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Osteogenetic changes in elongated styloid processes of Eagle syndrome patients
Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
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Osteogenetic changes in elongated styloid processes of Eagle syndrome patients Soung Min Kim a, Mi Hyun Seo a, Hoon Myoung a, Jin Young Choi a, Yeon Sook Kim b, Suk Keun Lee c, * a Department of Oral and Maxillofacial Surgery (Head: Prof. Jin Young Choi), Dental Research Institute, School of Dentistry, Seoul National University, Seoul, South Korea b Department of Dental Hygiene (Head: Prof. Yeon Sook Kim), Cheongju University, Cheongju, South Korea c Department of Oral Pathology (Head: Prof. Suk Keun Lee), College of Dentistry, Gangneung-Wonju National University, 123 Chibyun-dong, Gangneung 210702, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 November 2012 Accepted 13 September 2013
Abnormal elongation of the styloid process, or Eagle syndrome, can be painful, and is associated with differential diagnoses including cranio-facial malformations and vasculo-neurological disturbances. The precise molecular mechanism leading to styloid process elongation is unknown. In this study, elongated styloid processes with periosteal fibrous ligament tissue were obtained from three patients with Eagle syndrome and examined by immunohistochemical methods using different antisera. In all cases, marked bony deposition was found at the apex of the styloid process. The osteogenetic proteins, such as osteonectin, osteocalcin, BMP-2, BMP-4, and RANKL were strongly positive by immunohistochemistry in both the ligament fibers and the periosteal membrane attached to the styloid process apex. Staining for protective proteins, HO-1, HSP-70, and HSP-90 was also positive. These results suggest that styloid process elongation is related to increased expression of osteogenetic and protective proteins. Therefore, we propose that Eagle syndrome results from a protective response to increased tensile stress in the ligament attached to the styloid process, which could also signal osteogenetic protein expression in the periosteal fibrous tissue. Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Eagle syndrome Immunohistochemistry Osteogenetic proteins Styloid process Tensile stress
1. Introduction The stylohyoid complex is composed of the styloid process, the stylohyoid ligament and the lesser horn of the hyoid bone. The styloid process is a long cylindrical, cartilaginous extension of the temporal bone. Embryologically, the anatomical structures of the stylohyoid complex originate from Reichert’s cartilage, or the second branchial arch. The second branchial arch gives rise to the stylohyoid chain and contains potential ossification centers which eventually mineralize to varying degrees (Camarda et al., 1989a, 1989b). The normal styloid process length is approximately 20e 30 mm (Gokce et al., 2008), with a mean length of 21.6 mm on the right side and 21.2 mm on the left side. There are three groups of styloid processes: short (under 21 mm), normal (21e30 mm) and elongated (more than 30 mm). This classification is both
* Corresponding author. Tel.: þ82 33 640 2228; fax: þ82 33 642 6410. E-mail address: [email protected] (S.K. Lee).
biologically based and clinically relevant (Sokler and Sandev, 2001; Fini et al., 2000). In the general population, the frequency of an elongated styloid process is estimated to be 4%, of which only 4% show clinical manifestations. This suggests that the incidence of styloid syndrome is 0.16% (about 16,000 persons in Serbia). Deviation of the styloid process causes external or internal carotid impingement and pain that radiates along the arterial trunk (Petrovic et al., 2008a, 2008b). Styloid process deviation is considered important, because it is clinically similar to other painful cranio-facial syndromes, making diagnosis for treatment, difficult (Petrovic et al., 2008b). The American otolaryngologist Eagle was the first to describe this styloid syndrome in 1937. Eagle syndrome is a rare condition in which an elongated styloid process (more than 30 mm) impinges on adjacent anatomical structures (Mortellaro et al., 2002; Karam and Koussa, 2007). Ossification of the stylohyoid chain leads to a progressive decrease in elasticity and the potential for associated clinical symptoms. Theories about the stimulus for ossification include aging and reactive healing following surgical or
1010-5182/$ e see front matter Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2013.09.012
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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S.M. Kim et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
nonsurgical trauma (Gokce et al., 2008; Keur et al., 1986). The aetiologic triggering factor for Eagle syndrome has not yet been discovered. Two forms of Eagle syndrome exist: classic and vascular form. Patients with classic “Eagle syndrome” present with unilateral sore throat, dysphagia, tinnitus, unilateral facial and neck pain, and otalgia (Murtagh et al., 2001; Thot et al., 2000). In patients with vascular “Eagle syndrome”, the elongated styloid process is in contact with the extracranial internal carotid artery; this can cause compression while turning the head or dissection of the carotid artery, causing a transient ischemic accident or a stroke (Gokce et al., 2008). Classical stylohyoid syndrome is found after tonsillectomy and is characterized by pharyngeal, cervical, and facial pain and headache (Leong et al., 2007; Lorman and Biggs, 1983; Kim et al., 2008). Stylo-carotid syndrome is the consequence of irritation of the pericarotid sympathetic fibers and compression of the carotid artery (Bafaqeeh, 2000; Farhat et al., 2009; Chuang et al., 2007). Clinical manifestations are most frequent after headturning and neck compression. Eagle syndrome is defined as clinical symptoms of the neck with cervicofacial pain caused by an elongated styloid process that compresses neural and vascular structures including the glossopharyngeal nerve. Eagle syndrome can also cause stroke from carotid artery compression (Gokce et al., 2008). Eagle syndrome can be classified as an entrapment syndrome requiring neurosurgical attention (Karam and Koussa, 2007; Slavin, 2002). This study aimed to elucidate the molecular mechanism of styloid process elongation in Eagle syndrome through immunohistochemical detection of osteogenetic and protective proteins in the apical areas of surgically removed styloid processes. 2. Material and Methods Four elongated styloid processes including the periosteal fibrous ligament from three Eagle syndrome patients were studied with approval of the Institutional Review Board of Gangneung-Wonju National University (Table 1). One patient had surgery on both sides via an extraoral submandibular approach. After incision of the skin and identification and division of the posterior extension of the platysma muscle, blunt dissection was performed to divide the posterior border of the mandible and identify a portion of the external carotid artery. Immediately below the investing fascia of the external carotid or internal maxillary artery, the styloid process was identified and easily palpated. Once the fascia was removed from the surface of the styloid process, an incision was made in the periosteum, facilitating reflection of the periosteum and muscle attachments. The styloid process was removed near its base with dissection of the stylohyoid ligament at a point distal to the calcified portion. The wound was closed with a traditional layer suture. The other two patients had surgery on each side via an intraoral trans-tonsillar approach. With the patient in a hyperextended open-mouth position, a trans-tonsillar incision was made in the
Table 1 Eagle syndrome patients in this study. Age/ Symptoms Site gender Case 1 37/M (S2001-72a) Case 2 23/M (S2002-148) Case 3 41/M (S2008-452)
Operation F/U period Complications
Neck pain, Both Extraoral 2 years otalgia Neck pain Left Intraoral 2.5 years Neck pain, Right Intraoral dysphagia
2 years
None None None
a Biopsy number registered in the Department of Oral Pathology, GangneungWonju National University Dental Hospital.
pharyngeal mucosa, and the fascial plane was identified by blunt dissection of the involved muscle with a non-sharpened dissection scissors, palpating the hard tip of the calcified styloid process in the lateral pharyngeal space. Removed specimens were fixed in 10% neutral formalin and decalcified with 0.5-M EDTA solution (pH 8.0), embedded in paraffin, and cut into 4 m sections. Microsections were stained with hematoxylin and eosin, followed by immunohistochemical staining using antibodies against osteonectin, osteocalcin, BMP-2, BMP-4, RANKL, HO-1, HSP-70, or HSP-90. All immunostaining was performed with an indirect triple sandwich method as previously described (Lee et al., 2005; Kim et al., 2009). Background crossreaction was minimized by negative control staining without primary antibodies using the same procedures. Histological images of representative samples were captured with a digital camera (DP70Ò, Olympus Co., Japan). Each slide was evaluated for intensity of positive immunostaining, graded as þþþþ, þþþ, þþ, þ, þ/, , corresponding to extremely strong, strong, moderate, weak, rare and negative, respectively. A rare grade of þ/ was defined as a focal or questionable weakly positive signal. Images were analyzed and compared by ANOVA using SPSS for WindowsÒ (Version 12.0, SPSS Inc., USA) and mean values with 95% confidence intervals. Correlations were analyzed by Pearson’s correlation test (p < 0.05). 3. Results Removed styloid processes were mainly composed of compact bone with sparse marrow space. Processes were covered with a thin fibrous periosteal membrane that was thickened in the apical area (Figs. 1b1 and 3a1eb5). All four cases of elongated styloid processes showed active osteophytic bone deposition at the apical end with rare bony deposition on the lateral side (Figs. 1b1, 2a and 3c1ed5). Immunohistochemical localization of osteogenetic proteins and protective proteins was characteristic of the periosteal membrane, osteoid matrix, ligament fibers, and muscle bundles of each styloid process apex (Table 2). Immunoreaction for the osteogenetic protein BMP-2 was strongly positive in the apical periosteal membrane, and accentuated in the ligament fibers attached to the apical end of the styloid processes. Immunoreaction for RANKL was relatively weak in the periosteum but consistently positive in the ligament fibers attached to the apical end of the styloid processes (Figs. 1b2, c and 2b, i). Immunoreaction for BMP-4, a marker of chondroid differentiation, was rare in the apical area of the styloid processes (Fig. 2g). Staining for the bone matrix proteins osteonectin and osteocalcin was positive at the bone deposition site of the styloid process apex, with osteonectin staining strongly in the periosteum and osteoid matrix (Fig. 2d), and osteocalcin staining relatively weak in the periosteum but consistently positive in the bone deposition site of the styloid process apex (Fig. 2e). Staining for the protective proteins HSP-70 and HO-1 was clearly positive in the apical areas of the styloid processes. HSP-70 was strongly positive in the muscle fibers attached to the apical end and consistently positive in the periosteum near apical bone deposition (Fig. 2f). HO-1 was localized in the apical periosteum of the styloid processes (Fig. 2h). Both elongated styloid processes from one of the patients showed increased axial growth with multiple callus formation in the proximal areas of the styloid processes (Fig. 3). The callus of hyalinized chondroid tissue was clearly distinguishable by Masson trichrome stain (Fig. 3b2eb4), and contained hypertrophic chondrocytes (Fig. 3c2, c4, d2, d4). Multiple calluses appeared in the marrow space, growth plate-like thickening occurred in the proximal area of the styloid process (Fig. 3c, d). The growth plate-like callus consequently produced fibrous immature bone that stained
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 1. a: Panoramic view of Case 2 patient. Left elongated styloid process, irregularly curved (arrows) compared to the right. b1: Hematoxylin and eosin stain of styloid process mainly composed of compact bone (B) with sparse marrow space and thick fibrous periosteum (P) at the apical area. b2: Immunostaining for BMP-2, in the periosteal membrane of the styloid process, accentuated in the apical area (arrow). c: Immunostaining for BMP-2. c1: Higher magnification of square in panel b2. Thick fibrous bundles (arrows) attached to the styloid process apex staining for BMP-2. c2: Higher magnification of c1. BMP-2 in the fibrous periosteal tissue (arrow heads) and ligament fibers (arrows) at the center. c3: Higher magnification of c2. BMP-2 in ligament fibers (arrows). d: Immunostaining for RANKL. d1: Higher magnification of the square in b2. RANKL in the fibrous tissue at the apex of the styloid process. d2: Higher magnification of d1. RANKL in ligament fibers (arrows) in the center and peripheral fibrous tissue (arrow heads). d3: Higher magnification of d2. RANKL in ligament fibers (arrows; see BMP-2 in c3).
blue with Masson trichrome stain and aligned parallel to the axial direction of the styloid process (Fig. 3b2, d3, d5). 4. Discussion The incidence of Eagle syndrome is controversial (Rogers and Chang, 2007). Eagle estimated the incidence of an elongated styloid process in the general population to be 4%, with only 4% of those displaying symptoms (Fritz, 1940; Eagle, 1948, 1958, 1962). Kaufman et al. (Kaufman et al., 1970) reported elongated styloid processes in 28% of patients, while Correll et al. (Correll et al., 1979)
examined 1771 panoramic view radiographs and estimated an incidence of 18.2%, of which 93% exhibited bilateral elongation. Only a relatively small number of patients (8 of 1771) (Correll et al., 1979), exhibited symptoms related to Eagle syndrome, and most were unilateral (Harma, 1967; Jan, 1989). Although, styloid process elongation is not abnormal, styloid processes longer than 3 cm are generally considered to be elongated (Palesy et al., 2000). If an elongated styloid process is not adapted to regional neuromuscular functions, it will become symptomatic (Thot et al., 2000). Mineralization and elongation of the styloid process and Eagle syndrome show similar styloid elongation in which mineralization
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 2. Osteogenetic expression for Case 3. a: Hematoxylin and eosin stain. a1: Muscle bundles attached to the apical area of the styloid process. a2: Higher magnification of a1, with fibrous periosteum (P) and osteophytic bone formation (arrows) in the apical area. a3: Higher magnification of a2. Bone deposition and mineralization (arrows). b1: Immunostaining for BMP-2, localized to the periosteal membrane (P) (arrows). b2: Higher magnification of b1, fibrous periosteal tissue attached to bone (B) (arrows). c: BMP-4 immunostaining. d1: Osteonectin immunostaining in the periosteum of the styloid process apical area. d2: Osteonectin in osteoblasts (arrows) (B). e1: Osteocalcin immunostaining in the periosteum (P) and apical bone (B) of the styloid process. f1: HSP-70 immunostaining in the periosteal muscle (M) and apical new bone area. f2: Higher magnification of f1. HSP-70 in the proximal periosteum (arrows) of the styloid process apex, and apical end (arrow heads). g: HSP-90 immunostaining. h1: HO-1 immunostaining in the periosteal tissue (arrows) of the styloid process apical area. h2: Higher magnification of h1. HO-1 in the periosteal membrane (arrows) attached to the apical bone. i: RANKL immunostaining in the periosteal tissue of apical bone. j: Negative control staining without primary antibody.
of the stylohyoid ligament progresses to the styloid process of the temporal bone (Yetiser et al., 1997; Babad, 1995; Piagkou et al., 2009; Mupparapu and Robinson, 2005). A styloid process that is simply mineralized and elongated is different from Eagle syndrome in the symptoms displayed and the treatments sought. Mineralization and elongation of the styloid process is associated with unilateral or bilateral elongation of the styloid process that does not result in significant pain, discomfort, or limitation of neck
movement. Mineralization and elongation often remains asymptomatic until it is discovered on extraoral radiographs. Eagle syndrome involves pain and discomfort in the cervicofacial region resulting specifically from an elongated styloid process (Mupparapu and Robinson, 2005). Although the mechanism that causes an increase in tensile stress on the styloid ligament is unclear, this study demonstrated that the fibrous ligament attached to the styloid process apex is
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 3. Axial growth of styloid process in Case 1. a and b: Right, c and d: Left, a and c: Hematoxylin and eosin, b and d: Masson trichrome stain. a1 and b1: Two growth plate-like calluses (arrows) and an intramedullary callus (*) in the axis of the styloid process. a2ea4 and b2eb4: Callus (*) of chondrocytes. a5 and b5: Apical bony growth. c1 and d1: Axial growth with a growth plate-like callus (arrows) and multiple intramedullary calluses (*). c1, c4, d2, d4: Callus of hypertrophic chondrocytes. c3, c5, d3, d5: Fibrous immature bone (IB, blue) formed by endochondral ossification.
immunoreactive for HSP-70 and HO-1, which are known to be related to mechanical stress. Immunoreaction was coincident with positive staining for the osteogenetic proteins BMP-2, RANKL, osteonectin, and osteocalcin, in conjunction with limited reaction for BMP-4, a marker of chondrogenic cells, and HSP-90, a marker for osteoclasts (Sojo et al., 2005; Chaves et al., 2012; Ramazanoglu et al., 2013). HO-1 is involved in vascular disease, transplantation, and inflammation (Alcaraz et al., 2000; Orozco et al., 2007; Takahashi et al., 2004) and negatively regulates osteoclastogenesis, leading to upregulation of osteogenic differentiation for new bone deposition (Zwerina et al., 2005; Kook et al., 2009). In our
study, dense staining for HO-1 in the apical periosteum of the styloid processes suggested upregulation of osteogenesis in the apical ends of the styloid processes in Eagle syndrome. The major protective stress response protein, HSP-70, is expressed in response to stress as well as to osteogenic differentiation potential (Mauney et al., 2004; Tiffee et al., 2000). The strong positive reaction for HSP70 in the apical end of the styloid processes in our study could indicate active new bone deposition incited by mechanical stress, likely tensile stress transmitted through ligament fibers. The exact etiology of styloid process elongation and the role of ectopic calcification, which might be involved in styloid process
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Table 2 Immunohistochemical findings for elongated styloid processes in Eagle syndrome patients. Area Apical end
Lateral surface
BMP-2 RANKL Osteonectin Osteocalcin HSP-70 PM þ (4)a þ/ (2) þ (1) OR þ (4) (1) þ (2) LF þþ (4) þ (4)
MB þ/ (2) þ (1)
(4)
þ (4)
þ/ (2) þ (1)
HSP-90 HO-1
þþ (2)
þ (2)
þþ (2)
þþþ (1) þþ (4)
þþ (1) þ (4)
þþþ (4)
þ (2) þþ (1)
þ (2)
þ (4)
þþþ (1) þ/ (1) þþ (2) (4) þ (4) þþþ (1) þþ (4) (4) þ (2) þþ (1) þþ (2) (2) þ (2)
þþ (1)
(2)
þ (4)
þþþ (1) þ/ (1) þþ (1)
þ (2)
þ/ (2)
þþ (1)
þ (1)
þ (4)
(4)
(2) þ/ (1)
Abbreviations: PM, periosteal membrane; OR, osteoid matrix; LF, ligament fiber; MB, muscle bundle (: negative, þ/: rare, þ: mild, þþ: moderate, þþþ: severe in histological observation). a Expressed case number from 4 cases.
elongation, are still unknown. Abnormal metabolism of calcium, phosphorus and vitamin D is common in patients with end-stage renal disease. This calcification in non-osseous soft tissue related to abnormal serum calcium and phosphorus levels could be associated with Eagle syndrome (Gokce et al., 2008). In our study, osteophytic bone deposition in the apical end of the styloid processes was irregular and dystrophic in its calcification. The osteogenetic proteins BMP-2, RANKL, osteonectin, and osteocalcin were widely distributed in the fibrous periosteal membrane, ligament fibers, and osteoid matrix at the styloid process apices. These results suggest that styloid process elongation can be accelerated by ectopic calcification originating from systemic diseases, especially end-stage renal disease, and that the dystrophic nature of ectopic calcification could be related to the greater incidence of Eagle syndrome in patients older than 40 years (Keur et al., 1986; Thot et al., 2000). The developmental origin of the styloid process is the cartilaginous tissue (Camarda et al., 1989a, 1989b). Therefore, elongation of the styloid process might tend to result in callus-induced endochondral ossification rather than intramembranous bone ossification after an, osteogenetic signal such as a tensile stress on the styloid process. The bilateral elongation of the styloid process in the Case 1 patient showed dominant callus formation on axial growth but relatively weak osteophytic bony deposition on the apical growth. Multiple intramedullary calluses and growth plate-like endochondral ossification appeared in the proximal styloid process. This supports the hypothesis that the styloid process has the potential for callus-induced endochondral ossification for axial growth and intramembranous ossification for apical growth after application of tensile stress from the stylohyoid ligament. More studies should be needed with large numbers of Eagle syndrome patients in order to elucidate the different mechanisms of styloid process elongation in Eagle syndrome. However the results of this study suggest that apical and axial growth of styloid processes is directly affected by tensile stress induced by the pulling forces of the stylohyoid ligament. 5. Conclusion Our study examined elongated styloid processes from patients with Eagle syndrome. We used histological and immunohistochemical methods to evaluate the expression of osteogenetic and
protective proteins. We found that osteophytic bone deposition in the apical end of the styloid process was closely related to the expression of osteogenetic and protective proteins. We propose that Eagle syndrome results from a protective response to increased tensile stress on the ligament attached to the styloid process, which may signal osteogenetic protein expression in the periosteal fibrous tissue of the styloid process. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20120002538, -0002997). References Alcaraz MJ, Habib A, Lebret M, Creminon C, Levy-Toledano S, Maclouf J: Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts. Br J Pharmacol 130: 57e64, 2000 Babad MS: Eagle syndrome caused by traumatic fracture of a mineralized stylohyoid ligament e literature review and a case report. Cranio 13: 188e192, 1995 Bafaqeeh SA: Eagle syndrome: classic and carotid artery types. J Otolaryngol 29: 88e94, 2000 Camarda AJ, Deschamps C, Forest D: I. Stylohyoid chain ossification: a discussion of etiology. Oral Surg Oral Med Oral Pathol 67: 508e514, 1989a Camarda AJ, Deschamps C, Forest D: II. Stylohyoid chain ossification: a discussion of etiology. Oral Surg Oral Med Oral Pathol 67: 515e520, 1989b Chaves MD, de Souza Nunes LS, de Oliveira RV, Holgado LA, Filho HN, Matsumoto MA, et al: Bovine hydroxyapatite (Bio-OssÒ) induces osteocalcin, RANK-L and osteoprotegerin expression in sinus lift of rabbits. J Craniomaxillofac Surg 40: e315e320, 2012 Chuang WC, Short JH, McKinney AM, Anker L, Knoll B, McKinney ZJ: Reversible left hemispheric ischemia secondary to carotid compression in Eagle syndrome: surgical and CT angiographic correlation. Am J Neuroradiol 28: 143e 145, 2007 Correll RW, Jensen JL, Taylor JB, Rhyne RR: Mineralization of the stylohyoidestylomandibular ligament complex. A radiographic incidence study. Oral Surg Oral Med Oral Pathol 48: 286e291, 1979 Eagle WW: Elongated styloid process; further observations and a new syndrome. Arch Otolaryngol 47: 630e640, 1948 Eagle WW: Elongated styloid process; symptoms and treatment. AMA Arch Otolaryngol 67: 172e176, 1958 Eagle WW: The symptoms, diagnosis and treatment of the elongated styloid process. Am Surg 28: 1e5, 1962 Farhat HI, Elhammady MS, Ziayee H, Aziz-Sultan MA, Heros RC: Eagle syndrome as a cause of transient ischemic attacks. J Neurosurg 110: 90e93, 2009 Fini G, Gasparini G, Filippini F, Becelli R, Marcotullio D: The long styloid process syndrome of Eagle syndrome. J Craniomaxillofac Surg 28: 123e127, 2000 Fritz M: Elongated styloid process: cause of obscure throat symptoms. J Craniomaxillofac Surg 28: 123e127, 1940 Gokce C, Sisman Y, Sipahioglu M: Styloid process elongation or Eagle syndrome: is there any role for ectopic calcification? Eur J Dent 2: 224e228, 2008 Harma RS: Clinical experiences of 52 cases. Acta Otolaryngol (Stockh) 224: 149e 155, 1967 Jan A: Stylohyoid syndrome. J Pak Med Assoc 39: 23, 1989 Karam C, Koussa S: Eagle syndrome: the role of CT scan with 3D reconstructions. J Neuroradiol 34: 344e345, 2007 Kaufman SM, Elzay RP, Irish EF: Styloid process variation. Radiologic and clinical study. Arch Otolaryngol 91: 460e463, 1970 Keur JJ, Campbell JP, McCarthy JF, Ralph WJ: The clinical significance of the elongated styloid process. Oral Surg Oral Med Oral Pathol 61: 399e404, 1986 Kim E, Hansen K, Frizzi J: Eagle syndrome: case report and review of the literature. Ear Nose Throat J 87: 631e633, 2008 Kim SM, Myoung H, Choung PH, Kim MJ, Lee SK, Lee JH: Metastatic leiomyosarcoma in the oral cavity: case report with protein expression profiles. J Craniomaxillofac Surg 37: 454e460, 2009 Kook YA, Lee SK, Son DH, Kim Y, Kang KH, Cho JH, et al: Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells. Cell Biol Int 33: 424e428, 2009 Lee SK, Kim YS, Lee YJ, Lee SS, Song IS, Park SC, et al: Transglutaminase 2 expression in the salivary myoepithelial cells of mouse embryo. Arch Oral Biol 50: 301e 308, 2005 Leong SC, Karkos PD, Papouliakos SM, Apostolidou MT: Unusual complications of tonsillectomy: a systematic review. Am J Otolaryngol 28: 419e422, 2007 Lorman JG, Biggs JR: The Eagle syndrome. Am J Roentgenol 140: 881e882, 1983 Mauney JR, Kaplan DL, Volloch V: Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion. Biomaterials 25: 3233e3243, 2004
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Osteogenetic changes in elongated styloid processes of Eagle syndrome patients Soung Min Kim a, Mi Hyun Seo a, Hoon Myoung a, Jin Young Choi a, Yeon Sook Kim b, Suk Keun Lee c, * a Department of Oral and Maxillofacial Surgery (Head: Prof. Jin Young Choi), Dental Research Institute, School of Dentistry, Seoul National University, Seoul, South Korea b Department of Dental Hygiene (Head: Prof. Yeon Sook Kim), Cheongju University, Cheongju, South Korea c Department of Oral Pathology (Head: Prof. Suk Keun Lee), College of Dentistry, Gangneung-Wonju National University, 123 Chibyun-dong, Gangneung 210702, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 November 2012 Accepted 13 September 2013
Abnormal elongation of the styloid process, or Eagle syndrome, can be painful, and is associated with differential diagnoses including cranio-facial malformations and vasculo-neurological disturbances. The precise molecular mechanism leading to styloid process elongation is unknown. In this study, elongated styloid processes with periosteal fibrous ligament tissue were obtained from three patients with Eagle syndrome and examined by immunohistochemical methods using different antisera. In all cases, marked bony deposition was found at the apex of the styloid process. The osteogenetic proteins, such as osteonectin, osteocalcin, BMP-2, BMP-4, and RANKL were strongly positive by immunohistochemistry in both the ligament fibers and the periosteal membrane attached to the styloid process apex. Staining for protective proteins, HO-1, HSP-70, and HSP-90 was also positive. These results suggest that styloid process elongation is related to increased expression of osteogenetic and protective proteins. Therefore, we propose that Eagle syndrome results from a protective response to increased tensile stress in the ligament attached to the styloid process, which could also signal osteogenetic protein expression in the periosteal fibrous tissue. Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Eagle syndrome Immunohistochemistry Osteogenetic proteins Styloid process Tensile stress
1. Introduction The stylohyoid complex is composed of the styloid process, the stylohyoid ligament and the lesser horn of the hyoid bone. The styloid process is a long cylindrical, cartilaginous extension of the temporal bone. Embryologically, the anatomical structures of the stylohyoid complex originate from Reichert’s cartilage, or the second branchial arch. The second branchial arch gives rise to the stylohyoid chain and contains potential ossification centers which eventually mineralize to varying degrees (Camarda et al., 1989a, 1989b). The normal styloid process length is approximately 20e 30 mm (Gokce et al., 2008), with a mean length of 21.6 mm on the right side and 21.2 mm on the left side. There are three groups of styloid processes: short (under 21 mm), normal (21e30 mm) and elongated (more than 30 mm). This classification is both
* Corresponding author. Tel.: þ82 33 640 2228; fax: þ82 33 642 6410. E-mail address: [email protected] (S.K. Lee).
biologically based and clinically relevant (Sokler and Sandev, 2001; Fini et al., 2000). In the general population, the frequency of an elongated styloid process is estimated to be 4%, of which only 4% show clinical manifestations. This suggests that the incidence of styloid syndrome is 0.16% (about 16,000 persons in Serbia). Deviation of the styloid process causes external or internal carotid impingement and pain that radiates along the arterial trunk (Petrovic et al., 2008a, 2008b). Styloid process deviation is considered important, because it is clinically similar to other painful cranio-facial syndromes, making diagnosis for treatment, difficult (Petrovic et al., 2008b). The American otolaryngologist Eagle was the first to describe this styloid syndrome in 1937. Eagle syndrome is a rare condition in which an elongated styloid process (more than 30 mm) impinges on adjacent anatomical structures (Mortellaro et al., 2002; Karam and Koussa, 2007). Ossification of the stylohyoid chain leads to a progressive decrease in elasticity and the potential for associated clinical symptoms. Theories about the stimulus for ossification include aging and reactive healing following surgical or
1010-5182/$ e see front matter Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2013.09.012
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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nonsurgical trauma (Gokce et al., 2008; Keur et al., 1986). The aetiologic triggering factor for Eagle syndrome has not yet been discovered. Two forms of Eagle syndrome exist: classic and vascular form. Patients with classic “Eagle syndrome” present with unilateral sore throat, dysphagia, tinnitus, unilateral facial and neck pain, and otalgia (Murtagh et al., 2001; Thot et al., 2000). In patients with vascular “Eagle syndrome”, the elongated styloid process is in contact with the extracranial internal carotid artery; this can cause compression while turning the head or dissection of the carotid artery, causing a transient ischemic accident or a stroke (Gokce et al., 2008). Classical stylohyoid syndrome is found after tonsillectomy and is characterized by pharyngeal, cervical, and facial pain and headache (Leong et al., 2007; Lorman and Biggs, 1983; Kim et al., 2008). Stylo-carotid syndrome is the consequence of irritation of the pericarotid sympathetic fibers and compression of the carotid artery (Bafaqeeh, 2000; Farhat et al., 2009; Chuang et al., 2007). Clinical manifestations are most frequent after headturning and neck compression. Eagle syndrome is defined as clinical symptoms of the neck with cervicofacial pain caused by an elongated styloid process that compresses neural and vascular structures including the glossopharyngeal nerve. Eagle syndrome can also cause stroke from carotid artery compression (Gokce et al., 2008). Eagle syndrome can be classified as an entrapment syndrome requiring neurosurgical attention (Karam and Koussa, 2007; Slavin, 2002). This study aimed to elucidate the molecular mechanism of styloid process elongation in Eagle syndrome through immunohistochemical detection of osteogenetic and protective proteins in the apical areas of surgically removed styloid processes. 2. Material and Methods Four elongated styloid processes including the periosteal fibrous ligament from three Eagle syndrome patients were studied with approval of the Institutional Review Board of Gangneung-Wonju National University (Table 1). One patient had surgery on both sides via an extraoral submandibular approach. After incision of the skin and identification and division of the posterior extension of the platysma muscle, blunt dissection was performed to divide the posterior border of the mandible and identify a portion of the external carotid artery. Immediately below the investing fascia of the external carotid or internal maxillary artery, the styloid process was identified and easily palpated. Once the fascia was removed from the surface of the styloid process, an incision was made in the periosteum, facilitating reflection of the periosteum and muscle attachments. The styloid process was removed near its base with dissection of the stylohyoid ligament at a point distal to the calcified portion. The wound was closed with a traditional layer suture. The other two patients had surgery on each side via an intraoral trans-tonsillar approach. With the patient in a hyperextended open-mouth position, a trans-tonsillar incision was made in the
Table 1 Eagle syndrome patients in this study. Age/ Symptoms Site gender Case 1 37/M (S2001-72a) Case 2 23/M (S2002-148) Case 3 41/M (S2008-452)
Operation F/U period Complications
Neck pain, Both Extraoral 2 years otalgia Neck pain Left Intraoral 2.5 years Neck pain, Right Intraoral dysphagia
2 years
None None None
a Biopsy number registered in the Department of Oral Pathology, GangneungWonju National University Dental Hospital.
pharyngeal mucosa, and the fascial plane was identified by blunt dissection of the involved muscle with a non-sharpened dissection scissors, palpating the hard tip of the calcified styloid process in the lateral pharyngeal space. Removed specimens were fixed in 10% neutral formalin and decalcified with 0.5-M EDTA solution (pH 8.0), embedded in paraffin, and cut into 4 m sections. Microsections were stained with hematoxylin and eosin, followed by immunohistochemical staining using antibodies against osteonectin, osteocalcin, BMP-2, BMP-4, RANKL, HO-1, HSP-70, or HSP-90. All immunostaining was performed with an indirect triple sandwich method as previously described (Lee et al., 2005; Kim et al., 2009). Background crossreaction was minimized by negative control staining without primary antibodies using the same procedures. Histological images of representative samples were captured with a digital camera (DP70Ò, Olympus Co., Japan). Each slide was evaluated for intensity of positive immunostaining, graded as þþþþ, þþþ, þþ, þ, þ/, , corresponding to extremely strong, strong, moderate, weak, rare and negative, respectively. A rare grade of þ/ was defined as a focal or questionable weakly positive signal. Images were analyzed and compared by ANOVA using SPSS for WindowsÒ (Version 12.0, SPSS Inc., USA) and mean values with 95% confidence intervals. Correlations were analyzed by Pearson’s correlation test (p < 0.05). 3. Results Removed styloid processes were mainly composed of compact bone with sparse marrow space. Processes were covered with a thin fibrous periosteal membrane that was thickened in the apical area (Figs. 1b1 and 3a1eb5). All four cases of elongated styloid processes showed active osteophytic bone deposition at the apical end with rare bony deposition on the lateral side (Figs. 1b1, 2a and 3c1ed5). Immunohistochemical localization of osteogenetic proteins and protective proteins was characteristic of the periosteal membrane, osteoid matrix, ligament fibers, and muscle bundles of each styloid process apex (Table 2). Immunoreaction for the osteogenetic protein BMP-2 was strongly positive in the apical periosteal membrane, and accentuated in the ligament fibers attached to the apical end of the styloid processes. Immunoreaction for RANKL was relatively weak in the periosteum but consistently positive in the ligament fibers attached to the apical end of the styloid processes (Figs. 1b2, c and 2b, i). Immunoreaction for BMP-4, a marker of chondroid differentiation, was rare in the apical area of the styloid processes (Fig. 2g). Staining for the bone matrix proteins osteonectin and osteocalcin was positive at the bone deposition site of the styloid process apex, with osteonectin staining strongly in the periosteum and osteoid matrix (Fig. 2d), and osteocalcin staining relatively weak in the periosteum but consistently positive in the bone deposition site of the styloid process apex (Fig. 2e). Staining for the protective proteins HSP-70 and HO-1 was clearly positive in the apical areas of the styloid processes. HSP-70 was strongly positive in the muscle fibers attached to the apical end and consistently positive in the periosteum near apical bone deposition (Fig. 2f). HO-1 was localized in the apical periosteum of the styloid processes (Fig. 2h). Both elongated styloid processes from one of the patients showed increased axial growth with multiple callus formation in the proximal areas of the styloid processes (Fig. 3). The callus of hyalinized chondroid tissue was clearly distinguishable by Masson trichrome stain (Fig. 3b2eb4), and contained hypertrophic chondrocytes (Fig. 3c2, c4, d2, d4). Multiple calluses appeared in the marrow space, growth plate-like thickening occurred in the proximal area of the styloid process (Fig. 3c, d). The growth plate-like callus consequently produced fibrous immature bone that stained
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 1. a: Panoramic view of Case 2 patient. Left elongated styloid process, irregularly curved (arrows) compared to the right. b1: Hematoxylin and eosin stain of styloid process mainly composed of compact bone (B) with sparse marrow space and thick fibrous periosteum (P) at the apical area. b2: Immunostaining for BMP-2, in the periosteal membrane of the styloid process, accentuated in the apical area (arrow). c: Immunostaining for BMP-2. c1: Higher magnification of square in panel b2. Thick fibrous bundles (arrows) attached to the styloid process apex staining for BMP-2. c2: Higher magnification of c1. BMP-2 in the fibrous periosteal tissue (arrow heads) and ligament fibers (arrows) at the center. c3: Higher magnification of c2. BMP-2 in ligament fibers (arrows). d: Immunostaining for RANKL. d1: Higher magnification of the square in b2. RANKL in the fibrous tissue at the apex of the styloid process. d2: Higher magnification of d1. RANKL in ligament fibers (arrows) in the center and peripheral fibrous tissue (arrow heads). d3: Higher magnification of d2. RANKL in ligament fibers (arrows; see BMP-2 in c3).
blue with Masson trichrome stain and aligned parallel to the axial direction of the styloid process (Fig. 3b2, d3, d5). 4. Discussion The incidence of Eagle syndrome is controversial (Rogers and Chang, 2007). Eagle estimated the incidence of an elongated styloid process in the general population to be 4%, with only 4% of those displaying symptoms (Fritz, 1940; Eagle, 1948, 1958, 1962). Kaufman et al. (Kaufman et al., 1970) reported elongated styloid processes in 28% of patients, while Correll et al. (Correll et al., 1979)
examined 1771 panoramic view radiographs and estimated an incidence of 18.2%, of which 93% exhibited bilateral elongation. Only a relatively small number of patients (8 of 1771) (Correll et al., 1979), exhibited symptoms related to Eagle syndrome, and most were unilateral (Harma, 1967; Jan, 1989). Although, styloid process elongation is not abnormal, styloid processes longer than 3 cm are generally considered to be elongated (Palesy et al., 2000). If an elongated styloid process is not adapted to regional neuromuscular functions, it will become symptomatic (Thot et al., 2000). Mineralization and elongation of the styloid process and Eagle syndrome show similar styloid elongation in which mineralization
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 2. Osteogenetic expression for Case 3. a: Hematoxylin and eosin stain. a1: Muscle bundles attached to the apical area of the styloid process. a2: Higher magnification of a1, with fibrous periosteum (P) and osteophytic bone formation (arrows) in the apical area. a3: Higher magnification of a2. Bone deposition and mineralization (arrows). b1: Immunostaining for BMP-2, localized to the periosteal membrane (P) (arrows). b2: Higher magnification of b1, fibrous periosteal tissue attached to bone (B) (arrows). c: BMP-4 immunostaining. d1: Osteonectin immunostaining in the periosteum of the styloid process apical area. d2: Osteonectin in osteoblasts (arrows) (B). e1: Osteocalcin immunostaining in the periosteum (P) and apical bone (B) of the styloid process. f1: HSP-70 immunostaining in the periosteal muscle (M) and apical new bone area. f2: Higher magnification of f1. HSP-70 in the proximal periosteum (arrows) of the styloid process apex, and apical end (arrow heads). g: HSP-90 immunostaining. h1: HO-1 immunostaining in the periosteal tissue (arrows) of the styloid process apical area. h2: Higher magnification of h1. HO-1 in the periosteal membrane (arrows) attached to the apical bone. i: RANKL immunostaining in the periosteal tissue of apical bone. j: Negative control staining without primary antibody.
of the stylohyoid ligament progresses to the styloid process of the temporal bone (Yetiser et al., 1997; Babad, 1995; Piagkou et al., 2009; Mupparapu and Robinson, 2005). A styloid process that is simply mineralized and elongated is different from Eagle syndrome in the symptoms displayed and the treatments sought. Mineralization and elongation of the styloid process is associated with unilateral or bilateral elongation of the styloid process that does not result in significant pain, discomfort, or limitation of neck
movement. Mineralization and elongation often remains asymptomatic until it is discovered on extraoral radiographs. Eagle syndrome involves pain and discomfort in the cervicofacial region resulting specifically from an elongated styloid process (Mupparapu and Robinson, 2005). Although the mechanism that causes an increase in tensile stress on the styloid ligament is unclear, this study demonstrated that the fibrous ligament attached to the styloid process apex is
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Fig. 3. Axial growth of styloid process in Case 1. a and b: Right, c and d: Left, a and c: Hematoxylin and eosin, b and d: Masson trichrome stain. a1 and b1: Two growth plate-like calluses (arrows) and an intramedullary callus (*) in the axis of the styloid process. a2ea4 and b2eb4: Callus (*) of chondrocytes. a5 and b5: Apical bony growth. c1 and d1: Axial growth with a growth plate-like callus (arrows) and multiple intramedullary calluses (*). c1, c4, d2, d4: Callus of hypertrophic chondrocytes. c3, c5, d3, d5: Fibrous immature bone (IB, blue) formed by endochondral ossification.
immunoreactive for HSP-70 and HO-1, which are known to be related to mechanical stress. Immunoreaction was coincident with positive staining for the osteogenetic proteins BMP-2, RANKL, osteonectin, and osteocalcin, in conjunction with limited reaction for BMP-4, a marker of chondrogenic cells, and HSP-90, a marker for osteoclasts (Sojo et al., 2005; Chaves et al., 2012; Ramazanoglu et al., 2013). HO-1 is involved in vascular disease, transplantation, and inflammation (Alcaraz et al., 2000; Orozco et al., 2007; Takahashi et al., 2004) and negatively regulates osteoclastogenesis, leading to upregulation of osteogenic differentiation for new bone deposition (Zwerina et al., 2005; Kook et al., 2009). In our
study, dense staining for HO-1 in the apical periosteum of the styloid processes suggested upregulation of osteogenesis in the apical ends of the styloid processes in Eagle syndrome. The major protective stress response protein, HSP-70, is expressed in response to stress as well as to osteogenic differentiation potential (Mauney et al., 2004; Tiffee et al., 2000). The strong positive reaction for HSP70 in the apical end of the styloid processes in our study could indicate active new bone deposition incited by mechanical stress, likely tensile stress transmitted through ligament fibers. The exact etiology of styloid process elongation and the role of ectopic calcification, which might be involved in styloid process
Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012
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Table 2 Immunohistochemical findings for elongated styloid processes in Eagle syndrome patients. Area Apical end
Lateral surface
BMP-2 RANKL Osteonectin Osteocalcin HSP-70 PM þ (4)a þ/ (2) þ (1) OR þ (4) (1) þ (2) LF þþ (4) þ (4)
MB þ/ (2) þ (1)
(4)
þ (4)
þ/ (2) þ (1)
HSP-90 HO-1
þþ (2)
þ (2)
þþ (2)
þþþ (1) þþ (4)
þþ (1) þ (4)
þþþ (4)
þ (2) þþ (1)
þ (2)
þ (4)
þþþ (1) þ/ (1) þþ (2) (4) þ (4) þþþ (1) þþ (4) (4) þ (2) þþ (1) þþ (2) (2) þ (2)
þþ (1)
(2)
þ (4)
þþþ (1) þ/ (1) þþ (1)
þ (2)
þ/ (2)
þþ (1)
þ (1)
þ (4)
(4)
(2) þ/ (1)
Abbreviations: PM, periosteal membrane; OR, osteoid matrix; LF, ligament fiber; MB, muscle bundle (: negative, þ/: rare, þ: mild, þþ: moderate, þþþ: severe in histological observation). a Expressed case number from 4 cases.
elongation, are still unknown. Abnormal metabolism of calcium, phosphorus and vitamin D is common in patients with end-stage renal disease. This calcification in non-osseous soft tissue related to abnormal serum calcium and phosphorus levels could be associated with Eagle syndrome (Gokce et al., 2008). In our study, osteophytic bone deposition in the apical end of the styloid processes was irregular and dystrophic in its calcification. The osteogenetic proteins BMP-2, RANKL, osteonectin, and osteocalcin were widely distributed in the fibrous periosteal membrane, ligament fibers, and osteoid matrix at the styloid process apices. These results suggest that styloid process elongation can be accelerated by ectopic calcification originating from systemic diseases, especially end-stage renal disease, and that the dystrophic nature of ectopic calcification could be related to the greater incidence of Eagle syndrome in patients older than 40 years (Keur et al., 1986; Thot et al., 2000). The developmental origin of the styloid process is the cartilaginous tissue (Camarda et al., 1989a, 1989b). Therefore, elongation of the styloid process might tend to result in callus-induced endochondral ossification rather than intramembranous bone ossification after an, osteogenetic signal such as a tensile stress on the styloid process. The bilateral elongation of the styloid process in the Case 1 patient showed dominant callus formation on axial growth but relatively weak osteophytic bony deposition on the apical growth. Multiple intramedullary calluses and growth plate-like endochondral ossification appeared in the proximal styloid process. This supports the hypothesis that the styloid process has the potential for callus-induced endochondral ossification for axial growth and intramembranous ossification for apical growth after application of tensile stress from the stylohyoid ligament. More studies should be needed with large numbers of Eagle syndrome patients in order to elucidate the different mechanisms of styloid process elongation in Eagle syndrome. However the results of this study suggest that apical and axial growth of styloid processes is directly affected by tensile stress induced by the pulling forces of the stylohyoid ligament. 5. Conclusion Our study examined elongated styloid processes from patients with Eagle syndrome. We used histological and immunohistochemical methods to evaluate the expression of osteogenetic and
protective proteins. We found that osteophytic bone deposition in the apical end of the styloid process was closely related to the expression of osteogenetic and protective proteins. We propose that Eagle syndrome results from a protective response to increased tensile stress on the ligament attached to the styloid process, which may signal osteogenetic protein expression in the periosteal fibrous tissue of the styloid process. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20120002538, -0002997). References Alcaraz MJ, Habib A, Lebret M, Creminon C, Levy-Toledano S, Maclouf J: Enhanced expression of haem oxygenase-1 by nitric oxide and antiinflammatory drugs in NIH 3T3 fibroblasts. Br J Pharmacol 130: 57e64, 2000 Babad MS: Eagle syndrome caused by traumatic fracture of a mineralized stylohyoid ligament e literature review and a case report. Cranio 13: 188e192, 1995 Bafaqeeh SA: Eagle syndrome: classic and carotid artery types. J Otolaryngol 29: 88e94, 2000 Camarda AJ, Deschamps C, Forest D: I. Stylohyoid chain ossification: a discussion of etiology. Oral Surg Oral Med Oral Pathol 67: 508e514, 1989a Camarda AJ, Deschamps C, Forest D: II. Stylohyoid chain ossification: a discussion of etiology. Oral Surg Oral Med Oral Pathol 67: 515e520, 1989b Chaves MD, de Souza Nunes LS, de Oliveira RV, Holgado LA, Filho HN, Matsumoto MA, et al: Bovine hydroxyapatite (Bio-OssÒ) induces osteocalcin, RANK-L and osteoprotegerin expression in sinus lift of rabbits. J Craniomaxillofac Surg 40: e315e320, 2012 Chuang WC, Short JH, McKinney AM, Anker L, Knoll B, McKinney ZJ: Reversible left hemispheric ischemia secondary to carotid compression in Eagle syndrome: surgical and CT angiographic correlation. Am J Neuroradiol 28: 143e 145, 2007 Correll RW, Jensen JL, Taylor JB, Rhyne RR: Mineralization of the stylohyoidestylomandibular ligament complex. A radiographic incidence study. Oral Surg Oral Med Oral Pathol 48: 286e291, 1979 Eagle WW: Elongated styloid process; further observations and a new syndrome. Arch Otolaryngol 47: 630e640, 1948 Eagle WW: Elongated styloid process; symptoms and treatment. AMA Arch Otolaryngol 67: 172e176, 1958 Eagle WW: The symptoms, diagnosis and treatment of the elongated styloid process. Am Surg 28: 1e5, 1962 Farhat HI, Elhammady MS, Ziayee H, Aziz-Sultan MA, Heros RC: Eagle syndrome as a cause of transient ischemic attacks. J Neurosurg 110: 90e93, 2009 Fini G, Gasparini G, Filippini F, Becelli R, Marcotullio D: The long styloid process syndrome of Eagle syndrome. J Craniomaxillofac Surg 28: 123e127, 2000 Fritz M: Elongated styloid process: cause of obscure throat symptoms. J Craniomaxillofac Surg 28: 123e127, 1940 Gokce C, Sisman Y, Sipahioglu M: Styloid process elongation or Eagle syndrome: is there any role for ectopic calcification? Eur J Dent 2: 224e228, 2008 Harma RS: Clinical experiences of 52 cases. Acta Otolaryngol (Stockh) 224: 149e 155, 1967 Jan A: Stylohyoid syndrome. J Pak Med Assoc 39: 23, 1989 Karam C, Koussa S: Eagle syndrome: the role of CT scan with 3D reconstructions. J Neuroradiol 34: 344e345, 2007 Kaufman SM, Elzay RP, Irish EF: Styloid process variation. Radiologic and clinical study. Arch Otolaryngol 91: 460e463, 1970 Keur JJ, Campbell JP, McCarthy JF, Ralph WJ: The clinical significance of the elongated styloid process. Oral Surg Oral Med Oral Pathol 61: 399e404, 1986 Kim E, Hansen K, Frizzi J: Eagle syndrome: case report and review of the literature. Ear Nose Throat J 87: 631e633, 2008 Kim SM, Myoung H, Choung PH, Kim MJ, Lee SK, Lee JH: Metastatic leiomyosarcoma in the oral cavity: case report with protein expression profiles. J Craniomaxillofac Surg 37: 454e460, 2009 Kook YA, Lee SK, Son DH, Kim Y, Kang KH, Cho JH, et al: Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells. Cell Biol Int 33: 424e428, 2009 Lee SK, Kim YS, Lee YJ, Lee SS, Song IS, Park SC, et al: Transglutaminase 2 expression in the salivary myoepithelial cells of mouse embryo. Arch Oral Biol 50: 301e 308, 2005 Leong SC, Karkos PD, Papouliakos SM, Apostolidou MT: Unusual complications of tonsillectomy: a systematic review. Am J Otolaryngol 28: 419e422, 2007 Lorman JG, Biggs JR: The Eagle syndrome. Am J Roentgenol 140: 881e882, 1983 Mauney JR, Kaplan DL, Volloch V: Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion. Biomaterials 25: 3233e3243, 2004
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Please cite this article in press as: Kim SM, et al., Osteogenetic changes in elongated styloid processes of Eagle syndrome patients, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.09.012