Giovanni Vozzi

Criticality of the Biological and Physical Stimuli Array Inducing Resident Cardiac Stem Cell Determination

The replacement of injured cardiac contractile cells with stem cell‐derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine‐tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues.

Enzymatically crosslinked porous composite matrices for bone tissue regeneration

Three-dimensional porous hydroxyapatite/collagen (HA/Coll) composites with a random pore structure were obtained by freeze-drying and crosslinked by an enzymatic treatment using microbial transglutaminase (mTGase). The procedure resulted in improved mechanical strength and thermal stability of the scaffolds. The scaffolds were characterized in terms of their stability (Coll release, swelling, collagenase-mediated degradation), thermal properties (thermogravimetric analysis, differential scanning calorimetry), mechanical behavior under compression and cell compatibility. Enzymatic treatment stabilized the sponges to water vapors, with measurable swelling ratio between 100% for HA/Coll/mTGase 0/100 to 5% for HA/Coll/mTGase 80/20. Weight loss in water due to Coll release was between 2 and 10% in mTGase-crosslinked samples and decreased with increasing HA content. Cultures of MG63 osteoblast-like cells and human umbilical vein endothelial cells (HUVEC) showed good adhesion and proliferation on the scaffolds, good viability (through MTT test, 100-150% of control), and good differentiation (alkaline phosphatase, up to 40 UI/L with respect to 35 UI/L for control).

Organic field effect transistors for textile applications

In this paper, several issues concerning the development of textiles endowed with electronic functions will be discussed. In particular, issues concerning materials, structures, electronic models, and the mechanical constraints due to textile technologies will be detailed. The idea starts from an already developed organic field-effect transistor that is realized on a flexible film that can be applied, after the assembly, on whatever kind of substrate, in particular, on textiles. This could pave the way to a variety of applications aimed to conjugate the favorable mechanical properties of textiles with the electronic functions of transistors. Furthermore, a possible perspective for the developments of organic sensors based on this structure are described.

HEMET: Mathematical model of biochemical pathways for simulation and prediction of HEpatocyte METabolism

Many computer studies and models have been developed in order to simulate cell biochemical pathways. The difficulty of integrating all the biochemical reactions that occur in a cell in a single model is the main reason for the poor results in the prediction and simulation of cell behaviour under different chemical and physical stimuli. In this paper we have translated biochemical reactions into differential equations for the development of modular model of metabolism of a hepatocyte cultured in static and standard conditions (in a plastic multiwell placed in an incubator at 37 degrees C with 5% of CO(2)). Using biochemical equations and energetic considerations a set of non-linear differential equations has been derived and implemented in Simulink. This set of equations mimics some of the principal metabolic pathways of biomolecules present in the culture medium. The software platform developed is subdivided into separate modules, each one describing a different metabolic pathway; they constitute a library which can be used for developing new modules and models to project, predict and validate cell behaviour in vitro.

Automated software for analysis of ciliary beat frequency and metachronal wave orientation in primary ciliary dyskinesia

Patients with primary ciliary dyskinesia (PCD) have structural and/or functional alterations of cilia that imply deficits in mucociliary clearance and different respiratory pathologies. A useful indicator for the difficult diagnosis is the ciliary beat frequency (CBF) that is significantly lower in pathological cases than in physiological ones. The CBF computation is not rapid, therefore, the aim of this study is to propose an automated method to evaluate it directly from videos of ciliated cells. The cells are taken from inferior nasal turbinates and videos of ciliary movements are registered and eventually processed by the developed software. The software consists in the extraction of features from videos (written with C++ language) and the computation of the frequency (written with Matlab language). This system was tested both on the samples of nasal cavity and software models, and the results were really promising because in a few seconds, it can compute a reliable frequency if compared with that measured with visual methods. It is to be noticed that the reliability of the computation increases with the quality of acquisition system and especially with the sampling frequency. It is concluded that the developed software could be a useful mean for PCD diagnosis.

Optimization of PAM Scaffolds for Neural Tissue Engineering: Preliminary Study on an SH-SY5Y Cell Line

Engineering neural tissue is one of the most challenging goals of tissue engineering. Neural tissue is highly complex and possesses an organized three-dimensional (3D) distribution that is essential for tissue function. An optimal scaffold for tissue engineering has to provide this distribution until the cells are able to activate their normal functions and develop neural connections with the host tissue. Different strategies such as gene therapy and cell transplantation particularly in retinal tissue have been tested, but so far they have only induced retinal degeneration in animals. The objective of this work was to study neural cell assembly as a function of scaffold features and surface chemistry for application in retinal tissue engineering using microfabricated patterns with a well-defined geometry. Because retinal neurons are known to be arranged in hexagonal arrays, hexagonal scaffolds of poly(DL-lactide-co-glycolide) acid were fabricated using a pressure-assisted microsyringe (PAM) system. The behavior of a model cell, neuroblastoma originating from human retina (SH-SY5Y), was analyzed after seeding on the scaffolds, measuring cell density as a function of line width and length of the scaffold to identify the optimal hexagonal geometry. We also evaluated the influence of scaffold on cell metabolism using the methyl thiazolyl tetrazolium assay and on neurite extension. As far as two-dimensional scaffolds are concerned, the results show that although metabolic activity per cell remains constant, hexagons with sides of 500 microm and line widths of 20 +/- 5 microm are optimum for neural cell adhesion in terms of cell density. On 3D scaffolds, cell metabolism is about three times higher than controls, and the optimum number of layers in the scaffold is three or four.

Microfabricated electroactive carbon nanotube actuators

A variety of microfabrication techniques have been developed at the University of Pisa. They are based either on pressure or piston actuated microsyringes or modified ink-jet printers. This work present the results of a study aimed at fabricating carbon nanotube (NT) actuators using micro-syringes. In order to prevent the nanotubes from aggregating into clumps, they were enclosed in a partially cross-linked polyvinylalcohol - polyallylamine matrix. After sonication the solution remained homogenously dispersed for about 40 minutes, which was sufficient time for deposition. Small strips of NT, about 5 mm across and 15 mm long were deposited. Following deposition, the films were baked at 80 degree(s)C and their thickness, impedance and mechanical resistance measured. The results indicate that 50 minutes of baking time is sufficient to give a constant resistivity of 1.12 x 10-2 (Omega) m per layer similar to a typical semiconductor, and each layer has a thickness of about 6 micrometers .

A free-standing hydrostatic bioreactor for neural tissue culture

A hydrostatic bioreactor for the study of cells and tissue is described. The system, which was developed to determine the effects of increased pressure on neural tissue, is capable of applying pressures of up to 100 mmHg with an error of /spl plusmn/2 mmHg in an enclosed chamber. On-line pH, pressure and temperature control enables the system to be monitored continuously and to be independent of bulky cell culture incubators.

Rapid prototyping composite and complex scaffolds with PAM2.

To create composite synthetic scaffolds with the same degree of complexity and multilevel organization as biological tissue, we need to integrate multilevel biomaterial processing in rapid prototyping systems. The scaffolds then encompass the entire range of properties, which characterize biological tissue. A multilevel microfabrication system, PAM(2), has been developed to address this gap in material processing. It is equipped with different modules, each covering a range of material properties and spatial resolutions. Together, the modules in PAM(2) can be used to realize complex and composite scaffolds for tissue engineering, bringing us a step closer to real clinical applications. This chapter describes the PAM(2) system and discusses some of the practical issues associated with scaffold microfabrication and biomaterial processing.

Molecular Imprinted Nanostructures In Biomedical Applications

Molecular imprinting is an emerging technology that allows to introduce nanostructured cavities into a polymer. In preparing molecular imprinted polymers (MIPs), the functional monomer(s) is first prearranged around the template molecule by specific interactions; the polymerisation is then carried out with a high percentage of cross-linking agent (which “freezes” the macromolecular network). Molecular mechanics and dynamics can be used to gain indications on the best monomers to be used in order to maximize interactions with the template. Once the polymerization reaction has been completed, the template is removed from the rigid three-dimensional network, leaving free recognition cavities available for the successive selective rebinding of the template itself. Precipitation polymerisation in dilute solutions involves the spontaneous formation of submicron scale polymer particles, which result suitable for recognition-rebinding application. Therapeutic applications: The recognition mechanism by MIPs relies mainly on the establishment of reversible hydrogen bonding interactions. It is clear that the efficiency of this mechanism is endangered in aqueous environments. MIPs working in water solutions are clearly of great interest in the medical and food industry and in sensor applications. We recently overcame these difficulties by the realisation of a system where cross-linked MI methylmethacrylate-methacrylic acid nanospheres where loaded on the surface or inside the matrix of porous membranes created by phase inversion. E.g. membranes were modified by adding cholesterol imprinted nanoparticles. Rebinding performances of nanoparticles modified membranes in buffer solution were tested showing a specific recognition of 14.09 mg of cholesterol/g of system (membrane and nanoparticles), indicating maintained binding capacity of supported particles as well. Tissue engineering: The engineering of functionalised polymeric structures for the study of cell activity is essential to the development of biological substitutes containing vital cells capable of regenerating or enhancing tissue function. Cells are organised within a complex matrix consisting of high molecular weight protein and polysaccharides known as the Extracellular Matrix (ECM). Two approaches are described to explore the possibility to provide scaffolds with specific and selective recognition of peptide sequences or proteins involved in cell adhesion mechanisms: one approach consists in the modification of porous structures with nanoparticles imprinted with aminoacid sequences (epitopes) of ECM proteins or transmembrane integrins, while the other consists in the combination of Soft Litography and Molecular Imprinting technologies (SOFT-MI). This technology allows to create imprinting nanocavities selective towards ECM proteins in microfabricated scaffolds, and in particular it permits to realise patterns with a well defined microscale geometry in polymethylmethacrylate (PMMA) scaffolds providing them with cell adhesion properties that were missing in the non-imprinted scaffold.Copyright