Yosuke Fujikawa

Proinflammatory cytokine (TNFalpha/IL-1alpha) induction of human osteoclast formation.

TNFα and IL‐1α are potent stimulators of bone resorption in vivo and in vitro. Recently, it has been demonstrated that these two cytokines directly induce osteoclastogenesis in mouse marrow cultures. This study determined whether TNFα (± IL‐1α) is also capable of inducing human osteoclastogenesis. The CD14+ monocyte fraction of human peripheral mononuclear cells was cultured with TNFα ± IL‐1α in the presence of M‐CSF. TNFα induced the formation of multinucleated cells (MNCs) which were positive for TRAP, VNR and cathepsin K and showed evidence of resorption pit formation. IL‐1α stimulated TNFα‐induced lacunar resorption two‐ to four‐fold. Osteoprotegerin, the decoy receptor for RANKL, did not inhibit this process. Anti‐human IL‐1α neutralizing antibodies significantly inhibited resorption without inhibiting the formation of TRAP+/VNR+ MNCs. These results suggest that, in the presence of M‐CSF, TNFα is sufficient for inducing human osteoclast differentiation from circulating precursors by a process which is distinct from the RANK/RANKL signalling pathway. Copyright

Human osteoclast formation and bone resorption by monocytes and synovial macrophages in rheumatoid arthritis.

OBJECTIVE: To determine whether synovial macrophages and monocytes isolated from patients with rheumatoid arthritis patients are capable of differentiating into osteoclastic bone resorbing cells; and the cellular and humoral conditions required for this to occur. METHODS: Macrophages isolated from the synovium and monocytes from the peripheral blood of rheumatoid arthritis patients were cultured on bone slices and coverslips, in the presence and absence of UMR 106 rat osteoblast-like cells, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and macrophage colony stimulating factor (M-CSF), and assessed for cytochemical and functional evidence of osteoclast differentiation. RESULTS: Isolated calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), and vitronectin receptor (VNR) negative, CD11b and CD14 positive monocytes and macrophages differentiated into CTR, TRAP, and VNR positive multinucleated cells capable of extensive lacunar bone resorption when co-cultured for 14 d with UMR 106 cells in the presence 1,25(OH)2D3 and M-CSF. CONCLUSIONS: Mononuclear phagocytes (monocytes and macrophages) from rheumatoid arthritis patients are capable of differentiating into multinucleated cells showing all the cytochemical and functional criteria of mature osteoclasts. Synovial macrophage-osteoclast differentiation may represent an important cellular mechanism in the bone destruction associated with rheumatoid arthritis.

Human arthroplasty derived macrophages differentiate into osteoclastic bone resorbing cells.

OBJECTIVE In aseptic loosening, a heavy macrophage response to biomaterial wear particles is commonly found in arthroplasty tissues. The aim of this study was to discover if these cells contribute to the bone resorption of aseptic loosening by differentiating into osteoclasts. METHODS Macrophages were isolated from the pseudocapsule and pseudomembrane of loose cemented and uncemented hip arthroplasties at the time of revision surgery and then co-cultured on glass coverslips and dentine slices with UMR 106 rat osteoblast-like cells, both in the presence and absence of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. Macrophages isolated from the synovial membrane of patients with osteoarthritis (OA) undergoing hip replacements were similarly studied as a control group. RESULTS After 24 hours incubation, most cells isolated from the above periprosthetic tissues strongly expressed macrophage (CD11b, CD14) but not osteoclast markers. However, after 14 days incubation, numerous multinucleated cells showing the phenotypic features of osteoclasts (that is, positive for tartrate resistant acid phosphatase, the vitronectin receptor, and capable of extensive lacunar resorption) formed in co-cultures of arthroplasty derived macrophages and UMR 106 cells, in the presence of 1,25(OH)2D3. The addition of an antibody to macrophage colony stimulating factor (M-CSF) considerably reduced macrophage-osteoclast differentiation and hence the lacunar resorption seen in these co-cultures. In contrast, OA synovial macrophage/UMR 106 co-cultures showed little or no evidence of macrophage-osteoclast differentiation and this was only seen when human M-CSF was added to the co-cultures. CONCLUSION This is the first report showing that human macrophages isolated directly from periprosthetic tissues surrounding loosened implants can differentiate into multinucleated cells showing all the functional and cytochemical characteristics of osteoclasts. In contrast with other macrophage populations, exogenous M-CSF is not required for this to occur. In the context of the heavy macrophage response to wear particles in periprosthetic tissues macrophage-osteoclast differentiation may represent an important cellular mechanism whereby osteolysis is effected in aseptic loosening.

Human Osteoclast Formation from Blood Monocytes, Peritoneal Macrophages, and Bone Marrow Cells

Abstract. Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.

RADIO-OPAQUE AGENTS IN BONE CEMENT INCREASE BONE RESORPTION

A heavy infiltrate of foreign-body macrophages is commonly seen in the fibrous membrane which surrounds an aseptically loose cemented implant. This is in response to particles of polymethylmethacrylate (PMMA) bone cement and other biomaterials. We have previously shown that monocytes and macrophages responding to particles of bone cement are capable of differentiating into osteoclastic cells which resorb bone. To determine whether the radio-opaque additives barium sulphate (BaSO4) and zirconium dioxide (ZrO2) influence this process, particles of PMMA with and without these agents were added to mouse monocytes and cocultured with osteoblast-like cells on bone slices. Osteoclast differentiation, as shown by the presence of the osteoclast-associated enzyme tartrate-resistant acid phosphatase (TRAP) and lacunar bone resorption, was observed in all cocultures. The addition of PMMA alone to these cocultures caused no increase in TRAP expression or bone resorption relative to control cocultures. Adding PMMA particles containing BaSO4 or ZrO2, however, caused an increase in TRAP expression and a highly significant increase in bone resorption. Particles containing BaSO4 were associated with 50% more bone resorption than those containing ZrO2. Our results suggest that radio-opaque agents in bone cement may contribute to the bone resorption of aseptic loosening by enhancing macrophage-osteoclast differentiation, and that PMMA containing BaSO4 is likely to be associated with more osteolysis than that containing ZrO2.

Bisphosphonates in bone cement inhibit PMMA particle induced bone resorption

OBJECTIVE Wear particle induced bone resorption is thought to be one of the mechanisms that contribute to implant loosening. It has previously been shown that macrophages, in response to polymethylmethacrylate (PMMA) particles, differentiate into bone resorbing osteoclasts, and that this process is inhibited by a bisphosphonate, etidronate (EHDP). The aim of this study was to determine whether incorporating EHDP in bone cement could reduce PMMA associated bone resorption. METHODS Two concentrations of EHDP were mixed with PMMA monomer before polymerisation. Particles of PMMA (1–10 μm) were generated then added to mouse monocytes cocultured with UMR106 rat osteoblast-like cells and the extent of osteoclast differentiation was determined by assessing the extent of tartrate resistant acid phosphatase (TRAP) staining and measuring the amount of lacunar bone resorption. RESULTS The addition of PMMA to monocyte-UMR106 cocultures resulted in a marked increase in the number of TRAP positive osteoclast-like cells and a significant increase in the number of lacunar resorption pits compared with control cultures to which no particles had been added. After the addition of particles of PMMA + 20 mg EHDP, significantly fewer lacunar pits (p=0.00006) and fewer TRAP positive cells were noted compared with cocultures containing PMMA particles alone. CONCLUSIONS These results indicate that by mixing a bisphosphonate with bone cement, it is possible to inhibit PMMA particle induced bone resorption. This bisphosphonate inhibition of PMMA biomaterial wear particle containing macrophage-osteoclast differentiation and bone resorption may provide a possible therapeutic strategy to prevent or to control the osteolysis of aseptic loosening.

Increased osteoclastic differentiation by PMMA particle-associated macrophages. Inhibitory effect by interleukin 4 and leukemia inhibitory factor.

To determine the influence of polymethylmethacrylate (PMMA) wear particles on macrophage-osteoclast differentiation, PMMA particles were added to mouse monocytes which were cocultured with UMR 106 osteoblast-like cells in the presence of 1,25 dihydroxy vitamin D3[1,25(OH)2D3] for up to 7 days on glass coverslips and for up to 14 days on human cortical bone slices. An increase in osteoclast differentiation, as evidenced by the expression of the osteoclast-associated enzyme tartrate-resistant acid phosphatase (TRAP) and the extent of lacunar bone resorption, was observed in monocyte cultures to which PMMA had been added. Interleukin 4 (IL-4) and Leukemia Inhibitory Factor (LIF) added to these cocultures caused considerably less expression of TRAP and significant inhibition of lacunar bone resorption. This inhibitory effect was reversed by the addition of specific neutralizing antibodies to LIF and IL-4. These findings show that PMMA-wear particle-associated macrophages exhibit an enhanced capacity for differentiation to osteoclastic bone-resorbing cells.

Interleukin-1 receptor antagonist production in cultured synovial cells from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE--To measure the amounts of interleukin-1 receptor antagonist (IL-1ra) protein produced by cultured synovial cells obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS--Synovial cells obtained from patients with either RA or OA were cultured and the supernatants were measured for IL-1ra by enzyme linked immunosorbent assay. RESULTS--The synovial cells obtained from patients with RA produced significantly smaller amounts of IL-1ra than did those obtained from patients with OA, in a late passage (third to fifth) without stimulation and a first passage both with and without stimulation (p < 0.025, respectively). In addition, when the patients with RA were divided into two groups according to the maximum number of lining cell layers, the amounts of IL-1ra produced by the proliferative type were smaller than those produced by the less proliferative type (p < 0.025). CONCLUSIONS--The above findings suggest that IL-1ra production in RA synovial cells is suppressed, and that reduced IL-1ra protein production is one of the causes which leads to the proliferation of lining cells and persistent joint inflammation.

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-κB 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.

Role of multinuclear cells in granulation tissue in osteomyelitis Immunohistochemistry in 66 patients

We investigated the origin of multinuclear cells (MNCs) in the granulation tissue in osteomyelitis by immunohistochemical techniques in 66 patients. 12 samples were analyzed for the presence of CD68, cathepsin K, CD11b and tartrate-resistant acid phosphatase (TRAP) activity. Many MNCs were present in the granulation tissue adjacent to a sequestrum. MNCs in contact with the sequestrum were also noted, however, no osteoblasts were found. Immunohistochemically, CD68, cathepsin K and TRAP were strongly expressed in most of the MNCs, while CD11b positive cells were not found. MNCs remote from and in contact with the sequestrum showed the same immunohistochemical features which are characteristic of osteoclasts. Further, MNCs in contact with the sequestrum had originally developed in the granulation tissue and directly infiltrated towards the sequestrum without cellto-cell interaction with osteoblasts.