The aim of this pilot study was to evaluate the effect of the timing of postoperative orthodontic force application on bone remodeling during tooth movement into surgical alveolar defects with bone grafts in beagle dogs.
Six beagle dogs underwent surgery for buccal dehiscence-type defects (width, 5 mm; height, 6 mm) on the distal root of maxillary second premolars bilaterally for 12 defects. After 1-month healing, bone-augmentation procedures were undertaken at the dehiscence defects. The second premolars were protracted buccally for 6 weeks into the surgical sites immediately (F-0), at 4 weeks (F-4), or 8 weeks (F-8) after grafting. Orthodontic tooth movement was monitored using digital models. Remodeling of alveolar bone was evaluated by histology, histomorphometry, immunohistochemistry, microcomputed tomography, and fluorescence microscopy.
Group F-0 showed significant expansion (mean, 2.42 mm) and tipping (mean, 9.03°) after completing orthodontic tooth treatment. The vertical bone defect was significantly lower in groups F-4 and F-8 than that in group F-0 (mean, 2.1, 2.7, and 4.5 mm, respectively). In group F-4, the formation of new bone and mineralization were significantly greater than those in groups F-0 and F-8 ( P <0.05). Group F-4 showed a minimal amount of bone-material remnants. Immunohistochemistry showed the highest expression of collagen-1 and osteopontin in group F-4, followed by group F-8 and group F-0, which demonstrated high osteoblast activity and enhanced bone remodeling in group F-4.
Orthodontic force application at 4 weeks after an augmentation procedure provided the best functional stimulation for an alveolar bone graft. This strategy enhanced new-bone regeneration and degradation of bone substitutes and, eventually, promoted bone remodeling in the bone-grafted area.
The effect of timing of force application on bone graft regeneration was studied.
Force application at 4 weeks provided the best functional stimulation for a bone graft.
Force application at 4 weeks enhanced bone regeneration of bone substitutes.
Force application at 4 weeks increased bone degradation of bone substitutes.
Force application at 4 weeks promoted bone remodeling in the bone-grafted area.
Patients with narrow alveolar bone or severe skeletal discrepancies are likely to exhibit alveolar defects. Moving a tooth into a region of an alveolar defect without sufficient height and width of bone is difficult. The defect in the osseous coverage of the dental root may be due to fenestration or dehiscence. Studies have shown a higher prevalence of alveolar defects on the buccal surface than on the lingual surface. , The presence of such buccal alveolar bone defects reduces the bony support of the teeth. As a result, dehiscence and fenestration of the buccal cortical plate have been correlated to the buccal movement of teeth, such as protrusion and arch expansion. When the tooth is about to challenge the “orthodontic walls” of a dense cortex, this often causes gingival recessions, alveolar/teeth-root resorption, or damage to the pulp vitality. Even the movement of a single tooth in a buccolingual direction can produce the same effect. An orthodontic treatment is not accomplished unless the reestablishment of the alveolar bony volume has been achieved.
Bone grafting has been suggested to restore the appropriate alveolar dimensions after the application of orthodontic forces (OFs). , It enhances the possible range of tooth movements with increases in alveolar bone volume and a more structurally complete periodontium. , , Tooth movement might affect the remodeling of bone graft substitutes if the postoperative orthodontic adjustment is not carried out appropriately. Few scholars have analyzed the effects of buccal tooth movement and graft substitutes on surgical defects under different loading durations. Several clinical studies with a limited number of patients have advocated that an immediate, heavy OF should be applied to activate tooth movement and that in all patients, OF initiation should be undertaken within 2 weeks after surgery. , Nevertheless, those studies lacked a control group, and this suggestion was only based on clinical experience or imaging analyses. Ahn et al reported that, according to histology studies, the immediate application of a force after bone grafting increases the periodontal regeneration and reduces the risk of inhibited movement of the orthodontic teeth compared with a delayed force application. However, they focused on the mesiodistal movement of teeth, and it was also unclear whether bone grafting combined with buccal tooth movement would help to repair bony defects or if the timing of force application affecting the healing progression of surgical sites in which bone grafting had been done.
Animal experiments would present the best way to evaluate the biologic effects of bone grafting and help to obtain the guidelines for postoperative OF application. Therefore, this study aimed to (1) investigate the timing of buccal OF loading on an augmented area of an alveolar ridge; and (2) evaluate bone remodeling when OF was applied. The null hypothesis was that the timing of buccal OF application had no effect on bone remodeling with bone grafting at surgical alveolar defects in beagle dogs.
Material and methods
This study was implemented and accomplished in accordance with regional Ethics Committee guidelines. The experimental protocol was approved by the Animal Experimental Ethical Committee of our hospital (HKDL502).
Six skeletally mature (1.5 years) male beagle dogs (15-16 kg) were included in the study. The surgical procedure was carried out under general anesthesia with atropine sulfate (0.04 mg/kg body weight, intramuscular [IM]), ketamine (2.5 mg/kg, IM), and a 4% solution of pentobarbital sodium (25 mg/kg, intravenous), in addition to local anesthesia with lidocaine hydrochloride (2% with 1:100,000 epinephrine). Reflection of a full-thickness flap on the buccal side of the maxillary second premolars (P2s) bilaterally was done. Buccal dehiscence-type defects of 6 mm height from the cementoenamel junction and 5 mm width mesiodistally at the top were created. All periodontal ligament (PDL) tissues and cementum were removed from the root surface at the defect area using a curette. As a result, 12 buccal dehiscence-type defects were surgically created with an exposure of root surfaces ( Fig 1 , A ). After 1 month of tissue healing, computed tomography (CT) was performed to confirm this buccal dehiscence defect ( Fig 1 , B ). Each defect was then filled with deproteinized bovine bone mineral (Bio-Oss; Geistlich Pharma, Wolhuser, Switzerland) ( Fig 1 , C ). Subsequently, a resorbable collagen membrane (Bio-Guide; Geistlich Pharma) was positioned over the grafts ( Fig 1 , D ), and the flap was repositioned and sutured without excessive tension.
To prevent postoperative infection, we administered antibiotics daily for 1 week. In addition, a plaque-control regimen of 0.12% chlorhexidine gluconate was applied twice weekly throughout the entire study period. The animals were maintained on a soft diet during the study period.
Using a computer-generated random-number table, we divided all the surgical sites randomly into 3 groups according to the timing of postoperative OF application: immediately (F-0), 4 weeks (F-4), and 8 weeks (F-8) after grafting. First, target teeth were etched with 37% phosphoric acid. After sealant application, orthodontic brackets (0.022-in) were bonded onto the labial surfaces of the maxillary canine, the P2, and the fourth premolar (P4) with Transbond X (3M Unitek, 3M Dental Products, Monrovia, Calif) and were then light-cured for 10 seconds. A stainless-steel wire (0.021 × 0.025-in) was bent and placed passively through the bracket slots of the canine and P4. Then, P2 was buccally moved into the grafted area by a nickel-titanium closed coil spring and exerted a constant 50 g of force per side (Dentaurum, Ispringen, Germany) ( Fig 1 , E ).
Fluorochrome bone markers were injected (IM) with 25 mg/kg tetracycline (TE) hydrochloride, 20 mg/kg calcein (CA), and 30 mg/kg Alizarin Red (AL) to label the deposition of new bone around the P2 at 2, 4, and 6 weeks, respectively. Oral hygiene and OF magnitude were checked once a week. Six weeks after the appliance placement, the dogs were humanely killed by an anesthesia overdose. The timeline of experiments in this study is shown in Figure 2 . Tissue blocks (including the maxillary P2) and surgical defects were harvested and fixed immediately in 10% neutral buffered formalin for 48 hours.
An alginate impression (GC, Tokyo, Japan) of the maxilla was taken initially and at the end of treatment with a custom-made tray, and the model was poured using dental stone (GC Europe, Leuven, Belgium). To accurately quantify model analyses, the dental casts were imaged with a 3-dimensional (3D) scanner (D-250; 3Shape, Copenhagen, Denmark). Angulation of the P2 was calculated using the following references: line a = the line connecting the P2 cusp tip with the midpoint of the P2 palatal gingival margin; line b = a perpendicular line from the midpoint of the P2 palatal gingival margin to the midline of the palate. The internal angle was formed by both lines a and b . The buccal tooth movement was the perpendicular distance from the P2 cusp tip to the midline of the palate ( Fig 3 , A ). Pre- and posttreatment digital dental models were superimposed using the midpalatal raphe as a reference ( Fig 3 , B ).
Microcomputed tomography (microCT) was undertaken to evaluate the healing conditions of the defects. Each specimen was fixed in a cylindrical holder and imaged with a desktop microCT system (μCT-80; Scanco Medical, Wangen-Brüttisellen, Switzerland) in a high-resolution scanning mode (pixel matrix, 1,024 × 1,024; voxel size, 20 μm; slice thickness, 20 μm). The region of interest was restricted to the P2, with a width of 10 mm, length of 10 mm, and height of 12 mm. Then, the scanned data were converted into digital images using 3D image-reconstruction software (Scanco Medical). The microstructural parameters of bone mineral density (BMD), bone volume fraction (bone volume/total volume), and trabecular thickness of new bone (Tb.N) were calculated directly. These are the key indicators in the 3D characterization of cancellous bone that can reflect the formation of new bone and the ability of bone regeneration in the defected area under OF stimulation. The vertical bone defect of P2 was measured on the long axis of the buccolingual section using microCT. This defect was defined as the vertical distance from the crestal area of the bone defect to the cementoenamel junction of P2 ( Fig 4 ).
Half of the blocks were prepared as nondecalcified specimens. Samples were divided into the same 3 regions, which were dehydrated in alcohol with standard ascending concentration from 70% to 100%, and then embedded in polymethylmethacrylate. First, samples were taken for fluorescent labeling using confocal laser scanning microscopy using an Olympus (Tokyo, Japan) system. The region of interest was located along the maximal buccolingual section of the distal root of the P2. Fluorescent staining for evaluation of new-bone formation was quantified. The number of pixels labeled yellow (TE), green (CA), and red (AL) was determined as a percentage of bone formation area at 2, 4, and 6 weeks (24 hours before the dogs were killed), respectively, after OF application. Mineral apposition rate (MAR; the ratio of the distance between labels to the interlabel period) was calculated and analyzed. Finally, samples were stained with van Gieson’s picrofuchsin for histology. The percentage of newly formed bone and the residual materials in the defect area was quantified from the serial sections collected from each sample using Image-Pro Plus (version 6.0; Media Cybernetics, Rockville, Md).
The other half of the blocks were decalcified with 10% of ethylenediaminetetraacetic acid-2Na (pH 7.4) at 37.8°C for 9 months and dehydrated in ethyl alcohol with gradually increasing concentrations from 70% to 100%. After paraffin embedding, samples were sectioned in the sagittal plane at a thickness of 5 μm. Three sections of the maximal central part of the specimen were selected, stained with hematoxylin and eosin, and examined under a light microscope to evaluate alveolar bone around the experimental tooth and surgical defect.
To measure the expression of bone remodeling markers in the grafted area, we detected type-I collagen (Col-I) and osteopontin (OPN) by immunohistochemistry. For epitope retrieval, sections were deparaffinized and heat-treated with citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes at room temperature, and the tissue nonspecific-binding sites were blocked with 10% goat serum for 15 minutes. Then, sections were incubated with antibodies against anti–Col-I and OPN (1:100 dilution; Abcam, Cambridge, United Kingdom) for 24 hours at 4°C. Then, biotinylated secondary antibodies were added for 30 minutes. Diaminobenzene staining was viewed under the microscope. Counterstaining was carried out with hematoxylin. Immunolabeling of sections with omission of the primary antibody was used as a negative control.
Statistical analyses were undertaken using SPSS (version 18.0; IBM, Armonk, NY). Each variable was measured twice, and a comparison of the second measurement with the first one was done using the Pearson correlation coefficient (>0.98 at the 95% confidence interval). Therefore, the mean value of both datasets (with variables presented as the mean ± standard deviation) was used for further analyses. Normality of the data distribution at the tension side was confirmed using the Shapiro-Wilk test. For comparison between the 3 subgroups, 1-way analysis of variance and the Bonferroni post-hoc test were undertaken. P <0.05 was considered statistically significant.
All sites showed uneventful healing without marked swelling or tissue damage after the surgical procedure.
During 6 weeks of active OF, the mean expansion of P2 was 2.42 mm in group F-0, which was significantly greater than that in group F-4 (1.25 mm) and group F-8 (1.62 mm). All groups had uncontrolled buccal tipping of the P2. The P2 tipped 9.03° ± 1.02°, 5.32° ± 2.19°, and 3.24° ± 1.27° in groups F-0, F-4, and F-8, respectively, with a significant difference between group F-0 and group F-4 ( P <0.05) and group F-0 and group F-8 ( P <0.05) ( Fig 5 ).