Gene expression and distribution of connective tissue growth factor (CCN2/CTGF) during secondary ossification center formation.

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.     

Relationship between the cartilage canal and the perichondrium in the rat proximal tibial epiphysis

One of the most widespread hypotheses for chondral canal morphogenesis suggests that the canal is an extension of the perichondrium. To study the possible relation between perichondrium and chondral canal morphology, the proximal epiphyses in the tibias of 42 rats were studied from birth to their 29th day. The study was divided into three periods: from birth to the 4th day before canal appearance; from the 5th day, the moment of canal appearance, until the appearance of the secondary ossification center of the epiphysis on the 9th day; the 3rd ran from this point on the 10th day until its full development. We have also divided the canal into three regions: entrance, neck and bottom. The central portion (lumen) and canal wall were analyzed in each region. Our results show the perichondrium to be a complex structure, composed of a series of cellular layers in a biphasic extracellular matrix (eosinophil and basophil). The canal walls are lined by a layer of elongated cells. In the lumen there are many different cell types: fibroblasts, histiocytes, multinuclear giant cells and multivacuolated cells. Our study of the canal, its walls and lumen show no morphological structure that is reminiscent of the perichondrium. These results suggest that the canal is not itself a continuation of the perichondrium.     

Maturation process of secondary ossification centers in the rat and assessment of bone age.

The maturation process from the appearance to the fusion of the secondary ossification centers of extremities was studied in Wistar rats aged 0 to 134 weeks. The examination of the secondary ossification centers made by radiography. The assessment of the stage of development was made in accordance with the criteria proposed by Ohwada and Sutow. The secondary ossification center was found to be take one of the following three types of maturation processes : (1) the acute ossification, (2) the delayed ossification, and (3) the incomplete ossification. No fusion was observed up to 134 weeks in certain epiphyses of the rat. This type of ossification designated as the incomplete ossification may be specific to the mouse and rat. The relative lengths of time required for appearance and fusion in the average prospective life were obtained for the rat. They were compared with those of the mouse and man. The relative length of time necessary for maturity of the secondary ossification centers was shown to be the shortest in the rat and the longest in man. The results suggested that the rat may reach maturity in the bone age at 17 to 21 weeks of age. The rat at this age may be regarded as being adult corresponding to age 17 weeks in mice and 18 to 24 years in man.     

Epigenetic mechanical factors in the evolution of long bone epiphyses

In developing vertebrate long bones in which endochondral ossification occurs, it is preceded or accompanied by perichondral ossification. The speed and extent of perichondral apposition relative to endochondral ossification varies in different taxa. Perichondral ossification dominates early long bone development in extinct basal tetrapods and dinosaurs, extant bony fish, amphibians, and birds. In mammals and lizards, perichondral and endochondral ossification proceed more synchronously. One of the most important epigenetic factors in skeletogenesis is mechanical loading caused by muscle contractions which begin in utero or in ovo . It has been previously shown that the stress distributions created perinatally in the chondroepiphysis during human skeletal development can influence the appearance of secondary ossification centres. Using finite element computer models representing bones near birth or hatching, we demonstrate that in vertebrates in which perichondral ossification significantly precedes endochondral ossification, the distribution of mechanical stresses in the ossifying cartilage anlagen tends to inhibit the appearance of secondary ossification centres in the ends of long bones. In models representing vertebrates in which endochondral ossification keeps pace with perichondral apposition, the appearance of secondary centres is promoted. The appearance of secondary centres leads to the formation of bony epiphyses and growth plates, which are most common in mammals and extant lizards. We postulate that genotypic factors influencing the relative speed and extent of perichondral and endochondral ossification interact with mechanical epigenetic factors early in development to account for many of the morphological differences observed in vertebrate skeletons.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

Positional changes of the frontoparietal ossification centers in perinatal craniosynostotic rabbits.

It has been suggested that craniosynostosis is caused by abnormally located ossification centers (i.e., bony tubers) in the developing skull prior to suture formation [Mathijssen et al., 1996, 1997]. The present study was designed to test this hypothesis in a rabbit model of human familial, nonsyndromic coronal suture (CS) synostosis. Calvariae were taken from 99 New Zealand White rabbit perinates (55 normal controls, 15 with delayed-onset CS synostosis, and 29 with bilateral or unilateral CS synostosis), ranging in age from 23 to 34 days postconception (synostosis occurs at approximately 23 days in this model). Frontoparietal, interfrontal, and interparietal ossification center distances were obtained using a Wild microscope with camera lucida attachment and a 2-D computer digitization technique. Linear regression analysis was used to compare age-related changes in the perinatal ossification centers among groups. Results revealed that frontoparietal ossification center regression line slopes had similar start points (24-day intercepts) with significantly (P < 0.05) diverging slopes over time. Normal and delayed-onset ossification center distance increased more rapidly than in synostosed perinates. No significant (P > 0.05) differences were noted in regression line slopes among groups for interparietal or interfrontal ossification center distances. Results demonstrated that, in synostosed perinates, frontoparietal ossification center location was similar to normals around the time of synostosis and became displaced later. These findings suggest that ossification center (i.e., bony tuber) displacement seen in infants with craniosynostosis is probably a secondary and compensatory, postsynostotic change and not a primary causal factor of synostosis in this rabbit model.     

Microvascularization of the sternum in children

46 sternums originating from 1-day- to 17-year-old children were injected with India ink and transparified. The intraosteal and medullary vasculature is described at different stages, as well as cartilage canals, vessels of the isolated ossification center, vessels of the ossification center connected with peripheral vascular structures or neighboring cartilage canals, and finally the transition to the adult pattern. With age, the centrifugal vascular distribution develops to a centripetal pattern.     

Development of the supraorbital and mandibular lateral line canals in the cichlid,Archocentrus nigrofasciatus

The development of two of the cranial lateral line canals is described in the cichlid, Archocentrus nigrofasciatus. Four stages of canal morphogenesis are defined based on histological analysis of the supraorbital and mandibular canals. "Canal enclosure" and "canal ossification" are defined as two discrete stages in lateral line canal development, which differ in duration, an observation that has interesting implications for the ontogeny of lateral line function. Canal diameter in the vicinity of individual neuromasts begins to increase before ossification of the canal roof in each canal segment; this increase in canal diameter is accompanied by an increase in canal neuromast size. The mandibular canal generally develops later than the supraorbital canal in this species, but in both of these canals development of the different canal segments contained within a single dermal bone is asynchronous. These observations suggest that a dynamic process requiring integration and interaction among different tissues, in both space and time, underlies the development of the cranial lateral line canal system. The supraorbital and mandibular canals appear to demonstrate a "one-component" pattern of development in Archocentrus nigrofasciatus, where the walls of each canal segment grow up from the underlying dermal bone and then fuse to form the bony canal roof. This is contrary to numerous published reports that describe a "two-component" pattern of development in teleosts where the bony canal ossifies separately and then fuses with an underlying dermal bone. A survey of the literature in which lateral line canal development is described using histological analysis suggests that the occurrence of two different patterns of canal morphogenesis ("one-component" and "two-component") may be due to phylogenetic variation in the pattern of the development of the lateral line canals.     

A tale of two cities: The genetic mechanisms governing calvarial bone development

The skull bones must grow in a coordinated, three‐dimensional manner to coalesce and form the head and face. Mammalian skull bones have a dual embryonic origin from cranial neural crest cells (CNCC) and paraxial mesoderm (PM) and ossify through intramembranous ossification. The calvarial bones, the bones of the cranium which cover the brain, are derived from the supraorbital arch (SOA) region mesenchyme. The SOA is the site of frontal and parietal bone morphogenesis and primary center of ossification. The objective of this review is to frame our current in vivo understanding of the morphogenesis of the calvarial bones and the gene networks regulating calvarial bone initiation in the SOA mesenchyme.     

Development of the cartilage canals and the secondary center of ossification in the distal chondroepiphysis of the prenatal human femur.

The cartilaginous epiphysis of the distal femur is vascularized by a network of cartilage canals during prenatal development. The vascular invasion of the epiphysis begins at approximately eight to ten weeks of gestation with the initiation of cartilage canal formation. A complex vascular system develops within the canals and is well defined by fourteen weeks of gestation. The vascular system is fully developed several months prior to the development of the secondary center of ossification. The formation of the secondary center of ossification within the distal femoral epiphysis is preceded by changes that occur simultaneously within both the chondrocytes in the central portion of the epiphysis and the vascular and perivascular elements contained within the cartilage canals in the central portion of the epiphysis. These concurrent changes in the cellular morphology of the central chondrocytes and in the cellular structure of the central cartilage canals appear to be linked with the initiation of the process of osteogenesis.     

A tissue culture system supporting cartilage cell differentiation, extracellular mineralization, and subsequent bone formation, using mouse condylar progenitor cells

Undifferentiated progenitor cells of mandibular condyles of neonatal mice were kept in a tissue culture system for up to 9 days. After 2 days in culture, new chondroblasts developed within the explants, whereas the peripheries of the latter were occupied by undifferentiated cells undergoing mitosis. By 4 days in culture, many of the cartilage cells showed signs of hypertrophy, while the matrix revealed positive reactivity for type II collagen and matrix metachromasia. The process of maturation of cartilage cells appeared to have reached its final stages after 6 days in culture, at a time when the initial loci of matrix mineralization could be easily identified. Concomitantly, peripheral areas bordering the cartilaginous core, as well as portions of the cartilage, reacted positively for type I collagen and fibronectin. Light microscopy examination showed signs of new bone formation after 9 days in culture. The extracellular matrix at the upper portion of the explant, facing the medium-air interface, reacted intensely with antibodies against type I collagen and fibronectin, but not with antibodies against type II collagen. Further, the newly formed osteoid was found to have undergone mineralization, thus forming an expanded layer of new bony tissue. A close spatial association was found between mature, mineralized cartilage and new bone. Hence, we hereby introduce a new in vitro system serving as an experimental model for studies related to the differentiation of progenitor cells into chondroblasts, which in turn promote ossification.     

An ultrastructural investigation of nephrotoxicity in rats induced by feeding cultures of Penicillium verrucosum var. cyclopium.

A renal tubular lesion was induced in male rats by giving them a culture homogenate or culture filtrate of Penicillium verrucosum var. cyclopium by gastric gavage for 20 days. The fungus was obtained from stored maize in an area of endemic nephropathy in Bulgaria. Changes in the proximal convoluted tubules were studied by light and electron microscopy. The lesion was confined to the pars recta in the outer stripe of the outer zone of the medulla. It consisted of degeneration and necrosis of epithelial cells, prominent karyomegaly, arrested mitotic divisions and production of binucleate and tetranucleate tubular cells. Two patterns of degeneration occurred with comparable frequency: a vesicular form with pyknotic nucleus and electron lucent degeneration. Nuclei of the epithelial cells in affected tubules contained segregated nucleoli. The necrotic cells were replaced by actively regenerating cells derived from adjacent viable epithelium. The similarity between the tubular lesions induced in rats and the changes found in patients with Balkan endemic nephropathy is discussed.     

Palate morphogenesis. I. Immunological and ultrastructural analyses of mouse palate

Midpalate was analyzed for the presence of nonmuscle contractile systems. The results indicate that increased amounts of actin and myosin are present in cells of regions 2 and 3. A localization of the contractile proteins in cellular projections (filopodia) and in the peripheral cytoplasm of the cell body was confirmed by indirect immunofluorescence studies, using antibodies directed against smooth muscle myosin and against skeletal muscle actin. Specificity of the immunofluorescence reactions was ascertained by immunoabsorption studies using purified myosin and actin. Electron microscopic observations of the mesenchymal cells in region 2 revealed 70A microfilaments along the cell periphery and packed in fliopodia-like projections which course between the cells. These cells, which surround a small ossification center, show no orientation, but extend up to the cranial base perichondrium and down into the shelf between the tongue side epithelium and the ossification center. The cells and projections are attached to each other by adherens and tight-like junctions, forming a putative cohesive contractile network. Putative contractile cells in region 3 are strikingly aligned perpendicular to the oral epithelium and extend one-third of the distance into the shelf. Projections from region 3 cells are contiguous with basement membrane material of the oral epithelium. Axonal bundles and single axons were commonly observed coursing through regions 2 and 3, often seen in close association with the mesenchymal cells. Both clear and dense-core vesicles were found in the axons and cells of these regions. The possible role of these putative nonmuscle contractile cells in palate morphogenesis is discussed.     

Promoting effect of feeder cells in maintenance of adult rat hepatocytes

A procedure is described for maintaining primary cultures of adult rat hepatocytes for prolonged periods of time on layer of irradiated mouse fibroblast cell line (C3H/1OT1/2) and on a secondary lung fibroblasts obtained from Sprague Dawley rats. Morphologically and ultrastructurally the cocultivated hepatocytes retained many characteristics of hepatocytes in vivo. Within 24 hours after seeding, the individual cells were attached on the feeder cell layer and the in vivo polarity of the liver cells reappeared. Electron microscope studies demonstrated the appearance of newly developed bile ducts and junctions between hepatocytes as well as between hepatocytes and feeder cells. Histochemically, these cells were positive for glucose-6-phosphatase and for glycogen. After 14 days in culture the hepatocytes could be reseeded onto fresh C3H1OT1/2 cells. In contrast, hepatocytes maintained on plastic substrate lost their glycogen content and the epithelial character of the liver cells after 5 days in culture, and by day 10 this culture became predominantly fibroblastic. It is suggested that hepatocytes maintained on an irradiated fibroblast feeder layer provide a valuable approach for studying the morphogenesis, cytotoxicity, or the metabolism of different chemicals in vitro.     

The primary enamel knot determines the position of the first buccal cusp in developing mice molars

The enamel knot (EK), which is located in the center of bud and cap stage tooth germs, is a transitory cluster of non-dividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps by inducing secondary EKs. The morphological, cellular, and molecular events leading to the relationship between the primary and secondary EKs have not been described clearly. This study investigated the relationship between the primary and secondary EKs in the maxillary and mandibular first molars of mice. The location of the primary EK and secondary EKs was investigated by chasing Fgf4 expression patterns in tooth germ at some intervals of in vitro culture, and the relationship between the primary EK and secondary EK was examined by tracing the primary EK cells in the E13.5 tooth germs which were frontally half sliced to expose the primary EK. After 48 hr, the primary EK cells in the sliced tooth germs were located on the buccal secondary EKs, which correspond to the future paracone in maxilla and protoconid in mandible. The Bmp4 expression in buccal part of the dental mesenchyme might be related with the lower growth in buccal epithelium than in lingual epithelium, and the Msx2 expressing area in epithelium was overlapped with the enamel cord (or septum) and cell dense area. The enamel cord might connect the primary EK with enamel navel to fix the location of the primary EK in the buccal side during the cap to bell stages. Overall, these results suggest that primary EK cells strictly contribute to form the paracone or protoconid, which are the main cusps of the tooth in the maxilla or mandible.     

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

The fate of Meckel's cartilage chondrocytes in ocular culture

Modulation of the chondrocyte phenotype was observed in an organ culture system using Meckel's cartilage. First branchial arch cartilage was dissected from fetal rats of 16- and 17-day gestation. Perichondrium was mechanically removed, cartilage was split at the rostral process, and each half was grafted into the anterior chamber of an adult rat eye. The observed pattern of development in nonirradiated specimens was the following: hypertrophy of the rostral process and endochondral-type ossification, fibrous atrophy in the midsection, and mineralization of the malleus and incus. A change in matrix composition of the implanted cartilage was demonstrated with immunofluorescence staining for cartilage-specific proteoglycan (CSPG). After 15 days of culture, CSPG was found in the auricular process but not in the midsection or rostral process. In order to mark the implanted cells and follow their fate, cartilage was labeled in vitro with [3H]thymidine [3H]TdR). Immediately after labeling 20% of the chondrocytes contained [3H]TdR. After culturing for 5 days, 20% of the chondrocytes were still labeled and 10% of the osteogenic cells also contained radioactive label. The labeling index decreased in both cell types with increased duration of culture. Multinucleated clast-type cells did not contain label. Additional cartilages not labeled with [3H]TdR were exposed to between 20000 and 6000 rad of gamma irradiation before ocular implantation. Irradiated cartilage did not hypertrophy or form bone but a fibrous region developed in the midsection. Cells of the host animal were not induced to form bone around the irradiated cartilage. Our studies suggest that fully differentiated chondrocytes of Meckel's cartilage have the capacity to become osteocytes, osteoblasts, and fibroblasts.     

Similar growth pattern of mouse mammary epithelium cultivated in collagen matrix in vivo and in vitro

Mouse mammary ductal cells cultured in type I collagen gels give rise to three-dimensional multicellular outgrowths consisting of thin spikes which are often branched, and which may have pointed or blunt ends. The significance of these spikes to normal ductal morphogenesis has been unclear, since identical structures are not known to occur in vivo; conversely, it has not been possible to maintain in gel culture the highly structured end buds which are characteristic of ductal elongation in the animal. In order to evaluate whether the pattern of radiating spikes observed in collagen gel cultures results from chemical or physical peculiarities of the culture environment, a small volume of unpolymerized type I collagen solution was injected into mammary gland-free fat pads of young adult mice. After the bubble of collagen had polymerized, an implant of mammary ductal epithelium was introduced into the center of the gel. Histological examination of the implants after 3 to 6 days of growth revealed numerous small epithelial spikes, similar to those observed in gel culture, extending into the fibrous matrix. The early stages of regeneration of mammary implants placed in gland-free fat pads were then examined without the addition of exogenous collagen. In cases where the epithelium happened to contact a fibrous region of the fatty stroma, spikes were also seen to form in these natural collagenous substrates. Whether or not exogenous collagen was used, normal end buds were formed only when epithelial spikes contacted adipocytes. It was concluded that the three-dimensional pattern of radiating tubules in collagen gels in vitro is not merely an artifact of culture, but has a counterpart in vivo whereever regenerating mammary epithelium is surrounded by fibrous stroma. A model is presented in which the pattern of epithelial outgrowth is determined by the physical characteristics of the surrounding stroma; in collagen matrix a comparatively primitive and unspecialized type of morphogenesis occurs which may not require the participation of stromal cells. In contrast, epithelial-adipocyte interactions appear to be necessary for the formation of end buds and subsequent morphogenesis of fully structured mammary ducts.     

Changes in membrane-bound aminopeptidase on bone marrow-derived macrophages during their maturation in vitro

Myeloid colonies obtained by culturing mouse bone marrow cells with mouse lung conditioned medium were kept for up to 21 days in culture and the aminopeptidase content in single cells measured after staining with leucine 4-methoxy-2-naphthylamide. The enzyme was detectable only in mononuclear and not in granulocytic cells. The number of cells carrying the enzyme and the concentration of the enzyme in the mononuclear cells taken from whole dishes or single colonies increased remarkably but not uniformly from 7 days to maximal values at 13 days of culture, and then decreased again. The timing varied for individual colonies. Maximal enzyme concentrations were found in cells intermediate between the center and the fringe of a colony. However, most cells in a given colony showed concentration increases up to 13 days of culturing. During its life span in culture the mononuclear cell appears to gain aminopeptidase at the cell membrane and lose it again prior to death.