The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin- and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50-60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes

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Image analysis has gained new effort in the scientific community due to the chance of investigating morphological properties of three dimensional structures starting from their bi-dimensional gray-scale representation. Such ability makes it particularly interesting for tissue engineering (TE) purposes. Indeed, the capability of obtaining and interpreting images of tissue scaffolds, extracting morphological and structural information, is essential to the characterization and design of engineered porous systems. In this work, the traditional image analysis approach has been coupled with a probabilistic based percolation method to outline a general procedure for analysing tissue scaffold SEM micrographs. To this aim a case study constituted by PCL multi-scaled porous scaffolds was adopted. Moreover, the resulting data were compared with the outputs of conventionally used techniques, such as mercury intrusion porosimetry. Results indicate that image processing methods well fit the porosity features of PCL scaffolds, overcoming the limits of the more invasive porosimetry techniques. Also the cut off resolution of such IP methods was discussed. Moreover, the fractal dimension of percolating clusters, within the pore populations, was addressed as a good indication of the interconnection degree of PCL bi-modal scaffolds. Such findings represent (i) the bases for a novel approach complementary to the conventional experimental procedure used for the morphological analysis of TE scaffolds, in particular offering a valid method for the analysis of soft materials (i.e., gels); also (ii) providing a new perspective for further studies integrating to the structural and morphological data, fluid-dynamics and transport properties modelling.

The traditional paradigm of tissue engineering of regenerating in vitro tissue or organs, through the combination of an artificial matrix and a cellular population has progressively changed direction. The most recent concept is the realization of a fully functional biohybrid, where both, the artificial and the biotic phase, concur in the formation of the novel organic matter. In this direction, interest is growing in approaches taking advantage of the control at micro- and nano-scale of cell material interaction based on the realization of elementary tassels of cells and materials which constitute the beginning point for the expansion of 3D more complex structures. Since a spontaneous assembly of all these components is expected, however, it becomes more fundamental than ever to define the features influencing cellular behavior, either they were material functional properties, or material architecture. In this work, it has been investigated the direct effect of electrospun fiber sizes on oxygen metabolism of h-MSC cells, when any other culture parameter was kept constant. To this aim, thin PCL electrospun membranes, with micro- and nano-scale texturing, were layered between two collagen slices up to create a sandwich structure (µC-PCL-C and nC-PCL-C). Cells were seeded on membranes, and the oxygen consumption was determined by a phosphorescence quenching technique. Results indicate a strong effect of the architecture of scaffolds on cell metabolism, also revealed by the increasing of HIF1-α gene expression in nC-PCL-C. These findings offer new insights into the role of materials in specific cell activities, also implying the existence of very interesting criteria for the control of tissue growth through the tuning of scaffold architecture.

The use of scaffold-aided strategies for the regeneration of biological tissues requires the fulfilment of an accurate architectural design, that is, micro and macrostructure, with the final goal of realizing architectures to adopt as guidance for those cell activities specific to the formation of novel tissues. Here, highly porous scaffolds made up of biodegradable poly(ε-caprolactone) (PCL) have been realized by thermally induced phase separation (TIPS). Two different polymer/solvent systems, derived by the dissolution of PCL in dioxane and DMSO respectively, were investigated. The aim was to demonstrate the high potential of TIPS technique, in imprinting specific pore features to the polymer matrices, by a conscious selection of polymer/solvent systems. The investigation of pore architecture by SEM/mercury intrusion porosimetry/image analyses, firstly allow to detect remarkable variations in porosity (from 92% to 78%,) and pore sizes, ranging from micro-scale (ca 10 µm) to macro-scale (greater than 100 µm) as a function of the used polymer/solvent systems. Moreover, experimental and theoretical evidences referred to scaffold shaped in custom-made molds--a thin Teflon ring between two copper plates--allow exploring how the sensitivity of polymer solution features (i.e., crystallinity, thermal inertia) to the cooling temperature can affect the alignment of polymer phases and, ultimately, scaffold pore anisotropy. Analytical results supported by preliminary biological studies demonstrate the higher ability of PCL/dioxane solution to promote the formation of aligned pores which provide a morphological guidance to cell advance during the preliminary stage of culture. These findings, taken as a whole, put the basis for a better informed regeneration of structurally complex tissues based on the modeling of scaffold micro and macro-architecture by thermodynamic forces.

Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. In this chapter, we examine the promise and shortcomings of "top-down" and "bottom-up" approaches for creating engineered biological tissues. In top-down approaches, the cells are expected to populate the scaffold and create the appropriate extracellular matrix and microarchitecture often with the aid of a bioreactor that furnish the set of stimuli required for an optimal cellular viability. Specifically, we survey the role of cell material interaction on oxygen metabolism in three-dimensional (3D) in vitro cultures as well as the time and space evolution of the transport and biophysical properties during the development of de novo synthesized tissue-engineered constructs. We show how to monitor and control the evolution of these parameters that is of crucial importance to process biohybrid constructs in vitro as well as to elaborate reliable mathematical model to forecast tissue growth under specific culture conditions. Furthermore, novel strategies such as bottom-up approaches to build tissue constructs in vitro are examined. In this fashion, tissue building blocks with specific microarchitectural features are used as modular units to engineer biological tissues from the bottom up. In particular, the attention will be focused on the use of cell seeded microbeads as functional building blocks to realize 3D complex tissue. Finally, a challenge will be the potential integration of bottom-up techniques with more traditional top-down approaches to create more complex tissues than are currently achievable using either technique alone by optimizing the advantages of each technique.

the proposed investigation confirms that polymer blending is a promising approach to realize structurally organized platforms able to guide the regeneration processes of hierarchically organized tissues (i.e, tendons, muscles, ligaments and nerves).

Measuring oxygen concentration in three-dimensional cultures, without interfering with cellular activities, is a fundamental request of tissue engineering research. Among the other techniques, it has been demonstrated that phosphorescence quenching microscopy (PQM) represents a valid tool for the detection of oxygen concentration in 3D environments. Indeed, it is not invasive, with high spatial and temporal resolution, and, once calibrated, it is not affected by the presence of extracellular matrix components and other environmental factors. In this work, a description of the PQM experimental set up for oxygen measurements in solutions and 3D polymer-based cellular constructs is provided. Moreover, the advantage and the limits in the use of this technique are critically discussed to provide a technical note for future applications.