Maxillary ossification in a series of six human embryos and fetuses aged from 9 to 12 weeks of amenorrhea: clinical implications

An anatomic controversy exists concerning the number of nuclei of ossification of the maxilla in human embryos and fetuses. Many authors have described two maxillary ossification nuclei, an incisor nucleus and a maxillary nucleus properly so-called. Some of these have explained congenital labiomaxillary clefts by a defect of bony fusion between these two ossification nuclei. Others consider that there is only a single nucleus for maxillary ossification. In order to settle this question we performed a histologic study in six heads of embryos and fetuses aged from 9 to 12 weeks of amenorrhea (WA) to demonstrate the presence of one or more sites of maxillary ossification at the earliest stages. Our study revealed, on each side, a single zone of maxillary ossification, present from 9 WA, in the form of a sheet of osteoid tissue situated in close contact with the dental lamina. Maxillary ossification then progressed in the latero-medial direction, preserving a median transverse and ventral mesenchymal septum. This septum may constitute the phylogenetic remnant of an ancestral premaxilla.     

Static and dynamic osteogenesis: two different types of bone formation

The onset and development of intramembranous ossification centers in the cranial vault and around the shaft of long bones in five newborn rabbits and six chick embryos were studied by light (LM) and transmission electron microscopy (TEM). Two subsequent different types of bone formation were observed. We respectively named them static and dynamic osteogenesis, because the former is characterized by pluristratified cords of unexpectedly stationary osteoblasts, which differentiate at a fairly constant distance (28+/-0.4 microm) from the blood capillaries, and the latter by the well-known typical monostratified laminae of movable osteoblasts. No significant structural and ultrastructural differences were found between stationary and movable osteoblasts, all being polarized secretory cells joined by gap junctions. However, unlike in typical movable osteoblastic laminae, stationary osteoblasts inside the cords are irregularly arranged, variously polarized and transform into osteocytes, clustered within confluent lacunae, in the same place where they differentiate. Static osteogenesis is devoted to the building of the first trabecular bony framework having, with respect to the subsequent bone apposition by typical movable osteoblasts, the same supporting function as calcified trabeculae in endochondral ossification. In conclusion, it appears that while static osteogenesis increases the bone external size, dynamic osteogenesis is mainly involved in bone compaction, i.e., in filling primary haversian spaces with primary osteons.     

Leptin increases growth of primary ossification centers in fetal mice

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg⁻¹) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.     

Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.     

Effect of systemic calcium deficiency on the expression of transforming growth factor-beta in chick embryonic calvaria.

The developmental process of intramembranous ossification involves bone formation directly from mesenchymal differentiation without a cartilage intermediate. We have previously observed that systemic calcium deficiency in the developing chick embryo, produced by long-term shell-less culture, results in the appearance of chondrocyte-like cells in the calvarium, a parietal bone which normally develops via intramembranous ossification. This investigation aims to analyze the mechanism underlying this calcium deficiency-related, aberrant appearance of cartilage phenotype in the chick embryonic calvarium. In view of the reported involvement of transforming growth factor beta (TGF-beta) in osteogenesis and chondrogenesis, we have examined and compared here the expression of TGF-beta in the chick embryonic calvaria of normal (in ovo development, NL), shell-less (SL), and calcium-supplemented SL (SL+Ca) embryos. TGF-beta expression was analyzed at the mRNA level by blot and in situ cDNA hybridization, and at the protein level by immunohistochemistry and immunoblotting. The results presented here indicate that: 1) TGF-beta is expressed in the chick embryonic calvarium by both periosteal cells and osteocytes, as revealed by in situ hybridization and immunohistochemistry; 2) TGF-beta expression is significantly increased in SL calvarium compared to NL calvarium, at both protein and mRNA levels; 3) the number of TGF-beta expressing cells increases in the SL calvarium, particularly along the central, subcambial core region of the bone; and 4) exogenous calcium repletion to the SL embryo affects the expression of TGF-beta such that the pattern approaches that in the NL embryo. Taken together, these results indicate that altered TGF-beta expression accompanies the aberrant appearance of cartilage phenotype caused by systemic calcium deficiency. We postulate that normal cellular differentiation along the osteogenic pathway during embryonic intramembranous ossification is crucially dependent on regulated TGF-beta expression.     

Chondroid tissue in the early facial morphogenesis of the chick embryo

The calcified tissues involved in the early morphogenesis of the so-called intramembranous bones of the facial skeleton were studied by microradiographic and histological techniques in 22 chick embryos at the 9th, 12th and 14 th days of incubation. On the 9th day, the bones of the upper face and palatal vault are made up of thin sheets of chondroid tissue, deposited in their respective mesenchymal condensations. Woven and lamellar bone formation subsequently takes place in each of them from the 12th day of incubation, mainly on the external side of their chondroid primordia. The same phenomena occur in the lower facial and mandibular bones. These facts indicate that the primitive facial desmocranium of the chick embryo, which is classically considered to be formed by intramembranous ossification, first consists of chondroid tissue. As in the cranial vault, this tissue thus represents the initial modality of the skeletogenic differentiation within the avian facial mesenchyme.     

Autoradiographic investigation of calvarial growth in the rat

Results of autoradiographic investigations utilizing tritiated proline reveal an intricate mechanism of rat calvarial growth and reshaping from age two days through 75 days. Generalized intramembranous bone growth dominates the growth process from two through eight days; however, this simple bony enlargement is terminated early. At eight days a differential apposition-resorption process begins dorsally in the calvarium and progresses rostrally within a ten day period. This process includes differential apposition on the ectocranial and endocranial periosteal surfaces accompanied by a differential apposition-resorption pattern on the endosteal surfaces. The wave-like process begins in the occipital bone and progresses ventrally to the frontal bone resulting in a flattening of the bony components. At 35 days bone accretion is again generalized on most calvarial surfacse. However, at 40 days another change in the growth process evolves on the occipital and frontal bones. These bones are now seemingly displaced in a superior direction as bone apposition continues on their superior surfaces, namely, the ectocranial periosteal and endocranial endosteal surfaces and resorption progresses on the two inferior surfaces. The magnitude and duration of the process described in this paper is sufficient to account for calvarial flattening in the absence of bony spatial reorientation due to bending at sutures. The findings described here when compared to proposed in vitro studies can help put the importance of the so-called “functional matrix” related to bone growth into its proper perspective.     

Expression of NG2 proteoglycan during endochondral and intramembranous ossification.

We have used immunohistochemistry to study the distribution of the NG2 proteoglycan during bone development in the mouse. At embryonic day 15.5, NG2 was strongly detected in the immature cartilage of developing limbs. After transient down-regulation in mature chondrocytes, NG2 was up-regulated during primary ossification, colocalizing with alkaline phosphatase and tenascin C. In the epiphyseal growth plates of newborn mouse tibia, NG2 and alkaline phosphatase exhibited overlapping patterns of expression by hypertrophic chondrocytes and by osteoblasts surrounding newly formed bone trabeculae. NG2 was down-regulated after puberty, being only faintly detectable in the tibial growth plates of 3-month-old mice. In cranial sutures, NG2 was strongly labeled in osteogenic bone fronts and in the suture matrix. Our results indicate that NG2 expression is up-regulated during both endochondral and intramembranous ossification, but is down-regulated as ossification is completed.     

Histological study on the ossification of the yellow ligament in the thoracolumbar spines of the cadavers. Especially on the early stage of the ossification

The occurrence process of ossification of the yellow ligament (OYL) was studied radiographically and histologically by using the thoracolumbar spines of the cadavers taken out as en bloc. The subjects were from 44 to 89 years at age (average 66.1 years). After taking roentgenographs, the specimens were sectioned sagittally for histological observations with H-E stain, elastica.van-Gieson stain, safranin-O stain and alcian blue stain. The findings were as follows. 1. Roentgenographically, OYL was evidently found in 6 cases with average age of 71.5 years, while not found in 4 cases with average age of 58.0 years. OYL was more frequently recognized in elderly cadavers. This fact is presumed to be one of the physiological aging processes. 2. Histologically, ossification was found to start in the superior margin of the lamina. Ligament fibers of the yellow ligament attached to the bone tissue piercing through the uncalcified cartilage zone, tide mark and calcified cartilage zone in order. This structure is called enthesis. In the early stage of ossification, cartilage zone became wide being followed by endochondral ossification. Elastic fibers in the cartilage zone became sparse and fine granular calcification was focally found in the uncalcified cartilage zone. Then, these changes also occurred in the ligamentous attachment of the superior lamina, but they were more evident in the superior margin of the inferior lamina. 3. In the advanced stage of ossification, there were a widening of tide mark and an increase of chondrocytes in number. Linear calcified deposits were observed along the ligamentous fibers. Elastic fibers were reduced in the cartilage zone, enlarged and deranged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prenatal development of the human mandible.

In an effort to better understand the interrelationship of the growth and development pattern of the mandible and condyle, a sequential growth pattern of human mandibles in 38 embryos and 111 fetuses were examined by serial histological sections and soft X-ray views. The basic growth pattern of the mandibular body and condyle appeared in week 7 of fertilization. Histologically, the embryonal mandible originated from primary intramembranous ossification in the fibrous mesenchymal tissue around the Meckel cartilage. From this initial ossification, the ramifying trabecular bones developed forward, backward and upward, to form the symphysis, mandibular body, and coronoid process, respectively. We named this initial ossification site of embryonal mandible as the mandibular primary growth center (MdPGC). During week 8 of fertilization, the trabecular bone of the mandibular body grew rapidly to form muscular attachments to the masseter, temporalis, and pterygoid muscles. The mandible was then rapidly separated from the Meckel cartilage and formed a condyle blastema at the posterior end of linear mandibular trabeculae. The condyle blastema, attached to the upper part of pterygoid muscle, grew backward and upward and concurrent endochondral ossification resulted in the formation of the condyle. From week 14 of fertilization, the growth of conical structure of condyle became apparent on histological and radiological examinations. The mandibular body showed a conspicuous radiating trabecular growth pattern centered at the MdPGC, located around the apical area of deciduous first molar. The condyle growth showed characteristic conical structure and abundant hematopoietic tissue in the marrow. The growth of the proximal end of condyle was also approximated to the MdPGC on radiograms. Taken together, we hypothesized that the MdPGC has an important morphogenetic affect for the development of the human mandible, providing a growth center for the trabecular bone of mandibular body and also indicating the initial growth of endochondral ossification of the condyle.     

Making and shaping endochondral and intramembranous bones

Skeletal elements have a diverse range of shapes and sizes specialized to their various roles including protecting internal organs, locomotion, feeding, hearing, and vocalization. The precise positioning, size, and shape of skeletal elements is therefore critical for their function. During embryonic development, bone forms by endochondral or intramembranous ossification and can arise from the paraxial and lateral plate mesoderm or neural crest. This review describes inductive mechanisms to position and pattern bones within the developing embryo, compares and contrasts the intrinsic vs extrinsic mechanisms of endochondral and intramembranous skeletal development, and details known cellular processes that precisely determine skeletal shape and size. Key cellular mechanisms are employed at distinct stages of ossification, many of which occur in response to mechanical cues (eg, joint formation) or preempting future load-bearing requirements. Rapid shape changes occur during cellular condensation and template establishment. Specialized cellular behaviors, such as chondrocyte hypertrophy in endochondral bone and secondary cartilage on intramembranous bones, also dramatically change template shape. Once ossification is complete, bone shape undergoes functional adaptation through (re)modeling. We also highlight how alterations in these cellular processes contribute to evolutionary change and how differences in the embryonic origin of bones can influence postnatal bone repair.     

The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-β3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-β3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-β3-expressing ASCs/gelatin, filling ≈86% of the defect area and ≈61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.     

Prenatal development of the human mandible

In an effort to better understand the interrelationship of the growth and development pattern of the mandible and condyle, a sequential growth pattern of human mandibles in 38 embryos and 111 fetuses were examined by serial histological sections and soft X‐ray views. The basic growth pattern of the mandibular body and condyle appeared in week 7 of fertilization. Histologically, the embryonal mandible originated from primary intramembranous ossification in the fibrous mesenchymal tissue around the Meckel cartilage. From this initial ossification, the ramifying trabecular bones developed forward, backward and upward, to form the symphysis, mandibular body, and coronoid process, respectively. We named this initial ossification site of embryonal mandible as the mandibular primary growth center (MdPGC). During week 8 of fertilization, the trabecular bone of the mandibular body grew rapidly to form muscular attachments to the masseter, temporalis, and pterygoid muscles. The mandible was then rapidly separated from the Meckel cartilage and formed a condyle blastema at the posterior end of linear mandibular trabeculae. The condyle blastema, attached to the upper part of pterygoid muscle, grew backward and upward and concurrent endochondral ossification resulted in the formation of the condyle. From week 14 of fertilization, the growth of conical structure of condyle became apparent on histological and radiological examinations. The mandibular body showed a conspicuous radiating trabecular growth pattern centered at the MdPGC, located around the apical area of deciduous first molar. The condyle growth showed characteristic conical structure and abundant hematopoietic tissue in the marrow. The growth of the proximal end of condyle was also approximated to the MdPGC on radiograms. Taken together, we hypothesized that the MdPGC has an important morphogenetic affect for the development of the human mandible, providing a growth center for the trabecular bone of mandibular body and also indicating the initial growth of endochondral ossification of the condyle. Anat Rec 263:314–325, 2001. © 2001 Wiley‐Liss, Inc.     

Histological Development of the Fused Mandibular Symphysis in the Pig

The development of the mandibular symphysis in late fetal and postnatal pigs, Sus scrofa dom. (n = 17), was studied as a model for the early fusing symphysis of anthropoid primates, including humans. The suture-like ligaments occurring in species that retain a mobile symphysis are not present in the pig. Instead, cartilage is the predominant tissue in the mandibular symphysis prior to fusion. In late fetuses the rostrum of the fused Meckel's cartilages forms a minor posterior component of the symphysis whereas the major component is secondary cartilage, developing bilaterally and joined at the midline with mesenchyme. This remnant of Meckel's cartilage likely fuses with the flanking secondary cartilage. The overall composition of pig symphyseal histology in fetal and infant animals varies regionally and individually. Regions where the paired secondary cartilages abut in the midline resemble double growth plates. Chondrogenic growth in width of the symphysis is likely important in early stages, and central proliferation of mesenchyme is the probable source of new chondrocytes. Laterally, the chondrocytes hypertrophy near the bone fronts and are replaced by alveolar bone. Complete synostosis except for a small cartilage remnant had occurred in one 8-week-old postnatal specimen and all older specimens. Surprisingly, however, the initial phase of symphyseal fusion, observed in a 5-week-old postnatal specimen, involved intramembranous ossification of midline mesenchyme rather than endochondral ossification. Subsequently, fusion progresses rapidly at the anterior and labial aspects of the symphysis, leaving only a small postero-lingual cartilage pad that persists for at least several months. Anat Rec, 302:1372–1388, 2019. © 2018 Wiley Periodicals, Inc.     

Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18

Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. The ligands interacting with FGF receptors (FGFRs) in developing bone have remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are not known. Here we show that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibited expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone.     

Histological structure of antlers in castrated male fallow deer (Dama dama)

Antlers are periodically replaced cranial appendages that, except for the reindeer, are grown only by male deer. The annual antler cycle is controlled by seasonal fluctuations of sex steroid concentrations in the blood, and accordingly castration of male deer causes deviations from normal antler growth. The present study investigated antler histology of castrated fallow bucks (Dama dama). Castration in early spring was followed by casting of the hard antlers carried by the bucks and the growth of a new set of antlers, which remained in velvet permanently. In the following year, numerous bony protuberances developed from the original antler surface. Further growth of these protuberances, which were formed by subperiosteal intramembranous ossification, led to a marked increase in antler diameter in the affected areas. Compared to antlers of intact bucks, the antlers of the castrates showed histological signs of immaturity, suggestive of a reduced bone remodeling and an impairment of the mineralization process. These changes point to the dependence of the above processes on a stimulation by higher levels of sex steroids. Two years after castration, the antlers also developed integumental thickening and showed an initial formation of skin outgrowths. Cystic structures were present in the skin, which were often filled with a presumably sebaceous and/or keratinous material. Formation of intradermal bone or cartilage was not observed in the antlers of the castrated fallow bucks. The histological structure of the skin outgrowths suggested that they were caused by a hypertrophy of the dermal component of the velvet. Due to the localized bone overgrowth, resulting from the periosteal bone apposition onto the original antler surface, skin-lined infoldings originated, which reached deep into the newly formed bone. Our study revealed no indication of invasive/destructive bone growth in the antlers, i.e., of a penetration of the newly formed bone tissue into the pre-existing bone. The hypertrophic bone growth in the antlers of the castrates is compared with other forms of periosteally derived hypertrophic bone formation, including osteomas, in the mammalian skeleton. It is discussed whether the skin and bone outgrowths of the antlers of castrated fallow bucks may be classified as benign tumors.     

The nature and role of periosteum in bone and cartilage regeneration

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium > radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial > radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.     

Histochemical and Ultrastructural Study of Developing Gonial Bone With Reference to Initial Ossification of the Malleus and Reduction of Meckel's Cartilage in Mice

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916–1933, 2019. © 2019 American Association for Anatomy