Prx1 and Prx2 cooperatively regulate the morphogenesis of the medial region of the mandibular process

Mice lacking both Prx1 and Prx2 display severe abnormalities in the mandible. Our analysis showed that complete loss of Prx gene products leads to growth abnormalities in the mandibular processes evident as early as embryonic day (E) 10.5 associated with changes in the survival of the mesenchyme in the medial region. Changes in the gene expression in the medial and lateral regions were related to gradual loss of a subpopulation of mesenchyme in the medial region expressing eHand. Our analysis also showed that Prx gene products are required for the initiation and maintenance of chondrogenesis and terminal differentiation of the chondrocytes in the caudal and rostral ends of Meckel's cartilage. The fusion of the mandibular processes in the Prx1/Prx2 double mutants is caused by accelerated ossification. These observations together show that, during mandibular morphogenesis, Prx gene products play multiple roles including the cell survival, the region‐specific terminal differentiation of Meckelian chondrocytes and osteogenesis. Developmental Dynamics 238: 2599–2613, 2009. © 2009 Wiley‐Liss, Inc.     

Distribution of Matrix Proteins in Perichondrium and Periosteum During the Incorporation of Meckel's Cartilage into Ossifying Mandible in Midterm Human Fetuses: An Immunohistochemical Study

Immunohistochemical localization of versican and tenascin‐C were performed; the periosteum of ossifying mandible and the perichondrium of Meckel's cartilage, of vertebral cartilage, and of mandibular condylar cartilage were examined in midterm human fetuses. Versican immunoreactivity was restricted and evident only in perichondrium of Meckel's cartilage and vertebral cartilage; conversely, tenascin‐C immunoreactivity was only evident in periosteum. Therefore, versican and tenascin‐C can be used as molecular markers for human fetal perichondrium and fetal periosteum, respectively. Meckel's cartilage underwent endochondral ossification when it was incorporated into the ossifying mandible at the deciduous lateral incisor region. Versican immunoreactivity in the perichondrium gradually became weak toward the anterior primary bone marrow. Tenascin‐C immunoreactivity in the primary bone marrow was also weak, but tenascin‐C positive areas did not overlap with versican‐positive areas; therefore, degradation of the perichondrium probably progressed slowly. Meanwhile, versican‐positive perichondrium and tenascin‐C‐positive periosteum around the bone collar in vertebral cartilage were clearly discriminated. Therefore, the degradation of Meckel's cartilage perichondrium during endochondral ossification occurred at a different rate than did degradation of vertebral cartilage perichondrium. Additionally, the perichondrium of mandibular condylar cartilage showed tenascin‐C immunoreactivity, but not versican immunoreactivity. That perichondrium of mandibular condylar cartilage has immunoreactivity characteristic of other periosteum tissues may indicate that this cartilage is actually distinct from primary cartilage and representative of secondary cartilage. Anat Rec, 297:1208–1217, 2014. © 2014 Wiley Periodicals, Inc.     

Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fish: A light and TEM study

The present paper presents ultrastructural details of chondrogenesis of Meckel's cartilage and of ossification of its associated peri- and parachondral bones in a teleost fish, the cichlid Hemichromis bimaculatus. We have distinguished four stages during chondrogenesis, each of which is characterized by specific cellular and matrix features: blastema, primordium, differentiated cartilage and cartilage surrounded by perichondral bone. The blastema is characterized by prechondroblasts and the lack of cartilage matrix; the primordium by chondroblasts and the onset of secretion of matrix of fibrillar and granular nature; differentiated cartilage is characterized by chondrocytes and larger amounts of typical hyaline cartilage matrix. Once perichondral bone is laid down, the chondrocytes show degenerative features but not true hypertrophy. Differentiation of the cartilage cells is attended with cytoplasmic changes indicative of an increasing secretory activity. There is a regional calcification of the cartilage matrix by fusion of calcospherites. Chondrogenesis of the symphyseal area is continuous with that of the rami but starts slightly later. Formation of perichondral bone at the cartilage surface is attended with the deposition of a transitional zone apparently containing a mixture of the two matrices. The role of the perichondral cells is discussed and it is proposed that they may contribute to the formation of the two matrices. The transitional zone may then result either from a diffusion process or from the simultaneous deposition of elements of the two matrices. Growth of the cartilage is argued to be largely the result of matrix secretion, except in the symphyseal area where appositional growth probably occurs until the region is completely covered by perichondral bone. This paper provides a basis for further studies on the developmental interactions between cartilage, bone and teeth during mandibular development in cichlids. © 1992 Wiley-Liss, Inc.     

Anatomy and Ontogeny of the Mandibular Symphysis in Alligator mississippiensis

Crocodylians evolved some of the most characteristic skulls of the animal kingdom with specializations for semiaquatic and ambush lifestyles, resulting in a feeding apparatus capable of tolerating high biomechanical loads and bite forces and a head with a derived sense of trigeminal-nerve-mediated touch. The mandibular symphysis accommodates these specializations being both at the end of a biomechanical lever and an antenna for sensation. Little is known about the anatomy of the crocodylian mandibular symphysis, hampering our understanding of form, function, and evolution of the joint in extant and extinct lineages. We explore mandibular symphysis anatomy of an ontogenetic series of Alligator mississippiensis using imaging, histology, and whole mount methods. Complex sutural ligaments emanating about a midline-fused Meckel's cartilage bridge the symphysis. These tissues organize during days 37–42 of in ovo development. However, interdigitations do not manifest until after hatching. These soft tissues leave a hub and spoke-like bony morphology of the symphyseal plate, which never fuses. Interdigitation morphology varies within the symphysis suggesting differential loading about the joint. Neurovascular canals extend throughout the mandibles to alveoli, integument, and bone adjacent to the symphysis. These features suggest the Alligator mandibular symphysis offers compliance in an otherwise rigid skull. We hypothesize a fused Meckel's cartilage offers stiffness in hatchling mandibles prior to the development of organized sutural ligaments and mineralized bone while offering a scaffold for somatic growth. The porosity of the dentaries due to neurovascular tissues likely allows transmission of sensory and proprioceptive information from the surroundings and the loaded symphysis. Anat Rec, 302:1696–1708, 2019. © 2019 American Association for Anatomy     

Epithelial influences on skeletogenesis in the mandible of the embryonic chick

Intact mandibular processes and the enzymatically separated mesenchymal and epithelial components of the mandible from embryonic chicks of 2.5- to 5-day incubation (Hamburger and Hamilton, '51: stages 16-25) were grown individually, either in organ culture or as grafts to the chorioallantoic membranes of host embryos. The differentiation of cultured and grafted intact mandibular processes was histologically normal, but the time of histodifferentiation differed from that in vivo. The histodifferentiation of cultured and grafted mandibular mesenchyme grown isolated from its epithelium depended upon the age of the embryo from which the mesenchyme had been obtained. Intramembranous ossification producing membrane bones of the mandible occurred in mesenchyme isolated from 4.5- to 5-day embryos (HH 24–25), but did not occur in mesenchyme isolated from younger embryos. Cartilage (Meckel's) and subperichondrial bone in the articular process of Meckel's cartilage differentiated in mesenchyme isolated from embryos of all age groups tested (HH 16–25). Mandibular mesenchyme, therefore, requires the presence of epithelium until 4.5 days of incubation if the membrane bones of the mandible are to differentiate; if epithelial influences are required for Meckel's cartilage and subperichondrial bone formation, they are not required beyond 2.5 days of incubation. Mandibular epithelium isolated from its mesenchyme became layers of squamous cells in culture; but when grafted onto the chorioallantoic membrane, the epithelium became underlain by host fibroblasts and differentiated into a stratified squamous epithelium. Mandibular epithelium, therefore, is capable of differentiation in the presence of foreign fibroblasts derived from the chorioallantoic membrane.     

Differential changes in cartilage cell proliferation and cell density in the rat craniofacial complex during secondary palate development

During mammalian secondary palate formation sagittal growth of the lower face has been shown to be more rapid than that of the upper face, and the tongue and mandible extend beneath the primary palate. In order to identify factors contributing to this differential growth pattern, cellular and morphologic growth of the major cartilages of the upper and lower facial regions were studied in radioautographic sections labeled with tritiated thymidine. Evaluation of cell-density recordings, labeling indices, and structural dimensions revealed significant differences between Meckel's cartilage in the lower face, and the nasal cartilage and anterior cranial base cartilage in the upper face. After formation of the precartilaginous blastema, labeling indices were high in Meckel's cartilage (20–30%), but very low in the nasal cartilage and the anterior cranial base (0–2%). During secondary palate formation of the volume of Meckel's cartilage increased more rapidly than the other cartilages and its growth was primarily in the sagittal direction. Between days 15 and 17, the increase in the length of Meckel's cartilage (165%) was approximately twice as great as the increase in the combined length of the nasal cartilage and the anterior cranial base (77%). During this period induction of cleft palate with some teratogens has been shown to severely retard growth of Meckel's cartilage and produce mandibular retrognathia that contributes to delayed elevation of the palatal shelves. Therefore, extensive cell proliferation in Meckel's cartilage, during a period of limited proliferation in other craniofacial cartilages, appears to contribute to its rapid growth and its differential sensitivity to growth inhibition.     

Meckel's cartilage in the human embryo and fetus

This work studied the development of the ventral part of Meckel's cartilage in a series of human embryos (classified in stages) and fetuses. These stages appeared particularly important: stage 16, appearance of Meckel's cartilage; stage 20, beginning of membranous ossification of mandible; and stage 23, end of the embryonic period (8th week). The primitive bony nodule which develops from the embryonic mesenchyme appears as a double bony layer forming a groove containing the neurovascular bundle, into which the dental lamina is also invaginated. It was concluded that during the fetal period, the cartilage participates in the formation of the body of the mandible in an area close to the mental foramen via endochondral ossification. The cartilage disappears in parallel with the development of ossification by the sixth month. © 1994 Wiley-Liss, Inc.     

Microanatomy of the Mandibular Symphysis in Lizards: Patterns in Fiber Orientation and Meckel's Cartilage and Their Significance in Cranial Evolution

Although the mandibular symphysis is a functionally and evolutionarily important feature of the vertebrate skull, little is known about the soft‐tissue morphology of the joint in squamate reptiles. Lizards evolved a diversity of skull shapes and feeding behaviors, thus it is expected that the morphology of the symphysis will correspond with functional patterns. Here, we present new histological data illustrating the morphology of the joint in a number of taxa including iguanians, geckos, scincomorphs, lacertoids, and anguimorphs. The symphyses of all taxa exhibit dorsal and ventral fibrous portions of the joints that possess an array of parallel and woven collagen fibers. The middle and ventral portions of the joints are complemented by contributions of Meckel's cartilage. Kinetic taxa have more loosely built symphyses with large domains of parallel‐oriented fibers whereas hard biting and akinetic taxa have symphyses primarily composed of dense, woven fibers. Whereas most taxa maintain unfused Meckel's cartilages, iguanians, and geckos independently evolved fused Meckel's cartilages; however, the joint's morphologies suggest different developmental mechanisms. Fused Meckel's cartilages may be associated with the apomorphic lingual behaviors exhibited by iguanians (tongue translation) and geckos (drinking). These morphological data shed new light on the functional, developmental, and evolutionary patterns displayed by the heads of lizards. Anat Rec 293:1350–1359, 2010. © 2010 Wiley‐Liss, Inc.     

On the presence of a secondary cartilage in the mental symphyseal region of human embryos and fetuses

Summary The presence of a secondary cartilage in the mental symphyseal region was examined in this study. A double-staining method -with alcian blue and alizarin red S -was performed on both whole human embryos and fetuses (developmental age between 8 and 17 weeks, crown-rump length, CRL, between 37 and 124 mm) and their disjointed mandibles. Histological and histochemical techniques were applied to transverse serial sections of whole disjointed fetal heads. The ossification process observed in the mental symphysis is quite different from that of the mandibular body, whose membranous ossification is induced by the contiguous Meckel's cartilage. No evidence of any fusion of Meckel's cartilage with the symphyseal cartilage, that lies within the symphyseal space, was detected. On the basis of these findings, we suggested that the mental secondary cartilage is able to change into bone according to an endochondral ossification process. Moreover, the role of mechanical causes in the development of the mental symphysis was hypothesized.

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A propos de la présence d'un cartilage secondaire dans la région de la symphyse mentonnière sur les embryons et foetus humains

Résumé La présence d'un cartilage secondaire dans la région de la symphyse mentonnière a été étudiée dans ce travail. Une double coloration (avec le bleu alcian et le rouge alizarine S) a été réalisée sur 32 embryons et foetus humains (âgés de 8 à 17 semaines, longueur crâniocaudale- CRL - entre 37 et 124 mm) et sur leurs mandibules désarticulées. Les techniques histologiques et histochimiques ont été appliquées aux coupes sériées transversales de toutes les têtes foetales désarticulées. Le processus d'ossification observé au niveau de la symphyse mentonnière est tout à fait différent de celui du corps de la mandibule dont l'ossification membraneuse est induite par le cartilage de Meckel contigu. Nous n'avons détecté aucun signe de fusion du cartilage de Meckel avec le cartilage symphysaire qui se trouve dans l'espace symphysaire. Sur la base de nos constatations, nous suggérons que le cartilage secondaire mentonnier est capable de se transformer en os selon un processus d'ossification enchondrale. De plus le rôle des facteurs mécaniques dans le développement de la symphyse mentonnière est suggéré.

     

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     

Clonal proliferation and cell density of chondrocytes isolated from human fetal epiphyseal, human adult articular and nasal septal cartilage. Influence of hormones and growth factors

The present study characterizes the cell density and cellular growth characteristics of functionally and morphogenetically different human cartilages. In fetal epiphyseal, in postnatal nasal septal and in articular cartilage the influence of aging on cell density and in vitro growth characteristics were investigated. Cell density was highest in fetal epiphyseal cartilage and lowest in articular cartilage. In vitro growth of isolated chondrocytes as measured by clonal growth in a semisolid assay was almost identically high in fetal epiphyseal and septal cartilage and significantly lower in articular cartilage. Cell density increased with age in the nasal septal cartilage whereas no age dependency was found in the other cartilages. The stimulatory effects of human biosynthetic insulin, human growth hormone and partially purified IGF I on clonal growth of chrondrocytes were assessed. Only IGF I stimulated clonal growth of chondrocytes and its stimulatory effect was significantly higher in postnatal than in fetal chondrocytes.     

Stage-specific expression patterns of alkaline phosphatase during development of the first arch skeleton in inbred C57BL/6 mouse embryos

Timing and pattern of expression of alkaline phosphatase was examined during early differentiation of the 1st arch skeleton in inbred C57BL/6 mice. Embryos were recovered between 10 and 18 d of gestation and staged using a detailed staging table of craniofacial development prior to histochemical examination. Expression of alkaline phosphatase is initiated at stage 20.2 in the plasma membrane of mesenchymal cells in the distal region of the first arch. Expression is strongest in osteoid (unmineralised bone matrix) and presumptive periosteum at stage 21.32. Mineralisation begins at stage E23. Expression is present in the mineralised bone matrix. Secondary cartilages form in the condylar and angular processes by stage M24. The cartilaginous cells and surrounding cells in the processes are all alkaline phosphatase-positive and surrounded by the common periosteum, suggesting that progenitor cells of the processes, dentary ramus and secondary cartilages all originate from a common pool. Nonhypertrophied chondrocytes of Meckel's cartilage express alkaline phosphatase at stage M23. Expression in these chondrocytes is preceded by the expression in their adjacent perichondrium. This is true of chondrocytes in all other cranial cartilages examined. 3-D reconstruction of expression in Meckel's cartilage also revealed that the chondrocytes of Meckel's cartilage which express alkaline phosphatase and the matrix of which undergoes mineralisation are those surrounded by the alkaline phosphatase-positive dentary ramus. By stage 25, coincident with mineralisation in the distal section of Meckel's cartilage, most chondrocytes are strongly positive. The perichondria of malleus and incus cartilages express alkaline phosphatase at stage M24. Nonhypertrophied chondrocytes along these perichondria also express alkaline phosphatase. Superficial and deep cells in the dental laminae of incisor and 1st molar teeth become alkaline phosphatase-positive at the bud stage, stages 21.16 and 21.32, respectively. Dental papillae are negative until stage M24 when alkaline phosphatase expression begins in the dental papillae and follicles of the incisor teeth and the dental follicles of the 1st molar teeth. The dental papillae of the 1st molar teeth express alkaline phosphatase at stage 25. Expression in the dental papillae and follicles appears to coincide with cellular differentiation of follicle from papilla. The presumptive squamosal, ectotympanic and gonial membrane bones, lingual oral epithelial cells connected to the dental laminae of the incisor teeth, hair follicle papillae and sheath and surrounding dermis all express alkaline phosphatase in a stage-specific manner.     

Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fish: a light and TEM study.

The present paper presents ultrastructural details of chondrogenesis of Meckel's cartilage and of ossification of its associated peri- and parachondral bones in a teleost fish, the cichlid Hemichromis bimaculatus. We have distinguished four stages during chondrogenesis, each of which is characterized by specific cellular and matrix features: blastema, primordium, differentiated cartilage and cartilage surrounded by perichondral bone. The blastema is characterized by prechondroblasts and the lack of cartilage matrix; the primordium by chondroblasts and the onset of secretion of matrix of fibrillar and granular nature; differentiated cartilage is characterized by chondrocytes and larger amounts of typical hyaline cartilage matrix. Once perichondral bone is laid down, the chondrocytes show degenerative features but not true hypertrophy. Differentiation of the cartilage cells is attended with cytoplasmic changes indicative of an increasing secretory activity. There is a regional calcification of the cartilage matrix by fusion of calcospherites. Chondrogenesis of the symphyseal area is continuous with that of the rami but starts slightly later. Formation of perichondral bone at the cartilage surface is attended with the deposition of a transitional zone apparently containing a mixture of the two matrices. The role of the perichondral cells is discussed and it is proposed that they may contribute to the formation of the two matrices. The transitional zone may then result either from a diffusion process or from the simultaneous deposition of elements of the two matrices. Growth of the cartilage is argued to be largely the result of matrix secretion, except in the symphyseal area where appositional growth probably occurs until the region is completely covered by perichondral bone. This paper provides a basis for further studies on the developmental interactions between cartilage, bone and teeth during mandibular development in cichlids.     

Temporal and spatial localization of type I and II collagens in human thyroid cartilage

Thyroid cartilages of various ages were investigated by immunofluorescence staining for localization of the fibrillar collagen types I and II in order to understand the tissue remodeling occurring during the mineralization and ossification of thyroid cartilage. In fetal and juvenile thyroid cartilages, type I collagen was restricted to the inner and outer perichondrium, while type II collagen was localized in the matrix of hyaline cartilage. However, in advanced ages, type I collagen was also localized in the pericellular and in the interterritorial matrix of intermediate and central chondrocytes of thyroid cartilage. The matrix of peripheral chondrocytes was negative for type I collagen. This suggests that some chondrocytes in thyroid cartilage undergo a differentiation to type I collagen-producing chondrocytes. At the beginning of ossification, bone-related type I collagen was chiefly detected in the central cartilage layer, but was never deposited first from the perichondrium in the direction to the subperichondrial cartilage. This observation confirmed previous findings showing that osteogenesis mainly follows an endochondral ossification pattern. Interterritorial matrix failed to react with the type II collagen antibody in men from the beginning of the third decade, and later still in women, even after treatment with hyaluronidase. These observations indicate that major matrix changes occur faster in male than in female thyroid cartilage.Dedicated to Professor Dr. W. Kühnel on the occasion of his 60th birthday     

Differential responses to parathyroid hormone‐related protein (PTHrP) deficiency in the various craniofacial cartilages

PTHrP null mutant mice exhibit skeletal abnormalities both in the craniofacial region and limbs. In the growth plate cartilage of the null mutant, a diminished number of proliferating chondrocytes and accelerated chondrocytic differentiation are observed. In order to examine the effect of PTHrP deficiency on the craniofacial morphology and highlight the differential feature of the composing cartilages, we examined the various cartilages in the craniofacial region of neonatal PTHrP deficient mice. The major part of the cartilaginous anterior cranial base appeared to be normal in the homozygous PTHrP deficient mice. However, acceleration of chondrocytic differentiation and endochondral bone formation was observed in the posterior part of the anterior cranial base and in the cranial base synchondroses. Ectopic bone formation was observed in the soft tissue‐running mid‐portion of the Meckel's cartilage, where the cartilage degenerates and converts to ligament in the course of normal development. The zonal structure of the mandibular condylar cartilage was scarcely affected, but the whole condyle was reduced in size. These results suggest the effect of PTHrP deficiency varies widely between the craniofacial cartilages, according to the differential features of each cartilage. Anat Rec 255:452–457, 1999. © 1999 Wiley‐Liss, Inc.     

Different expression of 25-kDa heat-shock protein (Hsp25) in Meckel's cartilage compared with other cartilages in the mouse

The 25-kDa heat-shock protein (Hsp25) is expressed in the cartilage of the growth plate and suggested to function in chondrocyte differentiation and degeneration. Using immunohistochemistry, we examined the temporal and spatial occurrence of Hsp25 in Meckel's cartilage in embryonic mice mandibles, and in other types of cartilage in both embryonic and adult mice. In adults, Hsp25 immunoreactivity was detected in the hypertrophic chondrocytes located in growth plates of long bones and in non-osteogenic laryngeal and tracheal cartilages. No chondrocytes in the resting or proliferating phase exhibited Hsp25 immunoreactivity. In the embryonic mandibles, resting and proliferating chondrocytes in the anterior and intermediate portions of Meckel's cartilage showed Hsp25 immunoreactivity from the 12th day of gestation (E12) through E15, whereas those in the posterior portion showed little or no immunoreactivity. After E16, the overall Hsp25 immunoreactivity in Meckel's cartilage substantially reduced in intensity, and little or no immunoreactivity was detected in the hypertrophic chondrocytes located in the degenerating portions of Meckel's cartilage. The antisense oligonucleotide for Hsp25 mRNA applied to the culture media of the mandibular explants from E10 embryos caused significant inhibition of the development of the anterior and middle portions of Meckel's cartilage. These results suggested that Hsp25 is essential for the development of Meckel's cartilage and plays different roles in Meckel's cartilage from those in the permanent cartilages and the cartilages undergoing endochondral ossification.     

Lack of association between avian cartilages of different embryological origins when maintained in vitro

The possibility that cartilages of differing embryological origins behave as separate types with respect to cell-to-cell associations was tested by placing the cut ends of transversely sectioned embryonic chick tibial cartilages (of mesodermal origin) in apposition to transversely sectioned Meckel's cartilages (a neural crest (ectodermal) cartilage) on the surface of a semi-solid organ culture medium and maintaining the combinations in vitro for five to ten days. Tibia-tibia and Meckel's cartilage-Meckel's cartilage (homotypic) combinations, which served as controls, became united by a common extracellular matrix and by the proliferation of chondroblasts. Analysis of combinations where one partner had been prelabelled with ³H-thymidine indicated that chondroblasts intermingled at the contact zone. In contrast, tibia-Meckel's cartilage (heterotypic) combinations became separated by a layer of fibrous tissue. The chondroblasts at the contact zone failed to intermingle. We conclude that avian embryonic chondrocytes are not all equivalent and that part of their non-equivalence could be related to their embryological origin either from the mesoderm or from the ectodermal neural crest.     

Bone morphogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice

Secondary cartilages including mandibular condylar cartilage have unique characteristics. They originate from alkaline phosphatase (ALP)-positive progenitor cells of the periosteum, and exhibit characteristic modes of differentiation. They also have a unique extracellular matrix, and coexpress type I, II and X collagens. We have previously shown that there is a total absence of secondary cartilages in Runx2-deficient (Runx2-/-) mice. To clarify whether Runx2 is essential for chondrocytic differentiation of secondary cartilages, we performed an organ culture system using mandibular explants derived from Runx2-/- mice at embryonic day 18.0. Since mRNA for bone morphogenetic protein 2 (BMP2) was strongly expressed in osteoblasts of condylar anlagen in wild-type mice, and was down-regulated in those of Runx2-/- mice, we chose to investigate BMP2 effects on secondary cartilage formation. Condensed mesenchymal cells of mandibular condylar anlagen in precultured explants were ALP-positive and expressed type I collagen and Sox9. After culture with recombinant human (rh) BMP2, chondrocytic cells showing ALP activity and expressing Sox5, Sox9, and type I and II collagens, appeared from mesenchymal condensation. This expression profile was comparable with the reported pattern of chondrocytes in mouse secondary cartilages. However, chondrocyte hypertrophy was not observed in the explants. These findings indicate that BMP2 partially rescued chondrocyte differentiation but not chondrocyte hypertrophy in secondary cartilage formation in Runx2-/- mice. Runx2 is required for chondrocyte hypertrophy in secondary cartilage formation, and it is likely that BMP2, which is abundantly secreted by osteoblasts in condylar anlagen, contributes to the early process of secondary cartilage formation.     

Development of the microcirculation of the secondary ossification center in rat humeral head

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed.     

Autophagy Prior to Chondrocyte Cell Death During the Degeneration of Meckel's Cartilage

The central portion of Meckel's cartilage degenerates almost immediately after birth. Whether autophagy is involved in this process remains unclear. Thus, to explore the role of autophagy during this process, we have detected the expression of autophagy and apoptosis‐related markers in embryonic mice. In E15, Beclin1 and LC3 expressions were weak and negative in Meckel's cartilage, respectively. In E16, chondrocytes of the central portion became hypertrophic. Moderate immunoreactivities of Beclin1 and LC3 were observed in prehypertrophic and hypertrophic chondrocytes of the central portion. In E17, the degradation occurred in the central portion and expanded anteriorly and posteriorly. Beclin1 expression was observed in Meckel's cartilage with an increase in the hypertrophic chondrocytes of the central portion. The expression of LC3 was detected specifically in terminally differentiated hypertrophic chondrocytes. The mRNA expressions of LC3 and Beclin1 from E15 to E17 significantly increased. This result is in accord with the histologic findings. Terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick‐end labeling assay and Caspase 3 expression demonstrated that apoptosis was detected in the lateral part of terminal hypertrophic chondrocytes along the degeneration area of Meckel's cartilage. In addition, Bcl2 expression increased significantly from E15 to E17. These results indicate that autophagy is involved in hypertrophic chondrocytes during the degradation of Meckel's cartilage and occurs prior to chondrocyte cell death during this process. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.