Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles.

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure.

Correlation study between osteoporosis and hematopoiesis in the context of adjuvant chemotherapy for breast cancer.

This retrospective study attempts to establish if a correlation exists between osteoporosis and hematopoiesis before and after adjuvant chemotherapy in the context of non-metastatic breast cancer. Osteoporosis is interpreted both as a direct marker of osteoblastic decline and as an indirect marker of increased bone marrow adiposity within the hematopoietic microenvironment. Patients from the "Centre du Sein" at CHUV (Centre Hospitalier Universitaire Vaudois) undergoing adjuvant chemotherapy were included in this study. Evolution of blood counts was studied in correlation with the osteoporosis status. Toxicity of chemotherapy was coded according to published probability of febrile neutropenia. One hundred forty-three women were included: mean age 52.1 ± 12.5 years, mean BMI (body mass index) 24.4 ± 4.1. BMD (bone mineral density) scored osteoporotic in 32% and osteopenic in 45%. Prior to chemotherapy, BMD was positively correlated with neutrophil (p < 0.001) and thrombocyte (p = 0.01) count; TBS (trabecular bone score) was not correlated with blood count. After the first cycle of chemotherapy, an increase of one point in TBS correlated with a decrease of 57% on the time to reach leucocyte nadir (p = 0.004). There was a positive correlation between BMD and risk of infection (p < 0.001). Our data demonstrates an association between osteoporosis and lower blood counts in a younger cohort than previously published, extending it for the first time to neutrophil counts in females. Our results suggest that the healthier the bone, the earlier the lowest leucocyte count value, prompting further research on this area.

Beneficial role of the phytoestrogen genistein on vascular calcification.

Although soy phytoestrogen are proposed to prevent or improve postmenopausal vascular and bone diseases, the currently available data are controversial and unclear. In this study we evaluated the molecular and biochemical action of genistein on the cellular events involved in vascular calcification. Rat monocytes, aortic vascular cell and osteoblasts cultures in vitro exposed to Gen were employed. Gen down regulated the expression of cell adhesion molecules involved in stable leukocyte attachment. Using flow cytometry we found that the PE significantly diminished monocyte integrins CD11b, CD11c and CD18 expression either under basal and pro-inflammatory environment. At endothelial level, Gen also reduced Intercellular Adhesion Molecule 1 mRNA expression. On vascular muscle cells, the PE markedly reduced cell proliferation and migration. When vascular calcification was studied, muscle cells transdifferentiation into osteoblasts like cells was evaluated. Cells were cultured in osteogenic medium for 21 days. The expression of alkaline phosphatase and the presence of calcified nodules in the extracellular matrix were selected as features of muscle transdifferentiation. Calcified muscle cells exhibited higher levels of alkaline phosphatase activity and enhanced deposition of calcium nodules respect to native cells. Both osteoblastic markers were significantly reduced after Gen treatment. In contrast to this anti-osteogenic action, on bone cells Gen promoted osteoblasts growth, enhanced alkaline phosphatase activity and increased matrix mineralization. Its mitogenic action on osteoblasts directly depends on nitric oxide endothelial production stimulated by the PE. The data presented suppose a beneficial role of Gen on bone and vascular cells, with a cross link between both systems.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.

The endocannabinoid system and its therapeutic implications in rheumatoid arthritis.

Since the discovery of the endogenous receptor for Δ(9)-tetrahydrocannabinol, a main constituent of marijuana, the endocannabinoid system (comprising cannabinoid receptors and their endogenous ligands, as well as the enzymes involved in their metabolic processes) has been implicated as having multiple regulatory functions in many central and peripheral conditions, including rheumatoid arthritis (RA). RA is an immune-mediated inflammatory disease that is associated with the involvement of many kinds of cells (such as fibroblastlike synoviocytes [FLSs], osteoclasts, T cells, B cells, and macrophages) and molecules (such as interleukin-1ß, tumor necrosis factor-α, interleukin-6, matrix metalloproteinases [MMPs], and chemokines). Increasing evidence suggests that the endocannabinoid system, especially cannabinoid receptor 2 (CB2), has an important role in the pathophysiology of RA. Many members of the endocannabinoid system are reported to inhibit synovial inflammation, hyperplasia, and cartilage destruction in RA. In particular, specific activation of CB2 may relieve RA by inhibiting not only the production of autoantibodies, proinflammatory cytokines, and MMPs, but also bone erosion, immune response mediated by T cells, and the proliferation of FLSs. In this review, we will discuss the possible functions of the endocannabinoid system in the modulation of RA, which may be a potential target for treatment.

Ginsenoside Rg1, a novel glucocorticoid receptor agonist of plant origin, maintains glucocorticoid efficacy with reduced side effects.

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.

Obesity-mediated inflammatory microenvironment stimulates osteoclastogenesis and bone loss in mice.

Clinical evidence indicates that fat is inversely proportional to bone mass in elderly obese women. However, it remains unclear whether obesity accelerates bone loss. In this report we present evidence that increased visceral fat leads to inflammation and subsequent bone loss in 12-month-old C57BL/6J mice that were fed 10% corn oil (CO)-based diet and a control lab chow (LC) for 6 months. As expected from our previous work, CO-fed mice demonstrated increased visceral fat and enhanced total body fat mass compared to LC. The adipocyte-specific PPARγ and bone marrow (BM) adiposity were increased in CO-fed mice. In correlation with those modifications, inflammatory cytokines (IL-1ß, IL-6, TNF-α) were significantly elevated in CO-fed mice compared to LC-fed mice. This inflammatory BM microenvironment resulted in increased superoxide production in osteoclasts and undifferentiated BM cells. In CO-fed mice, the increased number of osteoclasts per trabecular bone length and the increased osteoclastogenesis assessed ex-vivo suggest that CO diet induces bone resorption. Additionally, the up-regulation of osteoclast-specific cathepsin k and RANKL expression and down-regulation of osteoblast-specific RUNX2/Cbfa1 supports this bone resorption in CO-fed mice. Also, CO-fed mice exhibited lower trabecular bone volume in the distal femoral metaphysis and had reduced OPG expression. Collectively, our results suggest that increased bone resorption in mice fed a CO-enriched diet is possibly due to increased inflammation mediated by the accumulation of adipocytes in the BM microenvironment. This inflammation may consequently increase osteoclastogenesis, while reducing osteoblast development in CO-fed mice.

Dehydroepiandrosterone inhibited the bone resorption through the upregulation of OPG/RANKL.

The plasma level of dehydroepiandrosterone (DHEA) decreases gradually along with aging. The beneficial effects of DHEA as an anti-aging steroid, such as the stimulatory effect on immune system, anti-diabetes mellitus, anti-atherosclerosis, anti-dementia, anti-obesity and anti-osteoporosis have been demonstrated in experiment both in vitro and in vivo. It is important to investigate the effective mechanism of DHEA in therapeutics for postmenopausal osteoporosis. Having isolated and cultured osteoblasts (OBs) and osteoclasts (OCs), we analysed the effect of DHEA on osteoblastic viability, regulation of DHEA on the expression of osteoprotegerin (OPG)/receptor activator of NF-kappaB ligand (RANKL) mRNA in OBs, and then observed the action of DHEA on bone resorption of OCs in the presence or absence of OBs. The results showed that DHEA improved viability of OBs within the concentration range of 0.01-1 microM, especially at the concentration of 0.1 microM. DHEA could apparently increase the ratio of OPG/RANKL mRNA in OBs. In the presence of OBs, DHEA could decrease the number and area of absorption lacuna of specula. We concluded, therefore, only in the presence of OBs, DHEA could inhibit the bone resorption of OCs, which may be mediated by OPG/RANKL of OBs.

Effects of Drynariae rhizoma on the proliferation of human bone cells and the immunomodulatory activity.

Drynariae Rhizoma (DR), a traditional Korea medicine, which is known for its effect to strengthen myoskeletal systems, frequently appears as the main ingredient in prescriptions for bone injuries. However, it is unclear how it pharmacologically contributes to the reformation of bone. In this study, the effect of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of DR and the cell proliferation was studied. When the concentration of DR was < or = 120 microg ml(-1), the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by DR with the concentrations of > 250 microg ml(-1). Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox-2) protein; slightly intensified band showed at the highest DR concentration, 120 microg ml(-1) during the course of culture. On the other hand, we investigated the immunomodulatory activity of DR on cellular and humoral immunity. When different doses of ethanolic and water extracts of DR was administered to mice, it was dose-dependently potentiated the delayed-type hypersensitivity reaction induced by both sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titre in mice in response to SRBC. But, DR did not any effect on macrophage phagocytosis. Prolonged administration of DR significantly ameliorated the total white blood cell count and also restored the immunosuppressive effects induced by cyclophosphamide. The present investigation reveals that DR possesses immunomodulatory activity. From the results, it was concluded that DR directly stimulated the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner, and stimulated both the cellular and the humoral immunity.

Articular cartilage degradation and de-differentiation of chondrocytes by the systemic administration of retinyl acetate-ectopic production of osteoblast stimulating factor-1 by chondrocytes in mice.

OBJECTIVE: Vitamin A derivatives are widely used therapeutic agents for the treatment of dermatological and rheumatological disorders. Long-standing administration of these drugs, in turn, causes skeletal changes including ossification of ligaments, premature fusion of epiphyses and abnormalities of modeling. Recent in vitro experiments have further suggested that retinoid treatment of cultured chondrocytes may cause apoptotic cell death. The present study aims to address detailed cartilage changes associated with in vivo administration of vitamin A derivatives. METHODS: Retinyl acetate was administrated to experimental mice, C3H-Heston, for more than 12 months. Modified morphometry on the articular cartilage and fluorescent labeling of the subchondral bone were carried out to address the changes in the articular cartilage and subchondral bone. In order to address the detailed chondrocytes phenotypes, electron microscopy was carried out. Since findings of these studies suggested that biological properties of the cartilage matrix might be altered, the present study also immunolocalized functional matrix molecules, type I collagen and osteoblast-stimulating factor-1 (OSF-1). RESULTS: Histomorphometry demonstrated that retinoid administration lead to progressive atrophy of the articular cartilage with concomitant proliferation of subchondral bone. Furthermore, detailed light and electron microscopy suggested that the subchondral bone proliferates into the degenerating cartilage. The affected articular cartilage also resembled that of osteoarthritis in terms of ectopic type I collagen production. Furthermore, the affected articular cartilage produced a developmentally regulated matrix molecule, osteoblast-stimulating factor-1 (OSF-1) that is normally expressed in both the fetal cartilage and the epiphyseal growth plate cartilage but not in the articular cartilage. CONCLUSION: The present results indicate that the systemic retinoid administration may alter the biological properties of the articular cartilage.

Inflammatory microcrystals alter the functional phenotype of human osteoblast-like cells in vitro: synergism with IL-1 to overexpress cyclooxygenase-2.

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE(2) in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D(3)-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.

Effects of polyethylene and alpha-alumina particles on IL-6 expression and secretion in primary cultures of human osteoblastic cells.

The effect of two biomaterials, polyethylene and alpha-alumina, on interleukin-6 (IL-6) secretion and expression has been studied in human osteoblasts in primary culture. Human osteoblastic cells were derived from fresh trabecular bone explants removed during total knee arthroplasty. On reaching confluence, cells were subcultured in 6 well plates; the resulting subcultures were incubated until confluence and polyethylene or alpha-alumina particles were added to some while the rest were left as controls. The IL-6 mRNA levels were assessed by reverse transcription (RT) followed by polymerase chain reaction (PCR). IL-6 secretion was measured in the conditioned medium. The IL-6 expression was higher in the presence of both biomaterials. Maximum expression occurred in response to a dose of 50 mg particles well with both biomaterials and was greater after polyethylene particle addition than after alpha-alumina particle addition at this dose. The maximum IL-6 secretion elicited by alpha-alumina was produced at 10 mg particles well while maximum response with polyethylene required 50 mg well. At a dose of 10 mg/well, alpha-alumina particles induced more secretion than 10 mg of polyethylene particles. Nevertheless, at a dose of 50 mg/well maximum secretion was produced with polyethylene particles. In conclusion and in our experimental conditions, polyethylene as well as alpha-alumina increased both the expression and the secretion of IL-6 in human osteoblastic cells in primary culture and stimulation from polyethylene appears stronger than that from alpha-alumina at the same dose.

Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes.

A new marker for cells of the osteoblastic lineage was identified by raising monoclonal antibodies against an immortalized rat osteoblastic cell line. Among the different antibodies one was selected which, on tissue sections, strongly reacts with osteoblasts, preosteocytes, and osteocytes. This antibody, designated E11, recognizes an antigen localized at the cell surface. The cDNA encoding the E11 antigen was cloned from a cDNA library prepared from ROS 17/2.8 cells, using a eukaryotic expression system. The E11 cDNA sequence revealed homology with the murine OTS-8/gp38 sequence. In situ hybridization confirmed that E11 mRNA expression in bone is restricted to osteoblasts and osteocytes. The tissue specificity of the E11 expression was studied by immunohistochemistry and Northern blot analysis. Apart from bone, E11-positive cells were also found in lung: namely, the alveolar cells of type I. Epithelial cells of the choroid plexus and endothelial cells of lymphatic vessels were also labeled with mAb E11. These results were confirmed by Northern blot, as the 1.8 kb E11 mRNA transcript was detected in bone and also in lung, brain, and skin. In conclusion, we describe a novel osteoblastic product which is expressed by mature osteoblasts and newly formed osteocytes.

The modulation of the expression of IL-6 and its receptor in human osteoblasts in vitro.

Interleukin 6 (IL-6) probably plays a central role in the acute phase response and in haemopoiesis and may be involved in the control of bone turnover. We have studied the release of IL-6 from human trabecular bone cells treated with a variety of stimuli using a specific bioassay. In serum free medium, unstimulated human osteoblast-like cells produced IL-6 in the range of 1000-2050 pg/ml/24 h. Recombinant human interleukin 1 (IL-1 alpha) (10(-13)-10(-11) M), tumor necrosis factor alpha (TNF alpha) (10(-9)-10(-7) M) and lipopolysaccharide (5-500 ng/ml) all stimulated release of IL-6 from human bone cells. Maximal levels of 17,000 pg/ml were observed using the highest concentration of IL-1. 1,25(OH)2D3 and PTH did not stimulate IL-6 release. Using a specific sheep antihuman IL-6 antibody, all IL-6 activity could be neutralized. In parallel studies, ROS 17/2.8 rat osteosarcoma cells released around 50 pg/ml of IL-6 under basal conditions which were increased to a maximum of 900 pg/ml by treatment with PTH (10(-9) M). The cytokines were less effective and 1,25(OH)2D3 again had no effect. Modulation of expression of IL-6 mRNA in human osteoblast cells was examined using a human complementary deoxyribonucleic acid probe. The mRNA was constitutively expressed, and IL-1 (10(-11) M) and TNF (10(-7) M) induced further mRNA expression within 2 h, which was sustained over 24 h. 1,25(OH)2D3 (10(-7) M), IL-6 (2000 pg/ml), and PTH (10(-9) M) exerted no effects at any time point. Dexamethasone (10(-6) M) suppressed both basal and IL-1- and TNF-induced IL-6 mRNA expression. IL-6 receptor mRNA was constitutively expressed but was not regulated by any of the above agents. It is clear that rodent and human osteoblasts differ in their production of IL-6 and its modulation. These data support the hypothesis that IL-6 is produced locally in human bone by osteoblasts under the direction of other cytokines. This could have implications in bone remodeling, haemopoiesis, and systemic responses to local injury.