Defects in the cartilaginous growth plates of brachymorphic mice

Homozygous brachymorphic (bm/bm) mice are characterized by disproportionately short stature. Newborn bm/bm epiphyseal cartilages are shorter than normal although the cells in the different zones of growth are relatively well organized. The extracellular matrix reacts poorly with stains specific for sulfated glycosaminoglycans. The ultrastructural appearance of the cartilage matrix indicates normal collagen fibrils; however, proteoglycan aggregate granules are smaller than normal and are present in reduced numbers, particularly in the columnar and hypertrophic zones of the growth plate. In addition, a prominent network of fine filaments, which are extractable in 4 M guanidine hydrochloride, are present in the bm/bm cartilage matrix. These findings suggest that a defect affecting the proteoglycan component of cartilage occurs in bm/bm mice.     

ELECTRON MICROSCOPY OF CARTILAGE AND BONE MATRIX AT THE DISTAL EPIPHYSEAL LINE OF THE FEMUR IN THE NEWBORN INFANT

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.     

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.     

Ultrastructure of elastic cartilage in the rat external ear

Summary The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy.The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40–100 nm) vesicles. Scarce glycogen granules and bundles of 6–7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant.Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments.The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a stellate reticulum. The complex of these two structures appears in the scanning electron micrographs as a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.     

Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues

A monoclonal antibody to a core-protein-related epitope of a small dermatan sulfate-rich proteoglycan (DS-PGII) isolated from adult bovine articular cartilage (22) was used to localize this molecule, or molecules containing this epitope, in bovine articular cartilages, in cartilage growth plate, and in other connective tissues. Using an indirect method employing peroxidase-labeled pig anti-mouse immunoglobulin G, DS-PGII was shown to be present mainly in the superficial zone of adult articular condylar cartilage of the metacarpal-phalangeal joint. In fetal articular and epiphyseal cartilages, the molecule was uniformly distributed throughout the matrix. By approximately 10 months of age it was confined mainly to the superficial and middle zones of articular cartilage and the inter-territorial and pericellular matrix of the deep zone. DS-PGII was not detected in the primary growth plate of the fetus except in the proliferative zone, where it was sometimes present in trace amounts. In contrast, it was present throughout the adjacent matrix of developing epiphyseal cartilage. In the trabeculae of the metaphysis, strong staining for DS-PGII was seen in decalcified osteoid and bone immediately adjacent to osteoblasts. Staining was also observed on collagen fibrils in skin, tendon, and ligament and in the adventitia of the aorta and of smaller arterial vessels in the skin. These observations indicate that DS-PGII and/or molecules containing this epitope are widely distributed in collagenous tissues, where the molecule is intimately associated with collagen fibrils; in adult cartilage this association is limited mainly to the narrow parallel arrays of fibrils which are found in the superficial zone at the articular surface. From its intimate association and other studies, this molecule may play an important role in determining the sizes and tensile properties of collagen fibrils; it may also be involved in the calcification of osteoid but not of cartilage.     

Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentation principle.

The critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0 M MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH. Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones. A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5 M MgCl2. In these cases, however, the stained material exhibited a ribbon-like configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0 M resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size. The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.     

Undersulfated chondroitin sulfate in the cartilage matrix of brachymorphic mice.

Homozygous brachymorphic ($\text{bm}\text{bm}$) mice have a disproportionately short stature, similar to human achondroplasia. We previously showed that each zone of growth in young $\text{bm}\text{bm}$ epiphyseal cartilages is smaller than normal and that the extracellular matrix appears to contain normal collagen fibrils, but smaller and reduced numbers of proteoglycan matrix granules. Our studies reported here indicate that mutant, like normal cartilage, synthesizes type II collagen and contains normal quantities of glycosaminoglycans as judged by uronic acid content. However, the glycosaminoglycans from the mutant differ from the normal in their chromatographic and electrophoretic properties. Further studies established that glycosaminoglycans from cartilages of brachymorphic animals were undersulfated. Whereas chondroitinase digests of glycosaminoglycans from cartilage of normal $\text{C57Bl}\text{6J}$ 5-day-old mice contained predominantly disaccharides sulfated in the 4-position, that of the mutant contained appreciable unsulfated disaccharides as well.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus: fine structure and immunohistochemical detection of elastin

Immunohistochemical and ultrastructural methods were used to examine the distribution of elastin and the fine structure of the trabecular, nasal, branchial, and pericardial cartilages in the sea lamprey, Petromyzon marinus. The cells and matrix, as well as the overall organization of these components, in larval and adult trabecular cartilage resemble those of adult annular and piston cartilages (Wright and Youson: Am. J. Anat., 167:59-70, 1983) Chondrocytes are similar to those in hyaline cartilage. Lamprin fibrils and matrix granules, but no collagen fibrils, are found in a matrix arranged into pericellular, territorial, and interterritorial zones. Branchial, pericardial, and nasal cartilages differ from trabecular, annular, and piston cartilages in the organization of their matrix and in the structural components of their matrix and perichondria. Furthermore, immunoreactive elastin-like material is present within the perichondria and peripheral matrices of nasal, branchial, and pericardial cartilages in both larval and adult lampreys. Oxytalan, elaunin, and elastic-like fibers are dispersed between collagen fibers in the perichondrium. The matrix contains lamprin fibrils, matrix granules, and a band of amorphous material, which is reminiscent of elastin, in the periphery bordering the perichondrium. The presence of elastic-like fibers and elastin-like material within some lamprey cartilages implies that this protein may have evolved earlier in vertebrate history than has been previously suggested.     

The immunohistochemical localization of superoxide dismutase activity in the avian epithelial growth plate

Summary Superoxide dismutase (SOD) is a scavenger enzyme which catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. Light microscopy employing immunohistochemistry was utilized for localizing SOD activity in the chick epiphyseal cartilage. Antibodies to mammalian liver CuZn—SOD were prepared and the avidin—biotin—peroxidase technique (ABC complex) was utilized to localize activity for this enzyme in the growth plate cartilage. The localization of enzyme activity varied in accordance with the characteristic zonation pattern of the growth plate (zone of proliferation, zone of maturation, zone of cell hypertrophy and zone of matrix calcification). In the upper regions of the epiphyseal cartilage (the zones of proliferation and maturation), where the vascularity is poor and the oxygen tension low, SOD activity was localized within the chondrocytes. No extracellular activity was observed. However, in the lower regions of the growth plate (the zones of cell hypertrophy and matrix calcification), where both the vascularity and the oxygen tensions are increased, SOD activity was intense in both the chondrocytes and the surrounding extracellular matrix. Thus, the distribution of SOD enzyme activity in this tissue seems to vary in accordance with the level of oxygen present. The significance of the extracellular SOD activity, seen in the lower aspects of the growth plate cartilage, may indicate the sensitivity of matrix components, especially collagen, to toxic free radicals such as the superoxide anion.     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

The structure of the integument of the sea cucumber,Thyone briareus

The integument and podia of the sea cucumber Thyone briareus were examined by bright field and electron microscopy. The epidermal surface was found to be covered by an acellular, PAS positive cuticle which appeared to be secreted by the underlying epidermal cells. Although the superficial portion of the cuticle contains numerous fine filaments, their ultrastructure bears no resemblance to collagen fibers. The epidermal cells are widely spaced and have long apical processes that extend along the under surface of the cuticle forming a contiguous epithelium. The apical expansions of the epidermal cells are attached to one another by means of septate desmosomes which may run continuously around all epidermal cells. Special attachment structures within these apical expansions appear to bind the cuticle to the dermis. The epidermal cells and their apical expansions are separated from the dermis by an 800 Å thick basement membrane. Granule containing cells in the upper dermis send processes up to the cuticle where they are bound to the typical epidermal cells by septate desmosomes. The abundant membrane bound granules of the cells enter villous-like processes which pass through the cuticle. The function of these cells may be to produce an adhesive material on the podia or they may be pigment cells. The thick dermis consists of a superficial zone, containing largely ground substance; a middle or laminated zone containing laminae of collagen fibers arranged in an orthogonal fashion; and a hypodermis consisting largely of ground substance and reticular fibers. Fibroblasts are abundant in the superficial dermis and between the collagen laminae. Wandering coelomocytes, or morula cells, accumulate between the collagen laminae and in the hypodermis. They may also become an integral part of the epidermis by forming septate desmosomes with epidermal cells. Morula cells contain highly specialized spherules whose tinctorial properties and electron microscopic appearance suggest that they contain protein and mucopolysaccharide.     

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.     

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.     

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.     

Ultrastructure of odontogenic cells during enameloid matrix synthesis in tooth buds from an elasmobranch, Raja erinacae

The ultrastructure of the inner dental epithelial cells (IDE) and odontoblasts in elasmobranch (Raja erinacae) tooth buds was investigated by transmission electron microscopy to determine what contribution each cell type makes to the forming enameloid matrix. Row II, early stage, IDE cells contained few organelles associated with protein synthesis, whereas preodontoblasts appeared competent to initiate extracellular matrix production. Row III IDE cells are also devoid of organelles related to secretory protein synthesis, although these IDE cells accumulated large pools of intracellular glycogen. The glycogen appeared to be packaged into vesicles and exocytosed into the lateral extracellular space toward the forming enameloid matrix. Row III odontoblasts had a morphology consistent with an active protein secretory cell. No procollagen granules were present within the odontoblasts, however, nor were many collagen fibers observed in the enameloid matrix. Instead, non-collagenous "giant" fibers having 17.5-nm periodic cross striations were associated with the invaginations of odontoblast cell processes. Giant fibers, which spanned a clear zone adjacent to the odontoblasts, terminated within the enameloid matrix. Smaller 25-nm-wide "unit" fibers emanated from the giant fiber tips to form the bulk of the enameloid matrix. The clear zone, which separated the odontoblasts from the enameloid matrix at early stages, diminished in size at later stages until the odontoblast processes were completely embedded in the enameloid matrix. Nascent enameloid crystallites were observed only after a layer of unmineralized predentin was deposited beneath fully formed enameloid matrix. The results suggest that the major constituent of the enameloid matrix in skates is a non-collagenous protein derived from the odontoblasts. The inner dental epithelial cells appear to contribute large quantities of carbohydrates to the forming enameloid matrix.     

Structural and histochemical aspects of the pericellular environment in cartilage.

Mature cartilage contains pericellular regions of matrix of fine texture, consisting of filamentous material and granules containing proteoglycan. Intercellular matrix contains collagen fibres with structural elements resembling those of the pericellular regions in the spaces between the fibres. Membrane bound bodies may be present at the margin of the pericellular region. Histochemically, chondroitin sulphate is found in the pericellular region in all zones but keratan sulphate is similarly stainable only in the deep zones of ageing cartilage.     

Chondrocyte-specific phenotype confers susceptibility of rat chondrocytes to lysis by NK cells

Normal chondrocytes are targets for natural killer (NK) cells. Since the mechanism of this phenomenon remains unknown, the present study was aimed at testing whether it is associated with chondrocyte-specific phenotype defined as ability of cartilage cells to produce sulfated glycosaminoglycans (GAG) and express collagen II and aggrecan mRNA. Lysis of rat epiphyseal chondrocytes by syngeneic spleen mononuclear cells (SMCs) was evaluated by ⁵¹Cr-release assay. Loss of chondrocyte phenotype following long-term culture resulted in their decreased susceptibility to lysis. Similar effect was also observed after suppression of chondrocyte phenotype by TNF. On the other hand, stimulation of cartilage-specific matrix component synthesis by IGF-1 resulted in increased chondrocyte killing and exogenous chondroitin sulfate A stimulated NK cell-mediated cytotoxicity against chondrocytes and human K562 cells. This suggests that chondrocyte susceptibility to lysis by NK cells depends on chondrocyte-specific phenotype, especially sulfated GAG production.     

Chondrocyte differentiation and bone growth during the development of the cartilaginous skeleton of chickens]

Certain local alterations in functional and reproductive activity of chondrocytes were stated at the development of the cartilage skeleton. In epiphyses chondrocytes gradually pass into the phase of rest (G0) with subsequent multiplication during the process of skeletal development. In these structures biosynthesis of nonsulfated proteoglycans predominate, in time, while in other cartilage zones--that of sulfated ones. Proofs are furnished on gradual transition of epiphyseal chondrocytes into the state peculiar for cells of the proliferative zone accompanied by an intensified biosynthesis of sulfated proteoglycans and collagenous proteins. Owing to these peculiarities they can be compared with the cells of the reserve zone in the mammalian metaepiphyseal cartilage. It was stated that intensity of chondrogenesis and growth of bones are affected by several processes: intensity of chondrocyte multiplication, the rate of their repeated division in the proliferative zone, the velosity with which the cells transfer into the state of hypertrophy and the rate of the periostal bone formation at the border-line of metaphysis and diaphysis.     

Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification

Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.