Role of CTGF/HCS24/ecogenin in skeletal growth control

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called "ecogenin: endochondral ossification genetic factor." In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions.     

Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf(-/-) mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.     

Ovarian maturation and oogenesis in the blue swimmer crab,Portunus pelagicus (Decapoda: Portunidae)

The study was aimed at understanding the process of reproduction and the changes happening in the ovary of Portunus pelagicus during maturation, which would be useful for its broodstock development for hatchery purposes. For that, tissue samples from different regions of the ovary at various stages of maturation were subjected to light and electron microscopy, and based on the changes revealed and the differences in ovarian morphology, the ovary was divided into five stages such as immature (previtellogenic oocytes), early maturing (early vitellogenic oocytes), late maturing (late vitellogenic oocytes), mature (vitellogenic oocytes), and spent (resorbing oocytes). The ovarian wall comprised of an outermost thin pavement epithelium, a middle layer of connective tissue, and an innermost layer of germinal epithelium. The oocytes matured as they moved from the centrally placed germinal zone toward the ovarian wall. The peripheral arrangement of nucleolar materials and the high incidence of cell organelles during the initial stages indicated vitellogenesis I. Movement of follicle cells toward oocytes in the early maturing stage and low incidence of mitochondria and endoplasmic reticulum in the ooplasm during late vitellogenic stage marked the commencement and end of vitellogenesis II, respectively. Yolk granules at various stages of development were seen in the ooplasm from late vitellogenic stage onwards. The spent ovary had an area with resorbing oocytes and empty follicle cells denoting the end of one reproductive cycle and another area with oogonial cells and previtellogenic oocytes indicating the beginning of the next.     

MicroRNA-26a Regulates RANKL-Induced Osteoclast Formation

Osteoclasts are unique cells responsible for the resorption of bone matrix. MicroRNAs (miRNAs) are involved in the regulation of a wide range of physiological processes. Here, we examined the role of miR-26a in RANKL-induced osteoclastogenesis. The expression of miR-26a was up-regulated by RANKL at the late stage of osteoclastogenesis. Ectopic expression of an miR-26a mimic in osteoclast precursor cells attenuated osteoclast formation, actin-ring formation, and bone resorption by suppressing the expression of connective tissue growth factor/CCN family 2 (CTGF/CCN2), which can promote osteoclast formation via up-regulation of dendritic cell-specific transmembrane protein (DC-STAMP). On the other hand, overexpression of miR-26a inhibitor enhanced RANKL-induced osteoclast formation and function as well as CTGF expression. In addition, the inhibitory effect of miR-26a on osteoclast formation and function was prevented by treatment with recombinant CTGF. Collectively, our results suggest that miR-26a modulates osteoclast formation and function through the regulation of CTGF.     

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.     

Abundant retention and release of connective tissue growth factor (CTGF/CCN2) by platelets

Wound healing and tissue regeneration are usually initiated by coagulation followed by fibrous tissue formation. In the present study, we discovered an abundance of connective tissue growth factor (CTGF/CCN2) in human platelets, which was released along with the coagulation process. The CTGF/CCN2 content in platelets was 10-fold higher than that in arterial tissue. Furthermore, the CTGF/CCN2 content in a single platelet was computed to be more than 20-fold higher than that of any other growth factor reported. Considering that CTGF/CCN2 promotes angiogenesis, cartilage regeneration, fibrosis and platelet adhesion, it may be now regarded as one of the major functional components of platelets.     

BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme (Bei, M. and Maas, R. (1998) Development 125, 4325-4333). To define the tissue requirements for Msx1 function, we performed tissue recombinations between wild-type and Msx1 mutant dental epithelium and mesenchyme. We show that through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although our experiments identify a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Previously, using an in vitro culture system, we showed that BMP4 stimulated the growth of Msx1 mutant dental epithelium (Chen, Y., Bei, M. Woo, I., Satokata, I. and Maas, R. (1996). Development 122, 3035-3044). Using the more powerful kidney capsule grafting procedure, we now show that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp.     

Xenopus cadherin-6 regulates growth and epithelial development of the retina

Cadherins are crucial for tissue cohesion, separation of cell layers and cell migration during embryogenesis. To investigate the role of classical type II Xcadherin-6 (Xcad-6), we performed loss-of-function studies by morpholino oligonucleotide injections. This resulted in severe eye defects which could be rescued with murine cadherin-6. In the absence of Xcadherin-6, morphological alterations and a decrease in cell proliferation were observed with eye cup formation. Eye field transplantations of Xcadherin-6 depleted donors yielded grafts that failed to form a proper neuroepithelium in a wildtype environment. At later developmental stages Xcadherin-6 deficient eyes showed lamination defects in the outer neural retina, a reduced thickness of the ganglion cell layer (GCL) and a fragmented retina pigment epithelium (RPE). Thus, Xcadherin-6 is essential early in eye development for structural organization and growth of the neuroepithelium before it differentiates into neural retina and RPE.     

Observations on tooth development and implantation in the upper pharyngeal jaws in Astatotilapia elegans (Teleostei,Cichlidae)

Prominent stages in the development of teeth, associated with the upper pharyngeal jaws in early postembryonic stages of the mouth brooding cichlid A statotilapia elegans were studied on semithin sections in relation to changes in the underlying endoskeletal parts and to the formation of the dentigerous bone. Because the pattern of tooth implantation on infrapharyngobranchial III-IV is constant, at least in early postembryonic stages, it is possible to trace the life history of a given tooth by tracing its homologue throughout the ontogenetic series. A probable causal relationship exists between tooth development and erosion of the underlying cartilage. Fully developed, though unerupted teeth, differentiate annular bone of attachment, which, depending on its position, is formed either outside the cartilage or within the previously induced erosion cavities. Attachment bone of adjacent teeth fuses to build up the dentigerous bone, which, as a result, may be situated within the area previously occupied by cartilage. As soon as the tooth has built up its bone of attachment, it may erupt. The collagenous matrix between tooth and attachment bone persists and gives rise to the movable connection between both. Differentiation of teeth on infrapharyngobranchial III-IV, together with enlargement of the dentigerous bone, proceeds from the lateral and the rostral border, where new germs constantly form. The appearance of new germs on infrapharyngobranchial II is more unpredictable.     

Endochondral ossification of costal cartilage is arrested after chondrocytes have reached hypertrophic stage of late differentiation

Late cartilage differentiation during endochondral bone formation is a multistep process. Chondrocytes transit through a differentiation cascade under the direction of environmental signals that either stimulate or repress progression from one step to the next. In human costal cartilage, chondrocytes reach very advanced stages of late differentiation and express collagen X. However, remodeling of the tissue into bone is strongly repressed. The second hypertrophy marker, alkaline phosphatase, is not expressed before puberty. Upon sexual maturity, both alkaline phosphatase and collagen X activity levels are increased and slow ossification takes place. Thus, the expression of the two hypertrophy markers is widely separated in time in costal cartilage. Progression of endochondral ossification in this tissue beyond the stage of hypertrophic cartilage appears to be associated with the expression of alkaline phosphatase activity. Costal chondrocytes in culture are stimulated by parathyroid hormone in a PTH/PTHrP receptor-mediated manner to express the fully differentiated hypertrophic phenotype. In addition, the hormone stimulates hypertrophic development even more powerfully through its carboxyterminal domain, presumably by interaction with receptors distinct from PTH/PTHrP receptors. Therefore, PTH can support late cartilage differentiation at very advanced stages, whereas the same signal negatively controls the process at earlier stages.     

Determining the most important cellular characteristics for fracture healing using design of experiments methods

Computational models are employed as tools to investigate possible mechanoregulation pathways for tissue differentiation and bone healing. However, current models do not account for the uncertainty in input parameters, and often include assumptions about parameter values that are not yet established. The objective of this study was to determine the most important cellular characteristics of a mechanoregulatory model describing both cell phenotype-specific and mechanobiological processes that are active during bone healing using a statistical approach. The computational model included an adaptive two-dimensional finite element model of a fractured long bone. Three different outcome criteria were quantified: (1) ability to predict sequential healing events, (2) amount of bone formation at early, mid and late stages of healing and (3) the total time until complete healing. For the statistical analysis, first a resolution IV fractional factorial design (L₆₄) was used to identify the most significant factors. Thereafter, a three-level Taguchi orthogonal array (L₂₇) was employed to study the curvature (non-linearity) of the 10 identified most important parameters. The results show that the ability of the model to predict the sequences of normal fracture healing was predominantly influenced by the rate of matrix production of bone, followed by cartilage degradation (replacement). The amount of bone formation at early stages was solely dependent on matrix production of bone and the proliferation rate of osteoblasts. However, the amount of bone formation at mid and late phases had the rate of matrix production of cartilage as the most influential parameter. The time to complete healing was primarily dependent on the rate of cartilage degradation during endochondral ossification, followed by the rate of cartilage formation. The analyses of the curvature revealed a linear response for parameters related to bone, where higher rates of formation were more beneficial to healing. In contrast, parameters related to fibrous tissue and cartilage showed optimum levels. Some fibrous connective tissue- and cartilage formation was beneficial to bone healing, but too much of either tissue delayed bone formation. The identified significant parameters and processes are further confirmed by in vivo animal experiments in the literature. This study illustrates the potential of design of experiments methods for evaluating computational mechanobiological model parameters and suggests that further experiments should preferably focus at establishing values of parameters related to cartilage formation and degradation.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.     

Late skeletal development at the articulation between upper pharyngeal jaws and neurocranial base in the fish, Astatotilapia elegans, with the participation of a chondroid form of bone

This paper presents light-microscopical details of the late development of skeletal tissues at the joint between upper pharyngeal jaws (UPJs) and neurocranial base (parasphenoid and basioccipital bones) in the acellular-boned teleost Astatotilapia elegans. On each of the supporting elements, a bone tissue (AB) is deposited that is anomalous because of its retention of cells within the matrix. Later, this layer is gradually replaced by the anomalous large-celled chondroid kind of bone (CB). Both AB and CB probably grow by apposition from the overlying fibrous layer. Osteoblastlike cells secrete osteoid, which soon calcifies and traps the cells. As in young cellular membrane bone, cells in the AB have a wide, elongate shape and lie amidst sparse, calcified, bonelike matrix but lack a canalicular system. Later generations of enclosed cells have a more vesicular shape, with at least some cells remaining alive in the calcified matrix. Appositional growth of the chondroid bone at its articular side is matched from a certain stage onward by erosion at its basal side. On the upper pharyngeal jaws this resorption is clearly related to the development of new teeth. Although in older stages and adults the chondroid tissue resembles a secondary cartilage, the term chondroid bone (CB) was preferred because of (1) the continuing formation by osteoblastlike cells; (2) the staining affinities of its matrix with that of bone; and (3) its formation both on cartilage bone (the infrapharyngobranchials III-IV and basioccipital bone) and on membrane bone (the parasphenoid bone).     

Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard, Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. In C. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin–enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes processes and enamel prisms are absent.     

A novel population of neuronal cells expressing the olfactory marker protein (OMP) in the anterior/dorsal region of the nasal cavity

The olfactory marker protein (OMP) is expressed in mature chemosensory neurons in the nasal neuroepithelium. Here, we report the identification of a novel population of OMP-expressing neurons located bilaterally in the anterior/dorsal region of each nasal cavity at the septum. These cells are clearly separated from the regio olfactoria, harboring the olfactory sensory neurons. During mouse development, the arrangement of the anterior OMP-cells undergoes considerable change. They appear at about stage E13 and are localized in the nasal epithelium during early stages; by epithelial budding, ganglion-shaped clusters are formed in the mesenchyme during the perinatal phase, and a filiform layer directly underneath the nasal epithelium is established in adults. The anterior OMP-cells extend long axonal processes which form bundles and project towards the brain. The data suggest that the newly discovered group of OMP-cells in the anterior region of the nasal cavity may serve a distinct sensory function.     

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.     

Intrinsic innervation in the tracheal smooth muscle of the large flying fox (Pteropus vampyrus): an immunohistochemical study

Structure and innervation of the trachea of the large flying fox (Pteropus vampyrus) were examined by mean hematoxylin-eosin staining and immunohistochemical methods. The tracheal rings were predominantly comprised of bone with a small amount of hyaline cartilage. Bone contained bone marrow and hyaline cartilage attached at the ends of the tracheal rings. The tracheal rings in the anterior third of the trachea were connected at each end with connective tissue to almost form a ring. A narrow membranous wall comprising a single layer of smooth muscle was present in the middle third, while inner and outer layers of smooth muscle were present in the posterior third. Protein gene product 9.5 (PGP 9.5)-, nitric oxide synthase (NOS)-, galanin- and vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers were present in the epithelium, lamina propria, smooth muscle layer and tunica adventitia. In the tunica adventitia, many PGP 9.5- and NOS-immunoreactive neurons were found singly or in small ganglia, while some VIP- and galanin-immunoreactive neurons were observed. A few PGP 9.5- and NOS-immunoreactive neurons were found in the submucosal layer. These results suggest that the trachea of the large flying fox has a rigid structure of tracheal rings and that the posterior two-thirds of the trachea probably contract via the actions of smooth muscles innervated with peptidergic or nitrenergic neurons that are located in tracheal ganglia, whereas the anterior third is non-mobile.     

Structure and development of first-generation teeth in the cichlid Hemichromis bimaculatus (Teleostei, Cichlidae)

In order to build a reference system to assess results of ongoing in vitro experiments on the study of epithelial-mesenchymal interactions during odontogenesis in actinopterygians, we have chosen to study the first-generation teeth of the cichlid Hemichromis bimaculatus from initiation until attachment both at the light and transmission electron microscopical level. Although their development follows the general pattern of teleost tooth formation, first-generation teeth show peculiarities compared with later tooth generations, including their size, bare emergence from the epithelium, absence of dentinal tubules and of nerves and capillaries in the pulp cavity, and organization of the outer dental epithelium. Four developmental stages (a to d) prior to attachment (stage e) have been distinguished. The oral epithelium invaginates into the underlying mesenchyme (stage a) and is later folded to form a bell-shaped dental organ (stage b) without any primordial thickening, or any other morphological indication of imminent invagination. Then, the collagenous enameloid matrix is laid down, most probably by the odontoblasts (early stage c), soon followed by predentine deposition and the beginning of enameloid mineralization (late stage c). With ongoing dentinogenesis, the enameloid matrix matures (stage d), i.e. the organic constituents are removed and the matrix further mineralizes. Finally (stage e), an annular collar of attachment bone is deposited to fix the tooth onto the underlying bone.     

Leukaemia inhibitory factor (lif) is expressed in hypertrophic chondrocytes and vascular sprouts during osteogenesis

Avascular cartilage is replaced by highly vascularized bone tissue during endochondral ossification, a process involving capillary invasion of calcified hypertrophic cartilage in association with apoptosis of hypertrophic chondrocytes, degradation of cartilage matrix and deposition of bone matrix. All of these events are closely controlled, especially by cytokines and growth factors. Leukaemia inhibitory factor (LIF), a member of the gp130 cytokine family, is involved in osteoarticular tissue metabolism and might participate in osteogenesis. Immunohistochemical staining showed that LIF is expressed in hypertrophic chondrocytes and vascular sprouts of cartilage and bone during rat and human osteogenesis. LIF is also present in osteoblasts but not in osteoclasts. Observations in a rat endochondral ossification model were confirmed by studies of human cartilage biopsies from foetuses with osteogenesis imperfecta. LIF was never detected in adult articular chondrocytes and bone-marrow mesenchymal cells. These results and other data in the literature suggest that LIF is involved in the delicate balance between the rate of formation of calcified cartilage and its vascularization for bone development.