Figure 2 - uploaded by Silvia Marino
Content may be subject to copyright.
Schematic illustration of isolation of osteoclast precursors from new-born mice (a) and adult mice (b) peripheral blood (c). See text under Isolation of osteoclast precursors in Materials and methods section for more details.
Source publication
Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution...
Contexts in source publication
Context 1
... step is repeated five times using fresh medium each time and the five fractions of bone marrow cells (containing both hae- matopoietic and stromal cells) are collected and combined in a 15 ml tube. The bone marrow cell suspension is centrifuged at 300 g for 3 min, the supernatant is discarded and the pellet is resuspended in 1 ml of culture medium 23 ( Figure 2a). ...
Context 2
... single cell suspension is obtained by passing the flushed bone marrow through needles of decreasing size (19G-21-25G) and transferring it into a conical 15 ml tube. The cell suspension is then centrifuged at 300 g for 3 min, the supernatant is discarded and the pellet is resuspended in 1 ml of culture medium ( Figure 2b). ...
Citations
... For osteoclastic differentiation, 7,000 cells per well of a 24-well plate were seeded and after 24 h stimulated with 100 ng/ml murine RANKL (day 0). Optimal RANKL concentration of 100 ng/ml was evaluated by testing a concentration series ranging from 10 to 200 ng/ml RANKL as suggested by Marino et al. [26]. After an additional 3 days, the medium and RANKL were renewed. ...
... Subsequently, adherent cells were stimulated with 30 ng/ml murine M-CSF for 7 days, and the medium was changed on days 3 and 5, followed by the addition of 100 ng/ml murine RANKL. Optimal concentrations for RANKL and M-CSF were evaluated by testing a concentration series ranging from 10 to 200 ng/ml for RANKL and 20-50 ng/ml for M-CSF as previously recommended [26]. The medium was changed on day 9 and day 11, and the medium volume was increased from 500 µl to 1000 µl. ...
Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.
... 36,37 Therefore, supplementing the culture medium with receptor activator of nuclear factor-kB ligand (RANKL) is sufficient to differentiate RAW264.7 cells into mature osteoclasts. 37,38 The differentiated RAW264.7 cells can then be cultured on bone tissue or biomaterials to assess interactions with osteoclasts and resorption in vitro, 38 an important feature for successful bone graft material considering osteointegration and bone remodeling. A significant body of literature exists around the interactions of osteoclasts with titanium and ceramics. ...
... 36,37 Therefore, supplementing the culture medium with receptor activator of nuclear factor-kB ligand (RANKL) is sufficient to differentiate RAW264.7 cells into mature osteoclasts. 37,38 The differentiated RAW264.7 cells can then be cultured on bone tissue or biomaterials to assess interactions with osteoclasts and resorption in vitro, 38 an important feature for successful bone graft material considering osteointegration and bone remodeling. A significant body of literature exists around the interactions of osteoclasts with titanium and ceramics. ...
Critically‐sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D‐printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D‐printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3‐E1 osteoblast (dMC3T3‐OB) and differentiated RAW264.7 osteoclast cells with 3D‐printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3‐OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast‐like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO‐based nanocomposites containing higher amounts of nHA have better interactions with osteoblast‐like and osteoclast‐like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair.
... To establish such a triple culture, media conditions are needed, allowing the simultaneous differentiation of OCys and OCs from their progenitors in the presence of OBs. Main obstacles are the different media requirements for OCy-differentiation (low serum) [25] and OC-formation from PBMC (10 % serum) [26]. ...
Bone remodelling, important for homeostasis and regeneration involves the controlled action of osteoblasts, osteocytes and osteoclasts. The present study established a three-dimensional human in vitro bone model as triple culture with simultaneously differentiating osteocytes and osteoclasts, in the presence of osteoblasts. Since high sulfated hyaluronan (sHA3) was reported as a biomaterial to enhance osteogenesis as well as to dampen osteoclastogenesis, the triple culture was exposed to sHA3 to investigate cellular responses compared to the respective bone cell monocultures. Osteoclast formation and marker expression was stimulated by sHA3 only in triple culture. Osteoprotegerin (OPG) gene expression and protein secretion, but not receptor activator of NF-κB ligand (RANKL) or sclerostin (SOST), were strongly enhanced, suggesting an important role of sHA3 itself in osteoclastogenesis with other targets than indirect modulation of the RANKL/OPG ratio. Furthermore, sHA3 upregulated osteocalcin (BGLAP) in osteocytes and osteoblasts in triple culture, while alkaline phosphatase (ALP) was downregulated.
... In Vitro Experiments. Mouse primary osteoclasts were differentiated from bone marrow-derived monocytes (BMMs) through the stimulation of M-CSF and RANKL, as previously described (53). Detailed protocols of in vitro osteoclast Fig. 7. Graphical abstract. ...
Long non-coding RNA (lncRNA) serves as a vital regulator of bone metabolism, but its role in pathologically overactive osteoclast differentiation remains elusive. Here, we identify lncRNA Dancr (Differentiation Antagonizing Non-protein Coding RNA) as a critical suppressor of osteoclastogenesis and bone resorption, which is down-regulated in response to estrogen deficiency. Global or osteoclast-specific Dancr Knockout mice display significant trabecular bone deterioration and enhanced osteoclast activity, but minimal alteration of bone formation. Moreover, the bone-targeted delivery of Dancr by Adeno-associated viral remarkably attenuates ovariectomy-induced osteopenia in mice. Mechanistically, Dancr establishes a direct interaction with Brahma-related gene 1 to prevent its binding and preserve H3K27me3 enrichment at the nuclear factor of activated T cells 1 and proliferator-activated receptor gamma coactivator 1-beta promoters, thereby maintaining appropriate expression of osteoclastic genes and metabolic programs during osteoclastogenesis. These results demonstrate that Dancr is a key molecule maintaining proper osteoclast differentiation and bone homeostasis under physiological conditions, and Dancr overexpression constitutes a potential strategy for treating osteoporosis.
... Bone marrow cells derived from WT mice or CX 3 CR1-EGFP/TRAP-tdTomato mice were cultured with 10 ng/mL M-CSF (R&D Systems) in α-MEM containing 10% fetal calf serum for 3 days. Then bone marrow macrophages were cultured for 3 days in the presence of 50 ng/mL RANKL (PeproTech) and 10 ng/mL M-CSF to induce differentiation into osteoclasts 58 . Human/mouse recombinant activin A (R&D Systems), mouse recombinant SFRP2 (R&D Systems), and mouse recombinant IGF2 (R&D Systems) were added at the concentrations indicated in Figure legends. ...
Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing loss and brain abscess formation. However, the pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a single-cell RNA sequencing analysis on human cholesteatoma samples and identify a pathogenic fibroblast subset characterized by abundant expression of inhibin βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in these fibroblasts results in decreased osteoclast differentiation in a murine model of cholesteatoma. Moreover, follistatin, an antagonist of activin A, reduces osteoclastogenesis and resultant bone erosion in cholesteatoma. Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone destruction via the induction of local osteoclastogenesis, suggesting a potential therapeutic target.
... Osteoclast precursors were obtained from CD11b + -enriched cells (with CD11b-Microbeads, Milteniy Biotec, France) from the flushed marrow fraction of WT mice as previously described 86 . They were added, in co-culture, to WT or mutant expanded osteogenic cells (5 × 10 4 cell/well) and stimulated with 1,25-dihydroxy vitamin D3 (vitD3, 10 nM), prostaglandin E2 (PGE2, 1 µM) and Dexamethasone (Dex, 50 nM) as previously described 65 in order to stimulate osteogenic cells to produce RankL/ MCSF and to inhibit OPG production 88,89 . OCL differentiation (multinucleated TRAP + cells) was quantified at day 8 after TRAP coloration as indicated above. ...
WHIM Syndrome is a rare immunodeficiency caused by gain-of-function CXCR4 mutations. Here we report a decrease in bone mineral density in 25% of WHIM patients and bone defects leading to osteoporosis in a WHIM mouse model. Imbalanced bone tissue is observed in mutant mice combining reduced osteoprogenitor cells and increased osteoclast numbers. Mechanistically, impaired CXCR4 desensitization disrupts cell cycle progression and osteogenic commitment of skeletal stromal/stem cells, while increasing their pro-osteoclastogenic capacities. Impaired osteogenic differentiation is evidenced in primary bone marrow stromal cells from WHIM patients. In mice, chronic treatment with the CXCR4 antagonist AMD3100 normalizes in vitro osteogenic fate of mutant skeletal stromal/stem cells and reverses in vivo the loss of skeletal cells, demonstrating that proper CXCR4 desensitization is required for the osteogenic specification of skeletal stromal/stem cells. Our study provides mechanistic insights into how CXCR4 signaling regulates the osteogenic fate of skeletal cells and the balance between bone formation and resorption.
... Then, cells were washed, and cell viability was evaluated with 1% trypan blue staining. Although no specific test was carried out in order to assess the conversion of THP-1 cells into macrophages, these cells, according to previous publications [16], were considered macrophages. ...
Objective
To analyze the possible in vitro effect of the cytokine RANKL and bacteria involved in apical periodontitis on the differentiation of macrophages into osteoclasts.
Material and methods
Bacteria were isolated (mainly E. faecium and E. faecalis) from the root canal of fifty patients with apical periodontitis, the possible effect of these bacteria on the phagocytic activity of the monocyte cell line THP-1 was analyzed by flow cytometry. Furthermore, the effect of these bacteria (alone or in combination with the cytokine RANKL) on the differentiation of THP-1 macrophages into osteoclasts was analyzed through the expression of the receptor RANK and the tartrate-resistant acid phosphatase TRAP. Finally, the release of different cytokines (IL-1β, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) by THP-1 cells induced to differentiate into osteoclasts was also analyzed.
Results
We observed a significant proportion of THP-1 cells were able to internalize E. faecium and E. faecalis. Furthermore, these bacteria were able to induce (alone or in combination with RANKL) a significant expression of RANK by THP-1 macrophages; accordingly, E. faecium and E. faecalis induced very significant levels of TRAP in these cells. Finally, during the differentiation of THP-1 macrophages induced by RANKL or bacteria, a significant release of the pro-inflammatory cytokines IL-6 and TNF-α was observed.
Conclusions and clinical relevance
Our data suggest that the causative agents of apical periodontitis can induce the differentiation of osteoclasts as well as the release of pro-inflammatory cytokines, phenomena that may have an important role in the bone damage observed in this condition.
... Osteoclasts were generated by the methods previously described [64,65]. Primary rat bone marrow macrophages (BMMs) from the whole bone marrow were isolated from the femur and tibia cavities of 4-week-old Sprague-Dawley rats. ...
Osteoimmunology mediators are critical to balance osteoblastogenesis and osteoclastogenesis to maintain bone homeostasis. A lot of the osteoimmunology mediators are regulated by interleukin-20 (IL-20). However, little is known about the role of IL-20 in bone remodeling. Here, we showed that IL-20 expression was correlated with osteoclast (OC) activity in remodeled alveolar bone during orthodontic tooth movement (OTM). Ovariectomize (OVX) in rats promoted OC activity and enhanced IL-20 expression, while blocking OC inhibited IL-20 expression in osteoclasts. In vitro, IL-20 treatment promoted survival, inhibited apoptosis of the preosteoclast at the early stages of osteoclast differentiation, and boosted the formation of osteoclasts and their bone resorption function at the late stages. More importantly, anti-IL-20 antibody treatment blocked IL-20-induced osteoclastogenesis and the subsequent bone resorption function. Mechanistically, we showed that IL-20 synergistically acts with RANKL to activate the NF-κB signaling pathway to promote the expression of c-Fos and NFATc1 to promote osteoclastogenesis. Moreover, we found that local injection of IL-20 or anti-IL-20 antibody enhanced osteoclast activity and accelerated OTM in rats, while blocking IL-20 reversed this phenomenon. This study revealed a previously unknown role of IL-20 in regulating alveolar bone remodeling and implies the application of IL-20 to accelerated OTM.
... In the bone, receptor activator of NF-κB (RANK) and RANK ligand (RANKL) signalling induces downstream activation of nuclear factor of activated T cells 1 (NFATc1), which collaborates with other transcription factors such as microphthalmia-associated transcription factor (MITF) to regulate osteoclast-specific genes and maintain osteoclast identity [52]. Osteoclast culture in 2D is well established and as a minimum requires RANKL together with CSF1 [53]; it would therefore follow that these molecules should be considered for the 3D culture of osteoclasts. While microglia and osteoclasts have reasonably well-defined in vitro cytokine requirements, it is less clear for other TRMs. ...
The advent of 3D cell culture technology promises to enhance understanding of cell biology within tissue microenvironments. Whilst traditional cell culturing methods have been a reliable tool for decades, they inadequately portray the complex environments in which cells inhabit in vivo. The need for better disease models has pushed the development of effective 3D cell models, providing more accurate drug screening assays. There has been great progress in developing 3D tissue models in fields such as cancer research and regenerative medicine, driven by desires to recreate the tumour microenvironment for the discovery of new chemotherapies, or development of artificial tissues or scaffolds for transplantation. Immunology is one field that lacks optimised 3D models and the biology of tissue resident immune cells such as macrophages has yet to be fully explored. This review aims to highlight the benefits of 3D cell culturing for greater understanding of macrophage biology. We review current knowledge of macrophage interactions with their tissue microenvironment and highlight the potential of 3D macrophage models in the development of more effective treatments for disease.
... In vitro studies on osteoclast differentiation, activity, and their interaction with other cell types such as osteoblasts make use of cell culture medium containing FBS [16]- [19]. Studies on osteoclast differentiation in a serum substitute medium are still in their infancy probably due to the limited availability of osteoclasts, their complex differentiation process, high donor variability, and short lifespan which make studying osteoclasts challenging in general [20]- [22]. ...
Fetal bovine serum (FBS) is a widely used supplement in cell culture media despite its known drawbacks, including ethical, safety, and scientific issues. To overcome the drawbacks of using FBS in cell culture, a defined serum substitute medium needs to be developed. The development of such a medium depends on the cell type, which makes it impossible to use one universal serum substitute medium for all cells. Osteoclasts are large, multinucleated cells originated from the hematopoietic stem cell lineage that play an important role in regulating bone mass and quality. To date, no defined serum substitute medium formulations have been reported for osteoclast differentiation of monocytes derived from peripheral blood mononuclear cells (PBMCs). Here, we have attempted to develop such a serum substitute medium for the osteoclastogenesis process in a stepwise approach. Essential components were added to the medium while monocytes were cultured in 96-well plates and in Osteo-Assay well plates to analyze the formation of tartrate resistant acid phosphatase (TRAP) expressing multinucleated osteoclasts with distinct actin ring and to analyze the resorption activity of mature osteoclasts for 21 days, respectively. The serum substitute medium was aimed at supporting monocyte and later osteoclast survival, differentiation of monocytes towards multinucleated osteoclasts, and the resorption of mineralized matrix as a measure of functionality. All points were achieved after 21 days of culture in the developed serum substitute medium. This serum substitute medium could potentially replace FBS in osteoclastogenesis studies eliminating its debated use. Moreover, the well-defined serum substitute environment simplifies the study of factors released by the cells that were so far overwhelmed by the complexity of FBS.
