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Induced Premaxillary Suture Fusion: Class III Malocclusion Model
1 Department of Stomatology, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China; Correspondence: * corresponding author, jborke{at}mail.mcg.edu
The etiology of class III malocclusion remains unknown. The present study investigates the relationship between craniofacial morphology and premaxillary suture fusion to test the hypothesis that class III malocclusion may be related to premaxillary suture fusion. Cyanoacrylate was applied to immobilize the left premaxillary suture in the experimental group. Sham surgeries in rats were used for controls. Dental impressions and radiographs were taken before and after surgery for comparison of craniofacial differences between groups. Overall cranial base lengths, craniofacial widths, and craniofacial angulations related to the anterior base showed significant differences between groups. At the end of the experiment, the growth of the snout in the experimental group was inhibited and deviated to the treated side, while no obvious change was seen in the control group. The results show that induced premaxillary suture fusion can affect craniofacial morphology and indicate that premature premaxillary suture fusion may result in class III malocclusion.
Key Words: animal model • artificial premaxillary suture fusion • class III malocclusion • craniofacial morphology
Class III malocclusion is common in children, especially Asian children (Mak, 1969). This malocclusion has distinctive features, such as concave midface and prognathism. Conventional cephalometry shows that the anterior cranial base length is shorter in class III persons when compared with their class I counterparts (Chan, 1974; Kerr and Adams, 1988). Singh et al.(1997), however, implied that developmental deficiency of the posterior cranial base could be associated with the development of class III malocclusion. Animal studies suggest that midfacial concavity is due to growth deficiencies in the posterior region of the anterior cranial base (Lozanoff et al., 1994; Ma and Lozanoff, 1996). Cephalometric studies suggest that, in children with this malocclusion, an acute basal angle exists that can push the mandible forward (Ellis and McNamara, 1984; Baccetti et al., 2004). Many researchers have shown that premature synostosis of the cranial sutures affects craniofacial morphology (Ulgen et al., 1996; Stelnicki et al., 1998; Itoh, 2000). In one rat study, unilateral artificial synostosis of the frontonasal and frontopremaxillary sutures caused the snout to bend toward the treated side (Xenakis et al., 1995). Currently, no reports show a relationship between premaxillary suture fusion and craniofacial morphology. Human premaxillary suture fusion begins around birth; however, the labial side is the only area fused at that time. The other suture areas remain patent much later (Singh, 1999). Thus, some authors suggest that class III malocclusion can be treated by pushing this bone block forward (Haskell and Farman, 1985). This study was designed to describe the craniofacial morphology changes produced by artificial fusion of the premaxillary suture with cyanoacrylate, and to test our hypothesis that class III malocclusion may be related to premaxillary suture fusion.
Thirty female Sprague-Dawley rats, aged 3 wks (Harlan, Indianapolis, IN, USA), were evenly divided into 2 groups, an experimental and a control. The groups were further separated into 3 subgroups (n = 5) according to different time periods, 3 wks, 5 wks, or 8 wks. The animal protocol was reviewed and approved by the Committee for Care and Use of Laboratory Animals at the Medical College of Georgia. Before surgery, all animals received intramuscular injections of an anesthetic cocktail with ketamine hydrochloride, xylazine, and acepromazine at a dosage of 0.4 to 0.6 mL/kg.
All rats received a 1- to 2-cm incision along the left premaxillary suture (Fig. 1
Weights, dental impressions, and radiographs were taken pre-operatively and post-operatively weekly for both groups under general anesthesia, except for the eight-week subgroups, which excluded measurements at the 5th and 7th wks. We used Superfast Regisil® Rigid polyvinylsiloxane (Dentsply, Milford, DE, USA) to take dental impressions of each maxilla. We used a Faxitron Series 43807N soft x-ray system (Hewlett-Packard, Palo Alto, CA, USA) and Kodak Insight dental film (Rochester, NY, USA) to take lateral and dorsoventral cephalograms. Soft x-rays were taken for 5 sec at 2.0 milliamperes and 45 Kvp. The head of each animal was fixed with a custom-made frame oriented vertically to the median sagittal plane.
Preparation of Sections
Histology and Immunohistochemistry
Morphometric Analysis
Statistical Analyses Because the craniofacial structure displays three-dimensional interconnectivity, several correlated measurements were merged into specific functional groups to illustrate the changes of each function unit as a whole (Fig. 2  ). For example, the anterior cranial base length (E-S), the posterior cranial base length (S-Ba), and the total cranial base length (E-Ba) were grouped to form the functional unit of the cranial base (Figs. 2A, and Appendix Figs. 2A and 2B). A description of the contributing measurements of each of these functional units can be found in the online APPENDIX. Because these are complex units, we used multivariate analysis of variance (MANOVA) to determine the growth differences of the functional groups between the control and experimental groups, and reported these differences as a comparison of the areas under the curves across the different timepoints. Wilks’ lambda test was also used as the appropriate test, in conjunction with MANOVA, to test whether there were differences between the means of control and experimental groups related to the combination of dependent variables. Group differences were also investigated between measurements at the 8th week. Areas under the curves (lines) connecting time zero and 8 wks were also compared to correct for initial variability between individual animals. The one exception was the measurement of differences in midfacial height (N-Mu). This was the only measurement in this functional unit, and therefore a two tailed t test was used for this analysis (Appendix Figs. 2A, 2B). Repeated measurements were performed on lateral and prone radiographs and impressions from 10 rats. The paired t test was used to compare the series of measurements to test for systematic error, and no statistically significant differences were found (p < 0.05). The Dahlberg coefficient was used to evaluate the method error (Bister et al., 2002). The range of Dahlberg values for all 28 measurements was from 0.05 mm to 0.65 mm, while the range for angular measures was 0.78° through 3.59°. All statistical analyses were performed with SAS software 9.1.3 (SAS Institute, Cary, NC, USA), and statistical significance was assessed at 5%.
Rat body weights increased continuously, without significant differences between groups.
Morphological Changes
At the 8th week timepoint (Appendix Fig. 2B), only the maxillary region displayed a statistically significant effect (p = 0.0287). The control group had higher overall areas under the curve for maxillary lengths than did the experimental group, with the maxillary length (Bu-Mu) being significantly different for the individual tests (p = 0.0065). The individual comparisons, however, showed a significant difference for measurements of the anterior base length (E-S), the upper incisor edge position (S-Iu), the alveolar ridge position of the upper incisor (S-Bu), the left dental arch length (I-FMp1), and the zygomatic width (Z1-Z2) (Appendix Fig. 2B). Also significant was the premaxillary inclination (Pm-Bu/S-E) (Appendix Fig. 3B). The experimental rat snouts had a significantly higher deviated angle (1.51 ± 0.65) toward the treated side than did the controls (–0.79 ± 0.68, p < 0.0001, t test, Figs. 4A, 4B
The growth of the dental arch was inhibited after immobilization of the unilateral premaxillary suture (Appendix Fig. 2C). Interestingly, both sides of the dental arch length were shorter in the experimental groups than in the sham groups from week 4. This result indicates that overall maxillary growth could be inhibited, even though only one side of the premaxillary suture was immobilized. Compared with the untreated side, the dental arch of the treated side for the control group did not show a significant difference, while statistical differences existed for the experimental group. At the end of the experiment, the average absolute increase in length for the control group (n = 5) on the left side was 4.87 mm, but only 3.94 mm for the experimental group (n = 5). Therefore, the lag growth rate was –19.10% for the experimental arch dental growth. In addition, the absolute length increase for the control on the right side was 4.85 mm, but only 4.11 mm for the experimental group. Thus, the lag growth rate was –15.26%. It is obvious that the treated side was inhibited more than the untreated side, which induced the snout deviation toward the treated side (Figs. 4A, 4B ). The same trend existed for the growth of premaxillary bone and palatal bone during the experiment. Like the palatal bone, premaxillary bone length for the experimental group (n = 5) was significantly shorter than that of the controls (n = 5) from the 4th wk until the end of the experiment (p = 0.032 at the 4th wk, p = 0.036 at the 6th wk, and p = 0.024 at the 8th wk).
Histological and Immunohistochemical Findings
Many studies have shown that the premaxilla and premaxillary suture are related to the development of the human face (Barteczko and Jacob, 2004). This is further supported by the observation that the premaxillary area was malformed in all holoprosencephalic fetuses (Kjær et al., 1991). This is also demonstrated by the present study, which shows that craniofacial morphology is altered after immobilization of the premaxillary suture. Compared with controls, the overall areas under the curve for the experimental cranial base lengths were significantly smaller. In addition, the overall areas under the curve for the angle related to the anterior base revealed statistically significant differences between the groups. These results showed a significant additive effect for these constituents. Some researchers found shorter anterior base lengths (Chan, 1974; Kerr and Adams, 1988) or shorter posterior base lengths (Singh et al., 1997) in persons with class III malocclusion. Our results show that the premaxillary suture perturbation can induce changes in both the anterior and the posterior cranial base. Thus, abnormal premature fusion of the premaxillary suture region could produce global and interconnective craniofacial dysmorphogenesis. Like premature cranial suture fusion (Xenakis et al., 1995; Ulgen et al., 1996; Stelnicki et al., 1998; Itoh, 2000), premaxillary suture fusion can also affect the local area growth. The growth of the snout was obviously inhibited 8 wks after surgery on the treated side of the experiment group. After the suture was glued, the palatal and premaxillary lengths became significantly shorter, and the dental arch length of the treated side at the 8h wk shortened, resulting in a left-deviated snout. Therefore, the immobilization of the premaxillary suture can inhibit the development of the palate and the premaxilla and give rise to a reduced length of the corresponding bone, which concurs with class III malocclusion, where the maxilla usually experiences hypoplasia. Presumably, the potential for osteogenesis of the premaxillary suture is lessened after immobilization. The current study did not show a significant difference in the midfacial height (N-Mu) across the entire experimental period. This observation differed from that of Sims et al.(1996), who found abnormal cranial vertical height expansion, with no different horizontal cranial width after fusion of the unilateral coronal sutures. For the experimental group, gross observations showed obvious inhibition of palatal and premaxillary development and a left-deviated snout at the 8th wk. The routine H&E staining showed that, after 8 wks, new bone tissue was forming in the premaxillary suture for the experimental rats, while the control illustrated no new bone and regular suture shape. Histological images from the earlier three- and five-week timepoints were highly variable and are not shown. Presentation of a single representative image from these timepoints would be misleading, since a few images from the 5th wk show areas of focal fusion, but most at 3 and 5 wks do not. At the 8th wk, however, the fusion of sutures and the formation of bone bridges were consistent features. However, even at 8 wks, the histology and immunohistochemistry showed fusing, rather than completely fused sutures. The absence of a completely fused suture may be explained by the use of older rats (with a rate of bone formation slower than in younger rats), or a short observation period of only 8 wks, or both. In summary, applying cyanoacrylate to the premaxillary suture can result in global craniofacial morphological changes in growing rats. These changes are similar to changes associated with class III malocclusion and imply that abnormal premaxillary suture function can cause class III malocclusion.
This study was supported by the Departments of Oral Biology and Surgery of the Medical College of Georgia. We also thank Dentsply for supplying the impression material.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/9/856/DC1. Received for publication October 16, 2006. Revision received May 1, 2008. Accepted for publication May 30, 2008.
Journal of Dental Research, Vol. 87, No. 9,
856-860 (2008)
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