Selective Effects of Cold Atmospheric Plasma on Bone Sarcoma Cells and Human Osteoblasts.

BACKGROUND: The use of cold atmospheric plasma (CAP) in oncology has been intensively investigated over the past 15 years as it inhibits the growth of many tumor cells. It is known that reactive oxidative species (ROS) produced in CAP are responsible for this effect. However, to translate the use of CAP into medical practice, it is essential to know how CAP treatment affects non-malignant cells. Thus, the current in vitro study deals with the effect of CAP on human bone cancer cells and human osteoblasts. Here, identical CAP treatment regimens were applied to the malignant and non-malignant bone cells and their impact was compared. METHODS: Two different human bone cancer cell types, U2-OS (osteosarcoma) and A673 (Ewing's sarcoma), and non-malignant primary osteoblasts (HOB) were used. The CAP treatment was performed with the clinically approved kINPen MED. After CAP treatment, growth kinetics and a viability assay were performed. For detecting apoptosis, a caspase-3/7 assay and a TUNEL assay were used. Accumulated ROS was measured in cell culture medium and intracellular. To investigate the influence of CAP on cell motility, a scratch assay was carried out. RESULTS: The CAP treatment showed strong inhibition of cell growth and viability in bone cancer cells. Apoptotic processes were enhanced in the malignant cells. Osteoblasts showed a higher potential for ROS resistance in comparison to malignant cells. There was no difference in cell motility between benign and malignant cells following CAP treatment. CONCLUSIONS: Osteoblasts show better tolerance to CAP treatment, indicated by less affected viability compared to CAP-treated bone cancer cells. This points toward the selective effect of CAP on sarcoma cells and represents a further step toward the clinical application of CAP.

Fluorosilicic acid and cotinine, separately and in combination, induce genotoxicity and telomeric reduction in human osteoblast cell line MG63.

Skeletal fluorosis is a severe case in which bone deformations and bone tissue weakening occur due to excessive fluorine deposition. Recently, data on smoking have been published that smoke constituents can indirectly influence bone mass and interfere in the metabolism of fluorides in humans. Thus, the present in vitro study aimed to assess the genetic instability in human osteoblast MG63 cells exposed to fluorosilicic acid (FA) and cotinine (COT), separately and in combination, in concentrations found in human plasma. For this, cell cytotoxicity was performed by MTT assay; DNA damage was performed by alkaline comet assay (CA), modified by repair endonucleases (+FPG); micronuclei test (MN) using CBMN-Cyt assay; and telomere length (TL) by qPCR in MG63 cells. No cytotoxicity was observed for all concentrations tested in this study. Alkaline CA results showed a significant increase in DNA damage at all FA concentrations (0.03125-0.300 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and the highest concentration of FA+COT (0.300 mg/L+250 ng/mL). Alkaline CA+FPG test was used to detect oxidized nucleobases, which occurred at the two highest concentrations of FA, COT, and FA+COT. Micronuclei test showed an increase in the frequency of MN at all concentrations of FA (0.075-0.300 mg/L) except in the lowest concentration (0.03125 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and all concentrations of FA+COT. There was no significant difference in nuclear division index, binucleated cells, nucleoplasmic bridge, and nuclear bud. A TL reduction was observed in cells treated with the highest concentrations of FA alone (0.300 mg/L) and FA+COT (0.300 mg/L+250 ng/mL). Finally, our study showed that FA and COT (mainly alone) at concentrations found in human plasma induced oxidative damage and genetic instability in human osteoblast cells.

Manufacture and mechanical properties of knee implants using SWCNTs/UHMWPE composites.

This article focuses on obtaining ultra high molecular weight polyethylene (UHMWPE) material reinforced with functionalized single-walled carbon nanotubes (f-SWCNTs) and the manufacturing of unicompartmental knee implants via Single-Point Incremental Forming process (SPIF). The physicochemical properties of the developed UHMWPE reinforced with 0.01 and 0.1 wt% concentrations of f-SWCNTs are investigated using Raman and Thermogravimetic Analysis (TGA). Tensile mechanical tests performed in the nanocomposite material samples reveal a 12% improvement in their Young's modulus when compare to that of the pure UHMWPE material samples. Furthermore, the surface biocompatibility of the UHMWPE reinforced with f-SWCNTs materials samples was evaluated with human osteoblast cells. Results show cell viability enhancement with good cell growth and differentiation after 14 incubation days, that validates the usefulness of the developed nanocomposite material in the production of hip and knee artificial implants, and other biomedical applications.

Compounds isolated from Euonymus spraguei Hayata induce ossification through multiple pathways.

The process of bone metabolism includes catabolism of old or mature bone and anabolism of new bone, carried out by osteoclasts and osteoblasts respectively. Any imbalance in this process results in loss of bone mass or osteoporosis. Drugs available to combat osteoporosis have certain adverse effects and are unable to improve bone formation, hence identifying new agents to fulfil these therapeutic gaps is required. To expand the scope of potential agents that enhance bone formation, we identified Euonymus spraguei Hayata as a plant material that possesses robust osteogenic potential using human osteoblast cells. We isolated three compounds, syringaresinol (1), syringin (2), and (-)-epicatechin (3), from E. spraguei. Results demonstrated that syringin (2), and (-)-epicatechin (3), increased alkaline phosphatase activity significantly up to 131.01% and 130.67%, respectively; they also elevated mineral deposition with respective values of up to 139.39% and 138.33%. In addition, 2 and 3 modulated autophagy and the bone morphogenetic protein (BMP)-2 signaling pathway. Our findings demonstrated that 2 and 3 induced osteogenesis by targeting multiple pathways and therefore can be considered as potent multi-targeted drugs for bone formation against osteoporosis.

Cytocompatibility studies of titania-doped calcium borosilicate bioactive glasses in-vitro.

The present study aims to elucidate the applications of Titania (TiO2) doped calcium borosilicate glass as a biocompatible material in regenerative orthopedic applications. In this context, we have examined the bioactivity of various concentrations of TiO2 doped glasses with the help of simulated body fluid (SBF). Cytocompatibility, cell proliferation, and protein expression studies revealed the potential candidature of TiO2 doped glasses on osteoblast cell lines (MG-63). We hypothesized that TiO2 doped calcium borosilicate glasses are most cytocompatible material for bone implants. Glasses with composition 31B2O3-20SiO2-24.5Na2O-(24.5-x) CaO- x TiO2 (x=0,0.5,1,2) have been prepared by the conventional melt-quenching technique. After immersion of glasses in the SBF, formation of hydroxyapatite layer on the surface was confirmed by X-ray Diffractometer (XRD), Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) analysis. Significant change in the pH of the body fluid was observed with the addition of titania. Degradation test was performed as per the ISO 10993. The results showed that partial substitution of TiO2 with CaO negatively influenced bioactivity; it decreased with increase in concentration of TiO2. Vickers hardness tester was used to measure the microhardness values of the prepared glasses. With the increasing of TiO2 content, the microhardness of the glass samples was increased from 545Hv to 576Hv. Cytocompatibility has been evaluated with MG-63 cells by using MTT assay. Further, we observed that there was no change in expressions of cyclin levels even after the incorporation of titania. The antibacterial properties were examined against E. coli and S. aureus. Strong antibacterial efficacy was observed for 2% TiO2 in the system. Hence it can be concluded that titania-doped borosilicate glasses may be used as potential materials in bone tissue engineering.

Formulation and in vitro and in vivo evaluation of a new osteoprotegerin-chitosan gel for bone tissue regeneration.

The osteoprotegerin (OPG) system plays a critical role in bone remodelling by regulating osteoclast formation and activity. The study aimed to determine the physicochemical properties and biocompatibility of a newly formulated OPG-chitosan gel. The OPG-chitosan gel was formulated using human OPG protein and water-soluble chitosan. The physicochemical properties were determined using Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Gel morphology was determined using scanning electron microscopy (SEM) and then it was subjected to a protein release assay and biodegradability test. An in vitro cytotoxicity test on normal human periodontal ligament (NHPL) fibroblasts and normal human (NH) osteoblasts was carried out using the AlamarBlue assay. In vivo evaluation in a rabbit model involved creating critical-sized defects in calvarial bone, filling with the OPG-chitosan gel and sacrificing at 12 weeks. In vitro results demonstrated that the 25 kDa OPG-chitosan gel had the highest rate of protein release and achieved 90% degradation in 28 days. At 12 weeks, the defects filled with 25 kDa OPG-chitosan gel showed significant (p < 0.05) new bone formation and the highest expression of osteocalcin and osteopontin compared to controls. Thus, the 25 kDa OPG-chitosan gel could be a promising new biomaterial for tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 398-407, 2017.

Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.

Preparation of multicompartment sub-micron particles using a triple-needle electrohydrodynamic device.

Control over the size and morphology of polymeric carriers for drug delivery systems is essential to optimize their functionality. In the current study, we demonstrate the feasibility of using an electrohydrodynamic process with a triple-needle device to prepare nearly mono-dispersed, spherical, tri-layered sub-micron particles. Three biocompatible polymer solutions of poly (lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and polymethylsilsesquioxane (PMSQ) were used to prepare particles with three distinct layers. Optimized particles were shown to be spherical with an average size ranging from 320 nm (±80 nm) to 220 (±8 nm), which varied with a change in the working distance in the electrohydrodynamic processing. The surface and internal structure and morphology were studied using confocal, transmission and scanning electron microscopy combined with focused ion beam sectioning. Cytotoxicity was shown to be negligible in an in vitro assay. The ability to fabricate such multilayered particles in a single step, under ambient conditions has considerable potential for a range of applications in particular controlled release drug delivery system.