Osteoclast Differentiation Of Monocytes Research Articles

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significanceDelivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

Regulation of osteoclastogenesis by mast cell in rheumatoid arthritis

BackgroundIn the pathogenesis of rheumatoid arthritis (RA), the role of mast cells has not been revealed clearly. We aimed to define the inflammatory and tissue-destructive roles of mast cells in rheumatoid arthritis (RA).MethodsSerum and synovial fluid (SF) concentration levels of tryptase, chymase, and histamine were quantified using ELISA. After activating mast cells using IL-33, the production of TNF-α, IL-1β, IL-6, IL-17, RANKL, and MMPs was determined using real-time PCR and ELISA. Osteoclastogenesis was assessed in CD14+ monocytes from peripheral blood and SF, which were cultured with IL-33-activated mast cells, by counting TRAP-positive multinucleated cells.ResultsThe concentration levels of serum tryptase, chymase, and histamine and SF histamine were higher in patients with RA than in controls. FcεR1 and c-kit-positive mast cells were higher in RA synovium than in osteoarthritic (OA) synovium. Stimulation of mast cells by IL-33 increased the number of trypatse+chymase− and tryptase+chymase+ mast cells. IL-33 stimulation also increased the gene expression levels of TNF-α, IL-1β, IL-6, IL-17, RANKL, and MMP-9 in mast cells. Furthermore, IL-33 stimulated human CD14+ monocytes to differentiate into TRAP+ multinucleated osteoclasts. When CD14+ monocytes were co-cultured with mast cells, osteoclast differentiation was increased. Additionally, IL-33-activated mast cells stimulated osteoclast differentiation. The inhibition of intercellular contact between mast cells and monocytes using inserts reduced osteoclast differentiation.ConclusionsIL-33 increased inflammatory and tissue-destructive cytokines by activation of mast cells. Mast cells stimulated osteoclast differentiation in monocytes. Mast cells could stimulate osteoclastogenesis indirectly through production of tissue-destructive cytokines and directly through stimulation of osteoclast precursors.

Delivery of miR-146a to Ly6Chigh Monocytes Inhibits Pathogenic Bone Erosion in Inflammatory Arthritis.

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored.Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice.Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions.Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.

Effects of Dihydrophaseic Acid 3'-O-β-d-Glucopyranoside Isolated from Lycii radicis Cortex on Osteoblast Differentiation.

Our previous study showed that ethanol extract of Lycii radicis cortex (LRC) prevented the loss of bone mineral density in ovariectomized mice by promoting the differentiation of osteoblast linage cells. Here, we performed fractionation and isolation of the bioactive compound(s) responsible for the bone formation–enhancing effect of LRC extract. A known sesquiterpene glucoside, (1′R,3′S,5′R,8′S,2Z,4E)-dihydrophaseic acid 3′-O-β-d-glucopyranoside (abbreviated as DPA3G), was isolated from LRC extract and identified as a candidate constituent. We investigated the effects of DPA3G on osteoblast and osteoclast differentiation, which play fundamental roles in bone formation and bone resorption, respectively, during bone remodeling. The DPA3G fraction treatment in mesenchymal stem cell line C3H10T1/2 and preosteoblast cell line MC3T3-E1 significantly enhanced cell proliferation and alkaline phosphatase activity in both cell lines compared to the untreated control cells. Furthermore, DPA3G significantly increased mineralized nodule formation and the mRNA expression of osteoblastogenesis markers, Alpl, Runx2, and Bglap, in MC3T3-E1 cells. The DPA3G treatment, however, did not influence osteoclast differentiation in primary-cultured monocytes of mouse bone marrow. Because osteoblastic and osteoclastic precursor cells coexist in vivo, we tested the DPA3G effects under the co-culture condition of MC3T3-E1 cells and monocytes. Remarkably, DPA3G enhanced not only osteoblast differentiation of MC3T3-El cells but also osteoclast differentiation of monocytes, indicating that DPA3G plays a role in the maintenance of the normal bone remodeling balance. Our results suggest that DPA3G may be a good candidate for the treatment of osteoporosis.

CD14− mononuclear stromal cells support (CD14+) monocyte–osteoclast differentiation in aneurysmal bone cyst

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14− mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/− macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14−, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14−, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14− cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14− mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.

Hypoxia‐inducible factor is expressed in giant cell tumour of bone and mediates paracrine effects of hypoxia on monocyte–osteoclast differentiation via induction of VEGF

Hypoxia is an important regulator of bone biology and stimulates osteoclast differentiation from monocytic precursors. Hypoxia-inducible factor (HIF) is a key pro-tumourigenic transcription factor mediating pathways of hypoxia-inducible gene expression. We have described expression of HIF-1alpha and HIF-2alpha in the multi-nucleated, osteoclast-like giant cells and the mononuclear stromal component of giant cell tumour of bone (GCTB), a locally osteolytic primary bone tumour. HIF induction was observed in culture in the osteoblastic MG-63 cell line, primary GCTB stromal cells, and monocyte-derived osteoclasts following stimulation with hypoxia (0.1% O2) or the osteoclastogenic cytokines hepatocyte growth factor (HGF) and macrophage colony-stimulating factor (M-CSF). This was accompanied by increased expression of the downstream target genes Bcl-2/adenovirus E1B 19 kD-interacting protein 3 (BNIP3), Glut-1, and vascular endothelial growth factor (VEGF). As VEGF can substitute for M-CSF to support osteoclastogenesis in the presence of receptor activator for nuclear factor kappaB ligand (RANKL), we assessed the effect of MG-63 hypoxic conditioned media on osteoclast differentiation. In the presence of RANKL, hypoxic conditioned media induced the formation of active osteoclasts, as assessed from the numbers of TRAP-positive multi-nucleated cells and the area of lacunar bone resorption, which was inhibited by co-incubation with a neutralizing anti-VEGF antibody. Targeted siRNA ablated HIF-1alpha and/or HIF-2alpha expression in MG-63 cells and reduced hypoxic secretion of VEGF. Hypoxic conditioned media from cells treated with siRNA for (HIF-1alpha + HIF-2alpha) produced a significant decrease in osteoclast number (p < 0.005) and activity (p < 0.05) in comparison with the scrambled siRNA control. These results suggest that local hypoxia could indirectly influence osteoclastogenesis via autocrine and paracrine secretion of VEGF under the control of HIF. This is potentially an important mechanism of pathogenesis for GCTB and other osteolytic lesions.

Hepatocyte growth factor can substitute for M-CSF to support osteoclastogenesis

Osteopetrotic mice lacking functional macrophage-colony stimulating factor (M-CSF) recover with ageing, suggesting that alternative osteoclastogenesis pathways exist. Hepatocyte growth factor (HGF) and M-CSF signal through tyrosine kinase receptors and phosphorylate common transducers and effectors such as Src, Grb2, and PI3-Kinase. HGF is known to play a role in osteoclast formation, and in this study we have determined whether HGF could replace M-CSF to support human osteoclastogenesis. We found that the HGF receptor, c-Met, is expressed by the CD14 + monocyte fraction of human peripheral blood mononuclear cells (PBMC). HGF was able to support monocyte-osteoclast differentiation in the presence of receptor activator for nuclear factor κB ligand as evidenced by the formation of numerous multinucleated tartrate-resistant acid phosphatase and vitronectin receptor positive cells which formed F-actin rings and were capable of lacunar resorption. The addition of a neutralising antibody to M-CSF did not inhibit osteoclast differentiation. HGF is a well-established survival factor and viability assays and live/dead staining showed that it promoted the survival and proliferation of monocytes and osteoclasts in a manner similar to M-CSF. Our findings indicate that HGF can substitute for M-CSF to support human osteoclast formation.

Tacrolimus and cyclosporine A inhibit human osteoclast formation via targeting the calcineurin-dependent NFAT pathway and an activation pathway for c-Jun or MITF in rheumatoid arthritis

In the present study, we aimed to determine whether tacrolimus (FK506) and cyclosporine A act directly on human osteoclast precursors obtained from patients with rheumatoid arthritis (RA) and influence monocyte-osteoclast differentiation induced by receptor activator of NF-kappaB ligand (RANKL) in vitro, the stage at which differentiation was affected and the manner in which tacrolimus or cyclosporine A affected the osteoclast signaling pathway. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF). Tacrolimus or cyclosporine A was added to these cultures to determine the effect on the osteoclast differentiation. Osteoclast formation was determined by assessing the number of tartrate resistant acid phosphatase (TRAP) staining cells and measuring the extent of lacunar resorption. The expression of osteoclast transcription factors, such as TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells c1 (NFATc1), c-Fos, c-Jun, microphthalmia transcription factor (MITF) and PU.1 in mononuclear cells (MNCs) was assayed by quantitative reverse transcription-polymerase chain reaction. Addition of tacrolimus or cyclosporine A resulted in a decrease in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and a decrease in the extent of lacunar resorption pit formation as compared to the control cultures; thus, human monocyte-osteoclast differentiation was more effectively inhibited at the late stage and addition of tacrolimus or cyclosporine A resulted in a decrease in the mRNA expression of NFATc1, c-Jun, and MITF at the late stage. Our results suggest that tacrolimus or cyclosporine A acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte-osteoclast formation via targeting both the calcineurin-dependent NFAT pathway and activation pathway for c-Jun or MITF.

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor κB ligand in vitro

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor kappaB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor alpha (TNFalpha), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP(+) MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFalpha antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP(+) MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor κB ligand in vitro

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor α (TNFα), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFα antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP+ MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

Human osteoblasts support human monocyte-osteoclast differentiation: evidence of prostaglandin stimulation

Human osteoblasts support human monocyte-osteoclast differentiation: evidence of prostaglandin stimulation

The Effect of Interleukin-1β, Tumour Necrosis Factor a and Particle Phagocytosis on Monocyte-Osteoclast Differentiation

Interleukin-1 (IL-1) and tumour necrosis factor (TNF) are inflammatory cytokines which are known to influence osteoclastic bone resorption. To determine the effect of these cytokines on inflammatory macrophage-osteoclast differentiation, we added IL-1ß and TNFα to long term co-cultures of mouse monocytes and osteoblastic cells. IL-1ß (10 &amp; 100 pg/ml) stimulated osteoclast formation as measured by expression of TRAP and the extent of lacunar bone resorption whereas TNFα (10 &amp; 100 ng/ml) inhibited macrophage-osteoclast differentiation and bone resorption. Particle phagocytosis (which is known to stimulate the release of these cytokines) did not influence the extent of osteoclast formation. IL-1ß stimulation of osteoclast formation may in part account for the increase in bone resorption associated with this inflammatory cytokine; TNFα inhibition of osteoclast formation may be due to interruption of the interaction between osteoblasts and osteoclast precursors.