Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages.

Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due to their immunomodulatory properties. The overall immunomodulatory response of hMSCs resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages (Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into improved Candida albicans phagocytosis activity of macrophages. Taken together, these results demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated immunomodulation with increased CD163 expression and decreased gene expression of matrix metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.

Nirogacestat suppresses RANKL-Induced osteoclast formation in vitro and attenuates LPS-Induced bone resorption in vivo.

Bone resorption, initiated by osteoclasts (OCs), plays an essential role in bone homeostasis. The abnormalities of bone resorption may induce a series of diseases, including osteoarthritis, osteoporosis and aseptic peri-implant loosening. Nirogacestat (PF-03084014, PF), a novel gamma-secretase inhibitor, has been used in phase II clinical trial for treatment of desmoid tumor. However, whether it has the therapeutic effect on abnormal bone resorption remains to be evaluated. In this study, we investigated the role of PF in the regulation of receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis in vitro, and the lipopolysaccharide (LPS)-induced bone resorption in vivo. It was found that PF could suppress the formation of osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, inhibit bone resorption and downregulate the mRNA level of osteoclast-specific markers, including calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), cathepsin K (CTSK), dendritic cell-specific transmembrane protein (Dc-stamp), Atp6v0d2 (V-ATPase d2) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Furthermore, Notch2 signaling, as well as RANKL-induced AKT signaling was significantly inhibited in BMMs. Consistent with in vitro observation, we found that PF greatly ameliorated LPS-induced bone resorption. Taken together, our study demonstrated that PF has a great potential to be used in management of osteolytic diseases.

2-O-digalloyl-1,3,4,6-tetra-O-galloyl-ß-D-glucose isolated from Galla Rhois suppresses osteoclast differentiation and function by inhibiting NF-κB signaling.

Natural compounds isolated from medicinal herbs and plants have immense significance in maintaining bone health. Hydrolysable tannins have been shown to possess a variety of medicinal properties including antiviral, anticancer, and anti-osteoclastogenic activities. As a part of a study on the discovery of alternative agent against skeletal diseases, we isolated a hydrolysable tannin, 2-O-digalloyl-1,3,4,6-tetra-Ogalloyl- ß-D-glucose (DTOGG), from Galla Rhois and examined the effect on osteoclast formation and function. We found that DTOGG significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by downregulating the expression of the key regulator in osteoclastogenesis as well as osteoclast-related genes. Analysis of RANKL/RANK signaling revealed that DTOGG impaired activation of IκBα and p65 in the nuclear factor kappa-lightchain- enhancer of activated B cells (NF-κB) signaling pathway. Furthermore, DTOGG reduced bone resorbing activity of osteoclasts, compared to the vehicle-treated control. These results suggest that DTOGG could be a useful natural compound to manage osteoclast-mediated skeletal diseases. [BMB Reports 2019; 52(6): 409-414].

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

M2C Polarization by Baicalin Enhances Efferocytosis via Upregulation of MERTK Receptor.

Baicalin is the main active ingredient primary isolated from the Chinese herb, Scutellaria baicalensis Georgi. Although baicalin can induce M2 macrophage polarization, we still do not know the subtype of macrophages polarized by baicalin. In this study, we characterized that murine bone marrow derived macrophages induced by M-CSF can be further polarized into M2C phenotype by baicalin. The signatures of M2C macrophages for mRNA expression like interferon regulatory factor 4 (IRF4), interleukin-10 (IL-10), MERTK and PTX3 were up-regulated. Moreover, we observed the concomitantly decreasing of tumor necrosis factor alpha (TNF- α ), interferon regulatory factor 5 (IRF5), IL-6. In contrast, M2 macrophages polarized by IL-4 increased gene transcript of arginase-1 (Arg-1) and surface marker of CD206 indicates that their identity as M2A rather than M2C subtypes. Interestingly, the phagocytosis as well as efferocytosis activity were significantly enhanced in M2C macrophage polarized by baicalin and these capacities were associated with the expression of MERTK receptor. Finally, we conclude that baicalin induced M2C macrophages polarization with both elevations of efferocytosis and anti-inflammatory activity.

Psoralen and Bakuchiol Ameliorate M-CSF Plus RANKL-Induced Osteoclast Differentiation and Bone Resorption Via Inhibition of AKT and AP-1 Pathways in Vitro.

BACKGROUND/AIMS: Psoralen and bakuchiol are the main active compounds found in the traditional Chinese medicine Psoralea corylifolia L., and have been used to treat osteoporosis. This study aims to investigate the anti-osteoporosis effects of these two compounds using osteoclasts precursor differentiation and bone absorption assays in vitro. METHODS: Primary mouse osteoclasts precursor cells were induced by M-CSF (macrophage colony stimulating factor) plus RANKL (receptor activator of nuclear factor kappa-B ligand) in vitro. TRACP (tartrate-resistant acid phosphatase) enzyme activity and toluidine blue staining were used to observe the effects of psoralen and bakuchiol on osteoclast differentiation and bone resorption, respectively. Gelatin zymography was used to assess MMP (matrix metalloproteinase) activity, and ELISA was performed to measure cathepsin K activity. Western blotting analysis for expression of phosphorylated AKT, ERK, NF-kB, and c-jun; and immunofluorescence analysis for c-jun and p65 nuclear translocation in induced osteoclasts were then used to determine the mechanism of anti-bone resorption of psoralen and bakuchiol. RESULTS: Mature osteoclasts were induced by M-CSF plus RANKL from primary bone marrow macrophages in vitro. Both psoralen and bakuchiol significantly inhibited TRACP enzyme activity and slightly decreased the number of TRACP+ multinuclear osteoclasts induced by M-CSF plus RANKL. Bakuchiol significantly decreased bone lacunae area and attenuated MMP-2 activity induced by M-CSF plus RANKL in osteoclasts. Both psoralen and bakuchiol significantly decreased the expression and nuclear translocation of phosphorylated c-jun stimulated by M-CSF plus RANKL, but no significant effect on p65 translocation was observed in osteoclasts. Additionally, bakuchiol significantly attenuated the increased of M-CSF plus RANKL-induced phosphorylation of AKT in osteoclasts. CONCLUSIONS: Psoralen and bakuchiol ameliorated M-CSF plus RANKL-induced osteoclast differentiation and bone resorption via inhibition of AKT and AP-1 pathways activation in vitro.

Granulocyte-macrophage colony-stimulating factor-dependent CD11c-positive cells differentiate into active osteoclasts.

Levels of circulating cytokines are elevated in inflammatory diseases. Previously, it was shown that interleukin (IL-)17A, in synergism with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and tumor necrosis factor α (TNFα), induces the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) by murine osteoblasts in vitro. In this study, we further analyzed the effects of GM-CSF on osteoclast development in vitro. The effects of IL-17A, TNFα, and 1,25(OH)2D3 on the regulation of osteoclast development were investigated in cocultures of bone marrow-derived osteoclast progenitor cells (OPC) and mouse calvarial osteoblasts. Additionally, OPC were grown for 3days in media containing macrophage colony-stimulating factor (M-CSF), GM-CSF, or M-CSF/GM-CSF. Subsequently, the osteoclastogenic potential and the capacity to dissolve amorphous calcium phosphate were assessed in each of the three populations of OPC. IL-17A, in synergism with TNFα and 1,25(OH)2D3, inhibited the development of osteoclasts in cocultures by stimulating the osteoblast lineage cells to release GM-CSF. GM-CSF-treated OPC expressed traits characteristic of dendritic cells. Upon removal of GM-CSF and supplementation of the culture media with M-CSF/RANKL, the cells lost their dendritic cell characteristics and differentiated into osteoclasts. OPC pretreated with GM-CSF and M-CSF/GM-CSF exhibited delayed development to osteoclasts and an extended proliferation phase. Elevated levels of GM-CSF in systemic inflammatory diseases may cause an expansion of the OPC pools in the bone, bone marrow, and blood. Upon homing to the bone, this may lead to an increase in the number of osteoclasts and in bone resorption.

Effects of 10-hydroxycamptothecin on differentiation of RAW264.7 cells into osteoclasts.

This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) and 30 ng/ml recombinant macrophage colony stimulating factor (M-CSF), and 10-HCPT with different solubilities were added. After five-day cultivation, tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts. mRNA expression of osteoclast-specific genes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detected by real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity of RAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPT with a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPT could inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the number of osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86±11.14), 2 ng/ml (66.67±7.51), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference was statistically significant (all P=0, P less than 0.05); mRNA expression of osteoclast-specific gene TRAP [1 ng/ml (24.38±0.68), 2 ng/ml (20.09±1.86), 5 ng/ml (6.23±0.53)], CTSK [1 ng/ml (10.08±0.81), 2 ng/ml (7.30±0.30), 5 ng/ml (3.20±0.56)] and MMP-9 [1 ng/ml (43.54±6.96), 2 ng/ml (28.28±5.83), 5 ng/ml (11.07±2.53)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P less than 0.05), and with the increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasing tendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing the expression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.

Defective Self-Renewal and Differentiation of GBA-Deficient Neural Stem Cells Can Be Restored By Macrophage Colony-Stimulating Factor.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucosylceramidase (GCase). Deficiency in GCase leads to characteristic visceral pathology and lethal neurological manifestations in some patients. Investigations into neurogenesis have suggested that neurodegenerative disorders, such as GD, could be overcome or at least ameliorated by the generation of new neurons. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are potential candidates for use in the treatment of neurodegenerative disorders because of their ability to promote neurogenesis. Our objective was to examine the mechanism of neurogenesis by BM-MSCs in GD. We found that neural stem cells (NSCs) derived from a neuronopathic GD model exhibited decreased ability for self-renewal and neuronal differentiation. Co-culture of GBA-deficient NSCs with BM-MSCs resulted in an enhanced capacity for self-renewal, and an increased ability for differentiation into neurons or oligodendrocytes. Enhanced proliferation and neuronal differentiation of GBA-deficient NSCs was associated with elevated release of macrophage colony-stimulating factor (M-CSF) from BM-MSCs. Our findings suggest that soluble M-CSF derived from BM-MSCs can modulate GBA-deficient NSCs, resulting in their improved proliferation and neuronal differentiation.

Regulation of the differentiation of osteoblasts and osteoclasts by a hot-water extract of adzuki beans (Vigna angularis).

Osteoporosis is a global public health problem thought to be caused by an imbalance in bone metabolism. We examined in this study the 40% ethanol fraction of HP-20 resin in combination with a hot-water adzuki extract (EtEx.40) for its effect on osteoblast and osteoclast differentiation. EtEx.40-treated murine preosteoblast MC3T3-E1 cells exhibited significantly elevated alkaline phosphatase activity and mineralization. EtEx.40 facilitated osteoblast differentiation by up-regulating such osteoblast differentiation-related molecules as runt-related transcription factor 2, distal-less homeobox 5, and osterix via p38 mitogen-activated protein kinase. EtEx.40 also suppressed the formation of large tartrate-resistant acid phosphatase-positive multinucleated cells in RAW264.7 cells that had been stimulated with the receptor activator of the nuclear factor κB ligand/macrophage colony-stimulating factor. EtEx.40 significantly inhibited NF-κB activation, thus reducing the expression of such downstream molecules as c-Fos and NFATc1. Our findings suggest that EtEx.40 could be used to maintain bone mass.

N-truncation and pyroglutaminylation enhances the opsonizing capacity of Aß-peptides and facilitates phagocytosis by macrophages and microglia.

Abnormal accumulations of amyloid-ß (Aß)-peptides are one of the pathological hallmarks of Alzheimer's disease (AD). The precursor of the Aß-peptides, the amyloid precursor protein (APP), is also found in peripheral blood cells, but its function in these cells remains elusive. We previously observed that mononuclear phagocytes release Aß-peptides during activation and phagocytosis, suggesting a physiologic role in inflammatory processes. Here, we show that supplementing the media with soluble N-terminally truncated Aß(2-40) and Aß(2-42) as well as Aß(1-42) induced the phagocytosis of polystyrene particles (PSPs) by primary human monocytes. If the PSPs were pre-incubated with Aß-peptides, phagocytosis was induced by all tested Aß-peptide species. N-terminally truncated Aß(x-42) induced the phagocytosis of PSPs significantly more effectively than did Aß(x-40). Similarly, the phagocytosis of Escherichia coli by GM-CSF- and M-CSF-elicited macrophages as well as microglia was particularly facilitated by pre-incubation with N-terminally truncated Aß(x-42). The proinflammatory polarization of monocytes was indicated by the reduced MSRI expression and IL-10 secretion after phagocytosis of PSPs coated with Aß(1-42), Aß(2-42) and Aß(3p-42). Polarization of the macrophages by GM-CSF reduced the phagocytic activity, but it did not affect the capabilities of Aß-peptides to opsonize prey. Taken together, Aß-peptides support phagocytosis as soluble factors and act as opsonins. Differential effects among the Aß-peptide variants point to distinct mechanisms of interaction among monocytes/macrophages, prey and Aß-peptides. A proinflammatory polarization induced by the phagocytosis of Aß-peptide coated particles may provide a model for the chronic inflammatory reaction and sustained plaque deposition in AD.

Spontaneous and induced osteoclastogenic behaviour of human peripheral blood mononuclear cells and their CD14(+) and CD14(-) cell fractions.

OBJECTIVES: Osteoclasts are descended from the CD14(+) monocyte/macrophage lineage, but influence of other haematopoietic cells on osteoclastic commitment of their precursors has remained poorly understood. In this study, osteoclastogenic behaviour of peripheral blood mononuclear cells (PBMC) and their CD14(+) and CD14(-) subpopulations has been accessed, in the absence or presence of M-CSF and RANKL. MATERIALS AND METHODS: Cell cultures were characterized for presence of actin rings and vitronectin and calcitonin receptors, TRAP activity and calcium phosphate resorbing activity, expression of osteoclast-related genes and secretion of M-CSF and RANKL. RESULTS: In the absence of growth factors, PBMC and CD14(+) cultures had some degree of cell survival, and some spontaneous osteoclastogenesis was observed, only on cultures of the former. Supplementation with M-CSF and RANKL significantly increased osteoclastogenic behaviour of cell cultures, particularly CD14(+) cell cultures. Nevertheless, PBMC derived a higher degree of osteoclastogenesis, either as absolute values or after normalization by protein content. It was observed that unlike CD14(+) cells, PBMC were able to express M-CSF and RANKL, which increased following growth factor treatment. Also, expression of TNF-α, GM-CSF, IL-1ß, IL-6 and IL-17 was higher in PBMC cultures. Finally, CD14(-) cultures exhibited limited cell survival and did not reveal any osteoclast features. CONCLUSIONS: Results show that although osteoclastic precursors reside in the CD14(+) cell subpopulation, other populations (such as CD14(-) cells) derived from PBMC, have the ability to modulate osteoclastogenesis positively.

Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo.

BACKGROUND AND OBJECTIVE: Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS: Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS: The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION: These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

Role of MKP-1 in osteoclasts and bone homeostasis.

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

Grape seed proanthocyanidin extract (GSPE) attenuates collagen-induced arthritis.

To examine whether grape seed proanthocyanidin extract (GSPE) which is known to act as an antioxidant has therapeutic effect on collagen-induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. Mice were treated with an intraperitoneal injection of GSPE (10, 50, or 100 mg/kg) or saline. Clinical, histological, and biochemical parameters were assessed. The effects of GSPE on osteoclastogenesis were determined by tartrate-resistant acid phosphatase (TRAP) staining of the inflamed joints and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were determined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflammation, cartilage erosion, bone erosion, and the number of TRAP+ osteoclasts. GSPE treatment significantly reduced the numbers of tumor necrosis factor alpha (TNF-alpha)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-alpha and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen peroxide production by anti-CD3-monoclonal-antibody-stimulated CD4+ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis.

Administration of high-dose macrophage colony-stimulating factor increases bone turnover and trabecular volume fraction.

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that plays a critical role in early osteoclastogenesis. To characterize the skeletal effects of M-CSF, we administered soluble M-CSF to mice. It was hypothesized that M-CSF would stimulate bone formation through coupled activity of osteoclasts and osteoblasts. Twenty-four male C57BL/6 J mice (n = 12/group, aged 7 weeks) received subcutaneous injections of human M-CSF [5 mg/(kg day)] or inert vehicle (VEH) for 21 days. M-CSF increased serum bone turnover markers (+57% TRAP-5b and +44% osteocalcin). Microcomputed tomography revealed an anabolic effect on tibial trabecular bone, with higher bone volume fraction (+35%), connectivity density (+79%), and number (+18%), as well as lower trabecular separation (-18%). M-CSF had no significant effect on cortical bone mineral content, geometry, or strength. There was no change in quantitative histomorphometry parameters of femoral cortical bone. These results reveal the complex, site-specific effects of M-CSF. In particular, we have demonstrated an anabolic effect of M-CSF on trabecular bone achieved through coupled activation of osteoblasts. However, in contrast to previous studies, M-CSF was found to have no effect on cortical bone. M-CSF was demonstrated to significantly influence both bone modeling and remodeling in relatively young animals.

Inhibitory effect of (-)-saucerneol on osteoclast differentiation and bone pit formation.

In this study, the effect of (-)-saucerneol, one of the lignans isolated from Saururus chinensis, on osteoclast differentiation and bone resorption was evaluated in two in vitro models for osteoclast differentiation, the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-treated RAW264.7 cells and mouse BMMs treated with both RANKL and macrophage-colony stimulating factor. (-)-Saucerneol significantly inhibited the RANKL-induced activity of tartrate-resistance acid phosphatase (TRAP, an early marker of osteoclast formation) and formation of osteoclasts in a dose-dependent manner. Interestingly, (-)-saucerneol was shown to inhibit the RANKL-induced activation of extracellular signal-regulated kinase in both in vitro models. In addition, (-)-saucerneol inhibited the bone resorptive activity and the expression of transcription factors and genes essential for osteoclast formation and bone resorption as well. In conclusion, (-)-saucerneol has a potential to inhibit the osteoclast differentiation via preventing the activation of ERK signaling pathway. In addition, its activity to inhibit the bone resorption activities of osteoclasts could result from its potential to inhibit RANKL-induced expression levels of transcription factors and genes essential for bone resorption.

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways.

UNLABELLED: Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-kappaB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. INTRODUCTION: A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. METHODS: The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-kappaB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-kappaB activity. RESULTS: Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-kappaB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. CONCLUSIONS: Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-kappaB activation.

Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells. We now report the effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light, transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells. Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear differentiation and activation of bone marrow progenitor and stromal cells.