Any striated muscle fibers conduct the construction load to the target such as bones via muscle-associated fibrous tissues including the tendon, tendon-muscle junction complex, epimysium, perimysium and endomysium [1, 2]. Simultaneously, the mechanical forces are co-ordinated and passed between adjacent muscle cells via cell-matrix interactions and the endomysial connective tissue that links the cells together [3-5].

In the head and neck, some striated muscles insert to soft tissues including another muscle. Moreover, therein, striated muscle fibers of two different muscles, not only synergic muscles but a muscle and its antagonist muscle, are likely to “cross” at almost right angle at the angle of mouth [6-8], the soft palate [9-14], the pharyngeal wall near the palatine tonsil [15, 16] and the tongue intrinsic muscles [17, 18]. Actually, the pelvic floor also contained two striated muscle sphincters (urethral rhabdosphincter and external anal sphincter) attaching to another striated muscle levator ani, but a smooth muscle-rich, specific interface tissue is present between the sphincter and levator ani muscle [19-21]. Consequently, using near-term fetuses, we aimed to identify which tissues interpose between muscles fibers crossing each other at almost right angle. If there is no specific interface, we speculated that a striated muscle fiber should provide a friction stress, or even sharing stress, to another muscle fiber.

We used histological sections of the head and pelvic floor from 12 near-term human fetuses (gestational age approximately 26–40 weeks; crown-rump length 215–334 mm). These sections, 0.1-mm interval, had been previously prepared by our group for studies of the palate and tongue [18, 22, 23] as well as of the pelvic floor [21, 24]. The near-term fetuses were part of the collection kept at the Department of Anatomy, Akita University, Akita, Japan. They had been donated by their families to the Department in 1975–1985 and preserved in 10% w/w neutral formalin solution for more than 30 years. Data for 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. The use of these specimens for research was approved by the Akita University Ethics Committee (No. 1428). Before routine procedures for paraffin embedding, the fetal specimens were decalcified by incubating them at room temperature in Plank-Rychlo solution (AlCl₂/6H₂O, 7.0 w/v%; HCl, 3.6; HCOOH, 4.6) for 3–7 days. The sections were stained with hematoxylin and eosin (H&E) and emeritus professor of the Akita University kindly gifted the sections to the present fifth author, Shin-ichi Abe.

In addition, we observed whole tongue frontal sections from 5 elderly cadavers (3 females and 2 males; 75–85 years old when he/she died). They had been donated to Tokyo Dental College for research and education on human anatomy, and Tokyo Dental College Committee approved their use for research (No. 922-2). The cadavers had been fixed by arterial injection of 10% neutral formalin solution in aqua and stored in 50% ethanol for more than three months. The histological sections (7–10 micron in thickness; H&E staining or Azan staining) had been prepared for our recent study [18]. All photographs for histology were taken with a Nikon Eclipse 80 (Nikon).

Although lots of muscles converged to the angle of mouth, most combinations of them did not show a crossing at an almost right angle but a merging or joining: at the merging (Fig. 1A), a muscle (e.g., platysma muscle [PM]) gradually changed the direction same as another muscle (e.g., the levator angli oris muscle [LAOM]). Fibrous tissues including the perimysium and endomysium were associated with the merging (Fig. 1B). Actually, the LAOM of which wide muscle sheet was evident, “crossed at right angle” its antagonist, i.e., the depressor angli oris muscle. However, at the crossing, the depressor muscle fibers were always bundled by the perimysium although the elevator was likely to issue a solitary muscle fiber to insert between depressor muscle bundles covered by the perimysium (Fig. 1C). Likewise, in the soft palate (Fig. 1D), the levator veli palatini muscle fibers were bundled by the perimysium (Fig. 1E), whereas a solitary muscle fiber of the uveae muscle was likely to insert between levator muscle bundles (Fig. 1F). Therefore, conversely, we did not find a muscle fiber interdigitation with the antagonist with a solitary-to-solitary manner.

Figure 1. Angle of mouth and the soft palate. H&E staining. Panels (A–C) (a fetus of 310 mm; a single frontal section) display the angle of mouth. Panels (D–F) (a fetus of 328 mm; a single sagittal section) exhibit the soft palate. Panels (B, C) (or E, F) are higher magnification views of squares in panel (A) (or D, E). The platysma muscle (PM) joins the levator angli oris muscle (LAOM) and, at the meeting site, a fibrous tissue appears to connect these muscle fibers (B). The levator muscle crosses the depressor angli oris muscle (DAOM), but a crossing of a solitary-to-solitary muscle fiber is unlikely because the depressor muscle fibers are bundled (C). Likewise, in the soft palate (D), a solitary muscle fiber of the uveae muscle (UM) crosses a bundled fibers of the levator veli palatini muscle (LVPM). (A, C–E) Scale bars=1 mm, (B, F) scale bars=0.1 mm. OOM, orbicularis oris muscle; PB, palatine bone.

The stylopharyngeus muscle was thick and inserted into the constrictor pharyngis superior muscle of the lateral pharyngeal wall at right angle (Fig. 2A) although these muscles have different functional vector (i.e., elevator vs. constrictor). However, at the muscle crossing, the constrictor was always comprised of clusters of muscle fibers bundled by the perimysium (Fig. 2B). Although a solitary muscle fiber of the levator was likely to insert to the pharyngeal wall (Fig. 2C), there was not a solitary-to-solitary muscle fiber crossing.

Figure 2. Lateral wall of the pharynx and the face including the external nasal orifice. H&E staining. Panels (A–C) (a fetus of 274 mm; a single horizontal section) display the lateral wall of the pharynx including the palatine tonsil. Panels (D, E) (a fetus of 334 mm; a single frontal section) exhibit the face including the external nasal orifice. Panels (B, C) are higher magnification views of squares in panels (A, B), respectively. A square in panel (D) is shown in panel (E) at the higher magnification. The stylopharyngeus muscle (SPM) inserts into the pharyngeal wall (A) and a solitary muscle fiber of the former crosses bundles of the constrictor pharynges superior muscle (CPSM) fibers (C). Near the external nasal orifice, a buccal stretch receptor (arrows in E) is embedded in the platysma, levator labii superioris and orbicularis oris muscles (PM, LLSM, OOM). (A, B, D) Scale bars=1 mm, (C, E) scale bars=0.1 mm. GN, glossopharyngeal nerve; ICA, internal carotid artery; PMM, pterygoideus medialis muscle; NM, nasalis muscle; NS, nasal septum.

In contrast, in the face near the external nasal orifice (Fig. 3A) as well as in the tongue intrinsic muscles (Fig. 4A), a solitary striated muscle fiber often (multiple sites per section) provided a crossing at almost right angle with another solitary fiber. The combinations of muscles at crossing were 1) the nasalis muscle (NM) vs. the PM (Fig. 3B–G) and, 2) the vertical intrinsic muscle vs. the transverse intrinsic muscle or inferior longitudinal muscle (Fig. 4B–E). Therein, a solitary muscle fiber appeared to “attach to” another solitary fiber since the endomysium was difficult to identify or underdeveloped. Consequently, there was a solitary-to-solitary muscle fiber crossing with the endomysium-to-endomysium contact in the head region.

Figure 3. Nasalis and platysma muscles (NM, PM) near the external nasal orifice. H&E staining. A single frontal section of a fetus of 334 mm (a section 0.8 mm superficial to ). Panels (B, D, F) display higher magnification views of three squares in panel (A). Squares in panels (B, D, E) are shown in panels (C, E, G) at the higher magnification, respectively. The nasalis muscle fibers cross the platysma muscle fibers with no or few interface tissue. Panels (B, D, F) or panels (C, E, G) were prepared at the same magnification. (A) Scale bars=1 mm, (B, C) scale bars=0.1 mm. NS, nasal septum.

Figure 4. Tongue intrinsic muscles at near-term. H&E staining. A single horizontal section of a fetus of 271 mm. Panels (B, D) display higher magnification views of squares in panel (A). Squares in panels (B, D) are shown in panels (C, E) at the higher magnification, respectively. The vertical muscle (VM) fibers cross muscle fibers of the inferior longitudinal muscle (ILM) at a manner between solitary-to-solitary muscle fibers with few tissue at the interface (C, E). These muscles also cross the transverse muscle (TM), but the latter muscle fibers are bundled (B, D). Panels (B, D) or panels (C, E) were prepared at the same magnification. (A) Scale bars=1 mm, (B, C) scale bars=0.1 mm.

We did not find muscle spindles at and near the angle of mouth, soft palate, pharyngeal wall and intrinsic tongue muscle layer. In the superficial side of the muscle crossing between the NM and PM, we rarely found a buccal stretch receptor (Fig. 2D, E).

Adults’ tongue sections sometimes (0–2 sites per section; Fig. 5A) contained muscle crossings in which the endomysium-to-endomysium contact was suggested (Fig. 5D, F). The combination of the muscle crossing was limited to the vertical and transverse intrinsic muscles. Both muscles were usually bundled (Fig. 5B, E), but the perimysium was unclear (Fig. 5C, D, F, G).

Figure 5. Tongue intrinsic muscles in adults. Azan staining. Two frontal sections from a 76 years old female (A–D) and an 82 years old male (E–G). Panel (B) displays a higher magnification view of a square in panel (A, C, D) correspond to squares in panel (B). Panels (F, G) are higher magnification views of circles in panel (E). The intrinsic vertical muscle (VM) fibers cross muscle fibers of the transverse muscle (TM) at a manner suggesting the endomysium-to-endomysium contact (D, F). The transverse muscle fibers appear to join in panel (C). The transverse muscle fibers appear to take a wavy course to avoid contact to the vertical muscle in panel (G). (A, B, E) Scale bars=1 mm, (C, D, F, G) scale bars=0.1 mm. SLM, superior longitudinal muscle.

At the beginning of this study, we aimed to describe an interface morphology between two muscles in a muscle crossing at almost right angle. Actually, lots of combinations of muscles provided such a muscle crossing in the head region. However, in most of the combinations, 1) a muscle fiber bundle, that was surrounded by the perimysium, crossed the other muscle bundle or, 2) a muscle bundle crossed a solitary muscle fiber (Fig. 6 middle). When a muscle and its antagonist met (e.g., levator and depressor), both or either of the muscle pair were/was bundled by the perimysium at any sites examined. Therefore, conversely, a solitary-to-solitary muscle fiber interdigitation seemed unlikely between synergic and antagonistic muscle groups or muscles.

Figure 6. Schematic representations showing three types of muscle fiber crossing. The crossing is hypothesized between two muscles with quite different vectors (the vectors cross at almost right angle) such as seen in the levator and lateral tractor. A striated muscle fiber or muscle cell (black) is covered by the basal lamina and surrounded by the endomysium (orange). Usually, when two muscles cross, 1) muscle fibers are bundled by the perimysium (green) and the bundles cross or 2) a bundle crosses a solitary muscle fiber. However, in limited sites, a solitary muscle fiber is likely to cross another fiber with the endomysium-to endomysium contact.

Notably, between two muscles even with quite different vectors, such as seen in the levator and lateral tractor (Fig. 6 right-hand side), a solitary muscle fiber was likely to cross another fiber with a manner of the endomysium-to-endomysium contact. This morphology was seen in the tongue intrinsic muscle layer and the face near the external nasal orifice. Because the endomysium-to-endomysium contact seemed to be unusual, we had considered the morphology as a transient morphology to the cell death in fetuses. Indeed the incidence or density became low, but elderly intrinsic tongue muscles also contained a crossing suggesting the endomysium-to-endomysium contact. This “unusual” contact was likely to maintain during postnatal life. To identify the arrangement of collagen fibrils at the interface between muscle fibers, a further observation is required using transmission electron microscope.

To avoid tissue injury, transmission of muscle contraction force to a tendon or fascia requires specific structures to buffer the spike-like load. Trotter [1] and Huijing [2] noted a folded end of the muscle cell as well as a complex of the epimysium and basal membrane. In the latter, non-fibril-forming collagens such as type VI collagen connect between a muscle cell and the extracellular matrix [3-5]. Muscle force is transmitted not only via the tendinous or muscular insertion but via a fascia covering the muscle group or an epimysium: the latter vector of transmission is likely to occur between synergic and antagonist muscle groups and direct laterally being different from the usual vector along the tendon [25, 26]. Those excellent studies seemed not to hypothesize either a solitary-to-solitary muscle fiber crossing or an endomysium-to-endomysium contact at the crossing.

At a solitary-to-solitary muscle fiber crossing with an endomysium-to-endomysium contact, a complete relaxation of a muscle cell seemed ineffective when another cell contracts because of the unstable fixation for the contrasted cell and because of a possible overstretch of another cell at the resting state. To minimize the mechanical stress, a delicate nervous control of the time, duration and strength of muscle contraction seemed to be necessary. Strangely, mechanoreceptors including a muscle spindle might be few in number, or even absent, at and near the muscle crossing. Histological studies on mechanoreceptors in and near facial muscles are very limited [27, 28]: this fact suggests little contribution of such receptors to maintenance of the suitable muscle tension.

Conceptualization: JHK, GM, SA. Data acquisition: JHK, KK, YH. Data analysis: JHK, GM, KK, SA. Drafting of the manuscript: JHK, YH, GM. Critical revision of the manuscript: KK, YH, SA. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

None.