G-292 Cells Research Articles

Synthesis and investigation on microstructural, mechanical features of mesoporous hardystonite/reduced graphene oxide nanocomposite for medical applications.

The use of hardystonite (Ca2ZnSi2O7, HT)-based composites could be one the main strategies to improve mechanical properties closing to natural bone. However, there are a few reports in this regard. Recent findings indicate that graphene is a promising biocompatible additive in ceramic-based composite. Here, we propose a simple approach for the synthesis of porous nano- and microstructured hardystonite/reduced graphene oxide (HT/RGO) composite using a sol-gel method followed by ultrasonic and hydrothermal processes. Integrating GO to the pure HT increased the bending strength and toughness values about 27.59% and 34.33%, respectively. It also allowed the increment of compressive strength and compressive modulus by about 8.18% and 86%, respectively, and improvement in the fracture toughness about 11.8 times compared to pure HT. The formation of HT/RGO nanocomposites with different RGO weight percentages ranging from 0 to 5.0 has been investigated by scanning electron microscopy (SEM) and X-ray diffraction and the efficient incorporation of GO nanosheets into HT nanocomposite as well as the mesoporous structural properties were also confirmed by Raman, FTIR and BET analyses. The cell viability of HT/RGO composite scaffolds was assayed by methyl thiazole tetrazolium (MTT) test in vitro. In this regard, the alkaline phosphatase (ALP) activity and the proliferation rate of mouse osteoblastic cells (MC3T3-E1) on the HT/1wt. % RGO composite scaffold enhanced in comparison with the pure HT ceramic. The adhesion of osteoblastic cells on the 1% wt. HT/RGO scaffold was interesting as well. In addition, the effect of 1% wt. HT/RGO extract on the proliferation of osteoblast human G-292 cells was successfully evaluated and remarkable observations were obtained. All together it can be said that the proposed bioceramic hardystonite/reduced graphene oxide composites can be a promising candidate for designing hard tissue implants.

Mechanical, electrochemical, and biological properties of YSZ-Mo: A new class of bio-composites

Polycrystalline tetragonal zirconia (3YTZP) has high strength and fracture toughness (K1C) among the other ceramics. However, this amount is still low compared to metals, limiting the use of these monolithic- parts. This study explores the effect of molybdenum on the mechanical, electrochemical, biological, and antibacterial properties of 3Y-TZP-based composites. Three samples of YSZ, YSZ-10wt.%Mo, and YSZ-20wt.%Mo were fabricated by the SPS method at 1450 °C. The microstructure of the mentioned bio-composites was examined by FESEM and XRD analysis. Three-point flexural strength, fracture toughness test, and Vickers microhardness test were also performed. YSZ-10Mo and YSZ-20Mo samples were subjected to Tafel polarization and electrochemical impedance spectroscopy (EIS) analysis. MTT assay was also used to assess the compatibility of the samples with G-292 cells. The antibacterial behavior of the specimens was assessed by the shake flask test. Microstructural results proved the complete densification of the samples. A rise in the molybdenum content slightly decreased the flexural strength (from 1185 ± 52 MPa for YSZ to 841 ± 63 for YSZ-20Mo) and hardness (from 13.35 ± 0.41 GPa for YSZ to 8.10 ± 0.24 GPa for YSZ-20Mo). However, the opposite behavior was observed for the fracture toughness of the samples (from 7.25 ± 0.67 MPa.m1/2 for YSZ to 14.90 ± 0.32 MPa.m1/2 for YSZ-20Mo). The corrosion test indicated the excellent corrosion resistance of the YSZ-10Mo composite. Enhancement of the molybdenum content had a destructive effect on the corrosion resistance of the composites. The results of the MTT test proved the excellent biocompatibility of the samples. An increment in Mo content also remarkably improved the biocompatibility and antimicrobial activity of the YSZ-20Mo sample.

Elimination of Induced Hypoxic Regions in Depth of 3D Porous Silk Scaffolds by the Introduction of Channel Configuration.

Development of large, clinically sized tissue constructs with efficient mass transport is a tremendous need in tissue engineering. One major challenge in large tissue-engineered constructs is to support homogeneous delivery of oxygen and nutrients throughout the tissue scaffold while eliminating induced hypoxic regions in depth. To address this goal, we introduced an especial channeled architecture on porous silk-based tissue scaffolds to improve supplying of oxygen to the cells in central regions of the scaffolds. Oxygen gradients were measured and evaluated in three scaffold prototypes, namely, one unchanneled and two channeled scaffolds with different channel diameters (500 μm and 1000 μm). The channels were introduced into the constructs using stainless-steel rods arranged uniformly in stainless-steel mold, a fabrication method that enables precise control over channel diameter and the distance between channels. During 2-week culture of G292 cells, the 1000 μm channeled scaffolds demonstrated higher oxygen concentration at the center compared to 500 μm channeled prototype; however, the oxygen concentration approached the same level around the last days of culture. Nevertheless, homogenous oxygen distribution throughout the 1000 μm channeled constructs and the consequence of higher cell proliferation at day 14 postseeding corroborate the efficient elimination of induced hypoxic regions; and therefore, it holds promise for clinically relevant sized scaffold especially in bone tissue engineering.

Development of a novel poly (lactic-co-glycolic acid) based composite scaffold for bone tissue engineering

The objective of this research was to synthesize and evaluate novel three dimensional scaffolds composed of poly (lactic-co-glycolic acid) (PLGA)/layered double hydroxide (LDH) composite microspheres for potential bone substitute applications. The mechanical properties of the scaffolds were found to be modulated by the sintering conditions, such as sintering temperature and sintering time, and by the PLGA/LDH ratio. After optimization, the newly developed PLGA/LDH (4:1) scaffold sintered at 90 °C for 2 h exhibited the highest mechanical properties; they were higher than those of the pure polymeric scaffold and also had a desirable pore structure for bone tissue engineering applications. The preliminary examination using L929 cells carried out and no signs of cytotoxicity were observed. The culture of G-292 cells on the optimized samples via the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay method demonstrated increased cell proliferation compared with the pure PLGA scaffolds and the control group (a cell culture plate). Additionally, when the cells were cultured on the composite scaffolds, the osteoblasts had significantly increased alkaline phosphatase (ALP) activity relative to those on the pure PLGA scaffolds and the control group. Thus, the obtained results indicated that the PLGA/LDH microsphere-sintered scaffolds deserve attention for bone tissue engineering applications.

Effects of Cabozantinib on Human G292 Osteosarcoma Cells

Cabozantinib (CBZ) N-(4-((6,7-dimethoxyquinolin-4-yl) oxy) phenyl)-N-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide) (XL184), an inhibitor of MET and vascular endothelial growth factor receptor (VEGFR-2), is an agent approved for the treatment of several types of carcinoma such as medullary thyroid and renal. Recent studies are encouraging for the effectiveness of CBZ in the treatment of osteosarcoma. Because of the complex nature of the microenvironment of osteosarcoma cell sites, in order to better understand the direct effects of CBZ on osteosarcoma cells, in vitro studies were conducted with the human osteosarcoma cell line, G292. Experiments were focused on the effects of CBZ on cell metabolic activity, differentiation, and apoptosis as well as the modulation of responses to growth factors such as platelet-derived growth factor (PDGF) and insulin like growth factor (IGF-I). The results indicate that CBZ can increase the activity of caspase 3/7 as an indicator of apoptosis as well as decrease cellular activity, measured by MTT assay and differentiation assessed by alkaline phosphatase activity. The drug partially downregulated the effects of PDGF on MTT activity and had significant inhibitory effects on the G292 cells response to IGF-I and production of VEGF. This study presents original information on responses of G292 human osteosarcoma cells to the chemotherapy agent, CBZ, and provides in vitro data consistent with the potential therapeutic effects of this agent for osteosarcoma.

Cyanidin 3-O-Glucoside Induces the Apoptosis in the Osteosarcoma Cells through Upregulation of the PPARγ and P21: An In Vitro Study.

Osteosarcoma (OS) is known as the malignant tumors in the bone. Cyanidin 3-OGlucoside (C3G) has a potential to induce the apoptotic cell death in different cancer cells; however, the mechanisms of action for C3G have not been clarified yet. In this study, the apoptotic effects of C3G on three different osteosarcoma cell lines including Saso-2, MG-63, and G-292 (clone A141B1) were investigated. The 24-hr IC50 of C3G for Saso-2, G-292, and MG-63 cells was evaluated by the MTT assay. Apoptosis induction in these cell lines after treatment with the C3G was approved by the Annexin V/PI flow cytometry. Changes at the mRNA expression level of PPARγ, P21, Bax, and Bcl-xl genes were investigated by real-time Polymerase Chain Reaction (PCR) technique, and P21 expression was further confirmed by the western blotting. The MTT assay results demonstrated that the 24-hr IC50 of C3G was equal to 110μg/ml for Saso-2 and G-292 cells while it was about 140μg/ml for the MG-63 cells. The results of real-time PCR clearly showed that treatment of the cells with 24hrs IC50 of C3G caused the upregulation of PPARγ, P21, and Bax genes. Moreover, western blot analysis confirmed that P21 protein overexpressed endogenously after treatment of the cells with the C3G, and it was more upregulated in the MG-63 cells compared to the other cell lines. According to the findings of the study, the C3G is a novel anti-osteosarcoma agent with the ability to induce the apoptosis in different osteosarcoma cells through upregulation of the PPARγ and P21 genes.

Sr-doped bioactive glasses for biological applications

In the present study, sol–gel derived BGs based on 60% SiO2-(36-x) CaO-4P2O5-x SrO (where x = 2, 4, 6 and 8 mol%) system were synthesized. The effect of Sr on bioactivity and proliferation of G292 cells was investigated. X-ray diffraction analysis (XRD), Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were utilized to study the obtained phases, HA morphology, and its functional groups, respectively. The XRD and FTIR tests showed that the rate of hydroxyapatite formation on sample 2S was higher than that of other samples. The 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after one day immersion in SBF, showed that the viability of the samples containing 6 mol% Sr (6S) increases by increasing immersion time, while the sample containing 8 mol% Sr (8S) showed a decrease in bioactivity in osteoblast G292 cells proliferation. According to the results 6S BG specimen with 6 mol% SrO exhibited appropriate bioactivity and cell proliferation.

MiR-384 Inhibits Malignant Biological Behavior Such as Proliferation and Invasion of Osteosarcoma by Regulating IGFBP3.

Osteosarcoma is the most common primary malignant bone tumor in the clinic. It is more common in children and adolescents. It has high malignancy, early metastasis rate, rapid disease progression, and high mortality. Although past years have witnessed the great improvement in the treatments of osteosarcoma, there remains a long way to go. MicroRNAs affect the malignant biological behaviors such as tumor proliferation and metastasis by regulating their target genes. In this study, we investigated the role and mechanism of miR-384 in osteosarcoma. Quantitative real-time polymerase chain reaction assay was performed to detect the expression of miR-384 and insulin-like growth factor binding protein 3 in osteosarcoma tissues and cell lines and established its correlation with osteosarcoma tumor progression and metastasis. To probe whether miR-384 played a tumor suppression role in osteosarcoma, we carried out gain-of-function and loss-of-function assays. Cell Counting Kit-8, cell colony formation, and transwell assays were carried out to determine the cells proliferation and invasion, respectively. Western blot was used to detect the changes of epithelial–mesenchymal transition marker proteins and insulin-like growth factor binding protein 3. MiR-384 was downregulated in osteosarcoma tissues and cell lines. MiR-384 was overexpressed in G292 cells transfected with miR-384 mimics and knocked down in Saos-2 cells with small hairpin RNA targeting miR-384. Ectopic expression of miR-384 inhibited osteosarcoma cell proliferation, colony formation, and invasion. E-cadherin was brought to a decrease whereas N-cadherin and Snail to an increase under the silent expression of miR-384, while overexpression of miR-384 led to an opposite result. MiR-384 could regulate insulin-like growth factor binding protein 3 expression in osteosarcoma. Quantitative polymerase chain reaction and Western blotting results validated that miR-384 knockdown downgrades both messenger RNA and protein levels of insulin-like growth factor binding protein 3 in G292 cells, while miR-384 upregulation exerted an opposite effect in Saos-2 cells. Insulin-like growth factor binding protein 3 was upregulated in osteosarcoma tissues and osteosarcoma cell lines compared with normal ones. Through the bioinformatics database found that the upstream transcriptional regulator of insulin-like growth factor binding protein 3 is MECP2. So miR-384 can directly inhibit MECP2 and then promote the expression of insulin-like growth factor binding protein 3. These results suggested that miR-384 might be a potential therapeutic targets and biomarker in osteosarcoma.

The physical, mechanical, and biological properties of silk fibroin/chitosan/reduced graphene oxide composite membranes for guided bone regeneration

In this study, blends of chitosan (CS)/silk fibroin (SF) and reduced graphene oxide (rGO) as guided bone regeneration membranes were fabricated by solvent casting method and their architectural features, hydrophilicity, porosity, swelling, degradation, and mechanical properties were investigated. The influence of CS, SF, and rGO on osteoblasts spreading and attachment was also determined by scanning electron microscopy (SEM) images and DAPI (4′, 6-diamidoino-2-phenylindole) staining assay. The osteogenic differentiation was determined by the alizarin red staining and alkaline phosphatase activity. The results demonstrated that, the hydrophilicity, swelling and degradability decreases with the increase of SF content, whereas tensile strength increases accordingly. It was confirmed that with increasing the rGO concentration, the porosity and tensile strength decreased but the hydrophilicity was improved. The cell behaviors of G-292 cells were enhanced by increasing the CS content. According to the results, it can be concluded that, SF/CS/rGO blended membranes are promising candidates for bone tissue engineering and optimum results were obtained for the membrane composed of SF:CS: rGO with 84:7:9 weight ratio. It should be noted that the optimized membranes should be further studied for clinical applications.

In vitro bioactivity and cytocompatablity of an injectable calcium phosphate cement/silanated gelatin microsphere composite bone cement

Different weight ratios of microemulsion-based gelatin uniform microspheres (GMs) were added to the tetra calcium phosphate (TTCP)-based cement (CPC) to investigate the physical, mechanical and apatite-forming ability of the CPC. The GMs contained 20 wt% of glycidoxypropyltrimethoxysilane (EPPTMS) as a crosslinking agent. The average size of GMs was on the order of 35 μm. For CPC, the initial and final setting times were 10 min and 21 min, respectively. These values were prolonged up to 18 min and 28 min, respectively, for CPC containing 10 wt% of GM (CPC-10GM). The highest injectability percentage and injection time was related to the CPC-10GM. The highest mechanical values were related to CPC-5GM while CPC-10GM possessed the lowest compressive strength and Young's modulus. Apatite formation on the surfaces of all samples was confirmed by acellular in vitro bioactivity experiment after various periods of soaking time in simulated body fluid (SBF). Needle-like HA precipitates were manifested on the surface of CPC-5GM. According to MTT colorimetric assay, the viability rate of cells on the surfaces of CPC-5GM and CPC increases significantly with time. An appropriate attachment of G-292 cells on the surface of both samples after 48 h was observed by DAPI staining and SEM imaging methods. There was considerable difference in the ALP activity per cell basis between CPC and CPC-5GM at intervals of 7–21 days. These results indicate the potential of the CPC–5GM composites to be used in bone replacement.

Biocomposites based on hydroxyapatite matrix reinforced with nanostructured monticellite (CaMgSiO4) for biomedical application: Synthesis, characterization, and biological studies

In this study, a simple and facile strategy was developed for the synthesis of novel hydroxyapatite (HA)/nanostructured monticellite ceramic composites by mechanical method. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive x-ray spectroscopy (EDS) were used to peruse the phase structure, and morphology of soaked ceramic composites in simulated body fluid (SBF). The in vitro bioactivity of HA-based ceramic composites with nanostructured monticellite ranging from 0 to 50 wt% was evaluated via investigating the formation ability of bone-like calcium phosphates in SBF and the effect of obtained extracts from composites dissolution on osteoblast-like G-292 cell line. Moreover, In vitro cytocompatibility of the HA/monticellite ceramic composites was investigated by MTT, cell growth & adhesion and alkaline phosphatase (ALP) activity assays, and quantitative real-time PCR analysis. The results showed that HA/nanostructured monticellite ceramic composites could induce apatite formation in SBF. The cell proliferation and growth exposed to ceramic composites extracts were significantly stimulated and promoted at a certain concentration range compared to control for various time periods of cell culture. The optimized composite extract enhanced considerably gene expression of G-292 type X collagen (COLX) at different days. Also, G-292 cells were spread and adhered well on the ceramic composite disc. Furthermore, ALP activity of G-292 cells exposed to ceramic composites extracts was dramatically enhanced in comparison with pure HA extract (as control) at different concentrations for various time periods of cell culture. The results suggest that the optimized HA/nanostructured monticellite composite is promising biomaterial for clinical applications such as orthopedic and dentistry.

MiR-193a regulates chemoresistance of human osteosarcoma cells via repression of IRS2

Chemoresistance prevents curative potential of chemotherapy in most cases. MicroRNAs (miRNAs) are key players in regulating chemoresistance in osteosarcoma, which is the most common primary bone cancer. Bisulfite sequencing and quantitative real time PCR analyses showed that miR-193a expression is downregulated by DNA hypermethylation at its promoter region in a chemoresistant cell line, SJSA-1, compared to a chemosensitive cell line G-292. Introduction of a miR-193a mimic in SJSA-1 cells or an antagomir into G-292 cells confirmed the role of miR-193a in osteosarcoma chemoresistance. Bioinformatics together with biochemical assays showed that insulin receptor substrate 2 (IRS2) is a target of miR-193a. Our data concludes that miR-193a plays a role in the osteosarcoma chemoresistance and thus might serve as a useful biomarker for osteosarcoma prognosis.

Monomer-Induced Customization of UV-Cured Atelocollagen Hydrogel Networks.

The covalent functionalization of type I atelocollagen with either 4-vinylbenzyl or methacrylamide residues is presented as a simple synthetic strategy to achieve customizable, cell-friendly UV-cured hydrogel networks with widespread clinical applicability. Molecular parameters, i.e., the type of monomer, degree of atelocollagen functionalization and UV-curing solution, have been systematically varied and their effect on gelation kinetics, swelling behavior, elastic properties, and enzymatic degradability investigated. UV-cured hydrogel networks deriving from atelocollagen precursors functionalized with equivalent molar content of 4-vinylbenzyl (F4VBC = 18 ± 1 mol.%) and methacrylamide (FMA = 19 ± 2 mol.%) adducts proved to display remarkably-different swelling ratio (SR = 1963 ± 58–5202 ± 401 wt.%), storage modulus (G′ = 17 ± 3–390 ± 99 Pa) and collagenase resistance (μrel = 18 ± 5–56 ± 5 wt.%), similarly to the case of UV-cured hydrogel networks obtained with the same type of methacrylamide adduct, but varied degree of functionalization (FMA = 19 ± 2 – 88 ± 1 mol.%). UV-induced network formation of 4VBC-functionalized atelocollagen molecules yielded hydrogels with increased stiffness and enzymatic stability, attributed to the molecular rigidity of resulting aromatized crosslinking segment, whilst no toxic response was observed with osteosarcoma G292 cells. Although to a lesser extent, the pH of the UV-curing solution also proved to affect macroscopic hydrogel properties, likely due to the altered organization of atelocollagen molecules during network formation. By leveraging the knowledge gained with classic synthetic networks, this study highlights how the type of monomer can be conveniently exploited to realize customizable atelocollagen hydrogels for personalized medicine, whereby the structure-property relationships can be controlled to meet the requirements of unmet clinical applications.

Profiling targetable immune checkpoints in osteosarcoma

ABSTRACTOsteosarcomas are aggressive bone tumors for which therapeutic advances have not improved over several decades. Unlike most pediatric tumors, the osteosarcoma genome is remarkably unstable, characterized by numerous copy number alterations and chromosomal structural aberrations. In this study, we asked if the targetable immune checkpoints CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3) and IDO1 are impacted by copy number alterations in osteosarcoma. Of the 215 osteosarcoma samples investigated, PD-L1/PD-L2, B7-H3 and IDO1 were independently gained at frequencies of approximately 8–9%, with a cumulative frequency of approximately 24%. RNA sequencing data from two independent cohorts revealed that B7-H3 is the most highly expressed immune checkpoint gene among the four investigated. We also show that IDO1 is preferentially expressed in pediatric solid tumors and that increased protein expression of B7-H3 and IDO1 are significantly associated with inferior survival in patient samples. Using human osteosarcoma cell lines, we demonstrate that IDO1 is gained in MG63 and G292 cells and that the IDO1 inhibitor, epacadostat, inhibits the enzymatic activity of IDO1 in a dose-dependent manner in these cells. Together, these data reveal the genomic and transcriptomic profiles of PD-L1, PD-L2, B7-H3 and IDO1 in osteosarcoma and identifies a potential context for targeted immunotherapeutic intervention in a subset of patients.

Effects of Chromeceptin on Insulin-like Growth Factor II in Human Osteoblastic and Osteosarcoma Cells

The insulin-like growth factor system is a complex regulatory system of insulin-like growth factors I and II (IGF-I and IGF-II) as well as their cell surface receptors and a family of insulin growth factor binding proteins (IGF-BPs). Despite extensive research on the IGF system in skeletal tissue, there is still not a complete understanding particularly of the role of IGF-II in the regulation of normal and pathological osseous cells in human skeletal biology. The purpose of this study was to further delineate the possible involvement of IGF-II in the regulation of human normal calvarial osteoblasts and G292 human osteosarcoma cellular activity using exogenously added IGF-II as well as a drug, Chromeceptin that has been shown in some other cellular systems to down regulate endogenous IGF-II levels.The results presented here show a similar dose dependent effect of Chromeceptin on inhibition of endogenous IGF-II levels (measured with an immunoassay) and cellular activity (measured with the MTT assay) in both the normal and osteosarcoma cells while small significant increases in activity with exogenously added IGF-II were observed only in the osteosarcoma cells. Moreover, IGF-BP1 levels (measured with an immunoassay) were shown to significantly increase in the G292 cells in a similar manner previously reported in other cell systems with only minor non-significant effects of exogenously added IGF-BP1 in the osteosarcoma cells studied here. These studies suggest that endogenous IGF-II is a critical regulator of activity in both normal and osteosarcoma cells and Chromeceptin can be an effective pharmacological agent for further studies on the role of the growth factor in human skeletal systems.

MiR-20a-5p represses the multi-drug resistance of osteosarcoma by targeting the SDC2 gene

BackgroundAs one of the hallmarks of cancer, chemoresistance hinders curative cancer chemotherapy in osteosarcoma (OS). MicroRNAs (miRNAs) act as key regulators of gene expression in diverse biological processes including the multi-chemoresistance of cancers.MethodsBased on the CCK8 experiments, we performed an RNA-seq-based miR-omic analysis of osteosarcoma (OS) cells (a multi-chemosensitive OS cell line G-292 and a multi-chemoresistant OS cell line SJSA-1) to detect the levels of miR-20a-5p. We predicted Homo sapiens syndecan 2 (SDC2) as one of the target genes of miR-20a-5p via several websites, which was further validated by detecting their expression of both mRNA and protein level in both the miR-20a-5p-mimic transfected G-292 and miR-20a-5p-antagomiR transfected SJSA-1 cells. The involvement of SDC2 with OS chemoresistance was checked by siRNA-mediated repression or overexpression of SDC2 gene. Cell viability was assessed by CCK8 assay.ResultsWe found that the miR-20a-5p level was higher in G-292 cells than in SJSA-1 cells. Forced expression of miR-20a-5p counteracted OS chemoresistance in both cell culture and tumor xenografts in nude mice. As one of miR-20a-5p’s targets, SDC2 was found to mediate the miR-20a-5p-induced repression of OS chemoresistance.ConclusionsOur results suggest that miR-20a-5p and SDC2 contribute to OS chemoresistance. The key players in the miR-20a-5p/SDC2 axis may be a potential diagnostic biomarker and therapeutic target for OS patients.

Mechanism of tribofilm formation on Ti6Al4V oxygen diffusion layer in a simulated body fluid

An Oxygen Diffusion Layer (ODL) was generated on the surface of Ti6Al4V alloy by thermal oxidation treatment. In vitro tests for cytotoxicity were performed in the presence of Ti6Al4V and the ODL samples with the culture of G292 Cells using MTT assay. The results showed a similar cell viability in the presence of the both samples. Wear behaviour of Ti6Al4V and the ODL samples was investigated in a phosphate buffered saline solution under a normal load of 30N at a sliding velocity of 0.1m/s. The worn surface and subsurface of the samples were studied using SEM, STEM, TEM, XPS, AFM, nano-hardness and surface profilometry. A bio-tribofilm was observed on the worn surface of the ODL. TEM studies showed that the tribofilm had an amorphous structure and contained oxygen and phosphorous as confirmed by XPS and EDS analysis. AFM images also revealed that the tribofilm consisted of compacted fine debris. The formation of the tribofilm on the ODL with higher hardness and strength resulted in a decrease of about 95% in the wear rate compared with Ti6Al4V alloy.

Engineered electrospun polycaprolactone (PCL)/octacalcium phosphate (OCP) scaffold for bone tissue engineering

The main challenge in bone tissue engineering is to find suitable biological substitutes which act as scaffolds. Hence, in this work, a novel scaffold composed of octacalcium phosphate (OCP) particles fabricated by co-precipitation method and polycaprolactone (PCL) using electrospinning technique was introduced. The electrospun scaffolds were characterized by SEM, FTIR, XRD, DSC and TGA analysis. The mechanical properties of the composite scaffolds including maximum tensile stress, strain at break and Young modulus were measured. The bioactivity of the scaffolds was determined by soaking in simulated body fluid (SBF). The osteoblast human G-292 cells were seeded on the scaffold's surface for in vitro studies including cell culture and MTT assay. The FTIR and XRD results showed that OCP component has an appropriate incorporation into the polymeric PCL matrix. The SEM analysis exhibited a significant reduction in the fiber size thanks to the OCP. The results of tensile test confirmed that the PCL/OCP composite introduced suitable mechanical properties. Furthermore, the OCP particles led to form hydroxyapatite layer on the scaffold's surface in the vicinity of SBF solution. The obtained results from the MTT assay described that OCP particles have a positive impact on the growth of the osteoblast human G-292 cells on the scaffolds. Overall, aforesaid features of the PCL/OCP composite scaffold make it a great candidate for the bone tissue engineering application.

Thiol-Ene Photo-Click Collagen-PEG Hydrogels: Impact of Water-Soluble Photoinitiators on Cell Viability, Gelation Kinetics and Rheological Properties.

Thiol-ene photo-click hydrogels were prepared via step-growth polymerisation using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene-terminated (PEG-NB), as a potential injectable regenerative device. Type-I collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80 Abs. % functionalisation and 90 RPN% triple helical preservation were recorded via 2,4,6-Trinitrobenzenesulfonic acid (TNBS) colorimetric assay and circular dichroism (CD). Type, i.e., either 2-Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator-induced cytotoxicity and to enable thiol-ene network formation of collagen-PEG mixtures. The viability of G292 cells following 24 h culture in photoinitiator-supplemented media was largely affected by the photoinitiator concentration, with I2959-supplemented media observed to induce higher toxic response (0.1 → 0.5% (w/v) I2959, cell survival: 62 → 2 Abs. %) compared to LAP-supplemented media (cell survival: 86 → 8 Abs. %). In line with the in vitro study, selected photoinitiator concentrations were used to prepare thiol-ene photo-click hydrogels. Gelation kinetics proved to be largely affected by the specific photoinitiator, with LAP-containing thiol-ene mixtures leading to significantly reduced complete gelation time (τ: 187 s) with respect to I2959-containing mixtures (τ: 1683 s). Other than the specific photoinitiator, the photoinitiator concentration was key to adjusting the hydrogel storage modulus (G’), whereby 15-fold G’ increase (232 → 3360 Pa) was observed in samples prepared with 0.5% (w/v) compared to 0.1% (w/v) LAP. Further thiol-ene formulations with 0.5% (w/v) LAP and varied content of PEG-NB were tested to prepare photo-click hydrogels with porous architecture, as well as tunable storage modulus (G’: 540–4810 Pa), gelation time (τ: 73–300 s) and swelling ratio (SR: 1530–2840 wt %). The photoinitiator-gelation-cytotoxicity relationships established in this study will be instrumental to the design of orthogonal collagen-based niches for regenerative medicine.

Wnt/β-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of β-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.