Otilia Zarnescu

Adapted response of the antioxidant defense system to oxidative stress induced by deoxynivalenol in Hek-293 cells

The mycotoxin deoxynivalenol (DON), a contaminant of certain foods and feeds, is cytotoxic and genotoxic to mammalians cells. Exposure of human embryonic kidney (Hek-293) cells to DON led to a dose- and time-dependent decrease in cell viability, with an IC(50) about 7.6 μM. The DON effects on Hek-293 morphology, reactive oxygen species, lipid peroxidation and antioxidative system and caspase 3 and bcl-2 expression were studied. Cells became round and in some are progressive loss of cell attachment appeared. These biochemical parameters were assessed after 6, 12 and 24 h of treatment with 2.5 and 5 μM DON. An increase in superoxide dismutase activity within the interval 6-12 h and almost complete recovery by the end of experiment for both concentrations was observed, whereas the profile of catalase activity was the same with the superoxide dismutase one for 2.5 μM and decreased in a time-dependent manner for 5 μM. A temporary activation of glutathione peroxidase and glutathione reductase was recorded at 12 h post-exposure, while the glutathione-S-transferase activity was unchanged for both concentrations. The NADP(+)-dependent isocitrate dehydrogenase activity showed a transient increase at the 12 h post-exposure. The caspase 3 expression remained unchanged and the bcl-2 one decreased after 24 h of exposure for the two concentrations. Our results showed the dose- and time specific changes in the antioxidants system of Hek-293 cells, which could not counteract efficiently the effects DON exposure. The different types of cell death which could be activated by this DON induced changes are mentioned.

Immunohistochemical localization of caspase-3, caspase-9 and Bax in U87 glioblastoma xenografts.

Development of new therapies for glioblastoma requires animal models that mimic the biological characteristics of human brain tumors. On the other hand, potential antitumoral effects of a new therapeutic strategy are often established by evaluation of tumor cells apoptosis. Caspases are key mediators in the regulation and execution of apoptosis. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria while caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Bax is a pro-apoptotic member of the Bcl-2 family that play key roles in the regulation of intrinsic apoptotic signaling. In the present study we investigated the immunohistochemical distribution of caspase 3, 9 and Bax in intracranial U87 glioblastoma xenograft. Immunohistochemistry showed that the glioblastoma xenografts contain cells positive for caspase-3, caspase-9, and Bax.

Structural and Oxidative Changes in the Kidney of Crucian Carp Induced by Silicon-Based Quantum Dots

Silicon-based quantum dots were intraperitoneally injected in Carassius auratus gibelio specimens and, over one week, the effects on renal tissue were investigated by following their distribution and histological effects, as well as antioxidative system modifications. After three and seven days, detached epithelial cells from the basal lamina, dilated tubules and debris in the lumen of tubules were observed. At day 7, nephrogenesis was noticed. The reduced glutathione (GSH) concentration decreased in the first three days and started to rise later on. The superoxide dismutase (SOD) activity increased only after one week, whereas catalase (CAT) was up-regulated in a time-dependent manner. The activities of glutathione reductase (GR) and glutathione peroxidise (GPX) decreased dramatically by approximately 50% compared to control, whereas the glutathione-S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) increased significantly after 3 and 7 days of treatment. Oxidative modifications of proteins and the time-dependent increase of Hsp70 expression were also registered. Our data suggest that silicon-based quantum dots induced oxidative stress followed by structural damages. However, renal tissue is capable of restoring its integrity by nephron development.

Preparation and characterization of a collagen-liposome-chondroitin sulfate matrix with potential application for inflammatory disorders treatment

Smart drug delivery systems with controllable properties play an important role in targeted therapy and tissue regeneration. The aim of our study was the preparation and in vitro evaluation of a collagen (Col) matrix embedding a liposomal formulation of chondroitin sulfate (L-CS) for the treatment of inflammatory disorders. Structural studies using Oil Red O specific staining for lipids and scanning electron microscopy showed an alveolar network of nanosized Col fibrils decorated with deposits of L-CS at both periphery and inner of the matrix. The porosity and density of Col-L-CS matrix were similar to those of Col matrix, while its mean pore size and biodegradability had significantly higher and lower values (P < 0.05), respectively. In vitro cytotoxicity assays showed that the matrix system induced high cell viability and stimulated cell metabolism in L929 fibroblast cell culture. Light and electron micrographs of the cell-matrix construct showed that cells clustered into the porous structure at 72 h of cultivation. In vitro diffusion test indicated that the quantity of released CS was significantly lower (P < 0.05) after embedment of L-CS within Col matrix. All these results indicated that the biocompatible and biodegradable Col-L-CS matrix might be a promising delivery system for local treatment of inflamed site.

A bioactive collagen-β tricalcium phosphate scaffold for tissue engineering

Collagen-phosphate composites (COL/β-TCP) are novel materials that have the potential to be used as bone analogues. The aim of our study was to develop a porous bioactive material composed of type I collagen, the main bone protein and tricalcium phosphate, the mineral phase of natural bone, and investigate their in vitro biocompatibility in a human dermal fibroblast culture system. In order to obtain the bioactive materials, type I collagen was isolated from bovine tendon and characterized by physicochemical methods. β-TCP was obtained from calcium carbonate by thermal decomposition at 900 °C temperature. The powder was examined with X-ray diffraction. Two variants of COL/β-TCP scaffolds (P1 and P2) were prepared and examined by scanning electron microscopy. Our results revealed a microporous structure with small white aggregates of β-TCP, non-homogenous scattered in the collagen framework without any preferential orientation. The biocompatibility of the obtained scaffolds was tested by biochemical and histological methods on human fibroblast cultures. Both materials acted as good subtrates for human dermal fibroblast proliferation and migration.

Co-localization of PCNA, VCAM-1 and caspase-3 with nestin in xenografts derived from human anaplastic astrocytoma and glioblastoma multiforme tumor spheres.

The cancer stem cell hypothesis proposes that tumors contain a small subset of cancer cells, the cancer stem cells, which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor. Markers that define cancer stem cells that are capable of recapitulating brain tumors as xenografts in mice has not been described. We investigated the relationship between expression of nestin and that of PCNA, VCAM-1 and caspase-3 in the xenografts developed from human anaplastic astrocytoma and glioblastoma tumor-derived spheres in the brain of nude mouse. Xenografts obtained from astrocytoma tumor stem cells (ATSC) and glioblastoma tumor stem cells (GTSC) have showed a large number of cells positive for both PCNA and the nestin, demonstrating that nestin expressing cells have a high rate of proliferation. Xenografts from GTSC showed heterogeneous staining pattern with cells that express both nestin and VCAM-1, whereas others cells remained complete negative. In this case it was noticed that most tumor cells with large or multinucleated nuclei coexpress nestin and VCAM-1. In xenografts from ATSC most cells positive for nestin express VCAM-1 and in this case the two proteins appear to occupy the same cytoplasmic region. Both GTSC and ATSC derived xenografts showed cells positive for both caspase-3 and for nestin detected mainly as single cells and as cell clusters located near or around a blood vessel.

Magnesium substitution effect on porous scaffolds for bone repair

Of great interest in developing artificial bone is the incorporation of magnesium (Mg) ions into the ceramic lattice in order to improve the physico-chemical and structural properties of the material and to increase its morphological affinity towards newly formed osseous tissue. In the present study, we evaluated the morphological and biological properties of composite scaffolds fabricated by mixing a nanopowder of Mg-substituted beta-tricalcium phosphate with collagen type I in two dry weight ratios (variant I and II). We used biochemical methods, and electron and light microscopy to investigate their porosity, biodegradability and morphology. Osteoblast cell culture behavior in the presence of nanocomposite variants was also examined. Variant I scaffold presented a higher percentage of cross-links and a better resistance to collagenase degradation compared to variant II scaffold. Their porosity did not vary significantly. Osteoblasts cultivated in the presence of nanocomposite scaffolds for 72 h exhibited good cell viability and a normal morphology. When osteoblasts were injected into the scaffolds, a slightly higher proportion of adhered cells were observed for Mg-substituted samples after 7 days of cultivation. All these results showed that Mg-containing porous composite scaffolds had controlled degradation, allowed osteoblast proliferation and adhesion and are good candidates for bone repair.

Collagen-Chondroitin Sulphate-Hydroxyapatite Porous Composites: A Histochemical and Electron Microscopy Approach

In this study the structure of collagen-chondroitin sulphate-hydroxyapatite porous composites is investigated by histochemical (Von Kossa staining), immunohistochemical, and transmission electron microscopy. Examination of composites on picrosirius red stained sections showed that polarization colors of collagen were generally in the range of orange-red. Immunofluorescence data indicate that chondroitin sulphate was either chemically incorporated into the bulk structure of collagen scaffolds or attached on surfaces of collagen bundles. Depending on the ratio between collagen:chondroitin sulphate:hydroxyapatite, von Kossa histochemical staining showed a progressive loading of collagen-chondroitin sulphate bundles with hydroxyapatite. Transmission electron microscopy studies have shown that composites contain mostly collagen fibrils aggregated with random orientation with very few collagen fibers showing the 67-nm banding pattern. Hydroxyapatite deposits of various sizes occurred among the collagen fibrils.

Liposomes-entrapped chondroitin sulphate: Ultrastructural characterization and in vitro biocompatibility

The purpose of this study was ultrastructural characterization of liposomes-entrapped chondroitin sulphate and to prove their in vitro biocompatibility in a human dermal fibroblast culture system, in order to use liposome-entrapped chondroitin sulphate in the treatment of inflammatory disorders. Chondroitin sulphate entrapped in liposomes appears as electron-dense particles in ultra-thin section. Comparative studies using chondroitin sulphate, empty liposomes and liposome-chondroitin sulphate systems were performed in order to evaluate their effect on growth and morphology of fibroblasts after 48 h of culture. Light microscopy indicated that chondroitin sulphate, empty liposomes and liposome-chondroitin sulphate systems do not induce appreciable cytotoxic effects, and cells maintain normal morphology when compared to control fibroblasts.

In vitro behaviour of osteoblast cells seeded into a COL/β-TCP composite scaffold

The purpose of the present study was to investigate the effect of a collagen/β-tricalcium phosphate (COL/β-TCP) composite on osteoblast growth and proliferation. The COL/β-TCP composite was prepared by mixing COL type I with β-TCP, in 1:1 (w/w) ratio and conditioned as sponge by freeze-drying. The osteoblast culture was obtained from rat calvaria bones by enzymatic digestion and cells were seeded in the COL/β-TCP composite. The cell morphology and viability, alkaline phosphatase and osteocalcin, as markers of osteoblast proliferation were evaluated at 3, 7 and 25 days of culture. Histological sections revealed that cell colonization progressively increased inside the COL/β-TCP scaffold, and osteoblasts had a random distribution throughout the scaffold. Cells cultured into the COL/β-TCP scaffold presented osteoblast phenotype, intense staining of alkaline phosphatase and increased production of osteocalcin. Transmission electron micrographs revealed intimate contacts between osteoblasts and the scaffold. MTT test indicated that the viability of the cells cultivated in the presence of COL/β-TCP scaffold was similar to that of the control. All these results show that our COL/β-TCP composite act as a good substrate for rat osteoblast proliferation and migration and could be a promising substitute for bone repair.