Effects of the unilateral removal and dissection of the masseter muscle on the facial growth of young rats

This study analyzed the effects of the unilateral removal and dissection of the masseter muscle on the facial growth of young rats. A total of 30 one-month-old Wistar rats were used. Unilateral complete removal of the masseter muscle was performed in the removal group, and detachment followed by repositioning of the masseter muscle was performed in the dissection group, while only surgical access was performed in the sham-operated group. The animals were sacrifi ced at three months of age. Axial radiographic projections of the skulls and lateral projections of the hemimandibles were taken. Cephalometric evaluations were made and the values obtained were submitted to statistical analyses. In the removal group, there were contour alterations of the angular process, and a signifi cant homolateral difference in the length of the maxilla and a signifi cant bilateral difference in the height of the mandibular body and the length of the mandible were observed. Comparison among groups revealed signifi cance only in the removal group. It was concluded that the experimental removal of the masseter muscle during the growing period in rats induced atrophic changes in the angular process, as well as asymmetry of the maxilla and shortening of the whole mandible. Descriptors: Masseter muscle; Maxillofacial development; Growth and development. Lucimar Rodrigues(a) Andréia Aparecida Traina(a) Luiz Felipe Nakamai(b) João Gualberto de Cerqueira Luz(c) (a) MS, Doctorate Student; (b)Graduate Student; (c)Associate Professor – Department of Maxillofacial Surgery, Prosthodontics and Traumatology, School of Dentistry, University of São Paulo. Surgery Corresponding author: João Gualberto de Cerqueira Luz Depto. de Cirurgia, Faculdade de Odontologia, Universidade de São Paulo Av. Prof. Lineu Prestes, 2227 Cidade Universitária São Paulo SP Brazil CEP: 05508-000 E-mail: jgcluz@usp.br Received for publication on 18 Sep, 2007 Accepted for publication on 21 Jan, 2008 Effects of the unilateral removal and dissection of the masseter muscle on the facial growth of young rats Braz Oral Res 2009;23(1):89-95 90 Introduction The dynamics of normal postnatal growth of the craniofacial skeletal system is incompletely understood.1 Studies have experimentally demonstrated the effect of bony or cartilaginous lesions on facial growth. Unilateral resection of the mandibular condyle induced severe facial asymmetry.2,3 Resection of the cartilaginous nasal septum promoted severe facial deformity.4 Also, osteotomies that simulated a mandibular fracture in the growing period had as consequences asymmetries of the maxilla and the mandible,5 or lessening of the mandible.6 It has been demonstrated that the masticatory muscle function is a determinant of the quality of bone of the growing mandible.7 The elevator muscles infl uence the transversal and the vertical dimensions of the face.8 Bilateral resections of the masseter and temporal muscles induced deformities in the mandible which were related to the amount of tissue removed.9 An open bite occurs in rats after bilateral removal of the masseter.10 Also, unilateral masticatory function caused asymmetric craniofacial growth.11 Surgical procedures on the mandible are frequently carried out with muscle detachment. The reattachment process includes superfi cial new bone formation.12 Experimental alterations of the masseter muscle length can change the gonial region.13 However, the role of the musculature in the process of bone formation is not well understood.14 The purpose of this study was to analyze the effects of the unilateral removal and dissection of the masseter muscle on the facial growth of young rats. Materials and Methods The study animals were 30 one-month-old female Wistar rats. All animals were fed an ordinary diet of rodent feed (Labina, Agribands Purina, Paulínia, SP, Brazil), and water. They were distributed into removal (n = 10), dissection (n = 10), and sham-operated (n = 10) groups. Under general anesthesia, induced by xylazine hydrochloride (Rompum, Bayer, Porto Alegre, RS, Brazil), 10 mg/kg of body weight, and ketamine hydrochloride (Ketalar, Parke-Davis, Rio de Janeiro, RJ, Brazil), 25 mg/kg of body weight, the right side was shaved and cleansed with a povidone-iodine solution (Riodeíne, Rioquímica, São José do Rio Preto, SP, Brazil). A submandibular incision of 1 cm was made, followed by exposure of the masseter muscle. The removal group was submitted to complete resection of the masseter muscle. The dissection group was submitted to subperiosteal complete elevation of the masseter muscle, followed by repositioning of this structure. The sham-operated group was submitted to exposure of the masseter muscle. The procedures were concluded by suturing in layers. The animals were sacrifi ced at three months of age. The specimens were evaluated macroscopically. Their heads and mandibles were macerated. Following formalin fi xation, radiographs in axial projections of the skull and lateral projection of the hemimandibles were obtained. These were taken with a dental X-ray machine (Spectro II, Dabi-Atlante, Ribeirão Preto, SP, Brazil) at 56 kV and 10 mA, with an exposure time of 0.4 s for the skull and 0.3 s for the hemimandibles. Periapical fi lms were used (Ektaspeed Plus, Eastman Kodak Co, Rochester, NY, USA). The radiographs were subjected to computerized cephalometric evaluation. They were digitized using an optical reader (Fotovix II, Tamron Co., Tokyo, Japan). Measurements were obtained with Imagelab software (Softium Informática Ltda., São Paulo, SP, Brazil). Using the skull radiographs, the following distances were measured bilaterally: TB-MR tympanic bulla to the mesial root of the fi rst molar; TBIF tympanic bulla to infraorbital foramen; and IFIP infraorbital foramen to incisal point (Figure 1). On the radiographs of the hemimandibles, the following distances were measured bilaterally: CP-AP condylar process to angular process; TM-AN distal face of the third molar to antegonial notch; II-CP lower insertion of incisor to condylar process; and II-AP lower insertion of incisor to angular process (Figure 2). To evaluate the differences between the mean values from the right and left sides in each group, the paired Student’s t-test was used, whilst the analysis of variance (ANOVA) and Tukey’s tests were used for the mean values of the three groups. The level of signifi cance was set at 5% (p ≤ 0.050). Rodrigues L, Traina AA, Nakamai LF, Luz JGC Braz Oral Res 2009;23(1):89-95 91 Results Macroscopic examination of the specimens revealed facial asymmetry in the removal group, with deviation of the mandible to the right side, and discrete asymmetry in the dissection group. Contour alterations with irregularities and signs of atrophy of the angular process were noted only in the removal group (Figure 3). The mean values of the distances found on the radiographs of the skulls of the removal, dissection and sham-operated groups are listed in Table 1. In the removal and dissection groups there was a signifi cant difference between sides for all measurements: tympanic bulla to mesial root of the fi rst molar (TB-MR), tympanic bulla to infraorbital foramen (TB-IF) and infraorbital foramen to incisal point (IF-IP). However, in the sham-operated group there was no signifi cant difference between sides for all measurements. Figure 2 Computerized cephalometric landmarks used in a lateral radiograph of the hemimandibles of a specimen from the removal group. CP = Condylar process, AP = Angular process, II = Lower insertion of incisor, AN = Antegonial notch, TM = Distal face of the third molar. II AN TM


Introduction
The dynamics of normal postnatal growth of the craniofacial skeletal system is incompletely understood. 1 Studies have experimentally demonstrated the effect of bony or cartilaginous lesions on facial growth.Unilateral resection of the mandibular condyle induced severe facial asymmetry. 2,3Resection of the cartilaginous nasal septum promoted severe facial deformity. 4Also, osteotomies that simulated a mandibular fracture in the growing period had as consequences asymmetries of the maxilla and the mandible, 5 or lessening of the mandible. 6t has been demonstrated that the masticatory muscle function is a determinant of the quality of bone of the growing mandible. 7The elevator muscles infl uence the transversal and the vertical dimensions of the face. 8Bilateral resections of the masseter and temporal muscles induced deformities in the mandible which were related to the amount of tissue removed. 9An open bite occurs in rats after bilateral removal of the masseter. 10Also, unilateral masticatory function caused asymmetric craniofacial growth. 11urgical procedures on the mandible are frequently carried out with muscle detachment.The reattachment process includes superfi cial new bone formation. 12Experimental alterations of the masseter muscle length can change the gonial region. 13owever, the role of the musculature in the process of bone formation is not well understood. 14he purpose of this study was to analyze the effects of the unilateral removal and dissection of the masseter muscle on the facial growth of young rats.

Materials and Methods
The study animals were 30 one-month-old female Wistar rats.All animals were fed an ordinary diet of rodent feed (Labina, Agribands Purina, Paulínia, SP, Brazil), and water.They were distributed into removal (n = 10), dissection (n = 10), and sham-operated (n = 10) groups.
Under general anesthesia, induced by xylazine hydrochloride (Rompum, Bayer, Porto Alegre, RS, Brazil), 10 mg/kg of body weight, and ketamine hydrochloride (Ketalar, Parke-Davis, Rio de Janeiro, RJ, Brazil), 25 mg/kg of body weight, the right side was shaved and cleansed with a povidone-iodine solution (Riodeíne, Rioquímica, São José do Rio Preto, SP, Brazil).A submandibular incision of 1 cm was made, followed by exposure of the masseter muscle.The removal group was submitted to complete resection of the masseter muscle.The dissection group was submitted to subperiosteal complete elevation of the masseter muscle, followed by repositioning of this structure.The sham-operated group was submitted to exposure of the masseter muscle.The procedures were concluded by suturing in layers.The animals were sacrifi ced at three months of age.The specimens were evaluated macroscopically.Their heads and mandibles were macerated.Following formalin fi xation, radiographs in axial projections of the skull and lateral projection of the hemimandibles were obtained.These were taken with a dental X-ray machine (Spectro II, Dabi-Atlante, Ribeirão Preto, SP, Brazil) at 56 kV and 10 mA, with an exposure time of 0.4 s for the skull and 0.3 s for the hemimandibles.Periapical fi lms were used (Ektaspeed Plus, Eastman Kodak Co, Rochester, NY, USA).
The radiographs were subjected to computerized cephalometric evaluation.They were digitized using an optical reader (Fotovix II, Tamron Co., Tokyo, Japan).Measurements were obtained with Imagelab software (Softium Informática Ltda., São Paulo, SP, Brazil).Using the skull radiographs, the following distances were measured bilaterally: TB-MR -tympanic bulla to the mesial root of the fi rst molar; TB-IF -tympanic bulla to infraorbital foramen; and IF-IP -infraorbital foramen to incisal point (Figure 1).On the radiographs of the hemimandibles, the following distances were measured bilaterally: CP-AP -condylar process to angular process; TM-AN -distal face of the third molar to antegonial notch; II-CP -lower insertion of incisor to condylar process; and II-AP -lower insertion of incisor to angular process (Figure 2).
To evaluate the differences between the mean values from the right and left sides in each group, the paired Student's t-test was used, whilst the analysis of variance (ANOVA) and Tukey's tests were used for the mean values of the three groups.The level of signifi cance was set at 5% (p ≤ 0.050).

Results
Macroscopic examination of the specimens revealed facial asymmetry in the removal group, with deviation of the mandible to the right side, and discrete asymmetry in the dissection group.Contour alterations with irregularities and signs of atrophy of the angular process were noted only in the removal group (Figure 3).
The mean values of the distances found on the radiographs of the skulls of the removal, dissection and sham-operated groups are listed in Table 1.In the removal and dissection groups there was a signifi cant difference between sides for all measurements: tympanic bulla to mesial root of the fi rst molar (TB-MR), tympanic bulla to infraorbital foramen (TB-IF) and infraorbital foramen to incisal point (IF-IP).However, in the sham-operated group there was no signifi cant difference between sides for all measurements.

IF IF MR MR TB TB IP IP
In the comparison of the maxillary measurements among groups, the ANOVA demonstrated that there were differences in two measurements: TB-IF on the right side and IF-IP on the right side (Table 2).With the use of the Tukey test, the following signifi cances between groups were found: TB-IF on the right side and IF-IP on the right side in the removal versus sham-operated groups.
The mean values of the distances found on the radiographs of the hemimandibles of the removal, dissection and sham-operated groups are listed in Table 3.In the removal group there was a signifi cant difference between sides in lower insertion of incisor to angular process (II-AP), but not in the remaining measurements.In the dissection group there was a signifi cant difference between sides in lower insertion of incisor to condylar process (II-CP), and lower insertion of incisor to angular process (II-AP), but not in the remaining measurements.However, in the sham-operated group there was no signifi cant difference between sides for all measurements.
In the comparison of the mandibular measurements among groups, the ANOVA demonstrated that there were differences in most measurements: TM-AN on the right side, TM-AN on the left side, II-AP on the right side, II-AP on the left side, II-CP on the right side, and II-CP on the left side (Table 4).Using the Tukey test, the following signifi cances between groups were found: TM-AN on the right side in the removal versus dissection groups, TM-AN on the right side in the removal versus sham-operated groups, TM-AN on the left side in the removal versus dissection groups, TM-AN on the left side in the removal versus sham-operated groups, II-AP on the right side in the removal versus sham-operated groups, II-AP on the left side in the removal versus sham-operated groups, II-CP on the right side in the removal versus sham-operated groups, II-CP on the left side in the removal versus dissection groups, and II-CP on the left side in the removal versus shamoperated groups.

Discussion
The present study has shown that the removal of  the masseter muscle in growing rats induced shortening of the mandible with consequences to the maxilla.Thus, the fi nal result was a shortened mandible associated with an atrophic angular process on the right side.The cephalometric evaluations also identifi ed asymmetric areas in the maxilla.On the other hand, some asymmetry in the length of the maxilla and mandible was found when comparing sides in the detachment of the masseter muscle, but not among groups.Mandibular asymmetries have been related to the experimental unilateral removal of the masseter muscle. 15,16Undersized mandibles are associated with bilateral resections of the masseter muscle. 9,10owever, this study detected a shortening of the whole mandible, probably due to a refi ned mensuration method and to the statistical analyses used.Another signifi cant fi nding was a shortening of the maxilla.The possibility of maxillary growth infl uencing mandibular growth and contrariwise by occlusal intercuspation has been described. 17Occlusal disturbances were considered the main cause of asymmetry of the midface after experimental mandibular fractures. 5,18Gross modifi cations in the form of the mandible occurred after resections of the masseter and temporal muscles in the young rat, supporting the functional matrix theory of the mandibular growth. 9,19Similar fi ndings are reported after the denervation of the masseter muscle. 20,21How-ever, the possibility that the removal of the muscles from the mandible eliminates the blood supply to the regions of insertion may be considered. 22ephalometric evaluations from radiographs of dissected specimens using a computer system lead to reliable measurements. 23The distances used in this study were similar to those used in other experiments. 23,24The distances shared signifi cant differences between sides in the dissection group, but not among groups.This may be attributable to the    detachment followed by repositioning of the masseter muscle.The possibility of a decrease in growth caused by the periosteal lesion should be considered. 25The effects of muscle reattachment comprise new bone formation to envelop the reorganizing tendon. 12Similar fi ndings were observed after transposition of the masseter muscle. 26ontour alterations of the angular process in the removal group were another important fi nding.After unilateral masseterectomy in the young rat, the angular process became blunt. 15,16Similar alterations on the angular process occurred after denervation of the masseter muscle. 20A study of the changes in the gonial region induced by alterations of the masseter muscle length concluded that blood supply and function infl uence the shape of the gonial region. 13

Conclusion
The present experimental study demonstrated that the unilateral removal of the masseter muscle in the growing period induced atrophic changes in the angular process, asymmetry of the maxilla, and shortening of the mandible.Dissection of this muscle induced asymmetry of the maxilla and the mandible which were not signifi cant when comparing among groups.

Figure 2 -
Figure 2 -Computerized cephalometric landmarks used in a lateral radiograph of the hemimandibles of a specimen from the removal group.CP = Condylar process, AP = Angular process, II = Lower insertion of incisor, AN = Antegonial notch, TM = Distal face of the third molar.

Figure 3 -
Figure 3 -Macroscopic view of the hemimandibles of a specimen from the removal group with atrophy, contour alterations and irregularities of the right (R) angular process.L = left side (control).

Figure 1 -
Figure 1 -Computerized cephalometric landmarks used in an axial radiograph of the skull of a specimen from the removal group.IP = Incisal point, IF = Infraorbital foramen, MR = Mesial root of the first molar, TB = Tympanic bulla.

Table 1 -
Mean values (mm) of the distances found on the axial radiographs of all the groups and significance of the paired Student's t-test.

Table 2 -
Significance of the Analysis of Variance of the measurements from the axial radiographs.

Table 4 -
Significance of the Analysis of Variance of the measurements from the lateral radiographs of the hemimandibles.

Table 3 -
Mean values (mm) of the distances found on the lateral radiographs of the hemimandibles of all the groups and significance of the paired Student's t-test.