Human Osteoblastic Cell Line Research Articles

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.

LINC01123 enhances osteosarcoma cell growth by activating the Hedgehog pathway via the miR-516b-5p/Gli1 axis.

The lncRNA LINC01123 has been reported to act as an oncogene in many human cancers. Nevertheless, the function and underlying mechanism of LINC01123 in osteosarcoma (OS) remain unclear. This study aimed to explore the roles and mechanisms of LINC01123 in OS progression. In this study, the expression of LINC01123 was significantly upregulated in OS cell lines than in a human osteoblast cell line. Furthermore, in vitro and in vivo experiments confirmed that knockdown of LINC01123 suppressed cell progression. Mechanistically, LINC01123 acted as a competing endogenous RNA by sponging miR‐516b‐5p, thus, increasing Gli1 expression by directly targeting its 3ʹUTR. Taken together, LINC01123 enhances OS proliferation and metastasis via the miR‐516b‐5p/Gli1 axis. Therefore, LINC01123 may be a potential therapeutic target for OS treatment.

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.

RETRACTED ARTICLE: Rb(I)/Se(IV) co-dopant into hydroxyapatite; their structural, morphological, and antibacterial effectiveness for biomedical applications

Co-substituted hydroxyapatite (HAP) with cationic rubidium (Rb+) and anionic selenite ( $${\text{SeO}}_{3}^{2 - }$$ ) was synthesized at different contributions of Rb+ ions. The chemical formula of the composition is [RbxCa(10–0.5x)(PO4)5(SeO3)(OH)2], where 0 ≤ x ≤ 0.8 with step 0.2. The structure and morphology of the as-prepared compositions were investigated upon the concentration of (Rb+) ions. It was mentioned that the roughness average (Ra) starts from 26.2 nm and grows up, reaching 42.6 nm. This noticeable roughness degree due to Rb insertions is reflected in adhesive ability in the biological environment. Rubidium dopant has a significant effect on particle size that inhibits size growth. The internal porosity could be classified as micropores except at x = 0.6 and 0.8, which are considered macro-pores. Rb/Se-HAP compositions' cell viability was investigated through the human osteoblast cell line (HFB4), and the life ratio achieved around 98.1 ± 3.9%. Furthermore, the additional Rb+ ions promote antibacterial activity, whereas the inhibition zone started from 1.6 ± 0.2 and 1.3 ± 0.3 mm and grew up to 5.3 ± 0.9 and 4.9 ± 0.8 mm for x = 0.0 and 0.8, against E. coli and S. aureus, respectively. Thus, the reported stoichiometric compositions are considered to be promising materials for biomedical applications.

Metabolic utilization of human osteoblast cell line hFOB 1.19 under normoxic and hypoxic conditions: A phenotypic microarray analysis.

Osteoblasts play an important role in bone regeneration and repair. The hypoxia condition in bone occurs when bone undergoes fracture, and this will trigger a series of biochemical and mechanical changes to enable bone repair. Hence, it is interesting to observe the metabolites and metabolism changes when osteoblasts are exposed to hypoxic condition. This study has looked into the response of human osteoblast hFOB 1.19 under normoxic and hypoxic conditions by observing the cell growth and utilization of metabolites via Phenotype MicroArrays™ under these two different oxygen concentrations. The cell growth of hFOB 1.19 under hypoxic condition showed better growth compared to hFOB 1.19 under normal condition. In this study, osteoblast used glycolysis as the main pathway to produce energy as hFOB 1.19 in both hypoxic and normoxic conditions showed cell growth in well containing dextrin, glycogen, maltotriose, D-maltose, D-glucose-6-phospate, D-glucose, D-mannose, D-Turanose, D-fructose-6-phosphate, D-galactose, uridine, adenosine, inosine and α-keto-glutaric acid. In hypoxia, the cells have utilized additional metabolites such as α-D-glucose-1-phosphate and D-fructose, indicating possible activation of glycogen synthesis and glycogenolysis to metabolize α-D-glucose-1-phosphate. Meanwhile, during normoxia, D-L-α-glycerol phosphate was used, and this implies that the osteoblast may use glycerol-3-phosphate shuttle and oxidative phosphorylation to metabolize glycerol-3-phosphate.

Development of Novel Thermal Sprayed Hydroxyapatite-Rare Earth (HA-Re) Coatings for Potential Antimicrobial Applications in Orthopedics

Biofilm-associated infections and the lack of successful tissue integration of biomaterial surfaces are the two main barriers to the long-term service of implanted biomaterials. Development of novel biocompatible antimicrobial materials has provided insights into their potential biomedical applications. Many clinical studies have successfully proved that hydroxyapatite coating has excellent osteogenic activity but lacks antibacterial infection in the early stages after implantation. Rare earth (Re) elements have become promising antibacterial biocides and bone-forming effects. Antibacterial capacity of 14 rare earth elements (Eu, Gd, Ce, Nd, Y, La, Pr, Er, Sm, Ho, Tb, Yb, Lu, Dy) was assayed. The gadolinium (Gd) showed outstanding broad-spectrum antibacterial activity against both Gram-positive and Gram-positive bacteria. Here, we report Gd-HA coatings deposited on titanium (Ti) substrate by liquid thermal spraying. The grain size of Gd-HA decreased slightly after Gd3+ incorporation. The antibacterial properties of Gd-HA composite coatings were determined against Gram-negative pathogens Escherichia coli and Gram-positive pathogens Staphylococcus epidermidis. The anti-infection performances were assessed by examining bacteria adhesion and biofilm formation on the coatings. The in vitro cytotoxicity of the Gd-doped HA coatings was further measured on human osteoblast cell line by CCK-8 method. The thermal sprayed HA-Re composite coatings show improved antimicrobial and biocompatible properties and great applicable potential in orthopedics.

Morphological, ultrasonic mechanical and biological properties of hydroxyapatite layers deposited by pulsed laser deposition on alumina substrates

The development of biomaterials innovative compositions to be appropriate for hard tissue regeneration is vital and wished to improve the quality of life worldwide. In this work, using the pulsed laser deposition (PLD) technique, hydroxyapatite (HAP) was sputtered on a dense substrate of alumina at different times of exposure. The investigation of surface morphology indicated that grains of alumina were configured with dimensions of about 1.3–2.94 μm, while high content of porosity was observed. Moreover, the data revealed a significant plunge of surface roughness, whereas the average roughness decreased from 53 nm to 29 nm, and the maximum roughness valley depth decreased from 281 to 248 nm, recorded for 5 to 20 min of exposure time. The mechanical properties were examined non-destructively using ultrasonic waves, and it was noticed that the microhardness changed significantly from 24.7 ± 0.7 GPa to 27.2 ± 0.8 GPa for the compositions compared to 0 and 20 min samples. The attachment behavior of human osteoblasts cell line towards the obtained scaffolds was examined in vitro and prove that cells were proliferated and spread to cover the scaffold surface. This elucidates that manipulation of an innovative scaffold design can be executed based on tailoring of bioactive material (HAP) depositions on an inert biomaterial (alumina) to combine both mechanical and bioactivity, with less degradation rate.

Expression level of UCMA as a candidate molecular target in osteoarthritis

Aim: Osteoarthritis (OA) is a degenerative joint disorder that damages cartilage, synovium and subchondral bone, and there is yet no effective treatment for OA. The identification of novel therapeutic methods is crucially needed for better treatment of OA. Upper zone of growth plate and cartilage matrix associated (UCMA) was discovered as a chondrocyte specific protein in 2008, but its expression is solely not specific to cartilage tissue. Although UCMA is implicated in cartilage and bone metabolic processes, the molecular function of UCMA in OA is not elucidated yet. We aimed to examine the potential effect of UCMA in osteoblast metabolism associated with OA.Materials and Methods: We created an in vitro OA model by inducing osteoblast cell line with IL-1β. The expression levels of 12 related genes were determined using the qRT-PCR method. The MMP1 and OPG expression levels in the supernatants of cells were detected with ELISA.Results: We found that there was a dramatic increase in the levels of UCMA expression and other OA-related markers. We showed that IL-1β triggered the expression of main transcription factors playing a role during bone formation. MMP1 and OPG synthesis and secretions were increased in IL-1β induced-hFOB1.19 cell line significantly.Conclusion: Our study, as the first one using the human osteoblast cell line, provides good evidence about the potential value of UCMA in the pathophysiology of OA and shows that UCMA can be a promising molecular target to develop therapeutic approaches for OA.

Study on proanthocyanidins crosslinked collagen membrane for guided bone tissue regeneration.

The goal of this study is to understand the ability of a newly developed barrier membrane to enhance bone tissue regeneration. Here in this study we present the in vitro characterization of the barrier membrane made from type I collagen and crosslinked by oligomeric proanthocyanidins (OPCs). The effects of the membrane (P-C film) on cell cycle, proliferation, alkaline phosphatase activity, and mineralization were evaluated using the human osteoblast cell line MG-63, while the barrier ability was examined using MG-63 cells, as well as the human skin fibroblast cell line WS-1. The pore size is one of the factors that plays a key role in tissue regeneration, therefore, we evaluated the pore size of the membrane using a capillary flow porometer. Our results showed that the mean pore size of the P-C film was approximately 7-9 µm, the size known to inhibit cell migration across the membrane. The P-C film also demonstrated excellent cell viability and good biocompatibility, since the cell number increased with time, with MG-63 cells proliferating faster on the P-C film than in the cell culture flask. Furthermore, the P-C film promoted osteoblast differentiation, resulting in higher alkaline phosphatase activity and mineralization. Therefore, our results suggest that this P-C film has a great potential to be used in guided bone regeneration during periodontal regeneration and bone tissue engineering.

The influence of zinc and silicate ions on biological properties of hydroxyapatite synthesized by a mechanochemical method

In this study, a mechanochemical method was applied to the synthesis of hydroxyapatite with double and triple substitution. Simultaneous replacement of phosphate ions by carbonate and silicate ions was implemented in the case of double substitution. The triple substitution involved additional replacement of a calcium ion by a zinc ion. The synthesized materials were characterized by X-ray diffraction and Fourier transform infrared spectroscopy. Samples of co-substituted hydroxyapatites in powder form were evaluated in vitro using human osteoblast cell line MG-63 and in vivo via implantation into a bone defect in the rat cranial vault. After 4 days of incubation in vitro, the hydroxyapatite containing zinc, silicon, and carbonate ions with composition Ca9.9Zn0.1(PO4)5.6(SiO4)0.2(CO3)0.2(OH)2 significantly increased cell proliferation in comparison with the other compositions. Four months after the implantation in vivo, this sample showed the greatest ability to integrate into rat skull bones, as evidenced by a morphological analysis of bone sections.

Biosynthesis of innovative calcium phosphate/hydrogel composites: physicochemical and biological characterisation

The goal of supporting and directing tissue regeneration requires the design of new, advanced materials, with features like biocompatibility, biodegradability and adequate mechanical properties. Our work was focused on developing a new injectable biomimetic composite material, based on a peptidic hydrogel and calcium phosphates with the aim of mimicking the chemical composition of natural bone tissue. Arg-Gly-Asp-grafted chitosan was used to promote cell adhesion. The obtained composite hydrogel was characterized with differential scanning calorimetry measurements, rheological analysis, field emission scanning electron microscopy, Fourier transform infrared spectroscopy and nuclear magnetic resonance measurements. The biological responsiveness was assessed using the MG-63 human osteoblast cell line.

Effect of Bioactivity of Surface Topography and Coating Forming by Infrared Light-Induced on Titanium for Bone Repair

Calcium ions and phosphate ions are usually present in biological organisms and human bodies. Different ratios of calcium to phosphorus result in different types of calcium-to-phosphorus crystals. Hydroxyapatite (HA) is the main component of human hard bony tissues. It has good biocompatibility and is often used in bone repair. With the addition of cobalt ions, it can act as a hypoxia-inducing factor to accelerate the regeneration of hard bony tissues. At present, the laser-assisted biomimetic (LAB) method can very quickly deposit calcium phosphate coatings, which can be used on polymer and titanium surfaces. In this study, we first used anodization treatment (with TiO2 nanotubes (TNTs)), alkali treatment (with NaOH), and acid treatment (with HCl) to form nanopore structures on titanium surfaces in the laboratory. Subsequently, LAB treatment was used to deposit calcium phosphate and cobalt-substituted hydroxyapatite onto titanium pieces with different surface treatments. The results showed that smaller holes resulted in better deposition (TNTs), and controlling the pH value in the solution changed the crystal morphology. LAB treatment imbued the titanium surface with super-hydrophilic properties and improved biocompatibility. A human osteoblast cell line (MG-63) used for the cell viability test showed that LAB treatment can improve cell growth. In particular, TNT-CoHA (cobalt-substituted hydroxyapatite) cells grew the best. Immersion in simulated bodily fluid confirmed that LAB treatment with a CoHA solution improved the hydrophilicity, biocompatibility, and bioactivity of titanium surfaces. It is hoped that this study provides useful information for surface coating of biomedical materials in the future.

Thiostrepton and miR-216b synergistically promote osteosarcoma cell cytotoxicity and apoptosis by targeting FoxM1.

Osteosarcoma is a common primary bone cancer that there are currently no effective treatment strategies for. Forkhead box M1 (FoxM1) is key in the development of osteosarcoma, and microRNA (miR)-216b serves an antitumor role by targeting FoxM1. Moreover, thiostrepton (TST), a natural thiazole antibiotic, induces antitumor effects and specifically targets FoxM1. Therefore, the present study investigated whether thiostrepton and miR-216b synergistically inhibited osteosarcoma cells by targeting FoxM1. The MTT assay, reverse transcription-quantitative PCR, a dual-luciferase reporter assay and flow cytometry were performed. Compared with the human osteoblast cell line hFOB1.19, miR-216b expression was significantly downregulated in the osteosarcoma cell lines U2OS, MG63 and Saos-2. By contrast, FoxM1 expression was significantly upregulated in osteosarcoma cell lines compared with the hFOB1.19 cell line. The results indicated that miR-216b targeted the 3′-untranslated region of FoxM1. Moreover, the results suggested that miR-216b cooperated with TST to decrease cell cytotoxicity and increase cell apoptosis. In addition, miR-216b cooperated with TST to increase Bax expression and decrease Bcl-2 expression. In conclusion, the combination of TST and miR-216b synergistically promoted osteosarcoma cell cytotoxicity and apoptosis by targeting FoxM1. Therefore, the present study suggested that the combination of TST and miR-216b may serve as a promising therapeutic strategy for osteosarcoma.

Therapeutic Applications of Biostable Silver Nanoparticles Synthesized Using Peel Extract of Benincasa hispida: Antibacterial and Anticancer Activities.

The purpose of this study was to fabricate biostable inorganic silver nanoparticles (AgNPs) using fresh peel (aqueous) extract of Benincasa hispida. A fast, robust, and eco-friendly approach was used for the synthesis of AgNPs, where bioactive components of peel extract of B. hispida acted as reducing and stabilizing agents. Synthesized AgNPs were characterized using a UV–Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and electron microscopy. The synthesized nanoparticles exhibited maximum absorption at 418 nm under the typical AgNPs surface plasmon resonance band range. They depicted a mean size of 26 ± 2 nm with a spherical shape. Their therapeutic prospective was determined by evaluating their antimicrobial and anticancer potential. The bio-synthesized silver nanoparticles exhibited strong antimicrobial activity with minimum inhibitory concentration (MIC 50) values of 14.5, 8.6, 6.063, and 13.4 μg/mL against Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 14593), Escherichia coli (ATCC 25922), and Klebsiella pneumonia (ATCC 13883), respectively. The biosynthesized AgNPs showed potent in vitro cytotoxicity against human cervical cancer cell line with a half maximal inhibitory concentration (IC50) value of 0.066 μg/mL; however, no cytotoxic effect was observed on normal human primary osteoblasts cell line. This study explored B. hispida extract and confirmed its effectiveness as a promising source in producing AgNPs that could be employed for several therapeutic applications.

Ghrelin uses the GHS-R1a/Gi/cAMP pathway and induces differentiation only in mature osteoblasts. This ghrelin pathway is impaired in AIS patients

We have examined the Acylated Ghrelin (AG)/Gi pathway in different human osteoblastic cell lines. We have found that: 1) AG induces differentiation/mineralization only in mature osteoblasts; 2) the expression of GHS-R1a increases up to the mature cell stage, 3) the action is mediated via the GHS-R/Gi/cAMP pathway only in mature osteoblasts, and 4) osteoblastic cells from adolescent idiopathic scoliosis (AIS) are resistant to the AG/Gi/cAMP pathway. Altogether, these results suggested that AG uses the GHS-R1a/Gi/cAMP pathway to induce differentiation in mature osteoblasts only. This pathway is impaired in AIS osteoblasts. Understanding AG-specific pathways involved in normal and pathological osteoblasts may be useful for developing new treatments for pathologies such as AIS or osteoporosis.

Hsa_circ_0008934 promotes the proliferation and migration of osteosarcoma cells by targeting miR-145-5p to enhance E2F3 expression.

To investigate the role of hsa_circ_0008934 in osteosarcoma and the molecular mechanism involved in the regulation of the occurrence and development of osteosarcoma METHODS: Differentially expressed circRNAs in the osteosarcoma cell lines SaOS2 and MG63 and in the normal human osteoblast cell line hFOB1.19 were identified via next-generation RNA sequencing. The expression and circular morphology of hsa_circ_0008934 were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and RT-PCR analysis, respectively. Proliferation, apoptosis, cell cycle progression, migration, and invasion of SaOS2 and MG63 cells with hsa_circ_0008934 silencing or overexpression were assessed using the MTS method, colony formation assay, flow cytometry, and the transwell system, respectively. The subcellular distribution of hsa_circ_0008934 was revealed via fluorescence in situ hybridization. The binding of hsa_circ_0008934 with microRNAs was confirmed using the dual-luciferase reporter assay. The oncogenic roles of hsa_circ_0008934 in osteosarcoma were determined using an in vivo tumorigenesis assay with nude mice. qRT-PCR, western blotting, TUNEL assay, and immunohistochemistry (IHC) were used to detect the tumorigenicity of hsa_circ_0008934 in osteosarcoma cells. Many circRNAs were differentially expressed in SaOS2 and MG63 cells than in hFOB1.19 cells. Hsa_circ_0008934 expression was significantly elevated in SaOS2 and MG63 cells. Hsa_circ_0008934 silencing significantly reduced proliferation, enhanced apoptosis, blocked cell cycle progression, and impaired migration and invasion capacities of SaOS2 cells. Opposite cellular alterations were achieved by overexpressing hsa_circ_0008934 in MG63 cells. Hsa_circ_0008934 was mainly distributed in the cytosol and positively regulated E2F3 expression in osteosarcoma cells. In addition, it directly bound with miR-145-5p to repress E2F3 expression and enhanced the tumorigenesis of MG63 cells in nude mice. qRT-PCR revealed that the intracellular injection of hsa_circ_0008934 lentivirus resulted in hsa_circ_0008934 overexpression and miR-145-5p downregulation. Western blotting confirmed that E2F3 was upregulated. Moreover, the TUNEL assay showed that hsa_circ_0008934 overexpression inhibited the apoptosis of tumor cells. IHC detection revealed that the hsa_circ_0008934 overexpression could promote the expression of Ki67 and PCNA. Elevated hsa_circ_0008934 expression promotes the proliferation and migration of osteosarcoma cells by sponging miR-145-5p to enhance E2F3 expression.

Influence of bismuth oxide nanoparticles on bystander effects in MCF-7 and hFOB 1.19 cells under 10 MV photon beam irradiation

BackgroundNanomaterials have been applied as radiosensitizer in an effort to improve the effectiveness of radiotherapy in killing cancer cells while simultaneously sparing the healthy normal tissue. Increase in radiotoxicity to the cancerous region might also influence the non-targeted cells through radiation-induced bystander effect (RIBE) mechanism. In this study, we implemented Bi2O3 NPs as radiosensitizer in combination with megavoltage radiotherapy and probe into the RIBE consequences in the non-targeted cells. AimTo investigate the effects of bismuth oxide nanoparticles (Bi2O3 NPs) on RIBE triggered in MCF-7 and hFOB 1.19 after irradiation with 10 MV clinical photon beam. Materials and methodsThe MCF-7 (human breast cancer) and hFOB 1.19 (human fetal osteoblast) cell lines were incubated with and without Bi2O3 NPs prior to irradiation. The treated cells were irradiated with radiation doses of 0 to 12 Gy using 10 MV photon beam in a single exposure. The irradiated-cell conditioned medium (ICCM) were collected from the targeted cells and transferred into the non-targeted cells. Reactive oxygen species (ROS), cell viability and colony forming assay was employed to evaluate the effect. ResultsThe present study demonstrated that the MCF-7 and hFOB 1.19 bystander cells are able to maintain their cell viability for more than 80% after 48 h incubation with ICCM treated with Bi2O3 NPs at 2 Gy radiation dose. The percentage of cell survival fraction of hFOB 1.19 cells which received ICCM with Bi2O3 NPs decreased to 86.8%, in contrast to MCF-7 bystander cells which show an increment in their cells survival after treatment with Bi2O3 NPs. Our results show that the ROS level was increased in the bystander cells, but the addition of Bi2O3 NPs did not significantly increase the ROS level. ConclusionsThe application of nanoparticles for radiosensitization during radiotherapy must also considered the RIBE responses in the non-irradiated cells. These findings provide evidence that the use Bi2O3 NPs as radiosensitizer in radiotherapy is safe and do not significantly increase the RIBE responses in non-targeted cells.

Fabrication of a hydroxyapatite-PDMS microfluidic chip for bone-related cell culture and drug screening

Bone is an important part of the human body structure and plays a vital role in human health. A microfluidic chip that can simulate the structure and function of bone will provide a platform for bone-related biomedical research. Hydroxyapatite (HA), a bioactive ceramic material, has a similar structure and composition to bone mineralization products. In this study, we used HA as a microfluidic chip component to provide a highly bionic bone environment. HA substrates with different microchannel structures were printed by using ceramic stereolithography (SLA) technology, and the minimum trench width was 50 μm. The HA substrate with microchannels was sealed by a thin polydimethylsiloxane (PDMS) layer to make a HA-PDMS microfluidic chip. Cell culture experiments demonstrated that compared with PDMS, HA was more conducive to the proliferation and osteogenic differentiation of the human foetal osteoblast cell line (hFOB). In addition, the concentration gradient of the model drug doxorubicin hydrochloride (DOX) was successfully generated on a Christmas tree structure HA-PDMS chip, and the half maximal inhibitory concentration (IC50) of DOX was determined. The findings of this study indicate that the HA-PDMS microfluidic chip has great potential in the field of high-throughput bone-related drug screening and bone-related research.