Luisa Mancuso

Despite the growing interest in nanoparticles (NPs), the evaluation of their safety use has to be deeply considered, but standardized procedures for the evaluation of their toxicity have not been defined. In vitro methods are ideal in toxicology research because they can rapidly provide reproducible results while preventing the use of animals. Primary cells are considered a better option as model systems for predicting toxicological behavior, although several cell types do not survive enough in culture and isolated cells can have substantial variability when obtained from different donors. Recently, a new test for acute toxicity based on the use of human bone marrow mesenchymal stem cells (hBMMSCs) has been developed and successfully tested in our laboratory following the ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) guidelines [1]. Along these lines, the aim of this study is to evaluate the acute cytotoxicity of ZnO nanoparticles using the new toxicity test based on hBMMSCs, while comparing their behavior with respect to the toxicity of ZnO micrometer ones. For this reason, we assessed the citotoxicity by performing Neutral Red assay, the cellular uptake by transmission electron microscopy and the effects on hBMMSCs cycle by FACS analysis. Furthermore, we also analyzed by means of GC-MS the polar metabolite profile of hBMMSCs samples treated with ZnO micro- and nanoparticles. Our results show that despite the slight differences in terms of cytotoxicity, nano and microparticles show a very different behavior with respect to their effects on hBMMSCs cycle, metabolite profile and cellular uptake.

Human exposure to copper oxide (CuO) nanoparticles (NPs) is rapidly increasing and for this reason reliable toxicity test systems are urgently needed. Recently, the acute cytotoxicity of CuO NPs using the new toxicity test based on human bone marrow mesenchymal stem cells (hBMMSCs) has been evaluated. It was shown that CuO NPs are much more toxic when compared to CuO microparticles (MPs). Several studies associate CuO toxicity to a possible alteration of reactive oxygen species (ROS) system. Unluckily, the mechanism that causes the toxicity is still not clear. In this work, the polar metabolite pool of treated cells, at the corresponding IC₅₀ value, for CuO micro and NPs has been studied by gas chromatography coupled to mass spectrometry (GC-MS) and multivariate statistical data analysis. By the same means, differences due to different treatments, on samples, were investigated. Results of discriminant analysis were considered with the aim of finding the relevant metabolites unique for each class. Serine, glyceric acid, and succinic acid were upregulated on samples treated with CuO microparticles, while glutamine was the only discriminant metabolite for the class of samples treated with nanoparticles.

Rationale : Chronic elevation of 3′-5′-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. Objective : How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results : Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. Conclusions : Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

The capacity of a composite vascular graft constituting polyurethane (PU) and gelatin to support cell growth was investigated using human mesenchymal stem cells (hMSCs). Gelatin-based polyurethane grafts were fabricated by co-spraying polyurethane and gelatin using a spray, phase-inversion technique. Graft microstructure was investigated by light and scanning electron microscopy. Uniaxial tensile tests were performed to assess the grafts’ mechanical properties in longitudinal and circumferential directions. hMSCs obtained from bone marrow aspirate were seeded onto flat graft samples. After 24, 48, and 72 h of incubation, cell morphology was evaluated by Giemsa staining and cell viability was calculated by XTT assay. SEM analysis evidenced that PU samples display a microporous structure, whereas the gelatin-based PU samples show a fibrillar appearance. The presence of cross-linked gelatin produced a significant increase of ultimate tensile strength and ultimate elongation in circumferential directions compared to PU material. Qualitative analysis of hMSC adhesion onto the grafts revealed remarkable differences between gelatin-based PU and control graft. hMSCs grown onto gelatin-based PU graft form a monolayer that reached confluence at 72 h, whereas cells seeded onto the control graft were not able to undergo appropriate spreading. hMSCs grown onto gelatin-based PU graft showed significantly higher viability than cells seeded onto bare PU at all time points. In conclusion, a composite vascular graft was successfully manufactured by simultaneous co-spraying of a synthetic polymer and a protein to obtain a scaffold that combines the mechanical characteristics of polyurethanes with the favorable cell interaction features of gelatin.

Despite the growing interest in nanoparticles (NPs), standardized procedures for the evaluation of their toxicity have not been defined. The risk of human exposure is rapidly increasing and reliable toxicity test systems are urgently needed. In vitro methods are ideal in toxicology research because they can rapidly provide reproducible results while preventing the use of animals. Recently, a new test for acute toxicity based on the use of human bone marrow mesenchymal stem cells (hBMMSCs) has been developed and successfully tested in our laboratory following the Interagency Coordinating Committee on the Validation of Alternative Methods guidelines. Along these lines, the aim of this study is to evaluate the acute cytotoxicity of copper oxide (CuO) NPs using the new toxicity test based on hBMMSCs. Our results show that CuO NPs are much more toxic compared to micrometer ones. Specifically, CuO NP exposure exhibits a significant cytotoxicity at all the concentrations used, with an IC50 value of 2.5 ± 0.53 µg/ml. On the other hand, CuO microsized particle exposure exhibits a very low cytotoxicity at the same concentrations, with an IC50 value of 72.13 ± 16.2 µg/ml.

Atherosclerosis and its complications still represent the leading cause of death in the developed countries. While autologous blood vessels may be regarded as the best solution for peripheral and coronary bypass, they are unavailable in most patients. Even though tissue engineering techniques are often applied to the development of small-diameter vascular grafts, limiting factors of this approach are represented by the lack of essential extracellular matrix proteins and/or poor biomechanical properties of the scaffolds used. Along these lines, the aim of this study was to develop a decellularization protocol for ovine carotids to be used as suitable small-diameter vascular grafts. Samples were treated either with sodium dodecyl sulphate (SDS) or with Trypsin and Triton X-100; a final nuclease digestion was performed for both protocols. Morphological analyses demonstrate complete removal of nuclei and cellular components in treated vessels, also confirmed by significant reduction in wall thickness and DNA content. Essential extracellular matrix proteins such as collagen, elastin, and fibronectin are well preserved after decellularization. From a mechanical point of view, Trypsin and Triton X-100 treated arteries show elastic modules and compliance comparable to native carotids, whereas the use of SDS makes samples stiffer, with a significant decrease in the compliance mean value and an increase in longitudinal and circumferential Young's modules. It is demonstrated that the treatment where Trypsin and Triton X-100 are combined guarantees complete decellularization of carotids, with no significant alteration of biomechanical and structural properties, thus preserving a suitable environment for adhesion, proliferation, and migration of cells.

Objectives: This study focuses on experimental analysis and corresponding mathematical simulation ofin vitroHUVECs (human umbilical vein endothelial cells) proliferation in the presence of various types of drugs.Materials and methods: HUVECs, once seeded in Petri dishes, were expanded to confluence. Temporal profiles of total count obtained by classic haemocytometry and cell size distribution measured using an electronic Coulter counter, are quantitatively simulated by a suitable model based on the population balance approach. Influence of drugs on cell proliferation is also properly simulated by accounting for suitable kinetic equations.Results and discussion: The models’ parameters have been determined by comparison with experimental data related to cell population expansion and cell size distribution in the absence of drugs. Inhibition constant for each type of drug has been estimated by comparing the experimental data with model results concerning temporal profiles of total cell count. The reliability of the model and its predictive capability have been tested by simulating cell size distribution for experiments performed in the presence of drugs. The proposed model will be useful in interpreting effects of selected drugs on expansion of readily available human cells.

This study focuses on analysis of in vitro cultures of chondrocytes from ovine articular cartilage. Isolated cells were seeded in Petri dishes, then expanded to confluence and phenotypically characterized by flow cytometry. The sigmoidal temporal profile of total counts was obtained by classic haemocytometry and corresponding cell size distributions were measured electronically using a Coulter Counter. A mathematical model recently proposed () was adopted for quantitative interpretation of these experimental data. The model is based on a 1‐D (that is, mass‐structured), single‐staged population balance approach capable of taking into account contact inhibition at confluence. The model’s parameters were determined by fitting measured total cell counts and size distributions. Model reliability was verified by predicting cell proliferation counts and corresponding size distributions at culture times longer than those used when tuning the model’s parameters. It was found that adoption of cell mass as the intrinsic characteristic of a growing chondrocyte population enables sigmoidal temporal profiles of total counts in the Petri dish, as well as cell size distributions at ‘balanced growth’, to be adequately predicted.