International Journal of Pediatric Otorhinolaryngology 133 (2020) 109973 Contents lists available at ScienceDirect International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl 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 carti- lage 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 sur- rounds 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 fe- tuses 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]).