STRUCTURAL DETERMINISTIC MODELING DESIGN AND FABRICATION OF ELECTROSPUN SCAFFOLDS FOR SOFT TISSUE ENGINEERING

The research fields of tissue engineering, biomechanics and regenerative medicine continue to evolve in response to the ever growing need for tissue replacement options. These fields aim to restore, maintain, or improve tissue or whole organ function. This doctoral studies focus on the development and experimental validation of a structural deterministic modeling strategy to: A) guide tissue engineering scaffold design, B) provide a better understanding of cellular mechanical and metabolic response to local micro-structural deformations. Targeted clinical application was the pulmonary heart valve. Electrospinning was selected as the optimal platform technology to implement, validate and test the presented designing strategy. An innovative custom made software was developed and tested on Electrospun poly (ester urethane) urea scaffolds (ES-PEUU), decellularized native tissues and collagen gels to fully characterized engineered constructs morphology. These structural information were adopted to feed and assist the mechanical modeling Two previously unevaluated fabrication modalities were investigated throughout both mechanical testing and image analysis in order to explore further how the electrospinning fabrication process can alter the structure and mechanical response: variation of mandrel translation velocity and concurrent electrospraying of cell culture medium with or without cells or rigid particulates. These fabrication parameters were studied to enrich control in the electrospinning process. 8 The detected material topology and mechanical equi-biaxial data were adopted to generate statistically equivalent scaffold mechanical models. The structural determinist approach was applied to ES-PEUU scaffolds, validated and mechanical response at organ and cell level was produced through FEM simulation. Prediction included: membrane tension vs. stretch relation, elasticity moduli, Nuclear Aspect Ratio vs. stretch relation for the cells micro-integrated into the scaffold. A three weeks in vivo - study on an ovine model was performed to demonstrated the feasibility of the adoption of ES-PEUU for TEHVs and more generally this material potential for soft tissue regeneration. Explants analysis showed surgical feasibility and acceptable valve functionality. The developed design strategies combining image analysis and structural deterministic modeling enabled the material topology to be both quantified and reproduced. Material fabrication parameters were related to material micro-architecture Similarly, the micro-architecture was related to macro scale mechanical responses such as in-plane reactions or flexural rigidity, and complex meso – micro scales mechanisms such NAR – stretch relation. In conclusion, the modeling approach introduced in this work bridges for the first time the scaffold fabrication parameters with the mechanical response at different scale length. The developed paradigm will be utilized to identify the optimal scaffold for a given soft tissue engineering application

Digital Service Act: garantire un ambiente online sicuro e responsabile

Il Digita Service Act (DSA) è una delle due parti di una proposta di legge che si prefigge come obiettivo quello di modernizzare e rendere più efficace la Direttiva sul commercio elettronico. I sevizi online non sempre sono usati in modo consono: basti pensare alle tecniche di “nudging” (il pilotare le scelte di un utente inconsciamente) o la numerosa presenza di contenuti illegali e disinformativi presenti sulle piattaforme di social media. La proposta di legge, avanzata dalla Commissione europea, si pone come obiettivo la risoluzione di queste problematiche, proponendo una serie di obblighi e restrizioni per evitare che l’utenza online subisca un’esperienza negativa. Allo scopo di capire quali siano i pensieri degli stakeholders riguardo la proposta del DSA, è stata condotta un’analisi statistica su un dataset di 110 file, ognuno dei quali rappresenta un feedback di uno stakeholder, tramite gli algoritmi Wordfish ed MCA

Systems Level Approach Reveals the Correlation of Endoderm Differentiation of Mouse Embryonic Stem Cells with Specific Microstructural Cues of Fibrin Gels.

Stem cells receive numerous cues from their associated substrate that help to govern their behaviour. However, identification of influential substrate characteristics poses difficulties because of their complex nature. In this study, we developed an integrated experimental and systems level modelling approach to investigate and identify specific substrate features influencing differentiation of mouse embryonic stem cells (mESCs) on a model fibrous substrate, fibrin. We synthesized a range of fibrin gels by varying fibrinogen and thrombin concentrations, which led to a range of substrate stiffness and microstructure. mESCs were cultured on each of these gels, and characterization of the differentiated cells revealed a strong influence of substrate modulation on gene expression patterning. To identify specific substrate features influencing differentiation, the substrate microstructure was quantified by image analysis and correlated with stem cell gene expression patterns using a statistical model. Significant correlations were observed between differentiation and microstructure features, specifically fibre alignment. Furthermore, this relationship occurred in a lineage-specific manner towards endoderm. This systems level approach allows for identification of specific substrate features from a complex material which are influential to cellular behaviour. Such analysis may be effective in guiding the design of scaffolds with specific properties for tissue engineering applications

Engineering in-plane mechanics of electrospun polyurethane scaffolds for cardiovascular tissue applications

Effective cardiovascular tissue surrogates require high control of scaffold structural and mechanical features to match native tissue properties, which are dependent on tissue-specific mechanics, function heterogenicity, and morphology. Bridging scaffold processing variables with native tissue properties is recognized as a priority for advancing the biomechanical performance of biomedical materials and when translated to clinical practice, their efficacy. Accordingly, this study selected electrospinning on a rotating cylindrical target as an apparatus of broad application and mapped the relationship between key processing variables and scaffold mechanics and structure. This information was combined with mechanical anisotropy ranges of interest for the three main categories of tissue surrogated in cardiovascular tissue engineering: heart valve leaflets, ventricle wall, and large diameter blood vessels. Specifically, three processing variables have been considered: the rotational velocity and the rastering velocity of the mandrel and the dry (single nozzle – polymer only) vs wet (double nozzle – polymer plus phosphate buffer saline solution) fabrication configuration. While the dry configuration is generally utilized to obtain micro-fiber based polymeric mats, the wet fabrication is representative of processing conditions utilized to incorporate cells, growth factors, or micro-particles within the fibrous scaffold matrix. Dry and wet-processed electrospun mats were fabricated with tangential and rastering velocities within the 0.3-9.0 m/s and 0.16-8 cm/s range respectively. Biaxial mechanics, fiber network, and pore microarchitectures were measured for each combination of velocities and for each fabrication modality (dry and wet). Results allowed identification of the precise combination of rotational and rastering velocities, for both dry and wet conditions, that is able to recapitulate the native cardiovascular tissue anisotropy ratio. By adopting a simple and broadly utilized electrospinning layout, this study is meant to provide a repeatable and easy to access methodology to improve biomimicry of the in plane-mechanics of heart valve leaflets, ventricular wall, and large diameter blood vessels

Gender-related differences in lifestyle may affect health status

Consistent epidemiological and clinical evidence strongly indicates that chronic noncommunicable diseases are largely associated with four lifestyle risk factors: inadequatediet, physical inactivity, tobacco use, and excessive alcohol use. Notably, obesity, a worldwide-growing pathological condition determined by the combination between inadequate diet and insufficient physical activity, is now considered a main risk factor for most chronic diseases. Dietary habits and physical activity are strongly influenced by gender attitudes and behaviors that promote different patterns of healthy or unhealthy lifestyles among women and men. Furthermore, different roles and unequal relations between genders strongly interact with differences in social and economic aspects as well as cultural and societal environment. Because of the complex network of factors involved in determining the risk for chronic diseases, it has been promoting a systemic approach that, by integrating sex and gender analysis, explores how sex-specific biological factors and gender-related social factors can interact to influence the health status

Identification of miR-9-5p as direct regulator of ABCA1 and HDL-driven reverse cholesterol transport in circulating CD14+ cells of patients with metabolic syndrome.

AIMS: Metabolic syndrome (MS) is a cluster of cardio-metabolic risk factors associated with atherosclerosis and low-grade inflammation. Using unbiased expression screenings in peripheral blood mononuclear cells, we depict here a novel expression chart of 678 genes and 84 microRNAs (miRNAs) controlling inflammatory, immune and metabolic responses. In order to further elucidate the link between inflammation and the HDL cholesterol pathway in MS, we focussed on the regulation of the ATP-binding cassette transporter A1 (ABCA1), a key player in cholesterol efflux (CE). METHODS AND RESULTS: ABCA1 mRNA levels are suppressed in CD14+ cells of MS patients and are negatively correlated to body mass index (BMI), insulin-resistance (HOMA-IR) and cardiovascular risk, and positively to HDL cholesterol and CE. miRNA target in silico prediction identified a putative modulatory role of ABCA1 for the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) target miR-9-5p, whose expression pattern was up-regulated in CD14+ cells of MS patients, positively correlated to BMI, HOMA-IR, and triglycerides, and negatively to ABCA1 mRNA levels, HDL cholesterol and CE. Ectopic gain and loss of miR-9-5p function in macrophages modulated ABCA1 mRNA and protein levels, ABCA1 miRNA 3'-untranslated region target sequence reporter assay, and CE into HDL, thus confirming ABCA1 as a target of miR-9-5p. CONCLUSIONS: We identified the NF-κB target miR-9-5p as a negative regulator of ABCA1 adding a novel target pathway in the relationship between inflammation and HDL-driven reverse cholesterol transport for prevention or treatment of atherosclerosis in MS.N/

Identification of miR-9-5p as direct regulator of ABCA1 and HDL-driven reverse cholesterol transport in circulating CD14+ cells of patients with metabolic syndrome.

Aims: Metabolic syndrome (MS) is a cluster of cardio-metabolic risk factors associated with atherosclerosis and low-grade inflammation. Using unbiased expression screenings in peripheral blood mononuclear cells, we depict here a novel expression chart of 678 genes and 84 microRNAs (miRNAs) controlling inflammatory, immune and metabolic responses. In order to further elucidate the link between inflammation and the HDL cholesterol pathway in MS, we focussed on the regulation of the ATP-binding cassette transporter A1 (ABCA1), a key player in cholesterol efflux (CE). Methods and results: ABCA1 mRNA levels are suppressed in CD14+ cells of MS patients and are negatively correlated to body mass index (BMI), insulin-resistance (HOMA-IR) and cardiovascular risk, and positively to HDL cholesterol and CE. miRNA target in silico prediction identified a putative modulatory role of ABCA1 for the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) target miR-9-5p, whose expression pattern was up-regulated in CD14+ cells of MS patients, positively correlated to BMI, HOMA-IR, and triglycerides, and negatively to ABCA1 mRNA levels, HDL cholesterol and CE. Ectopic gain and loss of miR-9-5p function in macrophages modulated ABCA1 mRNA and protein levels, ABCA1 miRNA 3'-untranslated region target sequence reporter assay, and CE into HDL, thus confirming ABCA1 as a target of miR-9-5p. Conclusions: We identified the NF-κB target miR-9-5p as a negative regulator of ABCA1 adding a novel target pathway in the relationship between inflammation and HDL-driven reverse cholesterol transport for prevention or treatment of atherosclerosis in MS.N/

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy

Unseeded Elastomeric Single Leaflets Retain Function and Remodel After Implant In Ovine Pulmonary Outflow Tract

Current materials for heart valve replacement and repair are limited by the inability to grow or remodel. Tissue engineered valves offer the potential to overcome these disadvantages by creating living structures, but is limited by the availability of biocompatible scaffold materials with desirable biomechanical properties. We assessed the in vivo performance of a novel scaffold poly(carbonate urethane) urea (PCUU), fabricated by electrospinning and implanted in the pulmonary outflow tract of sheep. PCUU was electrospun into elastomeric sheets of thickness ranging from 120-180 μm. Using cardiopulmonary bypass we replaced the native anterior pulmonary leaflet with an acellular PCUU leaflet. Valve function was evaluated by epicardial echocardiography at implant and explant at weeks 1 (n=3), 3 (n=3), 6 (n=3) and 16 (n=3). Histological, immunohistochemical, molecular imaging analyses and multi-photon imaging were performed on the explanted leaflets. Echocardiography demonstrated mobile functioning leaflets, with zero to mild pulmonary regurgitation. Molecular imaging showed increased levels of proteolytic activity and macrophage accumulation. Histology showed persistence of scaffold material up to 16 weeks with cellular infiltration throughout the leaflet. Picrosirius red revealed mature collagen deposition along the arterial surface of the construct at 6 and 16 weeks. These findings were corroborated by multi-photon analysis showing highly aligned collagen fibers across the leaflets. Both surfaces of the engineered leaflets were consistently covered with CD31 positive cells. The majority of cells expressed α-SMA and MMP2. CD45 positive cells, suggesting hematogenous origin, were found throughout the leaflet. These results suggest that: 1) PCUU can be a suitable polymer for valve bioengineering; 2) cell pre-seeding may not be required for tissue formation or remodeling for a functional engineered valve; 3) host cells seem to populate the leaflet either by migration from adjacent tissue or by attachment from circulating blood; 4) mature matrix orientation and increased proteolytic activity suggests active tissue remodeling. Longer term implants and the role of scaffold pre-seeding will require further study

Surgical treatment of primitive gastro-intestinal lymphomas: a systematic review

Primitive Gastrointestinal Lymphomas (PGIL) are uncommon tumours, although time-trend analyses have demonstrated an increase. The role of surgery in the management of lymphoproliferative diseases has changed over the past 40 years. Nowadays their management is centred on systemic treatments as chemo-/radio- therapy. Surgery is restricted to very selected indications, always discussed in a multidisciplinary setting. The aim of this systematic review is to evaluate the actual role of surgery in the treatment of PGIL