Morphological association between the muscles and bones in the craniofacial region.

The strains of inbred laboratory mice are isogenic and homogeneous for over 98.6% of their genomes. However, geometric morphometric studies have demonstrated clear differences among the skull shapes of various mice strains. The question now arises: why are skull shapes different among the mice strains? Epigenetic processes, such as morphological interaction between the muscles and bones, may cause differences in the skull shapes among various mice strains. To test these predictions, the objective of this study is to examine the morphological association between a specific part of the skull and its adjacent muscle. We examined C57BL6J, BALB/cA, and ICR mice on embryonic days (E) 12.5 and 16.5 as well as on postnatal days (P) 0, 10, and 90. As a result, we found morphological differences between C57BL6J and BALB/cA mice with respect to the inferior spine of the hypophyseal cartilage or basisphenoid (SP) and the tensor veli palatini muscle (TVP) during the prenatal and postnatal periods. There was a morphological correlation between the SP and the TVP in the C57BL6J, BALB/cA, and ICR mice during E15 and P0. However, there were not correlation between the TVP and the SP during P10. After discectomy, bone deformation was associated with a change in the shape of the adjacent muscle. Therefore, epigenetic modifications linked to the interaction between the muscles and bones might occur easily during the prenatal period, and inflammation seems to allow epigenetic modifications between the two to occur.

Developmental mechanism of muscle-tendon-bone complex in the fetal soft palate.

OBJECTIVE: This study was performed to investigate how the palatine aponeurosis, medial pterygoid process (MPP) of the sphenoid bone, and tensor veli palatini (TVP) muscle form the pulley: muscle-tendon-bone complex. DESIGN: Mice at embryonic day (ED) 14-17 were used as sample in this study. Azan staining was performed to observe the morphology, and immunohistochemical staining of desmin was performed to closely observe the development of the myotendinous junction. To confirm the bone formation process, immunohistochemical staining of type II collagen (col II), tartrate-resistant acid phosphatase (TRAP), and alkaline phosphatase (ALP) staining were performed. Furthermore, to objectively evaluate bone formation, the major axis and width of the MPP were measured, and osteoclasts that appeared in the MPP were counted. RESULTS: At ED 14 and 14.5, ALP showed a reaction throughout the MPP. The col II-positive area expanded until ED 16.5, but it was markedly reduced at ED 17. The TVP initially contacted with the palatine aponeurosis at ED 16.5. The major axis and width of the MPP and the number of TRAP-positive osteoclasts were significantly increased as the TVP and palatine aponeurosis joined. CONCLUSIONS: Therefore, in addition to the tissue units: muscle, tendon, and bone, the interaction in organogenesis promotes rapid growth of the pulley: muscle-tendon-bone complex.

Fetal Tendinous Connection Between the Tensor Tympani and Tensor Veli Palatini Muscles: A Single Digastric Muscle Acting for Morphogenesis of the Cranial Base.

Some researchers contend that in adults the tensor tympani muscle (TT) connects with the tensor veli palatini muscle (TVP) by an intermediate tendon, in disagreement with the other researchers. To resolve this controversy, we examined serial sections of 50 human embryos and fetuses at 6-17 weeks of development. At 6 weeks, in the first pharyngeal arch, a mesenchymal connection was found first to divide a single anlage into the TT and TVP. At and after 7 weeks, the TT was connected continuously with the TVP by a definite tendinous tissue mediolaterally crossing the pharyngotympanic tube. At 11 weeks another fascia was visible covering the cranial and lateral sides of the tube. This "gonial fascia" had two thickened borders: the superior one corresponded to a part of the connecting tendon between the TT and TVP; the inferior one was a fibrous band ending at the os goniale near the lateral end of the TVP. In association with the gonial fascia, the fetal TT and TVP seemed to provide a functional complex. The TT-TVP complex might first help elevate the palatal shelves in association with the developing tongue. Next, the tubal passage, maintained by contraction of the muscle complex, seems to facilitate the removal of loose mesenchymal tissues from the tympanic cavity. Third, the muscle complex most likely determined the final morphology of the pterygoid process. Consequently, despite the controversial morphologies in adults, the TT and TVP seemed to make a single digastric muscle acting for the morphogenesis of the cranial base.

Three-dimensional imaging of palatal muscles in the human embryo and fetus: Development of levator veli palatini and clinical importance of the lesser palatine nerve.

BACKGROUND: After palatoplasty, incomplete velopharyngeal closure in speech articulation sometimes persists, despite restoration of deglutition function. The levator veli palatini (LVP) is believed to be significantly involved with velopharyngeal function in articulation; however, the development and innervation of LVP remain obscure. The development of LVP in human embryos and fetuses has not been systematically analyzed using the Carnegie stage (CS) to standardize documentation of development. RESULTS: The anlage of LVP starts to develop at CS 21 beneath the aperture of the auditory tube (AT) to the pharynx. At CS 23, LVP runs along AT over its full length, as evidenced by three-dimensional image reconstruction. In the fetal stage, the lesser palatine nerve (LPN) is in contact with LVP. CONCLUSIONS: The positional relationship between LVP and AT three-dimensionally, suggesting that LVP might be derived from the second branchial arch. Based on histological evidence, we hypothesize that motor components from the facial nerve may run along LPN, believed to be purely sensory. The multiple innervation of LVP by LPN and pharyngeal plexus may explain the postpalatoplasty discrepancy between the partial impairment in articulation vs. the functional restoration of deglutition. That is, the contribution of LPN is greater in articulation than in deglutition.

TCDD disrupts posterior palatogenesis and causes cleft palate.

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.

Development of the human tensor veli palatini: specimens measuring 13.6-137 mm greatest length; weeks 6-16 of development.

The present study seeks to determine the main events that occur in the development of the tensor veli palatini (TVP). A light microscope was used on serial sections of 60 human specimens from weeks 6 to 16 of development. The TVP becomes visible in an embryo of 14.5 mm greatest length (GL; week 6) from a common blastema with the medial pterygoid muscle. In embryos of Carnegie stage 20 (week 7), the TVP is differentiated and relates to the anlage of the pterygoid hamulus. At week 8 of development, when the palatal shelves become horizontal, the presence of the anlage of the palatine aponeurosisis distinguished and is reached by the TPV. In an embryo of 30 mm GL, the chondrification nucleus of the pterygoid hamulus and the synovial bursa of the TVP are identifiable. At week 9, the TVP is continuous with the palatine aponeurosis. At week 13, a connective tissue lamina appears between the TVP and the intramembranous ossification center for the anterior process of the malleus, which we know as the goniale and interpret as an attachment of the muscle to the primary vertebrate jaw or incudomalleal joint. The TVP from its origin, innervation and relation to the goniale appears to be a muscle of mastication that, at the end of the embryonic period, reaches the palatine aponeurosis anlage and the mesenchyme of the auditory tube and specializes in the movements of the soft palate and the auditory tube.

New details on the clefted uvular muscle: analyzing its role at histological scale by model-based deformation analyses.

OBJECTIVE: As an initial step to a complex reconstruction model for virtual surgery, the present study was carried out to provide data on the prenatal cleft lip and palate uvular muscle in eight specimens. METHOD: Serial sections of viscerocrania of 18 aborted embryos and fetuses were studied microscopically and segmented manually. Registration, three-dimensional reconstruction, and finite element analyses were conducted. RESULTS: Incompletely clefted uvulae showed anterior fusion and dorsal fission of the bilateral uvular muscle bodies. A complete cleft lip and palate specimen evidenced single bilateral uvular muscle bodies lying medially and orally below the cleft shelf, its central longitudinal fibers running beneath the oral-median mucosa. In incompletely clefted uvulae, 10% to 50% of circular peripheral fibers crossed the midline within the central third of the anterioposterior muscle, behind the levator loop. Of the fibers, 30% to 60% crossed to the ipsilateral palatopharyngeus muscle. Fibers inserted into the uvular basal membrane in a 60% nasal and 40% oral distribution at the middle third of the macroscopically clefted uvula. The macroscopic uvula itself consisted of loose connective tissue and salivary glands. Deformation analysis did disclose local stress, suggesting the uvular muscle contributes to velopharyngeal closure in normal anatomy and extends the cleft edges in cleft palate. CONCLUSION: Cleft lip and palate reconstruction should reasonably use the uvular muscle to augment the velar midline bulk. Uvular muscle deformation calculation was successful, permitting functional insight on the basis of microanatomical specimens, so far a bigger complete velar model can be ventured.

Histology and function: analyzing the uvular muscle.

OBJECTIVE: Virtual surgery and virtual patients necessitate quantitative data on the area of interest. The study was conducted to exactly describe the embryonic and fetal uvular muscle (MU), relevant for clinical as well as virtual surgery and virtual patient generation. METHOD: Serially sectioned viscerocrania of 10 aborted embryos and fetuses underwent three-dimensional reconstruction to obtain detailed anatomic data and perform finite element analyses. RESULTS: The MU was paired in 80% of cases, while 20% allowed no clear-cut distinction. The MU merged with the levator muscle beneath the palatal aponeurosis without a hard palate insertion. Superior longitudinal central fibers ran below the nasal mucosa, and few circular peripheral fibers crossed in the central third to the contralateral side. This was seen in 30% of the paired muscles and in all cases when no differentiation was possible; about 40% to 80% MU fibers crossed to the ipsilateral and contralateral palatopharyngeus muscle behind the levator loop. MU fibers inserted 60% nasal and 40% oral to the basal membrane at the middle third of the macroscopic uvula, made of loose connective tissue and salivary glands. The results of the finite element simulation of the uvula showed no distinct patterns or distributions of local stress. CONCLUSIONS: Detailed anatomical study supported the concept of mediocranial MU repositioning during corrective surgery, although the impact is minor to the levator muscle's action. Future mathematical models describing effects of such a maneuver should integrate surrounding structures.

Myogenic determination and differentiation of the mouse palatal muscle in relation to the developing mandibular nerve.

The vertebrate palatal muscles are derived from the cranial paraxial mesoderm and start myogenesis by the expression of myogenic regulatory factors (MRFs). Predetermined myogenic cells migrate from the cranial paraxial mesoderm into the branchial arches, followed by myogenic differentiation. The objective of this study was to elucidate whether the determination, migration, and differentiation of myogenic cells during the myogenesis of the palatal muscles, particularly the tensor veli palatini (TVP), are related to the extending mandibular nerve in mouse embryos. By immunohistochemical staining at embryonic day (E) 9.5, MyoD1 and myogenin have been expressed in the mandibular arch, into which the mandibular nerve had not yet extended. At E11.5, these myogenic cells encircled the extending mandibular nerve and were distributed from the distal and lateral to the trigeminal ganglion and into the mandibular arch to form the muscle plate, a girdle-like structure. By E12.5, these myogenic cells lost their girdle-like pattern, vacated the trunk area of the mandibular nerve, and were separated into several incompletely divided masses encircling the collateral branches of the mandibular nerve. The TVP started differentiation at E13.5 with the appearance of myofilaments and acetylcholinesterase (AchE), whereas the other palatal muscles began differentiation at E14.5. We defined the differentiation process of mouse palatal muscles into five stages based on the present findings. These results suggest that the determination and initial migration of the palatal myogenic cells into the mandibular arch occur before the mandibular nerve extends out of the trigeminal ganglion, whereas the myogenic cells migrating into the final sites of differentiation intimately relate to the extending nerve.

Histomorphologic analysis of the soft palate musculature in prenatal cleft and noncleft A/Jax mice.

The two specific aims of this study were as follows: to evaluate the appropriateness of the A/Jax mouse model in the investigation of the key cellular stages in prenatal soft palate morphogenesis and myogenesis; and to describe structural differences in the histomorphology of the soft palate anatomy from cleft and noncleft mice prior to, during, and after palatogenesis. Cleft-induced and control groups of A/Jax mouse embryos from timed pregnancies were harvested sequentially on gestational days 15 to 19. Embryos were weighed and staged for external body morphology. The heads were removed and fixed for light microscopy, sectioned serially in the frontal plane at 10 microns and stained with hematoxylin-eosin to characterize and compare the soft palate musculature. All observations were made at the head depth of the trigeminal ganglion in both age- and stage-matched embryos. The following findings were made: (1) the A/Jax mouse is a suitable animal model for the study of soft palate myogenesis; (2) there were no discernible morphologic differences between the soft palate muscles in cleft and noncleft A/Jax mice when viewed under light microscopy; (3) the soft palate and related muscles were identifiable as muscle fields, in both the cleft and noncleft fetuses, as early as gestational day 15 and as specific muscles at gestational day 18; (4) in both the cleft and noncleft A/Jax fetuses, the soft palate muscles appeared in a sequential anatomic fashion (the palatine aponeurosis appeared first, next the tensor palatini, and then the levator palatini muscles); and (5) in the cleft palate fetuses, both pterygoid plates were angulated and displaced laterally.

Patterns of abnormal myogenesis in human cleft palates.

To test the hypothesis that soft palate muscles are abnormal in cleft palate, we compared soft palate morphogenesis in fetuses with cleft palate (n = 4) to age-matched (n = 3) and nonmatched (n = 1) control specimens. The morphologic status of all soft palate and masticatory structures were classified into one of six stages based on the level of histogenesis. At 54 mm crown-rump length (CRL), the levator veli palatini (L), palatopharyngeus (PP), and palatoglossus (PG) in cleft subjects demonstrated mesenchymal condensation into myoblastic fields, lagging behind the control specimens (97 mm CRL), which displayed definitive fields of myoblasts and myotube formation. In the 175 mm and 225 mm cleft and the 170 mm and 192 mm control specimens, muscular morphology was similar and had reached its postnatal appearance for the tensor veli palatini (175 m only) and L, PP, PG (225 mm only). Muscle fiber directions were, however, disoriented and disorganized, especially close to the medial epithelial edge of the cleft. The levator veli palatini, could not be distinguished as a discrete muscle in the cleft specimens, and what we believed to be the PP and PG seemed "normal" at the level of light microscopy, but malpositioned in a superior direction. This preliminary study demonstrates for the first time that early myogenesis in cleft palates differs from normal.

Soft-palate myogenesis: a developmental field paradigm.

Surgical correction of clefts of the soft palate leads to varying degrees of normal function although the repair itself is successful. Explanations for this include structural abnormalities of the muscles. Previous studies have focused primarily on gross anatomical features of late fetal and postnatal cleft palate musculature; however, infrequent reference has been made to early prenatal morphologic patterns of soft-palate development, beginning with the embryo. Thus we evaluated the chronology of prenatal myogenesis of the soft palate from its early mesenchymal phase through the appearance of definitive palatal muscles and associated structures in a sample of 22 human fetuses that represented postfertilization weeks 6.5 to 20.5 (18- to 192-mm crown-rump length). Specimens were histologically prepared for descriptive and morphometric light microscopy. Data were collected on the earliest appearance times of identifiable soft palate and associated structures within the mesenchymal field and on their individual stages of myogenesis (e.g., for muscles, from mesenchyme to myoblasts to fascicles). Analyses showed that (1) palatal muscles and related bony structures emerge sequentially as densely staining mesenchymal subfields within the larger mesenchymal soft-palate field during the 6- to 9-week period, with the tensor veli palatini muscle appearing earliest, and the musculus uvulae latest; (2) further morphogenesis of the soft palate and associated structures follows a definite timeline; and (3) by 16 to 17 weeks the postnatal palatal morphology is in place.

Three-dimensional computer reconstruction of the eustachian tube and paratubal muscles.

A new method of anatomic dissection and image reconstruction using computer techniques for better understanding of eustachian tube (ET) functioning is presented. Coronal sections of two noncleft fetal skulls were photographed and projected on a graphic tablet. Contours of the pertinent structures were digitized using a mouse. Coordinates of all digitized points were entered into a special computer program. The data were transformed into three-dimensional representations of anatomic structures. The levator veli palatini muscle (M-LVP) was found to have a close relationship with the ET running underneath it and passing at the inside of the pharyngeal edge of the medial cartilage before entering the soft palate. On M-LVP contraction, this part of the medial ET cartilage appears to be the sole point of impact for ET opening. The tensor veli palatini muscle (M-TVP) is connected with the lateral cartilage of the ET and leaves the ET, rounding the pterygoid hamulus before entering the palatal aponeurosis. On this anatomic basis, action of the muscle by isotonic contraction appears to be more likely than isometric contraction.

[Morphometric analysis of differentiation of skeletal muscle fibers of the maxillofacial region during the process of embryonic development]. / Morfometricheskii analizdifferentsirovki volokon skeletnykh myshts cheliustno-litsevoi oblasti v protsesse émbrional'nogo razvitiia.

The skeletal muscle tissue of the lip, tongue, soft palate and masticatory muscles have been morphometrically studied in 70 human fetuses (4-10-month-old). In the muscles mentioned decrease in the nuclear volume and in the nuclear-cytoplasmic ratio is observed. Increase of the muscle fibres in the area of transversal sections is especially intensive during last three months. The labial and glossal fibres reach the greatest thickness. On the 6th month and further, there is an essential difference in number of myons with nuclei in the transversal section of the tongue and lips, as compared with the masticatory muscle and soft palate. In 9-10-month-old fetuses the index on saturation of the glossal and labial muscle fasciculi with capillaries is much greater than that of the soft palate and the masticatory muscle. There exists a statistically important and rather high positive correlation between the area of transversal sections of the muscle fibres, the number of nuclei in them and the density of capillaries in the muscle fasciculi. The relation between histochemical profile of the muscles under investigation and the number of capillaries in them is discussed.

The morphology of M. palatoglossus in the 15-week human fetus.

The morphology of M. palatoglossus in the 15-week fetus was studied with the aid of 30-micron serial sections. The sample included 26 specimens, 9 each of which were sectioned in the coronal and sagittal planes and 8 which were sectioned transversely. M. Palatoglossus extends within the anterior pillar to attachments in both the soft palate and the tongue. Characteristically, the muscle fibers radiate within the velum, not only along its antero-posterior axis but superiorly as well, to intersect other palatal structures. Within the root of the tongue, some fibers of M. palatoglossus combine in a longitudinal muscular complex to course anteriorly toward the tip, others appear to join the transverse muscular system. These data are compared with the literature describing the morphology of this musculature in the adult.

[Embryogeny of facial, mastication, tongue, palate and neck muscles (author's transl)]. / L'embryogénie des muscles faciaux, masticateurs, de la langue, du voile et du cou.

The mesenchymal origin muscular tissues entailing some difficulties in the knowledge of certain muscles embryogeny, two methods are therefore applied: the comparison of the ontogenesis data with those of comparative anatomy and the use as a guide of the functional motor unit with which the muscle combines. In resorting to these methods necessary precautions are precisely defined. The origins of the concerned muscles are, at first, situated among those of the whole musculature subjected to will, which are of two types "somitic" and "branchial". The realities lying under these two misleading terms are analysed. Then the usual data on each of the muscular groups embryogeny reviewed and compared, if necessary, with recent works. For the facial muscles a confusion results from the use of the term platysma both in comparative anatomy and in embryology, in pursuance of transposition, exact on that particular point, of the philogenic development of these muscles in ontogenesis. The development of these muscles comes in the scope of the extensive general character of the superficial hyoïd arch derivatives, and their topographic and functional particularities should be brought together with the disposition of the facial nerve nucleus and its arising fibres. Among the muscles in action in the mastication, a link appears between their precise role in this function and their embryogeny. For the neck muscles, the spinal nerve systematisation and the variations of the sterno-mastoïd muscle in mammals agree in assigning to this muscle an entire somitic origin lying exactly at the junction with the so-called branchial muscles. The somitic origin of the tongue muscles and the sharing between the somitic and branchial origins of those of the soft palate bring to light the place in the organism of these two anatomic structures. Then, the conjunction of muscles proceeding thus from two origins in these anatomic structures carries a particular signification in man by reason of the language.

Anatomic relationshiphs between the human levator and tensor veli palatini and the eustachian tube.

To define the interrelationships of the human levator and tensor veli palatini muscles and the Eustachian tube, fetal heads were serially sectioned and anatomic reconstruction done. Cephalometric points on fetal and adult skulls were compared to evaluate the effect of growth and development on these interrelationships. Based on the results of this study, we propose a mechanism for Eustachian tube function in the normal and in the cleft palate patient. This mechanism offers some explanations for many previously unexplained and paradoxical clinical observations.

The longitudinal fibromuscular component of the soft palate in the fifteen-week human fetus: musculus uvulae and palatine raphe.

The structural relationships of the longitudinal fibromuscular component of the soft palate (musculus uvulae and raphe) were studied using histologic sections from 19 early human fetal specimens. Musculus uvulae arises in association with the palatine aponeurosis near the beginning of the second quadrant of the velum, follows a sigmoid course, and terminates near the base of the uvula. In addition, an occasional muscular loop may arise from the bony palate, arch downwards, and then recur into the uvular muscle. A complex relationship exists between the raphe in the velum and several palatal muscles. With regard to musculus uvulae, small muscular bundles arise from the raphe to embrace the muscle near its crest. These branches may aid in contouring the dorsal surface of the velum in the region of the levator eminence to complement the surface of the posterior pharyngeal wall and thus enhance the efficiency of the velopharyngeal seal.