Bone marrow mesenchymal stem cells provide an alternate pathway of osteoclast activation and bone destruction by cancer cells.

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells.

Sandblasted and acid-etched dental implants: a histologic study in rats.

Purpose: Current literature has revealed that surface etching of endosseous implants can improve bone-implant contact. The aim of this study was to evaluate the differences in bone-implant contact (BIC) between sandblasted/acid-etched and machined-surface implants.

Materials And Methods: Thirty-two Sprague-Dawley rats were used in this study.

Differential effects of interleukin-6 receptor activation on intracellular signaling and bone resorption by isolated rat osteoclasts.

The effects of the related cytokines interleukin-6 (IL-6), leukemia inhibitory factor (LIF) and oncostatin-M on bone resorption and cytosolic Ca(2+) signaling were compared in isolated rat osteoclasts. In the traditional disaggregated osteoclast (pit) assay, IL-6 and LIF, but not oncostatin-M, conserved the bone resorption otherwise inhibited by high extracellular [Ca(2+)] (15 mM). It produced a paradoxical, concentration-dependent stimulation of resorption by elevated extracellular Ca(2+).

Role of the osteoclast at the bone-implant interface.

A thorough understanding of the processes of healing, repair, and remodeling of bone is critical for the establishment and maintenance of osseointegration of dental implants. In this regard, much attention has been paid to the anabolic aspects of bone remodeling, including the cell biology of the osteoblast and the various cytokines and growth factors which regulate these processes. In contrast, there is little information on the bone-resorptive activity that occurs around implants during osseointegration, and of the role of osteoclasts, macrophages, and stromal cells in those catabolic processes associated with bone remodeling.

Expression of functional RANK on mature rat and human osteoclasts.

Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT-PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any.

Mode of action of interleukin-6 on mature osteoclasts. Novel interactions with extracellular Ca2+ sensing in the regulation of osteoclastic bone resorption.

We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti- IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry.

Contortrostatin, a homodimeric snake venom disintegrin, is a potent inhibitor of osteoclast attachment.

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-Asp (RGD) sequence near their carboxyl terminus. These polypeptides inhibit binding of adhesion molecules to their receptors (integrins) on the surface of cells. Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in interactions with the extracellular matrix (ECM), including attachment to bone and bone resorption.

Plasminogen activator system in osteoclasts.

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs. Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe.

Osteoclast molecular phenotyping by random cDNA sequencing.

The osteoclast is a cell type that is highly specialized for its bone resorption function. In order to decipher the numerous biochemical functions of osteoclasts, a description of the gene expression profile of osteoclasts would be beneficial. We have sought to identify genes that are highly expressed in osteoclasts by partially sequencing 194 randomly chosen cDNA clones from a representative rabbit osteoclast cDNA library.

Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping.

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts.

The effects of 1,25-dihydroxyvitamin D3 on osteoclast formation in fetal mouse metatarsal organ cultures.

We have previously described a model system, using 15-day fetal mouse metatarsals cultured in serumless medium, in which osteoclasts and their precursors develop from in situ progenitors in a manner which is similar, both temporally and spatially, to that which occurs in vivo. In this report we evaluate the role of the osteotropic hormone 1,25-dihydroxyvitamin D3 (1,25-D3) on osteoclast formation in this model system by characterizing its effects on proliferation, differentiation, and fusion of cells of the osteoclast lineage. Morphologic evaluation was used to enumerate osteoclast precursors, mono- and multinucleate osteoclasts, and osteoclast nuclei in serial paraffin sections.

Mammalian osteoclasts express a transient potassium channel with properties of Kv1.3.

Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel.

Calcitonin receptor expression and its regulation by 1 alpha-25-dihydroxyvitamin D3 during de novo osteoclast formation in organ cultures of fetal mouse metatarsals.

Organ cultures of 15-day embryonic mouse metatarsals cultured in serumless, chemically-defined medium were used to investigate the influence of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on calcitonin receptor (CTR) expression in osteoclasts formed from in situ progenitors. CTR expression was demonstrated in tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts and their precursors. The expression of TRAP preceded that of CTR and was first observed in cells identified as osteoclast precursors.

Skeletal development and formation of osteoclast-like cells from in situ progenitors in fetal mouse metatarsals cultured in chemically defined medium.

An in vitro model system is described, using metatarsal explants from 15-day mouse embryos (E15) cultured in serumless chemically defined medium, to study fetal skeletal development with particular emphasis on de novo osteoclast formation. The normal pattern of growth and differentiation observed in vitro, assessed by ultrastructure and morphometry, demonstrate a permissive local environment which replicates physiologic temporal and spatial relationships which exist in vivo. The population of committed osteoclast progenitors present in E15 metatarsals form osteoclasts and precursors which have cytochemical and ultrastructural features, as well as kinetics of formation, that are similar to that which occurs in vivo.

Bone remodeling in W/Wv mast cell deficient mice.

Strong experimental evidence exists for a relationship between mast cells and bone disease, but the role of mast cells in the regulation of bone remodeling is unknown. In order to address this question, mast cell deficient mice (W/Wv) were paired with their mast cell sufficient (+/+) littermates and evaluated for differences in response to an induced cycle of bone remodeling. This was achieved using a tooth egression protocol, in which a synchronous cycle of bone remodeling was induced in the mandibular buccal alveolar periosteum by extraction of the opposing dentition.

Mouse major histocompatibility complex (H-2) and fetal lung development: implications for human pulmonary maturation.

Using C57/10Sn (B10, H-2b) and B10.A/SgSn (B10.A,H-2a) congenic mice, we measured 1) the level of endogenous pulmonary corticosterone during mouse development; 2) the degree of lung morphological maturation on gestation day 17, with or without corticosteroid treatment; and 3) the maternal influence on normal lung development and fetal response to corticosteroids.

In vitro bone resorption activity produced by a hypercalcemic adenocarcinoma tumor line (CAC-8) in nude mice.

Tumor extracts and conditioned tissue culture media from a canine adenocarcinoma tumor line (CAC-8) propagated in nude mice significantly increased in vitro bone resorption in neonatal mouse calvaria as measured by release of previously incorporated 45Ca. In vitro bone resorption activity was induced in a dose-dependent manner, was not suppressible by indomethacin, and was heat- and acid-stable. Gel exclusion chromatography demonstrated peak bone resorbing activity at a relative molecular mass of approximately 28,000.

Inhibition of bone resorption in vitro by compound 48/80.

The mechanisms that control cycles of bone formation and bone resorption are not well understood. In this report we provide evidence that compound 48/80 is a potent inhibitor of bone resorption in vitro. Resorption was assessed by the release of calcium-45 from pre-labelled newborn mouse calvaria that were treated with compound 48/80 and/or parathyroid hormone (PTH) in organ culture.

Multiwell chamber chemotaxis assays: improved experimental design and data analysis.

The chemotaxis assay using the Boyden transfilter technique has become widely used in recent years for assessing migratory responses of a wide variety of cell types. In the study reported here we examined the migratory responses of mouse peritoneal macrophages using a multiwell chamber. The experiments were designed to analyze the components of variance in the assay method, to optimize the experimental design, and to develop objective statistical criteria for choosing among experiments with disparate results.

Bone-derived macrophage chemotactic factors: methods of extraction and further characterization.

Mononuclear phagocytes have been implicated as important cellular elements in the process of bone resorption. We have postulated that the recruitment and migration of mononuclear phagocytes to bone occurs via a mechanism(s) in which bone-derived chemotactic factors (BDCF) are released from foci undergoing resorption. In the experiments presented here we have used newborn mouse calvaria and examined a variety of extraction protocols, both dissociative and non-dissociative, as means of obtaining stable and reproducible chemotactic activity for mouse peritoneal macrophages.

Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function.

Organ cultures of newborn mouse calvaria were used to test the hypothesis that tartrate-resistant acid phosphatase might serve as a biochemical marker for osteoclast function. When bone resorption was stimulated in vitro with either parathyroid hormone or 1,25(OH)2D3, there was a significant increase in both tartrate-resistant and tartrate-sensitivity acid phosphatase activity in the medium relative to cultured controls. Tartrate-resistant activity was localized histochemically primarily over the osteoclast and appeared as three distinct activity bands when electrophoresed on polyacrylamide gels.

Immune function in congenital osteopetrosis: defective lymphocyte function in microphthalmic mice.

Osteopetrosis is a prominent feature of a congenital mutation described in microphthalmic mice and is thought to be due to defective osteoclast function which causes a generalized lack of bone resorption. Reversal of defective bone resorption in osteopetrotic mutants has been achieved by hematopoietic cell transplantations; and conversely, defective bone resorption has been transferred to normals by hematopoietic cells from osteopetrotic littermates. This suggested that osteopetrotic mutants might also demonstrate defective immune functions which could in turn be related to the lack of normal bone resorption.

Bone resorption and humoral hypercalcemia of malignancy: stimulation of bone resorption in vitro by tumor extracts is inhibited by prostaglandin synthesis inhibitors.

Extracts of tumors from patients with humoral hypercalcemia of malignancy (HHM) were tested using an in vitro bone resorption assay in order to investigate the pathogenesis of the hypercalcemia. Bone resorption was assessed by comparing the percent release of previously incorporated 45Ca from paired halves of newborn mouse calvaria. Saline extracts of three out of five tumors from HHM patients caused a significant increase in 45Ca release relative to controls.