Anatomical and molecular characterization of a regionally heterogeneous intramuscular tendon in the mouse masseter muscle

Abstract

The mouse masseter muscle comprises distinct superficial and deep layers, but the tendinous tissue located between these layers remains poorly characterized. In this study, we investigated the anatomical, molecular, and ultrastructural characteristics of this tissue. Serial anatomical and histological analyses demonstrated that the intramuscular tendon (IMT), located between the superficial and deep masseter layers, was anatomically distinct from the superficial aponeurosis–external tendon complex. These structures also exhibited different skeletal attachments: the aponeurosis–external tendon complex attached to the maxilla, whereas the IMT attached to the mandible. Spatial transcriptomic analysis revealed different gene‐expression patterns between the aponeurosis and IMT, with several extracellular matrix‐related transcripts, including Col16a1 , Col13a1 , Col5a2 , Tnn , Postn , and Col22a1 , showing relatively higher expression in the IMT. Within the IMT, several transcripts showed preferential distribution toward the bone‐side region, where immunofluorescence analysis demonstrated significantly higher POSTN immunoreactivity than in the middle‐ and lateral‐side regions. BGLAP exhibited a similar but statistically nonsignificant pattern, whereas COL22 was predominantly localized along the myotendinous junction. Transmission electron microscopy revealed distinct cellular characteristics between the middle‐ and lateral‐side regions. In the lateral‐side region, spindle‐shaped cells were observed, some of which were oriented toward the tendon interior. In contrast, the middle‐side region contained cells with elongated cytoplasmic processes extending toward neighboring cells, forming apparent intercellular contacts. IMT‐like tendinous structures were also observed in the jaw muscles of spiny dogfish, Cynops pyrrhogaster , and Xenopus laevis . Together, these findings demonstrate that the mouse masseter IMT is anatomically distinct from the superficial aponeurosis and exhibits regional molecular and ultrastructural heterogeneity. This characterization of the normal IMT may provide an anatomical and molecular reference for future investigations into masticatory muscle‐tendon‐aponeurosis hyperplasia (MMTAH).