Effects of Injury of the Posterior Belly of the Digastic Muscle on Mandibular Growth

Original Article

pISSN 2288-9272 eISSN 2383-8493 J Oral Med Pain 2017;42(4):109-115 JOMP https://doi.org/10.14476/jomp.2017.42.4.109 Journal of Oral Medicine and Pain

Seo-Jeong Hyun1, Hye-Youn Lim2, Kyung-Hee Kim2, Tae-Young Jung2, Sang-Jun Park2

1Department of Oral and Maxillofacial Surgery, Inje University Haeundae Paik Hospital, Busan, Korea 2Department of Oral and Maxillofacial Surgery, Inje University Busan Paik Hospital, Busan, Korea

Received September 13, 2017 Purpose: This study analyzed the effects of digastric muscle injury on mandibular growth in Revised December 14, 2017 young rats. Accepted December 27, 2017 Methods: Fourteen 4-week-old Wistar rats were divided into 3 groups; experimental group A (n=6) with unilateral (right) shortening of the posterior digastric muscle, experimental group B (n=6) with bilateral shortening of the posterior digastric muscles, and control group C (n=2) who underwent a sham operation. Eight weeks after the operation all animals were sacrificed and the outcomes were compared using body weight evaluation, mensurations on lateral radiograph of hemimandibles and histological evaluation. Results: There was no significant difference between groups A and B in body weight gain. Comparison of the mean values of hemimandible distance on radiograph was performed. The difference in group mean value of mandible-related distances was analyzed using the Wilcox- on test (rank sum test) and a comparison of the homonymous distances of group A and B was performed using the Mann-Whitney test. There were differences between sides in mandibular length in group B and mandibular ramus height and transverse width of the condyle in group A (all, p<0.05). There were differences in condylar height and mandibular length on the left side between groups A and B (p<0.05). Histologic examination of temporomandibular joint showed similar findings in all specimens. Correspondence to: Sang-Jun Park Conclusions: Injury of the posterior belly of the digastric muscle during the rat growth period Department of Oral and Maxillofacial induced shortening of mandibular ramus height and transverse width of the condyle. It can be Surgery, Inje University Busan Paik Hospital, 75 Bokji-ro, Busanjin-gu, inferred that trauma to the posterior belly of the digastric muscle affects horizontal and verti- Busan 47392, Korea cal growth of the mandible. Tel: +82-51-890-6369 Fax: +82-51-896-6675 E-mail: [email protected] Key Words: Digastic muscle; Mandible; Temporomandibular joint

INTRODUCTION inherent growth tendencies. The function of the masticatory muscle is related to the bone quality of the developing In the growing mandible, trauma and intra-articular dis- mandible and several studies have experimentally demon- eases of the temporomandibular joint are known to lead to strated the effect of masticatory muscle on facial growth. disc displacement, degenerative changes of the condyle, and The elevator muscles influence the transversal and the ver- inhibition of mandible growth. The appearance of condylar tical dimension of the face. The varied functional deformi- remodeling has been reported in experiments inflicting in- ties in the mandibles were related to resections of the mas- direct and direct injuries, such as condylar fracture, eminec- seter muscle and the posterior temporal muscle.7-9) tomy, and disc perforation at a temporomandibular joint.1-6) The digastric muscle in mastication interacts with the su- Also muscular activity is one of factors that can alter prahyoid muscle, and the intermediate tendon connects the

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www.journalomp.org 110 J Oral Med Pain Vol. 42 No. 4, December 2017 anterior and posterior belly. The digastric muscle is involved Length-shortening of the right posterior belly of the di- in mandibular retrusion or elevation of the hyoid bone, and gastric muscle was conducted in group A, and bilateral anatomical diversity as well as abnormal attachment of the shortening of the length of the posterior belly of each di- digastric muscle has been reported.10,11) gastric muscle was performed in group B. For the first ex- Despite the absence of articular change in experiments perimental animal in group B, length reduction of the right based on resection of the anterior belly of the digastric posterior belly of the digastric muscle and 2 mm cutting muscle in rats, patient reports have shown mandibular an- of the left posterior belly of the digastric muscle were per- terior and posterior displacement and atrophy or complete formed during the surgical procedure. The sham-operated agenesis of the digastric muscle anterior belly in 40% of group C was submitted to exposure of the bilateral digastric hemifacial microsomia patients.12-14) muscles. Surgical sites of all experimental animals were su- However, most research on the digastric muscle refers to tured with 4-0 silk (Black Silk) and showed normal wound the anterior belly, and studies on the posterior belly are in- healing after surgery. sufficient. This study evaluated the effect of posterior belly After 8 weeks, experimental animals were sacrificed us- injury on mandibular horizontal and vertical development ing CO2 gas (3.7 L/min within 5 minutes) and body weight using radiological analysis of rat mandible length and his- measurements of the experimental animals were recorded.18) tological analysis of the temporomandibular joint. Mandibles of sacrificed animals were separated from the cephalic region, and the isolated left and right mandibles MATERIALS AND METHODS were placed in 10% buffered formalin solution. After fixa- tion in formalin solution, a lateral radiograph of the uni- Fourteen 4-week-old male Wistar rats (SPF Outbred; lateral mandible of the experimental animal was collected Orientbio Inc., Seongnam, Korea) were included in this with a 0.2 second exposure using periapical film (RVG 6100 study. All experimental animals were fed common rodent size 2; Carestream Health Inc., New York, NY, USA) and forage (Cargill Agri Purina Inc., Seongnam, Korea) and dental radiology equipment (CS 2200; Carestream Health water.15) Inc.).19) Experimental animals were divided into 3 groups; ex- The relevant distances of the bilateral mandible were perimental group A (n=6) with unilateral (right) shortening measured from the lateral radiograph of the unilateral man- of the posterior belly of the digastric muscle, experimental dible on a PACS (Picture Archiving and Communication group B (n=6) with bilateral shortening of the posterior bel- System, m-view TM version 5.4; Infinitt Healthcare Co., ly of the digastric muscle, and control group C (n=2) with Ltd., Seoul, Korea) with reference to the studies of Teixeira a sham operation. This research was conducted with ap- and Toledo.1,19) The same experimenter performed the mea- proval of Inje University Animal Care and Use Committee surement 3 times. (2016-009). On the experimental animals’ lateral radiographs of the General anesthesia was induced with 10 mg/kg xylazine unilateral mandible, the mandibular ramus height was iden- hydrochloride (Rompun 23.3 mg/10 mL; Bayer Korea Ltd., tified as the distance between the vertex of the condyle (CP) Seoul, Korea) and isoflurane (4% at induction and 2% for and the vertex of the angular process of the mandible (AP). maintenance)16) and body weight measurements of the ex- The mandibular body height was measured as the distance perimental animals were recorded. from the intersecting point (TM) of the third molar’s distal A mid-ventricular incision from the inferior border of the surface and the mandibular ramus to the antegonial notch mid-mandible was performed. Skin and fascia were retract- (AN). The mandibular length was determined and measured ed to expose and identify the digastric muscle, which was as the distance from the frontal point of the intraosseous then subjected to shortening. Using a mosquito forceps, the insertion in the lower incisor to the vertex of the condyle posterior belly was lifted and ligated with 4-0 silk (Black (CP) (Fig. 1A). Silk; Ailee Co., Ltd., Busan, Korea).17) Other than the measured value of the above, the line

www.journalomp.org Seo-Jeong Hyun et al. Effects of Injury of the Posterior Belly of the Digastic Muscle 111

C1 C CP B

TM A2 II

AN AP A B D A1

Fig. 1. (A, B) Lateral radiograph of mandible. Lines represent the measurements made. AN, antegonial notch; AP, angular process; CP, condylar process; II, insertion of incisor; TM, distal face of third molar; B, condylar height; C1, coronoid height; C-D, transverse width of mandible; A2, transverse width of condyle.

Table 1. Mean values of body weight (g) examination and isolated. After the joints had been de- Pre-op 8 weeks Weight gain o Group mineralized in 4 C 10% EDTA for 4 weeks, they were in- (a) (b) (b-a) serted into paraffin and serially sliced into sagittal sec- A (right side injury) 137.17±2.48 480.67±17.00 343.50±16.16 tion by 2 μm. After staining with hematoxylin-eosin B (both side injury) 131.33±3.20 467.83±34.18 336.50±35.01 C (sham operated) 131.00±1.41 441.50±27.58 310.50±26.16 (H&E), the sample’s histological examination was con- Values are presented as mean±standard deviation. ducted at ×100 magnification, using Digital Pathology Slide Scanner (Aperio ScanScope CS.; Leica Biosystems, Wetzlar, connecting the sigmoid notch and the most forward point Germany) and Aperio ImageScope (version 12.0.05039; of the posterior surface of the mandible was defined as A1 Leica Biosystems).12,14) for distance measurement in association with the mandible. The distance of the vertical line B from line A1 to the cul- RESULTS minating point of the condyle was identified as condylar height, and the distance of the vertical line C1 from A1 to The body weight variation of groups A, B, and C was the culminating point of the coronoid process was set as analyzed preoperatively and at 8 weeks postoperative- coronoid height. The measured distance along line A1 be- ly. Results are shown in Table 1. According to the Mann- tween the vertical line C of the superior aspect of the coro- Whitney test, the preoperative body weight of group A was noid process and line D of the mandibular inferior mar- greater than that of group B (p=0.013), and there was no gin was defined as the transverse width of the mandible, significant difference in body weight 8 weeks after surgery and the measured distance of line A2, which is parallel to (p=0.337) or in weight gain (p=0.873) between groups A line A1 and divides line B into two, was set as the trans- and B. verse width of the condyle (Fig. 1B). The MedCalc (version The mean values of mandible-related distances were ana- 16.8.4; MedCalc Software bvba, Ostend, Belgium) statistical lyzed based on the lateral radiograph of the experimental program was used for statistical analysis of the measured animals’ unilateral mandibles (Table 2). The difference in values. group mean value of mandible-related distances was ana- The left and right temporomandibular joints of experi- lyzed using the Wilcoxon test (rank sum test). In group mental mandible was fixed in formalin for histological A with unilateral injury, the height was greater in the left

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Table 2. Distances found on lateral radiograph of hemimandibles Mandibular ramus (height [mm], CP-AP) Mandibular body (height [mm], TM-AN) Mandibular length (mm, Ⅱ-CP) Group Right Left Right Left Right Left

A (n=6) 8.32±0.30 8.87±0.43 5.17±0.23 5.42±0.14 26.48±0.95 26.08±0.84 B (n=6) 8.53±0.43 8.46±0.55 5.22±0.24 5.27±0.11 26.72±0.66 27.52±0.79 C (n=2) 8.71±0.27 8.39±0.24 5.28±0.04 5.54±0.27 27.47±0.01 26.92±0.13 CP, condylar process; AP, angular process; TM, distal face of third molar; AN, antegonial notch; II, insertion of incisor. Values are presented as mean±standard deviation.

Table 3. Measurements relative to ramus found on lateral radiograph of hemimandible Condylar height Coronoid process Transverse width of mandible Transverse width of condyle (mm) (height, mm) (mm) (mm)

Left A (n=6) 4.70±0.60 2.78±0.32 15.64±0.44 4.08±0.34 B (n=6) 5.33±0.38 2.95±0.24 15.68±0.48 4.06±0.18 C (n=2) 5.37±0.12 2.94±0.10 15.31±0.04 4.12±0.04 Right A (n=6) 5.08±0.52 2.90±0.20 15.54±0.42 3.82±0.24 B (n=5) 5.17±0.25 3.07±0.33 15.92±0.41 4.05±0.17 C (n=2) 5.59±0.39 2.99±0.25 15.62±0.54 4.04±0.24 Values are presented as mean±standard deviation. mandible (p=0.031) (Fig. 2A) In group B with bilateral inju- Statistical analysis was conducted after exclusion of the ry, the left mandible was longer than the right (p=0.031) (Fig. first experimental animal measurement in group B in which 2B). shortening of the right posterior belly and severing of the A comparison of the homonymous distances of group A left posterior belly were performed. Thus, a significant dif- and B was performed using the Mann-Whitney test. There ference (p=0.045) was shown in the left mandibular lengths was no significant difference between groups A and B in of groups A (26.08±0.84 mm) and B (27.55±0.88 mm). the right mandible, but left mandibular length was signifi- Histological examination offered similar anatomical and cantly longer in group B with bilateral injury (p=0.025) (Fig. histological perspectives in all specimens. The cartilage area 2C). of the condyle was observed in 4 different layers charac- Using the same method, the mean values of ramus-related terized as the articular zone, proliferation zone, maturation distances were analyzed based on the unilateral mandible zone, and hypertrophic zone. Degenerative changes, includ- lateral radiographs (Table 3). In the distance measurement ing osteoarthritic changes, were not observed (Fig. 3). of right mandible-related distance, after the right coronoid process was fractured and separated from the mandible dur- DISCUSSION ing the separation from the skull, the measurements of the first experimental animal in group B were excluded. The digastric muscle is an essential marker in head and The mean difference between left and right distances in neck surgery. The posterior belly of the digastric muscle the same experimental group was analyzed. In group A, the starts from the mastoid notch, which is in front of the tem- transverse width of the left condyle was longer than that of poral mastoid process, and runs downward to insert at the the right condyle (p=0.031) (Fig. 2D). For the mean hom- intermediate tendon. The functions of the posterior belly are onymous distance value between groups A and B, the left specialized for elevating the position of the hyoid bone with condylar heights were higher in group B with bilateral in- the anterior belly and for mandibular retrusion.20) jury (p=0.037) (Fig. 2E). The anterior belly of the digastric muscle embryologically

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AB 9.4 28.5 9.2 28.0 9.0

8.8 27.5 8.6

(mm) (mm) 8.4 27.0 8.2 26.5 8.0 7.8 26.0 ar (cp-ap) al (cp-ap) br (ll-cp) bl (ll-cp) C DE 28.5 4.6 6.0 28.0 4.4 5.5 27.5 4.2 27.0 5.0 26.5 4.0

(mm) (mm) (mm) 26.0 4.5 3.8 25.5 4.0 3.6 25.0 24.5 3.4 3.5 al (ll-cp) bl (ll-cp) ar (trans.con) al ()trans.con al (cond.h) bl ()cond.h Fig. 2. (A) Comparison of mandibular ramus heights of group A. ar (cp-ap), right mandibular ramus height; al (cp-ap), left mandibular ramus height. (B) Comparison of mandibular lengths of group B. br (ll-cp), right mandibular length; bl (ll-cp), left mandibular length. (C) Comparison of left mandibular lengths. al (ll-cp), left mandibular length of group A; bl (ll-cp), left mandibular length of group B. (D) Comparison of transverse width of condyles of group A. ar (trans.con), transverse width of right condyle; al (trans.con), transverse width of left condyle. (E) Comparison of left condylar heights. al (con.h), left condylar height of group A; bl (con.h), left condylar height of group B.

A B C 300 mm 200 mm 300mm

Fig. 3. (A) The mandibular condyle of rat C4-2 (sham operation, right condyle), showing normal component cartilage layers. (B) The mandibular condyle of rat A2-1 (unilateral operation, right condyle), showing normal component cartilage layers. (C) The mandibular condyle of rat B2-1 (bilateral operation, right condyle), showing normal component cartilage layers. A-C: H&E staining, higher magnification ×10. originates from the first pharyngeal arch and is innervated Also a difference in digastric muscle myofiber dispersion by the inferior alveolar nerve, and the posterior belly origi- has been reported, and Type I fiber was discovered in the nates from the second pharyngeal arch and is under the fa- anterior belly’s central part and the posterior belly’s core in cial nerve.21) rats. Type IIB fiber was also found at a high rate at the edge

www.journalomp.org 114 J Oral Med Pain Vol. 42 No. 4, December 2017 of the anterior belly of the digastric muscle. This heteroge- mandibular lengths were significantly different (p=0.045). neity in the fiber type compositions can be assumed to re- It is hypothesized that this result is due to the loss of digas- flect complex role of muscles during function.22) tric muscle posterior traction power to the mandibular sym- Several studies have experimentally demonstrated the ef- physis when the left posterior belly of the digastric muscle fect of anterior belly of digastric muscle on growth and de- of the first experimental animal in group B was severed. velopment of mandible. This result explains the left condylar height comparison of Ohura reported that the mandible steadily showed pos- groups A and B and the finding that the mandibular length terior rotation and anterior displacement after ant digas- of group B with bilateral injury was longer than that of tric muscle resection in 5-week-old rats, and no change in group A without shortening of the left digastric muscle. mandibular height or length was identified.12) The suprahyoid, including the digastric muscle, is in- In another study of mini pig’s anterior digastric muscle, 4 clined to adapt to limited length changes quickly and is not mini pigs underwent reduction of anterior belly length us- actively involved in the vertical development of the lower ing silk to form 2 loops and were sacrificed after 2 weeks. half of the face. The geniohyoid muscle adapted to its new The developmental change was analyzed by juxtaposing position after downward and forward displacement of the cephalograms before and after the surgical procedure. In 3 mandible. The anatomy and the structure of the digastric out of 4 experimental animals, the homonymous unilateral muscles are different and rats also have a symphysial artic- mandibular length, which is the distance from the condy- ulation, so that hemimandibles are capable of limited inde- lar head posterior point to the mandibular symphysis, was pendent movement. Although a change in length less than reduced, and remodeling of the mandibular symphysis area 30% is known to not interfere with condylar growth, as this was shown. However, hindrance of condylar growth was experiment shows, the horizontal and vertical growth of the not confirmed. Furthermore, unlike the unilateral length re- mandible are estimated to be affected.17) duction, shortening of the bilateral lengths deterred forward In this experiment, the shortening of the posterior belly growth of the symphysis.17) of the digastric muscle had distinct influence on the man- In our experiment, the mandibular height of group A, in dibular height and condylar transverse width. To improve which the right posterior belly of the digastric muscle was the statistical reliability of this experiment, it is necessary to shortened, was lower than the mandibular height of the un- confirm the variation of growth based on trauma with more touched left posterior belly of the digastric muscle, and the experimental animals. right condylar transverse width was shorter than that of the Histologically, the articular structure of rats is similar to left. There was not a significant difference in mandibular that of humans except absence of articular eminence.23) In length. this study, the histological examination was conducted only Group B with bilateral length reduction showed a signifi- with H&E staining. In all specimens the histologic charac- cant difference between left and right mandibular lengths. teristics were similar and had normal component cartilage This result was possible as, in contrast to mini pigs, the layers. It will benefit the further assessment of the effect of presence of symphysial articulation of rats enabled the left condylar growth to use different staining methods, such as and right unilateral mandibles to undergo limited but inde- 5-bromo-2-deoxyuridine staining for observation of cellu- pendent development.17) lar proliferation and TUNEL staining for observation of the To investigate the effects of the first experimental animal degree of cell death.24) in group B, after exclusion of this measurement, statisti- In conclusion, the effects of injured digastric muscle pos- cal analysis of left and right mandibular length, left con- terior belly on the mandibular development of growing rats dylar height, and left mandibular length were performed. were radiologically and histologically analyzed. Length re- A significant difference was not present (p>0.05) between duction of the posterior belly was observed to result in de- left and right mandibular lengths of group B or in the left creased mandibular height and condylar transverse width. condylar heights between groups A and B, whereas left We can hypothesize that trauma to the posterior belly of the

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