Arthroplasty membrane-derived fibroblasts directly induce osteoclast formation and osteolysis in aseptic loosening.

PURPOSE: Both macrophages and fibroblasts are the main cell types found in periprosthetic tissues surrounding failed joint arthroplasties. These fibroblasts are known to express RANKL and to produce TNFalpha, factors which promote osteoclast formation and bone resorption. In this study we have analysed the role that arthroplasty membrane-derived fibroblasts (AFb) play in inducing the generation of bone resorbing osteoclasts. METHODS: Fibroblasts were isolated from periprosthetic tissues and co-cultured with human monocytes in an osteoclast differentiation assay in the presence or absence of M-CSF and inhibitors of RANKL (OPG) and/or TNFalpha. RANKL expression by AFbs was determined by RT-PCR and the extent of osteoclast differentiation by the expression of TRAP, VNR and evidence of lacunar resorption. RESULTS: In the presence of M-CSF, large numbers of TRAP(+) and VNR(+) multinucleated cells capable of lacunar resorption, were noted in co-cultures of monocytes and RANKL-expressing AFbs. Cell-cell contact was required for osteoclast formation. The addition of OPG and anti-TNFalpha alone significantly reduced but did not abolish the extent of osteoclast formation, whereas the addition of both together abolished osteoclast formation and lacunar resorption. CONCLUSION: Our results indicate that fibroblasts in periprosthetic tissues are capable of inducing the differentiation of normal human peripheral blood mononuclear cells to mature osteoclasts by a mechanism that involves both RANKL and TNFalpha. Suppression of both RANKL and inflammatory cytokines is likely to be required to control periprosthetic osteolysis.

Transforming growth factor-beta induces osteoclast formation in the absence of RANKL.

Transforming growth factor beta (TGFbeta) is a multifunctional growth factor that is produced by many cells in bone and is abundant in the bone matrix. TGFbeta is known to regulate RANKL-induced osteoclast formation and bone resorbing activity. In this study we sought to determine whether TGFbeta could directly induce osteoclast formation by a RANKL-independent mechanism. We found that the addition of TGFbeta to cultures of human monocytes and RAW 264.7 cells (in the presence of M-CSF and the absence of RANKL, TNFalpha or IL-6/IL-11) was sufficient to induce the formation of TRAP+ and VNR+ cells, which formed actin rings and were capable of extensive lacunar resorption. The addition of osteoprotegerin or antibodies to TNFalpha and its receptors, as well as antibodies to gp130, did not inhibit lacunar resorption, indicating that TGFbeta did not act by stimulating RANKL, TNF or IL-6 production by monocytes. TGFbeta-induced osteoclast formation was qualitatively different from that induced by RANKL with numerous TRAP+/VNR+ mononuclear and small multinucleated cells being formed; these cells produced many small resorption lacunae. Our results indicate that TGFbeta, which is abundant in the bone matrix, can, in the presence of M-CSF, directly induce mononuclear phagocyte osteoclast precursors to differentiate into osteoclastic cells capable of lacunar resorption.

Menatetrenone (vitamin K2) acts directly on circulating human osteoclast precursors.

It is still not certain what the direct effect of menatetrenone is on osteoclast precursors. In the present study, we investigated whether menatetrenone has a direct effect on circulating osteoclast precursors to influence osteoclast differentiation. Monocytes isolated from human peripheral blood were cultured with receptor-activated NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Menatetrenone or vitamin K1 was then added to the cultures. Geranylgeraniol or phytol (the respective side chain) was also added to the cultures instead of menatetrenone or vitamin K1, respectively. After 7 and 14 days incubation, cultures were evaluated for cytochemical and functional evidence of osteoclast formation. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the percentage area of lacunar resorption induced by RANKL and M-CSF were decreased when menatetrenone or geranylgeraniol was added to the cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when the cultures were treated with menatetrenone or geranylgeraniol. In contrast, vitamin K1 or phytol did not affect the number of TRAP-positive MNCs nor the percentage area of lacunar resorption. These results indicate that menatetrenone not only influences osteoclast formation via bone stromal cells but also acts directly on circulating osteoclast precursors to influence osteoclast differentiation. These findings also suggest that geranylgeraniol, the side chain of menatetrenone, plays an important role in this inhibitory effect.

Cellular mechanisms of osteoclast formation and lacunar resorption in giant cell granuloma of the jaw.

BACKGROUND: Giant cell granuloma (GCG) is an osteolytic tumour of the jaw which is characterised by the presence of both mononuclear and multinucleated (osteoclast-like) giant cell components. The nature of these component cells and the pathogenesis of the extensive osteolysis associated with this lesion is uncertain. METHODS: Using cell culture techniques and immunohistochemistry, we defined the phenotypic characteristics of the mononuclear and multinucleated cells present in four cases of GCG of the jaw. We also analysed the cellular and humoral factors associated with osteoclast formation and osteolysis in these tumours and determined whether GCG stromal cells are capable of supporting osteoclast formation. RESULTS: GCG-derived giant cells expressed the phenotypic characteristics of osteoclasts (TRAP+, VNR+, and calcitonin responsive) and were capable of lacunar resorption. In addition to macrophages, the mononuclear cell population contained numerous spindle-shaped stromal cells which proliferated in culture and expressed RANKL; these GCG-stromal cells were capable of supporting human osteoclast formation from circulating monocyte precursors. CONCLUSION: Our findings indicate that the giant cells in GCG of the jaw are osteoclast-like and formed from monocyte/macrophage precursors which differentiate into osteoclasts under the influence of RANKL-expressing mononuclear stromal cells found in this lesion.

Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism.

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are known to influence osteoclast formation and bone resorption. In order to determine whether IL-6 and IL-11 could independently support human osteoclast formation, these factors were added to cultures of human peripheral blood mononuclear cells of the monocyte (CD14(+)) fraction in the presence of macrophage colony-stimulating factor (M-CSF). Under these conditions, IL-6 and IL-11 induced the formation of multinucleated cells which were positive for TRAP, VNR, and calcitonin receptor and capable of lacunar resorption. Osteoclastogenesis induced by IL-6 and IL-11 was inhibited by the addition of an anti-gp130 antibody but not by osteoprotegerin. These results indicate that IL-6 and IL-11, which are thought to play a role in several osteolytic bone disorders, are directly capable of inducing osteoclast formation by a RANKL-independent mechanism.

Two distinct cellular mechanisms of osteoclast formation and bone resorption in periprosthetic osteolysis.

PURPOSE: TNFalpha and IL-1alpha are proinflammatory cytokines that are abundant in periprosthetic tissues. These cytokines stimulate bone resorption and have recently been shown to directly induce osteoclast formation in mouse marrow cultures. We examined whether TNFalpha and IL-1alpha can directly induce osteoclast formation from human arthroplasty-derived (CD14(+)) macrophages by a mechanism independent of RANKL-induced osteoclastogenesis. METHODS: TNFalpha and M-CSF (+/-IL-1alpha) were added to cultures of magnetically sorted (CD14(+)) and unsorted (CD14(+)/CD14(-)) cells isolated from the pseudomembrane of loosened hip arthroplasties. Osteoprotegerin (OPG), RANK:Fc and antibodies to TNF receptors (p55 and p75) were added to these cultures to distinguish the pathway of osteoclastogenesis. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and lacunar resorption. RESULTS: The addition of TNFalpha (+/-IL-1alpha) resulted in differentiation of CD14(+) macrophages into TRAP(+) and VNR(+) multinucleated cells capable of extensive lacunar resorption. Both OPG and RANK:Fc (which inhibit RANKL-induced osteoclastogenesis) did not block osteoclastogenesis. The addition of antibodies directed against the p55 receptor subunit of TNF resulted in significant inhibition of osteoclast formation and lacunar resorption. CONCLUSIONS: Our results indicate that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from arthroplasty macrophages and that TNFalpha acts synergistically with IL-1alpha to stimulate lacunar resorption. This process is distinct from the RANK/RANKL signalling pathway and is likely to operate in periprosthetic tissues when there is heavy wear particle deposition and cytokine production.

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts.

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.

Osteoclast differentiation factor induces synovial macrophage-osteoclast differentiation in rheumatoid arthritis.

Abstract The aim of this study was to clarify the role of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) in synovial macrophage-osteoclast differentiation. Synovial macrophages were cultured in the presence of macrophage-colony-stimulating factor (M-CSF) and/or ODF. OPG was added to cocultures of synovial macrophages and UMR106. The cultures on glass coverslips were stained with osteoclast-associated markers, tartrate-resistant acid phosphatase (TRAP), and vitronectin receptor (VNR), as well as macrophage-associated markers CD11b and CD14. Functional evidence of osteoclast formation was determined by a resorption pit assay. To investigate whether rheumatoid arthritis (RA) synovial cells expressed messenger RNA (mRNA) for ODF, OPG, and the receptor activator of NF-κB (RANK), we performed a polymerase chain reaction (PCR) analysis. The addition of M-CSF or ODF alone induced TRAP-positive multinucleated cell formation. Resorption pits were rarely detected with M-CSF alone. ODF was capable of inducing bone resorption and enhancing osteoclastogenesis, as well as bone resorption in the presence of M-CSF. In the coculture system, both osteoclast formation and bone resorption were inhibited by OPG in a dose-dependent manner. In all experiments, synovial cells, including macrophages and fibroblasts, expressed the mRNA for RANK, ODF, and OPG. Our findings suggest that ODF plays a role in regulating RA synovial macrophage-osteoclast differentiation, and that synovial cells might have the ability to produce ODF. OPG might be further developed as a new strategy for treating bone destruction in RA joints.

Identification of 142 single nucleotide polymorphisms in 41 candidate genes for rheumatoid arthritis in the Japanese population.

Single-nucleotide polymorphisms (SNPs) can make an important contribution to our understanding of genetic backgrounds that may influence medical conditions and ethnic diversity. We undertook a systematic survey of genomic DNA for SNPs located not only in coding sequences but also in non-coding regions (e.g., introns and 5' flanking regions) of selected genes. Using DNA samples from 48 Japanese patients with rheumatoid arthritis (RA) as templates, we surveyed 41 genes that represent candidates for RA, screening a total of 104 kb of DNA (30 kb of coding sequences and 74 kb of non-coding DNA). Within this 104 kb of genomic sequences we identified 163 polymorphisms (1 per 638 bases on average), of which 142 were single-nucleotide substitutions and the remainder, insertions or deletions. Of the coding SNPs, 52% were non-synonymous substitutions, and non-conservative amino acid changes were observed in a quarter of those. Sixty-nine polymorphisms showed high frequencies for minor alleles (more than 15%) and 20 revealed low frequencies (<5%). Our results indicated a greater average distance between SNPs than others have reported, but this disparity may reflect the type of genes surveyed and/or the relative ethnic homogeneity of our test population.

[Two cases of Buschke-Löwenstein tumor].

We report two cases of Buschke-Löwenstein Tumor on 39- and 55-year-old males. Both patients presented with mass and pain of glans penis. Pathological diagnosis of biopsy specimens were condyloma acuminatum in both cases. Bleomycin was not effective. Therefore, tumor resection in the first case and partial penectomy on second case were performed. Pathological examination revealed hyperkeratosis, acanthosis and downward growth of the prickle cell layer. But cellular atypia were not seen. Pathological diagnosis was Buschke-Löwenstein tumor. Our two patients are alive with no evidence of disease after 10 years. Pathological and clinical problems of Buschke-Löwenstein tumor were discussed by review of the literature.