Morphology of epiphyseal apparatus of a ranid frog (Rana Esculenta)

Morphological, histochemical and ultrastructural investigations on epiphyseal apparatus of Rana Esculenta were made. The most important findings were the following: 1) metaphyseal cartilage is localized inside proximal diaphyseal compact bone as a plug; 2) metaphyseal cartilage do not reduce in thickness during ageing; 3) metaphyseal cartilage do not show vascular invasion and do not mineralize in degenerative zone; 4) trabecular bone was not at all evident in this animal; 5) external periosteum is well vascularized and proliferates in correspondence to marginal epiphyseal end of the diaphyseal. From these results the hypothesis that the ranid frog bone growth is not due to metaphyseal metabolism (as in avian and mammals) but to bone periosteal marginal mineralization is reached.     

Gender dependent effects of testosterone and 17β-estradiol on bone growth and modelling in young mice

This study examined the effects of estrogen (17β-estradiol) and testosterone on the growth of long bones in male and female mice, with and without gonadectomy. Weight and nose-to-tail length were determined at 3 weeks of age at time of gonadectomy, 7 days later at the onset of hormone therapy, and throughout the treatment period. Gonadectomized mice exhibited an initial weight gain during the pretreatment period but length was unaffected. Hormone treatment altered weight gain in surgical and intact animals in a gender- and hormone-dependent manner. Estradiol enhanced weight gain in intact mice, but inhibited weight gain in ovariectomized mice. Lower doses of estradiol increased weight gain in orchiectomized mice at early time points. Testosterone increased weight in intact females and males, but not in gonadectomized mice. Estradiol increased nose-to-tail length in intact females at early time points, but inhibited length in ovariectomized females at later times, and it decreased length in intact males. Testosterone increased length in normal females and normal males. Serum Ca was unaffected by ovariectomy, but orchiectomy resulted in decreased levels. Estradiol reduced serum Ca in gonadectomized animals; serum Ca was increased by estradiol treatment in intact females. Changes in tibial bone weight, ash weight and mineral composition, and relative sizes of epiphyseal and metaphyseal bone were gender-, gonadectomy- and hormone-specific. Bone weight was greater in ovariectomized mice. Ash weight per bone was comparable, but there was an increase in Ca and P content with ovariectomy. Estradiol increased bone weight, ash content, and bone Ca and P in ovariectomized and intact females. Orchiectomy alone did not alter bone weight, ash content, or Ca and P, but orchiectomized mice were sensitive to estradiol; all parameters were increased in the orchiectomized animals treated with estradiol. Analysis of the ash content and Ca and P per mg bone, rather than per bone, demonstrated estradiol and testosterone alter net bone formation, but not the amount of mineral per unit bone. Ovariectomy increased hypertrophic cartilage. While estradiol did not alter tibial area in ovariectomized mice, it caused an increase in intact females. The total amount of growth plate cartilage in ovariectomized animals was decreased by estradiol to levels typical of intact animals due to a greater decrease in the hypertrophic cartilage in the ovariectomized mice, as well as a greater increase in metaphyseal bone area. Testosterone had no effect on these parameters in the females. Orchiectomy decreased the amount of growth plate cartilage, but increased the hypertrophic zone. Estradiol increased growth plate cartilage in intact male mice, but decreased it in orchiectomized mice. This difference was also seen in the hypertrophic zone. Total growth plate cartilage and hypertrophic cartilage were increased by testosterone in intact males, whereas metaphyseal and epiphyseal bone area were decreased. The results show for the first time that there is a gender-specific response in both male and female mice to both estradiol and testosterone, whether or not the animals have been gonadectomized. For many parameters, orchiectomized mice behave like females in response to both sex steroids, indicating that the male gonad is needed for mouse bone to exhibit the male phenotypic response to estradiol and testosterone.     

Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

Autoradiographic Studies of the Utilization of Ca45 by the Chick Embryo

Calcium-45 was injected into the dense albumen of fertile hen's eggs, to the extent of 25 µc. per egg. The eggs were incubated under standard conditions and three or more embryos removed daily and fixed in 10 per cent neutral formalin. Stripping-film autoradiograms were prepared from paraffin sections of the tibiofibulae. Exposure varied with the isotope concentration. The tissue sections with their autoradiograms in place were stained with dilute Giemsa, while other sections were stained with hematoxylin-azure-eosin and by von Kossa to demonstrate bone salt. At about 9 days, Ca⁴⁵ is found in the cartilage template both intra- and extracellularly. Between 9 and 11 days, a primary diaphyseal lamella is deposited which is largely acellular. The lamella is eroded by capillaries from the periosteum and a resorption center is established in the cartilage. New lamellae of bone are deposited centrifugally in an imbricated pattern. Bone matrix formation precedes calcification by about 1 to ½ days, and calcification in a particular lamella is not uniform. Endochondral bone formation is described, as well as calcification of the epiphyseal/diaphyseal cartilage. Calcium-45 occurs intracellularly in the osteocyte during bone formation.     

Lysozyme in preosseous cartilage VI. Purification, characterization and localization of mammalian cartilage lysozyme

Lysozyme (mucopeptide-N-acetylmuramylhydrolase, EC 3.2.1.17) is present in mammalian cartilage. Lysozyme was isolated and purified from bovine and canine cartilage and from dog serum using various chromatographic steps and affinity chromatography on carboxymethylated chitin. Amino acid analysis of bovine cartilage lysozyme showed that it is similar to other mammalian lysozymes. Anti-canine lysozyme antibodies cross-react with calf lysozyme, but not with hen egg white or embryonic chick cartilage lysozyme. In the epiphyseal plate of the dog, 90-μm sections were analyzed for lysozyme and its was found that in the hypertrophic zone its concentration is approximately six times higher than it is in the resting zone. Using immunocytochemical techniques at the electromicroscopic level, lysozyme in the epiphyseal plate of the dog was localized extracellularly, mainly in the immediate vicinity of the chondrocytes, the territorial matrix.     

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18- 19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage matrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.     

Long bone growth during prolonged intermittent corticosteroid treatment and subsequent rehabilitation

Summary Immature A/J mice were treated for up to 7 weeks with intermittent doses of triamcinolone hexacetonide and were thereafter allowed to recover for 7 weeks. Qualitative and quantitative morphological measurements were performed on the epiphyseal cartilage plate and diaphyseal bone of the humerus. By the third injection significant structural changes were noted in the cartilaginous tissue followed by a complete cessation of bone growth. The hormonal inhibitory effect on long bone growth lasted throughout the experimental period. However, at the end of the recovery period the length of the humerus was 96% of the normal. In contrast, the humeral width at midshaft and the width of its medullary cavity revealed slower recovery, achieving only 80% of the control values. Following rehabilitation, the growth of experimental epiphyseal plates exceeded that of nontreated animals as their width and the number of hypertrophic chondrocytes were 131% and 125% of their controls respectively. Thus, in A/J mice (a highly susceptible inbred strain of mice) intermittent (every four days) administration of a long-acting corticosteroid hormone arrested endochondral and periosteal bone formation; the former, however, underwent full recovery following the termination of the hormonal treatment.Supported in part by the Hy and Ann Natovich Orthopaedic and Rehabilitation Research FoundationPart of this work was presented in abstract form at the Annual Meeting of the American Association of Anatomists, 1979The authors are grateful to Miss Aviva Valensi, Miss Dorit Licht and Mrs. Pessia Shenzer for their excellent technical assistance     

Calmodulin localization in bone and cartilage

The localization of calmodulin (CaM), a calcium-dependent regulatory protein, was demonstrated in the following rat skeletal tissues by the indirect immunofluorescence method: a) growth plate cartilage of fetal and juvenile long bones, b) fetal epiphyseal cartilage, c) juvenile hyaline costal cartilage, d) neonatal mandibular condylar cartilage, e) neonatal diaphyseal lamellar bone. CaM was not detected in perichondrial and periosteal cells. Mature and mineralizing chondrocytes demonstrated the highest labelling intensities.     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis.A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation.These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.     

Role of mitochondria in calcification, Mitochondrial activity distribution in the epiphyseal plate and accumulation of calcium and phosphate ions by chondrocyte mitochondria

Histologically homogeneous sections corresponding to resting, columnar and hypertrophic zones of the epiphyseal plate of calf scapula were homogenized and assayed for mitochondrial, lysosomal and others important to calcification activities. These activities were found to be significantly higher in the columnar and hypertrophic zones as compared with those in the resting zone. Mitochondria obtained from the hypertrophic zone of the epiphyseal plate of calf scapula or calf costal chondral junction were resolved into a “light” and a “heavy” population by isopycnic centrifugation presumably due to difference in the content of granule forming calcium phosphate. Finally mitochondria from resting cartilage can give rise only to a “light” mitochondrial population, unless they are allowed to accumulate calcium and phosphate ions from the medium during respiration where a “light” and a “heavy” population results upon centrifugation.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Connective tissue alterations following neonatal thymectomy. VI. Calcium histochemical, growth-dynamical and thermoanalytical investigations on bone tissue of thymectomized rats.

Thymectomy was performed in newborn rats and the changes occurring in the epiphyseal cartilage and bone were investigated by Ca histochemical and thermoanalytical methods, one, two and six weeks following operation. Formation of Ca complexes was slowed down in the epiphyseal cartilage and the rate of growth decreased. At the same time the inorganic substance content decreased considerably in the bone tissue of operated rats as compared to the controls.     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: II. Localization of type I and type II collagen during long bone development

The transition of type I and type II collagens during cartilage and bone development in the chick embryo was studied by immunofluorescence using antibodies against type I or type II collagens. Type II collagen was found in all cartilaginous structures which showed metachromatic staining. Type I collagen appeared in the perichondrium of the tibia at stage 28 and was also found in osteoid, periosteal and enchondral bone after decalcification, periosteum, and tendons, ligaments, and capsules.Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis. During enchondral ossification type I collagen is deposited onto the eroded surface of cartilage. It partially diffuses into the cartilage matrix forming a “hybrid” collagen matrix with type II collagen, which is a site for subsequent ossification. During appositional growth of diaphyseal cartilage and differentiation of epiphyseal perichondrium into articular cartilage, perichondral cells switch from type I to type II collagen synthesis when differentiating into chondroblasts. In the transition zones, chondroblasts are imbedded in a “hybrid” matrix consisting of a mixture of type I and type II collagens.