Human Osteoblast Cell Line hFOB Research Articles

Effect of luteolin on oxidative stress and inflammation in the human osteoblast cell line hFOB1.19 in an inflammatory microenvironment

BackgroundPeriapical lesions are characterized by periapical inflammation and damage to periapical tissues and eventually lead to bone resorption and even tooth loss. H2O2 is widely used in root canal therapy for patients with periapical inflammation. Luteolin possesses high anti-inflammatory, antioxidant, and anticancer potential. However, the underlying mechanism of the efficacy of H2O2 and luteolin on oxidative stress and inflammatory tissue has not been previously addressed. We aimed to investigate the anti-inflammatory and antioxidative effects of luteolin on H2O2-induced cellular oxidative inflammation.MethodsAfter human osteoblasts (hFOB1.19) were treated with lipopolysaccharide (LPS), luteolin, or H2O2, cell proliferation was analysed by using a cell counting kit-8 (CCK-8), cell apoptosis was measured by using flow cytometry, the production of reactive oxygen species (ROS) was evaluated by using an oxidation-sensitive probe DCFH-DA ROS assay kit, and the expression of genes and proteins was detected by using reverse transcription quantitative polymerase chain reaction (RT‒qPCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA).ResultsWe demonstrated that inflammation is closely related to oxidative stress and that the oxidative stress level in the inflammatory environment is increased. Luteolin inhibited the H2O2-induced increase in the expression of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor α (TNF-α) and significantly repressed the H2O2-induced increase in ROS, as well as markedly strengthened superoxide dismutase (SOD) activity in hFOB1.19 cells. Moreover, we detected that luteolin may inhibit H2O2-induced hFOB1.19 cell injury by suppressing the NF-κB pathway.ConclusionWe elucidated that luteolin protected human osteoblasts (hFOB1.19) from H2O2-induced cell injury and inhibited the production of proinflammatory cytokines by suppressing the NF-κB signalling pathway. Our findings provide a potential drug for treating H2O2-induced periodontitis and cell injury.

Negative Regulation of LINC01013 by METTL3 and YTHDF2 Enhances the Osteogenic Differentiation of Senescent Pre-Osteoblast Cells Induced by Hydrogen Peroxide.

Senescent pre-osteoblasts have a reduced ability to differentiate, which leads to a reduction in bone formation. It is critical to identify the keys that regulate the differentiation fate of senescent pre-osteoblasts. LINC01013 has an essential role in cell stemness, differentiation, and senescence regulation. This study aims to examine the role and mechanism of LINC01013 in regulating osteogenic differentiation in senescent human embryonic osteoblast cell line (hFOB1.19) cells induced by hydrogen peroxide (H2O2). The results show that LINC01013 decreased alkaline phosphatase activity, mineralization of hFOB1.19 cells in vitro, and the expression of collagen II, osteocalcin, and bone sialoprotein. LINC01013 knockdown enhances the osteogenesis of hFOB1.19 cells and rescues osteogenic differentiation impaired by H2O2. METTL3 negatively regulates LINC01013 expression, enhancing hFOB1.19 cells' osteogenesis in vitro and in vivo. METTL3 overexpression can enhance hFOB1.19 cells' osteogenic differentiation impaired by H2O2. YTHDF2 promotes LINC01013 decay, facilitating osteogenic differentiation. YTHDF2 overexpression rescues hFOB1.19 cells osteogenic differentiation impaired by H2O2. Taken together, METTL3 upregulates osteogenic differentiation by inhibiting LINC01013, and YTHDF2 accelerates LINC01013 degradation, reducing its inhibitory effect. This study highlights LINC01013 as a key regulator in the fate switching process of senescent hFOB1.19 cells, impacting osteogenic differentiation.

Characterization of hFOB 1.19 Cell Line for Studying Zn-Based Degradable Metallic Biomaterials.

In vitro testing is the first important step in the development of new biomaterials. The human fetal osteoblast cell line hFOB 1.19 is a very promising cell model; however, there are vast discrepancies in cultivation protocols, especially in the cultivation temperature and the presence of the selection reagent, geneticin (G418). We intended to use hFOB 1.19 for the testing of Zn-based degradable metallic materials. However, the sensitivity of hFOB 1.19 to zinc ions has not yet been studied. Therefore, we compared the toxicity of zinc towards hFOB 1.19 under different conditions and compared it with that of the L929 mouse fibroblast cell line. We also tested the cytotoxicity of three types of Zn-based biomaterials in two types of media. The presence of G418 used as a selection reagent decreased the sensitivity of hFOB 1.19 to Zn2+. hFOB 1.19 cell line was more sensitive to Zn2+ at elevated (restrictive) temperatures. hFOB 1.19 cell line was less sensitive to Zn2+ than L929 cell line (both as ZnCl2 and extracts of alloys). Therefore, the appropriate cultivation conditions of hFOB 1.19 during biomaterial testing should be chosen with caution.

Preosteoclast plays a pathogenic role in syndesmophyte formation of ankylosing spondylitis through the secreted PDGFB — GRB2/ERK/RUNX2 pathway

ObjectivesAnkylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the sacroiliac joint and spine. However, the real mechanisms of immune cells acting on syndesmophyte formation in AS are not well identified. We aimed to find the key AS-associated cytokine and assess its pathogenic role in AS.MethodsA protein array with 1000 cytokines was performed in five AS patients with the first diagnosis and five age- and gender-matched healthy controls to discover the differentially expressed cytokines. The candidate differentially expressed cytokines were further quantified by multiplex protein quantitation (3 AS-associated cytokines and 3 PDGF-pathway cytokines) and ELISA (PDGFB) in independent samples (a total of 140 AS patients vs 140 healthy controls). The effects of PDGFB, the candidate cytokine, were examined by using adipose-derived stem cells (ADSCs) and human fetal osteoblast cell line (hFOB1.19) as in vitro mesenchymal cell and preosteoblast models, respectively. Furthermore, whole-transcriptome sequencing and enrichment of phosphorylated peptides were performed by using cell models to explore the underlying mechanisms of PDGFB. The xCELLigence system was applied to examine the proliferation, chemotaxis, and migration abilities of PDGFB-stimulated or PDGFB-unstimulated cells.ResultsThe PDGF pathway was observed to have abnormal expression in the protein array, and PDGFB expression was further found to be up-regulated in 140 Chinese AS patients. Importantly, PDGFB expression was significantly correlated with BASFI (Pearson coefficient/p value = 0.62/6.70E − 8) and with the variance of the mSASSS score (mSASSS 2 years − baseline, Pearson coefficient/p value = 0.76/8.75E − 10). In AS patients, preosteoclasts secreted more PDGFB than the healthy controls (p value = 1.16E − 2), which could promote ADSCs osteogenesis and enhance collagen synthesis (COLI and COLIII) of osteoblasts (hFOB 1.19). In addition, PDGFB promoted the proliferation, chemotaxis, and migration of ADSCs. Mechanismly, in ADSCs, PDGFB stimulated ERK phosphorylation by upregulating GRB2 expression and then increased the expression of RUNX2 to promote osteoblastogenesis of ADSCs.ConclusionPDGFB stimulates the GRB2/ERK/RUNX2 pathway in ADSCs, promotes osteoblastogenesis of ADSCs, and enhances the extracellular matrix of osteoblasts, which may contribute to pathological bone formation in AS.

Multifunctional Zn and Ag co-doped bioactive glass nanoparticles for bone therapeutic and regeneration

Bone cancer has traditionally been treated using surgery, radiotherapy, and/or chemotherapy. The nonspecific distribution of chemotherapy and implantable infections are significant risk factors for the failure of the bone to heal. Multifunctional zinc and silver co-doped bioactive glass nanoparticles (yAg–xZn-BGNPs) with a diameter of 150 ± 30 nm were successfully synthesized using modified sol–gel and two-step post-functionalization processes, tailored to provide antibacterial and anticancer activity whilst maintaining osteogenesis ability. Co-doped BGNPs with Zn and Ag did not significantly alter physicochemical properties, including size, morphology, glass network, and amorphous nature. Apatite-like layer was observed on the surface of yAg–xZn-BGNPs and resorbed in the simulated body fluid solution, which could increase their bioactivity. Human fetal osteoblast cell line (hFOB 1.19) treated with particles showed calcified tissue formation and alkaline phosphatase activity in the absence of osteogenic supplements in vitro, especially with 0.5Ag–1Zn-BGNPs. Moreover, these particles preferentially disrupted the metabolic activity of bone cancer cells (MG-63) and had an antibacterial effect against B. subtilis, E. coli, and S. aureus via the disc diffusion method. This novel 0.5Ag–1Zn-BGNP and 1Ag–1Zn-BGNPs, with wide-ranging ability to stimulate bone regeneration, to inhibit bone cancer cell proliferation, and to prevent bacterial growth properties, may provide a feasible strategy for bone cancer treatment. The 0.5Ag–1Zn-BGNPs and 1Ag–1Zn-BGNPs can be applied for the preparation of scaffolds or filler composites using in bone tissue engineering.

Abstract 3609: Interaction of interleukin-6/8/18 with prostate cancer cell secreted growth factors are crucial for osteoblast proliferation and differentiation

Abstract There is currently no effective therapy for bone metastatic prostate cancer, which is the main cause of mortality in people with prostate cancer. Poor prognosis of bone metastasis has limitations in current therapeutics mainly due to the complexity of microenvironment. Our focus was on the interactions between prostate cancer cells and the bone microenvironment, including osteoclasts and osteoblasts in regard to growth factors and cytokines, as the bone microenvironment plays a significant role in this process. The present study shows the downstream effects of Stavudine (KB-S); a known antiviral compound along with 5-fluoro-1-((1R,4R)-4-((tetrahydro-2H-pyran-2-yl)oxy)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KB-G) and 1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KB-H) on bone metastatic prostate cancer. Effects of KB-G/H and S on HEK-293 control cells were determined as moderate under tested conditions. A series of KB-G/H and S (10, 25, 50, 75 and 100 µM) was given for a 4-day period (with 24 h intervals) and the prostate cancer cell population was determined at the end of the 4-day treatment period. Preliminary results indicated that all 3 compounds significantly decreased DU-145 and PC-3 cell population compared to the effects on HEK-293 cells. Results suggested that all 3 compounds can decrease the prostate cancer cell progression by enhancing apoptosis. Our data also suggested that all 3 compounds downregulated interleukin (IL)-18, IL-8, IL-6 and C-X-C motif ligand-1(CXCL-1) in prostate cancer cells (PC-3 and DU-145). A human osteoblast cell line (hFOB 1.19) is being used to study the effect of cytokines in relation to transforming growth factor β (TGFβ), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) that are secreted by prostate cancer cells. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence microscope, cytokine profile array, real time qPCR and Western blot techniques are being used in this study to further analyze role of cytokines and growth factors on osteoblast differentiation. Our preliminary results suggest that all 3 compounds can be used to disrupt the main steps of prostate cancer bone metastasis. Results suggested that KB-G/H and S can be targeted to develop customized, tailored therapies for bone metastatic prostate cancer which merit further research. Citation Format: Aaron Jeshua Todman, Wishrawana S. Ratnayake, Luke Lajmi, Sloan Breedy, Kirpal Bisht, Mildred Acevedo-Duncan. Interaction of interleukin-6/8/18 with prostate cancer cell secreted growth factors are crucial for osteoblast proliferation and differentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3609.

Development of nanoparticles for delivering recombinant human secretory leukocyte protease inhibitor (rhSLPI) for enhancing human osteoblast differentiation

The global number of elderly people has been increasing and the world can now be considered as an aging society. One of the most concerning health problems in the elderly is a musculoskeletal disease, in which fractures and osteoporosis are the most common problems. The deteriorating bone quality in the elderly is a limitation for treatment and for patient recovery. Several drugs and biomolecules have been studied for enhancing osteoblast properties and differentiation. The secretory leukocyte protease inhibitor (SLPI), which is a serine protease inhibitory protein, has been reported to enhance osteoblast cell adhesion, proliferation, and differentiation. However, the application of SLPI in real clinical settings is limited due to its short half-life in circulation and the fact that it can be destroyed by circulating protease enzymes. Therefore, the application of nanoparticle encapsulation might be of benefit for SLPI delivery. This study aims to fabricate the liposome nanoparticles encapsulating recombinant human SLPI or rhSLPI (rhSLPI-LNPs) for augmenting the half-life and enhancing human osteoblast differentiation. The liposome nanoparticles (LNPs) were fabricated by the thin film hydration method. Encapsulation of rhSLPI was performed by adding 0.33 µg/mL rhSLPI in ultrapure water to the thin film lipid and then rhSLPI-LNPs formed into vesicles. The size and zeta potential of blank-LNPs or rhSLPI-LNPs were measured by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The results showed that the size of both blank-LNPs and rhSLPI-LNPs was approximately 200 nm, and the zeta potentials were −55.6 ± 16.90 mV and −64 ± 7.86 mV, respectively. The rhSLPI encapsulation was confirmed by ELISA. The results showed that liposome nanoparticles could encapsulate rhSLPI. For cytotoxicity, rhSLPI-LNPs treatment did not cause any toxicity to the human osteoblast cell line (hFOB 1.19). In addition, pre-incubation of hFOB 1.19 with rhSLPI-LNPs could significantly enhance osteoblast adhesion when compared with the untreated group.

Rspo1 inhibited apoptosis of glucocorticoid-induced osteoblasts via Wnt/β-catenin pathway in Legg-Calve-Perthes disease.

The pathogenesis of Legg-Calve-Perthes disease (LCPD), a juvenile form of avascular necrosis of the femoral head (ANFH), is not fully understood. The purpose of this work was to study the regulatory effect of R-spondin 1 (Rspo1) on osteoblastic apoptosis and evaluate the pre-clinical efficacy of recombinant human protein Rspo1 (rhRspo1) in treatment of LCPD. This is an experimental study. In vivo rabbit ANFH model was established. Human osteoblast cell line hFOB1.19 (hFOB) was used to overexpress and silence Rspo1 in vitro. Additionally, hFOB cells were induced with glucocorticoid (GC) and methylprednisolone (MP), and treated with rhRspo1. The expressions of Rspo1, β-catenin, Dkk-1, Bcl-2, and caspase-3, and the apoptosis rate of hFOB cells were examined. The expressions of Rspo1 and β-catenin were lower in ANFH rabbits. The expression of Rspo1 was decreased in GC-induced hFOB cells. Compared to the control group, after 1 μM MP induction for 72 h, the expressions of β-catenin and Bcl-2 were higher, while Dkk-1, caspase-3 and cleaved caspase-3 expressions were lower in Rspo1 overexpression and rhRspo1-treated groups. The apoptosis rate of GC-induced hFOB cells was decreased in Rspo1 overexpression and rhRspo1-treated groups compared to the control group. R-spondin 1 inhibited GC-induced osteoblast apoptosis via Wnt/β-catenin pathway, which might be associated with the development of ANFH. Moreover, rhRspo1 had a potential pre-clinical therapeutic effect on LCPD.

Microrna-206 induces hypoxic necrosis of femoral head by inhibiting VEGF/PI3K/AKT signaling pathway.

The most common form of non-traumatic necrosis of the femoral head is anoxic necrosis of the femoral head, which is a metabolic disease, mainly involving young and middle-aged people. Apoptosis and its related signal regulation pathway play an important role in the occurrence and development of hypoxic necrosis of the femoral head. In order to investigate the possible pathological manifestations of miR-206 and VEGF/PI3K/AKT signal pathway genes and their interactions in hypoxic necrosis of the femoral head, this paper intended to systematically study the expression and regulation mechanism of miR-206 and VEGF/PI3K/AKT signal pathway genes. The interaction between miR-206 and VEGF/PI3K/AKT signaling pathway and its regulation on apoptosis, differentiation and proliferation of human osteoblast cell line hFOB1.19 (SV40 transfer of human osteoblasts) were studied by double luciferase reporter gene analysis, overexpression and inhibition of miR-206, and gene silencing of VEGF/PI3K/AKT signaling pathway. After 24h and 48h of intervention with MicroRNA 206 on osteoblasts, it was found that the fluorescence intensity of caspase-3 was higher than that of 0h group (p < 0.05). This paper has provided an important research basis for the research of femoral head necrosis and the development of new diagnosis and therapeutic drugs for this kind of disease. It also has provided a reference for the further promotion of the chemotherapy drug delivery system.

Magnetic Separation of Cell-Secreted Vesicles with Tailored Magnetic Particles and Downstream Applications.

The analysis of the receptors on the surface of the cell-secreted vesicles provides valuable information of the cell signature and may also offer diagnosis and/or prognosis of a wide range of diseases, including cancer.Due to their low concentration, conventional procedures for extracellular vesicle (EV) detection usually require relatively large sample volumes, involving preliminary purification or preconcentration steps from complex specimens. Here, we describe the separation and preconcentration in magnetic particles of extracellular vesicles obtained from cell culture supernatants from MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines, human fetal osteoblastic cell line (hFOB), and human neuroblastoma SH-SY5Y cell line, as well as exosomes from human serum. The first approach involves the covalent immobilization for the exosomes directly on micro (4.5μm)-sized magnetic particles. The second approach is based on tailored magnetic particles modified with antibodies for further immunomagnetic separation of the exosomes. In these instances, micro (4.5μm)-sized magnetic particles are modified with different commercial antibodies against selected receptors, including the general tetraspanins CD9, CD63, and CD81 and the specific receptors (CD24, CD44, CD54, CD326, CD340, and CD171). The magnetic separation can be easily coupled with downstream characterization and quantification methods, including molecular biology techniques such as immunoassays, confocal microscopy, or flow cytometry.

Physicochemical degradation of calcium magnesium phosphate (stanfieldite) based bone replacement materials and the effect on their cytocompatibility

Regenerative bone implants should be completely replaced by new bone within a period of time corresponding to the growth rate of native bone. To meet this requirement, suitable biomaterials must be biodegradable and promote osteogenesis. The combination of slowly degrading but osteoconductive calcium phosphates (CPs) with rapidly degrading and mechanically more resilient magnesium phosphates represents a promising material class for this purpose. In order to create the best possible conditions for optimal implant integration, microporous calcium magnesium phosphate (CMP) cements were processed using 3D powder printing. This technique enables the production of a defect-adapted implant with an optimal fit and a high degree of open porosity to promote bone ingrowth. Four different compositions of 3D printed CMP ceramics were investigated with regard to essential properties of bone implants, including chemical composition, porosity, microstructure, mechanical strength, and cytocompatibility. The ceramics consisted of farringtonite (Mg3(PO4)2) and stanfieldite (Ca4Mg5(PO4)6), with either struvite (NH4MgPO4·6H2O) or newberyite (MgHPO4·3H2O) and brushite (CaHPO4·2H2O) as additional phases. The CMP materials showed open porosities between 13 and 28% and compressive strengths between 11 and 17 MPa, which was significantly higher, as compared with clinically established CP. The cytocompatibility was evaluated with the human fetal osteoblast cell line hFOB 1.19 and was proven to be equal or to even exceed that of tricalcium phosphate. Furthermore, a release of 4–8 mg magnesium and phosphate ions per mg scaffold material could be determined for CMPs over a period of 21 d. In the case of struvite containing CMPs the chemical dissolution of the cement matrix was combined with a physical degradation, which resulted in a mass loss of up to 3.1 wt%. In addition to its beneficial physical and biological properties, the proven continuous chemical degradation and bioactivity in the form of CP precipitation indicate an enhanced bone regeneration potential of CMPs.

LncRNA ZFAS1 contributes to osteosarcoma progression via miR-520b and miR-520e-mediated inhibition of RHOC signaling

ObjectivesWe examined the expression of Lnc-ZFAS1 in osteosarcoma and comprehensively evaluated its effects on osteosarcoma in vitro and vivo. Moreover, we revealed the regulatory mechanism between Lnc-ZFAS1 and miR-520b/miR-520e-mediated RHOC and provided a novel clue for ameliorating osteosarcoma. MethodThe expression of Long non-coding RNA Zinc Finger Antisense 1 (LncRNA ZFAS1) osteosarcoma tissues and normal tissues in the TCGA database was analyzed. Then, LncRNA ZFAS1 expression was further verified in clinical samples and osteosarcoma cell lines (U2OS and KHOS), as well as the human osteoblast cell line hFOB1.19 by qRT-PCR. Thereafter, LncRNA ZFAS1 was overexpressed or silenced to explore its effects on cell proliferation, apoptosis, migration, invasion, and Epithelial-Mesenchymal Transition (EMT). The fundamental mechanism through which Lnc-ZFAS1 affects osteosarcoma progression was further investigated and verified. ResultsWe found that LncRNA ZFAS1 was upregulated in osteosarcoma, and Lnc-ZFAS1 overexpression facilitated osteosarcoma cells proliferation, migration, invasion and EMT, while Lnc-ZFAS1 silence exerted reverse influence. Mechanistically, Lnc-ZFAS1 functionally acted as a sponger of microRNA-520b (miR-520b) and microRNA-520e (miR-520e) to up-regulate Ras Homologue C (RHOC). In addition, depleted Lnc-ZFAS1 restrained osteosarcoma cells proliferation, migration, and invasion, which could be rescued by RHOC overexpression. Lnc-ZFAS1 was upregulated in osteosarcoma and Lnc-ZFAS1 could exert promoted impact upon osteosarcoma cells proliferation, migration, invasion, and EMT in vitro. ConclusionsLnc-ZFAS1 acted sponger of miR-520b and miR-520e to promote RHOC, indicating that Lnc-ZFAS1/miR-520b/RHOC and Lnc-ZFAS1/miR-520e/RHOC axes might serve as potential therapeutic strategies against osteosarcoma.

Mir-Oz Kegulates Osteoblast Apoptosis, Proliferation and Differentiation through Targeted Inhibition of Runx2 in Osteoporosis Running Title: Role and Potential Mechanism of Mir-152 in Osteoporosis

Objective: To explore the role and potential mechanism of miR-152 in osteoporosis. Methods: Fifty-four osteoporotic patients and 54 healthy subjects were recruited from August 2017 to January 2019. Serum samples of the two groups were obtained, and the miR-152 expression in serum was detected and compared. The human osteoblast cell line hFOB1.19 was obtained and miR-152 in cells was increased. The biological behavior changes such as cell proliferation, apoptosis and differentiation were observed by MTT, flow cytometry and detection of osteoblast differentiation markers (ALP, OCN). Results: miR-152 was elevated in osteoporosis patients, and AUC value of serum miR-152 in diagnosing osteoporosis was 0.939. After miR-152 in osteoblasts was elevated, cell proliferation was inhibited, cell apoptosis rate increased, and ALP and OCN content in cells reduced, while increasing cell RUNX2 simultaneously was totally different. Dual luciferase report showed that RUNX2 could be targeted and regulated by miR-152. Conclusion: miR-152 is elevated in serum of osteoporosis patients and can be used as a biological indicator for diagnosing osteoporosis. In addition, miR-152 can inhibit osteoblast proliferation, differentiation and induce apoptosis through negative regulation of RUNX2.

Silencing of LINC00707 suppresses cell proliferation, migration, and invasion of osteosarcoma cells by modulating miR-338-3p/AHSA1 axis.

Osteosarcoma is the most common type of primary malignant tumor of the bone, with a high metastatic rate and poor prognosis. Therefore, it is important to further elucidate the molecular mechanisms involved in the development of osteosarcoma and explore new molecular therapeutic targets. Long intergenic nonprotein-coding RNA 707 (LINC00707) is an oncogenic gene in several cancers. In this study, we further clarified its role and regulatory mechanism in osteosarcoma. We found that LINC00707 levels are significantly higher in the osteosarcoma cell lines SW 1353, HOS, U-2 OS, MG-63, and Saos-2 compared to those in human fetal osteoblastic cell line hFOB1.19. LINC00707 silencing suppressed cell proliferation, migration, and invasion of MG-63 and Saos-2 cells. Moreover, LINC00707 can act as a competitive endogenous RNA of miR-338-3p, and miR-338-3p inhibitor and AHSA1 overexpression alleviated the effect of LINC00707 silencing. In conclusion, we demonstrated high expression of LINC00707 in osteosarcoma cell lines and that silencing LINC00707 suppresses cell proliferation, migration, and invasion by targeting the miR-338-3p/AHSA1 axis in MG-63 and Saos-2 cells. These findings suggest that LINC00707 may serve as a potential target for osteosarcoma treatment.

NPR3, transcriptionally regulated by POU2F1, inhibits osteosarcoma cell growth through blocking the PI3K/AKT pathway

Natriuretic peptide receptor 3 (NPR3), mediates natriuretic peptides degradation, was reported to act as a tumor suppressor or promoter in some types of cancer. Previous studies showed that NPR3 was significantly decreased in osteosarcoma (OS) samples. However, the function and potential regulatory mechanism of NPR3 in OS development are unknown. By analyzing the protein expression of NPR3 in OS cell lines (n = 5) and human osteoblast cell line hFOB 1.19, we found that NPR3 expression was also significantly decreased in OS cells. The loss/gain-of-function analysis indicated that NPR3 overexpression observably decreased OS cell viability, arrested cell cycle, and induced apoptosis. However, NPR3 knockdown further enhanced the malignant phenotype of OS cells. Furthermore, NPR3 downregulation activated the PI3K/AKT pathway in OS cells, and the effects of NPR3 silencing on cell proliferation were reversed by the blockade of PI3K/AKT pathway. Of note, dual-luciferase reported assay and site-directed mutagenesis assay indicated that transcription factor POU domain class 2 transcription factor 1 (POU2F1) was proved to suppress NPR3 promoter activity by mainly binding to the −900 to −800 bp region of NPR3 promoter. Moreover, NPR3 overexpression inversed the promotion effect of POU2F1 on cell proliferation. In vivo experiments confirmed that NPR3 overexpression suppressed the growth of xenograft tumors. Taken together, the present study demonstrates that NPR3 may serve as a novel tumor suppressive factor through blocking the PI3K/AKT pathway and transcriptionally regulated by POU2F1.

LncRNA RUSC1-AS1 promotes osteosarcoma progression through regulating the miR-340-5p and PI3K/AKT pathway.

Dysregulation of long noncoding RNA (lncRNA) is frequently involved in the progression and development of osteosarcoma. LncRNA RUSC1-AS1 is reported to be upregulated and acts as an oncogene in hepatocellular carcinoma, cervical cancer and breast cancer. However, its role in osteosarcoma has not been studied yet. In the present study, we investigated the role of RUSC1-AS1 in osteosarcoma both in vitro and in vivo. The results showed that the expression of RUSC1-AS1 was significantly upregulated in osteosarcoma cell line U2OS and HOS compared to that in human osteoblast cell line hFOB1.19. Similar results were found in human samples. Silencing RUSC1-AS1 by siRNA significantly inhibited U2OS and HOS cell proliferation and invasion, measured by CCK-8 and transwell assay. Besides, knockdown of RUSC1-AS1 increased cell apoptosis in osteosarcoma cell lines. In addition, RUSC1-AS1 promoted the epithelial-mesenchymal transition (EMT) process of osteosarcoma cells. In vivo experiments confirmed that RUSC1-AS1 knockdown had an inhibitory effect on osteosarcoma tumor growth. Mechanically, we showed that RUSC1-AS1 directly binds to and inhibits miR-340-5p and activates the PI3K/AKT signaling pathway. In conclusion, our study demonstrated that RUSC1-AS1 promoted osteosarcoma development both in vitro and in vivo through sponging to miR-340-5p and activating the PI3K/AKT signaling pathway. Therefore, RUSC1-AS1 becomes a potential therapeutic target for osteosarcoma.

Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells

The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

Probiotic Propionibacterium freudenreichii MJ2 Enhances Osteoblast Differentiation and Mineralization by Increasing the OPG/RANKL Ratio.

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins.

Aqueous Polyphenol Semi-Purified Fractions from Galls of Quercus infectoria Increase ALP Activity and Mineralisation of hFOB 1.19 Cells

The galls of Quercus infectoria (QI) have been reported to possess numerous medicinal values and give a positive effect on bone metabolism. This study investigated the effects of semi-purified fractions of QI gall extract on the proliferation, alkaline phosphatase (ALP) activity, and mineralisation of human foetal osteoblast cell line (hFOB1.19). The semi-purified fractions (fraction A and B) were prepared by column chromatography methods. MTT assay was used to measure cell proliferation activity to obtain half maximal concentration (EC50) of the cells treated with fraction A (phenolic components and contained amide), fraction B (phenolic components with the presence of alkene), and pamidronate (drug control). The most potent or lowest EC50 was further used to measure ALP activity in the treated and untreated cells at day 1, 3, 7, 10, and 14 by ELISA. Cell mineralisation was determined by von Kossa staining for phosphate depositions and Alizarin Red S staining for calcium depositions. The EC50 values for fraction A and B were 8.86 and 9.92 μg/mL, respectively, which showed a greater effect compared to the pamidronate (15.27 μg/mL). The ALPactivity of both fractions in the treated cells were also greater compared to the two control groups (cells treated with pamidronate and untreated cells), starting from day 3 onwards. The calcium depositions appeared as red spots, while phosphate depositions appeared as black spots. Interestingly, the calcium depositions of cells treated with both fractions were higher than those of the two control groups. In conclusion, semi-purified fractions of QI gall extract enhanced proliferation, improved mineralisation, and increased ALP activity of hFOB 1.19 cells.