Development and Growth of Auricular Cartilage and Muscles a Study

Home , Antihelix, Antitragus, Auricle (anatomy), Helix (ear), Intertragic notch, Tragus (ear)

International Journal of Pediatric Otorhinolaryngology 133 (2020) 109973

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International Journal of Pediatric Otorhinolaryngology

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Development and growth of auricular cartilage and muscles: A study using T human fetuses ∗ Yohei Honkuraa, Shogo Hayashib, , Ji Hyun Kimc, Gen Murakamid, Hiroshi Abee, José Francisco Rodríguez-Vázquezf, Yukio Katoria a Department of Otolaryngology-Head and Neck Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan b Department of Anatomy, School of Medicine, International University of Health and Welfare, Narita, Japan c Department of Anatomy, Chonbuk National University Medical School, Jeonju, South Korea d Division of Internal Medicine, Jikou-kai Clinic of Home Visits, Sapporo, Japan e Department of Anatomy, Akita University School of Medicine, Akita, Japan f Department of Anatomy and Embryology, School of Medicine, Complutense University, Madrid, Spain

ARTICLE INFO ABSTRACT

Keywords: Objectives: The auricle is a key target in pediatric plastic surgery and is considered to develop from a ring- or Auricle funnel-like arrangement of six hillocks in the embryo. However, there has been no report showing the Auricular anterior muscle morphologies of the auricular muscle and cartilage after midterm in humans. Cartilage Methods: We examined histological sections of 20 near-term human fetuses (29–40 weeks) and those from 7 Helices major muscle midterm fetuses (15–16 weeks). Human fetus Results: At midterm, the auricular cartilage was a single wavy plate with the helicis major muscle (HMM). The Tragus superior and posterior auricular muscles (SAM, PAM) were inserted into the middle parts, and the anterior auricular muscle (AAM) was inserted into the lowest part of the cartilage plate, while the tragus and antitragus were not clearly identified. In near-term fetuses, the cartilage plate varied in size and shape between specimens. The scapha and antihelix were separated from the cartilage plate with major or minor involvement of the HMM from the initial mass along the helix. The SAM inserted to the crus helix or the developing scapha, while the insertion sites of the AAM and PAM into the helix were stable. The tragus–antitragus cartilages were well- developed and they sandwiched a deep notch of skin below the helix tail. The antitragicus muscle was more evident than the tragicus muscle. An unnamed muscle was evident along the external acoustic meatus. The other intrinsic muscles, including the transverse and oblique muscles, might develop from the HMM after birth. Conclusions: Development of the auricle was advanced after midterm. However, a single wavy plate-like cartilage was maintained until late-stage. Near term, the antihelix and scapha developed from the plate-like core of the auricle and the tragus and antitragus were added in the antero-inferior side of the cartilage plate. Establishment of muscle arrangements was markedly delayed compared to cartilage development. Altogether, the classical concept of an initial funnel-like arrangement of cartilage anlagen might have been biased by studies of adult morphology.

1. Introduction distribution of intrinsic muscles near term and in newborns were shown by Oda [1], who examined not only 14 adult specimens, but also spe- Although the auricle is one of the major targets of pediatric plastic cimens from 3 newborns and 7 near-term fetuses. However, in his in- surgery, anatomical information about it in newborns and children tricate line-drawings, the shapes of the growing auricular cartilage were seems to be limited to its external views. There are no or few reports depicted in the same way, irrespective of the age of the specimens, and providing topographical information of the internal cartilages and no attention seemed to have been paid to a likely difference in the muscles. Insertions of the extrinsic auricular muscle and detailed shape of the growing cartilage.

∗ Corresponding author. Department of Anatomy, School of Medicine, International University of Health and Welfare, 4-3 Kozunomori, Narita, Chiba, 286-8686, Japan. E-mail addresses: [email protected] (Y. Honkura), [email protected] (S. Hayashi), [email protected] (J.H. Kim), [email protected] (G. Murakami), [email protected] (H. Abe), [email protected] (J.F. Rodríguez-Vázquez), [email protected] (Y. Katori). https://doi.org/10.1016/j.ijporl.2020.109973 Received 17 October 2019; Accepted 27 February 2020 Available online 03 March 2020 0165-5876/ © 2020 Elsevier B.V. All rights reserved. Y. Honkura, et al. International Journal of Pediatric Otorhinolaryngology 133 (2020) 109973

although the size is markedly smaller than that in adults. Therefore, at midterm, a ring- or funnel-like arrangement of cartilage anlagen surrounds the future concha or an opening of the external acoustic meatus (Fig. 1B). Notably, according to the classical concept, a wide intertragic notch corresponds to an early space between the anterior and posterior ends of the initial cartilage plate (or a site between hillocks-1 and -6; Fig. 1A). Consequently, we aimed to describe the topographical anatomy of the cartilage and muscles using auricular specimens from human fetuses at midterm and near-term. Although the shape of cartilage is most likely to change markedly during the late fetal period, we hypothesized that extrinsic muscle insertions as well as intrinsic muscle attachments (Table 1) would be stable and unchanged on the growing auricular cartilage. Therefore, we expected that muscle insertion and attachments could be used as landmarks on the developing cartilage. In the present study, we first compared the auricular morphology between midterm and near-term human fetuses, and then identified parts of the growing cartilage according to its topographical relationship with extrinsic and intrinsic muscles. Fig. 1. Schematic representation of auricular cartilage development: A classical concept. Panel A displays the initial morphology in embryos; panel B displays the 2. Materials and methods morphology at midterm. Six hillocks (Nos. 1–6) are the anlagen of the auricular cartilages. A wide in- The study was performed in accordance with the provisions of the tertragic notch forms as an opening of the semicircular cartilage (arrow in panel Declaration of Helsinki of 1995 (as revised in Fortaleza in 2013). We A). The helix (colored red) corresponds to a superficial margin of the funnel- examined paraffin-embedded histological sections from 20 near-term shaped cartilage. The antihelix (colored yellow) develops from hillocks 3–5 at or until midterm. (For interpretation of the references to color in this figure fetuses (approximately 29–40 weeks of gestational age; 243–330 mm legend, the reader is referred to the Web version of this article.) crown–rump length [CRL]) and 7 midterm fetuses (CRL 113–125 mm; approximately 15–16 weeks). All near-term fetuses formed part of the collection of the Department of Anatomy, Akita University, Akita, Japan. These speci- Table 1 mens had been donated by the families to the Department from 1975 to Muscle insertions to the auricular cartilage. 1985 and had been preserved in 10% (w/w) neutral formalin solution (Extrinsic muscles) for more than 30 years. The available data were limited to the date of Anterior auricular muscle spine of the helix donation and gestational age, and there was no information on family Superior auricular muscle upper part of auricle via a tendon name, the name of the obstetrician or hospital, or the reason for Posterior auricular muscle ponticulus on the conchal eminence abortion. The use of these fetuses for research was approved by the ethics committee of Akita University (No. 1428). (Intrinsic muscles) The left or right auricle was removed from the fetus's head, taking Helicis major muscle anterior margin of the helix care not to detach the lateral part of the external acoustic meatus from Helicis minor muscle crus helicis the specimen. After routine procedures for paraffin-embedded his- Tragicus muscle tragus tology, we prepared semi-serial sections (100–200-μm interval; 10-μm Antitragicus muscle antitragus to the tail of helix Transverse auricular muscle conchal eminence thickness) and stained these with hematoxylin and eosin (HE). The Oblique auricular muscle conchal eminence to triangular eminence sectional planes were horizontal (12 fetuses) or frontal (8 fetuses). A specimen for horizontal (or frontal) sections was divided into upper and According to Gray's Anatomy edited by Williams (1995). lower (or anterior and posterior) halves. Thus, we made two paraffin blocks for each of the auricles. Serial sections (5–7-μm thick) from 7 midterm fetuses were re- Previous studies on development of the auricle seemed to con- trieved from a part of the large collection maintained at the Department centrate on the contributions of six hillocks originating from the first of Anatomy and Embryology, School of Medicine, Universidad and second pharyngeal arches [2–7]. In short, hillock-1 is considered to Complutense, Madrid. These fetuses were donated after miscarriages differentiate into the tragus, hillock-2 into the crus helicis, hillock-3 and ectopic pregnancies at the Department of Obstetrics of that uni- into the ascending helix, hillocks-4 and 5 into the helix, scapha, and versity. Four of the 7 fetuses had been sectioned frontally, and the other antihelix, and hillock-6 into the helix and antitragus. Fusion of these 3 horizontally. Most sections were stained with HE, while some were hillocks occurs at 6–8 weeks of development (Fig. 1A) and it is followed stained with azan, orange G, or silver stain. The use of the midterm by changes in the topographical relationship between parts of the specimens was approved by Complutense University ethics committee auricle as a result of differential growth, i.e., there is a considerable (B08/374). difference in the growth rate between parts (reviewed by Kagurasho All observations and photographs were obtained with a Nikon et al. [8]). According to an excellent review by Moneta and Quintanilla- Eclipse 80 camera (Nikon, Minato, Japan). Dieck [9], the final topographical anatomy is established at midterm,

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Fig. 2. Horizontal sections of a specimen of CRL 115 mm (approximately 15 weeks). Panel A displays the most inferior site in the figure, while panel H shows the most superior site. Intervals between panels are 0.2 mm (A–B, B–C), 0.4 mm (C–D, D–E, E–F, F–G) and 0.5 mm (G–H), respectively. Panel A shows the external acoustic meatus (EAM) filled with a cornifi- cation plug. Panel B exhibits the tragus and antitragus, but the intrinsic muscle is concentrated on the antitragus. The superior and posterior auricular muscles (SAM, PAM) provide a muscle bundle, respectively (panels C and D), whereas sparsely-dis- tributed muscle fibers attach to a tubercle of the cartilage in the anterior auricular muscle (AAM; panel E). The helicis major muscle (HMM) is seen in the upper half of the auricle (panels D–F). Panel I is a schematic representation of the shape of auricular cartilage. The posterolateral thickness is made by a curved inferolateral end of the cartilage including the crus of helix. Panels A–H were prepared at the same magnification (scale bar in panel A, 1mm).

3. Results portion of the cartilage plate. Because of these tubercles, as well as due to the wavy helix, there was a posterolateral thickening in the cartilage 3.1. Observations of midterm fetuses plate (Fig. 2i). Insertions of extrinsic auricular muscles, i.e., the superior, anterior, The auricle at midterm was identified as a short superolateral pro- and posterior auricular muscles (SAM, AAM, and PAM) were arranged jection at the temporal region of the head. Auricular cartilages were in the antero-inferior half of the cartilage plate. The SAM was inserted identified as a wavy plate extending downward from the skin projection at the lower portion of the cartilage plate, the PAM was inserted at a to the subcutaneous tissue, along the temporal bone or temporalis site immediately above the SAM, and the AAM was inserted at the muscle (Figs. 2 and 3). The cartilage plate demonstrated two thick parts anterior end of the mid-portion of the cartilage plate. Conversely, the or tubercles: 1) an inferior tubercle near the external acoustic meatus, extrinsic muscle insertions were not seen in the skin projection. They which possibly corresponded to the tragus–antitragus cartilages were located within 0.6–1.0 mm in the supero-inferior axis. The SAM (Fig. 2B) and 2) another tubercle at the base of the skin projection, insertion carried a long tendon (Figs. 2C and 3A), while the PAM in- which possibly corresponded to the future crus of the helix (Fig. 2D). sertion was identified as an attachment of a fan-like or band-like array Thus, the candidate for the crus of the helix was located at the mid- of muscle fibers (Figs. 2D and 3B).

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Fig. 3. Development and growth of the auricular cartilage from 15 weeks to 31 weeks: Frontal sections of two specimens. Panel A–C, a specimen of CRL 116 mm (approximately 15 weeks); panels D–H, CRL 256 mm (approximately 31 weeks). Panel A or D displays the most anterior site in the specimen. Intervals between panels are 2.8 mm (A–B), 1.2 mm (B–C), 1.4 mm (D–E), 3.2 mm (E–F), 3.5 mm (F–G) and 1.6 mm (G–H), respectively. At midterm, a wavy cartilage plate is C-shaped at the base of the skin projection (panels B and C) and a distinct fascia (arrows) suggests a medial margin of the future auricle. Near term, a long part of the cartilage plate is contained in the skin projection (panels G and H), but the scapha and antihelix are remained in the temporal subcutaneous tissue (panel D). A series of several loops continues to the external acoustic meatus (EAM). Panels A–C and panels D–H were prepared at the same magnification, respectively (scale bars in panels A and E, 1 mm). AAM, anterior auricular muscle; crus, crus of the helix; HMM, helix major muscle; PAM, posterior auricular muscle; SAM, superior auricular muscle.

In addition, two clusters of intrinsic auricular muscles were seen: 1) term fetuses. We did not evaluate a likely reduction in size by fixation the helicis major muscle along the upper half of the cartilage plate and dehydration. (Fig. 2D–F and 3A), and 2) the antitragicus muscle along the posterior As at midterm, the auricle of near-term fetuses was divided into an surface of the antitragus cartilage (Fig. 2B). upper part in the skin projection (Fig. 3F–H) and a lower part in the temporal subcutaneous tissue. The former was relatively simple and 3.2. Observations of near-term fetuses contained a wavy cartilage plate, while the latter subcutaneous part carried a complex of tubercles on the wavy cartilage plate, continuous Fig. 3 (frontal sections) shows the marked difference (ca. with the external auricular meatus cartilage (Fig. 3D–G). The skin 3 mm–12 mm) in height between auricles at midterm and near-term. A projection was identified as a posterior part of the auricle in horizontal height or a supero-inferior length from the top of the helix to the tra- sections (Figs. 4 and 6). Conversely, the subcutaneous or anterior part gus–antitragus was measured in frontal sections from 8 near-term fe- contained 1) a large tubercle of the crus helix; 2) the developing anti- tuses, while the width along the antero-posterior axis of the cartilage helix and scapha (Fig. 4), 3) extrinsic muscle insertions (Figs. 5 and 6), plate was obtained in horizontal sections from 12 fetuses (Figs. 4–7). and 4) the tragus–antitragus cartilages near the tail of the helix (Fig. 7). Although a greater height or width tended to be found in larger fetuses, The tragus cartilage was connected with the antitragus cartilage; individual variations were seen in similar sized fetuses. The maximum both were connected with and located below the tail of the helix car- height ranged from 11.5 mm (in a fetus of CRL 243 mm) to 16.0 mm (in tilage. The tragus–antitragus cartilages showed a Y-shaped arrangement a fetus of CRL 275 mm), while the maximum width varied between in horizontal sections (Fig. 7) and they sandwiched an intertragic in- 12.3 mm (in a fetus of CRL 258 mm) and 19.7 mm (in a fetus of CRL cisura, or notch of the skin, which faced the opening of the external 328 mm). Thus, the height and width did not differ markedly in near- acoustic meatus (Fig. 7BD). Thus, the tragus and antitragus were

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Fig. 4. Separation of the scapha and antihelix from the major part of the auricular cartilage, including the helix. Panels A–C displays a specimen of CRL 245 mm (approximately 30 weeks), while panels D–F CRL 282 mm (approximately 33 weeks). A loop along the internal or medial side of the cartilage plate or helix appears to provide an independent ring of cartilage, i.e., the future scapha and antihelix. During the process, a large or small part of the helix major muscle (HMM) is incorporated into the scapha side (arrowheads). All panels were prepared at the same magnification (scale bars in panel A, 1 mm). AM, anterior auricular muscle.

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auricular cartilage, including the helix. Notably, the developing antihelix, rather than the established helix, often received extrinsic auricular muscles. As in midterm fetuses, in near-term fetuses, a long and thick tendon was evident for the SAM insertion, in contrast to attachments of muscle fibers at insertions of the AAM and PAM(Figs. 5 and 6). Notably, near term, we found individual variations in the SAM insertion site in horizontal sections of 12 specimens: 1) at or near the crus of the helix (2 specimens; Fig. 5A), 2) a posterior part of the cartilage plate immediately above the crus of the helix (2 specimens; Fig. 5B), 3) the developing antihelix (8 specimens; Fig. 5C–E). The PAM insertion was located near the SAM insertion. Thus, the PAM also often inserted into the developing antihelix. Whether the former was anterior or posterior to the latter varied between specimens; the PAM insertion was located on the anterior side of the SAM insertion when the SAM inserted into a site at or near the crus of the helix (Fig. 5A and B). In contrast to the SAM, the AAM insertion site was invariably at the anterior end of the wavy cartilage plate (Fig. 6); it was sometimes comprised of two muscle bundles (Fig. 6A,D). Those three muscle insertions were located within 2.0 mm in the supero-inferior axis, but in the antero-posterior axis, the SAM insertion was located 3.5–8.0 mm posterior to the AAM insertion. Three clusters of intrinsic auricular muscles were evident: 1) the helicis major muscle along the upper half of the cartilage plate (Fig. 4A–C, 5A and B, and 6A and C), 2) the antitragicus muscle (Fig. 7C,E), and 3) an unnamed muscle mass attaching to the infero- posterior end of external acoustic meatus cartilages (Fig. 5A and B and 7A). The helicis major muscle consistently extended along the internal aspect of the helix, rather than along the external aspect or skin side. The helicis major muscle sometimes extended inferiorly to reach the crus of the helix (Fig. 6C); it appeared to correspond to a future helicis minor muscle. Notably, the helicis major muscle was likely to extend along the developing scapha and antihelix; this muscle cluster appeared to be separated from the major part along the helix (Fig. 4). Moreover, in some cases, the major part was markedly smaller than the separated muscles along the developing antihelix (Fig. 4D and E). In addition, ligaments at the crus of the helix and the antitragus were absent. The unnamed muscle along the external acoustic meatus extended medially Fig. 5. Individual variations in insertion site of the superior and posterior or deeply, and it was never connected with either the helicis major auricular muscles in horizontal sections from five near-term fetuses. muscle or the antitragicus muscle. We did not observe the transverse Panel A, a specimen of CRL 284 and oblique auricular muscles in these specimens. mm (approximately 33 weeks); panel B, CRL 282 mm (approximately 33 weeks); panel C, CRL 330 mm (approximately 40 weeks); panel D, CRL 248 4. Discussion mm (approximately 30 weeks); panel E, CRL 258 mm (approximately 31 weeks). A sequence of variations are seen in insertions The present study provided photographic demonstrations of auri- of the superior auricular muscle (SAM): at the crus of helix in panel A, near the cular cartilages and muscles in near-term human fetuses, which have crus in panel B, at the base of the developing scapha in panel C, at the mid–- not been reported to date. We provide evidence of the auricular mor- portion of the scapha in panel D and, near the top of the scapha in panel E. phology at birth; the skeleton of the auricle was most likely a markedly Insertion of the posterior auricular muscle (PAM) was seen in a section near or wavy cartilage plate with tubercles and a loop. Near term, a major part same as each of panels. Arrowheads in panels A and B indicate an unnamed of the plate was still embedded in the temporal subcutaneous tissue in muscle along the external acoustic meatus cartilage. All panels were prepared at contrast to a minor part projecting out of the skin. In the final auricle, the same magnification (scale bar in panel A, 1 mm). crus of the helix;HMM, there should be two large fossae or hollows: the scapha and concha. We helix major muscle; PAM, posterior auricular muscle; SAM, superior auricular muscle. propose a separation process from the helix to the antihelix at near term. Thus, the funnel-shaped scapha was surrounded by a primitive antihelix, which was absent at midterm. Even at near term, the concha was not fully developed and, instead, the tragus–antitragus cartilages unlikely to develop from different ends of the initial cartilage plate. A occupied the lower half of the auricle below the helix tail. Thus, the connection was seen between the tragus–antitragus cartilage complex conchal eminence, including the ponticulus, seemed to develop after and the external acoustic meatus cartilage (Fig. 2AB and 3 EF). In all birth. Therefore, the final insertion of the PAM into the ponticulus near-term specimens, a small or large loop of the cartilage plate created (Table 1) seemed to be established by “moving” from a site near the an independent ring of cartilage; the latter possibly corresponded to the SAM insertion (a change in muscle insertion: see the final paragraph of future scapha and antihelix (Fig. 4). Thus, the scapha and antihelix the Discussion). were most likely to be secondarily separated from major parts of the In the classical concept of auricular development, after midterm, a

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Fig. 6. Insertion site of the anterior auricular muscle is always located at the anterior end of the helix, without individual variation. Panel A, a specimen of CRL 328 mm (approximately 40 weeks); panel B, CRL 282 mm (approximately 33 weeks); panel C, CRL 258 mm (approximately 31 weeks); panel D, CRL 245 mm (approximately 30 weeks). Panel E is a higher magnification view of a square in panel C.The anterior auricular muscle often divides into two muscle bundles near the insertion (panels A and D). The muscle fibers insert into or intermingle witha fascia wrapping along the anterior end of the helix (arrows in panels B and E). The asterisk in panel C indicates an artificial space created during the histological procedure. Panels A–D were prepared at the same magnification (scale bars in panels Band E, 1 mm). AAM, anterior auricular muscle; crus, crus of the helix; HMM, helix major muscle.

ring- or funnel-like arrangement of cartilage anlagen surrounds the funnel-shaped arrangement of cartilages around the concha seemed to future concha or an opening of the external acoustic meatus (Fig. 1). be established after birth. The intertragic notch should also be evident at midterm. However, we We initially considered the extrinsic muscle insertions suitable as were not able to find either a funnel-like helix cartilage or apairof landmarks for identification of parts of the growing cartilage. However, independent cartilages (i.e., tragus and antitragus). An angled or con- we could not deny the possibility that the muscle insertions “move,” cave area on the cartilage plate at midterm was unlikely to correspond depending on changes in cartilage shape; this mechanism has been to either the concha or the intertragic notch, since it was distant from termed “differential growth” (reviewed by Kagurasho et al.[8]). Since the external acoustic meatus. Near term, the tragus–antitragus carti- the AAM continued on to a thick fascia wrapping along the helix at and lages were connected mutually and the intertragic notch was re- near its insertion, the muscle insertion seemed to be liable to move. We cognized as a deep groove in the covering skin, corresponding to an demonstrated that the developing antihelix often showed insertions of acute curve of the tragus–antitragus cartilage complex. A funnel-like the SAM and PAM; these are also likely to move, because the antihelix arrangement of cartilages in the classical concept of embryology might never receives extrinsic muscles in adults. Likewise, the helicis major be biased by the adult morphology, but the tragus and antitragus car- muscle separated from the initial muscle mass along the helix and tilages are connected at the lower end of the funnel, even in adults. might form the transverse and/or oblique auricular muscles on the in- In their study of variations of cartilages surrounding the human ternal or medial aspects of the auricular cartilage. The suggested external acoustic meatus, Ikari et al. [10] demonstrated that the tra- change in muscle origin or insertion depending on cartilage growth is gus–antitragus as well as the crus of the helix is located near the lateral similar to that of the inferior pharyngeal constrictor. Hayashi et al. [11] end of the external auditory meatus cartilages. As also found in the reported that, depending on growth of the cartilage protrusion on the present study, the concha was poorly developed even in CRL 255- and thyroid cartilage, the muscle insertion changes from the sternothyr- 275-mm specimens in their study. A connection between the tragu- oideus muscle bundle, via the cricothyroideus muscle surface, to the s–antitragus cartilage complex and the external acoustic meatus carti- linea obliqua and the inferior cornu of the thyroid cartilage. lage seemed to be important for development of the final concha. A

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Fig. 7. Tragus and antitragus cartilages at the infero-anterior end of the helix. Panels A–C displays a specimen of CRL 245 mm (approximately 30 weeks), while panels D–F shows a specimen of CRL 282 mm (approximately 33 weeks). The tragus and antitragus cartilages in combination with the tail of the helix takes on a reversed Y–shaped arrangement, including the intertragic incisura or notch. The antitragicus muscle is well developed, while little or no tragicus muscle can be seen. A lateral end of the cartilages along the external acoustic meatus (EAM) carries a well-developed muscle (star in panels A and B). All panels were prepared at the same magnification (scale bars in panel A, 1 mm). ATM, antitragicus muscle.

Funding Acknowledgements

This study was supported by Grants-in Aid for Scientific Research We are grateful to Ms. Hisako Shibata (Department of (No.18H02949 for Yukio Katori and No.18K16826 for Yohei Honkura) Otolaryngology-Head and Neck Surgery, Tohoku University Graduate from the Ministry of Education, Culture, Sports, Science, and School of Medicine, Japan) for her preparation of histological sections. Technology, Japan. References

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