Chondrocytes Of Epiphyseal Cartilage Research Articles

Epiphyseal cartilage canal architecture and extracellular matrix remodeling in mucopolysaccharidosis VII dogs at the onset of postnatal growth

ABSTRACT Purpose: Mucopolysaccharidosis (MPS) VII is a genetic, lysosomal storage disease characterized by abnormal accumulation of glycosaminoglycans in cells and tissues. MPS VII patients exhibit multiple failures of endochondral ossification during postnatal growth, including markedly delayed cartilage-to-bone conversion in the vertebrae and long bones. Cartilage canals provide the template for vascularization at the onset of secondary ossification. The objective of this study was to investigate whether abnormal cartilage canal architecture and enzyme-mediated extracellular matrix (ECM) remodeling contribute to delayed cartilage-to-bone conversion in MPS VII. Materials and Methods: The epiphyseal cartilage canal networks of 9-day-old healthy control and MPS VII-affected dog vertebrae were characterized using high-resolution, contrast-free quantitative susceptibility mapping magnetic resonance imaging. Relative expression levels of matrix metalloproteinases (MMPs) 9, 13 and 14 were examined using immunohistochemistry, while tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were examined using in situ enzyme staining. Results: Interestingly, the density, number, connectivity and thickness of cartilage canals was not significantly different between MPS VII and control vertebrae. Immunohistochemistry revealed diminished MMP-9, but normal MMP-13 and 14 expression by epiphyseal cartilage chondrocytes, while ALP and TRAP enzyme expression by chondrocytes and chondroclasts, respectively, were both diminished in MPS VII. Conclusions: Our findings suggest that while the epiphyseal cartilage canal network in MPS VII is normal at the onset of secondary ossification, expression of enzymes required for cartilage resorption and replacement with mineralized ECM, and initiation of angiogenesis, is impaired.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.

Identification of Fibroblast Growth Factor-18 as a Molecule to Protect Adult Articular Cartilage by Gene Expression Profiling

To identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions, we initially compared four types of chondrocytes: articular (AA) versus growth plate (AG) cartilage chondrocytes in adult rats, and superficial layer (IS) versus deep layer (ID) chondrocytes of epiphyseal cartilage in infant rats. Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The results were validated by real-time RT-PCR analysis. Among them, Hoxd1, Fgf18, and Esm1 were expressed more strongly in AA than in IS. Fgf18 was the extracellular and secreted factor that decreased glycosaminoglycan release and depletion from the cartilage, and enhanced proliferation of articular chondrocytes. Fgf18 was strongly expressed in the articular cartilage chondrocytes of adult rats. In a surgical rat osteoarthritis model, a once-weekly injection of recombinant human FGF18 (rhFGF18) given 3 weeks after surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with significant effect at 10 μg/week of rhFGF18. As the underlying mechanism, rhFGF18 strongly up-regulated Timp1 expression in the cell and organ cultures, and inhibition of aggrecan release by rhFGF18 was restored by addition of an antibody to Timp1. In conclusion, we have identified Fgf18 as a molecule that protects articular cartilage by gene expression profiling, and the anticatabolic effects may at least partially be mediated by the Timp1 expression.

Longitudinal growth in chronic hypokalemic disorders

Growth retardation remains a major complication in children with primary tubular disorders, despite adequate supplemental treatment with electrolytes, water and bicarbonate. Chronic hypokalemia, characteristic of some tubulopathies, impairs growth by mechanisms that are not well known. Association with growth hormone deficiency has been reported in patients with Bartter's or Gitelman's syndrome. Tissue-specific alterations of growth hormone and insulin-like growth factor I axis have been described in experimental models of potassium depletion. Hypokalemic rats gain less body length and weight than pair-fed normokalemic animals and, by contrast, develop renal hypertrophy. These rats have low circulating concentrations of insulin-like growth factor I, depressed messenger ribonucleic acid (mRNA) levels of this peptide in the tibial growth plate, and they are resistant to the longitudinal growth-promoting effects of exogenous growth hormone. The reason for this resistance remains to be defined. No alterations in the intracellular signaling for growth hormone have been found in the liver of hypokalemic rats. However, treatment with high doses of growth hormone is unable to normalize hypertrophy of the epiphyseal cartilage chondrocytes, which are severely disturbed in potassium depletion and likely play an important role in the pathogenia of growth impairment in this condition.

Molecular differentiation in epiphyseal and physeal cartilage. Prominent role for gremlin in maintaining hypertrophic chondrocytes in epiphyseal cartilage

We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.

The Expression of Connexins during Growth and Development of Skeletal Elements in the Dog

Efficient and well‐regulated intercellular communication is essential to organize the activities of the different cell populations involved during growth and concomitant structural maturation of the skeleton. For direct cell‐cell communication gap junctions composed of connexins (Cx) are present. So far 21 Cx types have been identified in mammals, which differ in molecular permeability as well as tissue expression pattern. To characterize the Cx distribution in the developing canine skeleton, samples from humerus, scapula and lumbar vertebrae of Beagle dogs aged 1.5, 3, 4.5 months and 1.5 years were fixed in Bouin's solution, decalcified with 25% EDTA and embedded in paraffin wax. Using immunohistochemistry and in situ hybridization, the expression of Cx 26, 30, 31, 32, 43, and 45 was studied in bone tissue plus associated epiphyseal and growth plate cartilage. Of all bone cells, osteoblasts showed the largest Cx expression repertoire: they were positive for Cx 26, 30, 32 and 43. After transformation into bone lining cells Cx43 expression was lost while in osteocytes only Cx 26 and 32 was detected. A wide variety of Cx reactions was also found in osteoclasts: Cx 26, 30, 32, and 43. In contrast, chondrocytes showed a much more restricted Cx expression pattern. Positive reactions were only seen in hypertrophic chondrocytes of epiphyseal cartilage (Cx 26, 31, 32) and in the proliferative (Cx32) and hypertrophic zone (Cx 26 and 32) of growth plate cartilage. Additionally, Cx31 was strongly expressed in the cellular layer of the periosteum covering areas of intensive bone modelling in the puppies. Age‐ and microlocation‐dependent variations in expression and/or staining intensity were also observed in the other Cx types. These results demonstrated that canine bone cells are capable of synthesizing a wide variety of Cx types during skeletal development, thus propagating different cellular signals via gap junctions in a differentially regulated and requirement orientated manner.

Osteopontin Distribution in the Canine Skeleton during Growth and Structural Maturation

The distribution of osteopontin (OPN) was studied immunohistochemically in cells and extracellular matrix in the humerus, scapula, and lumbar vertebrae of growing (age: 6 weeks, 12 weeks, 4.5 months) and adult dogs. OPN was expressed in hypertrophic chondrocytes of epiphyseal cartilage and in chondrocytes of the deep zone of mature articular cartilage, where extracellular matrix was also stained. OPN expression was strongest in 4.5-month-old puppies in cells of the osteoblastic lineage. It also varied with microlocation and was pronounced in areas prone to resorption due to modelling and remodelling activities. Osteoclasts were always strongly labelled with OPN. OPN deposition in extracellular bone matrix was detected particularly as a delineation of cartilage cores within secondary trabeculae and as a lining of the trabecular surfaces in resorption microlocations. The OPN distribution pattern is discussed here for each cell population with regard to its functional implications.

The tyrosine phosphatase, OST‐PTP, is expressed in mesenchymal progenitor cells early during skeletogenesis in the mouse

Osteotesticular protein tyrosine phosphatase (OST-PTP; OST), is a signaling molecule which catalyzes the removal of phosphates from tyrosine residues. It is known to be highly regulated in bone cells and has been shown to be important for the in vitro progression from a preosteoblast to a mature, mineralizing cell. However, the in vivo expression of this phosphatase during skeletogenesis has not been examined. Using Northern analysis and in situ hybridization (ISH), we have observed that this gene is strongly expressed early during the formation of the mouse skeleton. By 12.5 days postcoitum (dpc), expression of OST mRNA transcripts increases and is localized within the mesenchyme of craniofacial bones, ribs, limbs, and Meckel's cartilage. Following initiation of chondrogenesis, OST mRNA becomes restricted to the perichondrium of all endochondral elements. With ossification, this gene is also expressed by cells, presumably osteoblasts, at the chondro-osseous border and along cortical and trabecular bone surfaces. Unlike other bone markers examined such as Osterix and type II collagen, OST transcripts do not appear to be expressed by chondrocytes of epiphyseal cartilage or by non-hypertrophic or hypertrophic chondrocytes. Because the temporal expression patterns of OST and Runx2 were similar suggesting a potential interrelationship in bone regulation and function, OST expression was examined in transgenic mice lacking a functional Runx2/Cbfal protein (Runx2/Cbfal delta C (deltaC)) and possessing a cartilaginous skeleton. Interestingly, the OST gene was expressed with localization similar in wild-type, homozygous, and heterozygous embryos. These studies suggest that the expression of the OST gene may be important during skeletogenesis, potentially from commitment of mesenchymal cells to the ossification of new bones. Early in embryogenesis, regulation of OST expression may be independent of Runx2/Cbfa1.

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10al gene expression, we have isolated and sequenced the first exon and 5′ flanking promoter regions of bovine Col10al and human COL10al. Sequence comparisons, including those previously published for mouse Coll0al, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5′-regulatory sequences of human COL10al (hCOL10al) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upsteam of the transcription start point(s) of hCOL10al, which is distinctly different from that reported for the chick.

Human studies on the biological actions of IGF-1. Evidence suggesting that human fetal and postnatal epiphyseal cartilage is a target tissue for IGF-1 action.

Recent data show that in humans IGF-1 has insulin-like anabolic and growth promoting effects. However, the plasma levels at which IGF-1 exerts these effects differ. IGF-1 plasma levels similar to those in normal children and adults have a growth promoting effect, but higher levels are necessary for IGF-1 to exert insulin-like anabolic effects, and hypoglycemia is a potential risk. Longitudinal bone growth results from the proliferation and differentiation of chondrocytes in epiphyseal cartilage. We present our data from cultured human epiphyseal chondrocytes, which suggest that epiphyseal cartilage may be a target tissue for IGF-1 action during human fetal and postnatal development.

Glucose 6-phosphatase and glycogen phosphorylase activities in chondrocytes in epiphyseal cartilage of growing rats.

Glycogen, glycogen phosphorylase, and glucose 6-phosphatase (G6Pase) activities were examined cytochemically in chondrocytes of femoral epiphyseal cartilages and cartilaginous ribs of 3- and 7-day-old rats. G6Pase activity was also examined biochemically. Glycogen was abundant in chondrocytes of the reserve zone, while it became scarce in the cells of the proliferative zone. From the upper part (adjoining the proliferative zone) to the lower part of the hypertrophic zone, glycogen accumulated in chondrocytes and decreased in the cells of the degenerative zone. Inversely, glycogen phosphorylase a and G6Pase activities were relatively high in chondrocytes of the proliferative zone and upper hypertrophic zone and were low in the cells of the reserve zone, lower hypertrophic zone, and degenerative zone. The reaction product for G6Pase was present in the endoplasmic reticulum and nuclear envelope of all types of chondrocytes composing the cartilages, although the amounts of reaction product varied with the cell types in parallel with the histochemical results. Biochemical G6Pase activity was higher in epiphyseal cartilages than in cartilaginous ribs. The possible mechanism and significance of the accumulation and decrease of glycogen in chondrocytes of the epiphyseal cartilage were discussed.

Zonal analysis of metabolic profiles of articular-epiphyseal cartilage chondrocytes: a histochemical study.

Articular-epiphyseal cartilage from the femur of New Zealand rabbits was subjected to histochemistry for determination of the presence of metabolic enzymes along its zonal stratification. Glycolytic enzymes were strongly reactive in all of the zones. Krebs cycle enzymes, enzymes of the hexose monophosphate shunt and the respiratory chain enzymes showed a progressive increase in reactivity from the tangential zone through the top half of the epiphyseal zone. Indicators of lipid metabolism were fairly high in all regions of the cartilage.

Correlation of freeze-fracture and scanning electron microscopy of epiphyseal chondrocytes.

Chondrocytes in epiphyseal cartilage were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using freeze-fracture techniques. Freeze-fracture replicas showed large numbers of fingerlike, 0.11-0.15 micrometer diameter, projections from the chondrocyte surface, with numerous 95-180 A diameter intramembranous particles associated with both the cell membrane surface and these projections. With SEM, these cytoplasmic projections were also obvious, but appeared collapsed into clusters of globular-shaped projections on the surface of the chondrocytes. With freeze-fracture techniques, in which shrinkage artifacts were essentially eliminated, the cytoplasmic projections were often seen in intimate contact with the extracapsular matrix. However, with chondrocytes prepared by both SEM and conventional TEM, there was evidence of shrinkage, the cytoplasmic projections having little contact with the extracapsular matrix. These findings show that the cytoplasmic processes are not artifacts of tissue processing and provide morphological evidence in support of the hypothesis that matrix vesicles are of cellular origin.

Electron Microscopic Study of the Intracellular Activity of Alkaline Phosphatase in the Hypertrophic Chondrocytes of Epiphyseal Cartilage

Electron Microscopic Study of the Intracellular Activity of Alkaline Phosphatase in the Hypertrophic Chondrocytes of Epiphyseal Cartilage

A new chondrodystrophic mutant in mice. Electron microscopy of normal and abnormal chondrogenesis.

The occurrence of a new mutation affecting cartilage and bone in mice is reported. The gene is lethal, shows autosomal recessive inheritance, and has high penetrance. It is not allelic to shorthead and probably not to phocomelia or achondroplasia. It results in a foreshortened face, cleft palate, defective trachea, and shortened long bones with flared metaphyses. Chondrocytes of epiphyseal cartilage from the mutant are not aligned in columns, and there is a decrease in the usual staining of the cartilage matrix. Electron microscope observations show large, wide collagen fibrils with "native" banding in the matrix of mutant cartilage, which are not present in normal cartilage. Possible explanations for the expression of this genetic disorder of cartilage development are put forward.

Fine structure of epiphyseal cartilage

An examination of the fine structure of proximal tibia-epiphyseal cartilage (3-5 weeks-old rabbit) has ravealed the following information.1) Chondrocytes of epiphyseal cartilage contain numerous cytoplasmic processes, and in their cytoplasm many vacuoles. The so called cartilage lacuna, like a halo, which gradually proceeds to the cartilageons matrix is seen around the cell to a certain extent.2) The matrix appears to be irregularly intricated, possessing a felt-like structure, with fibre and then small fibres appear, without demonstrable periodicity, on the longitudinal septa.It is assumed that these fibres develop from the granules in the cartilage lacuna, in the cytoplasmic vacuoles.3) Chondrocytes involve changes in cytoplasmic hydration rather than changes in the organelles. After maximum hydration, chondrocytes undergo death and degeneration.4) Calcification initiates on the longitudinal matrix with relative electron-dense granules, gradually, forming itself into islets and granular specks. As they increase in number and volume, they fuse with each others to complete the formation of calcified cartilage.

Single cilia in chondrocytes

Electron microscopic investigation of embryonic mouse radii reveals that chondrocytes in epiphyseal cartilage can form single cilia. The cilia can be distinguished from ordinary microvilli by their greater length and the organization of their bases, which are formed by a centriole accompanied by a second one in the near neighborhood. Hitherto no tubular elements have been found in the interior of these cilia. In connection with this it may be assumed that the level of organization of the described single cilia is low in comparison with most other cilia. The literature available on the subject of single cilia is reviewed, and an attempt is made to classify the different types of cilia with respect to single or multiple occurrence, internal structure, and the number of centrioles associated with them.