Human Fetal Osteoblastic Cell Line Research Articles

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.

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.

STAT3 expression in patients with fragility fractures and its effect on the biological function of osteoblasts.

To determine the expression of signal transducer and activator of transcription 3 (STAT3) in patients with fragility fractures (FFs) and its effect on the biological function of osteoblasts. The study included 32 patients with FFs who were diagnosed and treated in the research group and 30 concurrent healthy individuals in the control group. We observed STAT3 mRNA expression in the patients with FFs and controls and altered STAT3 mRNA to detect changes in the proliferation, invasion, and apoptosis of osteoblasts. The patients with FFs presented higher serum STAT3 mRNA expression than the controls (P < 0.05). We plotted receiver operating characteristic curves based on the STAT3 mRNA expression and found that the area under the curve for STAT3 mRNA was 0.856 (P < 0.05). Transfection of STAT3 mRNA mimics resulted in increased STAT3 mRNA expression, inhibited cell proliferation as detected by an MTT assay, and increased apoptosis rate, which was determined using flow cytometry with human fetal osteoblastic cell line 1.19 cells. STAT3 mRNA expression was elevated in the serum of patients with FFs and can be used as a biomarker for the diagnosis of the disease. Regulating STAT3 mRNA can inhibit the proliferation and induce the osteoblasts apoptosis.

Sex Determining Region Y-Box 12 Promotes Proliferation, Migration and Invasion of Osteosarcoma Cells by Activating β-Catenin/T Cell Factor Axis

Objectives: This study aims to clarify the role of sex determining region Y-box 12 (SOX12) in accelerating the proliferative, migratory and invasive abilities of osteosarcoma (OS) via β-catenin/TCF axis. Materials and Methods: SOX12 levels in human osteosarcoma cell lines and human fetal osteoblastic cell line were determined by RT-qPCR. The proliferation rates of osteosarcoma cells were both determined by CCK-8 assay and EdU staining. In addition, osteosarcoma cell migration and migration were determined by wound healing assay and trans-well assay, respectively. TOPFlash/FOPFlash reporter activity assay and western blot assay were simultaneously performed for the detection of β-catenin/TCF axis. Results: SOX12 was elevated in osteosarcoma cell lines, developing the critical role in proliferation, migration and invasion of osteosarcoma cells. The β-catenin/TCF pathway was activated in osteosarcoma. SOX12 overexpression exerted promotive effects on activation of β-catenin/TCF pathway and SOX12 knockdown showed the opposite effects. Conclusions: SOX12 accelerates proliferation, migration and invasion of osteosarcoma cells by activating β-catenin/TCF axis, thus stimulating the progression of OS.

Naturally prefabricated 3D chitinous skeletal scaffold of marine demosponge origin, biomineralized ex vivo as a functional biomaterial

Solutions developed by nature for structural and functional optimization of three-dimensional (3D) skeletal structures provide unique windows not only into the evolutionary pathways of organisms, but also into bioinspired materials science and biomimetics. Great examples are naturally formed 3D chitinous scaffolds of marine sponge remain a focus of modern biomedicine and tissue engineering. Due to its properties like renewability, bioactivity, and biodegradability such constructs became very interesting players as components of organic-inorganic biocomposites. Herein, we developed chitin-based biocomposites by biomimetic ex vivo deposition of calcium carbonate particles using hemolymph from the cultivated mollusk Cornu aspersum and chitinous matrix from the marine demosponge Aplysina fistularis. The biological potential of the developed biofunctionalized scaffolds for bone tissue engineering was evaluated by investigating the spreading and viability of a human fetal osteoblast cell line has been determined for the first time. Performed analyses like dynamic mechanical analysis and atomic force microscopy shown that biofunctionalized scaffold possess about 4 times higher mechanical resistance. Moreover, several topographical changes have been observed, as e.g., surface roughness (Rq) increased from 31.75 ± 2.7 nm to 120.7 ± 0.3 nm. The results are indicating its potential for use in the modification of cell delivery systems in future biomedical applications.

Evaluation of the Growth and Differentiation of Human Fetal Osteoblasts (hFOB) Cells on Demineralized Bone Matrix (DBM)

ABSTRACT Cells with osteogenic potential are believed to be an ideal source for bone tissue bioengineering. Large bone defects require temporary substitution of the damaged parts. In this respect, the transplantation of bone cells cultured on osteogenic substrates has been investigated. To use the natural bone matrix, one approach is the so-called demineralized bone matrix (DBM). In this study, we evaluated the interaction of human fetal osteoblasts (hFOB 1.19 cells, a human fetal osteoblastic cell line) with DBM fragments. No additional bone differentiation inducer was used other than the DBM itself. The samples were processed, had adhesion pattern evaluated and analyzed by light microscopy (cytochemical and immunocytochemical analysis) and electron microscopy (scanning and transmission). The adhesion pattern of hFOB cells on DBM was similar to what was observed on the cell culture plate. Morphological analysis showed that the hFOB cells had emitted filopodia and cellular projections on both controls and DBM. On DBM, the adhered cells emitted prolongations and migrated into the matrix. The monolayer growth pattern was observed as well as the accumulation of filamentous and reticulate extracellular materials when hFOB cells were cultured on the DBM surface. EDS analysis revealed the deposition of calcium on DBM. Immunocytochemical data showed that the hFOB cells were able to secrete extracellular matrix molecules such as fibronectin and laminin on DBM. Our data indicate that DBM successfully stimulates the osteoblastic phenotype of osteoblast-like cells and corroborate with the fact that DBM is a considerable natural matrix that promotes fractured-bone healing.

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.

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.

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.

Effects of chitosan-loaded hydroxyapatite on osteoblasts and osteosarcoma for chemopreventative applications.

Osteosarcoma remains one of the most common malignant primary bone tumors. Post-surgical defect repair combined with tumor suppression remains a major clinical challenge. Investigations of alternative treatments for osteosarcoma, while promising, have led to multi-drug resistance. These constraints of common treatment strategies have triggered the need for new therapeutic candidates in bone cancer treatment. Chitosan, a common biopolymer utilized in bone and tissue engineering applications, has recently been studied as a pro-apoptotic agent in metastatic cell lines like breast cancer, but has not been utilized in bone cancer applications. In this study, chitosan was directly loaded onto HA disks to evaluate its in vitro release and effects on human fetal osteoblast (hFOB) and human osteosarcoma (MG-63) cell lines. It is hypothesized that the sustained release of chitosan will decrease osteosarcoma cell proliferation and enhance proliferation of osteoblast cells. Through morphological characterization and MTT assay analysis, chitosan showed no toxicity to human fetal osteoblast (hFOB) cells. Chitosan was also shown to decrease human osteosarcoma cell viability by up to 96% compared to control samples. This suggests a pro-apoptotic mechanism against osteosarcoma as well as the potential clinical application of chitosan as a drug candidate in ceramic scaffolds at tumor resected sites.

PI3K/Akt Signaling Involved with Osteoinductive Effects of Achatina fulica Mucus

Abstract Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.

Preparation and characterization of silk fibroin from four different species of Thai-local silk cocoon for Bone implanted applications

The metal interlocking nail is normally used in diaphyseal fractures. The bio-composite interlocking nail will be fabricated in the near future by using local silk fibroin reinforce Polylactic acid filament for 3D printing. Four species of local Thai Bombyx mori silk cocoons were selected to extract silk fibroin. The silk cocoon species consist of 1) Nangnoi Srisaket-I (NN), 2) Nanglai (NL), 3) Luang Saraburi (LS), and 4) J108. After the extraction, fibroins of each silk cocoon species were characterized and compared the physical property by using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDS) and Fourier Transform Infrared Spectroscopy (FT-IR). Then, the biological test was performed on cell viability and cytotoxicity with human fetal osteoblast cell line. The result presents that all of local silk cocoons species presented non-cytotoxicity ability which can be used in human or animal body without endangerment.

Proliferation study and microscopy evaluation on the effects of tannic acid in human fetal osteoblast cell line (hFOB 1.19).

Tannic acid (TA) is a phenolic compound that might act directly on osteoblast metabolism. The study was performed to investigate the effects of TA on the proliferation, mineralization, and morphology of human fetal osteoblast cells (hFOB 1.19). The cells were divided into TA-treated, untreated, and pamidronate-treated (control drug) groups. Half maximal effective concentration (EC50 ) values for TA and pamidronate were measured using MTT assay. The EC50 of hFOB 1.19 cells treated with TA was 2.94 M. This concentration was more effective compared to the pamidronate (15.27 M). Cell proliferation assay was performed to compare cell viability from Day 1 until Day 14. The morphology of hFOB 1.19 was observed via inverted microscope and scanning electron microscope. Calcium (Ca) and phosphate (P) were assessed using energy-dispersive X-ray (EDX) analysis. Furthermore, the mineralization of hFOB 1.19 was determined by von Kossa staining (P depositions) and Alizarin Red S staining (Ca depositions). The number of cells treated with TA was significantly higher than the two control groups at Day 10 and Day 14. The morphology of cells treated with TA was uniformly fusiform-shaped with filopodia extensions. Besides, globular-like structures of deposited minerals were observed in the TA-treated group. In line with other findings, EDX spectrum analysis confirmed the presence of Ca and P. The cells treated with TA had significantly higher percentage of both minerals at Day 3 and Day 10 compared to the two control groups. In conclusion, TA enhances cell proliferation and causes cell morphology changes, as well as improved mineralization.