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P Osdoby

Publications and source records attributed to P Osdoby.

At least 37 records · Page 2Linked to original sources
PubMed1991

Isolation of avian osteoclasts: improved techniques to preferentially purify viable cells.

Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg-laying hen maintained on a low-calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable. The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low-calcium diet. A corresponding decrease in bone-associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast-directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (greater than 99%) nonviable, but the third population contains 8-fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25-fold in comparison with the first two populations. The osteoclast-like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody-reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.

Animals↗
PubMed1991

Osteoclast-specific monoclonal antibodies coupled to magnetic beads provide a rapid and efficient method of purifying avian osteoclasts.

Osteoclasts are the major cell type responsible for normal and pathologic bone resorption. Obtaining highly purified populations of these multinucleated cells has been problematic, although such populations would greatly facilitate investigations of osteoclast regulation and activity. A new immunomagnetic protocol has been devised to surmount these difficulties, employing avian osteoclast-directed monoclonal antibodies (designated 121F, 35L, and 75B) surface coupled to uniformly small, magnetic polystyrene beads covalently conjugated with sheep antimouse IgG. Presentation of these antiosteoclast antibody-coated beads to mixed cell preparations derived from marrow-depleted, collagenase- and/or trypsin-treated chick tibiae and wing bones, followed by magnetic separation and washing, results in efficient and selective binding of osteoclasts to the immunomagnetic beads within minutes. The specific nature of this bead-cell interaction is further demonstrated by the progressive decline in antiosteoclast antibody-coated bead binding to osteoclasts by uncoated beads or beads coated with an irrelevant antibody. Under optimal conditions, these isolations typically yield more than a 100-fold enrichment and greater than a 90% purification of osteoclasts from subpopulations of either predominantly nonviable or viable osteoclasts. Although scanning electron microscopy reveals that immunomagnetically purified and cultured osteoclasts internalize large numbers of the antibody-coated beads, such cells appear unimpaired in their ability to attach to tissue culture plastic or devitalized cortical bone slices and to produce resorption pits characteristic for osteoclasts. Additional studies to ascertain the most effective method for removal (desorption) of antibody-coated beads from magnetically isolated osteoclasts demonstrate that moderate physical agitation is at present the most effective protocol to dislodge antibody-coated beads from the cell surface while maintaining osteoclast viability and function. This immunomagnetic technique therefore provides a gentle method for the isolation of highly purified populations of osteoclasts from heterogeneous bone cell populations in a rapid, efficient, and selective manner.

Animals↗
PubMed1991

Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase.

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast-directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane-associated glycoprotein. Western blot analysis on disulfide bond-reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS-polyacrylamide gels. Silver staining of the purified antigen on SDS-polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N-linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O-linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N-terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type-specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast-mediated bone resorption.

Amino Acid Sequence↗
PubMed1991

Evidence for an immunological and functional relationship between superoxide dismutase and a high molecular weight osteoclast plasma membrane glycoprotein.

Large multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less dtoxic H2O2 (Fridovich: J Biol Chem 264:7761-7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross-reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow-derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.

Animals↗
PubMed1991

Osteoclast formation is related to bone matrix age.

Little is known about the relationship between the age of the skeleton and the development of multinucleated bone-resorbing cells, osteoclasts. It has been shown that mineralized bone implanted onto the chick chorioallantoic membrane (CAM) is effective in the recruitment and differentiation of osteoclast precursors. In studies reported here we used the CAM system to examine the influence of bone matrix age on osteoclast formation. Devitalized mineralized bone particles (75-250 microns) were prepared from rats of various ages (2, 4, 9, 12, and 16 months). The particles were implanted onto the chick chorioallantoic membrane and 8 days later implants were harvested and processed for morphometric or immunohistochemical analysis. Osteoclast number, cell area, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen, defined by the 121F monoclonal antibody, were determined. Bone particles of each age group resulted in the formation of osteoclast-like giant cells. Compared with multinucleated cells that formed in response to bone particles obtained from 2-month-old rats, matrix from the oldest age group (16 months) elicited significantly fewer and smaller cells which contained a smaller number of nuclei. These data suggest that with aging, bone undergoes qualitative and/or quantitative changes that affect the recruitment and differentiation of osteoclast precursor cells.

Aging↗
PubMed1990

Correlation of an osteoclast antigen and ruffled border on giant cells formed in response to resorbable substrates.

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the organic and inorganic components of bone matrix. The functional and developmental relationship between osteoclasts and foreign body giant cells is unclear. The osteoclast plasma membrane ruffled border juxtaposed to the bone surface is a unique morphologic characteristic of active osteoclasts. In the studies reported here giant cell formation was induced in response to a variety of materials implanted onto the richly vascularized chick chorioallantoic membrane. Light and electron microscopic techniques were used to examine the morphologic characteristics of the giant cells. In addition, immunohistochemical methods were used to demonstrate the appearance of a 150 kD cell surface antigen on chicken osteoclasts recognized by monoclonal antibody 121F. Giant cells that formed in response to mineralized bone particles exhibited ruffled borders and stained positively with the 121F antibody. Many giant cells that formed in response to hydroxyapatite possessed ruffled borders similar to but not as extensive as those observed on giant cells formed on bone. Immunohistochemical localization of the 121F antigen on these cells suggested that the antigen was present, but staining intensity was reduced compared to that of bone-associated giant cells. The formation of mineral matrix complexes by the adsorption to hydroxyapatite of bone extract or osteocalcin enhanced ruffled borders and the presence of the 121F antigen on elicited giant cells. In contrast, giant cells that formed on non-resorbable materials, such as Sepharose beads, mica, and methacrylate, lacked ruffled borders and were negative for the 121F antigen. It appears that expression of the 121F osteoclast antigen correlates with the appearance and extent of ruffled membranes on giant cells. Furthermore, it appears that giant cell ruffled membrane development and the presence of the 121F osteoclast antigen are related to giant cell formation in response to resorbable materials that are subject to extracellular dissolution. Expression of this antigen may be indicative of the developmental and/or functional state of giant cells (osteoclasts) that form on resorbable substrates. In addition, components of the bone matrix, including osteocalcin, in association with bone mineral, lead to elevated levels of this osteoclast antigen.

Allantois↗
PubMed1990

An in vivo model system for the study of avian osteoclast recruitment and activity.

We have developed a model system for the study of osteoclast recruitment and activity using devitalized bovine cortical bone slices implanted onto the chorioallantoic membrane (CAM) of chicken embryos. Bone slices were examined after 3, 6, and 8 days of incubation on the CAM. A marked cellular reaction to the bone was observed, characterized by a prominent angiogenic response. Upon histological examination, numerous multinucleated giant cells were associated with the undersurface of the bone slice and concentrated towards its periphery. These multinucleated cells were often associated with resorption lacunae and demonstrated ruffled borders when viewed by transmission electron microscopy. Removal of the cells and examination of the bone surface by scanning electron microscopy revealed numerous resorption pits characteristic of osteoclastic activity. These pits were evident on day 3 of incubation and appeared to be more extensive by day 8. This work demonstrates that the cells recruited to such ectopically implanted devitalized bone slices are functional osteoclasts, and that this system may provide a useful model for the study of osteoclast recruitment and activity.

Animals↗
PubMed1990

Stimulation of craniofacial and intramedullary bone formation by negatively charged beads.

To test for their osteogenic stimulating capacity, charged beads were implanted into cranial or mandibular defects, used as an onlay on the nasal bone surface, or injected into femoral medullary cavities of young adult rats. One month later, negatively charged beads were found to have stimulated extensive bone formation resulting in closure of craniofacial defects, a new layer of bone on the nasal bone surface, and a bead-bone lattice within marrow cavities of long bones. Positively charged beads were nonosteogenic, but elicited a pronounced fibroblastic response in the craniofacial skeleton. Positively charged beads were found associated with multinucleated giant cells at all implantation sites. Uncharged beads failed to elicit formation of new bone and were associated with connective tissue that was less cellular and less organized than was seen with positively charged beads. It was concluded that beads that have ben chemically treated to confer either a negative or positive surface charge, when placed in contact with bone, evoke osteogenesis or formation of dense connective tissue, the response depending on the surface charge of the bead. The mechanism(s) by which the charged beads foster the osteogenic or fibroblastic response is not clear. The use, however, of alloplastic materials with charged surfaces in repair and augmentation of bone, and in wound repair, warrants further investigation.

Animals↗
PubMed1988

Osteoclast development: the cell surface and the bone environment.

Bone development and remodelling processes depend on complex interactions between bone cell precursors, mature bone cells, extracellular matrix molecules, growth factors, the immune system and humoral factors. The exact molecular nature of many of the cell-cell and cell-matrix interactions occurring during bone remodelling remains to be resolved. Cell surface molecules are likely to have important roles in both bone cell differentiation and regulatory processes. However, little is known about changes in the osteoclast cell surface during development and there is only limited information on the cell surface composition of the mature cell phenotype. We describe how one osteoclast-specific monoclonal antibody has been used to identify, characterize and purify a 96 kDa/140 kDa osteoclast membrane protein. The antibody has also been used as a phenotypic marker in studies designed to identify soluble and matrix-related bone factors involved in the terminal stages of osteoclast differentiation. In parallel studies using marrow-derived giant cells and the chick chorioallantoic membrane (CAM), immunohistochemical and enzyme-linked immunoassays (ELISA) have been used to investigate the influence of calvaria, calvaria-conditioned medium, bone matrix, and bone matrix components on osteoclast development. Marrow-derived giant cells express osteoclast-specific cell surface antigens when co-cultured with live calvariae or when exposed to calvaria-conditioned medium. In the richly vascularized and mesenchymal cell-containing CAM, intact bone matrix induces the formation of giant cells that express the osteoclast-specific antigens. In contrast, isolated bone matrix components implanted on the CAM recruit only mononuclear cells which are not recognized by the osteoclast-specific antibody.

Animals↗
PubMed1988

Charged beads: generation of bone and giant cells.

Based on reports of electrically induced bone formation and findings that some materials used to promote bone ingrowth are stimulatory in bead form, the osteogenic potential of beads with different surface charges was examined. In this preliminary study, three types of Sephadex beads were injected into chick femora: type I, DEAE beads, positively charged; type II, CM beads, negatively charged; type III, G-25, uncharged. Beads were injected into the femoral midshaft, and after 3 days, 4 days, and 1 week, birds were sacrificed and femora were processed for histology. Type I beads: at 3 days, were surrounded by multinucleated giant cells; by 4 days, patches of bead-associated new bone were present along with giant cells; after 1 week, occasional bead-associated multinucleated cells were seen, but now most beads were surrounded by new intramedullary bone, forming an extensive bead-bone lattice. With bead types II and III, bead-associated new bone was seen at 3 days and 4 days only when beads lodged near the endosteum or in the metaphysis. At 7 days, no bone was seen with either of these two bead types. The response to the type I beads may be likened to a remodeling phenomenon with large numbers of giant cells at 3 days, new bone and giant cells at 4 days, and evidence only of bone formation at 7 days.

Animals↗
PubMed1988

Osteoclast development in marrow cultured in calvaria-conditioned media.

The precise signals responsible for recruitment and differentiation of osteoclasts (OCs) from their mononuclear precursors are poorly understood. Marrow mononuclear cells, a reputed source of OC precursors, fuse in culture, forming multinucleated cells. These cells, although similar to OCs, differ from osteoclasts in cell-surface morphology and are not recognized by an OC-specific monoclonal antibody. We have used the expression of an osteoclast-specific membrane epitope designated by monoclonal antibody 121F to delineate OCs from marrow-derived giant cells (MAGC). In this report we describe a series of experiments designed to better define the role of the bone environment in the osteoclast differentiation process. Periosteum-free calvariae from hatchling chicks or their conditioned media were combined with adherent Day 1 cultured marrow cells. The time course of OC marker expression was monitored by ELISA and the requirement for live bone and PTH was investigated. Freshly isolated marrow, MAGC, and calvariae were devoid of OC expression. Antigen expression developed in cultured MAGC after 4 days of coplating with either live bone or live bone-conditioned media. The presence of PTH in the cocultures or conditioned media from PTH-treated calvariae did not significantly alter the level of expression. These data indicate that live bone is, in part, responsible for the production of osteoclasts from mononuclear precursors.

Animals↗
PubMed1985

Identification of osteoclast-specific monoclonal antibodies.

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.

Animals↗
PubMed1985

Growth behavior and lineage of isolated and cultured cells derived from giant cell granuloma of the mandible.

A central giant cell granuloma of the mandible was fractionated into its mononuclear and multinuclear cellular constituents. The cells were subsequently grown in tissue culture. Sections from the original lesion and the cultured cells were analysed histochemically and immunocytochemically. Acid phosphatase, non-specific esterase, Lysozyme and alpha-1-antitrypsin were employed as markers for cells of histiocytic origin and Factor VIII-related antigen served as an endothelial cell marker. The mononuclear cells were of 2 types; a spindle-shaped cell and a round macrophage-like cell. The giant cells and the macrophage-like cells had a limited life span in culture and survived for up to 2 and 5 weeks respectively. However, the spindle-shaped cells continued to proliferate with a doubling time of 48 h. The giant cells and the macrophage-like cells were identical in their staining characteristics and showed positive staining for all the histiocytic markers tested. In contrast, the spindle-shaped cells were negative for those markers. None of the 3 cell types stained positively for Factor VIII-related antigen. These findings suggest that the giant cells in giant cell granuloma of the jaw are reactive, fully differentiated end-cells that are probably derived from stromal macrophages. The histogenesis of the spindle-shaped cell is not yet known. It is also shown in this study that the histochemical and in vitro growth characteristics of the cells of central giant cell granuloma of the mandible are analogous to those of giant cell tumors of long bones.

Adult↗
PubMed1983

Orchestration of tooth movement.

Recent advances in the art and mechanics of delivering orthodontic forces have achieved greater precision and control of tooth movement. The exact mechanism by which these forces orchestrate tooth movement is not thoroughly understood. An accurate understanding and precise control over the factors responsible for initiating and carrying out the tissue reaction will ultimately optimize the rate of tooth movement. An integrated hypothetical model for the mechanism of tooth movement is discussed here. This model is based on the most recent body of information available to explain how various stimuli affect bone cells. The roles played by piezoelectric responses, prostaglandin production, and biochemical factors are discussed, with particular emphasis on their importance and contribution in terms of maximizing the rate of tooth movement. The intelligent use of this knowledge will permit us to modify the orthodontic appliance and treatment regimen in order to achieve an optimum tissue response.

Alveolar Process↗
PubMed1983

The development of embryonic bone and cartilage in tissue culture.

Embryonic chick long bone develops in a series of temporally controlled, cellular events and involves the integration of at least three distinctly different sets of cells: collar osteoblasts, core osteoblasts, and resorptive or osteoclastic cells. The morphology of the long bones is established by the developing cartilage rudiment or model. All of these events seem to be influenced by positional cues. The cultivation of all of these cells and their presumptive progenitor cells potentially allows a detailed analysis of their individual and collective phenotypic traits. Future studies can include how long bones form, how bone-forming and bone-resorbing cells interact, and how osteogenic cells influence each other throughout each stage of their respective developmental lineages.

Animals↗
PubMed1983

Isolation and characterization of chondrocytes and non-chondrocytes from high-density chick limb bud cell cultures.

This communication describes a replating technique for the separation of the chondrogenic and non-chondrogenic cells from stage-24 chick limb bud mesenchymal cell cultures by means of sequential digestion with collagenase. Four sub-populations of cells were obtained: The first consisted solely of non-chondrocytes and the next three were progressively enriched in chondrocytes. In addition to morphological differences, the four cell populations differed from each other in their rates of incorporation of sulfate into macromolecular material which were roughly proportional to the percentage of chondrocytes. The chondrocytes and non-chondrocytes no longer exhibited a density dependence of phenotype. In addition, the normal multilayered nodular morphology associated with cartilage development was not observed. These isolated cells have been used as starting material for detailed biochemical studies. Together, these studies indicate that the expressional program governing biosynthetic changes in chondrocytes is not controlled by the extracellular matrix.

Animals↗