Experimental Study Of Effect On Mandibular Growth And Development After Denervation And Chemodenervation Of Masseter Muscle

BackgroundFacial contour plasty has become one of the hot spots of plastic surgery with the development of craniofacial surgery these years. Prominent mandibular angle and masseteric hypertrophy is the most popular defect of the facial contour of the oriental nations. Mini-invasive technique is now the new trend of treatment for prominent mandibular angle and masseteric hypertrophy with the improvement of minimal invasive surgery and non-surgical cosmetic techniques, such as denervation of masseter muscle, radiofrequency volumetric reduction of masseter muscle, injection of botulinum toxin type A to the masseter muscle, etc. Hwang firstly reported four cases of selective neurectomy of masseteric nerve in 2004. Moore used botulinum toxin type A to treat masseteric hypertrophy in 1994. After that, excellent results were seen using domestic-made botulinum toxin type A to treat masseteric hypertrophy in China.The growth and development of mandible is a continual process. The growth and remodeling of the mandible depends on the balance between bone deposition and bone resorption, especially during the adolescent age. With the development of plastic and cosmetic surgery, plastic and cosmetic operations are safe now and there are very few significant complications. Attitudes about plastic and aesthetic surgery have changed over the past ten years. An increasing number of patients undergo plastic and cosmetic procedures every year. The number of teenagers choosing to have cosmetic procedures is increasing at a rapid rate during these years. The merits such as simple, short time, mini-invasion and rapid recovery of minimal invasive and non-surgical cosmetic techniques attract more and more teenagers. But new problems of facial contour surgery arise due to the succession of growth and development of mandible and the increasing teenagers. If denervation and chemodenervation are performed before puberty, are there any influences of the mandible during the adolescent age? What changes will occur to the mandibular morphology, histology, ultramicrostructures, BMD and biomechanics properties? Studies are urgently needed to solve these problems before widespread clinical apply of these mini-invasive techniques.ObjectiveThe objective of this study is to evaluate the effect of denervation and chemodenervation (injection of botulinum toxin type A) of masseter muscle on the growth and development of mandible. Animal models of denervation and chemodenervation of masseter muscle are established before puberty. In the denervation group, one side of the masseteric nerve is severed by microsurgery procedure. While botulinum toxin type A is injected into one side of the masseter muscle in the chemodenervation group. When the animals grow up and maturity is attained, studies are as below:measurement of the mandibular length, mandibular height and bigonial width by 3D reconstructive technique; observation of histology and ultramicrostructures; measurement of the BMD of the mandible; biomechanical analysis of the mandible using finite element method. This preliminary study is aim for providing some available information about mandibular growth and development after denervation and chemodenervation of masseter muscle.Method1. Experimental animals and grouping:Thirty 21 days old male Wistar rats were randomly divided into four groups:operation group (9 rats), sham group (9 rats) botulinum toxin group (6 rats) and control group (6 rats)2. Animal model of operation group and sham group:When the rats were 28 days old, the main trunk and initial branches of masseteric nerve on the right side were resected by microsurgery procedure in the operation group. The nerve was exposed, but not resected in the sham group.3. Animal model of botulinum toxin group:When the rats were 28 days old, the right side of masseter muscle was injected with botulinum toxin type A (0.1ml,2U) and the left side was injected with sterile saline (0.1m) in the botulinum toxin group. The control group was only anaesthetised.4. CT scan and anthropometric measurements:CT scan was taken when all rats were 75 days old. Bilateral mandibles were 3D reconstructed by PHILIPS Brilliance TM CT software. Seven cephalometric points were digitized as below:Cor (Coronoid point), Con (Condylion point), Go (Gonion point), GoT (Gonion tangent point), Ma (Mandibular alveolar point), Iia(Incisive inferior alveolar point), Me (Menton point). And selected seven linear distances were measured as below:Mandibular lengthⅠ(Cor-Iia), Mandibular lengthⅡ(Con-Iia), Mandibular lengthⅢ(Go-Iia), mandibular heightⅠ(Ma-Me), mandibular heightⅡ(Cor-GoT), mandibular heightⅢ(Con-GoT) and Bigonial width (GoT1-GoT2).5. Measurement of the masseter muscles:The rats were killed with an overdose of 10% chloral hydrate after CT scan and anthropometric measurements. Masseter muscles of each side were integrallty obtained and weighted on electronic balance immediately.6. Observation of histology, ultramicrostructures and IHC staining:Mandibles of each group were fixed with 4% paraformaldehyde solution for 1-2 hours and then were decalcified with 10% ethylene diamine tetra acetic acid (EDTA) for 3 weeks. HE staining was performed on the region of angle, ramus, and condyle of the mandible. Other mandibles of each group were washed with 0.1mmol/l phosphate buffered saline (PBS) and then were fixed with 2.5% glutaraldehyde solution for 24 hours. Dehydration, arescent at critical point and conductive coating were performed before observation with SEM. Some of the histological slides from each group were immunohistochemical stained. Reactions of the antibodies including IL-6 and TNF-a were analyzed in the mandible.7. Measurement of BMD:Bilateral mandibles of all four groups were scanned by GE lunar prodigy bone densitometer. ROI was defined in the region where the masseter muscle attached. Then the results of BMD were calculated with the software of small animals (en CORE 2006 V10.50.086).8. Finite element analysis:The DICOM files from the CT scan of the mandible were imported into Mimics. Preliminary mandibular 3D model was built after segmentation, thresholding, region growing and Calculate 3D. After the mandibular model was remeshed,3D tetrahedral network model of mandible was established by the software of MSC.Patran. According to the value of gray scale at different parts of the mandible from the CT sections in Mimics, the Elastic module can be obtained (maximum:1200MPa; minimum:1MPa). Poisson Ratio was 0.3. Then the three-points bending tests of mandibular ramus and mandibular angle were performed on the model using the software of MSC.Marc. The load of 200N was applied to the center of mandibular ramus and 10N was applied to the Gonion point. The load was exerted by ten steps in one second. FEA results included maximum equivalent Von Mises stress, maximum equivalent of total strain, maximum external force and maximum displacement.9. Statistical analysis:Statistical analyses were carried out with SPSS 13.0. All measurement data were presented as means with standard deviations. The differences between the left and right side to each group were determined by paired samples t-test. The differences among groups were determined by independent samples t-test and one-way ANOVA, and multiple comparisons were determined using Fisher's least significant difference t-test. The significance level was set at P<0.05.Results1. General status:All of the rats in operation and sham group are live after surgery. Dead does not occurred. Lateral deviated occlusion is observed on two rats in botulinum toxin group. Gross appearance:significant atrophy on the right side can be seen in both operation group and botulinum toxin group. Bilateral masseter muscles were symmetric in sham group and control group.2. Body weight and Quality of masseter muscle:Differences of the body weight between pre-operation and post-operation in operation group and sham group are not significant. Differences of the body weight between pre-injection and post-injection in botulinum toxin group and control group are not significant, except for the first week after injection. In the operation group, the quality of masseter muscle of the right side is less than that of the left side (P=0.000), and it is also less than that in sham group and control group. In the botulinum toxin group, the quality of masseter muscle of the right side is less than that of the left side (P=0.001); the quality of masseter muscle on both sides are less than that in control group (P=0.016,P=0.000) 3. Measurements of mandible3.1 Operation group and sham group(1) Comparison between two sides:Mandibular heightⅡ(12.0±0.3mm) and mandibular heightⅢ(9.4±0.4mm)of the right side are less than those of the left side (12.6±0.4mm,10.5±0.3mm, respectively) (P=0.001, P=0.000, respectively) in operation group. In sham group, mandibular heightⅢ(9.6±0.4mm) of the right side is less than that of the left side (10.6±0.4mm) (P=0.000)(2) Comparisons of right side among groups:Differences of mandibular length and mandibular heightⅠamong groups are not significant. Mandibular heightⅡ(12.0±0.3mm) of operation group is less than that of control group (P=0.017) Mandibular heightⅢof operation group and sham group (9.4±0.4mm,9.6±0.4mm, respectively) are all less than those of control group (all, P=0.000)(3) Comparisons of bigonial width:Bigonial width of operation group is less than that of control group (P=0.018)3.2 Botulinum toxin group(1) Comparison between two sides:Mandibular lengthⅢ(19.2±1.4mm) of the right side is less than that of the left side (20.4±1.4mm) (P=0.033). Mandibular heightⅡ(10.8±0.8mm) of the right side is less than that of the left side (12.0±0.9mm) (P=0.001)(2) Comparisons of right side among groups:Differences of Mandibular length and mandibular heightⅠamong groups are not significant. Mandibular heightⅡ(10.8±0.8mm)and mandibular heightⅢ(9.5±0.6mm)of botulinum toxin group are less than those of control group (P=0.007, P=0.005, respectively)(3) Comparisons of bigonial width:Bigonial width of botulinum toxin group is less than that of control group, but differences are not significant.4. Histology:In operation group, sham group and control group, all mandibles have compact cortex, abundant bone trabecula and uniform distributed bone lacuna. The cortical bone of mandible in botulinum toxin group is also compact. But bone trabecula is not as abundant as that of control group. Distributions of bone lacunas are inhomogeneous. The condylar cartilage of control group is stained and delaminated clearly. Whereas the cellulous layers of cartilage are not conspicuous in botulinum toxin group.5. Ultramicrostructures:The cortical bone of mandible in control group is compact. There are plenty of bone trabeculas on the condyle and thickness of them is uniform. A conferted 3D reticular structure is built by the bone trabeculas connecting to each other. Array of collagen fibers on the surface of trabecula are regular and compact. Parts of the fibers connect to each other forming a network. The ultramicrostructures of operation group and sham group is similar as that of control group. But array of collagen fibers are loose and irregular. In botulinum toxin group, thickness of bone trabecula is inhomogeneous. The interspaces among the trabeculas are a little wider. Array of collagen fibers are more irregular. The network of collagen fibers is only presented on partial region.6. IHC staining:Positive reaction is not found on osteoblast, osteocyte and osteoclast in all the four groups.7. BMD:Differences of the BMD between left and right side of mandible in all the four groups are not significant. Differences of the BMD on the right side of mandible among operation group, sham group, botulinum toxin group and control group are not significant.8. Finite element analysis:3D-finite element model of mandible was established in operation group, botulinum toxin group and control group. In botulinum toxin group, maximum equivalent Von Mises stress (630.5MPa) and maximum equivalent of total strain (1.847) at mandibular ramus are less than those of control group. And maximum displacement (2.466mm) at mandibular ramus is also less than that of control group. At mandibular angle, maximum equivalent Von Mises stress (66.4MPa) and maximum equivalent of total strain (0.2278) are less than those of control group. But maximum displacement (7.508mm) is greater than that of control group. The results at mandibular ramus and mandibular angle of operation group are similar as those of control group.Conclusion1. After resection of the masseteric nerve of rat before puberty, alterations of mandibular length and mandibular heightⅠare not seen when the rat grow up and maturity is attained. Mandibular heightⅡand bigonial width are decreased. Histology of cortical and trabecular bone of mandible are generally normal. Array of collagen fibers on the surface of trabecula are loose and irregular, and collagen network can be seen. BMD of mandible is not changed. The maximum equivalent Von Mises stress, maximum equivalent of total strain and maximum displacement at mandibular ramus and mandibular angle are similar as those of control group. The biomechanical behaviour of resisting elastic deformation of the mandible is not changed after neurectomy of masseter muscle.2. After injection of botulinum toxin type A to masseter muscle of rat before puberty, alterations of mandibular length, mandibular heightⅠand bigonial width are not seen when the rat grow up and maturity is attained. Mandibular heightⅡand mandibular heightⅢare decreased. The cortical bone of mandible is compact. The condylar cartilage is poorly developed. Array of collagen fibers on the surface of trabecula are more irregular. The network of collagen fibers is only presented on partial region. BMD of mandible is not changed. The maximum equivalent Von Mises stress, maximum equivalent of total strain and maximum displacement at mandibular ramus are less than those of control group. While the maximum equivalent Von Mises stress and maximum equivalent of total strain at mandibular angle are greater than those of control group. The biomechanical property of resisting elastic deformation of the mandible is not changed after neurectomy of masseter muscle. And displacement of the mandibular angle is greater than other groups. The biomechanical behaviour of resisting elastic deformation of the mandible is influenced by chemodenervation of masseter muscle. The strength of the mandibular angle is decreased.There are several new ideas brought by this study. Firstly, the animal models of denervation and chemodenervation of masseter muscle are established simultaneously. Systematic anthropometric measurements of the mandible are performed by 3D reconstruction. The two-dimensional limitation of X-ray cephalometric measurements is eliminated. And the bigonial width can be measured in 3D space. Secondly, theoretical support can be afforded to the biomechanical study of mandible on base of observation of histology and measurement of BMD. Thirdly, the mandible of rat is too small to undergo biomechanical test. FEM is an alternative method and FEA can afford reliable results.On the whole, although neurectomy of masseteric nerve and intramuscular injection of botulinum toxin type A are not extensively applied to improving facial contour, basic research is necessary for the development of plastic and cosmetic surgery. This study aims for affording available references about the safety, prediction of therapeutic effect and complications of denervation and chemodenervation of masseter muscle.