Human Osteosarcoma Saos-2 Cells Research Articles

Anti-tumor effects of cryptotanshinone (C19H20O3) in human osteosarcoma cell lines.

Osteosarcoma is the most prevalent malignant bone tumor and occurs most commonly in the adolescent and young adult population. Despite the recent advances in surgeries and chemotherapy, the overall survival in patients with resectable metastases is around 20%. This challenge in osteosarcoma is often attributed to the drastic differences in the tumorigenic profiles and mutations among patients. With diverse mutations and multiple oncogenes, it is necessary to identify the therapies that can attack various mutations and simultaneously have minor side-effects. In this paper, we constructed the osteosarcoma pathway from literature and modeled it using ordinary differential equations. We then simulated this network for every possible gene mutation and their combinations and ranked different drug combinations based on their efficacy to drive a mutated osteosarcoma network towards cell death. Our theoretical results predict that drug combinations with Cryptotanshinone (C19H20O3), a traditional Chinese herb derivative, have the best overall performance. Specifically, Cryptotanshinone in combination with Temsirolimus inhibit the JAK/STAT, MAPK/ERK, and PI3K/Akt/mTOR pathways and induce cell death in tumor cells. We corroborated our theoretical predictions using wet-lab experiments on SaOS2, 143B, G292, and HU03N1 human osteosarcoma cell lines, thereby demonstrating the potency of Cryptotanshinone in fighting osteosarcoma.

Biological Response to Bioinspired Microporous 3D-Printed Scaffolds for Bone Tissue Engineering.

The scaffold is a key element in the field of tissue engineering, especially when large defects or substitutions of pathological tissues or organs need to be clinically addressed. The expected outcome is strongly dependent on the cell–scaffold interaction and the integration with the surrounding biological tissue. Indeed, mimicking the natural extracellular matrix (ECM) of the tissue to be healed represents a further optimization that can limit a possible morphological mismatch between the scaffold and the tissue itself. For this aim, and referring to bone tissue engineering, polylactic acid (PLA) scaffolds were 3D printed with a microstructure inspired by the trabecular architecture and biologically evaluated by means of human osteosarcoma SAOS-2 cells. The cells were seeded on two types of scaffolds differing for the designed pore size (i.e., 400 and 600 µm), showing the same growth exponential trend found in the control and no significant alterations in the actin distribution. The microporous structure of the two tested samples enhanced the protein adsorption capability and mRNA expression of markers related to protein synthesis, proliferation, and osteoblast differentiation. Our findings demonstrate that 3D-printed scaffolds support the adhesion, growth, and differentiation of osteoblast-like cells and the microporous architecture, mimicking the natural bone hierarchical structure, and favoring greater bioactivity. These bioinspired scaffolds represent an interesting new tool for bone tissue engineering and regenerative medicine applications.

Cytotoxic Impact of N-Oleoylethanolamine on Bone Cancer Cells.

Cancer is a complex disease that derives from the uncontrolled proliferation of cells. Bone cancer is a type of prevalent cancer that occurs both in young and adults. Bone cancer is most common in the long bones of the pelvis, arms and legs. Statistically, more than 200 cases of osteosarcoma have been reported annually in our country. Classical treatment with chemotherapeutics remains ineffective in the cure of this cancer type. Recent studies have shown that ceramide induces apoptosis at its increased levels in the cells. Thus, many studies have been conducted to cause the accumulation of ceramide molecules in the cell by different ways to induce apoptosis. NOE (Noleoylethanolamine) is a specific inhibitor of ceramidase enzymes that hydrolyze intracellular ceramides and prevent apoptosis. This study investigates the cytotoxic and apoptosis-inducing activities of NOE on human osteosarcoma Saos-2 cells. Cytotoxic effects were investigated by MTT colorimetric assay. For the detection of morphological and ultrastructural indicators of apoptosis, confocal and TEM techniques were used. Our finding indicated that NOE is effective in the inhibition of the growth of Saos-2 cells. Confocal and TEM findings showed morphological and ultrastructural changes as chromatin condensation, fragmentation of nuclei and mitochondria as well as damaged cytoskeleton and cell shrinkage. The results revealed that NOE exerts its cytotoxicity on Saos-2 cells through changing the ultrastructure and morphology of cells with clear apoptotic sparks.

Facile One-Pot Method for All Aqueous Green Formation of Biocompatible Silk Fibroin-Poly(Ethylene Oxide) Fibers for Use in Tissue Engineering.

Silk fibroin (SF) fibers are highly regarded in tissue engineering because of their outstanding biocompatibility and tunable properties. A challenge remains in overcoming the trade-off between functioning and biocompatible fibers and the use of cytotoxic, environmentally harmful organic solvents in their processing and formation. The aim of this research was to produce biocompatible SF fibers without the use of cytotoxic solvents, via pressurized gyration (PG). Aqueous SF was blended with poly(ethylene oxide) (PEO) in ratios of 80:20 (labeled SF-PEO 80:20) and 90:10 (labeled SF-PEO 90:10) and spun into fibers using PG, assisted by a range of applied pressures and heat. Pure PEO (labeled PEO-Aq) and SF solubilized in hexafluoro-isopropanol (HFIP) (labeled SF-HFIP) and aqueous SF (labeled SF-Aq) were also prepared for comparison. The resulting fibers were characterized using SEM, TGA, and FTIR. Their in vitro cell behavior was analyzed using a Live/Dead assay and cell proliferation studies with the SaOS-2 human bone osteosarcoma cell line (ATCC, HTB-85) and human fetal osteoblast cells (hFob) (ATCC, CRL-11372) in 2D culture conditions. Fibers in the micrometer range were successfully produced using SF-PEO blends, SF-HFIP, and PEO-Aq. The fiber thickness ranged from 0.71 ± 0.17 μm for fibers produced using SF-PEO 90:10 with no applied pressure to 2.10 ± 0.78 μm for fibers produced using SF-PEO 80:10 with 0.3 MPa applied pressure. FTIR confirmed the presence of SF via amide I and amide II bands in the blend fibers because of a change in structural conformation. No difference was observed in thermogravimetric properties among varying pressures and no significant difference in fiber diameters for pressures. SaOS-2 cells and hFOb cell studies demonstrated higher cell densities and greater live cells on SF-PEO blends when compared to SF-HFIP. This research demonstrates a scalable and green method of producing SF-based constructs for use in bone-tissue engineering applications.

Biosynthesis and characterization of silver nanoparticles from Tricholoma ustale and Agaricus arvensis extracts and investigation of their antimicrobial, cytotoxic, and apoptotic potentials

In this study, silver nanoparticles (AgNPs) were synthesized using Tricholoma ustale and Agaricus arvensis extracts by the microwave-assisted method. The biosynthesized AgNPs were characterized by UV–Vis spectroscopy, Fourier transforms infrared spectroscopy (FTIR), X-ray Diffraction (XRD), energy dispersive spectroscopy (EDS), scanning transmission electron microscopy (STEM), and dynamic light scattering (DLS). The synthesized AgNPs were almost spherical shaped with average sizes below 20 nm. The anti-microbial activity of the synthesized AgNPs against the pathogen bacterial and fungal strains Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecalis, Candida albicans, and Candida utilis was determined by minimum inhibitory concentration and disc diffusion methods. The cytotoxicity of the AgNPs was examined on human breast cancer (MCF-7), lung cancer (A549), colon cancer (HT-29), and osteosarcoma (Saos-2) cells by MTT assay. The AgNPs exhibited an anti-proliferative effect on MCF-7, HT-29, and Saos-2 cells in a dose-depended manner via induction of intrinsic apoptotic signaling pathways. Hence, the synthesized AgNPs may be potential metal-based nanoparticle systems to treat infectious diseases and cancer in medicine.

Integrin α2β1 as a negative regulator of the laminin receptors α6β1 and α6β4

Integrin α2β1 is a widely expressed collagen I receptor which also mediates laminin-111 binding in some cell types, but the functional relevance of collagen versus laminin binding for different cell types is poorly understood. Here we use AFM-based singe-cell force spectroscopy (SCFS) to compare α2β1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2β1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2β1 as a receptor only for collagen in these cells. Inversely, the α2β1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2β1 expression suppresses laminin binding. Analogous results were obtained in a pair of SAOS-2 human osteosarcoma cell lines. Again, wildtype cells (SAOS-WT) adhered strongly to laminin and poorly to collagen, while expression of integrin α2β1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2β1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2β1 expression downregulated transcription and expression of integrin subunits α6 and β4, components of the main laminin-111 binding receptors integrin α6β1 and α6β4 in these cells. Integrin α6 and β4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2β1 expression negatively regulates integrin α6β1 and α6β4-mediated adhesion, spreading and invasion on laminin in different cancer cell types. In contrast to SAOS-WT, but similar to SAOS-A2 osteosarcoma cells, primary Human osteoblasts (HOB) cells express α2 but only low levels of β4 integrin, preferentially adhere to and spread on collagen over laminin and show suppressed laminin-dependent transmigration. By enhancing collagen binding directly and suppressing laminin binding indirectly through laminin receptor downregulation, α2β1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.

GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling.

Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078′s increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078′s apoptotic effects on human osteosarcoma cells.

MicroRNA-1226-3p has a tumor-promoting role in osteosarcoma.

Osteosarcoma is a malignant bone tumor that commonly occurs in young individuals. It accounts for 10% of solid tumors in those who are 15–19 years old. MicroRNA (miRNA/miR) dysregulation serves a crucial role in the molecular mechanism of osteosarcoma. The present study reported a novel miRNA (miR-1226-3p) and investigated its function in osteosarcoma. miR-1226-3p mimics and miR-1226-3p antisense oligonucleotides were transfected into human osteosarcoma SaOS-2 cells to alter miR-1226-3 expression, while the hFOB 1.19 cell line was used as the control. The apoptosis rate was analyzed using a dead cell apoptosis kit. TNF receptor-associated factor 3 (TRAF3) protein expression was assayed by western blotting. The results of bioinformatics and clinical specimen analyses revealed that higher expression levels of miR-1226-3p were associated with lower survival rates. Additionally, the results of experiments on cultured cells revealed that miR-1226-3p promoted the proliferation of SaOS-2 cells, while miR-1226-3p inhibition decreased cell proliferation and increased apoptosis. Furthermore, it was revealed that miR-1226-3p targeted TRAF3 in SaOS-2 cells. In conclusion, the present study suggested that miR-1226-3p promoted the proliferation of osteosarcoma cells.

In vitro cytotoxicity of biodegradable Zn-Mg surgical wires in tumor and healthy cells

In this work, we examined the in vitro cytotoxicity of new biodegradable surgical wires. The wires made of zinc with the addition of a small amount of magnesium (pure zinc, ZnMg 0.0026, ZnMg 0.0068, and ZnMg 0.08) have been investigated. The wires were produced using a technology based on extrusion and subsequent drawing. The resulting wires with a diameter of 0.8–1.0 mm are designed to be used in surgical operations related to bone joints. For cytotoxicity studies, we have selected human dental pulp stem cells (hDPSC) as the cell population representing normal osteoprogenitor cells. Considering that, after bone surgeries, the chance of osteosarcoma increases, we have compared the results obtained in hDPSC to those obtained with Saos-2 human osteosarcoma cell line. Cultured cells were exposed to the extracts obtained from the materials incubated in culture medium for 24 h with and without preincubation. Extracts of different ratios were examined. The results showed that the extracts obtained from wires made of ZnMg 0.0026 alloy exhibit high toxicity to Saos-2 osteosarcoma cells and low toxicity to hDPSC cells. This was in contrast to all reference materials, i.e., commercial surgical sutures made of steel and polymers, that did not display cytotoxicity toward osteosarcoma cells. Thus, the detected phenomenon for the ZnMg 0.0026 alloy can become the basis for creating biodegradable Zn-Mg surgical wires with antitumor activity.

Anti-tumour effect of tocilizumab for osteosarcoma cell lines.

AimsTocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines.MethodsWe used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay.ResultsThe OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2.ConclusionIn conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells.Cite this article: Bone Joint Res 2020;9(11):821–826.

Nanoparticle-directed and ionically forced polyphosphate coacervation: a versatile and reversible core\u2013shell system for drug delivery

A drug encapsulation/delivery system using a novel principle is described that is based on an intra-particle migration of calcium ions between a central Ca2+-enriched nanoparticle core and the surrounding shell compartment. The supply of Ca2+ is needed for the formation of a coacervate shell around the nanoparticles, acting as the core of drug-loadable core–shell particles, using the physiological inorganic polymer polyphosphate (polyP). This polyanion has the unique property to form, at an alkaline pH and in the presence of a stoichiometric surplus of calcium ions, water-insoluble and stabile amorphous nanoparticles. At neutral pH a coacervate, the biologically active form of the polymer, is obtained that is composed of polyP and Ca2+. The drug-loaded core–shell particles, built from the Ca–polyP core and the surrounding Ca–polyP shell, were fabricated in two successive steps. First, the formation of the nanoparticle core at pH 10 and a superstoichiometric 2:1 molar ratio between CaCl2 and Na–polyP into which dexamethasone, as a phosphate derivative, was incorporated. Second, the preparation of the coacervate shell, loaded with ascorbic acid, by exposure of the Ca–polyP core to soluble Na–polyP and L-ascorbate (calcium salt). EDX analysis revealed that during this step the Ca2+ ions required for coacervate formation migrate from the Ca–polyP core (with a high Ca:P ratio) to the shell. Electron microscopy of the particles show an electron-dense 150–200 nm sized core surrounded by a less sharply delimited electron-sparse shell. The core–shell particles exhibited strong osteogenic activity in vitro, based on the combined action of polyP and of dexamethasone and ascorbic acid, which reversibly bind to the anionic polyP via ionic Ca2+ bonds. Drug release from the particles occurs after contact with a peptide/protein-containing serum, a process which is almost complete after 10 days and accompanied by the conversion of the nanoparticles into a coacervate. Human osteosarcoma SaOS-2 cells cultivated onto or within an alginate hydrogel matrix showed increased growth/viability and mineralization when the hybrid particles containing dexamethasone and ascorbic acid were embedded in the matrix. The polyP-based core–shell particles have the potential to become a suitable, pH-responsive drug encapsulation/release system, especially for bone, cartilage and wound healing.

Cicer arietinum L. Sprouts' Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells.

In the present study, we investigated the biological activity of four extracts obtained from Cicer arietinum L. sprouts. The fermentation of the sprouts with Lactobacillus casei and their incubation with β-glucosidase elevated the concentrations of isoflavonoids, especially coumestrol, formononetin and biochanin A. To study the biological activity of C. arietinum, the human osteosarcoma Saos-2 and human breast cancer MCF-7 cell lines were used. The extracts obtained from fermented sprouts exhibited the strongest ability to decrease intracellular oxidative stress in both types of cells. They augmented mineralization and alkaline phosphatase activity in Saos-2 cells, as well as diminished the secretion of interleukin-6 and tumor necrosis factor α. Simultaneously, the extracts, at the same doses, inhibited the migration of MCF-7 cells. On the other hand, elevated concentrations of C. arietinum induced apoptosis in estrogen-dependent MCF-7 cells, while lower doses stimulated cell proliferation. These results are important for carefully considering the use of fermented C. arietinum sprouts as a dietary supplement component for the prevention of osteoporosis.

Flavonoid compound breviscapine suppresses human osteosarcoma Saos-2 progression property and induces apoptosis by regulating mitochondria-dependent pathway.

This study was aimed to investigate the ability of a flavonoid compound breviscapine (BVP) to suppress growth and elicit apoptosis in human osteosarcoma (OS) Saos‐2 cells. The cells were cultured in vitro and treated with three concentrations of BVP (80, 160, and 320 μg/ml). Moreover, C57 mice were injected with Saos‐2 cells to establish a subcutaneous xenograft model, and they were subsequently treated with three doses of BVP via intraperitoneal injection. The viability of the cells was examined by the Cell Counting Kit‐8 method. The apoptotic cells were assessed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The tumor volume and weight were monitored from day 3 through day 21 after the last injection. The expression of bax, bcl‐2, and cytochrome c (cyt c) mRNA was detected by a real‐time polymerase chain reaction. The protein levels of bax, bcl‐2, cyt c, caspase 3, and caspase 9 were evaluated by Western blot. The expression and distribution of bcl‐2 and bax in tissues were detected by immunohistochemistry. Compared with the control group, BVP treatment inhibited cell proliferation and induced apoptosis of Saos‐2 cells in vitro. Consistently, treatment of mice bearing transplanted tumors with BVP suppressed the growth of OS tumors and promoted cell apoptosis; it also reduced tumor volume and weight. Mechanistically, BVP‐induced apoptosis was mediated by the mitochondria‐dependent pathway, as evidenced by the increased expression of bax and cyt c and the decreased expression of bcl‐2, as well as activation of caspase 9 and caspase 3 in vitro and in vitro. Collectively, BVP inhibits growth and promotes apoptosis of OS by activating the mitochondrial apoptosis pathway.

Phenolic-Enriched Collagen Fibrillar Coatings on Titanium Alloy to Promote Osteogenic Differentiation and Reduce Inflammation.

The adsorption of biomolecules on biomaterial surfaces can promote their integration with surrounding tissue without changing their bulk properties. For biomaterials in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation are desirable. Fibrillar coatings are interesting because of fibrils’ high surface area-volume ratio, aiding adsorption and adhesion. Fibrils also serve as a matrix for the immobilization of biomolecules with biological activity, such as the phenolic compound phloroglucinol (PG), the subunit of marine polyphenols. The aim of this work was to investigate the influence of PG coatings on fibroblast- and osteoblast-like cells to increase the osseointegration of titanium implants. Collagen fibril coatings, containing PG at low and high concentrations, were produced on titanium alloy (Ti6Al4V) scaffolds generated by additive manufacturing (AM). These coatings, especially PG-enriched coatings, reduced hydrophobicity and modulated the behavior of human osteosarcoma SaOS-2 and mouse embryonic fibroblast 3T3 cell lines. Both osteoblastic and fibroblastic cells spread and adhered well on PG-enriched coatings. Coatings significantly reduced the inflammatory response. Moreover, osteogenic differentiation was promoted by collagen coatings with a high PG concentration. Thus, the enrichment of collagen fibril coatings with PG is a promising strategy to improve Ti6Al4V implants for bone contact in orthopedics and dentistry and is worthy of further investigation.

LncRNA LINC-PINT regulating proliferation and apoptosis of osteosarcoma cells by targeting miR-524-5p

Objective: To study the effect of long non-coding RNA LINC-PINT on proliferation and apoptosis of osteosarcoma cells. Methods: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expressions of LINC-PINT and miR-524-5p in normal osteoblast hFOB and human osteosarcoma cell lines HOS, MG63 and SAOS2 cells. The pcDNA plasmid, pcDNA-LINC-PINT plasmid, negative control siRNA (si-NC), si-LINC-PINT, negative control mimics (miR-NC), miR-524-5p mimics (miR-524-5p), pcDNA-LINC-PINT combined with miR-NC, pcDNA-LINC-PINT combined with miR-524-5p were transfected into HOS cells with liposome, respectively. The protein expressions of PCNA and cleaved-caspase-3 in the cells were detected by western blot. Cell proliferation ability was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. The apoptosis was detected by flow cytometry. The transcriptional activity was detected by double luciferase reporter assay. Results: Compared with normal osteoblast hFOB cell (1.00±0.08 vs 1.00±0.06), the expressions of LINC-PINT were down-regulated (0.18±0.01; 0.33±0.01; 0.42±0.01), while the expressions of miR-524-5p were up-regulated (2.65±0.23; 1.68±0.14; 1.51±0.13) in human osteosarcoma cell lines HOS, MG63 and SAOS2 cells, respectively. Overexpression of LINC-PINT significantly inhibited the proliferation (0.41±0.05 vs. 0.62±0.05 for 48 h; 0.57±0.05 vs. 1.06±0.09 for 72 h, both P<0.05) while promoted the apoptosis (25.28±2.15 vs. 9.01±0.17, P<0.01) of HOS cells. Knockdown of LINC-PINT or overexpression of miR-524-5p can significantly promote the proliferation and inhibit apoptosis of HOS cells. Moreover, miR-524-5p inhibited the fluorescence activity of wild-type LINC-PINT (0.31±0.03) in HOS cells when comparred with miR-NC (1.00±0.03) and was negatively regulated by LINC-PINT. Overexpression of miR-524-5p reversed the proliferation inhibition and apoptosis-promotion effects of LINC-PINT in HOS cells. Conclusions: Long non-coding RNA LINC-PINT can inhibit the proliferation and promote apoptosis of osteosarcoma cells through targeting miR-524-5p, which will provide a new target for the treatment of osteosarcoma.

Antibacterial and Osteoconductive Effects of Chitosan/Polyethylene Oxide (PEO)/Bioactive Glass Nanofibers for Orthopedic Applications

This study investigated the efficiency of chitosan/polyethylene oxide (PEO)-based nanofibers with incorporated bioactive glass particles as a coating for titanium alloy, in order to improve the bacteriostatic behavior and, concurrently, promote the production of mineralized tissue. Nanofibers with and without bioglass powder were fabricated by electrospinning technique and characterized using several microscopic and spectroscopic techniques in order to study their morphological and physiochemical properties. Subsequently, the substrates were tested in vitro against Staphylococcus epidermidis and SaOS-2 human osteosarcoma cell line. After in vitro testing, viability and CFU counting assays combined with fluorescence microscopy showed a clear decrease in bacterial growth on all substrates with increasing time. However, this trend was stronger for substrates coated with nanofibers. Formation of mineralized matrix upon exposure to osteoblasts was confirmed by means of SEM/EDX and the content/distribution of osteocalcin and osteopontin estimated by fluorescence microscopy. Incorporation of bioglass promoted biomineralization and stimulated osteoblasts to produce a higher amount of bone extracellular matrix. The present results suggest that a chitosan/PEO/bioactive glass nanofiber composite applied as coating on titanium alloys could concurrently improve antibacterial and osteoconductive properties and could be a potential candidate for dental and orthopedic applications.

Coating Ti6Al4V substrate with the triple-layer glass-ceramic compositions using sol–gel method; the critical effect of the composition of the layers on the mechanical and in vitro biological performance

In the present study, three-layer glass-ceramic coatings of different chemical compositions were synthesized and coated on the Ti6Al4V substrate using the sol–gel method. Hydroxyapatite, hydroxyapatite–wollastonite, and wollastonite–anorthite were coated on the Ti6Al4V substrate, respectively. X-ray diffraction results showed that crystallinity of the main phase in the first (hydroxyapatite (94 wt%)), second (wollastonite (88 wt%)), and third (anorthite (80 wt%)) layers were about 70%, 61%, and 55%, respectively. Scanning electron microscopy analysis showed that when the applied coatings were heat-treated at 800 °C, the morphology of the first layer was changed from spherical to needle-like morphology. The values of microhardness for the first, second, and third layers of the coating were measured as 400, 503.3, and 750.4 Hv, respectively. The mean values of the surface roughness for the mentioned layers, which was analyzed using atomic force microscopy (AFM), were 20, 54, and 63 nm, respectively. The results showed that the coated samples interacted with SBF medium, resulting in the increment of pH and the formation of hydroxyapatite on the coated substrate. MTT assay indicated that the coating process and the coating layers had no adverse effect on the viability of the human osteoblastic SaOS2 osteosarcoma cell line. Cell attachment results showed that the SaOS2 cell line was well attached and expanded on each layer of the coating.

Overexpression of Regucalcin Suppresses the Growth of Human Osteosarcoma Cells in Vitro: Repressive Effect of Extracellular Regucalcin

Regucalcin plays a pivotal role as a suppressor of human carcinogenesis, and downregulation of regucalcin expression may contribute to the promotion of human osteosarcoma. Overexpression of regucalcin suppressed the proliferation of Saos-2 human osteosarcoma cells in vitro and decreased the protein levels of multiple signaling components, transcription factors, and tumor suppressors. Interestingly, extracellular regucalcin repressed colony formation and proliferation of Saos-2 cells, and reduced the protein levels of multiple signaling components, cell cycle inhibitor, and various transcription factors. Thus, regucalcin suppressed the growth of human osteosarcoma cells, providing a novel strategy with the gene therapy for treatment of osteosarcoma.

Gukang Capsule Promotes Fracture Healing by Activating BMP/SMAD and Wnt/β-Catenin Signaling Pathways.

Background Gukang capsule (GKC) is a traditional Chinese medicine formulation which has been used extensively in the clinical treatment of bone fractures. However, the mechanisms underlying its effects on fracture healing remain unclear. Methods In this study we used a rabbit radius fracture model, and we measured the serum content of bone alkaline phosphatase (ALP), calcium, and phosphorus and examined pathology of the fracture site as indicators of the fracture healing effects of GKC. SaOS-2 human osteosarcoma cells were used to measure (i) ALP activity, (ii) ornithine transcarbamylase (OTC), calcium, and mineralization levels, (iii) the expression of osteogenic-related genes, that is, runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen I (COL-I), osteopontin (OPN), OTC, and osterix (Osx), and (iv) the expression of key proteins in the Wnt/β-catenin and BMP/SMAD signaling pathways to study the mechanisms by which GKC promotes fracture healing. Results We found that GKC effectively promotes radius fracture healing in rabbits and enhances ALP activity, increases OTC and calcium levels, and stimulates the formation of mineralized nodules in SaOS-2 cells. Moreover, COL-I, OTC, Osx, BMP2, and OPN expression levels were higher in SaOS-2 cells treated with GKC than control cells. GKC upregulates glycogen synthase kinase 3β (GSK3β) phosphorylation and Smad1/5 and β-catenin protein levels, thereby activating Wnt/β-catenin and BMP/Smad signaling pathways. Inhibitors of the Wnt/β-catenin and BMP/Smad signaling pathways (DKK1 and Noggin, respectively) suppress the osteogenic effects of GKC. Conclusions GKC promotes fracture healing by activating the Wnt/β-catenin and BMP/Smad signaling pathways and increasing osteoprotegerin (OPG) secretion by osteoblasts (OBs), which prevents receptor activator of nuclear factor kappa B ligand (RANKL) binding to RANK.

Design, synthesis and characterization of a PEGylated stanozolol for potential therapeutic applications.

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signaling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH2)10kDa, (MeO-PEG-NH)10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, 1H NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.