Chondrocytes are released as viable cells during cartilage resorption associated with the formation of intrachondral canals in the rat tibial epiphysis

The development of cartilage canals is the first event of the ossification of the epiphyses in mammals. Canal formation differs from vascular invasion during primary ossification, since the former involves resorption of resting cartilage and is uncoupled from bone deposition. To learn more about the fate of resorbed chondrocytes during this process, we have carried out structural, cell proliferation, and in situ hybridization studies during the first stages of ossification of the rat tibial proximal epiphysis. Results concerning the formation of the cartilage canals implied the release of resting chondrocytes from the cartilage matrix to the canal cavity. Released chondrocytes had a well-preserved structure, expressed type-II collagen, and maintained the capacity to divide. All these data suggested that chondrocytes released into the canals remained viable for a specific time. Analysis of the proliferative activity at different regions of the cartilage canals showed that the percentage of proliferative chondrocytes at areas of active cartilage resorption was significantly higher than that in zones of low resorption. These results are consistent with the hypothesis that resting chondrocytes surrounding canals have a role in supplying cells for the development of the secondary ossification center. Since released chondrocytes are at an early stage of differentiation greatly preceding their entry into the apoptotic pathway and are exposed to a specific matrix, cellular, and humoral microenvironment, they might differentiate to other cell types and contribute to the ossification of the epiphysis.This research was supported by the Ministerio de Ciencia y Tecnología (Spain), grant no. MCT-00-BMC-0446. The Instituto Universitario de Oncología is financed by Obra Social Cajastur-Asturias, Spain. J. Alvarez receives financial support from the Ministerio de Ciencia y Tecnología (CAJAL-03-06) and L. Costales from the Ministerio de Ciencia y Tecnología (MCYT, FP2000-5486).     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Immunolocalization of complement C1s and matrix metalloproteinase 9 (92kDa gelatinase/type IV collagenase) in the primary ossification center of the human femur

The first component of complement has been shown to degrade type I and type II collagens (Yamaguchi et al. 1990), the latter of which is a major constituent of the cartilage matrix. In order to understand the physiological roles of in cartilage resorption, the expression of C1s was examined by immunohistochemistry in the primary ossification center where the matrix is removed and replaced by bone marrow. Hypertrophic chondrocytes, endothelium and hematogenous elements in the capillary buds were intensely stained by a monoclonal antibody against C1s. Matrix metalloproteinase 9 (MMP-9, 92kDa gelatinase/type IV collagenase) was also immunolocalized in hypertrophic chondrocytes, mesenchymal cells in the primitive bone marrow and the cartilage matrix adjacent to the marrow. In addition, was found to activate the zymogen of MMP-9. These observations suggest that and MMP-9 coordinately participate in matrix degradation in cartilage.Abbreviations MMP Matrix metalloproteinase - APMA 4-aminophenylmercuric acetate - DFP diisopropyl fluorophosphate, HE hematoxylin and eosin - C1s inactive C1s - activated C1s     

Morphogenesis and secretory activity of mouse mammary cultures on EHS biomatrix

Summary The nature of the substratum profoundly influences the growth and function of epithelia in tissue culture. Mammospheres, hollow spherical structures, develop when epithelial clusters are plated on a biomatrix derived from the Engelbreth-Holm-Swarm murine tumour (EHS matrix). Morphologic examination of mammosphere development demonstrates that morphogenesis is a two stage process. Over the first 48 h the cells aggregate into spheres, drawing the matrix up and over themselves to become buried within the material. Changes in matrix morphology emphasize the importance of the quasi-fluid nature of the substratum on which the cells are plated. Lumen formation ensues over the next 2 to 5 days as the cells, polarized by basal contact with the matrix, differentiate. They form tight junctions at their apical borders and synthesize milk proteins, secreting caseins into the enlarging interior cavity and transferrin from their basal surfaces into the medium. These experiments demonstrate that the physical properties of the EHS matrix allow epithelial cells to develop the cuboidal shape necessary for secretory activity.Abbreviations EHS Engelbreth-Holm-Swarm biomatrix     

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Background

Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.

Methodology/Principal Findings

The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.

Conclusions/Significance

Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.     

Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells

Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O₂) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O₂ environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs. cartilage; bone     

Age changes in the localization and distribution of glycosaminoglycans in human hyaline cartilage

Summary The localization and distribution of acid glycosaminoglycans, glycoproteins and basic proteins have been studied in human costal cartilage, trachea and primary bronchi from the fetal period through old age. Alcian blue and PAS were used for the staining of acid glycosaminoglycans and glycoprotein and/or sugar-containing compounds respectively. Bromphenol blue and/or bromsulfalein were utilized for the detection of basic proteins. In a further attempt to histochemically identify the various polyanions staining was carried out with AB containing different concentrations of electrolytes. Mild acid hydrolysis, hyaluronidase and proteolytic digestions were also employed. It appeared that the distribution, localization and concentration of the different macromolecules vary according to age, to the type of hyaline cartilage and to the different areas of the same segment. The results also suggested that in developing tissues the areas of appositional growth synthesize mainly chondroitinsulphate while in the territory and interterritory this acidic polyanion is found with keratansulphate. With advancing age the latter slowly disappears from the interterritorial matrix and is nearly exclusively found in the matrix adjacent to the chondrocytes.The susceptibility of these tissues to enzyme digestion also varied according to age and to the type of hyaline cartilage. Proteolytic and hyaluronidase treatment, which were extremely effective in the prenatal period and in young subjects, had practically no effect on mature and old ribs. In the bronchi, instead, the substrates were easily extracted even at an old age. The problems related to the different chemical state of the various macromolecules, their localization and distribution and their possible effect on senescence of hyaline cartilage are discussed.Supported by Research Grant DE-01952(03) of the National Institutes of Public Health, Bethesda, Md.     

Differentiation-dependent glycosylation of gp190, an oncofetal crypt cell antigen expressed by Caco-2 cells

gp190 is a glycoprotein expressed on the cell surface of several human colon carcinoma cells in culture, on epithelial cells of fetal colon, but not on the normal mucosa of adult colon; thus it is referred to as an oncofetal crypt cell antigen. We report the characterisation of O[emsp4 ]-linked glycans carried by gp190 synthesised by [³H]glucosamine-labelled Caco-2 cells at the confluence (undifferentiated cells) and at three weeks of postconfluence (differentiated cells). By using a specific monoclonal antibody, gp190 was isolated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mobility of gp190 from differentiated cells was found to be lower than that from undifferentiated cells, suggesting a more extensive glycosylation process in the former glycoprotein. The major results of the glycan characterisation have been as follows: (i) gp190 carries mainly, if not exclusively, O-linked glycans with the core-2 structure; (ii) the elongation with N-acetyllactosamine units of the Gal1,4GlcNAc1,6(Gal1,3)GalNAc tetrasaccharide predominates in gp190 synthesised by differentiated cells, whereas the direct 2,3sialylation of the tetrasaccharide is prevalent in gp190 synthesised by undifferentiated cells. The increment in the core-2 1,6GlcNAc-transferase activity under the Caco-2 differentiation process may be relevant in producing the larger occurrence of polylactosaminoglycans in gp190 from differentiated cells. Since no change in the activity of the 2,3sialyltransferases upon cell differentiation was observed, we suggest that the lower 2,3sialylation in gp190 synthesised by polarised cells might be due to a changed transit-rate through the distal Golgi apparatus.     

Autophagocytosis in the larval midgut cells of Pieris brassicae during metamorphosis

Summary We observed three types of cells in the epithelial layer of the midgut of last instars of Pieris brassicae. The columnar and goblet cells degenerate during the second part of the last larval stage while the undifferentiated basal cells proliferate during this period and create the epithelium of the pupal midgut. The first morphological sign of involution is the formation of autophagic vacuoles and dense bodies in the cytoplasm of columnar and goblet cells which begins on day 4 of the stage. The number and size of autophagic vacuoles and dense bodies increase during the spinning period (85–96 h). Finally, at the end of the stage, the columnar and goblet cells become displaced by the growing pupal epithelium and reach the lumen where they disintegrate.Autophagocytosis was not seen in the cells during the feeding period (0–72 h). However, we observed many autophagic vacuoles in the columnar and goblet cells of 50-h-old instars 3 h after the administration of 30 g/g body weight of 20-hydroxyecdysone. The hormone treatment elevated by 100% the incorporation of ³H-leucine into the proteins of the midgut. Inhibitors of protein synthesis, cycloheximide and puromycin, in doses that supressed the incorporation of the amino acid by 60–70% either in hormone treated or untreated larvae, exerted diverse effects on the autophagic process. Puromycin did not block the hormone-induced formation of autophagic vacuoles while cycloheximide prevented it. Possible explanations for this diversity are discussed.     

Modularity of osteoclast behaviour and “mode-specific” inhibition of osteoclast function

This study is part of an attempt to understand the role of specific cellular activities in the bone resorptive process. Experiments were performed whereby known pharmacological agents were used to inhibit individual modes of osteoclastic activity, such as motility and secretion. The effects of such treatments on bone resorption were assessed by quantitative scanning electron microscopy. The compounds included colchicine, which was used to inhibit osteoclast motility; molybdate ions which were used to selectively inhibit the catalytic activity of secreted acid phosphatase, and omeprazole which was employed to inhibit the secretion of hydrogen ions. All compounds inhibited osteoclastic bone resorption, but singularly affected defined modes of activity. These findings suggest that each mode of osteoclastic activity is essential for the bone resorptive process, and that mode-specific inhibition may provide a means whereby excessive activity of the osteoclast can be regulated in disease.     

Correlative histochemical and morphological study on the maturation of sensory ganglion cells in the rat

Summary In neonatal rats the sensory ganglion cells are uniform in size and in their stainability with hematoxylin and eosin. At this stage the cells differ, however, in the intensity of staining for RNA and for various enzyme activities. With maturation the ganglion cells differentiate into light (mostly large) cells, and dark (mostly small) cells. The differentiation is accompanied by changes in intensity of various enzyme activities. In sections stained for acid phosphatases and acetylcholine esterase, maturation was associated with a higher activity in the small than in the large cells, whereas with thiamine pyrophosphatase it was associated with a higher activity in the large than in the small neurones. With non-specific cholinesterase, maturation of all cells was accompanied by loss of activity in perikarya and increased activity in axons and satellite cells. With monoamine oxidase, the changes during maturation differed in the trigeminal from the spinal ganglion cells.The findings indicate that the difference between small and large cells might have a functional significance, the nature of which is discussed.     

An osteoclastic protein-tyrosine phosphatase may play a role in differentiation and activity of human monocytic U-937 cell-derived, osteoclast-like cells

This study investigated if an osteoclastic protein-tyrosine phosphatase (PTP), PTP-oc, plays a role in the functional activity and differentiation of osteoclastic cells by determining the effects of overexpression of wild-type (WT)- or phosphatase-deficient (PD)-PTP-oc on bone resorption activity and differentiation of human promyelomonocytic U-937 cells, which could be induced to differentiate into "osteoclast-like" cells by phorbol ester/1,25(OH)₂D₃ treatment. U-937 cells overexpressing WT- or PD-PTP-oc were produced with a transposon-based vector. The size and depth of resorption pits created by WT-PTP-oc-overexpressing osteoclast-like cells were greater, while those by PD-PTP-oc-overexpressing osteoclast-like cells were less, than those created by control osteoclast-like cells. Overexpression of WT-PTP-oc also enhanced, while overexpression of PD-PTP-oc suppressed, their differentiation into osteoclast-like cells. Overexpression of WT-PTP-oc increased apoptosis and proliferation of U-937 cells, and overexpression of PD-PTP-oc reduced cell proliferation. Cells overexpressing WT-PTP-oc has also led to greater c-Src and NF- activation, whereas cells overexpressing PD-PTP-oc resulted in less c-Src and NF- activation. c-Src activation and NF- activation each correlated with resorption activity and differentiation into osteoclast-like cells. In summary, these results show that 1) PTP-oc regulates both the activity and the differentiation of osteoclast-like cells derived from U-937 cells; 2) PTP-oc enzymatic activity is important to these processes; 3) high PTP-oc enzymatic activity caused an increase in U-937 cell apoptosis and proliferation, leading to no significant changes in the number of viable cells; and 4) some of the PTP-oc actions are mediated in part by the c-Src and/or NF- pathways. osteoclast; resorption; nuclear factor-; c-Src     

Effect of cytokinin on morphological changes of suspension cultured cells of the moss, Barbula unguiculata

Suspension cultured cells of the moss, Barbula unguiculata, grow actively in both light and dark culture. Light-grown cells contain chlorophyll and exhibit an undifferentiated callus form. When cells are transferred to a dark condition, they develop into protonemata. Protonemata formation in the dark can be inhibited by the addition of 5 M benzyladenine or 6-furfurylaminopurine but is not affected by the addition of 5 M 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - kinetin 6-furfurylaminopurine - NAA naphthalene acetic acid     

Histological and histochemical studies of the cells of the human foetal adrenal and hypophysis in tissue culture

Summary The histological and histochemical changes taking place in expiants and outgrowing cells of the human foetal adrenals and hypophyses have been studied on the 5th, 10th, 20th, and 28th day of the culture period. In the outgrowth from adrenals, both the epithelial-type and the fibroblast-type cells contain 3-ol steroid dehydrogenase activity. The cells growing out from hypophyseal expiants appear to be morphologically undifferentiated. Expiants contain differentiated cells in the first 10 to 13 days of the culture period.     

The fine structure of the cartilage in the annelidSabella penicillum

Summary The fine structure of the tentacular cartilage cells of the annelidSabella penicillum has been studied by scanning (SEM) and transmission electron microscopy (TEM). The cells contain a large, transparent vacuole centrally, and a thin layer (>0.1 m) of cytoplasm around. Some granular endoplasmic reticulum, mitochondria and smaller clear vesicles are present in the cytoplasm. The nuclei are dense in the TEM preparations. The surrounding matrix is most dense close to the cells, and form a chondroid matrix, 0.2 to 0.5 m thick, consisting of a flocculate material. The boundary to the surrounding matrix is condensed in the central cartilaginous cells, but not in the pinnular. The rigid structure of the close chondroid matrix is demonstrated in the SEM. The structure is compared to other invertebrate cartilages so far described, and its functions are discussed.     

Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells

Background

Chondrogenesis is the complex process that leads to the establishment of cartilage and bone formation. Due to their ability to differentiate in vitro and mimic development, embryonic stem cells (ESCs) show great potential for investigating developmental processes. In this study, we used chondrogenic differentiation of ESCs as a model to analyze morphogenetic events during chondrogenesis.

Methodology/Principal Findings

ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.

Conclusions/Significance

We demonstrate that ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages. The study of chondrogenesis via ESC differentiation may be informative to our further understanding of skeletal growth and regeneration.     

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.     

Stem Cells: Properties and Prospective Medical Applications

The properties of some stem cells (SCs) that are most interesting in terms of their implications for medicine (embryonic, hematopoietic, and mesenchymal SCs) are considered. SCs are undifferentiated cells capable of both self-maintenance and differentiation into specialized cells. According to their origin, SCs are divided into embryonic and somatic ones. The former can be maintained in vitro for an infinitely long time and can differentiate into all cells of adult organisms. The latter have a limited capacity for differentiation and, probably, a limited proliferative potential. The plasticity of somatic SCs, i.e., their capacity for context-dependent differentiation into unrelated cell types, is of considerable therapeutic importance, although some researchers doubt this capacity. It is assumed that most types of SCs differentiate by the stepwise hierarchical maturation mechanism, one of the steps being rapidly proliferating progenitor cells. The use of SCs in medicine is currently at the stage of preclinical trials. Although embryonic SCs are promising for medicine, there are serious limitations of their use in therapy in the near future. However, the first clinical trials have demonstrated that the approaches involving autotransplantation of hematopoietic and mesenchymal SCs are effective for treating ischemia of extremities and the consequences of myocardial infarction. Obviously, the use of SCs in medicine promises dramatic progress in treating many severe diseases.     

Evidence for Phosphoprotein Microspheres in Bone

Bone sialoprotein and osteopontin are bone-specific phosphoproteins, but their function is uncertain and their ultrastructural associations remain unclear. Insight into their role was sought by special attention to their general distribution and specific morphology under the high-power optical microscope. Their extracellular staining characteristics were examined in cryosections of adult rat skeletal tissues using two immunohistochemical methods. The two proteins were clearly evident in immature woven bone of endochondral and intramembranous origin (although cartilage was negative, even when calcified). In mature lamellar bone, bone sialoprotein remained ubiquitous, while osteopontin was confined to cement lines and other relatively discrete sites of past and present resorption activity, particularly near blood vessels. In neither case was the distribution of the stain structureless and diffuse. Invariably (except when non-specific), it was sharply defined and had the form of microspheres measuring approximately 1 m in diameter. In both immature and mature regions, these objects appeared in sheets, chains or groups in a pattern that was evidently coincident with a similar structural arrangement found within the inorganic phase of bone. It was concluded that phosphoproteins are not randomly located throughout the collagenous matrix but are apparently integral to calcified microsphere populations, and it is suggested that these structures are well placed to control the chemical State of the mineral over their surfaces and influence remodelling.