Dynamics of Alloplastic Bone Grafts on an Early Stage of Corticotomy-Facilitated Orthodontic Tooth Movement in Beagle Dogs

Alveolar augmented corticotomy is effective in accelerating orthodontic tooth movement, but the effect only lasts for a relatively short time. Therefore, the purpose of this study was to investigate the underlying biology of the immediate periodontal response to orthodontic tooth movement after a corticotomy with alloplastic bone grafts. The results demonstrated that measurable tooth movement began as early as 3 days after the intervention in beagle dogs. Based on the results and histological findings, augmented corticotomy-facilitated orthodontic tooth movement might enhance the condition of the periodontal tissue and the stability of the outcomes of orthodontic treatment.


Introduction
New appliances, materials, and mechanics of orthodontic treatment are being developed every day, but much of the biology of orthodontic tooth movement still needs to be clarified. Many adjunctive modalities are available to accelerate orthodontic tooth movement in humans, such as corticotomy [1][2][3][4][5][6][7][8][9][10], distraction osteogenesis [11], mechanical vibration [12], medication with local prostaglandins [13], and low-level laser treatment [14,15]. Among these interventions, corticotomy is known to be the most effective means to accelerate orthodontic tooth movement [7,16]. In experiments that used a split-mouth design, with corticotomy performed on one side and the other side serving as the control, the velocity of the tooth movement was accelerated on the corticotomy side [1,[17][18][19], and the amount of movement doubled over the duration of the experiments [17][18][19][20].
The initial microscopic changes and early application of orthodontic force have been emphasized in corticotomyfacilitated orthodontic tooth movement [16], and it has been hypothesized that a corticotomy or an osteotomy can lead to intensified osteoclastic activity resulting in local osteopenia and increased bone remodeling [2,17,21,22]. To date, however, the immediate periodontal response has not been fully elucidated. Most researchers have either studied the small animals such as rats, cats, or rabbits [2,6,10,[20][21][22], for relatively long periods of 6-12 weeks [4,5], or carried out gross observational studies with no histologic measurements [1,2,9,19,23].
The purpose of this study was to investigate the immediate periodontal response to a corticotomy with alloplastic bone grafts in beagle dogs.

Animal Subjects.
Five adult male beagle dogs, weighing 10-13 kg, were used in the experiment, and their selection, care, and preparation, together with the surgical protocol, were carried out according to the guidelines for animal experiments (IRB no. KHMC-IACUC2012-024). They were caged separately under regulated conditions and fed a normal diet 2 BioMed Research International and water ad libitum to secure the experimental orthodontic appliances.
For the preparation processes and surgical procedures, the animals were anesthetized with a mixture of tiletaminezolazepam and xylazine, via intramuscular and intravenous injections using a catheter in the vessel of the ear.

Study Preparation.
Alginate impressions of each beagle were taken to make study models, and orthodontic appliances were custom-made for each model. The canine and fourth premolar teeth were banded to form anchor teeth, and a Ø 0.9 mm stainless steel wire was welded onto the buccal surface of the bands. The second and third premolars were banded with a lingual button. After 2 weeks, the animals were anesthetized to fit the orthodontic appliances to the teeth ( Figure 1).

Surgical Procedures for the Corticotomy and Alloplastic
Bone Graft. Under general anesthesia, 2% lidocaine with 1 : 100,000 epinephrine was also infiltrated to the surgical sites. An intrasulcus incision was performed with a no. 12 blade from the canine tooth to the first molar, and a full-thickness flap was lifted. The circumscribing corticotomy (Figure 2(a)) was performed with a round bur (Ø 1.5 mm) under sterile saline irrigation. Alloplastic bone material (MBCP+, Biomatlante, Vigneux de Bretagne, France), composed of 20% hydroxyl apatite and 80% -tri-calcium phosphate, was used for the graft. The graft bone was soaked with blood, and 1 g of the MBCP+ was grafted onto the surgical surface ( Figure 2(b)). The mucoperiosteal flaps were repositioned and sutured with 5-0 nylon and primary closure was obtained (Figure 2(c)). A closed coil spring made of nickel-titanium shape memory wire of 200 g force was applied to the second and third premolars in a buccolingual direction (Figure 2(d)).
All of the surgical procedures were performed under sterile conditions to prevent infection. After surgery, antibiotics and anti-inflammatory analgesics were administered by intramuscular injection twice a day for 6 days. A 1% chlorhexidine-gluconate solution dressing was applied simultaneously for infection control. A soft diet was supplied for 1 or 2 weeks and then a normal diet. Mechanical plaque control was performed once a week. The animals were euthanized with an overdose of thiopental sodium after 1 day, 3 days, 1 week, 2 weeks, and 4 weeks following the surgery (Figure 3).

Histological Processing and Analysis.
Following a predetermined time schedule, after the animals were killed, their maxillae and mandibles were dissected, and the sections containing the canine to the fourth premolar teeth were retrieved. The block specimens were rinsed in sterile saline and immediately immersed in 10% neutral-buffered formalin fixatives for 14 days. The block specimens were large, and rapid decalcification was performed for 6 days using 5% nitric acid because it is sufficiently strong [24]. Had this study been designed for immunohistology, the use of ethylenediamine-tetra-acetic acid or 10% aqueous or formic acid would have been suitable [25], but this was not the case [5,26].
The specimens were then dehydrated through a series of ethanol solutions of increasing concentrations and embedded in paraffin. Buccolingual sections were sliced with a microtome set at 5 m and stained with the Masson's trichrome solution. One slide was processed per experimental tooth.
Histological examinations were conducted using a light microscope (Olympus BX 51, Olympus, Tokyo, Japan) equipped with a DP controller 3.2.276.2 and DP manager 3.1.1.208 (Olympus, Tokyo, Japan). After microscopic examination, a photograph of each slide was taken with a digital camera (Olympus DP 71, Olympus, Tokyo, Japan). With imaging software (cellSens version 1.6, Olympus), we measured the buccal tipping angle ( ∘ ) and distance ( m). The buccal tipping angle was measured from the reversal line of the lingual/palatal bone wall to the lingual/palatal root surface. The buccal tipping distance was measured from the lingual/palatal alveolar crest to the shortest lingual/palatal root surface. A total of 40 slides were fabricated and examined. After taking photographs, the amount of tooth movement was measured three times in each slide for angular changes and linear displacement.

Statistical Data Analysis.
Statistical data analysis was performed using the R programming language [27]. The data on tooth movement did not fulfill the parametric conditions of normality and equality of variance after the D' Agostino normality test was performed. We therefore conducted the Kruskal-Wallis rank sum test to determine whether there existed a significant between-group difference in general and the Wilcoxon test to find a significant pair between two groups. The Bonferroni correction and the Type I error were applied to counteract the problem of multiple comparisons. The data were analyzed with a confidence level of 95%. Figure 4 shows the amount of tooth movement for each beagle dog. Due to the minimal tooth movement, the buccal tipping angle at 1 day could not be measured. No significant difference was observed between the teeth in the maxilla and those in the mandible or in the right or left part of the dentition. When the tipping movement was measured from the angular changes, no statistically significant difference was found among the experimental groups ( Figure 4(a)). However, the linear measurements demonstrated statistically significant differences between 1 day and 3 days and between 2 weeks and 4 weeks after the start of the experiment (Figure 4(b)).  Figure 1: Orthodontic appliances were custom-made for each study model. The canine and fourth premolar teeth were banded to form an anchor tooth, and a Ø 0.9 mm stainless steel wire was welded on the buccal surface of the bands. The second and third premolars were banded with a lingual button. After 4 weeks, the animals were anesthetized to fit the orthodontic appliances to the teeth. The arrows indicate the direction of force. buccal pressure side. The tension side at the lingual alveolar bone crest contained more cells than the pressure side and active osteoblasts forming a new bone ( Figure 6(h)). At 1 week after the start of the experiment, most of grafted MBCP+ particles were well maintained (Figure 7). On the pressure side, the PDL space was slightly widened compared with that at 3 days (Figure 7 (Figure 9(a)). Also, a new bone island was formed in the center of the bone-derived mesenchymal matrix (Figure 9(d)), and osteoblasts and osteocytes were observed (Figure 9(e)). On the buccal side, grafted MBCP+ particles were bridged with newly formed bone in the bone-derived mesenchymal matrix. Entrapped osteocytes and aggregated osteoblasts were observed (Figure 9(g)). The palatal crestal (Figure 9(j)) and apical (Figure 9(l)) tension sides showed aggregated osteoblasts and active forms of osteoblasts, and a new bone-forming buccal bone wall and crest were observed (Figure 9(o): native bone (red star) and new bone (yellow star)). New bone was formed on the outer and inner surfaces of the native bone (Figures 9(n) and 9(q)), and the outer portion of the bone-derived mesenchymal matrix could be seen (Figure 9(t)).

Discussion
The biological mechanism by which the tooth movement is facilitated after a corticotomy has been suggested to be mediated by a regional acceleratory phenomenon [28,29], which might boost the appearance of the macrophages that eliminate the hyaline as early as 1 week after the application of orthodontic force [17,21]. For this reason, we designed an experiment to determine immediate periodontal responses, which represents the first study to observe the immediate effect of corticotomy-facilitated orthodontic tooth movement. Two studies previously reported observations made 3 days after a corticotomy in rats [2,22]. However, the results obtained in small animals may differ from those in larger animals [16]. We believe that the current study is unique because we observed the histological responses of periodontal tissue as early as 1 day, 3 days, 1 week, 2 weeks, and 4 weeks after a corticotomy and force application for orthodontic tooth movement in larger animals.
In most experiments in dogs or rats, corticotomyfacilitated tooth movement was observed at a rate of about 1 mm per month, which was almost double that observed on the control side [17][18][19][20]. In this study, we observed significant tooth movement within 3 days (Figure 4). We measured the width of the PDL at the crest of the lingual/palatal sides, which does not represent the direct distance of clinical tooth movement. The significant tooth movement observed 4 weeks after the start of the experiment was to be expected, but this obviously occurred earlier in the experiment. The tooth movement measurements demonstrated a similar pattern in angular changes and linear displacement, as shown in Figure 4. We conjectured that this might imply that the pattern of rapid tooth movement was not a bodily translation in general but mostly occurred through tipping of the tooth. Therefore, during clinical orthodontic treatment, methods to control for unwanted tipping should also be considered. Despite the similar pattern between the angular and linear changes, the linear measurements exhibited a statistically significant difference while the angular changes did not. Linear measurements are assessed between two points while degrees of angles are measured between three points, from which a variation in angular measurements can be produced. This may be the cause of the larger variation in angular measurements than in linear measurements.
A typical cell-free zone on the pressure side and an inflammatory reaction were seen on the 1-and 3-day slides ( Figures 5 and 6). A force of 200 g may be strong for tipping movement, but it was difficult to determine the hyalinization layer in the 1-and 3-day slides. The corticotomy was probably responsible for this, but the underlying mechanism was not clear. The progenitor cells regarded as osteoblasts in the 3-day slides showed strong cellular activity.
On the 1-week slide, we observed that the PDL space was widened at the pressure side compared with that of the 3-day slide (Figure 7(b)). This could be explained by undermining resorptions. On the tension side, active osteoblasts forming a new bone were observed. This new bone formation is a common phenomenon in orthodontic tooth movement.
Interestingly, new bone surrounding the graft materials was observed on the compression side on the 2-week slides ( Figure 8(d)) and at the buccal sides distant from PDL (Figures 8(s), 8(t), and 8(u)). Graft particles were bridged by newly formed bone and osteoclastic and osteoblastic activities were both seen (Figure 8(u)). From the findings of the 4-week sections, the new bone formation in the center of the MBCP+ graft material on the buccal side was very distinctive (Figures 9(h) and 9(i)), and many entrapped osteocytes and aggregated osteoblasts were observed (Figure 9(g)). However, the cause of this new bone formation on the buccal sides around the graft materials was not certain. Further studies could provide the answer.
The slides taken at 2 and 4 weeks did not show a distinctive loss of periodontal attachment, and the small areas of root resorption that were seen were not significant.
Orthodontic patients have complained about the length of their treatment, and it has become necessary to develop adjunctive methods to tackle this problem [1,3,[7][8][9][10][11][12][13][14][15]. Alveolar corticotomy is effective in accelerating orthodontic tooth movement [2,7,16]. However, according to pertinent studies [1,16,19], the regional acceleratory phenomenon only persists for about 4 months, after which the tooth movement rate returns to normal. To make use of this "window" period in an effective, efficient, and efficacious way, it is imperative to understand the underlying biology of early periodontal responses to tooth movement after augmented corticotomy and to develop appropriate clinical procedures. Also, from the clinical point of view, it is important to determine how to reduce the length of total treatment time. When should the Although there is no doubt that this procedure could align teeth within a shorter period of time [1,3,8,16,[30][31][32][33], at the present time, we cannot expect augmented corticotomy-facilitated orthodontic tooth movement to reduce the entire length of orthodontic treatment noticeably in adult patients. In addition, there is a paucity of information in the available research to assert that grafting enhances the stability of orthodontic treatment [16]. To prove this, well-designed randomized controlled clinical trials should be performed. The combination of corticotomy with an alveolar graft was introduced by Wilcko et al. and is referred to as accelerated osteogenic orthodontics or periodontally accelerated osteogenic orthodontics [8,9,34]. They asserted that bone grafting of the labial and lingual cortical bones would increase the stability of orthodontic treatment, enhance the range of possible tooth movements, increase alveolar bone volume, and provide a more structurally stable periodontium. However, no convincing scientific or histological evidence was available other than clinical reports. In some case reports, increased volume of bone around the alveolus was observed after bone grafting [3,5,9]. The present study supported the hypothesis that tooth movement with an augmented corticotomy might enhance orthodontic tooth movement, because we found new bone formation at the buccal surface. However, it focused on initial responses, and further investigation is needed to clarify its findings.
Tooth movement that was thought to be difficult or impossible to produce in the past has become possible due to the development of orthodontic mechanics and new appliances and materials. Therefore, orthodontic treatment is mainly limited by the scope of the alveolar bone, and orthognathic surgery is required when this limit is exceeded. If an augmented corticotomy could increase the alveolar bone volume, this would help patients who have a limited amount of supporting alveolar bone.
Augmented corticotomy surgery is not free from some morbidity. It also requires a skilled clinician, and there may be some discomfort to patients and additional costs.

Conclusions
The findings from this study suggest that measurable tooth movement starts as early as 3 days after augmented corticotomy-facilitated orthodontic treatment and that this procedure might enhance the condition of periodontal tissue and the stability of orthodontic treatment outcomes.