Comparison of tooth movement and biological response resulting from different force magnitudes combined with osteoperforation in rabbits

Abstract Objective To compare tooth movement rate and histological responses with three different force magnitude designs under osteoperforation in rabbit models. Methodology 48 rabbits were divided into three groups: Group A, Group B, and Group C, with traction force of 50 g, 100 g, 150 g, respectively. Osteoperforation was performed at the mesial of the right mandibular first premolar, the left side was not affected. One mini-screw was inserted into bones between two central incisors. Coil springs were fixed to the first premolars and the mini-screw. Tooth movement distance was calculated, and immunohistochemical staining of PCNA, OCN, VEGF, and TGF-β1 was analyzed. Results The tooth movement distance on the surgical side was larger than the control side in all groups (P<0.01). No significant intergroup difference was observed for the surgical side in tooth movement distance among the three groups (P>0.05). For the control side, tooth movement distance in Group A was significantly smaller than Groups B and C (P<0.001); no significant difference in tooth movement distance between Group B and Group C was observed (P>0.05). On the tension area of the moving premolar, labeling of PCNA, OCN, VEGF and TGF-β1 were confirmed in alveolar bone and periodontal ligament in all groups. PCNA, OCN, VEGF and TGF-β1 on the surgical side was larger than the control side in all groups (P<0.001). Conclusion Osteoperforation could accelerate orthodontic tooth movement rate in rabbits. Fast osteoperforation-assisted tooth movement in rabbits was achieve with light 50 g traction.


Introduction
In clinical tooth extraction cases of protrusive adult orthodontics, it always takes approximately two and half years of traditional fixed appliance treatment.
Long-term comprehensive orthodontic treatments bring many risks to patients, such as root absorption, dental decalcification and periodontal disease. 1 Thus, the efforts to increase the rate of tooth movement and shorten the treatment period have always been a focus of clinical orthodontics. Furthermore, the growing number of adults requiring orthodontic treatment has brought more awareness to the matter. 2 In the last 20 years, many procedures to accelerate tooth movement and shorten the orthodontic treatment period, such as low-intensity laser treatments, lowintensity pulsed ultrasound, pulsed electromagnetic fields, corticotomy, and corticision, have been investigated. 3,4 Good-quality randomized clinical trials show that corticotomy is useful in increasing tooth movement rate. 5 Originally, corticotomy was composed of buccal and lingual flap reflections, followed by bone decortication.
Later, some modifications to the cortical cuts were performed, of which the recent more attractive and lower-trauma form was osteoperforation.
Osteoperforations are defined as surgical operations in which flaps are reflected and cortical bone perforations are performed, while the cancellous bone is reserved. 6 Köle 7 (1959) first proposed the theory of "bony block movement". Based on this theory, Köle successfully accelerated tooth movement by conducting a corticotomy around the moving tooth. Later, Wilcko et al. 8 (2001) indicated that the fundamental principle of accelerated tooth movement assisted by corticotomy might not be the phenomenon of "bony block movement"; instead, it was the regional acceleratory phenomenon (RAP). Since then, RAP is supposed to be the rationale behind the accelerated effect of tooth The experiments were based on the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978  A retentive horizontal groove around the crown of the mandibular first premolars was made using a slowspeed handpiece. One mini-screw (Protect, Hangzhou, Zhejiang, China) was inserted into bones between two mandibular central incisors using a manual driver. One nickel-titanium closed coil spring (Protect, Hangzhou, Zhejiang, China) was fixed to the right first premolar and the mini-screw with a 0.010-inch stainless steel ligature wire (Protect, Hangzhou, Zhejiang, China).
Another coil spring was fixed to the first left premolar and the mini-screw ( Figure 1). The coil spring would create 50 g/100 g/150 g continuous traction force; force was standardized with a dynamometer.

Measurement of Tooth Movement Distance
The tooth movement distance was acquired from the three-dimensional CT data of the rabbits. The mandible of rabbits was scanned before surgery (T1) and after two weeks of spring traction (T2). CT data were uploaded into Dolphin software (Version 11.9, Dolphin Digital Imaging, Chatsworth, Calif, USA).
The distance between the mesial alveolar crest of the second premolar and the distal alveolar crest of the first premolar on both sides was measured by a digital ruler at T1 and T2 ( Figure 2). Tooth movement distance was calculated by subtracting the measured distance at T1 from the measured distance at T2. Three independent investigators conducted the measurement, and the average of the values acquired by three investigators was obtained for further analysis.

Histological Staining
After two weeks, the rabbits were sacrificed by overdosing sodium pentobarbital. The specimens from the mandibular second premolars extending anteriorly to 10 mm mesial of the mandibular first premolars were obtained. The specimens were

Results
During the experiment, all springs remained intact.
The mesial moving of the mandibular first premolars on both sides were observed in the three groups and all the premolars moved in the proper direction.

Tooth Movement Distance and Rate
The ICC coefficients of tooth movement distance measurements were higher than 0.851, and the 95% confidence interval ranged from 0.656 to 0.993 among the measurements. The results of the exploratory chi-square statistical tests confirmed the hypothesis of normality of distribution for the tooth movement distance. The tooth movement distance on the surgical side was larger than the control side in all groups (P<0.01) ( Table 1). We found no significant intergroup difference in tooth movement distance among the three groups (P>0.05) for the surgical side. For the control side, the tooth movement distance in Group A was significantly smaller than those in Group B (P<0.001) and Group C (P<0.001). However, we found no significant difference in tooth movement distance between Group B and Group C (P>0.05) for the control side.
The average movement rate of the premolars Comparison of tooth movement and biological response resulting from different force magnitudes combined with osteoperforation in rabbits J Appl Oral Sci. 2021;29:e20200734 5/9 during the beginning two weeks was calculated to analyze the tooth movement in detail. In Group A, the tooth movement rate on the surgical side was 0.073 mm/day, and the rate on the control side was 0.037 mm/day. In Group B, the tooth movement rate on the surgical side was 0.073 mm/day, and the rate on the control side was 0.053 mm/day. In Group C, the tooth movement rate on the surgical side was 0.074 mm/day, and the rate on the control side was 0.052 mm/day.

Histological Analysis
We observed alveolar osteopenia on the compression area of the moving premolar in all groups. Visible periodontal cells and multinucleated osteoclast-like cells could be observed on the alveolar bone margin adjacent to the compressed periodontal ligament (PDL). We noted dilated blood vessels on the tension area of the moving premolar and newly formed woven bone with numerous osteocytes. (Figure 3).

Immunohistochemical Analysis
The ICC coefficients of mean optical density measurements were higher than 0.561, and the 95% confidence interval ranged from 0.411 to 0.999 among the measurements. The results of the exploratory chisquare statistical tests confirmed the hypothesis of normality of distribution for the mean optical density.
The data of immunohistochemical calculations in the three groups are presented in Table 2.
We found PCNA, OCN, VEGF, and TGF-β1 signals on the tension area of the moving premolar in the alveolar bone in all groups (Figure 4). The expression levels of PCNA, OCN, VEGF, and TGF-β1 on the surgical side were more expressive than the control side in all groups (P<0.001).  Based on these, we selected diameter 1.0 mm to osteoperforations in rabbit models. In one study, Kurohama et al.14 (2017) reported that the corticotomy was performed to the rats' maxilla using a round bur at a low speed of 100 rpm. Considering the density of the mandibular cortical bone, we supposed that the rpm should be higher for rabbit models than for rat models. Combined with the factor of injury to the bone during the drilling procedure, we finally selected 250 rpm to osteoperforations in rabbit models.

Control Side Surgical
The mandibular first premolar movement model applied in our study was improved from the model used formerly. As rabbits have small incisors with poor retention, a mini-screw inserted into the bone between two mandibular incisors was used as an anchor unit.
We used a nickel-titanium coil spring-assisted by the mini-screw to pull the mandibular first premolar and move it mesially.
Several studies have indicated that osteoperforation could accelerate orthodontic tooth movement. 15 Data are presented as mean±SD; PAB: the difference of mean optical density between Group A and Group B; PAC: the difference of mean optical density between Group A and Group C; PBC: the difference of mean optical density between Group B and Group C; P<0.05, P<0.01 and P<0.001=significant; *: P<0.05; † : P<0.01; ‡ : P<0.001.  However, in disagreement with our findings, Alkebsi, et al. 23 (2018) claimed no noticeable difference in tooth movement distance between the osteoperforation and the control groups. Differences between these studies might be due to differences in the study design, such osteoperforation itself, bone removal associated with osteoperforation, animal model, and magnitude of traction force.
The tooth movement rate can be regulated by the degrees of bone turnover, bone density and PDL hyalinization in response to the traction force. 24 Regarding bone turnover, the balance between bone regeneration and bone resorption during tooth movement will affect the rate of bone remodeling and subsequently influence the rate of tooth movement.
Our results were consistent with previous studies 25,26 showing greater bone apposition and increased localized bone turnover subjected to corticotomy and resulted in the acceleration of tooth movement.
We discovered that osteoperforation-assisted tooth movement distance under different force magnitudes was almost similar. We then inferred that the injury from traction force was much lower compared with cortical bone perforation, and the increase of force magnitude in the movement of osteoperforationassisted teeth would not increase alveolar bone resorption. 17 This discovery was similar to Zuppardo et al. 26 (2020), who reported that adding decortication to corticotomy would not improve its efficiency of accelerating tooth movement. Therefore, a low force magnitude of 50 g was enough to benefit fast osteoperforation-assisted tooth movement.
The immunohistochemical staining analysis can help us better understand the change in osteoblast activities on the tension area of the moving tooth.
PCNA is a cell cycle-associated nuclear protein that is maximally elevated in proliferating cells, and it is reportedly useful for assessing the proliferation activity of osteoblasts. 27 OCN is a mature marker of osteoblasts and present at high levels in a mineralized bone matrix during the late stage of bone formation. 28 In our experiment, after two weeks of osteoperforation, the mean optical densities of PCNA-positive osteoblasts and OCN-positive osteoblasts on the surgical side were significantly higher than the control side in all groups.
However, there was no difference in the expression of PCNA under osteoperforation among the three groups.
These discoveries indicated that osteoblasts around the moving tooth were active at the early stage under osteoperforation, and a lower force of 50 g was enough to activate the osteoblasts. Further studies applying micro-CT examination might be useful to provide more information. Conclusions 1. The application of osteoperforation could accelerate the orthodontic tooth movement rate in rabbits.
2. An application of a light force of 50 g was suitable to achieve fast osteoperforation-assisted tooth movement in rabbits.