Jose Oca-Cossio

Novel synthesis of cerium oxide nanoparticles for free radical scavenging

AIMSnThe aim of this article is to present a novel synthetic route to form CeO(2) nanoparticles that protects against the detrimental influence of oxidative stress in mammalian cells.nnnMETHODSnThe noncytotoxic surfactant lecithin was used to synthesize CeO(2) nanoparticles and the products were colloidally stabilized in a biocompatible tri-sodium citrate buffer. These nanoparticles were delivered into murine insulinoma betaTC-tet cells, and intracellular free radical concentrations responding to exposure to hydroquinone were measured in a variety of extracellular CeO(2) concentrations.nnnRESULTSnWell-dispersed, highly crystallized CeO(2) nanoparticles of 3.7 nm in size were achieved that are chemically and colloidally stable in Dulbeccos modified Eagles medium for extended periods of time. Treating betaTC-tet cells with these nanoparticles alleviated detrimental intracellular free radical levels down to the primary level.nnnCONCLUSIONnCeO(2) nanoparticles synthesized from this route are demonstrated to be effective free radical scavengers within betaTC-tet cells. Furthermore, it is shown that CeO(2) nanoparticles provide an effective means to improve cellular survival in settings wherein cell loss due to oxidative stress limits native function.

Pathophysiology and fate of hepatocytes in a mouse model of mitochondrial hepatopathies

Background: Although oxidative phosphorylation defects can affect the liver, these conditions are poorly understood, partially because of the lack of animal models. Aims: To create and characterise the pathophysiology of mitochondrial hepatopathies in a mouse model. Methods: A mouse model of mitochondrial hepatopathies was created by the conditional liver knockout (KO) of the COX10 gene, which is required for cytochrome c oxidase (COX) function. The onset and progression of biochemical, molecular and clinical phenotypes were analysed in several groups of animals, mostly at postnatal days 23, 56, 78 and 155. Results: Biochemical and histochemical analysis of liver samples from 23–56-day-old KO mice showed liver dysfunction, a severe COX deficiency, marked mitochondrial proliferation and lipid accumulation. Despite these defects, the COX-deficient hepatocytes were not immediately eliminated, and apoptosis followed by liver regeneration could be observed only at age 78 days. Hepatocytes from 56–78-day-old KO mice survived despite very low COX activity but showed a progressive depletion of glycogen stores. In most animals, hepatocytes that escaped COX10 ablation were able to proliferate and completely regenerate the liver between days 78 and 155. Conclusions: The results showed that when faced with a severe oxidative phosphorylation defect, hepatocytes in vivo can rely on glycolysis/glycogenolysis for their bioenergetic needs for relatively long periods. Ultimately, defective hepatocytes undergo apoptosis and are replaced by COX-positive cells first observed in the perivascular regions.

Reactive oxygen species scavenging properties of ZrO2–CeO2 solid solution nanoparticles

AIMSnThe hypothesis that an increase in defects in cerium oxide (CeO(2)) nanoparticles induced by solid solutions with differences in valency and ionic radius of the solute will yield superior reactive oxygen species (ROS) scavengers at room temperature will be tested.nnnMETHODSnSolid solutions of zirconium in CeO(2), that is, Ce(x)Zr(1-x)O(2) nanoparticles, were synthesized by a reverse micelle method. Their crystal structures, particle sizes and level of agglomeration were characterized. The nanoparticles activities to scavenge ROS were tested in response to hydrogen peroxide at physiological levels and room temperature using an enzyme peroxidase-based assay.nnnRESULTSnSolid solutions of Zr in CeO(2) nanoparticles enhanced ROS scavenging fourfold. The hypothesis is confirmed that more defects are formed and that the scavenging activities of Ce(x)Zr(1-x)O(2) correlate to the nanoparticles oxygen-storage capacity.nnnCONCLUSIONSnThe antioxidant efficacy of CeO(2) nanoparticles can be enhanced by dissolving zirconium in the CeO(2) lattice. The Ce(x)Zr(1-x)O(2) nanoparticles act as an enhanced catalyst at room temperature that scavenges ROS. Increased efficacy will enable lower nanoparticle dosages to protect cells from ROS, thus increasing the therapeutic width of these compounds.

Insights into the role of anaplerosis in insulin secretion : a 13C NMR study

Aims/hypothesisDefining mechanisms and enzymatic paths critical to fuel-regulated insulin secretion are key goals of diabetes research. In this study, 13C-nuclear magnetic resonance spectroscopy and isotopomer analysis were used to investigate the link between insulin secretion and metabolic pathways associated with the tricarboxylic acid (TCA) cycle.Materials and methodsTo this end, four insulinoma cell lines (βTC3, βTC-tet, INS-1 [832/13], R7T1) and porcine islets were examined under a variety of culture conditions (i.e. presence vs absence of amino acids and sera, and low vs high glucose).ResultsGlucose consumption, insulin release, and glutamate isotopomeric patterns were influenced by media complexity (e.g. PBS, plain culture media, fully supplemented culture media). The 13C-labelled metabolites increased with media complexity and increasing glucose concentration, with the notable exception of aspartate, which was always higher under low-glucose conditions. The 13C-glutamate isotopomeric fractions were fitted to metabolic models to estimate the relative metabolic fluxes to the TCA cycle through key enzymatic processes. These indices of metabolism were compared with insulin secretion to determine correlative links. A model containing a single pool of pyruvate, an entrance to the TCA cycle via the pyruvate dehydrogenase complex, and two anaplerotic entrances, one through pyruvate carboxylase and another through an undefined (by the modelling program) source, provided the best fit to the data under all conditions tested, for all cell lines.Conclusions/interpretationOn the basis of our findings, a strong correlation may exist between stimulated insulin secretion and non-pyruvate carboxylase anaplerosis for the four cell lines examined in this study.

Repurposed biological scaffolds: kidney to pancreas

ABSTRACT. Advances in organ regeneration have been facilitated by gentle decellularization protocols that maintain distinct tissue compartments, and thereby allow seeding of blood vessels with endothelial lineages separate from populations of the parenchyma with tissue-specific cells. We hypothesized that a reconstituted vasculature could serve as a novel platform for perfusing cells derived from a different organ: thus discordance of origin between the vascular and functional cells, leading to a hybrid repurposed organ. The need for a highly vascular bed is highlighted by tissue engineering approaches that involve transplantation of just cells, as attempted for insulin production to treat human diabetes. Those pancreatic islet cells present unique challenges since large numbers are needed to allow the cell-to-cell signaling required for viability and proper function; however, increasing their number is limited by inadequate perfusion and hypoxia. As proof of principle of the repurposed organ methodology we harnessed the vasculature of a kidney scaffold while seeding the collecting system with insulin-producing cells. Pig kidneys were decellularized by sequential detergent, enzymatic and rinsing steps. Maintenance of distinct vascular and collecting system compartments was demonstrated by both fluorescent 10 micron polystyrene microspheres and cell distributions in tissue sections. Sterilized acellular scaffolds underwent seeding separately via the artery (fibroblasts or endothelioma cells) and retrograde (murine βTC-tet cells) up the ureter. After three-day bioreactor incubation, histology confirmed separation of cells in the vasculature from those in the collecting system. βTC-tet clusters survived in tubules, glomerular Bowmans space, demonstrated insulin immunolabeling, and thereby supported the feasibility of kidney-to-pancreas repurposing.

Use of Magnetic Nanoparticles to Monitor Alginate-Encapsulated βTC-tet Cells

Noninvasive monitoring of tissue‐engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide‐based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for βTC‐tet cells encapsulated within alginate/poly‐L‐lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION‐labeled βTC‐tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed. Magn Reson Med 61:282–290, 2009.