Immunohistochemical and immunoblot analyses of collagens in the developing fibrocartilage in the glans penis of the rat

The distal segment of the os penis of rats is a fibrocartilage whose development is dependent on androgens. Electrophoretic and immunoblot analysis indicated that the fibrocartilage contained both type I and type II collagens. Immunohistochemically, type I collagen was detected in the fibrous matrix of the distal segment in all the stages examined. Type II collagen was detected first at 4 weeks in the extracellular matrix surrounding the mature chondrocytes distributed sparsely as single cells or small clusters among immature chondroblasts. The clusters reactive with anti-type II collagen serum increased in size and number at 6 weeks, and almost all the region of the distal segment became reactive with anti-type II collagen serum at 8 weeks.     

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.     

COMP mutations, chondrocyte function and cartilage matrix

Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.     

Light- and electron-microscopic observations on the mandibular condylar cartilages in growing rats on a low-calcium diet.

In order to obtain more insight into the physiologic mechanism of endochondral ossification, histological changes occurring in the mandibular condylar cartilage of growing rats fed on a low-calcium diet were investigated by light and electron microscopy. Twenty-three-day-old rats were fed on a normal diet or a low-calcium diet for 8 weeks. For the histological observations the mandibular condyles were dissected from each animal at 1, 2, 4, 5 and 8 weeks after the initiation of the experiment. Histological changes occurring in the mandibular condylar cartilages of the rats fed on a low-calcium diet were as follows: (1) narrow proliferative and mature cell zones and a wide hypertrophic cell zone, (2) inhibition of development of cell organelles in the mature chondrocytes, (3) decrease in dead cells in the proliferative zone, (4) decrease in glycogen accumulation in the chondrocytes and (5) inhibition of calcification in the extracellular matrix of the hypertrophic cell zone. Additionally at the end of the experimental period, the following findings were observed: (1) appearance of small light cells in the mature cell zone and the hypertrophic cell zone and (2) decrease in proteoglycan granules and appearance of large collagen fibrils in the pericellular region of the hypertrophic cell zone.     

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.     

Cartilaginous metaplasia of the thoracic aorta of control turkeys and exacerbation by beta-aminopropionitrile

Seventeen of sixty distal extremities of the thoracic aortas of 12-week-old control male turkeys and 37 of 40 distal extremities of the aortas of turkeys fed 0.07% beta-aminopropionitrile (BAPN) from 4 to 12 weeks of age contained areas of cartilaginous metaplasia when examined by light microscopy. The cartilaginous areas were generally elongated and located in the subendothelium of control turkeys, but a roundish area of cartilage was occasionally evident in the deep media. The magnitude of chondroplasia was enhanced by feeding BAPN; the extensive lesion usually extended from the subendothelium to deep in the media. Regardless of treatment, chondrocytes were pleomorphic, contained vacuoles, and had cytoplasmic processes. The cells were separated by pools of proteoglycans and connective tissue. The ultrastructure of chondrocytes in the aortas of both treatment groups was typical of this cell type. They had undulations or projections of the cell membranes. The cisternae of endoplasmic reticulum were dilated and contained electron-translucent material which was similar to extracellular proteoglycans. Golgi apparatus, free ribosomes, mitochondria, glycogen granules, filaments, and a centriole also were present in the cytoplasm. The extracellular matrix, which included collagenous and elastic fibers and also delicate fibrils and interconnecting matrix granules, separated adjacent chondrocytes by spaces of varying size.     

Cartilage calcification in the human thoracic column

A histological and microradiological study of the cartilage calcification processes in the human thoracic column of an old man has been performed. Two different types of cartilage mineralization have been identified. The first corresponds to a calcification of the hyaline cartilage ground substance where chondrocytes are apparently intact. The second is a real mineralization of the chondrocyte lacunae in an uncalcified matrix, which we have called cartilaginous necrosis.     

Ultrastructure of inclusion bodies in annulus cells in the degenerating human intervertebral disc

The rough endoplasmic reticulum (rER) of the cell has an architectural editing function that checks whether protein structure and three-dimensional assembly have occurred properly prior to export of newly synthesized material out of the cell. If these have been faulty, the material is retained within the rER as an inclusion body. Inclusion bodies have been identified previously in chondrocytes and osteoblasts in chondrodysplasias and osteogenesis imperfecta. Inclusion bodies in intervertebral disc cells, however, have only recently been recognized. Our objectives were to use transmission electron microscopy to analyze more fully inclusion bodies in the annulus pulposus and to study the extracellular matrix (ECM) surrounding cells containing inclusion bodies. ECM frequently encapsulated cells with inclusion bodies, and commonly contained prominent banded aggregates of Type VI collagen. Inclusion body material had several morphologies, including relatively smooth, homogeneous material, or a rougher, less homogeneous feature. Such findings expand our knowledge of the fine structure of the human disc cell and ECM during disc degeneration, and indicate the potential utility of ultrastructural identification of discs with intracellular inclusion bodies as a screening method for molecular studies directed toward identification of defective gene products in degenerating discs.     

The differentiation and calcification of chondrocytes in primary cell cultures.

The cartilage from a non-immobilized fracture undergoes a series of morphological and biochemical changes resembling the in vivo differentiation and calcification in the epiphyseal plate. The studies reported here demonstrate that a homogeneous population of chondrocytes isolated from fracture callus fibrocartilage undergoes the same changes in vitro. Chondrocyte primary cultures were grown for 28 days during which time the morphological, histological and histochemical properties of the cultures were studied. Demonstrated by various histological procedures, chondrocytes synthesized the characteristic cartilage matrix, and progressively calcified with increased culture age. This system can be used to elucidate the cellular and molecular mechanisms of calcification.     

Ultrastructure of neuromuscular contacts in the embryonic pond snail Lymnaea stagnalis L

Ultrastructural characteristics of muscle fibers and neuromuscular contacts were investigated during two stages of embryogenesis of the pulmonate snail Lymnaea stagnalis. The first muscle cells appear as early as during metamorphosis (50-55% of embryonic development), whereas previously, in the trochophore/veliger stages (25-45%), muscular elements cannot be detected at all. The first muscle fibers contain large amounts of free numbers, a well-developed rER system and only a few irregularly arranged contractile elements. The nucleus is densely packed with heterochromatine material. At 75% adult-like postmetamorphic stage, the frequency of muscle fibers increases significantly, but, bundles of muscle fibers cannot yet be observed. Furthermore the muscle cells are characterized by large numbers of free ribosomes and numerous rER elements. Fine axon bundles and single axon processes, both accompanied by glial elements, can already be found at this time. Axon varicosities with different vesicle and/or granule contents form membrane contacts with muscle fibers, but without revealing membrane specialization on the pre- or postsynaptic side. The late development of the muscle system and neuromuscular contacts during Lymnaea embryogenesis correlates well with the maturation of different forms of behavior of adult, free-living life, and also with the peripheral appearance of chemically identified components of the embryonic nervous system of central origin.     

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.     

Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones

Galectin 3 is a beta-galactoside binding protein which localizes to the cytoplasm of proliferative, mature, and hypertrophic chondrocytes in the growth plate cartilage of developing long bones. To elucidate the function of galectin 3 during bone development, we examined the epiphyseal femurs and tibias of fetal mice carrying a null mutation for the galectin 3 gene. Detailed histological and ultrastructural studies identified abnormalities in the cells of the proliferative, mature, and hypertrophic zones and in the extracellular matrix of the hypertrophic zone, as well as a reduction in the total number of hypertrophic chondrocytes. The expression patterns of several chondrocyte and bone cell markers were analyzed and revealed a subtle modification of Ihh expression in the galectin 3 mutant growth plate. A striking difference was observed at the chondrovascular junction where many empty lacunae are present. In addition, large numbers of condensed chondrocytes exhibiting characteristic signs of cell death were found in the late hypertrophic zone, indicating that the rate of chondrocyte death is increased in the mutants. These results suggest a role for galectin 3 as a regulator of chondrocyte survival. In addition, this unique phenotype shows that the elimination of chondrocytes and vascular invasion can be uncoupled and indicates that galectin 3 may play a role in the coordination between chondrocyte death and metaphyseal vascularization.     

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.     

The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.     

Expression of FAM20C in the Osteogenesis and Odontogenesis of Mouse

Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth. (J Histochem Cytochem 58:957–967, 2010)     

Body muscle-cell differentiation from coelomic stem cells in colonial tunicates

Body muscle-cell differentiation was ultrastructurally examined in palleal buds of the colonial tunicate Symplegma reptans. Undifferentiated coelomic cells accumulate near the primordial oral siphon and associate with the basal lamina beneath the epidermis. They initially display the characteristics of hemoblast cells that have a large nucleus with a prominent nucleolus and narrow cytoplasm filled with polysomes. However, they soon become unique due to the development of an indented contour of the nucleus. When the basal lamina of the epidermis develops into the fibrous extracellular matrix (ECM), the muscle precursor cell has the deeply-notched nucleus, and thick and thin filaments in the cytoplasm facing the ECM. Collagen fibril-like structures appear in the ECM. Myofilaments are arranged with the ratio of thick to thin filaments being 1:2.5. Dense bodies and plaques become evident before the oral siphon is perforated. These results show that in S. reptans, the sphincter muscle cells arise from undifferentiated hemoblasts, and that their differentiation begins with a morphological change in their nuclei. Epidermal cells and/or the ECM may have an inductive effect on muscle cell differentiation.     

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae)

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.     

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.