Three-dimensional tissue scaffolds from interbonded poly(e-caprolactone) fibrous matrices with controlled porosity

In this article, we report on the preparation and cell culture performance of a novel fibrous matrix that has an interbonded fiber architecture, excellent pore interconnectivity, and controlled pore size and porosity. The fibrous matrices were prepared by combining melt-bonding of short synthetic fibers with a template leaching technique. The microcomputed tomography and scanning electron microscopy imaging verified that the fibers in the matrix were highly bonded, forming unique isotropic pore architectures. The average pore size and porosity of the fibrous matrices were controlled by the fiber/template ratio. The matrices having the average pore size of 120, 207, 813, and 994 mm, with the respective porosity of 73%, 88%, 96%, and 97%, were investigated. The applicability of the matrix as a three-dimensional (3D) tissue scaffold for cell culture was demonstrated with two cell lines, rat skin fibroblast and Chinese hamster ovary, and the influences of the matrix porosity and surface area on the cell culture performance were examined. Both cell lines grew successfully in the matrices, but they showed different preferences in pore size and porosity. Compared with two-dimensional tissue culture plates, the cell number on 3D fibrous matrices was increased by 97.27% for the Chinese hamster ovary cells and 49.46% for the fibroblasts after 21 days of culture. The fibroblasts in the matrices not only grew along the fiber surface but also bridged among the fibers, which was much different from those on two-dimensional scaffolds. Such an interbonded fibrous matrix may be useful for developing new fiber-based 3D tissue scaffolds for various cell culture applications.

In vitro studies of cells grown on the superconductor PrOxFeAs

The recent discovery of arsenic-based high temperature superconductors has reignited interest in the study of superconductor: biological interfaces. However, the new superconductor materials involve the chemistry of arsenic and their toxicity remains unclear [Hand, E., 2008. Nature 452 (24), 922]. In this study the possible adverse effects of this new family of superconductors on cells have been examined. Cell culture studies in conjunction with microscopy and viability assays were employed to examine the influence of arsenic-based superconductor PrO_xFeAs (x = 0.75) material in vitro. Imaging data revealed that cells were well adhered and spread on the surface of the superconductor. Furthermore, cytotoxicity studies showed that cells were unaffected during the time-course of the experiments, providing support for the biocompatibility aspects of PrO_xFeAs-based superconductor material.

In vitro behavior of human osteoblast-like cells (SaOS2) cultured on surface modified titanium and titanium–zirconium alloy

In this study, titanium (Ti) and titanium-zirconium (TiZr) alloy samples fabricated through powder metallurgy were surface modified by alkali-heat treatment and calcium (Ca)-ion-deposition. The alteration of the surface morphology and the chemistry of the Ti and TiZr after surface modification were examined. The bioactivity of the Ti and TiZr alloys after the surface modification was demonstrated. Subsequently, the cytocompatibility of the surface modified Ti and TiZr was evaluated via in vitro cell culture using human osteoblast-like cells (SaOS2). The cellular attachment, adhesion and proliferation after cell culture for 14 days were characterized by scanning electron microscopy (SEM) and MTT assay. The relationship between surface morphology and chemical composition of the surface modified Ti and TiZr and cellular responses was investigated. Results indicated that the surface-modified Ti and TiZr alloys exhibited excellent in vitro cytocompatibility together with satisfactory bioactivity. Since osteoblast adhesion and proliferation are essential prerequisites for a successful implant in vivo, these results provide evidence that Ti and TiZr alloys after appropriate surface modification are promising biomaterials for hard tissue replacement.

8-modified-2'-deoxyadenosine analogues induce delayed polymerization arrest during HIV-1 reverse transcription

The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication.

Enzyme-assisted self-assembly under thermodynamic control

The production of functional molecular architectures through self-assembly is commonplace in biology, but despite advances^{1, 2, 3}, it is still a major challenge to achieve similar complexity in the laboratory. Self-assembled structures that are reproducible and virtually defect free are of interest for applications in three-dimensional cell culture^{4, 5}, templating⁶, biosensing⁷ and supramolecular electronics⁸. Here, we report the use of reversible enzyme-catalysed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives^{9, 10} provides a driving force that enables a protease enzyme to produce building blocks in a reversible and spatially confined manner. We demonstrate that this system combines three features: (i) self-correction—fully reversible self-assembly under thermodynamic control; (ii) component-selection—the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries^{11, 12, 13}; and (iii) spatiotemporal confinement of nucleation and structure growth. Enzyme-assisted self-assembly therefore provides control in bottom-up fabrication of nanomaterials that could ultimately lead to functional nanostructures with enhanced complexities and fewer defects.

On corrosion behaviour of magnesium alloy AZ31 in simulated body fluids and influence of ionic liquid pretreatments

This paper reports on the corrosion of Mg alloy AZ31 in simulated body fluid (SBF) using static immersion tests and electrochemical impedance spectroscopy. A preliminary study on the effect of flowing SBF on the corrosion behaviour of AZ31 has also been carried out. Low toxicity ionic liquids (ILs) trimethyl(butyl)phosphonium diphenyl phosphate P1444DPP and trihexyl(tetradecyl)-phosphonium bis-2,4,4trimethylpentyl-phosphinate [P66614][ i(C8) 2PO2] have been used to provide corrosion protection for AZ31 in SBF. Time dependent immersion tests indicate that under static conditions, AZ31 suffers severe localised corrosion in SBF, with pits developing predominantly beside the Al-Mn intermetallic phase in the α matrix. At longer immersion times, the corrosion product eventually precipitates and covers the entire specimen surface. When exposed to SBF under flowing conditions with a shear stress of 0·88 Pa, more uniform corrosion was observed. The optical profilometry results and electrochemical impedance spectroscopy analysis suggest that both P
1444DPP and [P66614][i(C8)2PO2] pretreatments can increase the corrosion resistance of AZ31 in SBF, in particular by decreasing the number of deeper pits found on the alloy surface. Cytotoxic test shows that the presence of the ILs P
1444DPP and [P66614][i(C8)2PO2] in cell culture media slightly inhibits the growth of human coronary artery endothelial cells in comparison with the good cell viability around the treated specimen. A pretreatment with IL is used in order to improve the corrosion resistance of this alloy in SBF. © 2012 Institute of Materials, Minerals and Mining.

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copper II

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper^II [Cu^II(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson’s disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu^II(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu^II(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu^II(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu^II(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from CuII(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O2 as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu^II(atsm) that increases cellular retention of the Cu.

A controllable simple method for production of polymeric nanofiberous composite via single nozzle jet electrospinning

A special Micro-Nano fiberous composite structure composed of nano- and micro-scale fiber of Polycaprolactone (PCL) and Gelatin produced by using single nozzle electrospinning instrument. By controlling the solution (polymer concentration and polymer composition percent) and processing parameters of electrospinning (feed rate and electrostatic field), different portion of nano and micro fibers in the structure is achieved. This method can result a one-stage method of fabrication of Micro-Nano fiberous composite structure instead of previously used twostage process or using additional facility to produce structure near-similar to this composite structure. The resulting materials finely mingle nano- and micro fibers together, rather than simply juxtaposing them, as is commonly found in the literature. The results obtained from SEM, Flow Porosimetry, and DMA led the authors to confirm that the structure has very versatile and improved properties for many applications like cell culture scaffolds. These favourable mechanical and structural properties can provide easier opening of spaces for cell penetration to deeper levels of the scaffold and withstand to tensions during to clinical handling.

Carbon Nanotube Biofiber Formation in a Polymer-Free Coagulation Bath

A novel solution spinning method to produce highly conducting carbon nanotube (CNT) biofibers is reported. In this process, carbon nanotubes are dispersed using biomolecules such as hyaluronic acid, chitosan, and DNA, and these dispersions are used as spinning solutions. Unlike previous reports in which a polymer binder is used in the coagulation bath, these dispersions can be converted into fibers simply by altering the nature of the coagulation bath via pH control, use of a crosslinking agent, or use of a biomolecule-precipitating solvent system. With strength comparable to most reported CNT fibers to date, these CNT biofibers demonstrate superior electrical conductivities. Cell culture experiments are performed to investigate the cytotoxicity of these fibers. This novel fiber spinning approach could simplify methodologies for creating electrically conducting and biocompatible platforms for a variety of biomedical applications, particularly in those systems where the application of an electrical field is advantageous?for example, in directed nerve and/or muscle repair.

Differentiation of cultured epithelial cells : response to toxic agents

Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

MicroRNA pathways : an emerging role in identification of therapeutic strategies

For years researchers have exerted every effort to improve the influential roles of microRNA (miRNA) in regulating genes that direct mammalian cell development and function. In spite of numerous advancements, many facets of miRNA generation remain unresolved due to the perplexing regulatory networks. The biogenesis of miRNA, eminently endures as a mystery as no universal pathway defines or explicates the variegation in the rise of miRNAs. Early evidence in biogenesis ignited specific steps of being omitted or replaced that eventuate in the individual miRNAs of different mechanisms. Understanding the basic foundation concerning how miRNAs are generated and function will help with diagnostic tools and therapeutic strategies. This review encompasses the canonical and the non-canonical pathways involved in miRNA biogenesis, while elucidating how miRNAs regulate genes at the nuclear level and also the mechanism that lies behind circulating miRNAs.

Expansion of human hematopoietic stem/progenitor cells on decellularized matrix scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic 'human-body niche-like' conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

Evaluation of polyvinyl alcohol composite membranes containing collagen and bone particles

Composite biomaterials provide alternative materials that improve on the properties of the individual components and can be used to replace or restore damaged or diseased tissues. Typically, a composite biomaterial consists of a matrix, often a polymer, with one or more fillers that can be made up of particles, sheets or fibres. The polymer matrix can be chosen from a wide range of compositions and can be fabricated easily and rapidly into complex shapes and structures. In the present study we have examined three size fractions of collagen-containing particles embedded at up to 60% w/w in a poly(vinyl alcohol) (PVA) matrix. The particles used were bone particles, which are a mineral-collagen composite and demineralised bone, which gives naturally cross-linked collagen particles. SEM showed well dispersed particles in the PVA matrix for all concentrations and sizes of particles, with FTIR suggesting collagen to PVA hydrogen bonding. Tg of membranes shifted to a slightly lower temperature with increasing collagen content, along with a minor amount of melting point depression. The modulus and tensile strength of membranes were improved with the addition of both particles up to 10 wt%, and were clearly strengthened by the addition, although this effect decreased with higher collagen loadings. Elongation at break decreased with collagen content. Cell adhesion to the membranes was observed associated with the collagen particles, indicating a lack of cytotoxicity.

Locked nucleic acid modified bi-specific aptamer-targeted nanoparticles carrying survivin antagonist towards effective colon cancer therapy

We investigated the anti-cancer activity of alginate coated chitosan nanoparticles (CHNP) encapsulating cell-permeable dominant negative survivin (SR9) with locked nucleic acid (LNA) aptamers targeting EpCAM and nucleolin (termed as "nanobullets") in vitro (2D and 3D cell culture models) and in vivo (colon cancer mouse xenograft model). We incorporated three LNA modifications in each sequence in order to enhance the stability of these aptamers. Confocal microscopy revealed binding of the LNA-aptamers to their specific markers with high affinity. The muco-adhesive nanobullets showed 6-fold higher internalization in cancer cells when compared to non-cancerous cells, suggesting a tumour specific uptake. A higher intensity of nanobullets was observed in both the periphery and the core of the multicellular tumour spheroids compared to non-targeted CHNP-SR9. The nanobullets were found to be the highly effective as they led to a 2.26 fold (p < 0.05) reduction at 24 h and a 4.95 fold reduction (p ≤ 0.001) in the spheroid size at 72 h. The tumour regression was 4 fold higher in mice fed on a nanobullet diet when compared to a control diet. The nanobullets were able to show a significantly high apoptotic (p ≤ 0.0005) and necrotic index in the tumour cell population (p ≤ 0.005) when compared to void NPs. Therefore, our nanoparticles have shown highly promising results and therefore deliver a new conduit towards the approach of cancer-targeted nanodelivery. This journal is