Establishment and validation of an efficient method for the 3D culture of osteoclasts in vitro.

INTRODUCTION: Osteoclasts (OCs) play a crucial role in maintaining bone health. Changes in OC activity are linked to different bone diseases, making them an intriguing focus for research. However, most studies on OCs have relied on 2D cultures, limiting our understanding of their behavior. Yet, there's a lack of knowledge regarding platforms that effectively support osteoclast formation in 3D cultures. METHODS: In our investigation, we explored the capacity of collagen and GelMA hydrogels to facilitate osteoclast development in 3D culture settings. We assessed the osteoclast development by using different hydrogels and cell seeding strategies and optimizing cell seeding density and cytokine concentration. The osteoclast development in 3D cultures was further validated by biochemical assays and immunochemical staining. RESULTS: Our findings revealed that 0.3 % (w/v) collagen was conducive to osteoclast formation in both 2D and 3D cultures, demonstrated by increased multinucleation and higher TRAP activity compared to 0.6 % collagen and 5 % to 10 % (w/v) GelMA hydrogels. Additionally, we devised a "sandwich" technique using collagen substrates and augmented the initial macrophage seeding density and doubling cytokine concentrations, significantly enhancing the efficiency of OC culture in 3D conditions. Notably, we validated osteoclasts derived from macrophages in our 3D cultures express key osteoclast markers like cathepsin K and TRAP. CONCLUSIONS: To conclude, our study contributes to establishing an effective method for cultivating osteoclasts in 3D environments in vitro. This innovative approach not only promises a more physiologically relevant platform to study osteoclast behavior during bone remodeling but also holds potential for applications in bone tissue engineering. CLINICAL SIGNIFICANCE: This study introduces an efficient method for cultivating osteoclasts in 3D environments in vitro. It offers a more physiologically relevant platform to investigate osteoclast behavior and holds promise to advance research in bone biology and regenerative dentistry.

Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFß1/SMAD3 signaling.

Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor ß1 (TGFß1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation.

Rg3 inhibits gemcitabine-induced lung cancer cell invasiveness through ROS-dependent, NF-κB- and HIF-1α-mediated downregulation of PTX3.

PTX3, a member of the long pentraxin subfamily, associated with innate immunity is indispensable for resistance to some cancer. Gemcitabine, an analog of cytosine arabinoside, has shown restrained benefits because of profound chemoresistance. The PTX3 expression on GEM in human lung cancer cells have not yet been clarified; the present study aimed to show reactive oxygen species (ROS) mediatory PTX3 expression through distinct mechanisms. Whereas ginsenoside Rg3 is a herbal medicine with strong antitumor activity. Furthermore, we tested the hypothesis; Rg3 abrogates GEM-induced production of ROS-mediated activation of Akt and extracellular signal-regulated kinase (ERK) pathways and inhibits nuclear piling-up of nuclear factor kappa B (NF-κB) and HIF-1α. On the basis of time and dose-dependent manner, our data demonstrated that GEM-induced PTX3 expression was dependent on ROS generation as it was abrogated by pretreatment of lung cancer cells with the free radical scavenger N-acetyl-l-cysteine. Our data demonstrated that PTX3 upregulation by GEM correlated with the time-dependent escalation of NF-κB and HIF-1α in the nucleus resulted from phosphorylation-induced degradation of IκBα, whereas HIF-1α upregulation was NF-κB-dependent. Increase in ROS expression in lung cancer cells on GEM treatment preceded the nuclear accumulation of NF-κB and HIF-1α and suppression of ROS diminished these effects. ERK1/2 and Akt activation mediated the effect of ROS on NF-κB and HIF-1α and their pharmacological inhibition suppressed GEM-induced PTX3. Our study findings reinforced the role regarding PTX3 signaling in GEM-induced resistance and pointed toward an unintended and undesired effect of chemotherapy and to get an active regimen; the synergy was associated with NF-κB downregulation in lung cancer.