A novel experimental model for studying transverse orthodontic tooth movement in the rat mandible
To establish a rat model of a one-piece mandible using the principles of gingivoperiosteoplasty and guided bone regeneration to fuse the midline symphyseal area. Twenty-four Sprague-Dawley female rats were divided into two groups: 12 experimental and 12 control. Both groups were imaged using in vivo micro-computed tomography at baseline and at end point (5 months). The experimental group received regenerative surgery at the symphysis area; the control group received no treatment. Outcomes were evaluated by radiographic examination of gross and volumetric bony changes in the symphyseal region of interest marked between the mental foramina bilaterally and the two central incisors near the most coronal margin of the alveolar crests. These landmarks were chosen as they can be reproduced on the computed tomography images at baseline and end point. Histologic examination was performed on all samples at a level 5 mm apical to the alveolar bone crest. Radiologic and histologic examinations of the experimental group revealed complete bony fusion of the symphyseal area in three subjects, partial fusion in five subjects, and thickening of the alveolar bony socket in three subjects; one rat died of anesthesia-related complications. No evidence of fusion or alveolar bone thickening was found in any of the controls. This surgical animal model demonstrates that a rat mandible can be surgically manipulated to mimic the one-piece human mandible. This novel model may prove useful in studying mandibular bone remodeling and orthodontic mandibular tooth movement.ABSTRACT
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Material and Methods:
Results:
Conclusions:
INTRODUCTION
The skeletal and dental effects of maxillary expansion treatments in humans have been well described.1–4 In addition to alveolar bone remodeling, maxillary expansion is facilitated by remodeling of the midface sutures, in particular the midpalatal suture. In contrast, the mandible is a single bone and orthodontic transverse expansion will likely only occur through tooth movement within the alveolar trough, in the buccolingual direction, and by subperiosteal bone remodeling (SBR). The current theoretical model of orthodontic tooth movement is mainly focused on alveolar bone modeling and remodeling at the periodontal ligament space. No evidence exists to demonstrate SBR at the outer surface of the alveolar bone in response to transverse orthodontic tooth movement.
Sequential micro-computed tomography(micro-CT) of small live animals such as rats allows nondestructive detailed analysis of bone remodeling. Tooth movement and bone remodeling experiments in rats have focused on maxillary bony changes. Few studies have used the rat model to investigate orthodontic tooth movement or bone remodeling in the mandible.5–11 One major limitation is that the mandible of the rat model does not resemble that of the human.7 The rat mandible is two bones linked at the midline with a soft tissue ligament reflected radiologically as a radiolucent area (Figure 1A,B).
Citation: The Angle Orthodontist 83, 5; 10.2319/112512-900.1
Although the rat mandible represents a suitable model for anterior-posterior tooth movement, the presence of the midline suture limits its usefulness for investigating transverse orthodontic tooth movement. The human mandible is a one-unit bony structure and any transverse tooth movement would involve remodeling at the periodontal and periosteal levels only. The presence of the soft tissue at the midline of the rat's mandible may affect the force system applied to posterior teeth in that the supporting bone may pivot or move partially or en masse in response to the applied transverse forces. If the suture were surgically fused, transverse changes due to sutural growth in response to orthodontic forces might be eliminated, isolating treatment effects to bone remodeling around teeth. The principle of gingivoperiosteoplasty (GPP) has been used in the treatment of cleft lip and palate defects in humans12–15 and to investigate bone formation in animals.16 The aim of this study was to use the same principle to generate bone in the symphysis of the rat mandible to create a one-piece mandibular structure.
MATERIALS AND METHODS
Animals
All experimental procedures met the standards of Canadian Council on Animal Care and the study protocol was approved by Animal Care and Use Committee for Health Sciences, University of Alberta. Twenty-four female Sprague-Dawley rats (246–299 g body weight, 6–8 weeks old) were randomly divided into two groups (12 in the experimental group and 12 in the control group) and caged with 12-hour dark and light cycles and fed ad libitum for 2 weeks. All rats were then anesthetized using 2% isoflurane/L and oxygen inhalation and baseline imaging was performed for 30 minutes using high-resolution micro-CT (Skyscan 1076 in vitro Micro CT, Kontich, Belgium).
Surgical Procedures
Experimental subjects underwent surgery using a strict aseptic surgical protocol. All surgical procedures were performed by a certified periodontist familiar with the regenerative procedure used in human periodontal surgeries. The surgical procedures were performed under general anesthesia (isoflurane 2% in oxygen) with local anesthesia (1∶100,000 2% lidocaine hydrochloride with adrenaline) and infiltration at the surgical site to reduce bleeding. A vertical incision using a15-C blade was done between the two mandibular central incisors. The periosteum at the symphyseal region was curetted (scraped) bilaterally using a sharp periodontal sickle scaler. Enamel matrix protein gel (Emdogain gel, Straumann, Burlington, Ontario, Canada) was then injected into the incised area to facilitate bone regeneration and wound healing.
The area was covered with a synthetic membrane (Atrisorb Gel, Citagenix Inc, Laval, Quebec, Canada) to prevent soft tissue growth into the symphyseal region during healing. The flap was sutured with chromic gut suture (6-0) and cyanoacrylate surgical adhesive (periacryl) was used to facilitate hemostasis. To ensure immobility and promote clot stability, the mandibular incisors were etched (35% orthophosphoric acid) and bonded together with composite resin. Postoperative analgesia through Metacam injection (0.6 mL per rat) (Metacam (meloxicam), Boehringer Ingelheim Vetmedica, Inc, St. Joseph, MO) was given subcutaneously. All animals except one (which died of anesthesia-related complications during surgery) tolerated the procedure and had no adverse conditions during the recovery period, other than initial weight loss, which was recovered in subsequent weeks. Animals received a soft diet for the first 2 postoperative weeks and were housed for 5 months.
Micro-CT Analysis
On day 150, all 23 rats were anesthetized with generic isoflurance for 35 minutes and imaged according to an identical protocol used at baseline. Images were examined using vendor-supplied software (CT-analyzer, Skyscan NV, Kontich, Belgium) to determine the total bone volume changes in order to detect the amount of bone formed in the symphysis region in each subject during the study period. Micro-CT images were binarized with a grayscale threshold of 40/255 and a pixel size of 17.16 µm. Two independent observers marked the region of interest (ROI) (Figure 1C).
Histology
Rats were then euthanized, body weights were recorded, and the decapitated heads were fixed in 10% buffered formalin acetate (Fisher Scientific, Ottawa, Ontario, Canada) for 1 week (with multiple changes of the solution). The mandibles were dissected, taking extra care not to twist the symphysis area, and decalcified using formic acid (Cal-Ex II, Fisher Scientific). The solution was changed every 24 hours for the first 3 days then once a week for 3 weeks. Frontal sections were obtained at the level of 5 mm apical to the most coronal margin of the alveolar bone between the two central incisors (Figure 1D). The samples were paraffin embedded and sectioned at 5 µm; the mounted slides were stained with hematoxylin and eosin. The specimens were examined under light microscopy to detect the evidence of new bone formation in the ROI in the animals in the experimental group compared with those in the control group.
Statistical Analysis
Statistical analyses were conducted using the SPSS statistical software (version 13.0, SPSS, IBM, Armonk, NY). For the quantitative measures of body weight and bone volume, unpaired t-tests were used to compare groups. A P value < .05 was considered significant.
RESULTS
General Observations and Body Weight
Rats in both groups were able to eat and function normally and gained an average of 110 g during the study period (Table 1). One rat showed lack of attrition of both of the mandibular incisors; however, there was no obvious body weight loss. No facial deformity was detected and all the rats had similar facial features at the 5-month period. Comparing the end point average weight gain between the two groups, there was no statistical significant difference (P < .05) (Table 1). This suggested that the surgical procedure and the fusion of the symphyseal area had no adverse effect on masticatory function.
Micro-CT Radiologic Examination
The control group demonstrated consistent evidence of a translucent area between the two halves of the mandible, coinciding with the soft tissue ligament suture (Figure 2A–C). The experimental group demonstrated three distinct patterns of new bone formation: (1) three subjects had through and through bony union in at least one region of the symphysis in any given level of the micro-CT sections (Figure 3A–C); (2) five subjects demonstrated partial union, which was defined as evidence of a dovetail pattern of radiopaque bony structures in areas that would normally have been radiolucent (Figures 3D–H); and (3) three subjects showed thickening of the alveolar bone around the two central incisors with no evidence of union or the dovetail bony pattern (Figures 3I–K). In all three patterns the teeth in the experimental group showed some evidence of rotation within the bony sockets compared with the control group.
Citation: The Angle Orthodontist 83, 5; 10.2319/112512-900.1
Citation: The Angle Orthodontist 83, 5; 10.2319/112512-900.1
Histologic Examinations
In the control group, the symphysis areas consisted of a dense, interwoven meshwork of collagen fibers (Figure 4A,B). No evidence of cartilaginous or bone remodeling was detected. The experimental group demonstrated the three distinct patterns of bone response described earlier. First, in three subjects complete bridging of the symphysis area was evidenced by new bone formation between the two halves of the hemimandible. The absence of the soft tissue fibers in the symphysis region was clear evidence of bone formation compared with the control group (Figure 4C,D). Second, in five subjects partial fusion of the symphyseal area was seen through bony union at the basal area of the mandible, and there was further evidence of bone formation at the coronal area. The latter may have been an outcome of post-union bone remodeling (Figure 4E,F). Third, three subjects showed thickening of the alveolar bone socket wall in the midline region (Figure 4G,H). No evidence of root resorption was detected in any of the subjects.
Citation: The Angle Orthodontist 83, 5; 10.2319/112512-900.1
Volumetric Bony Changes
Micro-CT results measured significantly (P < .05) increased bone volume in the ROI (from baseline to the end point) in both groups. The bone volume increase was significantly greater (P < .05) in the experimental group than in the control group (Table 2).
DISCUSSION
The elucidation of bone remodeling in response to orthodontic tooth movement in humans is constrained by the need to conduct those evaluations using noninvasive imaging procedures. Cone beam computed tomography is widely used in orthodontics and research. Unfortunately, the poor nominal resolution of conventional cone beam computed tomography is inadequate to accurately study how bone remodeling affects tooth movement. Despite the availability of higher-resolution units, radiation safety prohibits their application to humans.
Rats provide a cost-effective research model for several types of orthodontic interventions. However, the presence of a midline mandibular suture limits the use of rats to study the response to transverse orthodontic forces. GPP (scraping the periosteum in the midline region) seems to trigger the required cellular response in the area of the rat symphysis through which new bone formation can occur. GPP must recruit some bone mesenchymal stem cells from the periosteum, circulating blood, or vascular pericytes at the site. Moreover, three other factors might be of particular importance in facilitating new bone formation. First, according to Merzel and Salmon,17 enamel-related periodontium is an embryonic tissue that acts as a source of the osteogenic cells required for the process of bone remodeling during the continuous eruption of the rodent's incisor teeth. That tissue may have served as a source for osteogenic cells and cytokines as a result of the mechanical scraping of the area during the surgery. Second, the existence of cartilaginous cells in the symphyseal region of rats18,19 would theoretically be a precursor for endochondral ossification. Third, the use of Emdogain in this experiment may have had a major role in stimulating local osteogenic cells to lay down genuine bone to fuse the symphysis. Emdogain is an osteoinductive material extracted from porcine teeth and has been used in human guided bone regeneration surgeries to facilitate the osteogenic potential of periodontal and bone cells. The principle of applying Emdogain in this study differs from that used in other studies as no physical barrier was used to contain it in the region.
Although some studies have used the guided bone regeneration principle to form new bone outside the bony envelope of the mandible (on the surface of the ramus)16 or within the jaw in an artificially made bone defect,20 this study aimed to form new bone within the confines of the normal anatomy that is typically devoid of bone. Both models resulted in new bone formation in response to the application of Emdogain. Whether or not the bone would form in the symphysis area in response to the scraping of the periosteum without the use of Emdogain, remains to be investigated.
In this study, the GPP would likely have served as the stimulus for cellular migration to the site, triggering new bone formation. Although the surgical technique was blinded in that no direct visibility of the site was possible lingually because of the smaller anatomy of the rat mandible; nonetheless, it was anticipated that the alveolar mucosa acted as a barrier to maintain the Emdogain at the site. Otherwise, the effect of Emdogain would be limited by the fact that the animals would swallow the gel or spit it out. The immobilization of the incisor teeth through bonding was used as an adjunct to stabilize the clot at the site by splinting the hemimandible during healing, which may have helped initiate and maintain the osteogenic cellular activity. Although not specifically recorded, we observed that the splinting survived varying lengths of time. The duration of rigid splinting may have influenced the amount of bone formation witnessed at the end point, given the three phenotypically distinct outcomes; however, it was difficult to determine in vivo the timing of the loss of the fixation.
One of the rats that showed complete fusion also showed continued teeth eruption; this might feasibly occur because of a longer period of immobilization, which may have resulted in the absence of secondary bone remodeling in the midline region. Three rats in the experimental group maintained the bony fusion at the midline at the end point, eight rats showed evidence of interlocking pattern of bone from either side of the midline or complete splitting of the newly formed bone, and of these, three showed evidence of thickening of the bony socket walls. The latter may indicate that the process of secondary bone remodeling resumed after the fusion to open up the suture, or that complete fusion did not occur as the rats continued to function, which may have created a flexing moment at the suture. Rats with partially fused mandibles may still serve as effective animal models for studying mandibular transverse tooth movement as such fusion may prevent the independent movement of the hemimandible.
In summary, changes in bone volume were significantly greater in the experimental group than the control group. The resulting bony union was formed with genuine bone at the symphysis region, which overwhelmed the genetic predisposition of the intervening soft tissue. Further research is required to determine the minimum period of time required and to better control splinting of the hemimandible and other related factors needed for reliable complete bone fusion to occur in the symphysis using this technique.
CONCLUSIONS
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Genuine bone formation is induced in the rat symphysis region with GPP and guided tissue regeneration surgical techniques.
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The rat mandible can be modified to become a one-piece mandible. This novel animal model may enhance the future study of orthodontic tooth movement and bone remodeling in response to transverse forces in the mandible.
(A,B) The Sprague-Dawley rat hemimandible is divided into two pieces joined together by symphyseal soft tissue ligament. (C) Region of interest (ROI) marked as being the bony structure, excluding the tooth material, which extends from the level of the mental foramina bilaterally to the most crestal bone between the two rat central incisors. (D) The decalcified specimens were sectioned 5 mm apical to the level of the most coronal alveolar bone. Six sections were obtained from each side of the sample 5 µm apart (the arrows).
Control group: Coronal section at the most coronal region of the bony socket of the central incisors showing consistent radiolucent area at the symphysis at (A and B) baseline and (C) end point.
(A–C) Experimental group: Complete fusion occurred at different levels (white arrows). (D–H) Experimental group: New bone formation in the symphyseal area as evidenced by the dovetail pattern of the symphyseal joints not consistent with that seen in the control group. The alveolar bony socket walls are also thicker at the symphyseal side when compared at the same coronal level of the control subjects. (I–K) Experimental group: Thickening of the bony socket wall between the two central incisors compared with the control group.
(A and B) Control group: Absence of any bone or cartilage formation in the symphysis region of the hemimandible. Soft tissue ligament (arrow) separating the two sides of the hemimandible. (C and D) Experimental group: Evidence of bone bridging (arrow between the two sides of the hemimandible). (E–F) Experimental group: Complete bone union at the crestal area (arrow) with irregular bone formation at the rest of the symphyseal region. (G and H) Experimental group: Thickening of the alveolar bony walls around the incisor teeth with clear demarcation between the new and mature alveolar bone.
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