Branched ends of Reichert’s cartilage in the ear: a histological study using human near-term fetuses

Abstract

There is little information regarding whether the styloid process “inserts” deeply into the temporal bone petrosa. We examined the involvement of Reichert’s cartilage (RC) in the petrosa using sagittal or horizontal histological sections of the heads from 17 near-term fetuses (25–40 weeks). The cranial part of RC usually carried two short branches or protrusions (T-shaped appearance). One of the branches ended in or adjacent to the future tympanic cavity, while another branch ended near the facial nerve canal. Near the latter end, a part of the RC was usually fused with the petrosa, but it was rarely separated from the latter. When a bar toward the tympanic cavity was short or absent, RC displayed a reversed J-shaped course near the facial nerve canal and ended at the attachment to the petrosa (3 of 17 specimens). Overall, considerable variations were evident in the shape, length and topographical relation of RC in the ear. Therefore, an intra-otic root of the styloid process, if identified in adults, seemed not to be a simple bar inserting between the tympanic cavity and the vertical portion of the facial nerve canal. Considerable variations in the RC head suggested that the shape was likely to be determined by mechanical loads from the developing petrosa and/or tympanic bone. Those observations were clearly contrast to the degenerating Meckel’s cartilage that induces protrusions of the petrosa ant tympanic bone. After birth, endochondral ossification of the petrosa appeared to expand antero-inferiorly to involve all branches of RC.

Introduction

The cartilage of the second pharyngeal arch (Reichert’s cartilage [RC]) has become well known since its discovery [1]. It develops into the styloid process, hyoid lesser horn, and stylohyoid ligament. The styloid process accompanies a sheath-like bone termed the vagina of styloid (Fig. 1) at the skull base in adults.

Fig. 1.

Styloid process in the temporal bone. (A) Inferior view. (B) A cut surface along the styloid process. At the root, the styloid process is sandwiched between the stylomastoid foramen and jugular fossa (A). A bony ridge, the vagina of styloid process, separates the root from the tympanic bone. When the temporal bone is cut almost frontally along the styloid process, the cut surface (B) contains both the tympanic membrane (or tympanic bony ring) and a downward course of the facial nerve canal. Note that the styloid process inserted deeply into the ear near the tympanic cavity. Modified from [Anatomic atlas for medical students and clinical doctors (Vol. 1)]. Urban & Schwarzenberg, 1903 [13].

According to Rodríguez-Vázquez [2, 3], the stapes is not derived from RC itself, but instead, it arises from the second arch mesenchyme “before” RC differentiates. Thus, the stapes and RC appear simultaneously around stage 16 (crown-rump length [CRL]=10–11 mm), and both are connected by a mesenchymal condensation, i.e., the “interhyale” as named by Hanson et al. [4] and Louryan [5]. The interhyale becomes the insertion tendon of the stapedius muscle. The stapes and RC soon become cartilaginous at stage 18 (CRL=10–12 mm), and subsequently and throughout life, RC or its derivative (the styloid process) is believed to maintain the connection to the otic capsule cartilage and its derivative (the temporal bone petrosa). A hook-like angulated RC, termed the laterohyale by Hanson et al. [4], is located near and lateral to the facial nerve. At stage 20 (CRL=15–20 mm), the otic capsule cartilage issues an inferolateral protrusion, i.e., the crista parotica [6], which connects to the angulated RC. Instead, RC loses its connection to the stapes via the interhyale. These findings are consistent with recent studies using personal computer-assisted three-dimensional reconstruction [7, 8]. Our group confirmed the connection between RC and the otic capsule cartilage in early and midterm fetuses [9].

Although the late-stage specimens were limited in number (2 fetuses; 215 mm and 310 mm CRL), Bast et al. [10] demonstrated a simple connection between the cranial end of RC and the otic capsule cartilage or ossified petrosa at a site sandwiched by the tympanic cavity (anterior) and the vertical portion of the facial nerve canal (posterior). However, in our recent studies of the ear [11, 12], we found a fact that the simple connection of RC was rather rare. We considered that the fate of RC in the ear was still obscure. Toldt [13] provided a drawing of the intra-otic “root” of the styloid process in the ear (Fig. 1B). Notably, his-described position is consistent with a simple morphology shown by Bast et al. [10] in spite of the almost fifty years’ span of these publications. Consequently, to understand the role of RC for growth and ossification of the temporal bone petrosa, the aim of this study was to examine topographical anatomy of the late-stage RC in near-term fetuses.

Materials and Methods

The study was performed in accordance with the provisions of the Declaration of Helsinki of 1995 (as revised in 2013). We used histological sections from 17 near-term human fetuses (gestational age [GA]=25–40 weeks; CRL=201–372 mm). The near-term fetuses were part of the collection of the Department of Anatomy of Akita University (Akita, Japan), and they were donated by families 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 GA; however, the information did not include the names of the families, obstetricians, or hospitals, and the reason for abortion was also not available. The use of these specimens for this research was approved by Ethics Committees of Akita University (No. 1428) and the fifth author (Gen Murakami) was one of the research member.

Before routine procedures for embedding in paraffin, the fetal head specimens were decalcified by incubation at room temperature in Plank-Rychlo solution (AlCl₂/6H₂O, 7.0% w/v; hydrochloric acid, 3.6% w/v; formic acid, 4.6% w/v) for 3–7 days. Sagittal (10 specimens) or horizontal (seven specimens) sections of the head were prepared at 0.1–mm intervals to a thickness of 7–10 µm and stained with H&E or Azan. The specimen size and sectional plane are presented in Table 1. All photographs were captured using a Nikon Eclipse 80 microscope (Nikon). Parts of the present specimens were previously used by Rodríguez-Vázquez et al. [14] and Jin et al. [15]. Cavitation of the tympanic cavity was not fully finished in the present materials [16].

Table 1.

Variations in the T-shaped cranial end of Reichert’s cartilage at near-term

	Fusion site of RC with petrosa	Free end exposing to T cavity
201ha)	Inferior part attaching to petrosa	Superior end in the future T cavityb)
205sb)	No free end; RC cranial end simply fused with the ossified petrosa near the T cavity (Fig. 2)
210s	Medial part, cartilaginous	Lateral end in the future T cavity
228h	Inferior part near ossified petrosa	Superior end in the future T cavity
254s	Medial end reaching J foramenc)	Lateral end in the future T cavity (Fig. 3)
256s	No free end; RC cranial end simply fused with a cartilage in the petrosa ditant from the T cavity
262h	Inferolateral part, cartilaginous	Superomedial end in the future T cavity
264s	Medial part, cartilaginous	Lateral end close to the T cavity (Fig. 4)
270h	Inferolateral part, cartilaginous	Superomedial end in the future T cavity
271h	Inferolateral part, cartilaginous	Superomedial end in the future T cavity
280s	Medial part, cartilaginous	Lateral end in the future T cavity
281s	Lateral part, cartilaginous	Medial end in the future T cavity
282s	Medial part, 2 cartilage processes	Lateral end in the future T cavity (Fig. 5)
290h	Medial end, cartilaginous	Lateral acute flexion near the T cavity
310s	Medial part attaching to petrosa	Lateral end in the T cavity and fusedd) (Fig. 6)
312h	No free end; RC cranial end simply fused with a cartilage in the petrosa near the T cavity
372s	Lateral part, cartilaginous	Medial end in the future T cavity

RC, Reichert’s cartilage; T, tympanic; h, horizontal; s, sagittal; J, jugular. ^a)Specimen numbers indicate crown-rump length mm; h or s sections, ^b)T cavity after cavitation or its future recess (a mesenchymal tissue before cavitation at near-term), ^c)J foramen; the medial end did “not attach” to the petrosa, but a loose tissue separated between them, ^d)near the lateral end reaching the future T cavity, RC fused with the petrosa.

Results

Overview of the present figures

To easily understand the topographical anatomy between the styloid process (styloid part of RC) and the tympanic cavity, all present figures are based on sagittal sections and demonstrated from the small specimen to the large specimen). In each of the figure, panels are arranged from the medial plane to the lateral plane. We preliminarily observed a few specimens of frontal sections, but we omitted their description because the head of RC in the ear changed drastically among 2–3 sections (within a length of 0.2–0.3 mm along the anteroposterior axis). The thickness of RC was almost 0.1 mm at the skull base, and it reached 0.15 mm at the thickest part in the ear.

Observations of a simple morphology of Reichert’s cartilage

In 3 of the 17 specimens examined, we found a simple morphology of RC (Fig. 2, Table 1) as described Bast et al. [10] and Toldt [13] (Fig. 1B). The RC displayed a reversed J-shaped course along a site sandwiched by the tympanic cavity (anterior) and the vertical portion of the facial nerve canal (posterior). The free end of the “reversed J” was fused with the ossified petrosa (2 specimens) or a cartilage remained in the petrosa (1 specimen; 256 mm in Table 1). The curved part of the reversed J was located near or in the loose mesenchymal tissue or the future tympanic cavity (Fig. 2A–C). The RC was also near the stapedius muscle at the curved part near the exit of RC from the ear (Fig. 2D). A communicating branch between the facial and glossopharyngeal nerves ran along or crossed the lateral aspect of RC (Fig. 2E, F). Horizontal sections exhibited that, in a fetus of 312 mm CRL (Table 1), the curved part of the reversed J was very long (4 mm) and extended along the mediolateral axis (figure, not shown).

Fig. 2.

The reversed J-shaped Reichert’s cartilage (RC) in a fetus of 205 mm crown-rump length: the anterior connection to the ossified petrosa near the tympanic cavity. Sagittal sections. Azan staining. Panel (A) (or F) displays the most medial (or lateral) plane in the figure. Intervals between panels are 0.8 mm (A–B), 1.2 mm (B–C), 0.2 mm (C–D), 0.5 mm (D–E) and 1.0 mm (E–F). The most medial plane (A) displays a fusion between RC and the ossified petrosa. Because of no protrusion toward the tympanic cavity (C), RC head in the ear takes a reversed J-shaped appearance rather than T-shaped: a small diagram in (B) exhibits the shape of RC (arrowhead, parts near the tympanic cavity). RC is below the stapedius muscle (SM) and facial nerve (FN) (C, D). A communicating branch (nerve) between the facial and glossopharyngeal nerves is seen in (C, E, F). All panels were prepared at the same magnification. Scale bar: 1 mm in (A). TYB, tympanic bone; TM, tympanic membrane; EAM, external acoustic meatus; VNAB, auricular branch of the vagus nerve; PET, petrosa.

Observations of a branched Reichert’s cartilage

The cranial part of RC usually (14 of the 17 specimens; Table 1) carried two short branches or protrusions in the ear (Figs. 3–6). One of the branches ended in or adjacent to the future tympanic cavity, while another branch ended near the facial nerve canal. The branching pattern is shown schematically in each figure and, therein, an arrowhead indicates a RC end near the tympanic cavity. Near the latter end, a part of the RC was usually fused with the petrosa, but it was rarely separated from the latter (Fig. 3). The entire parts of the branched RC head were located below the facial nerve canal or the stapedius muscle (Figs. 4E, 5I, 6C) although the nerve or muscle is not always shown in figures (e.g., a concomitant artery of the nerve is shown in Fig. 3F).

Fig. 3.

The upper end of Reichert’s cartilage (RC) in a fetus with a crown-rump length of 254 mm: the medial end approaching the unossified petrosa and the lateral free end facing the tympanic cavity. The figure presents sagittal sections submitted to H&E staining. Panel (A) or (I) displays the most medial (or lateral) plane in the figure. The intervals between the panels are 0.7 mm (A–B), 0.3 mm (B–C), 0.2 mm (C–D), 0.3 mm (D–E), 0.4 mm (E–F), 0.9 mm (F–G), 0.4 mm (G–H), and 0.1 mm (H–I). The most medial plane (A) displays a wall of the internal jugular vein (IJV). Likewise, the arrowhead in (B) indicates loose tissue around the vein. Thus, the medial end (arrow in C) of the remnant of RC reaches the lateral margin of the jugular foramen. Black stars indicate the unossified parts of the petrosa. In addition to this cartilage mass in (E, F) a small cartilage bar (open star) is present near a communicating branch (nerve) between the facial and glossopharyngeal nerves. The superior part of RC is embedded in the petrosa (D–G), but the lateral end (arrow in I) is located at the posterior angle of the future recess of the tympanic cavity (triangle in H, I). A small diagram between panels (C) and (G) exhibits the shape of the cranial end of RC (arrowhead, the anterior free end). Artery, stylomastoid artery. All panels were prepared at the same magnification. Scale bar: 1 mm in (A). TM, tympanic membrane; EAM, external acoustic meatus; TYB, tympanic bone; SCM, sternocleidomastoid muscle; FN, facial nerve; PET, petrosa.

Fig. 4.

The upper end of Reichert’s cartilage (RC) in a fetus with a crown-rump length of 264 mm: the posteromedial connection to the unossified petrosa and the anterior free end facing the tympanic cavity. The figure presents sagittal sections subjected to H&E staining. Panel (A) (or H) displays the most medial (or lateral) plane in the figure. The intervals between the panels are 0.4 mm (A–B), 0.1 mm (B–C), 0.2 mm (C–D), 0.6 mm (D–E), 1.0 mm (E–F), 0.4 mm (F–G), and 0.5 mm (G–H). Panels (C–F) show the T-shaped end of Reichert’s cartilage. In the medial planes, the remnant of RC, presented at higher magnification in (I–K), connects to the unossified part of the petrosa (star in A, B, I). A nerve is seen communicating between the facial and glossopharyngeal nerves. By contrast, the lateral part of RC provides a free end in a recess of the tympanic cavity (triangle in G, H). A small diagram surrounded by (E, F, I) exhibits the shape of the cranial end of RC (arrowhead, the anterior free end). Panels (A–H) and panels (I–K) were prepared at the same magnification. Scale bars: 1 mm in (A, I). TYB, tympanic bone; SCM, sternocleidomastoid muscle; SM, stapedius muscle; TM, tympanic membrane; FN, facial nerve; PET, petrosa.

Fig. 5.

The upper end of Reichert’s cartilage (RC) in a fetus with a crown-rump length of 282 mm: the medial connection to the unossified petrosa and the anterior free end near the tympanic cavity. The figure present sagittal sections submitted to H&E staining. Panel (A) (or I) displays the most medial (or lateral) plane in the figure. Asterisk in (A) indicates an artifact space during histological procedure. The intervals between the panels are 0.4 mm (A–B), 0.2 mm (B–C, C–D, D–E), 0.4 mm (E–F), 0.7 mm (F–G), and 1.0 mm (G–H, H–I). In the medial planes, the remnant of RC connects to the unossified part of the petrosa (star in A–C, I). The stars in (D, E) indicate a crista parotica-like cartilage process. Meanwhile, the lateral part of RC provides a free end in a future recess of the tympanic cavity (triangle in H, I). The lateral part is near the facial nerve. A small diagram inserted in (I) presents the shape of the cranial end of RC (arrowhead, the anterior free end). All panels were prepared at the same magnification. Scale bar: 1 mm in (A). PSC, posterior semicircular canal; TYB, tympanic bone; SCM, sternocleidomastoid muscle; SPM, stylopharyngeus muscle; TM, tympanic membrane; FN, facial nerve; EAM, external acoustic meatus; LSC, lateral semicircular canal; PET, petrosa.

Fig. 6.

The upper end of Reichert’s cartilage (RC) in a fetus with a crown-rump length of 310 mm: the posteromedial attachment to the unossified petrosa and the anterior free end facing the tympanic cavity and connecting to the ossified petrous bone. The figure presents sagittal sections subjected to H&E staining. Panel (A) (or F) displays the most medial (or lateral) plane in the figure. The intervals between the panels are 0.3 mm (A–B), 0.2 mm (B–C), 1.1 mm (C–D), 0.7 mm (D–E), and 0.4 mm (E–F). Panels (G–I) are higher-magnification images of the squares in (B, C, F), respectively. The inset in (A) presents a higher-magnification image of a section near (A). In the medial planes (panel A and its inset), the posteromedial part of the remnant of RC (panel A and its inset) almost attaches to the unossified part of the petrosa (stars in A–C, G, H). Meanwhile, the lateral part of RC provides a free end facing the tympanic cavity and connecting to the ossified part of the petrosa (I). A small diagram inserted at the lower angle of (I) presents the shape of the cranial end of RC (arrowhead, the anterior free end). Panels (A–F) and panels (G–I) were prepared at the same magnification. Scale bars: 1 mm in (A, G, H), 0.1 mm in (I). SM, stapedius muscle; TYB, tympanic bone; TM, tympanic membrane; PET, petrosa.

A protrusion or branch of RC near the tympanic cavity reached a loose mesenchymal tissue corresponding to the future posterolateral recess of the tympanic cavity (Figs. 3I, 5H, I) or the real recess after cavitation (Figs. 4G, H, 6E, F). In a fetus of 310 mm CRL, this cranial and of RC was near the fusion between the petrosa and RC (Fig. 6F, I). However, near the facial nerve and stapedius muscle, another bar usually approached or attached to the petrosa (Figs. 3C, D, 4B, C, 5B, C): this position was also near the internal jugular vein (Fig. 3A, B) or the auricular branch of the vagus nerve (Fig. 2B). A communicating branch between the facial and glossopharyngeal nerves ran in or along the lateral aspect of the RC-petrosa junction (Fig. 4J). These thin nerves were easily identified in horizontal sections (figure, not shown). Horizontal sections also exhibited a close topographical relation between the cartilaginous occipital bone and RC.

Notably, in the branches or protrusions of RC in the ear, topographical relation between the free end toward the tympanic cavity and the site for fusion with the petrosa was classified into 2 types (Table 1): the fusion site was located in the medial side of the free end in 7 specimens, whereas the former was in the lateral and/or inferior side of the latter in the other 7 specimens. As stated, the residual 3 specimens did not have a free end, but the RC was fused with the petrosa at the cranial end of RC. Such a quite unstable topographical anatomy was very rare in the other components of the ear (e.g., cochlea, chorda tympani nerve and Meckel’s cartilage). For easy comparison between specimens, the free end is always drawn in the right-hand side of the fusion site in a small schema in each figure.

No ossification was observed in and along the perichondrium of RC at any site. Beyond our expectation, we found degenerative morphologies of RC such as sparsely distributing cartilage cells with a pale color only in a single specimen (262 mm CRL; horizontal sections). A sheath-like cartilaginous structure corresponding to the “vagina of the styloid process” (Fig. 1) was absent in near-term fetuses. Overall, without any correlation with age (Table 1), considerable variations were evident in the shape, thickness, length and topographical relation of the head of RC in the ear. Fig. 7 summarizes how RC connected to the petrosa and changed the upper end morphology.

Fig. 7.

Schematic representation illustrating the involvement of Reichert’s cartilage (RC) in the petrous temporal bone in fetuses. Medial view of the right ear (viewed from the tympanic cavity). Panels (A) and (B) are based on our previous studies (see the introduction). RC is first initially connected to the otic capsule cartilage (arrow). But it is transiently detached and fragmented. Arrowhead indicates a transient junction between the main part of RC and a fragment at the upper end. Panel (C) exhibits the late-term morphology shown in the present study. RC is usually branched and typically takes a T-shaped appearance at the cranial part: a transverse part of the “T” divides into posterior and anterior cartilage bars. The posterior bar fuses with the otic capsule remnant (green dotted area), whereas the anterior bar has a free end reaching the tympanic cavity. The latter free end is close to the tympanic membrane (TM), facial nerve (FN), and tympanic bone (TYB). The anterior bar appeared to become incorporated into the ossified petrous bone because of the inferior growth (curved arrows). The mastoid part of the temporal bone is not yet developed.

Discussion

The present study revealed the morphology of the head of RC (cranial parts of RC) in the ear. Although the vagina of the styloid process (a sheath-like bone; Fig. 1) is known to cover the process at the skull base, it appeared to be underdeveloped in near-term fetuses. Otherwise, the head of RC in the ear was usually branched or carried protrusions. We identified 3 protrusions: a part attaching to or fusing with the temporal bone petrosa; the second part ending at or near the future lateral recess of the tympanic cavity and, the third part or free end near the facial nerve or stapedius muscle. We regarded the cartilage shape as a “T-shaped” appearance although the real T was rare. Postnatally, endochondral ossification of the petrosa appeared to expand antero-inferiorly to involve the entire part of the T-shaped RC in the ear. Therefore, the entire RC head appeared to become absolutely embedded in or surrounded by the petrosa. The aforementioned lateral recess of the tympanic cavity in fetuses appeared to correspond to the hypotympanic recess in adults [17].

At adolescence, depending on pneumatization, the mastoid part of the temporal bone develops and enlarges to cover the superficial side of RC, although the latter might finish ossification. In adults, the mastoid air cells are connected to the tympanic cavity by the tympanic antrum, as described by Toldt [13] (Fig. 1B), or the “aditus ad antrum”, as noted by Mansour et al. [17]. This connecting part should be located immediately above the facial nerve as well as the lateral semicircular canal. Because the entire cranial parts of RC were located in an area below the facial nerve course, the mastoid was unlikely to attach to the remnant of RC even if the latter is persistent in adults. Otherwise, the RC seemed to provide bony elements around thin nerves (mastoid canaliculi) as well as an anterior wall of the facial nerve canal.

Our group recently demonstrated in near-term fetuses that the remnant of Meckel’s cartilage induces transformation of the tympanic ring or the other nearby bone to provide a cranial terminal for the sphenomandibular ligament, although the latter is believed to the sphenoid spine [15]. Meckel’s cartilage in the future mandibular base is also likely to play an inductive role in ossification [18]. Conversely, RC in the ear as well as the perichondrium of RC was unlikely to play an inductive or transformative role in nearby bones. The considerable variation in the head of RC in the ear suggested that its fate is not determined by RC itself, but instead, it is strongly influenced by surrounding structures such as the growing petrosa and tympanic bones. The latter bones’ ossification earlier than RC seemed to provide a mechanical pressure to determine various shapes of RC.

We rarely found RC degeneration in contrast to Meckel’s cartilage at near-term [15]. RC in the ear increased in thickness (0.05 mm to 0.1 mm) and length by almost 2-fold between 16 and 24 weeks (data, not shown). In the ear, there is a well-known site (fissula ante fenestram) in which a fetal cartilage long remains after birth and induces abnormal ossification of the stapes-cochlear junction [14]. This pathology, termed “ear sclerosis”, often causes hearing loss. Likewise, a part of the RC upper ends might remain postnatally to induce abnormally-delayed ossification near or along the facial nerve as well as the auricular branch of the vagus nerve. Because of the close topographical relation, the remnant RC might cause a partial facial paralysis with unknown reason and/or an abnormal sensory loss of the external ear.

Conclusive remark

Although it was completely embedded in the temporal bone petrosa the upper end of RC (or the future root of the styloid process) was long, double-headed and close to the facial nerve and tympanic cavity. The significant variation might be connected to a postnatal pathology.

Study limitation

The limited numbers of specimens represented an essential problem of this study, but it was extremely difficult to obtain near-term fetus heads available for histology. Another critical limitation was the fact that the present observations were not performed on serial sections, but instead, they were performed on semiserial sections at 0.1-mm intervals. We likely missed thin cartilage bars.

Funding Statement

Funding This study was supported by Wuxi Municipal Bureau on Science and Technology (N20202008) and Zhangjiagang Science and Technology Innovation Project (ZKCXY2123) in China.