Localization of collagen X in human fetal and juvenile articular cartilage and bone.

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

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.     

Perivascular cells in cartilage canals of the developing mouse epiphysis

Morphological variability among perivascular cells adjacent to cartilage matrix during the elongation of canals through both uncalcified and calcified matrix has not been reported. Cartilage canals were located in distal femoral epiphyses of 5- to 7-day-old mice and identified as vascular channels arising from perichondrial surfaces along the condyles and intercondylar fossae. Three stages of canal development were identified based on the length of canals and on characteristics of chondrocytes and matrix surrounding the canals. Superficial canals terminated in uncalcified matrix of resting cartilage; intermediate canals terminated in matrix containing hypertrophic chondrocytes; deep canals terminated in calcified matrix. The ultrastructural morphology of perivascular cells in contact with the matrix varied in the three stages. Cells resembling fibroblasts and vacuolated macrophages were present adjacent to the uncalcified matrix in superficial canals. At the tips of intermediate canals, cells resembling fibroblasts were larger, contained numerous lysosomes and phagolysosomes, and were in intimate contact with the matrix. At the tips of deep canals, chondroclasts with ruffled borders and clear zones contacted the calcified matrix. The results indicate that 1) mouse epiphyses provide a suitable model for studying cartilage-canal perivascular cells, 2) calcification of cartilage matrix occurs along the course of the canal, and 3) the morphology of perivascular cells in contact with the matrix may be determined, in part, by matrix calcification.     

Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage

The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.     

Morphology of normal and osteochondrotic porcine articular-epiphyseal cartilage. A study in the domestic pig and minipig of wild hog ancestry

Osteochondrosis is a generalized skeletal disorder that affects the growth cartilage in the growing domestic pig but not in the minipig of wild hog ancestry. In the present study, we compare the ultrastructure of the articular and epiphyseal growth cartilage in the domestic pig with that in the minipig. The domestic pigs had areas of enlarged epiphyseal cartilage with chondronecrosis, which had caused focal impairment of the endochondral ossification, with retention of cartilage in the subchondral bone. Areas of chondronecrosis close to blood vessels were found in the resting zone, with no evidence of thickened cartilage or impaired ossification. The chondronecrosis was surrounded by chondrocytes, organized in small clusters, containing many lipid droplets. The vascular channels adjacent to the chondronecrosis contained degenerated blood vessels. The minipigs showed no areas of enlarged epiphyseal cartilage. A few dead chondrocytes could be seen close to vascular channels which contained morphologically normal blood vessels. We conclude that restricted perivascular chondrolysis may occur in the pig without the presence of vascular degeneration and without progressing to osteochondrosis.     

Vitamin-D-dependent calcium-binding protein (CaBP-9K) in rat growth cartilage

Summary The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.     

Ultrastructural studies of initial stages of mineralization of long bones and vertebrae in human fetuses

We studied 27 embryos of 5-12 weeks gestational age where pregnancy was interrupted due to paramedical reasons, in order to find the developmental stages at which matrix vesicles appear in cartilage, and whether they are involved in the mineralization process. Specimens of long bones, lumbar and thoracic vertebral column were prepared for light, transmission and scanning electron microscopic studies. In the cartilaginous models of long bones, matrix vesicles were found amongst maturing and hypertrophic chondrocytes already by the 6th week after fertilization. By that stage, bone rudiments consisted of only cartilage that was not yet mineralized. In the vertebral column matrix, vesicles were found in the vertebral bodies amongst maturing and hypertrophic chondrocytes at the beginning of the 8th week. At that stage, although hypertrophy of chondrocytes was observed, mineralization was still absent. No matrix vesicles were found in the perichondrium, investing mesenchyme and intervertebral discs. Mineralization of cartilage in long bone rudiments started in the form of hydroxyapatite crystals within or around the matrix vesicles at 7 weeks of age and in the vertebral column at 11 weeks. As mineralization progressed, more hydroxyapatite crystals were observed around the matrix vesicles, forming typical calcospherites . Mineralization then progressed in the form described in other animals.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

Persistence of cartilage proteoglycan and link protein during matrix-induced endochondral bone development: An immunofluorescent study

Monospecific antibodies to cartilage proteoglycan monomer and link protein were employed with immunofluorescence microscopy to determine the tissue distribution of these constituents during matrix-induced endochondral bone development. Subcutaneous implantation of demineralized diaphyseal bone matrix resulted in new endochondral bone formation. On Day 3, the implant consisted of mesenchymal tissue which did not contain any demonstrable cartilage-related proteoglycan or link protein. With the onset of early chondrogenesis on Day 5, cartilage proteoglycan monomer and link protein were first localized together in the cartilage matrix, particularly around chondrocytes in territorial sites. Progressively more staining around cells was observed at Days 7 and 9. On Day 9, when mineralization was first observed, there was no evidence of a net loss of these molecules prior to mineralization of the cartilage matrix. On Day 11 and thereafter, bone formation was observed by appositional growth on calcified cartilage spicules. Whereas the osteoblasts and bone matrix were devoid of any staining for cartilage proteoglycan and link components, the residual, partly mineralized cartilage spicules still reacted with antibodies to cartilage proteoglycan monomer and link protein in territorial sites, but in reduced amounts, indicating a loss of these molecules associated with a loss of hypertrophic chondrocytes. Since mineral prevented the access of Fab' antibody subunits, demineralization after fixation was routinely employed. The results reveal that cartilage proteoglycan monomer and link protein are present around chondrocytes in hyaline cartilage during the early stages of endochondral bone formation and that there is no net loss of these molecules prior to mineralization of this cartilage matrix as was previously thought.     

Hypertrophic Chondrocytes in the Rabbit Growth Plate Can Proliferate and Differentiate into Osteogenic Cells when Capillary Invasion Is Interposed by a Membrane Filter

The fate of hypertrophic chondrocytes during endochondral ossification remains controversial. It has long been thought that the calcified cartilage is invaded by blood vessels and that new bone is deposited on the surface of the eroded cartilage by newly arrived cells. The present study was designed to determine whether hypertrophic chondrocytes were destined to die or could survive to participate in new bone formation. In a rabbit experiment, a membrane filter with a pore size of 1 µm was inserted in the middle of the hypertrophic zone of the distal growth plate of ulna. In 33 of 37 animals, vascular invasion was successfully interposed by the membrane filter. During 8 days, the cartilage growth plate was enlarged, making the thickness 3-fold greater than that of the nonoperated control side. Histological examination demonstrated that the hypertrophic zone was exclusively elongated. At the terminal end of the growth plate, hypertrophic chondrocytes extruded from their territorial matrix into the open cavity on the surface of the membrane filter. The progenies of hypertrophic chondrocytes (PHCs) were PCNA positive and caspase-3 negative. In situ hybridization studies demonstrated that PHCs did not express cartilage matrix proteins anymore but expressed bone matrix proteins. Immunohistochemical studies also demonstrated that the new matrix produced by PHCs contained type I collagen, osteonectin, and osteocalcin. Based on these results, we concluded that hypertrophic chondrocytes switched into bone-forming cells after vascular invasion was interposed in the normal growth plate.     

Ultrastructural analysis of cytochrome oxidase in chick epiphyseal growth plate cartilage

Histochemical detection of cytochrome oxidase activity in chicken growth plate revealed both positively and negatively stained mitochondria in chondrocytes of all zones, i.e., proliferative, pre-hypertrophic, hypertrophic, and calcifying zones. The proportion of positive to negative cells was lowest in the proliferative zone. As cytodifferentiation progressed, more positively stained cells were present. In positive cells all mitochondria were usually stained, and in negative cells all mitochondria were unstained. A few cells appeared to be in transition and contained both types of mitochondria. The results indicate that chondrocytes utilizing both aerobic and anaerobic metabolism are present in growth plate cartilage and that oxidative metabolism is favored in the more mature cells. The relationship of oxidative metabolism to calcification is discussed.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.     

Role of the Subchondral Vascular System in Endochondral Ossification: Endothelial Cells Specifically Derepress Late Differentiation in Resting Chondrocytesin Vitro

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophiedin vitroand expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.     

Electron microscopic observations on the fate of hypertrophic chondrocytes in condylar cartilage of rat mandible

The present study focused on the hypertrophic cell zone and the adjacent region of primary spongiosa in the mandibular condylar cartilage in growing rats (3 to 7 weeks old). In this cartilage, chondrocytes were not arranged in columns, and there was no clear distinction between longitudinal and transverse septum. The hypertrophic chondrocytes were not surrounded entirely by calcified matrix, and capillaries were in close contact with cartilage cells. The staining intensity of the pericellular matrix decreased in the lower hypertrophic cell zone in comparison with that in the upper part of the hypertrophic cell zone. Electron microscopic examinations indicated that the lowest hypertrophic cells contained lysosomes and pinocytotic vesicles. Some hypertrophic chondrocytes appeared to have been released from their lacunae and were observed in the region of the primary spongiosa. Hence it is suggested that the lowest hypertrophic chondrocytes in the rat mandibular condyle do not die but are released from their lacunae into the bone marrow. Further study is needed to determine whether or not these cells do indeed become osteoblasts and/or chondroclasts.     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often spotty distribution of collagen X with increasing cartilage degeneration associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and spotty collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

Periodic acid Schiff--p phenylenediamine staining of glycogen in chondrocytes. A new combination which improves both cellular detail and glycogen identification

This study reports a method whereby glycogen is identified in the chondrocytes of the secondary center of ossification prior to mineralization. The use of new fuchsin rather than basic fuchsin on one micron Spurr sections of femoral head cartilage fixed with potassium ferrocyanide-reduced osmium produced excellent identification of glycogen and when followed by p phenylenediamine, intensified cellular detail.