Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 µg/mL for 1, 2, 4 h; 5, 10, 20 µg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 µg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to examine in depth the effects induced by MWCNTs-COOH and, in particular, the timing of the MWCNT-COOH-cell interaction.

Tissue-engineered airway: a regenerative solution.

The use of synthetic degradable or permanent polymers and biomaterials has not yet helped to achieve successful clinical whole-airway replacement. A novel, clinically successful approach involves tissue engineering (TE) replacement using three-dimensional biologic scaffolds composed of allogeneic extracellular scaffolds derived from nonautologous sources and recellularized with autologous stem cells or differentiated cells. In this paper, we discuss this novel approach and review information that can lead to a better understanding of stem cell recruitment and/or mobilization and site-specific tissue protection, which can be pharmacologically boosted in humans.

Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies.

Increasing pancreatic islet survival and function is a starting point for obtaining a valuable bioartificial pancreas for the treatment of type 1 diabetes. In this context, decellularized matrices, obtained after the removal of tissue cellular part, are known to support in vitro adhesion, growth, and function of several cell types. We demonstrate that a homologous acellular pancreatic matrix is a suitable scaffold for rat islet cultures maintaining their long-term viability and function. Islets adhered to the pancreatic matrix showed a constant glucose-induced insulin release during long-term in vitro incubation, while islets cultured without a matrix or on the liver matrix showed a progressive reduction. In order to obtain implantable devices, acellular matrix/islet cultures were entrapped into poly(vinyl alcohol) (PVA)/ poly(ethylene glycol) (PEG) tubes obtained by the freezing/thawing procedure. Under this condition, an in vitro constant insulin release was detected. The devices were then implanted into diabetic rats where reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Indeed, immunofluorescence confirmed the presence of insulin- and glucagon-producing cells into the explanted devices. These data show that PVA/PEG semi-permeable membrane can obtain devices that restore, at least in part, insulin secretion.

In vitro biological activity of bovine milk ribonuclease-4.

Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.

Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation.