Annals of Anatomy 254 (2024) 152246 Available online 8 March 2024 0940-9602/© 2024 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). Growing stylohyoideus muscle insertion to the hyoid bone with special reference to its topographical relation to the intermediate tendon of digastricus muscle: A histological study using human fetuses Kei Kitamura a,*,1,2, Ryu Suzuki b,1, Satoshi Ishizuka c,3, Gen Murakami b,d, Jose Francisco Rodríguez-Vázquez e,4, Hitoshi Yamamoto a, Shin-ichi Abe b,5 a Department of Histology and Developmental Biology, Tokyo Dental College, Tokyo, Japan b Department of Anatomy, Tokyo Dental College, Tokyo, Japan c Department of Pharmacology, Tokyo Dental College, Tokyo, Japan d Division of Internal Medicine, Cupid Clinic, Iwamizawa, Japan e Department of Anatomy and Embryology, School of Medicine, Complutense University, Madrid, Spain A R T I C L E I N F O Keywords: Stylohyoideus muscle Intermediate tendon of digastricus muscle Tendon sheath Dynamic fixation Hyoid bone Human fetus A B S T R A C T Background: In adults, the intermediate tendon of digastricus muscle usually runs along the medial or lateral side of the stylohyoideus muscle insertion. To provide a better understanding of the variations, we examined the topographical anatomy of the muscle and tendon in fetuses. Methods: We examined histological sections from six early-term, 26 mid-term and six near-term fetuses (approximately 8–9, 12–18 weeks and 25–33 weeks). Results: At early-term, an initial sheath of intermediate tendon of digastricus muscle received the stylohyoideus muscle at the superior aspect. The muscle and tendon was distant from the hyoid. At mid-term, near the insertion to the hyoid greater horn, the stylohyoideus muscle consistently surrounded more than 2/3 of the intermediate tendon circumference. In contrast, we found no near-term specimen in which the stylohyoideus muscle sur rounded the intermediate tendon. The multilayered tendon sheath was fully developed until near-term and connected to the body of hyoid by an intermuscular septum between the thyrohyoideus muscle and one or two of suprahyoid muscles. Therefore, the hyoid insertion of the styloglossus muscle was a transient morphology at mid- term. Conclusion: The stylohyoideus muscle insertion was appeared to move from the tendon sheath to the hyoid greater horn and, until near-term, return to the tendon sheath. A fascia connecting the tendon sheath to the body of hyoid was strengthened by the suprahyoid and infrahyoid muscles. The latter muscles seemed to regulate fixation/relaxation of the intermediate tendon to the hyoid. The stylohyoideus muscle slips sandwiching the intermediate tendon might be a rare morphology. 1. Introduction According to textbooks of anatomy (Schaeffer, 1953; Standring, 2020), the stylohyoideus muscle (StH) divides into two parts near the insertion to the hyoid bone and the tendon slips sandwich the inter mediate tendon of the digastricus muscle (DiT). This structure is believed to play the role of a pulley to change the tendon direction (Fig. 1a). However, Fujita (1956) and Mori (1964) classified the tendon * Correspondence to: Department of Histology and Developmental Biology, Tokyo Dental College, 2-9-18 Kandamisaki-cho, Tokyo, Chiyoda-ku 101-0061, Japan. E-mail addresses: kitamurakei@tdc.ac.jp, keikitamura@tdc.ac.jp (K. Kitamura), ryu@suzuki-shika.or.jp (R. Suzuki), ishidukasatoshi@tdc.ac.jp (S. Ishizuka), g. murakamisaori@gmail.com (G. Murakami), jfrodvaz@ucm.es (J.F. Rodríguez-Vázquez), hyamamoto@tdc.ac.jp (H. Yamamoto), abesh@tdc.ac.jp (S.-i. Abe). 1 These authors contributed equally to this work. 2 ORCID: 0000-0003-3335-1440 3 ORCID: 0000-0002-1492-2300 4 ORCID: 0000-0001-5423-4492 5 ORCID: 0000-0003-4632-9736 Contents lists available at ScienceDirect Annals of Anatomy journal homepage: www.elsevier.com/locate/aanat https://doi.org/10.1016/j.aanat.2024.152246 Received 24 November 2023; Received in revised form 16 February 2024; Accepted 5 March 2024 mailto:kitamurakei@tdc.ac.jp mailto:keikitamura@tdc.ac.jp mailto:ryu@suzuki-shika.or.jp mailto:ishidukasatoshi@tdc.ac.jp mailto:g.murakamisaori@gmail.com mailto:g.murakamisaori@gmail.com mailto:jfrodvaz@ucm.es mailto:hyamamoto@tdc.ac.jp mailto:abesh@tdc.ac.jp www.sciencedirect.com/science/journal/09409602 https://www.elsevier.com/locate/aanat https://doi.org/10.1016/j.aanat.2024.152246 https://doi.org/10.1016/j.aanat.2024.152246 https://doi.org/10.1016/j.aanat.2024.152246 http://crossmark.crossref.org/dialog/?doi=10.1016/j.aanat.2024.152246&domain=pdf http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ Annals of Anatomy 254 (2024) 152246 2 course into the medial type (Fig. 1b), lateral type (Fig. 1c) and a type in which the tendon passing through the StH insertion (Fig. 1a), the inci dence of “two tendon slips sandwiching the DiT” is limited to 9.4% (Fujita, 1956) or 27.9% (Mori, 1964). In brief, the StH often or usually simply passes along the medial or lateral side of the DiT. Therefore, StH muscle activity may play a small role in mechanical support of the DiT. Any change in the direction of the DiT would seem to require “another” muscle connection. Otherwise, the thick posterior belly of the digas tricus muscle may direct its strong load directly to the hyoid for eleva tion and posterior traction. Near the StH insertion, the anterior belly of the digastricus muscle shows significant variations in attachment and shape, including dupli cated bellies (Schaeffer, 1953; Standring, 2020). The incidence of such variation is significant, exceeding 50% in Japanese cadavers (Sato and Akita, 2000) and 73% in Westerners (Bergman et al., 2006). These anterior variations seem to be due to a delayed joining of the anterior belly and long tendon of the posterior belly (i.e., future DiT) at 9 weeks of gestational age, which is much later than the appearance of the muscle anlage (Katori et al., 2011). Moreover, development of the hyoid bone starts early, but extends beyond 11–12 weeks of gestational age (Rodríguez-Vázquez et al., 2011; 2015). These developmental events are likely to determine the specific topographical relationship between the StH and the DiT. Consequently, to provide a better understanding of the pulley of the DiT in adults, the present study examined age-dependent changes in muscle anatomy around the hyoid, especially at the StH insertion. 2. Material and methods The study was performed in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in 2013). We used histological sections from six early-term, 26 mid-term and six near-term human fetuses. Paraffin-embedded sections of six early-term and 26 mid-term fetus heads (gestational age (GA) approximately 8–9 and 12–18 weeks of gestational age; crown-rump length (CRL) 30–42 and 80–150 mm) were part of the large collection kept at the Department of Anatomy and Embryology of Complutense University Madrid, and the resulting em bryos were obtained from miscarriages and ectopic pregnancies from the University Department of Obstetrics. No information on the genetic background of the embryos and/or abortion was available. The sectional planes were sagittal (10 fetuses), frontal (12) or transversal (10). The sections were stained with hematoxylin and eosin (HE), Azan, or Verde Luz-orange G-acid fuchsin (VOF). This study was approved by the Ethics Committees of Complutense University (B08/374) and Tokyo Dental College (No. 932–2). For comparison with observations of mid-term fetuses, we used transversal sections (HE staining) from six near-term fetus heads (GA approximately 25–33; CRL 201–280 mm) 0.1-mm interval, which had been prepared for our recent studies of the lower cranial nerves, middle ear and nose (Cho et al., 2023; Jin et al., 2023; Yamamoto et al., 2023). These near-term fetuses (approximately 28–35 weeks of gestational age) were part of the collection of the Department of Anatomy, Akita Uni versity, Akita, Japan, and had been donated by the families concerned to the Department in 1975–1985 and preserved in 10% w/w neutral formalin solution for more than 30 years. Data on these specimens included the date of donation and the number of gestational weeks, but did not include the name of the family, obstetrician or hospital or the reason for abortion. Use of the specimens for research by one of the authors (G.M.) was approved by the Akita University Ethics Committee (No. 1428). Before routine procedures for paraffin embedding, the fetus head specimens were decalcified by incubating them at room tempera ture in Plank-Rychlo solution (AlCl2/6 H2O, 7.0 w/v%; HCl, 3.6; HCOOH, 4.6) for 3–7 days. All histological photographs were taken with a Nikon Eclipse 80. 3. Results 3.1. Overview of present histological sections We observed sections that had been cut frontally (Figs. 2a-f, 3 and 6), sagittally (Fig. 2g-k and 4) and transversally (Figs. 5–10). Frontal and sagittal sections are able to show the supero-inferior axis on a single photo. Thus, these sectional plains seem to allow an easier under standing of the anatomy around the StH and DiT than transversal sec tions. In contrast, transversal sections seem to provide a clear demonstration of fasciae because the latter is likely to extend along the supero-inferior axis, such as along the suprahyoid and infrahyoid mus cles. However, in sagittal sections it is difficult to determine whether or not the StH sandwiches the DiT since the tendon slips are likely to run almost inferiorly toward the hyoid. Therefore, we suspected that ob servations of sagittal section would miss either the medial or lateral StH slips when both are present. To aid readers’ understanding, the present figures of sections are arranged in “frontal-sagittal- transversal” order from earlier to later age. Fig. 2 shows the early attachment between the StH and DiT, Figs. 3–5 show the topographical anatomy around the StH and DiT at mid-term, while Figs. 6 and 7 focus on the StH surrounding the DiT. Because of the limited number of available specimens, all near- Fig. 1. Topographical relationship between the stylohyoideus muscle and intermediate tendon of the digastricus muscle. A thick posterior belly of the digastricus muscle (DiP) is connected to the flat and thin anterior belly of the digastricus muscle (DiA) by the intermediate tendon of the digastricus muscle (DiT). Near the insertion to the hyoid, the stylohyoideus muscle (StH) is believed to sandwich the intermediate tendon to provide a pulley to change the tendon direction (Panel a). However, the muscle slip often or usually takes a “medial or lateral course” possibly without any mechanical support to the tendon (Panels b and c). For other abbreviations, see the list of common ab breviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor phar yngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 3 Fig. 2. Early-term of the intermediate tendon of the digastricus muscle and stylohyoideus muscle. Panels a-f display frontal sections from a fetus with 37 mm CRL (Azan staining). Panels g-k show sagittal sections of a fetus with 35 mm CRL (HE staining). In the former specimen panel a is 0.05 mm lateral to panel c. Panels d, e and f are higher magnification views of squares in panels a, b and c, respectively. In the latter specimen panel g is 0.1 mm lateral to panel i. Panels j and k are higher magnification views of squares in panels h and i, respectively. In both specimens, 1) the stylohyoideus muscle (StH) is located in the superior side of the intermediate tendon of the digastricus muscle (DiT; panels d and j) and 2) an initial sheath is seen around the tendon (panels e and j). Note a connection between the DiT and anterior belly of the digastricus muscle (DiA) contains an irregularly-arrayed fibrous tissue (sandwiched by arrowheads in panel k and surrounded by dotted line in panel f). Panels a-c and g-i (or Panels d-f, j and k) were prepared at the same magnification (scale bars, 1 mm in panel a; 0.1 mm in panel d). GN, glossopharyngeal nerve; MC, Meckel’s cartilage; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle. For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 4 Fig. 3. Tendon sheath attached to the hyoglossus and thyrohyoideus muscles and connecting with the sternohyoideus muscle by a thick fascia. Frontal sections. A specimen with a CRL of 113 mm. Azan staining. Panel a (or Panel g) displays the most posterior (or anterior) plane in the figure. Intervals between Panels are 1.4 mm (a-b), 1.0 mm (b-c, c-d), 1.5 mm (d-f) and 1.3 mm (f-g), respectively. Panels e, h and i are higher-magnification views of the squares in Panels a, c and f, respectively. The stylohyoideus muscle (StH) surrounds the intermediate tendon of the digastricus muscle (DiT) on the posterior side (Panels a and b) and changes to a tendon sheath on the anterior side (Panels c and d). The hyoglossus muscle (HG) is attached to both the tendon sheath and the greater horn of the hyoid (Panels e and h) and is connected to the thyrohyoideus muscle (TH) by a thick fascia (Panel h). The tendon sheath is connected to the sternohyoideus muscle by another thick fascia (stars in Panel i). “Median” in the tongue indicates the median septum (Panel f). Panels a-d, f and g were prepared at the same magnification (scale bars: 1 mm in Panel a; 0.1 mm in Panels e, h and i). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 5 Fig. 4. . intermediate tendon of the digastricus muscle suspended by a fascia inserting into the sternohyoideus muscle. Sagittal sections. Azan staining. A specimen with a CRL of 82 mm. Panels a-g show the left side of the oral floor including the hyoid, while Panels h-j show the right side. On the left, Panel a (or Panel f) displays the most lateral (or medial) plane. On the right, Panel h displays the most lateral plane. Intervals between Panels are 0.7 mm (a-b, b-c), 1.5 mm (c-d), 1.6 mm (d-e), 0.1 mm (e-f), 0.4 mm (h-i) and 0.7 mm (i-j). Panel g is a higher-magnification view of the square in Panel c. The inferior end of the stylohyoideus muscle (StH) is shown in Panels b and i. On the left side, the end of the StH as well as the anterior belly of the digastricus muscle (DiA) is connected to an intra muscular tendon (arrows in Panels b-e) of the sternohyoideus muscle (SH). The mylohyoideus muscle (MH) also joins the intramuscular tendon. On the right side, a fascia surrounding the intermediate tendon of the digastricus muscle (DiT; arrowheads in Panels i and j) is connected to the greater horn of the hyoid (GHH). This fascia is not clear on the left side (arrowheads in Panel b and c), but it provides a common attachment for the thyrohyoideus and hyoglossus muscles (TH, HG; Panel g). Panels a-f and h-j were prepared at the same magnification (scale bars: 1 mm in Panel a; 0.1 mm in Panel g). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, sty loglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyro hyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 6 Fig. 5. Stylohyoideus muscle inserting to a common fascial attachment for the thyrohyoideus and mylohyoideus muscle. Transversal sections. Azan staining (Panels a-c and e-h) and HE staining (Panel d). A specimen with a CRL of 93 mm. Panel a (or Panel g) shows the most superior (or inferior) plane in the figure. Intervals between Panels are 0.6 mm (a-b), 0.4 mm (b-c), 0.3 mm (c-e), 0.4 mm (e-f) and 0.2 mm (f-g). Panel h is a higher-magnification view of the square in Panel f. Panel d, a section near Panel e, contains an intermediate membrane (arrowheads) in a sheath of the intermediate tendon of the digastricus muscle (DiT). The stylohyoideus muscle (StH) covers and attaches to more than half the circumference of the tendon sheath (Panels a-c) and inserts to a fascia (stars in Panels f-h) that provides a common attachment for the geniohyoideus (GH) and thyrohyoideus muscles (TH). The hyoglossus and mylohyoideus muscles (Panels d and g) also use the fascia for a common attachment with the TH. Panels a-c and e-g were prepared at the same magnification (scale bars: 1 mm in Panels a and d; 0.1 mm in Panel e). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glosso pharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 7 Fig. 6. Stylohyoideus muscle surrounding the intermediate tendon of the digastricus muscle: individual differences and laterality: observations of frontal sections. Azan staining (Panels a, b, d-f and h) and VOF staining (Panels c and g). Panels a and e, an 80-mm CRL fetus; Panels b and f, 95-mm CRL fetus; Panels c and g, 107-mm CRL fetus; Panels d and h, 137-mm CRL fetus. Left-hand column (Panels a-d) shows the contralateral side of the right-hand column (Panels e-h) although we were unable to confirm which was left or right. The intermediate tendon of the digastricus muscle (DiT) is fully surrounded by the stylohyoideus muscle (StH) in Panels d and h, whereas the covering area is less than half the circumference of the tendon in Panel f. The stylohyoideus muscle does not face the medial (Panels a, b, e) or superior (Panels c and g) part of the tendon. Arrows in Panel c indicate an intermuscular septum between the hyoglossus and thyrohyoideus muscles (HG, TH). Arrowheads in Panel b indicate an intermediate membrane. These sections do not always show the tendon sheath attachment to the greater horn of the hyoid (GHH) since we chose sections containing the largest sectional area of the stylohyoideus muscle. All Panels were prepared at the same magnification (scale bars: 1 mm in Panel a). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 8 term sections were transversal (Figs. 8–10). 3.2. Observations of early-term specimens In the infero-anterior side of the submandibular gland (Fig. 2), the StH was located in the immediately superior or superolateral side of the DiT. The tendon was composed of a bundle of collagenous fibers running in parallel. The StH was separated from the DiT in the superior site (Fig. 2a, g), but it was attached to or inserted to a thin sheath of the tendon at and near the anterior end of the tendon (Fig. 2d, j). The muscle Fig. 7. Stylohyoideus muscle surrounding the intermediate tendon of the digastricus muscle: individual differences and laterality: observations of transversal sections. Azan staining (Panels a, b, e, f and h) and HE staining (Panels c, d and g). Panels a and e, a CRL 100-mm fetus; Panels b and f, 105 mm CRL; Panels c and g, 116 mm CRL; Panels d and h, 150 mm CRL. Left-hand column (Panels a-d) shows the contralateral side of the right-hand column (Panels e-h), although we were unable to ensure which was left or right. The intermediate tendon of the digastricus muscle (DiT) is fully surrounded by the stylohyoideus muscle (StH) in Panels a, e and g. The latter muscle dose not face the medial (Panels b and h), lateral (Panel d) or posterior (Panels c and f) part of the tendon. These sections do not always show the tendon sheath attachment to the greater horn of the hyoid (GHH) since we chose sections containing the largest sectional area of the stylohyoideus muscle. All Panels were prepared at the same magnification (scale bars: 1 mm in Panel a). For other abbreviations, see the list of common abbrevi ations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 9 insertion to the hyoid was absent and the greater horn of the hyoid was separated from the StH and DiT by the hypoglossal nerve in frontal sections (Fig. 2d). Because of the lateral position, sagittal sections con taining the StH and DiT did not show the hyoid (Fig. 2h, i). Notably, collagenous fibers of the DiT lost the regular arrangement at a junction between the tendon and the anterior belly of the digastricus muscle (Fig. 2f, k). Thus, the direction was different between the anterior belly muscle fiber and the DiT collagenous fiber. The junction site was located in the lateral side of the body of the hyoid (Fig. 2c). The hyoglossus and thyrohyoideus muscles were separated from the StH and DiT by a loose fibrous tissue. Fig. 8. Stylohyoideus muscle separated from the hyoid bone by the thyrohyoideus and hyoglossus muscles. Transversal sections. HE staining. A 201-mm CRL fetus. Panel a (or Panel g) displays the most superior (or inferior) plane in the figure. Intervals between Panels are 0.8 mm (a-b), 0.1 mm (b-c) and 0.2 mm (c-g). Panels d-f are higher-magnification views of the squares in Panels a-c, respectively. Panel g, a near section of Panel c, was also prepared at the higher magnification. The stylohyoideus muscle (StH) and the intermediate tendon of the digastricus muscle (DiT) are separated from the hyoid by the thyrohyoideus and hyoglossus muscles (TH, HG; Panels a-c): the distance between the StH and greater horn of the hyoid is shortest in Panel a. The stylohyoideus muscle is located on the ante romedial side of the intermediate tendon of the digastricus muscle (DiT) without sandwiching the tendon (Panels a-c). The muscle and tendon are connected to the hyoid by a fascia (stars in Panels d-g), which provides a common attachment for the TH and HG. Panels a-c and Panels d-g were prepared at the same magnification (scale bars: 1 mm in Panels a and d). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 10 3.3. Observations of mid-term specimens The StH became thin along the inferior course toward the hyoid and, to a greater or lesser degree, contained and associated fasciae when approaching or attaching to the DiT. The associated fasciae provided a tendon sheath containing the DiT and, in turn, the latter provided a major insertion to the StH muscle fibers (Figs. 3c,d, 4i, 5c-e, 6b and 7b, c). Conversely, StH muscle fibers appeared to insert to the hyoid only rarely (Fig. 3e). The StH appeared to lose the muscular insertion to the hyoid or, as seen in early-term fetuses, it failed to obtain a definite insertion to the hard tissue. We sometimes found a thin intermediate membrane between the sheath and tendon (Figs. 5d and 6b). The tendon sheath, attached to the hyoid, was located close to the hyoglossus and thyrohyoideus muscles and, notably, these muscles originated from or inserted to the tendon sheath and a fascia associated with the StH (Figs. 3h, 4b,i, 5f,g, 6b-d). At a slightly higher level, the constrictor pharyngis medius muscle was also close to the StH (Fig. 3a). Therefore, theses suprahyoid and infrahyoid Fig. 9. Hyoid distant from the intermediate tendon of the digastricus muscle and stylohyoideus muscle. Transversal sections. HE staining. A 271-mm CRL fetus. Panel a (or Panel f) shows the most superior (or inferior) plane in the figure. Intervals between Panels are 1.2 mm (a-b), 0.4 mm (b-c, c-d) and 0.6 mm (d-e). Panels f-h are higher-magnification views of the squares in Panels c-e, respectively. The inferior end of the stylohyoideus muscle (StH), shown in Panels e and h, is connected to the body of the hyoid (HB) by a fascia (stars in Panel h), which extends between the thyrohyoideus and hyoglossus muscles (TH, HG; Panel e). The stylohyoideus muscle is located on the medial side of the intermediate tendon of the digastricus muscle (DiT; Panels b-e). A thick sheath surrounds the tendon (arrows in Panels g and h). Panels a-e and Panels f-h were prepared at the same magnification (scale bars: 1 mm in Panels a and f). For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyro hyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 11 Fig. 10. Thick sheath of the intermediate tendon of the digastricus muscle in the lateral side of the stylohyoideus muscle. Transversal sections. HE staining. A 280-mm CRL fetus. Panel a (or Panel e) shows the most superior (or inferior) plane in the figure. Intervals between Panels are 0.2 mm (a-b), 0.6 mm (b-c), 0.2 mm (c-d) and 1.0 mm (d-e). Panels f-h are higher-magnification views of the squares in Panels c-e, respectively. A tendon sheath of the intermediate tendon of the digastricus muscle (DiT) is clearly seen (Panels c-e) and it accompanies intermediate membranes (arrowheads in Panel g and h). The stylohyoideus muscle (StH) is thick in the anterolateral side of the DiT (Panels a and b) and its inferior end (Panels e and h) insets to a connecting fascia between the tendon sheath and hyoid. The connecting fascia (stars) provides a common attachment to the thyrohyoideus and hyoglossus muscles (TH, HG; Panel f). Asterisks in Panel f indicate tissue damage during histological procedure. Panels a-e and Panels f-h were prepared at the same magnification (scale bars: 1 mm in Panel a; 0.1 mm in Panel g). For other ab breviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior muscle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, intermediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glossopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reichert’s cartilage; SCM, sterno cleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 12 muscles appeared to interfere with the StH insertion to the hyoid. The StH was always seen near the greater horn of the hyoid (Figs. 3a,b, 4b,i and 5c,d), and not the body of the hyoid. The lesser horn was located on the superior (Fig. 3f) or posteromedial (Fig. 5a) side of the StH insertion. Near the insertion to the hyoid, the StH muscle belly consistently surrounded more than 2/3 of the circumference of the DiT (Figs. 6 and 7). The “gap” of muscle surrounding the DiT (an area without muscle covering on the circumference of tendon) was usually present, but in three specimens (Figs. 6h and 7a,g), the StH surrounded the DiT completely and the muscle appeared to embed the tendon. The maximum degree of covering by the muscle was not seen at the inferior end of the StH muscle belly (Figs. 4b and 5f) but slightly higher (Figs. 4a and 5b). Notably, an insertion tendinous slip of the StH (Fig. 1; see also the Introduction) was not evident on either the medial or lateral side of the DiT. Thus, we were unable to find a candidate representing an in termediate morphological form of the muscular insertion from the hyoid to the tendon sheath. The StH or the DiT tendon sheath connected to an intermuscular septum between the thyrohyoideus and hyoglossus mus cles (Figs. 4g, 5h and 6c). The StH or sheath was also likely to issue a fascia to the sternohyoideus muscle (Fig. 4b) and the mylohyoideus muscle (Figs. 3f and 5g). The sternohyoideus muscle appeared to be connected to the mylohyoideus muscle by not only the covering fascia but also a common intramuscular septum (Fig. 3i and 4d,e). We sometimes observed left-right differences in the anatomy of the tendon sheath, such as 1) which muscle the sheath was attached to (e.g., Fig. 4c vs, Figs. 4i) and 2) whether or not there was a gap of the muscle covering DiT (e.g., Fig. 6a vs. Fig. 6e). Although a left-right difference in height of the sheath should be present, evaluation was difficult because slight tilting was likely in the sectional plane and, identification of left or right was often impossible since no sections of the trunk were available in more than half of the present specimens. 3.4. Observations of near-term specimens We found no specimen in which the StH muscle belly completely surrounded the DiT. In fact, the StH muscle fibers were usually attached to a small area of the tendon sheath in the anteromedial or medial side of the DiT (Figs. 8 and 9). Conversely, a single specimen had a StH on the lateral or superficial side of the DiT (Fig. 10a). In a single specimen, muscle fibers of the StH inserted directly to the hyoid (Fig. 9), while in another five, the StH muscle belly was separated from the hyoid by the hyoglossus and/or thyrohyoideus muscles (Figs. 8 and 10). Fig. 10 exhibits the largest covering of tendon by the StH: the area corresponded to an almost half circumference of the DiT (Fig. 10a, b). Notably, a connecting fascia between the tendon sheath and the hyoid provided a common origin (Figs. 8g and 10f) or an intermuscular septum (Fig. 8e, f) to these suprahyoid and infrahyoid muscles. Even in the single specimen showing the StH muscle insertion to the hyoid, at a lower level (Fig. 9e, h) a fascia was evident connecting the tendon sheath to the hyoid: this connecting fascia provided an intermuscular septum between the thyrohyoideus and the hyoglossus muscles. Unlike the tendon sheath at mid-term, a thick intermediate layer was evident at near-term in combination with the other thin inner mem branes (Figs. 9g and 10g,h). Blood capillaries were present in the in termediate layer around the DiT as well as the tendon itself. At many sections at near-term, the tendon sheath had not been cut sharply because of the hard tendon. When compared with sections of mid-term specimens (Figs. 6 and 7), therefore, the sheath did not appear clear and beautiful. Consequently, taken together with observations of early-term and mid-term specimens, it appeared that the StH muscle fibers usually had no direct insertion to the hyoid. Furthermore, a StH muscle insertion to the hyoid, if present, was likely to be a transient morphology seen at mid-term. Likewise, the StH muscle fibers surrounding the DiT were often or sometimes seen at mid-term but appeared unlikely to exist at near-term. Instead, at any stages of fetuses, the StH consistently attached to the tendon sheath of DiT and the latter was connected to the hyoid by a definite fascia. The connecting fascia provided an insertion or origin to the thyrohyoideus and one or two of suprahyoid muscles. 4. Discussion The present study demonstrated that no fetuses had a pair of StH tendon slips sandwiching the DiT: the usual morphology in adults sug gests that the insertion tendon slip may be established postnatally. In contrast to the StH muscle belly surrounding the DiT at mid-term, the StH muscle fibers were usually attached to a small area of the tendon sheath on the anteromedial or medial side of the DiT at near-term. Therefore, the number of StH muscle fibers would likely be reduced to when the insertion became fascial or tendinous during the stages be tween mid-term and near-term (i.e., GA 19–24 weeks of gestational age in the present materials). A strong digastricus muscle function might accelerate the hypothetical degeneration of StH muscle fibers. Although not widely known, delayed muscle fiber degeneration has been reported during fascial development in the deep flexor muscles of the elbow (Jin et al., 2016a), the gluteus maximus muscle (Cho et al., 2018; Shiraishi et al., 2018) and the plantaris muscle (Jin et al., 2021). Likewise, a delayed change of the muscle insertion or origin has been reported in the semimembranosus muscle at the knee (Jin et al., 2016b) and the constrictor pharyngis inferior muscle (Hayashi et al., 2020; Yamamoto et al., 2020). The StH insertion to the tendon sheath of DiT, including the hyoid attachment of the connecting fascia, appeared to move anteromedially from the greater horn of the hyoid to the body of the hyoid. The driving forces might be differences in growth rate between 1) the DiT and StH and 2) the StH and thyrohyoideus muscle. A traction by the thyro hyoideus muscle might also pull the StH insertion anteriorly. The con necting fascia provided a common attachment to the thyrohyoideus and multiple suprahyoid muscles. Therefore, not only the StH but also these suprahyoid and infrahyoid muscles strengthen and regulate the fixation/ relaxation of the tendon sheath to the hyoid. As typically known in the shoulder rotator cuff (Standring, 2020), the DiT fixation seemed not to be static but dynamic depending on the related muscle activity: contraction (or relaxation) of the suprahyoid and infrahyoid muscles seems to make the fixation strong (or loose) and shortens (or increases) the distance between the hyoid and the DiT. Therefore, due to the muscle cooperation involved in straightening the DiT, the posterior belly of digastricus muscle appeared to increase the posterior traction power of the hyoid. This function appeared to correspond to the phase of deglutition immediately after laryngeal elevation. The present study demonstrated, in mid-term fetuses, a spectrum of variations in tendon sheath morphology from StH muscle fibers sur rounding the DiT (Fig. 6h), via partly surrounding muscle fibers in combination with a half-circular sheath (Fig. 3h), and finally to a complete circular sheath (Figs. 9e,h and 10e). Since blood capillaries were contained, the thick intermediate layer (Figs. 9g and 10h) seemed to correspond to an endotendon by Benjamin et al. (2008) or a para tenon by van et al. (2011) and Stecco et al. (2013). According to Hayashi et al. (2023), the calcaneal tendon sheath is a monolayer at mid-term and becomes multilaminar up to near-term in human fetuses. Thus, earlier than the calcaneal tendon, the DiT carried a sheath near the anterior end. Therein, at early-term, composite fibers of the DiT lost the regular arrangement and they appeared not to continue to the anterior belly of the digastricus muscle (Fig. 2f, k). This fact seemed to support a hypothetical secondary connection between the DiT and the anterior belly (Katori et al., 2011). Taking these considerations as a whole, we hypothesized the following complex series of anatomical changes in the StH and DiT (Fig. 11): 1) at the initial stage, the StH inserts to a thin sheath of the DiT; 2) at the intermediate stage (Week 9–12 and also later possibly), we hypothesized muscle fibers of the StH obtaining an attachment to the hyoid greater horn. However, at and after mid-term, 3) the StH K. Kitamura et al. Annals of Anatomy 254 (2024) 152246 13 attachment was usually lost with degeneration of muscle fibers in combination with expansion of the thyrohyoideus and hyoglossus muscle attachment. Simultaneously, a gap of muscle covering the DiT might enlarge and a complete circular covering is lost. 4) At the final stage, a definite tendon sheath acts as a dynamic pulley of the DiT, since the thyrohyoideus and hyoglossus muscles strengthen the tendon sheath and regulate the distance between the hyoid and the DiT. During the aforementioned process, the attachment of the StH or tendon sheath to the hyoid greater horn was most likely to move anteriorly toward the body of the hyoid. Textbook descriptions of StH tendon slips sand wiching the DiT might be biased by a rare morphology. 4.1. Study limitation A major study limitation was the small number of specimens, espe cially those at near-term. Therefore, we have limited discussion of the near-term situation to the fact that in all six specimens, the StH did not surround the DiT but provided a bundle on the medial side of the latter. Ethical Statement This study was performed in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in 2013). This study was approved by Ethics Committees of Complutense University (B08/374), Tokyo Dental College (No. 932–2) and Ethics Committees of Akita University (No. 1428). CRediT authorship contribution statement Kei Kitamura: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. Ryu Suzuki: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. Jose Francisco Rodríguez-Vázquez: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. Hitoshi Yamamoto: Writing – review & editing, Visualization, Investigation, Data curation. Satoshi Ishizuka: Writing – review & editing, Validation, Project administration, Formal analysis. Gen Murakami: Writing – review & editing, Writing – original draft, Supervision, Investigation, Formal analysis. Shin-ichi Abe: Writing – review & editing, Visualization, Investigation, Data curation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This study was supported by Complutense University of Madrid and Tokyo Dental college in 2023. This study was supported by a Grant-in- Aid for Scientific Research (nos. 21K17049: Kei Kitamura) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Benjamin, M., Kaiser, E., Milz, S., 2008. Structure-function relationships in tendons: a review. J. Anat. 212 (3), 211–228. Bergman, R.A., Afifi, A.K., Miyauchi, R., 2006. Muscles of head and neck. In: Illustrated Encyclopedia of Human Anatomic Variation. 〈http://www.anatomyatlases.org/An atomicVariants/AnatomyHP.shtml〉. Cho, K.H., Jin, Z.W., Abe, H., Wilting, J., Murakami, G., Rodríguez-Vázquez, J.F., 2018. Tensor fasciae latae muscle in human fetuses with special reference to its contribution onto development of the iliotibial tract. Folia Morphol. 77 (4), 703–710. Cho, K.H., Honkura, Y., Kim, J.H., Hayashi, S., Kitamura, K., Murakami, G., Rodríguez- Vázquez, J.F., 2024. Topohistology of the cranial nerves IX–XII at the cranial base Fig. 11. Schematic representation showing development of a dynamic pulley system for the digasticus muscle. Panel a, showing the morphology of early term, represents an initial stage at which the stylohyoideus muscle (StH) inserts to a thin sheath of the intermediate tendon of the digastricus muscle (DiT). Panel b, showing the morphology at mid-term, represents an interme diate stage at which the stylohyoideus muscle fibers obtain insertion to the greater horn of the hyoid. The muscle is likely to surround completely the in termediate tendon of the digastricus muscle. Panel c exhibits three morphologic features: 1) muscle fiber degeneration in parts of the StH to provide the fascial insertion and 2) expansion of the fiber attachment of the suprahyoid and infrahyoid muscles toward the stylohyoideus fascial slips and; 3) anterior shift of the tendon sheath and stylohyoideus insertion toward the body of the hyoid. Panel d, a final stage seen at mid-term or near-term, shows the tendon sheath for which the fascia connecting to the hyoid is strengthened by a common attachment of the suprahyoid and infrahyoid muscles. For other abbreviations, see the list of common abbreviations. CPI, constrictor pharyngis inferior mus cle; CPM, constrictor pharyngis medius muscle; DiA, anterior belly of the digastricus muscle; DiP, posterior belly of the digastricus muscle; DiT, inter mediate tendon of the digastricus muscle; GH, genioglossus muscle; GN, glos sopharyngeal nerve; GHH, greater horn of the hyoid; HB, body of the hyoid; HG, hyoglossus muscle; HN, hypoglossal nerve; LA, lingual artery; LHH, lesser horn of hyoid; LN, lingual nerve; LX, larynx; MC, Meckel’s cartilage; MH, mylohyoideus muscle; PX, pharynx; MP, medial pterygoid muscle; RC, Reich ert’s cartilage; SCM, sternocleidomastoideus muscle; SG, styloglossus muscle; SH, sternohyoideus muscle; SMG, submandibular gland; StH, stylohyoideus muscle; TC, thyroid cartilage; TG, thyroid gland; TH, thyrohyoideus muscle;. K. Kitamura et al. http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref1 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref1 http://www.anatomyatlases.org/AnatomicVariants/AnatomyHP.shtml http://www.anatomyatlases.org/AnatomicVariants/AnatomyHP.shtml http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref2 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref2 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref2 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref2 Annals of Anatomy 254 (2024) 152246 14 and upper parapharyngeal space: a histological study using human fetuses (in press.). Anat. Rec.. https://doi.org/10.1002/ar.25355. Fujita, T., 1956. Anatomical study of suprahyoid muscles in Japanese. Bull. Anat. Tokyo. Dental. College 1, 1–20. Hayashi, S., Hirouchi, H., Murakami, G., Wilting, J., Rodríguez-Vázquez, J.F., Kim, J.H., 2020. Transient connection or origin of the inferior pharyngeal constrictor during fetal development: a study using human fetal sagittal sections (Epub). Ann. Anat. 228, 1–8. https://doi.org/10.1016/j.aanat.2019.151438. Hayashi, S., Kim, J., Jin, Z.W., Murakami, G., Rodríguez-Vázquez, J.F., Abe, H., 2023. Development and growth of the calcaneal tendon sheath with special reference to topographical relation to the plantaris tendon: a histological study using human fetuses. Surg. Radiol. Anat. 45 (3), 247–253. Jin, Z.W., Jin, Y., Yamamoto, M., Abe, H., Murakami, G., Yan, T.F., 2016a. Oblique cord (chorda obliqua) of the forearm and muscle-associated fibrous tissues at and around the elbow joint: a study using human fetal specimens. Folia Morphol. 75 (4), 493–502. Jin, Z.W., Abe, H., Jin, Y., Shibata, S., Murakami, G., Rodríguez-Vázquez, J.F., 2016b. Positional changes in tendon insertions from bone to fascia: development of the pes anserinus and semimembranosus muscle insertion in human foetuses. Folia Morphol. 75 (4), 503–511. Jin, Z.W., Kim, J.H., Suzuki, D., Sugai, N., Murakami, G., Abe, H., Rodríguez-Vázquez, J. F., 2021. Relationship of the fabella with the origins of the plantaris and gastrocnemius lateral head muscles in late-term fetuses: a histological study. Anat. Cell. Biol. 54 (2), 270–279. Jin, Z.W., Honkura, Y., Yamamoto, M., Jin, S., Murakami, G., Abe, H., Rodríguez- Vázquez, J.F., 2023. Sphenomandibular ligament and the degenerating Meckel’s cartilage revisited: sequential variations with the temporal bone deformity for attachment of the ligament in near-term human fetuses. J. Anat. in press. https:// doi.org/10.1111/joa.13974. Katori, Y., Kim, J.H., Rodriguez-Vazquez, J.F., Kawase, T., Murakami, G., Cho, B.H., 2011. Early fetal development of the intermediate tendon of the digastricus and omohyoideus muscles: a critical difference in histogenesis. Clin. Anat. 24 (7), 843–852. Mori, M., 1964. Statistics on the musculature of the Japanese. Okajimas. Fol. Anat. Jpn. 40, 195–300. Rodríguez-Vázquez, J.F., Kim, J.H., Verdugo-López, S., Murakami, G., Cho, K.H., Asakawa, S., Abe, S., 2011. Human fetal hyoid body origin revisited. J. Anat. 219 (2), 143–149. Rodríguez-Vázquez, J.F., Verdugo-López, S., Abe, H., Murakami, G., 2015. The origin of the variations of the hyoid apparatus in human. Anat. Rec. 298 (8), 1395–1407. Sato, T., Akita, K., 2000. University of Tokyo Press, Tokyo, p. 74. Japanese Body, Considerations of the Anatomical Variations, 1st Edition. Schaeffer, J.P., 1953. Morris’s Human Anatomy, 11th edition. Blakiston, New York, pp. 428–429. Shiraishi, Y., Jin, Z.W., Mitomo, K., Yamamoto, M., Murakami, G., Abe, H., Wilting, J., Abe, S.I., 2018. Fetal development of the human gluteus maximus muscle with special reference to its insertion to the tractus iliotibialis. Folia Morphol. 77 (1), 144–150. Standring, S., 2020. Gray’s Anatomy, 42nd edition, 632. Churchill Livingstone, London, p. 628. Stecco, C., Corradin, M., Macchi, V., Morra, A., Porzionato, A., Biz, C., De, Caro, R., 2013. Plantar fascia anatomy and its relationship with Achilles tendon and paratenon. J. Anat. 223 (6), 665–676. van, D., van, C.N., Sterkenburg, M.N., Wiegerinck, J.I., Karlsson, J., Maffulli, N., 2011. Terminology for achilles tendon related disorders. Knee. Surg. Sports Traumatol. Arthrosc. 19 (5), 835–841. Yamamoto, M., Hashimoto, K., Honkura, Y., Murakami, G., Abe, H., Rodríguez- Vázquez, J.F., Abe, S.I., 2020. Morphology of the upper esophageal sphincter or cricopharyngeus muscle revisited: a study using adult and fetal specimens. Clin. Anat. 33 (5), 782–794. Yamamoto, M., Hayashi, H., Honkura, Y., Hirano-Kawamoto, A., Katori, Y., Murakami, G., Rodríguez-Vázquez, J.F., 2023. Nasal capsule ossification: a histological study using human foetuses to find an association between the foetus and adult morphologies of the nasal wall. J. Anat. 243 (3), 517–533. K. Kitamura et al. https://doi.org/10.1002/ar.25355 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref4 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref4 https://doi.org/10.1016/j.aanat.2019.151438 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref6 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref6 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref6 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref6 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref7 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref7 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref7 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref7 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref8 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref8 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref8 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref8 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref9 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref9 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref9 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref9 https://doi.org/10.1111/joa.13974 https://doi.org/10.1111/joa.13974 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref11 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref11 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref11 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref11 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref12 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref12 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref13 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref13 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref13 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref14 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref14 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref15 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref15 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref16 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref16 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref17 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref17 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref17 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref17 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref18 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref18 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref19 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref19 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref19 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref20 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref20 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref20 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref21 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref21 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref21 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref21 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref22 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref22 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref22 http://refhub.elsevier.com/S0940-9602(24)00038-4/sbref22 Growing stylohyoideus muscle insertion to the hyoid bone with special reference to its topographical relation to the interm ... 1 Introduction 2 Material and methods 3 Results 3.1 Overview of present histological sections 3.2 Observations of early-term specimens 3.3 Observations of mid-term specimens 3.4 Observations of near-term specimens 4 Discussion 4.1 Study limitation Ethical Statement CRediT authorship contribution statement Declaration of Competing Interest Acknowledgment References