Foreign body giant cell induction in the CSF-1-deficient osteopetrotic (op/op) mouse

The osteopetrosis (op) mutation in mice is characterized by generalized skeletal sclerosis; reduced numbers of osteoclasts, macrophages, and monocytes; and failure to be cured by bone marrow transplantation. This mutation has been shown to result from an absence of colony-stimulating factor-1 (CSF-1) and reported to be cured by treatment with CSF-1. Macrophage polykaryons are known to be formed by fusion of mononuclear precursors and the presence of subcutaneous implants can elicit the formation of macrophage polykaryons. In order to determine if recruitment of foreign body giant cells is also impaired in osteopetrotic mice, tissue reactions to subcutaneously implanted polyvinyl sponges were studied and compared with normal mice. Our result showed that, in the op mouse, recruitment of macrophages and foreign body giant cells in response to the implants was quantitatively not different from that of normal mice. However, these cells were smaller and did not migrate as deeply into the implant as those seen in normal littermates. In contrast, resident macrophages obtained by peritoneal lavage were significantly reduced in op mice. These data indicate that there is a deficiency in the ability of op mice to mount a foreign body giant cell response to an implanted sponge characterized by a deficiency in the recruitment of precursor cells that are capable of either full development and spreading or migration into the implanted sponge. These data add to the emerging appreciation of the regional differences among macrophage populations in their dependence on CSF-1 for differentiation and survival.     

Modulation of Osteoclastogenesis with Macrophage M1- and M2-Inducing Stimuli

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.     

Biochemical effects of 12-O-tetradecanoylphorbol-13-acetate, retinoic acid, phenobarbital, and 5-azacytidine on a normal rat liver epithelial cell line

Compound 48/80 (poly-p-methoxyphenethylmethylamine), an agent commonly used to trigger degranulation of mast cells, at concentrations of 5-20 micrograms/ml suppresses the proliferation of L1210 and Friend leukemic cells in vitro, inducing the formation of giant cells, which are polykaryons. Both the proportion of polykaryons in cultures and their size (which reflects the number of nuclei per polykaryon) increase during growth in the presence of 48/80 up to 48 hr; thereafter, the cells lose viability. A predominant number of nuclei in these polykaryons contain a 4C, or higher DNA content. The data indicate that compound 48/80 impairs the cleavage (cytokinesis) and perhaps mitotic processes. Mechanisms by which compound 48/80 induces the described effects are unknown but may be related to the polycationic nature of the polymer and its interaction with the cell membrane. Certain attributes of compound 48/80 suggest that this or similar polymers may have value as research tools for the study of regulatory mechanisms involved in cell division.     

Comparison of the pathogenicity of three species of coagulase-negative Staphylococcus in a mouse model with and without a foreign body

Staphylococcus schleiferi, Staphylococcus lugdunensis, and Staphylococcus epidermidis produce a high incidence of abscesses in a mouse model with an implanted foreign body. We investigated the significance of the foreign body in this process. Fourteen strains of S. schleiferi, S. epidermidis, and S. lugdunensis were tested in our model. A preadhered foreign body was implanted into one mouse group, followed by injection of a test strain. Another group received injection without implant. Abscesses were assessed at 7 days; foreign bodies and infected tissues were cultured. The percent of samples that developed abscesses or were culture positive was compared for each strain. Nearly all mice infected with S. schleiferi developed abscesses and were culture positive. The foreign body made no difference in abscess formation for three of four S. schleiferi but increased the incidence of both organism recovery and abscess for three of five S. epidermidis. The foreign body enhanced abscess formation for four of five S. lugdunensis, with all five strains yielding significantly more culture recovery. Although the pathogenicity of nine strains was increased by the foreign body, five strains yielded high abscess and culture recovery rates that were not enhanced by its presence.     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

The characteristics of the course of mitosis in polykaryons formed during cell fusion]

A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.     

Distinct Regions of the Large Extracellular Domain of Tetraspanin CD9 Are Involved in the Control of Human Multinucleated Giant Cell Formation

Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role.     

PGS支架的合成、特性及在生物医学中应用的研究进展

聚癸二酸甘油酯(PGS)是一种生物可降解的高分子聚合弹性体,因其良好的性能,在许多生物医学研究中应用广泛。PGS支架的机械性能与机体软组织相似,依从性好,降解时以表面侵蚀的方式降解,不伴有膨胀或变形,周围组织炎症反应、纤维变性轻,与多种细胞相容性好。基于PGS良好的性能,主要应用于软组织替代和软组织工程,比如心肌、血管、神经、软骨、视网膜、鼓膜,另外也有用于药物转运载体、组织粘附材料的研究。     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.     

Application of poly-L-lactide screws in flat foot surgery: histological and radiological aspects of bio-absorption of degradable devices

The flat foot in childhood is a condition frequently observed in orthopedic practice but it is still debated when and in which patients surgical corrective treatment is appropriate; recently, the application of poly-L-lactic-acid (PLLA) screws was proposed. The present study investigates a group of 33 patients treated with PLLA expansion endorthesis in order to evaluate the deformity correction. Clinical and radiological outcomes in patients were correlated with: a) morphological characterization of screws both before and after being removed from patients, when necessary; b) histological and bio-molecular evaluation of degradation processes of the implants, focusing attention on the correlation between the cellular cohort involved in inflammatory reaction and the bio-absorption degree of PLLA screws. Deformity correction was mostly achieved, with minimal need of screw removal; the results obtained clearly show the occurrence of chronic rather than acute inflammation in removed screw specimens. At the histological level, after biomaterial implantation, the sequence of events occurring in the surrounding tissues ultimately ends in the formation of foreign body giant cells (FBGCs) at the tissue/material interface; but the mechanisms which influence the fate of screw implants, i.e. the resolution of acute inflammation rather than the progression towards chronic inflammation, are of crucial importance for biodegradable materials like "polylactic acid". In fact, the FBGC response ensures a long-term mechanism which eliminates the foreign material from the body, but at the same time the implications of prolonged FBGC responses, which generate negative side effects, could significantly impede the healing progress.     

RANKL induces formation of avian osteoclasts from macrophages but not from macrophage polykaryons

We have shown that chick macrophages express RANK at their surface and human RANKL (hRANKL) triggers the formation of osteoclasts able to degrade dentine. As described for mammalian osteoclasts, hRANKL also stimulates the resorbing activity of chick bone-derived osteoclasts. In other hands, in culture, chick macrophages spontaneously form polykaryons sharing most of the osteoclast markers but unable to resorb bone. Since both bone-resorbing osteoclasts and macrophage polykaryons found in inflammatory tissues are multinucleated cells deriving from the fusion of macrophages, we examined whether macrophage polykaryons could be induced toward bone-resorbing osteoclasts. Long-term exposure of macrophage polykaryons to hRANKL failed to activate any resorbing activity, indicating that although deriving from the same precursors macrophage polykaryons and osteoclasts are independent cell types and polykaryons are not immature osteoclasts.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.     

Cytokine-stimulating activity of lipopolysaccharide of gram-negative bacteria and its role in antitumor immunity

The review deals with the discussion of different mechanisms of the antitumor action of lipopolysaccharides (LPS), including the induction of endogenous cell mediators, the development of inflammatory and immune response and systemic metabolic changes. The complex action of these factors on the levels of cells, tissues, organs and the whole body led, in some cases, to the suppression or hemorrhagic necrosis of the tumor. These cases were regarded as analogous to the development of the septic syndrome when a cascade of reactions leading to the resorption of tumors was initiated and to chronic inflammation resulting, on the contrary, in the formation of pretumor status. The data on the role of cytokines in carcinogenesis are presented and the possible causes of established contradictions are discussed. The prospects of using LPS in the therapy of oncological diseases are discussed.     

Comparative histopathology of chromomycosis and cladosporiosis in the experiment

Conclusions 1. The reaction of the brain tissue and skin in cladosporiosis and chromomycosis (as well as in some other fungal affections) is in many respects similar and is manifested by the formation of microabscesses, containing giant cells, with phagocytized elements of the causative agents. The fungus contained in the cells at the late phase of the experiment retains its viability, which may be the cause of the dissemination of the process when the reactivity of the organism is lowered.2. The course of cladosporiosis of the brain is characterized by the absence of visible changes of the nerve cells even near the growth of the fungus. The observed edematous changes of neurons in the area of the infiltrations are connected with the foci of the purulent liquefaction of the brain tissue.3. The tissue form both in chromomycosis and cladosporiosis is a stage of the fungus development independent of the organ which it affects (skin, brain).4. The predominating involvement of the brain byCladosporium trichoides may be partly due to tis quicker development in the tissue of the skin as compared toHormodendron pedrosoi and to its ability to germinate through the walls of the blood vessesls, resulting in the penetration into their lumen and hematogenic dissemination.5. Under the conditions of the increased content of glucocorticoids in the organism, the macrophagic and giant cell reaction of the tissues is regularly inhibited, the formation of collagens is disturbed, which results in the generalization of the mycotic process.     

Effects of selective cyclooxygenase-2 inhibitor robenacoxib on primary cells derived from feline injection-site sarcoma

Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasms that arise from injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, there is a consensus that FISS is associated with chronic inflammation caused by irritation of injection-related trauma and foreign chemical substances. Chronic inflammation can provide a proper microenvironment for tumour development, which has been known as one of the risk factors of tumorigenesis in many tumours. To investigate the tumorigenesis of FISS and screen for its potential therapeutic targets, cyclooxygenase-2 (COX-2), an inflammation-enhancing enzyme, was selected as a target for this study. In vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. The results demonstrated that expression of COX-2 could be detected in formalin-fixed and paraffin-embedded FISS tissues and FISS-derived primary cells. Cell viability, migration and colony formation of FISS-derived primary cells were inhibited, and cell apoptosis was enhanced by robenacoxib in a dose-dependent manner. However, susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with COX-2 expression. Our results suggest that COX-2 inhibitors could be potential adjuvant therapeutics against FISSs.     

Alterations in the adhesion behavior of osteoblasts by titanium particle loading: inhibition of cell function and gene expression

Total joint replacement prostheses are required to withstand corrosive environments and sustain millions of loading and articulation cycles during their term of implantation. Wear debris generation has been implicated as one of the primary causes of periprosthetic osteolysis and subsequent implant loosening in total joint replacements. Particulate debris consisting of metals, polyethylene, ceramics, and bone cement have each been shown to provoke a biological response in joint tissues. The major cell types within the interfacial granulomatous fibrous tissues consist of fibroblasts, macrophages, lymphocytes, and foreign-body giant cells. Osteoblasts are one of the principal cell types in the bone tissue adjacent to prostheses, maintaining physiologic bone remodeling through the balanced coordination of bone formation and resorption in concert with osteoclasts. To date the phenomenon of osteoblast phagocytosis of titanium particles has been suggested, but has not been sufficiently studied or confirmed. This study seeks to clarify the influence of titanium particles on osteoblast adhesion, deformability, proliferation, and gene expression profile. These studies were accomplished by performing biorheological testing, Northern blot analysis and RNase protection assay. The uptake of metallic particles by the osteoblast resulted in a particle-filament complex formation, which induced a series of variations in cell function. Understanding these variations is critical to expanding our knowledge of implant loosening and elucidating the nature of prosthetic joint failure. This study suggests that the impact of titanium particles on osteoblast function and subsequent implant loosening may have been previously underestimated.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Ultrastructural evidence in vitro of osteoclast-induced degradation of calcium phosphate ceramic by simultaneous resorption and phagocytosis mechanisms

Osteoclasts are physiological polykaryons specialized in the resorption of calcified tissue. In the context of the clinical use of calcium-phosphate (CaP) ceramics as bone substitutes, this study used transmission electron microscopy to investigate the in vitro mechanisms of CaP ceramic degradation by osteoclastic cell types. Osteoclasts cultured on CaP ceramic developed typical ultrastructural features of bone osteoclasts, such as a polarized dome shape, a clear zone and a ruffled border. Modification of the shape and density of CaP crystals under the ruffled border indicated an acidic microenvironment. Moreover, osteoclasts were able to degrade ceramic by simultaneous resorption and phagocytosis mechanisms. Phagocytosis did not alter the ability of osteoclasts to resorb CaP ceramic. The phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. The common mechanism of phagocytosis described here is similar to that observed with the monocyte/macrophage lineage, confirming that osteoclasts are part of the mononuclear phagocyte system. Osteoclasts are thus clearly involved in CaP degradation by means of resorption and phagocytosis.