Morphological modifications during long-term survival of Meckel's cartilage hypertrophic chondrocytes transplanted in the mouse spleen.

To examine the ultimate fate of hypertrophic chondrocytes, Meckel's cartilage bars from 18-day-old mouse embryos were transplanted into isogenic mouse spleen for 3, 7, 14 and 21 days and observed at the light and electron microscopic levels. The midportions of these Meckel's cartilage bars were used as explants; they were characterized by many hypertrophic chondrocytes containing euchromatic round nuclei, a large amount of glycogen particles, and some vacuoles. Grafted cartilage adapted well to the splenic tissue, showing intense metachromasia around the territorial matrix. Ultrastructural observations indicated that the number of large vacuoles and glycogen aggregates in the hypertrophic cells became markedly reduced with grafting time, whereas the Golgi apparatus and rough endoplasmic reticulum were well-developed. Needle-like crystals showing initial apatite deposition appeared in association with matrix vesicles; these proliferated as time elapsed after transplantation. On day 14 after transplantation, cells displaying such various structural features as pyknotic nuclei, large vacuoles, and cytoplasmic shrinkage were noted in addition to intact hypertrophic chondrocytes. Following resorption of the calcified cartilage by multinucleated giant cells, many osteoblasts appeared along the border of the calcified matrix. Some remaining hypertrophic cells in the calcified matrix had transformed into osteocyte-like cells. On day 21, the resorbed area of the calcified cartilage was invaded by many blood vessels. Hypertrophic chondrocytes, now exposed from cellular lacunae, and the osteocyte-like cells in the calcified matrix displayed involutional changes. The present study showed that, although the hypertrophic chondrocytes in Meckel's cartilage essentially underwent regressive changes, they retained the ability to stimulate endochondral ossification within the microenvironment of the spleen. In addition, some of these cells were transformed into osteocyte-like cells.     

An electron microscope study of the distal segment of the os penis of the rat

The d-segment of the rat penile bone at 14 weeks is composed of an outer zone of atrophy, a middle zone of hypertrophy and an inner zone of ossification. Hypertrophic chondrocytes in the middle zone generally present a few secretory vesicles and a poorly developed Golgi apparatus and rough endoplasmic reticulum, suggesting a serious decrease in the secretion of territorial matrix substances. Instead, most of the cells contain prominent glycogen lakes, lipid droplets and/or fine cytoplasmic filaments. These and other findings indicate that the chondrocytes are in a resting state. In the calcified layer of the zone, and in addition to the hypertrophic or degenerating chondrocytes, another type of cell is recognizable. These sometimes remain viable through the process of cartilage degeneration and may be liberated from the besieging calcified interterritorial matrix and from their own lacuna by chondroclasts. These surviving cells are more similar in ultrastructure to bone cells than to the adjacent chondrocytes, surrounded by the lamina limitans characteristic of the resting osteocytes. However, they are directly covered by a typical territorial matrix inside the lamina limitans and do not extend slender cell processes into the calcified matrix beyond the lamina limitans, and the territorial matrix is never calcified even after calcification of the interterritorial matrix. The cells, therefore, are regarded as belonging to the chondrocytes.     

Endochondral calcification by hypertrophic chondrocytes in the Meckel's cartilage grafted into the isogenic mouse spleen

We found that when the midsection of Meckel's cartilage bars obtained from mice on the eighteenth day of gestation were grafted into isogenic mouse spleen, chondrocytes induced an endochondral calcification. Concurrent with the onset of calcification throughout Meckel's cartilage matrix, periodic banded thick collagen fibrils and matrix vesicles were observed around the chondrocytes. Although most of the chondrocytes prior to grafting were hypertrophic cells, they survived for seven days in the splenic tissue and had well-developed secretory organelles. The cells which were surrounded by calcified matrix were relatively small, spherical, and showed a morphology closely resembling that of osteocytes. These findings suggest that the life span of hypertrophic chondrocytes is influenced by the microenvironment of the spleen.     

Ultrastructural and histochemical studies of the epiphyseal plate in normal chicks

Background: Chondrocytes in the epiphyseal plate undergo a series of well-defined stages, each stage containing a morphologically homogeneous cell population. However, biochemical studies show that there are some functionally heterogeneous cell types in the calcifying zone of the chick epiphyseal plate. Methods: We studied the sequence of chondrocytic maturation in the normal chick epiphyseal plate ultrastructurally and histochemically. Chondrocytes in the calcifying zone were of three distinct types, the appearance of each cell type being closely related to the stage of matrix calcification. Results: Clear cells were observed in the upper calcifying region, stellate cells appeared in the middle calcifying region, and hypertrophic clear cells appeared in the lower calcifying region. Rough endoplasmic reticulum (RER) and lysosome-rich cells were found, these being limited to the outermost layers of the calcifying zone and containing ACPase-positive products. Osteoclasts were attached to the matrix near the RER and lysosomerich cells in the poorly calcified regions. Conclusion: We hypothesized that each cell type played a different role in the initiation, progression, and maintenance of cartilage calcification. RER and lysosome-rich cells may be responsible for the resorption of uncalcified cartilage matrix, this resulting in induction of the osteoclastic resorption of the calcified matrix. In addition, the fate of the chondrocytes was twofold: hypertrophic clear cells died, while the RER and lysosme-rich cells survived, suggesting that these cells were transformed into osteogenic cells. © 1995 Wiley-Liss, Inc.     

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.     

Ultramicroscopic aspects of the conversion of fibroblasts to chondrocytes in the mouse dorsal subfascia induced by bone morphogenetic protein (BMP)

Direct conversion of typical fibroblasts to chondrocytes in the mouse fibrous connective tissue induced by bone morphogenetic protein (BMP) was observed by light as well as electron microscopy. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the dorsal subfascia of 5 week-old mice. Until 3 days after implantation of BMP, all the connective tissue cells in the pellet region of the dorsal subfascia showed the fine structural features of typical fibroblasts. The cells in the pellet region changed their shape from spindle-like to polygonal by 5 days after implantation. At this time, small vacuoles 150-450 nm and vesicles 40-60 nm in diameter, containing a homogeneous substance of low electron density, appeared in the cytoplasm of the cells. A small amount of extracellular substance, showing metachromasia by toluidine blue staining, was seen around the cells. Moreover, autoradiography of 35S revealed the uptake of sulfur by the cells and its accumulation in the extracellular substance around the cells in the pellet region at 5 days. The rough endoplasmic reticulum and Golgi apparatus increasingly developed with time and after 7 days both elements were distributed throughout the cytoplasm. The cytoplasmic small vacuoles and vesicles also increased in number with time, and the metachromatic extracellular substance containing fine filamentous meshwork and many tiny particles, which was regarded as the matrix of cartilage, also increased rapidly in amount. By 9 days, the cells in the pellet region became oval or round in shape, showing many short cytoplasmic processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcification capacity of dental papilla mesenchymal cells transplanted in the isogenic mouse spleen

The capacity of the dental pulp to form calcified tissue was examined in papilla cells dissociated from first molar tooth germs of the neonatal mouse and isografted in the spleen for up to 7 days. To obtain papilla cell populations without odontoblasts, pulpal mesenchyme was isolated mechanically from the enamel organ after 0.1% trypsin treatment and rolled on a membrane filter. On day 3 after transplantation, the grafted papilla cells had changed into large, spindle-shaped cells, and initial calcification with needle-like crystals began in association with the collagenous matrix surrounding those cells. On day 7 after transplantation, the spindle cells transformed into odontoblast-like cells containing well-developed secretory organelles, and irregular, but nontubular, calcified tissues were commonly observed surrounding the extracellular collagenous matrix. The calcified tissue matrix with cellular inclusions displayed a structure similar to that of osteodentin. During this period, an intense positive reaction for alkaline phosphatase (ALPase) activity was demonstrated along the cell membranes of the odontoblast-like cells aligned at the periphery of forming calcified tissue. Enzymatic activity could not be detected on the cells incorporated completely into osteodentin-like matrix. The present results show that the papilla cell population transplanted into the spleen formed osteodentin-like material, thus demonstrating the capacity of papilla cells to produce calcified tissue.     

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.     

Spatiotemporal pattern of type X collagen gene expression and collagen deposition in embryonic chick vertebrae undergoing endochondral ossification

We examined the spatio-temporal pattern of type X collagen mRNA and its protein in the embryonic chick vertebrae undergoing ossification by in situ hybridization and immunohistochemistry. Hypertrophic chondrocytes, producing type X collagen, were developed as islands of cells in a few vertebral body segments of stage 36 embryos. These cells were increased in number at stages 37 and 38 and they expressed high levels of type X collagen mRNA and deposited its protein in the matrix. Blood vessels entered from the perichondrium at stage 37 and invaded deeply into hypertrophic cartilage at stage 38. As the vertebrae grew further at stage 40, the leading front of active hypertrophic chondrocytes with high levels of type X mRNA shifted from the midvertebral perivascular area towards intervertebral borders, while the perivascular area retained a number of inactive hypertrophic chondrocytes with low levels of type X mRNA. Type X collagen was found in large amounts throughout the matrix areas containing both active and inactive hypertrophic chondrocytes. Calcium was detected by von Kossa's technique in hypertrophic cartilage matrix in a small amount at stage 37, in parts of the matrix with type X collagen deposition in succeeding stages, and finally in almost the entire area of type X collagen deposition at stage 45. The vertebral segments of stage 45 embryos also showed a clearly reversed pattern of expression between type X collagen mRNA and types II and IX collagen mRNAs. The results demonstrate that the production of type X collagen by hypertrophic chondrocytes precedes both vascular invasion and mineralization of the matrix, suggesting that hypertrophic chondrocytes have an important role in regulating these events.     

Avian tibial dyschondroplasia. I. Ultrastructure.

Tibial dyschondroplasia is an abnormality of the growth cartilage that occurs in chickens and other rapidly growing animals. The disease is characterized by a mass of avascular opaque cartilage, which is continuous with the growth plate of the proximal tibia and extends into the metaphysis. In this study electron micrographs revealed that chondrocytes in the hypertrophic zone of the growth plate were normal in appearance with the exception that the cells did not undergo complete hypertrophy. In the proximal region of the lesion, cells began to undergo necrotic changes suggestive of an energy depletion. These changes included dilatation and vesiculation of the endoplasmic reticulum, enlargement of the paranuclear space, mitochondrial swelling with dilatation of the intracristal spaces and the appearance of electron-dense, flocculent material in the mitochondrial matrix, chromatin margination, and dilatation of the Golgi saccules. Chondrocytes also occurred with rarefied cytoplasm and atrophic Golgi saccules. A few cartilage cells in the proximal region of smaller lesions contained crescentic caps of condensed chromatin in the nuclei, which is indicative of apoptosis. These cells also exhibited dilated endoplasmic reticulum and lamellar bodies; and sometimes, in the proximal region of the lesion, they appeared to be condensed and convoluted. This process continued in the mid and distal regions. The condensed necrotic cells appeared as amorphous osmiophilic masses with karyorrhexic and pyknotic nuclei. Matrix vesicles were observed at all levels of the lesion, but calcified only at the distal edge of the lesion, where mineralization of both matrix and cells occurred. The resulting shell of mineral may act as a diffusion barrier.     

The influence of bone and marrow on cartilage hypertrophy and degradation during 30-day serum-free culture of the embryonic chick tibia.

In this study, an organ culture system is defined which demonstrates complete loss of cartilage matrix from embryonic chick tibiae. Efficient loss of the cartilage matrix occurs within 30 days of serum-free culture only when the intact tibiae containing bone, marrow, and cartilage tissue are cultured. During organ culture nonhypertrophic chondrocytes become hypertrophic and stain positively for type X collagen and alkaline phosphatase. The cartilage loses Safranin O staining, and finally all cartilage matrix disappears leaving the bony collar and marrow cells. If the tibial cartilage is separated from the bony collar and cultured alone in serum-free medium, the nonhypertrophic chondrocytes also hypertrophy; the matrix loses Safranin O staining; however, some components of the matrix including type X collagen still remain after 30 days. In the presence of serum, the chondrocytes will hypertrophy but cartilage degradation is not evident. The results of this study support the conclusions that 1) hypertrophy is inherently programmed in the chondrocyte and 2) while Safranin O staining of cartilage cultured alone is diminished in serum-free organ culture, the degradation of cartilage is complete only when bone and marrow are also present.     

Type?X collagen is not localized in hypertrophic or calcified cartilage in the developing rat trachea

 Our previous studies have shown that rat tracheal chondrocytes become larger and hypertrophic, and that the cartilage matrix calcifies during development. Type X collagen is a short collagen molecule identified in hypertrophic and calcified cartilage in the growth plate of long bones during endochondral ossification. The present study was designed to investigate the distribution of type X collagen in rat tracheal cartilage during development before and after hypertrophization and calcification. Tracheas from postnatal Wistar rats, newborn, and at 4, 8 and 10 weeks were fixed along with hind limbs from newborn rats. Serial sections were made and adjacent sections were processed for von Kossa staining or immunohistochemistry for type X collagen. In addition, the immunoreactivity to type II collagen was examined as a control. The anti-type X collagen antibody stained hypertrophic and/or calcified cartilage in the newborn rat tibia. The immunoreaction for type X collagen was localized in the uncalcified peripheral region of tracheal cartilage in 4, 8 and 10-week-old rats. In contrast, the anti-type X collagen antibody did not show immunoreactivity to hypertrophic or calcified cartilage in the central region of the 10-week-old rat tracheal cartilage. The present study has suggested that type X collagen is not involved in hypertrophization of chondrocytes or calcification of the matrix in developing rat tracheal cartilage. Accepted: 24 November 1997     

A functional agarose-hydroxyapatite scaffold for osteochondral interface regeneration

Regeneration of the osteochondral interface is critical for integrative and functional cartilage repair. This study focuses on the design and optimization of a hydrogel-ceramic composite scaffold of agarose and hydroxyapatite (HA) for calcified cartilage formation. The first study objective was to compare the effects of HA on non-hypertrophic and hypertrophic chondrocytes cultured in the composite scaffold. Specifically, cell growth, biosynthesis, hypertrophy, and scaffold mechanical properties were evaluated. Next, the ceramic phase of the scaffold was optimized in terms of particle size (200 nm vs. 25 μm) and dose (0-6 w/v%). It was observed that while deep zone chondrocyte (DZC) biosynthesis and hypertrophy remained unaffected, hypertrophic chondrocytes measured higher matrix deposition and mineralization potential with the addition of HA. Most importantly, higher matrix content translated into significant increases in both compressive and shear mechanical properties. While cell hypertrophy was independent of ceramic size, matrix deposition was higher only with the addition of micron-sized ceramic particles. In addition, the highest matrix content, mechanical properties and mineralization potential were found in scaffolds with 3% micro-HA, which approximates both the mineral aggregate size and content of the native interface. These results demonstrate that the biomimetic hydrogel-ceramic composite is optimal for calcified cartilage formation and is a promising design strategy for osteochondral interface regeneration.     

Morphological observations concerning the pattern of mineralization of the normal and the rachitic chick growth cartilage

Summary The entire calcified layer of the chick growth cartilage is penetrated by canals that contains blood vessel complexes: some of these canals pass through all the layers of the cartilage from the resorptive zone at the metaphysis, through the mineralizing, hypertrophic, proliferative and resting regions. This study aimed to provide more details of the 3-D microanatomy of this region and to establish whether there are differences in the process and progress of mineralization compared with the established mammalian model.Proximal tibial heads from 6 to 8 weeks old normal and vitamin D deficient chickens were rapidly frozen and prepared for scanning electron microscopy using freezefracture, freeze-drying, plasma ashing, and chemical deproteinization techniques. Cartilage samples were also embedded in PMMA and polished for BSE imaging. Other samples were prepared for light microscopy.Zones of (mineralized) cartilage several cells thick separate adjacent canals. At the mineralizing front, calcification of the matrix is most advanced close to the canals, but the matrix adjacent to the canal lumens does not calcify. Mineralisation of the cartilage matrix is incomplete and small fenestrae of unmineralized matrix connect chondrocyte lacunae. These discontinuities in matrix calcification could serve as a route for diffusion of nutrients, metabolites and dissolved gases.The calcified cartilage is more mineralized than the contiguous developing bone. Osteoblasts surrounded by bone were seen to occupy the lacunae of hypertrophic chondrocytes. We tentatively suggest that some osteoblasts represent a terminal stage in the differentiation of hypertrophic chondrocytes.The rachitic cartilage was disorganised. It was penetrated by irrugular vascular canals and exhibited a greatly expanded hypertrophic zone. The matrix was mineralized and mineral particles and clusters were spread throughout the matrix. However, these centres did not become continuous with adjacent or contiguous mineral. The results indicate that an absence of vitamin D affects crystal growth rather than initiation.     

Chondroclasts and endothelial cells collaborate in the process of cartilage resorption.

The condylar cartilage of the young rat is a major growth center of the craniofacial complex. Differences between the mechanism that results in bone formation from growth centers in the epiphyseal plates of long bones are dictated primarily by the different character of the mineralization of the cartilage. In this ultrastructural study we demonstrate that the terminal hypertrophic chondrocytes undergo apoptosis and disintegration while simultaneously chondroclasts dissolve gaps in the calcified cartilage that engulfs them. The latter are also phagocytizing debris of the chondrocytes. The chondroclasts are intimately followed by tube-forming endothelial cells that most probably coalesce to create extensions of the invading capillaries into the evacuated lacunae. The chondroclasts have ultrastructural features similar to osteoclasts. They are multinucleate, are rich in mitochondria and vacuoles, form clear zones that adhere to the spicules of the calcified cartilage, and also form a sort of ruffled border. The latter is not as elaborate and orderly arranged as is known from osteoclasts. The capillaries that follow orient the stroma cells to the evacuated lacunae and, together with the calcified cartilaginous scaffold, supply the adequate environmental conditions for the stroma cells to differentiate into osteoblasts and to build up trabecular bone.     

Chondroclasts and endothelial cells collaborate in the process of cartilage resorption

The condylar cartilage of the young rat is a major growth center of the craniofacial complex. Differences between the mechanism that results in bone formation from growth centers in the epiphyseal plates of long bones are dictated primarily by the different character of the mineralization of the cartilage. In this ultrastructural study we demonstrate that the terminal hypertrophic chondrocytes undergo apoptosis and disintegration while simultaneously chondroclasts dissolve gaps in the calcified cartilage that engulfs them. The latter are also phagocytizing debris of the chondrocytes. The chondroclasts are intimately followed by tube-forming endothelial cells that most probably coalesce to create extensions of the invading capillaries into the evacuated lacunae. The chondroclasts have ultrastructural features similar to osteoclasts. They are multinucleate, are rich in mitochondria and vacuoles, form clear zones that adhere to the spicules of the calcified cartilage, and also form a sort of ruffled border. The latter is not as elaborate and orderly arranged as is known from osteoclasts. The capillaries that follow orient the stroma cells to the evacuated lacunae and, together with the calcified cartilaginous scaffold, supply the adequate environmental conditions for the stroma cells to differentiate into osteoblasts and to build up trabecular bone. © 1992 Wiley-Liss, Inc.     

Transmission and scanning electron microscope studies of calcified cartilage resorption

The authors' previous report (Savostin-Asling and Asling, 1973) demonstrated that Meckel's cartilage is a favorable site for study of calcified cartilage resorption. In the present study the ultrastructural features at this resorption front have been examined by transmission and scanning electron microscopes (19-day rat fetus). Multinucleated giant cells (chondroclasts) dominated the erosion front. The many features which they showed in common with osteoclasts included abundant mitochondria, vacuolation, lysosomes, sparsity of roughsurfaced endoplasmic reticulum, and deep infoldings at loci of contact with calcified matrix. Crumbling of matrix (with mineral crystals penetrating between these foldings) and fragmentation of collagen fibrils were also seen. The propensity of chondroclasts for spanning several opened lacunae provided special opportunity to demonstrate cell surface modifications in presence or absence of matrix contact. Ameboid processes extending into lacunae were seen by both transmission and scanning procedures; they were sometimes tipped with a veil of filamentous processes as small as 0.3 μm in diameter. Most hypertrophic chondrocytes, when released from lacunae, appeared to be disintegrating. However, in accord with previous evidence of their possible merger with chondroclasts (in light microscopic studies) there was also evidence for breakdown of cell walls between a chondroclast and a chondrocyte in intimate contact, with possibility of cytoplasmic continuity.     

Association of the C-Propeptide of Type II Collagen with Mineralization of Embryonic Chick Long Bone and Sternal Development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation.

These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.     

Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation. These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.     

Ultrastructure of cartilage from young adult sea lamprey,Petromyzon marinus L: A new type of vertebrate cartilage

Ultrastructural observations of cartilage from adult sea lamprey, Petromyzon marinus, reveal a highly cellular cartilage with an unusual extracellular matrix. The avascular cartilage is surrounded by a vascular perichondrium, which consists of dense connective tissue containing fibroblasts, collagen fibrils, and microfibrils. The cells (chondrocytes) vary in morphology in different parts of the cartilage in a way that may reflect their state of activity. Chondrocytes within the peripheral cartilage contain tubulo-vesicular structures along the cell surface, an extensive lamellar rough endoplasmic reticulum, and a well-developed Golgi complex with associated vesicles and vacuoles. The presence of material within the Golgi elements that resembles components of the extracellular matrix suggests the involvement of the peripheral chondrocytes in the synthesis and secretion of the matrix components. Chondrocytes within the central cartilage are hypertrophied and contain a pale cytoplasm with a reduced number of organells that are widely spaced throughout the cell. The appearance of the organelles within these cells suggests that they are not as actively involved in the production of the matrix as those of the peripheral cartilage. The extracellular matrix consists of a dense network of randomly arranged, branched, noncollagenous matrix fibrils 15–40 nm in diameter and varying amounts of electron-dense matrix granules. Due to the unique nature of its extracellular matrix, the cartilage of the lamprey cannot be likened to any of the known vertebrate cartilages and, therefore, must be considered a new type of vertebrate cartilage.