Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial

ABSTRACT Introduction: Micro-osteoperforation is a minimally invasive technique that has been used to accelerate orthodontic tooth movement and reduce treatment duration. However, literature presents conflicting reports about this technique. Objective: To evaluate the effectiveness of micro-osteoperforations on the rate of canine retraction and expression of biomarkers in gingival crevicular fluid (GCF). Methods: This was a randomized clinical trial with split-mouth study design. Thirty adult subjects with age above 18 years (20.32 ± 1.96) who required fixed orthodontic treatment and extraction of maxillary first premolars were enrolled and randomly allocated to either the experimental or control group. Randomization was performed by block randomization method, with a 1:1 allocation ratio. The experimental group received three micro-ostoperforations (MOPs) distal to maxillary canine, using the Lance pilot drill. The retraction of maxillary canine was performed with NiTi coil-spring (150g) in both experimental and control groups. The primary outcome was the evaluation of canine retraction rate, measured on study models from the baseline to 16 weeks of canine retraction. Secondary outcomes were the estimation of alkaline and acid phosphates activity in GCF at 0, 1, 2, 3, and 4 weeks. Results: There was a statistically significant difference in the rate of canine retraction only after the first 4 weeks. Subsequently there was no statistically significant difference from the eighth to the sixteenth weeks between MOPs and control group. There was a statistically significant difference in alkaline and acid phosphates activity in GCF between MOPs and control groups during the initial 4 weeks of canine retraction. Conclusion: Micro-ostoperforation increased the rate of tooth movement only for the first 4 weeks; thereafter, no effect was observed on the rate of canine retraction during 8, 12 and 16 weeks. A marked increase in biomarker activity in the first month was observed.


INTRODUCTION
Over the past decade, accelerated orthodontic movement has become an encouraging area of research in the orthodontic field. Several techniques have claimed to improve orthodontic treatment efficiency, by reducing treatment duration in complex adult treatment. 1,2 Current research indicates that the most effective methods for the acceleration of tooth movement are the surgical approaches, including distraction osteogenesis, corticotomy, osteotomy, and piezocision technique. However, it is assumed that the surgical approaches have not been widely employed, due to the aggressiveness and associated complications. [3][4][5][6] Recently, less invasive and controlled micro-trauma through This evidence indicates that the MOPs increase the catabolic Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial and anabolic activities, thus reducing tooth movement resistance. These catabolic and anabolic activities can be measured by the expression of bone resorption and bone formation biomarkers in gingival crevicular fluid (GCF). 9 Teixeira et al. 8 conducted a study on rats and stated that minimum cortical perforations increased the inflammation and enhanced tooth movement. Recently, many studies have been conducted to evaluate the effect of MOPs on the rate of tooth movement. 10-14 Some of these studies have shown an increase in the rate of tooth movement in the experimental group of more than 2 folds, when compared to the control group. 10,11 However, contradictory results have also been reported by several studies. 12,13 According to a Cochrane review, 14 most of the randomized clinical trials presented small sample size and unclear risk of bias. A recently conducted meta-analysis indicated that there was a statistically significant difference in the rate of canine retraction after performing MOPs; however, clinically, it failed to show substantial outcomes. 15 Up to the present date, several articles have been published on accelerated orthodontics, but there is a lack of information on the relationship between bone catabolic and anabolic biomarkers. Ferguson et al. 9 conducted a systematic review to evaluate the effect of various surgical accelerating techniques Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial 6 on the expression of biomarkers, and found that most of the studies were done using animals. Assessing a human sample, only the report published by Alikhani et al. 4 was found.
The authors evaluated the effect of MOPs on the expression of inflammatory markers, and found that there was 2.3-fold increased rate of canine retraction, with increased expression of cytokines. The study comprised a small sample size, and follow-up was done for only 28 days; moreover, a possible conflict of interest should be discussed, because they used commercially available appliances, and randomization and allocation concealment was unreported. In addition, they used lateral incisor as a reference point to measure canine retraction, which is considered an unstable point, and used 0.016 x 0.022-in stainless wire in 0.022-in slots for canine retraction, a procedure that allows more tipping movement and could give a false perception of tooth movement acceleration. All these shortcomings signify that there is an urgent need for high-quality randomized controlled clinical trials that helps proving the effectiveness of MOPs and its correlation with the expression of bone biomarkers. Therefore, the present study is, as far as we know, the second study done with human sample, with a follow-up of 16 weeks, to evaluate the effect of MOPs on the rate of canine retraction and its correlation with expression of biomarkers in GCF.
Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial 7 The null hypothesis tested was that there is no difference in the rate of canine retraction and the level of the biomarkers in the GCF between control and micro-osteoperforated group.

SPECIFIC OBJECTIVES OR HYPOTHESES
The objectives of the present study were: » Evaluate the effect of MOPs on the rate of canine retraction for a period of 16 weeks.
» Evaluate the changes in the level of biomarkers in the GCF.

TRIAL DESIGN
The present study was a single-center randomized controlled clinical trial using a split-mouth design, with 1:1 allocation.
No changes were made after the initial trial.

PARTICIPANTS, ELIGIBILITY CRITERIA AND SETTINGS
Ethical approval was obtained from ethical reviewer board of the institute at Swami Vivekanand Subharti University (Meerut/India). The trial was also registered at ICMR with CTRI number 01516450. Subjects were screened from the depart-  Table 1. A detailed medical history was recorded for each patient, followed by a detailed clinical examination. Informed written consent was taken from patients or parents/legal guardians, after informing the study procedures.

SAMPLE SIZE CALCULATION
The sample size was calculated based on a type I error frequency of 5%. Power analysis with G*Power software showed that 27 subjects per group would be needed for a statistical power of more than 80% to detect a significant difference, with 0.66 effect size and 0.05 as the significance level.

11
Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial for retraction (Fig 1). The NiTi closed coil spring was attached from the canine power arm to the hook of a molar tube.
At each appointment, a Dontrix gauge was used to measure the retraction force. If the force level was found to be less than 150 g, then NiTi coil spring was activated to maintain the force level. The bite contact was raised in those subjects in which occlusal interferences were present.

Primary outcome
The rate of canine retraction was assessed as the primary outcome of the study. To monitor the rate of canine movement, alginate impressions were made before the canine retraction (T 0 ) and after four weeks (T 1 ), eight weeks (T 2 ), twelve weeks (T 3 ) and sixteen weeks (T 4 ); and study models were fabricated with Type-II dental stone. For the measurement of canine retraction on the study model, the method used by Loztof et al. 16 was applied, with a slight modification. In our method, an acrylic palatal plug with reference wires was fabricated on the first study model (T 0 ) over the Nance palatal button used for anchorage control, and was stabilized with pinheads on anterior teeth. Reference stainless steel wires (0.9 mm) were placed mesial to canine on both sides, and the terminal ends were embedded in the acrylic plug. A long axis of canine was drawn from cusp tip to cervical end, and the midpoint of this axis was marked. The base value was set by measuring the distance from this midpoint to the mesial reference wire on the first model (T 0 ) (Fig 2). The plug was then transferred to the subsequent study models (T 1 , T 2 , T 3 and T 4 ), and the distance that the canine has moved at every 4-week interval was measured. The palatal plug was considered as the reference device for all the study models of the same patient. All measurements were recorded by other blinded investigator, using a

RESULTS
Two subjects were excluded from the study due to irregular follow-up. Data from 28 subjects were analyzed (Fig 3). Subjects data including gender, age, amount of extraction space, and cephalometric analysis are listed in Table 2. Subject's age     Table 3). p ≤ 0.05 is significant (S), p > 0.05 is non-significant (NS).

SECONDARY OUTCOME
There was a statistically significant difference in the level of alkaline phosphatase (ALP; Table 4, Fig 4) and acid phosphatase (TRAP; Table 5, Fig 5), on both mesial and distal sides, between experimental and control groups at different time intervals. The level of alkaline phosphatase was found significantly higher on the mesial side, while the level of acid phosphatase was found significantly higher on the distal side.

DISCUSSION
The principle of micro-osteoperforations is a regional con- Nimeri et al. 19 has also documented that RANKL:OPG ratio is proportional to age, which affects the rate of bone remodeling and tooth movement. To minimize the effect of age, only adult subjects (age >17 years) were included in this study.
Strict discipline and clear instructions were given to maintain excellent oral hygiene. Occlusal interferences in the path of canine retraction were removed by raising the bite, when necessary. According to Yang et al. 24 , maximum stress during the canine retraction was distributed on the cervix at distolabial Since the purpose of MOPs is to perforate only the alveolar bone, a Lance pilot drill was used, because it has sharp edges, calibrated length and is designed to cut the bone effectively.
To effectively perforate the cancellous bone, the perforation depth was kept at least 5 mm, because average gingival thickness is 2 to 3 mm, and average cortical bone thickness is 1.5 to 2.0 mm. Although the thickness of attached gingival tissue may vary from patient to patient, for those patients in which attached gingival thickness was more than average, the perforation depth was increased to reach the cancellous bone.
After MOPs, a force of 150 g was used for canine retraction, as recommended by Samuels et al. 26 Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial 24 In this trial, the accelerating effect of MOPs was observed only during the first four weeks. In this period, the rate of canine Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial 25 The secondary outcome of the present study was to correlate the rate of tooth movement with the expression of biomarkers after MOPs. The systematic review conducted by Kapoor et al. 30 showed that there was a positive correlation between the levels of ALP and TRAP in the GCF and the velocity of orthodon- MOPs and control groups. The peak of ALP level occurred at the second week on mesial and distal sides in both MOPs and control groups, but there was a markedly increased ALP activity in MOPs group at the second, third and fourth weeks, when compared to control group. These results indicate that there was increased osteoblastic activity at the third and fourth week on mesial side (tension side), as a compensatory mechanism Raghav P, Khera AK, Preeti P, Jain S, Mohan S, Tiwari A -Effect of micro-osteoperforations on the rate of orthodontic tooth movement and expression of biomarkers: a randomized controlled clinical trial to increased osteoclastic activity (occurring in the initial two weeks on distal side). Increased osteoblastic activity on distal side (compression side) was due to homeostatic mechanism induced by micro-perforations. These biomarker activity shows that increased osteoclastic activity persist only for the first three weeks after the increased osteoblastic activity (Fig 6).
These results directly correlate, and may be the reason for the