movement over the mini-screws. The diode laser delivery system was an optical fibre with a detachable non-initiated 400 µm quartz tip presenting a Gaussian energy distribution. The aiming beam was a low-power visible red semiconductor diode laser (632-nm, power < 5 mW) and was transmitted coaxially along with the optical fibre. The laser beam was positioned over the mini-screw head at 22 mm height, covering an area of 0.8 cm 2 , with a power density of 1.2 W/cm 2 , a total energy of 50 joules, and an energy density with a movement of 8.3 J/cm 2 . The speed of movement was 1 mm/s. As for the P group, the irradiation through the placebo laser (0 W, 0 J/cm 2 ) was performed for 50 s.
The precision of the laser therapy parameter was secured by the Pronto-250 power meter (Gentec Electro-Optics, Inc., Québec, QC, Canada). Adverse events due to a possible undesirable thermal effect were avoided by monitoring the irradiation with a thermal camera FLIR ONE Pro-iOS (FLIR Systems, Inc. designs, Portland, OR, USA) (dynamic range: −20 °C/+400 °C; resolution 0.1 °C). All laser safety measurements were respected in accordance with the American National Standards Institute (ANSI) guideline [18,20].

Laser Application
Each patient in this study underwent a laser application on one of the mini-screws and a placebo irradiation on the other in the following manner: first at the day of the implantation (T0) followed by every other day for five days at T0 + 2 days, T0 + 4 days, and at each orthodontic visit (every three weeks) for a period of three months at T0 + 3 weeks, T0 + 6 weeks, T0 + 9 weeks, and T0 + 12 weeks.
For the L group, the laser device employed in the treatment was an 810-nm diode laser (Picasso Lite; AMD lasers, Indianapolis IN, USA). The laser energy was applied in continuous wave mode with an output power of 1 W for 50 s and was used in a sweeping movement over the mini-screws. The diode laser delivery system was an optical fibre with a detachable non-initiated 400 µm quartz tip presenting a Gaussian energy distribution. The aiming beam was a low-power visible red semiconductor diode laser (632-nm, power < 5 mW) and was transmitted coaxially along with the optical fibre. The laser beam was positioned over the mini-screw head at 22 mm height, covering an area of 0.8 cm 2 , with a power density of 1.2 W/cm 2 , a total energy of 50 joules, and an energy density with a movement of 8.3 J/cm 2 . The speed of movement was 1 mm/s.
As for the P group, the irradiation through the placebo laser (0 W, 0 J/cm 2 ) was performed for 50 s.
The precision of the laser therapy parameter was secured by the Pronto-250 power meter (Gentec Electro-Optics, Inc., Québec, QC, Canada). Adverse events due to a possible undesirable thermal effect were avoided by monitoring the irradiation with a thermal camera FLIR ONE Pro-iOS (FLIR Systems, Inc. designs, Portland, OR, USA) (dynamic range: −20 • C/+400 • C; resolution 0.1 • C). All laser safety measurements were respected in accordance with the American National Standards Institute (ANSI) guideline [18,20].

Orthodontic Intervention
At each orthodontic appointment, a hygiene level assessment was carried out, followed by tooth movement activation using well-controlled and identical forces on both sides. In fact, a dynamometer was used to measure the force delivered by the power chain, and when an open coil was inserted its length was calculated using the formula: inter-bracket Appl. Sci. 2023, 13, 8167 7 of 14 distance × 1.5 mm. Force was measured to assure that the same intensity was used on both sides.

Outcomes
The predictor variable was the 810-nm PBM's ability to interact with the cellular target involved in the energetic metabolic metabolism. The primary endpoint was the induced increased stability of the mini-screws.

Data Analysis
The measurements collected were statistically analysed while taking into consideration the mean and standard deviation of the measured data and the statistical power of the sample size.
The statistical software of IBM SPSS Statistics (version 26.0, USA) was used to analyse the data. The Kolmogorov-Smirnov test was used to assess the distribution normality of continuous variables and the reliability by calculating the Cronbach's coefficient (α < 0.05). To compare the ISQ (lower, upper, and distal) within times and between the presence or absence of laser treatment, repeated measurement analysis of variance (ANOVA) was used for within-subject comparisons.
This test was followed by univariate analysis and the Bonferroni multiple comparison test.
The level of significance was established at 95%. The value p < 0.05 was accepted as the significance level of the tests.
The sample size was calculated by G*Power Statistical Power Analyses Software version 3.1.9.7 [21]. The power analysis showed that to have a power of 80% with an effect size of (d = 0.8) and α error of 0.05, a sample size of 40 mini-screws for a within-subject study design was sufficient.

Participants and Randomization
Between January 2020 and February 2021, 22 patients were screened for the study resulting in two patients being deemed ineligible: one patient had Down syndrome and the other was a heavy smoker. Consequently, according to the established inclusion/exclusion criteria, 20 Caucasian patients (8 males and 12 females) with a skeletal and dental Class II relationship and a mean age of 22.9 years ± 4.78 participated in the study. The study followed a within-subject study design and the split-mouth technique; as such, two miniscrews were inserted in each patient: One was treated without laser (P group) and one was treated with laser (L group). This led to a total of 40 mini-screws (20 mini-screws per condition). A full subject medical history was obtained and a thorough oral examination was done prior to participation in the study. All twenty enrolled patients completed the treatments successfully. No adverse effects were reported.

Primary Outcome
Comparison of the Lower ISQ over Time with or without Laser The mean and the standard deviation of the lower ISQ measurement after activation with and without laser are illustrated in Table 1.

Comparison over Time
After laser treatment, lower ISQ significantly decreased over time (p < 0.001), significantly decreasing between T0 and T1 by 7.5 u, then significantly decreasing between T1 and T2 by 2.8 u, then significantly decreasing between T2 and T3 by 5.4 u (p < 0.05). The difference, however, was not significant between T3 and T4 (p = 1.000). Without laser, lower ISQ significantly decreased over time (p < 0.001), significantly decreasing between T0 and T1 by 4.3 u, then between T1 and T2 by 5.1 u, and then between T2 and T3 by 5.4 (p < 0.05). The difference, however, was not significant between T3 and T4 (p = 1.000).

Comparison between Treatments with or without Laser
The change in lower ISQ of laser-treated mini-implants was not significantly different than that of non-laser-treated mini-implants (p = 0.920; statistical interaction). The mean values of the lower ISQ were not significantly different between the presence and absence of laser, neither at baseline nor for the entire duration of the follow-up (p > 0.05).
The mean and the standard deviation of the upper ISQ measurement after activation with or without laser are illustrated in Table 2.

Comparison between Treatment with or without Laser
The variation of the upper ISQ of the mini-implants treated with laser was not significantly different than that of the mini-implants not treated with laser (p = 0.955; statistical interaction). In addition, the mean values of the upper ISQ were not significantly different between the presence and absence of laser at baseline and throughout the follow-up (p > 0.05).

Comparison of the Distal ISQ over Time with or without Laser
The mean and the standard deviation of the distal ISQ measurement after activation with or without laser are illustrated in Table 3.
Without laser, distal ISQ significantly decreased over time (p < 0.001), decreasing significantly between T0 and T1 by 4.7 u, then between T1 and T2 by 4.1 u, then between T2 and T3 by 4.8 u (p < 0.05). The difference, however, was not significant between T3 and T4 (p = 0.997).

Comparison between Treatments by Laser or Not by Laser
The variation of distal ISQ at the level of the mini-implants treated with laser was not significantly different than that of the mini-implants not treated with laser (p = 0.689; statistical interaction). The mean values of the distal ISQ were not significantly different between the treatment with or without laser at baseline and for the entire duration of the follow-up (p > 0.05).

Comparison of the Global ISQ over Time and with or without Laser
The mean and the standard deviation of the global ISQ measurement after activation with or without Laser are illustrated in Table 4.

Global Comparison between Treatments by Laser or Not by Laser
The change in global ISQ at the level of the mini-implants treated with laser was not significantly different than that of the mini-implants not treated with laser (p = 0.842; statistical interaction).
The mean values of the global ISQ were not significantly different between the treatment with or without laser at baseline and for the entire duration of the follow-up (p > 0.05).
Note that the lower, upper, and distal ISQ measurements were not significantly different from each other regardless of the follow-up time (p > 0.05).

Discussion
During the past few years, several animal studies dealing with the same subject have been conducted, and the results found have favoured the use of PBMt to increase miniscrew stability [27,28]. Human studies have also been conducted, but our study was the first to apply PBMt to assess the mini-screw stability for a period up to three months.
It is common knowledge that the stability of mini-screws decreases with time, especially in the first two weeks [29]. However, if the expected results are achieved, this stability decrease does not have a negative influence on the treatment. Instead, it is a physiological phenomenon that arises from several factors, among which immediate or delayed loading of the mini-screws can be cited. It has been suggested that immediate loading be performed because the primary stability of the mini-screw is sufficiently capable of fulfilling its clinical use [3,30]. In our clinical trial, we performed immediate loading, initially using light forces that were later increased progressively.
In fact, Derid Ure et al. demonstrated in their study that the stability of orthodontic mini-screws decreases during the first three weeks but then started to increase between weeks 3 and 5 [9]. Similarly, according to our results shown in Table 1, the stability of the irradiated and non-irradiated mini-screws decreased significantly up to two months post-implantation (T3 and T4) where the changes of ISQ values became non-significant, and the stability curve of mini-screws appears to stabilize. This observation was found interesting because the three-month treatment and follow-up represented the originality of our study. On a clinical basis, this finding could guide us in terms of force magnitude by indicating light forces for the first two months post-implantation to start and then continuing with higher increments in order to rule out one of the risk factors known to jeopardize the clinical stability of the mini-screw.
Furthermore, on the distal side of the L group mini-screws, another non-significant decrease of ISQ values were recorded between the third and twelfth weeks (T1 and T4) ( Table 1). Knowing that the distal side of a mini-screw is opposed to the force applied, it is probably the one that undergoes most of the cellular changes and reactions. This is where the PBMt could potentially be the most effective.
Similarly, in the L group between the third and sixth weeks of post-implantation (between T1 and T2), both the ISQ values recorded on the upper side of the mini-screws and the global ISQ values stopped decreasing significantly. As mentioned above, the first week following mini-screw insertion, the PBMt was in fact performed every other day and then carried out after two weeks (T1) during the first follow-up appointment. This likely means that the effect of PBMt was maximal during this period and that this positively influenced the stability of the mini-screws. Hence, PBMt seems to follow this rule: the more, the better. Results favouring the use of PBMt with mini-screws could be obtained if laser applications are more frequent. However, this frequency may seem impractical since orthodontic appointments are normally scheduled every three to four weeks.
Our clinical trial showed no significant difference between the stability of irradiated and non-irradiated mini-screws. While several clinical studies have targeted the same subject, different protocols were used, and different results have been found. In fact, a study by Maranon-Vasquez et al [31]. evaluated the effect of PBMt on the stability and displacement of mini-screws with immediate and delayed loading. Two types of lasers were used: a red emission laser with a wavelength of 660 nm that was applied only on the same day of implantation, and a second infra-red laser with a wavelength of 808 nm that was applied every two to three days for two weeks. This study included 35 mini-screws and showed a lower loss in stability than in the ones that received PBMt. Concerning mini-screw displacement, no statistically significant difference was found. The results of that study were not in accordance with ours, favouring the use of PBMt to increase mini-screw stability. In fact, they used two types of lasers. According to the same authors, the 660-nm wavelength laser was used immediately after the insertion of the mini-screw to enhance the healing of damaged tissue and to promote the initial inflammatory reaction. In the following days, the 808 nm wavelength laser was subsequently used because it had already been shown that PBMt boosts mini-screw bone healing during the intermediate postoperative period [32]. Another study by Matys et al. [33] used a diode laser at an 808 nm wavelength with an irradiance of 800 mW/cm 2 for a one-month period of PBMt. It measured the stability of the mini-screws for two months and found an increase after 30 to 60 days. These results proved the theory stating that better outcomes are obtained when a longer follow-up is carried out. The same result was obtained by Osman et al. [34] at a 910 nm laser wavelength with an average power not exceeding 0.7 W for 60s, by Matys et al. [35] in another study using a diode laser at a 635 nm wavelength, 100 mW and 20 J/cm 2 , and by Ekizer et al. [36] using a LED at an irradiance of 20 mW/cm 2 . In the literature, a large variability clearly exists among lasers, wavelengths, and energy densities [37]. In our study, an 810-nm diode laser wavelength at a power density of 1.2 W/cm 2 , a total energy of 50 joules, and an energy density with movement of 8.3 J/cm 2 delivered with a Gaussian optical fibre beam profile were used. According to the literature, the use of PBMt in patients involves almost exclusively the red and near infrared portion of the electromagnetic spectrum (650 nm-1100 nm). This range of wavelengths, known as the optical window, is where the effective tissue penetration of light is maximized [31]. The effective tissue penetration of light and the specific wavelength of light absorbed by photo-acceptors are two of the major parameters to be considered in light therapy [16].
As discussed earlier, PBMt possesses an anti-inflammatory effect that can increase blood flow, stimulate metabolic activities, and accelerate the healing process [14]. In fact, Yanaguizawa et al. reported in their study high levels of interleukins-8 for non-irradiated mini-screws after 24 h, 48 h, and 72 h of implantation [38]. Matys et al. also concluded that PBMt applied during the early healing phase positively modulates the whole procedure and can improve mini-screw stability after one to two months [33]. This could explain why, in our study, the ISQ values of irradiated mini-screws were stable between T1 and T2.
On the other hand, results similar to ours were obtained by Abohabib et al. [39] and by a meta-analysis conducted by Chen et al. [40] to conclude on this subject. In fact, after analysing all the clinical trials that studied the effect of PBMt on orthodontic mini-screws, they found that the existing data are inconclusive, and that further high-quality human studies are required to obtain scientifically convincing and clinically applicable conclusions. According to this meta-analysis, the study designs of several studies were not highly satisfactory. In fact, it has been demonstrated that the effect of PBMt is related to the treatment protocol, which is why unsuitable protocols may decrease the treatment efficacy. In addition, the wide variety of diode laser wavelengths assessed is characterized by a Gaussian beam profile delivery and the obtained results were inconsistent due to a lack of standardization in the applied laser protocols and techniques. According to the Arndt Schulz curve, when the power density of the laser light is insufficient or is applied for a short period of time, no effect is found on the tissue. Likewise, when using a high energy density or when the time of exposure is long, an inhibitory effect takes place. Thus, the optimal effects are obtained when a balance between the power density and the time of application is found [16]. This may explain the positive outcomes at early stages.
PBMt does induce osteogenesis since it stimulates cell proliferation, vascularization, and osteoblastic activity [41][42][43]. In fact, in our study the laser therapy promoted the stability of the mini-screws; however, it was not statistically different when compared to the placebo group.
The inconsistency in laser protocol and parameters clearly exists among published studies. Hence, it would be interesting to conduct a comparative study on the effect of different PBMt protocols on the stability of orthodontic mini-screws to finally reach the best combination.
Limits of the presents study include the absence of comparison of different laser applications and a relatively short observation period. Future studies will be necessary after having considered the current study's findings.

Conclusions
Based on the orthodontic therapy and scientific approach of this clinical trial, we can conclude that PBMt did not significantly increase the stability of orthodontic mini-screws over a period of three months.
When considering the limitations mentioned above, new strategies could better guide future research on this topic.