Mouse Marrow Cultures Research Articles

Effects of Alendronate and Dexamethasone on Osteoclast Gene Expression and Bone Resorption in Mouse Marrow Cultures.

Osteoclasts are cells whose main function is the resorption of bone matrix. However, several factors, including medications, can interfere with the resorption process. Alendronate (ALN), a nitrogen-containing type of bisphosphonate, and dexamethasone (DEX), a glucocorticoid, are drugs that may affect the resorption activity. The aim of this study is to investigate the effects of ALN, and/or DEX on osteoclast gene expression and resorption activity in primary mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D3, a model for the bone microenvironment. Cultures were treated only with ALN (10-5 M), DEX (10-6 M), and with a combination of both agents. Viability assays performed at days 5, 7, and 9 showed the highest number of viable cells at day 7. All the following assays were then performed at day 7 of cell culture: tartrate resistant acid phosphatase (TRAP) histochemistry, receptor activator of nuclear factor kappa B ligand (RANKL) immunofluorescence, osteoprotegerin (OPG), and RANKL gene expression by qPCR and resorption analysis by scanning electron microscopy. Treatment with ALN, DEX, and the combination of both did not promote significant changes in the number of TRAP+ cells, although larger giant cells were detected in groups treated with DEX. DEX treatment increased the gene expression of RANKL and reduced OPG. The treatment with ALN reduced the depth of the resorption pits, but their inhibitory effect was less effective when administered with DEX.

Radiation Resistance of Double Knockout (DKO) Smad3-/- Fancd2-/- (129/Sv) Mouse Bone Marrow Stromal Cell Lines

Introduction: Bone marrow progenitor cells from Fanconi Anemia (FA) patients have a hyperactive TGF-β signaling pathway which may explain hematopoietic stem cell depletion leading to bone marrow failure (Zhang, et al, Cell Stem Cell, 18:668-681, 2016). To determine whether blockade of TGF-B signaling by knockout of Smad3 normalized hematopoiesis in Fancd2-/- mice, we bred two double knockout mouse strains. The first (DKO#1) was derived by crossing C57BL/6 Fancd2 +/- mice with 129/Sv Smad3 +/- mice. The second (DKO#2) was derived by mating 129/Sv Fancd2 +/- mice with the same 129/Sv Smad3 +/- mice.Materials and Methods: Long term bone marrow cultures (LTBMCs) were established from DKO#1, DKO#2, parental mouse strains and F1 (129/Sv X C57Bl/6) control mice for DKO#1, 129/Sv Smad3 -/-, and C57BL/6 Smad3 -/- mice . The cultures were scored for weekly numbers of cobblestone islands as an indicator of stem cells in the adherent layer, weekly production of nonadherent cells, and cells producing Day 7 and Day 14 CFU-GEMM .Bone marrow stromal cell lines were derived from the adherent layer of LTBMCs and radiosensitivity measured in clonogenic radiation survival curves. Cells were irradiated to doses of 0 to 8 Gy, plated in 4 well linbro plates, incubated for 7 days at 37oC, stained with crystal violet and colonies of greater than 50 cells counted. Western analysis of the cell lines quantitated expression levels of proteins involved in the TGF-β signaling pathway, and DNA double strand break repair by homologous recombination and nonhomologous end-joining.Results: Both DKO1 and DKO2 LTBMCs showed decreased duration and magnitude of hematopoiesis, thus being similar to that observed with both C57BL/6 Fancd2-/- and 129/Sv Fancd2-/- mouse marrow cultures. Both were significantly lower than that for control mouse or Smad3-/- (129/Sv) marrow cultures. As expected, radiation survival curves showed that both C57Bl/6 Fancd2-/- and 129/Sv Fancd2-/- marrow stromal cell lines were radiosensitive compared to control cell lines including: F1 control, C57BL/6 control, and 129/Sv control (Berhane, et al, Rad Res. 181:76-89, 2014, Berhane et.al, Rad Res 182:35-49, 2014). In contrast marrow stromal cell lines from Smad3-/- (129/Sv) marrow cultures were radioresistant (Epperly, et al, Rad Res 165:671-677, 2006). Fresh marrow CFU-GEMM from both DKO#1 and DKO#2 mice showed resistance to abrogation of colony formation by increasing concentrations of TGF-B, (similar to the 129/Sv Smad3-/- cell line). In contrast, cell lines from all controls and both Fancd2-/- mice showed clear TGF-B mediated inhibition of hemopoietic colony formation. In striking contrast to the above similarities between DKO#1 and DKO#2 mice, marrow stromal cell lines from DKO#1 were radiosensitive (like theirFancd2-/- parent) while those from DKO#2 were radioresistant (like their Smad3-/- parent). Thus, DKO#1 retained the C57Bl/6 Fancd2-/- genotype cell line radiosensitivity: Do of 1.41 ± 0.03 Gy and 1.45 ± 0.05 Gy respectively, and were more radiosensitive than the control F1 bone marrow stromal cell line (p = 0.0230 and 0.0418, respectively) and DKO#2 retained the 129/Sv Smad3-/- cell line genotype radioresistance (Do = 2.15 ± 0.13 Gy) compared to control 129/Sv stromal cells (Do = 1.86 ± 0.04, p = 0.0054). Western analysis revealed that p21 was elevated in DKO#2 but not DKO#1 marrow stromal cell lines.Conclusions: While marrow from both DKO#1 and DKO#2 mice showed resistance to TGF-B signaling consistent with their smad3-/- genotype, only DKO#1 stromal cells retained the radiosensitivity of their Fancd2-/- genotype. Reduced p21 in irradiated DKO#1 marrow stromal cells may have allowed procession through G to S phase causing reduced time for DNA repair and radiosensitivity. The irradiated DKO#2 cells may have been blocked by p21 from passing through the G1 checkpoint and may have allowed DNA strand break repair and radioresistance. These DKO mice and derived cell lines should be valuable for analysis of the interaction of TGF-B signaling and FA pathways.Supported by NIAID/NIH U19-AI068021 DisclosuresNo relevant conflicts of interest to declare.

Characterization of Regulatory Extracellular Vesicles from Osteoclasts

Extracellular vesicles (EVs), which include exosomes and ectosomes/microvesicles, have emerged as important intercellular regulators. EVs can interact with surface receptors of target cells and can transport luminal components, including messenger RNAs (mRNAs), microRNAs, and enzymes, to the cytosol of the target cell. Here, we show that hematopoietic cells grown in culture shed exosome-like EVs as they differentiate from preosteoclasts into osteoclasts. These EVs were between 25 and 120 nm (mean, 40 nm) in diameter determined by transmission electron microscopy. The exosome-associated markers CD63 and EpCAM were enriched in the isolated EVs while markers of Golgi and endoplasmic reticulum were not detected. Treatment of isolated hematopoietic cells with EVs did not affect their receptor activator of nuclear factor κB–ligand (RANKL)–stimulated differentiation into osteoclasts. However, EVs from osteoclast precursors promoted 1,25-dihydroxyvitamin D3–dependent osteoclast formation in whole mouse marrow cultures, and EVs from osteoclast-enriched cultures inhibited osteoclastogenesis in the same cultures. These data suggested that osteoclast-derived EVs are paracrine regulators of osteoclastogenesis. EVs from mature osteoclasts contained receptor activator of nuclear factor κB (RANK). Immunogold labeling showed RANK was enriched in 1 in every 32 EVs isolated from osteoclast-enriched cultures. Depletion of RANK-rich EVs relieved the ability of osteoclast-derived EVs to inhibit osteoclast formation in 1,25-dihydroxyvitamin D3–stimulated marrow cultures. In summary, we show for the first time that EVs released by osteoclasts are novel regulators of osteoclastogenesis. Our data suggest that RANK in EVs may be mechanistically linked to the inhibition of osteoclast formation. RANK present in EVs may function by competitively inhibiting the stimulation of RANK on osteoclast surfaces by RANKL similar to osteoprotegerin. RANK-rich EVs may also take advantage of the RANK/RANKL interaction to target RANK-rich EVs to RANKL-bearing cells for the delivery of other regulatory molecules.

DNA Cross-Linking Agent Sensitivity of Fanconi Anemia (FA) Cells Is Preserved in Double Knockout (DKO) SMAD3-/- Fancd2-/- Mouse Cell Lines

Increased sensitivity to inhibition of hematopoiesis by TGF-β has been hypothesized to be a mechanism of hematopoietic failure in Fanconi Anemia (FA). To determine whether abrogation of TGF-β signaling rescued biologic parameters of hematopoiesis, we derived a novel DKO mouse strain (SMAD3-/- (129/Sv) Fancd2-/- (C57BL/6)) by breeding double heterozygote mice. The DKO mice were small at birth, but achieved normalized growth and development by six - eight weeks. Hematopoiesis in long-term bone marrow cultures derived from DKO mice, ceased generating day 14 CFU-GEMM hematopoietic progenitors by 18 weeks, similar to that observed with Fancd2-/- marrow cultures. In contrast, cultures from SMAD3-/- (129/Sv) demonstrated continuous hematopoiesis for over 30 weeks confirming results with marrow from SMAD3-/- (C57BL/6J) mice (Epperly, et al., Experimental Hematology, 33:353-362, 2005), and significantly longer than that in control 129/Sv X C57BL/6J F1 mouse long-term marrow cultures. Bone marrow stromal cell lines derived from long-term marrow cultures were tested for the diagnostic criterion of FA, increased sensitivity to DNA cross-linking agent, Mitomycin-C. Cells were incubated in Mitomycin-C at concentrations ranging from 0-20 ng/ml for four days and then plated in 4 well tissue culture plates, incubated for 7 days at 37o C in a CO2 incubator, stained with crystal violet, and colonies of greater than 50 cells counted. Similar to Fancd2-/- marrow stromal cells, DKO marrow stromal cells showed increased sensitivity to Mito-C, while marrow stromal cell lines from wild type or SMAD3-/- mouse marrow cultures were relatively resistant (p = 0.0086 comparing Fancd2-/- to control 129/Sv X C57BL/6J F1 cells, p = 0.0156 comparing DKO with control 129/Sv X C57BL/6J F1 cells, and p = 0.4676 comparing Smad3-/- cells to control 129/Sv X C57BL/6J F1 cells). Fresh bone marrow from DKO mice, similar to that from SMAD3-/- mice, demonstrated resistance to inhibition of hematopoietic cell colonies in vitro by increasing concentrations of TGF-β. Therefore, abrogation of TGF-β signaling in DKO cells did not alter the sensitivity of Fancd2-/- marrow stromal cells to Mitomycin-C.Supported by research grant NIAID/NIH, U19A168021. DisclosuresNo relevant conflicts of interest to declare.

Strontium potently inhibits mineralisation in bone-forming primary rat osteoblast cultures and reduces numbers of osteoclasts in mouse marrow cultures.

SummaryThe basic mechanisms by which strontium ranelate acts on bone are still unclear. We show that an important action of strontium salts is to block calcification in cultures of osteoblasts, the bone-forming cells. These results suggest that strontium treatment could have previously overlooked effects on bone.IntroductionThe basic mechanisms of action of strontium ranelate (SrR) on bone have remained unclear. We studied the direct actions of Sr2+ salts in functional cultures of osteoblasts and osteoclasts.MethodsCultures of primary osteoblasts from rat calvariae and osteoclast-forming mouse marrow cells were treated continuously with either SrR or strontium chloride (SrCl2).ResultsAbundant, discretely mineralised ‘trabecular’ bone structures formed in control osteoblast cultures after 14 days. SrR at 0.01, 0.1 and 1 mM inhibited mineralisation to 59, 98 and 100 % (all p < 0.001) of control values, respectively. SrCl2 at the same concentrations caused similar inhibitions. Osteoblast cell numbers and alkaline phosphatase activity were unaltered. SrR dose-dependently reduced the formation of multinucleated osteoclasts from marrow mononuclear cells cultured on dentine for 8 days in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL), with a 50 % inhibition occurring at 1 mM; SrCl2 was slightly less effective, eliciting a maximal 30 % inhibition. Corresponding decreases in total resorption pit formation were observed, suggesting Sr2+ salts affect osteoclast formation rather than resorptive activity.ConclusionOur findings are consistent with the documented physicochemical inhibitory action of Sr2+ on mineralisation but contrast with reports that Sr2+ increases osteoblast activity and number in vitro. Our results suggest that rather than acting as an agent that ‘uncouples’ bone formation and resorption, Sr2+ acts as a global inhibitor of bone cell function, with particularly marked effects on mineralisation. The potential effects of long-term Sr2+ on secondary mineralisation in bone deserve investigation.

Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

Propolis inhibits osteoclast maturation

Propolis, a natural product produced by the honey bee, has been successfully used in medicine as an anti-inflammatory and antimicrobial agent. Traumatic injuries to the teeth, especially avulsion injuries, present a challenging situation for the clinician because of post-treatment complications, such as inflammatory and/or replacement resorption. Agents that reduce osteoclast numbers and activity may be useful in the treatment of traumatic injuries to the teeth. In this study, we evaluated propolis as an anti-resorptive agent. Calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts, were exposed to the ethanol extracts of propolis or vehicle control and stained for tartrate-resistant acid phosphatase (TRAP)-activity to identify osteoclasts. A significant, dose-dependent reduction in multinuclear TRAP+ cells was demonstrated, although the propolis treatment accommodated cell growth and survival (P < 0.05). Propolis also reduced the formation of actin rings in pure cultures of RAW 264.7 osteoclast-like cells, suggesting that it exerts direct actions on osteoclast maturation. In summary, our data suggest that propolis inhibits late stages of osteoclast maturation including fusion of osteoclasts precursors to form giant cells and formation of actin rings. This supports the hypothesis that it may prove useful as a medicament to reduce resorption associated with traumatic injuries to the teeth.

Identification of Enoxacin as an Inhibitor of Osteoclast Formation and Bone Resorption by Structure-Based Virtual Screening

An interaction between the B2 subunit of vacuolar H(+)-ATPase (V-ATPase) and microfilaments is required for osteoclast bone resorption. An atomic homology model of the actin binding site on B2 was generated and molecular docking simulations were performed. Enoxacin, a fluoroquinolone antibiotic, was identified and in vitro testing demonstrated that enoxacin blocked binding between purified B2 and microfilaments. Enoxacin dose dependently reduced the number of osteoclasts differentiating in mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D(3), as well as markers of osteoclast activity, and the number of resorption lacunae formed on bone slices. Enoxacin inhibited osteoclast formation at concentrations where osteoblast formation was not altered. In summary, enoxacin is a novel small molecule inhibitor of osteoclast bone resorption that acts by an unique mechanism and is therefore an attractive lead molecule for the development of a new class of antiosteoclastic agents.

Ki-energy (life-energy) stimulates osteoblastic cells and inhibits the formation of osteoclast-like cells in bone cell culture models.

Some practitioners of the Nishino Breathing Method (NBM) were found to have a higher bone density than the average values of age- and gender-matched non-practitioners. Using bone cell culture models, we investigated a possible mechanism behind this observation. For the study of bone mineralization, we performed the following two experiments using cultured osteoblastic MC3T3-E1 cells: (i) Kozo Nishino, a Japanese Ki expert, sent Ki-energy to the cells once for 5 or 10 min after they were seeded in culture dishes in the presence of 10% fetal bovine serum (FBS). They were incubated for 72 h and the cells were counted. The number in the dish with 10-min Ki-exposure was significantly greater than that in the control (P < 0.01 with n = 8). We performed a reverse transcription-polymerase chain reaction (RT–PCR) study using these cells, but the mRNA expressions did not change significantly. (ii) After cells were incubated for 72 h without Ki-exposure (in the presence of FBS), they were further cultured for 48 h (in the absence of FBS) to promote differentiation. At the beginning of the second culture stage, Ki was applied once for 10 min. After 48 h, RT–PCR was performed. The mRNA expressions which are related to bone mineralization, such as Runx2, α1(I) collagen, alkaline phosphatase and osteocalcin, increased significantly (P < 0.05 and n = 4 for all). For the bone resorption study, we used mouse marrow cultures, which can form osteoclast-like cells in the presence of (1–34) parathyroid hormone (PTH), and stimulate resorption. We exposed these cells to Ki-energy twice for the duration of 5 or 10 min on day 0 and day 4. On day 7, the cells were counted. The number of osteoclast-like cells in dishes with Ki exposure was significantly smaller than those in control dishes (P < 0.05 with n = 5). The difference between 5-min exposure and 10-min exposure was not statistically significant. All of our data suggest that the Ki-effect on osteoporosis should be further explored.

Eosinophil chemotactic factor-L (ECF-L) enhances osteoclast formation by increasing in osteoclast precursors expression of LFA-1 and ICAM-1

ECF-L is a novel autocrine stimulator of osteoclast (OCL) formation that enhances the effects of 1,25-(OH) 2D 3 and RANK ligand (RANKL) and is increased in inflammatory conditions such as rheumatoid arthritis. ECF-L acts at the later stages of OCL formation and does not increase RANKL expression. Thus, its mechanism of action is unclear. Therefore, RAW 264.7 cells and M-CSF-dependent murine bone marrow macrophage (MDBM) cells were treated with RANKL and/or with recombinant ECF-L expressed as a Fc fusion protein (ECF-L-Fc) to determine their effects on NF-κB, AP-1 and JNK activity, and on the expression of the adhesion molecules that have been implicated in OCL formation. These parameters were measured by semiquantitative and PCR and Western blot analysis. In addition, the role of ICAM-1 was further assessed by treating normal mouse marrow cultures with ECF-L-Fc and 10 − 10 M 1,25-(OH) 2D 3 in the presence or absence of a blocking ICAM-1 antibody or treating marrow cultures from ICAM-1 knockout mice with ECF-L and 1,25-(OH) 2D 3. ECF-L-Fc by itself only modestly increased NF-κB binding and JNK activity in RAW 264.7 cells, which was further enhanced by RANKL. In contrast, ECF-L-Fc increased LFA-1α and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10 − 10 M 1,25-(OH) 2D 3 and ECF-L. Furthermore, ECF-L did not increase OCL formation in marrow cultures from ICAM-1 knockout mice. Taken together, these results demonstrate that ECF-L enhances RANKL and 1,25-(OH) 2D 3-induced OCL formation by increasing adhesive interactions between OCL precursors through increased expression of ICAM-1 and LFA-1.

Mechanisms involved in the enhancement of osteoclast formation by enamel matrix derivative

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, and it has been reported that EMD can induce the formation of osteoclasts in mouse marrow cultures. In the present study, we investigated the mechanisms of EMD-induced osteoclast formation using a mouse monocytic cell line, RAW 264.7. Bioactive fractions were purified from EMD by reverse-phase HPLC using a C18 hydrophobic support, following which RAW 264.7 cells were cultured with EMD or its purified fractions in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) for 8 d. Following staining with tartrate-resistant acid phosphatase (TRAP), TRAP-positive multinucleated cells were counted. The expression of receptor activator of nuclear factor-kappaB (RANK), as well as phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, in RAW 264.7 cells were detected using immunoblotting. To determine whether EMD has an effect on osteoclast function, differentiated RAW 264.7 cells were cultured on Osteologic Multitest slides with RANKL in the presence of EMD. Purified EMD fractions (fraction numbers 21-25; EMD peak 2) were found to enhance the formation and function of RAW 264.7 cells induced by RANKL. Moreover, EMD peak 2 enhanced the levels of phosphorylation of ERK p38 and RANK in RAW 264.7 cells stimulated with RANKL. Our results indicate that EMD induces the formation of osteoclasts through interaction with RANKL, while ERK and p38 MAPK may play a critical role in the enhancement of osteoclast formation in RAW 264.7 cells.

Eosinophil Chemotactic Factor‐L (ECF‐L) Enhances Osteoclast Formation by Increasing ICAM‐1 Expression

Eosinophil chemotactic factor-L (ECF-L) is a novel stimulator of osteoclast (OCL) formation that acts at the differentiation/fusion stage of OCL formation, and is a cofactor for RANK ligand (RANKL). We examined the effects of ECF-L on the intracellular signaling pathways utilized by RANKL, and on the expression of ICAM-1/LFA-1 to determine its mechanism of action. RAW 264.7 and bone marrow cells were treated with RANKL and/or ECF-L Fc protein to determine their effect on NF-kappaB and AP-1 activity. ECF-L by itself only modestly increased NF-kappaB binding and JNK activity in RAW 264.7 cells, which were further enhanced by RANKL. In contrast, ECF-L Fc increased LFA-1alpha and ICAM-1 mRNA levels 1.8-fold in mouse marrow cultures, and anti-ICAM-1 almost completely inhibited OCL formation induced by 10(-10) M 1,25-(OH)2D3, and ECF-L Fc. Furthermore, ECF-L Fc did not enhance OCL formation by ICAM-1 knockout (KO) cells. Increased expression of ICAM-1 by ECF-L appears to be critical for its effects on OCL formation.

Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.

Enamel matrix derivative promotes osteoclast-like cell formation by RANKL production in mouse marrow cultures

Enamel matrix derivative promotes osteoclast-like cell formation by RANKL production in mouse marrow cultures

Regucalcin stimulates osteoclast‐like cell formation in mouse marrow cultures

The effect of regucalcin, a regulatory protein in intracellular signaling, on osteoclastic cell formation in mouse bone marrow culture is investigated. The bone marrow cells were cultured for 7 days in an alpha-minimal essential medium containing either vehicle or regucalcin (10(-10)-10(-8)M). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of regucalcin (10(-10)-10(-8)M) caused a remarkable increase in osteoclast-like multinucleated cells (MNCs). The effect of regucalcin in stimulating osteoclast-like cell formation was significantly inhibited in the presence of calcitonin (CT; 10(-9)M), 17beta-estradiol (10(-9)M), beta-cryptoxanthin (CX; 10(-6)M), or zinc sulfate (10(-4)M), which is an anti-bone resorbing factor. The effect of regucalcin on osteoclast-like cell formation was not significantly blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. The effect of parathyroid hormone (10(-7)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), prostaglandin E(2) (10(-5)M), or tumor necrosis factor-alpha (10 ng/ml) in increasing osteoclast-like cell formation was significantly enhanced in the presence of regucalcin (10(-8)M). Moreover, when rat femoral-diaphyseal or -metaphyseal tissues were cultured for 48 h in the presence of regucalcin (10(-10)-10(-8)M), the diaphyseal or metaphyseal calcium content was significantly decreased in the presence of regucalcin (10(-10)-10(-8)M) in vitro. The consumption of glucose and the production of lactic acid in culture medium by the diaphyseal or metaphyseal tissues was significantly raised in the presence of regucalcin (10(-10)-10(-8)M). This study demonstrates that regucalcin directly stimulates osteoclast-like cell formation in mouse marrow culture in vitro, and that the protein stimulates bone resorption in rat femoral tissues in vitro.

Effects of basic fibroblast growth factor on osteoclasts and osteoclast-like cells

Mouse marrow, which contains osteoblast and osteoclast precursors, was grown in the presence of calcitriol and/or basic fibroblast growth factor (FGF-2). RAW 264.7 cells were differentiated into osteoclast-like cells in the presence of receptor activator of NF-κB-Ligand (RANK-L) and/or FGF-2. FGF-2 alone supported osteoclastogenesis in mouse marrow cultures, but not by RAW 264.7 cells alone. Although FGF-2 supported low levels of osteoclastogenesis in mouse marrow cultures, it strongly inhibited the high levels of osteoclastogenesis triggered by calcitriol. Adding excess recombinant-RANK-L to the cultures did not relieve this inhibition. After mouse marrow osteoclasts were differentiated, FGF-2 dose-dependently inhibited bone resorptive activity. FGF-2 increased the tendency of RAW 264.7 osteoclast-like cells to fuse into very large giant cells and induced reorganizations of the actin cytoskeleton in mature, RANK-L-induced RAW 264.7 osteoclast-like cells. These results suggest that FGF-2 has both direct and indirect effects on osteoclast formation and bone resorption.

Inhibitory effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow cultures

The carotenoid β-cryptoxanthin has been shown to have an inhibitory effect on bone-resorping factor-stimulated bone resorption in rat bone tissues in vitro. The effect of β-cryptoxanthin on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in α-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1–34) (PTH), prostaglandin E 2, 1,25-dihydroxyvitamin D 3, lipopolysaccharide, or tumor necrosis factor-α (TNFα)] with an effective concentration. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10 −7 M), prostaglandin E 2 (10 −5 M), 1,25-dihydroxyvitamin D 3 (10 −7 M), lipopolysaccharide (10 μg/mL), or TNFα (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of β-cryptoxanthin (10 −8 to 10 −6 M). β-Cryptoxanthin (10 −7 and 10 −6 M) significantly inhibited dibutyryl cyclic adenosine monophosphate (DcAMP) (10 −5 M) or phorbol 12-myristate 13-acetate (PMA) (10 −5 M), an activator of protein kinase C, induced osteoclast-like cell formation. Also, β-cryptoxanthin (10 −7 and 10 −6 M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-κB ligand (RANKL) (10 and 20 ng/mL) in the presence of macrophage colony-stimulating factor (M-CSF) (10 and 20 ng/mL). The stimulatory effect of RANKL and M-CSF on osteoclast-like cell formation was significantly enhanced in the presence of PMA, while such an effect was not seen by DcAMP. β-Cryptoxanthin (10 −6 M) significantly inhibited osteoclast-like cell formation induced by RANKL and M-CSF in the presence of PMA or DcAMP. Moreover, the inhibitory effect of β-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFα-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10 −7 M), an inhibitor of protein synthesis at translational process, or 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (10 −6 M), an inhibitor of transcription. This study demonstrates that β-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of β-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Effects of matrix metalloproteinase inhibitors on bone resorption and orthodontic tooth movement.

Matrix metalloproteinases are involved in the regulation of bone remodeling. The hypothesis that matrix metalloproteinase inhibitors may be useful for experimentally limiting orthodontic tooth movement, a process involving perturbations of normal bone remodeling, was tested. General matrix metalloproteinase inhibitors limited the resorption of bone slices by mouse marrow cultures stimulated by calcitriol, parathyroid hormone, and basic-fibroblast growth factor. Pre-coating dentin slices with short arginine-glycine aspartic acid (RGD) peptides, but not arginine-glycine-glutamic acid (RGE) controls, restored bone resorption in the presence of matrix metalloproteinase inhibitors. Orthodontic tooth movement was inhibited by local delivery of Ilomastat, a general matrix metalloproteinase inhibitor, with the use of ethylene-vinyl-acetate (ELVAX) 40, a non-biodegradable, non-inflammatory sustained-release polymer. This study shows that orthodontic tooth movement can be inhibited with the use of matrix metalloproteinase inhibitors, and suggests a mechanistic link between matrix metalloproteinase activity and the production of RGD peptides.

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

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

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

TNFalpha and IL-1alpha 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 TNFalpha (+/- IL-1alpha) is also capable of inducing human osteoclastogenesis. The CD14(+) monocyte fraction of human peripheral mononuclear cells was cultured with TNFalpha +/- IL-1alpha in the presence of M-CSF. TNFalpha induced the formation of multinucleated cells (MNCs) which were positive for TRAP, VNR and cathepsin K and showed evidence of resorption pit formation. IL-1alpha stimulated TNFalpha-induced lacunar resorption two- to four-fold. Osteoprotegerin, the decoy receptor for RANKL, did not inhibit this process. Anti-human IL-1alpha neutralizing antibodies significantly inhibited resorption without inhibiting the formation of TRAP(+)/VNR(+) MNCs. These results suggest that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from circulating precursors by a process which is distinct from the RANK/RANKL signalling pathway.