William Wei Ning Chen

Protein expression profiles in osteoblasts in response to differentially shaped hydroxyapatite nanoparticles.

The use of synthetic hydroxyapatite as bone substitute calls for the knowledge of the influence on adjacent cells. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of human osteoblast cell line incubated separately with various nano sized hydroxyapatite powders with different shapes and chemical compositions using iTRAQ-coupled 2D LC-MS/MS approach. In the present study, we investigated several intracellular signaling molecules involved in calcium regulation to analyze how osteoblast cells respond to dissimilar HA nanoparticles. It was found there was a significant decrease in cell population after adding the HA nanoparticles to the osteoblasts. Our results combining proteomics analysis and RT-PCR validation on targeted genes involved in calcium regulation confirmed the differences in the cellular response to dissimilar HA nanoparticles.

Cytotoxicity Evaluation of Oxidized Single-Walled Carbon Nanotubes and Graphene Oxide on Human Hepatoma HepG2 cells: An iTRAQ-Coupled 2D LC-MS/MS Proteome Analysis

Because of their attractive chemical and physical properties, graphitic nanomaterials and their derivatives have gained tremendous interest for applications in electronics, materials, and biomedical areas. However, few detailed studies have been performed to evaluate the potential cytotoxicity of these nanomaterials on living systems at the molecular level. In the present study, our group exploited the isobaric tagged relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) approach with the purpose of characterizing the cellular functions in response to these nanomaterials at the proteome level. Specifically, the human hepatoma HepG2 cells were selected as the in vitro model to study the potential cytotoxicity of oxidized single-walled carbon nanotubes (SWCNTs) and graphene oxide (GO) on the vital organ of liver. Overall, 30 differentially expressed proteins involved in metabolic pathway, redox regulation, cytoskeleton formation, and cell growth were identified. Based on the protein profile, we found oxidized SWCNTs induced oxidative stress and interfered with intracellular metabolic routes, protein synthesis, and cytoskeletal systems. Further functional assays confirmed that oxidized SWCNTs triggered elevated level of reactive oxygen species (ROS), perturbed the cell cycle, and resulted in a significant increase in the proportion of apoptotic cells. However, only moderate variation of protein levels for the cells treated with GO was observed and functional assays further confirmed that GO was less cytotoxic in comparison to oxidized SWCNTs. These finding suggested that GO was more biocompatible and could be a promising candidate for bio-related applications.

Ultrasound assisted extraction of carbohydrates from microalgae as feedstock for yeast fermentation.

Recently, carbohydrates biomass from microalgae is considered as a promising and inexpensive feedstock for biofeuls production by microorganism fermentation. The main obstacle of the process is microalgae pretreatment and carbohydrates extraction from algal cell. In this study, comparison of three pretreatment methods was performed and the results showed that ultrasonic assisted extraction (UAE) was very effective. The effects of four parameters (ultrasonic power, extraction time, flow rate and algal cell concentration, respectively) on extraction efficiency were also investigated. Additionally, in order to identify significant factors for glucose yield, combination of these four parameters was examined by using fractional factorial design (FFD) and the regression model was obtained. Meanwhile, the refined model was confirmed as a good fitting model via analysis of variance (ANOVA). After extraction, glucose obtained from microalgae was used as substrate for Rhodosporidium toruloides fermentation and yeast biomass was much higher than that of control culture.

Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials : an iTRAQ-coupled 2-D LC-MS/MS analysis

Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ‐coupled 2‐D LC‐MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.

Biogenic tellurium nanorods as a novel antivirulence agent inhibiting pyoverdine production in Pseudomonas aeruginosa

While antibiotic resistance in bacteria is rapidly increasing, the development of new antibiotics has decreased in recent years. Antivirulence drugs disarming rather than killing pathogens have been proposed to alleviate the problem of resistance inherent to existing biocidal antibiotics. Here, we report a nontoxic biogenic nanomaterial as a novel antivirulence agent to combat bacterial infections caused by Pseudomonas aeruginosa. We synthesized, in an environmentally benign fashion, tellurium nanorods (TeNRs) using the metal‐reducing bacterium Shewanella oneidensis, and found that the biogenic TeNRs could effectively inhibit the production of pyoverdine, one of the most important virulence factors in P. aeruginosa. Our results suggest that amyloids and extracellular polysaccharides Pel and Psl are not involved in the interactions between P. aeruginosa and the biogenic TeNRs, while flagellar movement plays an important role in the cell–TeNRs interaction. We further showed that the TeNRs (up to 100 µg/mL) did not exhibit cytotoxicity to human bronchial epithelial cells and murine macrophages. Thus, biogenic TeNRs hold promise as a novel antivirulence agent against P. aeruginosa. Biotechnol. Biotechnol. Bioeng. 2014;111: 858–865.

Investigation of Multiwall Carbon Nanotube Modified Hydroxyapatite on Human Osteoblast Cell Line Using iTRAQ Proteomics Technology

Hydroxyapatite (HA) is a bioactive ceramic material with a chemical composition similar to natural bone, and carbon nano tubes (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. This study reported an attempt to use a commercially multiwalled CNT strengthen brittle hydroxyapatite bioceramics. Using iTRAQ-coupled 2D LCMS/ MS analysis, we report the first study of protein profile in osteoblasts from human osteoblastic cell line incubated separately on HA with and without strengthening multiwall CNT surfaces. Sixty proteins were identified and quantified simultaneously at the initial culturing stage of 3 days. The results were validated by Western blotting for selected proteins: Fetuin-A, Elongation factor II and Peroxiredoxin VI. Fetuin-A showed up-regulation, and Peroxiredoxin VI gave down-regulation in the osteoblasts cultured on HA based ceramic surfaces. Similar regulation was expressed by the protein of Elongation factor II on the phase pure HA surfaces as compared to the control group cultured on the polystyrene substrate. Relatively high EF 2 expression was detected on the phase the surfaces of CNT strengthen HA samples.

A study of osteocalcin production in vitro on various hydroxyapatite based biomaterials

Hydroxyapatite based biomaterials were prepared by a spark plasma sintering technology. The human limb-derived osteoblasts were cultured on the various biomaterial surfaces (HA, RF21, 1SiHA and 5SiHA) for up to two weeks to investigate the cellular behaviors. The bone gammacarboxyglutamic protein or osteocalcin in the medium were determined at different periods of cell culture. The results indicated that a combined effect of bioceramic surface composition and surface morphology had influenced the osteoblast behaviors. The amount of osteocalcin in the medium increased in the initial periods of culture but decreased in the late periods of culture.

Proteomics Study of the Osteoblast Cells Proliferated on Nanostructured Hydroxyapatite Coatings

A big variety of bioceramics have been successfully utilized as implant materials for promoting fixation of bony tissues. Different bioceramics exhibited markedly different proliferation rates of the osteoblast cells in vitro. Clarification of the mechanism about the attachment and proliferation/differentiation of the cells would contribute to selecting suitable biomaterials for hard tissue replacement. Proteomics study was performed in this study employing the 2-dimensional electrophoresis assay with an aim of recognizing the changes in proteins. Nanostructured hydroxyapatite (HA) coatings have been fabricated and they have shown promising mechanical performances. Results showed that the nanostructured HA coatings promoted proliferation of the osteoblast cells. Alkaline phosphatase (ALP) assay revealed an increased ALP activity of the proliferated viable cells, and obviously the presence of the nanosized pores can enhance the anchoring and stretching of the cells. No obvious difference in the 2-D gel maps taken for the cells proliferated on the HA coating and for control can be found. This in turn suggests that the nanostructured HA coating induces minor changes in proteins of the cells.