3-D Fibrin Scaffold Improves Stemness of Human Peripheral Blood Endothelial Progenitor Cells

Aims Fibrin is a natural biopolymer appealing for cell-based regenerative therapies, because it can support growth, migration and differentiation of different cell types. Endothelial progenitor cells (EPC) represent a very interesting alternative cell source for mature endothelial cells; the fact that can easily isolated from the peripheral blood, thereby eliminating donor morbidity, makes them ideal in applications in the field of regenerative medicine. We have demonstrated that fibrin can support EPC viability and growth. Aim of this study was to evaluate if fibrin can affect EPC differentiation and stem cell markers expression. Methods Fibrin was prepared mixing commercially available (Kedrion S.p.A. Lucca, Italy) fibrinogen (9 mg/ml) and thrombin (25 U/ml). Clot ultrastructure was investigated by scanning electron microscopy (SEM) and cryogenic SEM (CRYO-SEM) to measure fibre diameter and density. Clot elasticity was evaluated by atomic force microscopy (AFM), measuring the tip-sample force by cantilever displacement. EPC were obtained from peripheral blood and cultured on fibrin at the concentration of 1x106cell/cm2. Fibronectin coating was used as a control. Metabolic activity was assessed after 7 and 14 days by WST1 assay and viability by confocal microscopy (calcein incorporation). The expression of both endothelial (CD31, KDR, vWF, Ve-Cadherin) and stem cell markers (nanog, oct-4) was assessed by flow cytometry, confocal microscopy and Real Time RT-PCR. Results SEM analysis revealed a nanometric fibrous structure, with mean fiber diameter of 165?4 nm and mean density of 95.9?0.2 %. CRYO-SEM suggested a reticulate structure with mesh-size up to 10 ?m. Fibrin clot elasticity was 1.78 MPa, as in literature. WST1 assay showed that fibrin increased EPC metabolic activity as compared to fibronectin (fibrin: 0.606?0.056 a.u. vs. fibronectin: 0.311?0.067). Calcein staining demonstrated that EPC were still viable at 14 days. Flow cytometry showed the expression of endothelial markers (CD31=41.8?8.4%; vWF=32.3?3.0%; KDR=89.3?3.7%; VE-Cadherin=41.2?3.8%), confirmed also by confocal microscopy and Real Time RT-PCR. Interestingly, nanog and oct-4 (embryonic stem cell markers) expression was significantly greater on fibrin (p<0.001) as compared to fibronectin. Conclusions These findings suggest that fibrin it is not only a suitable scaffold for EPC growth and viability but also induces EPC differentiation. The observation that Nanog, known as the most important marker of stemness, is maintained longer than on fibronectin, may offer a surplus value to stem cell-based therapies

Fibrin gel: a new scaffold for cardiovascular applications

Aims: Peripheral blood endothelial progenitor cells (EPC) are promising therapies for irreversible myocardial damage, heart failure and peripheral ischemia disease. Natural biopolymers as fibrin are appealing in tissue engineering, because fibrin is biocompatible and bioresorbable. In vitro studies indicate that fibrin can support the growth migration and proliferation of several cells types. Up to date numerous studies have proved the potential of fibrin based injectable cell delivery systems. No studies are available with fibrin as scaffold for EPC. The goal of this study was to investigate if fibrin is a suitable matrix for EPC culture as compared with fibronectin and if different concentrations of fibrinogen (Fb) and thrombin (Th) can influence fibrin structure and EPC behaviour. Methods: Fibrin (Kedrion S.p.a. Lucca, Italy) was prepared mixing Fb (final 4.5-9-18-36 mg/ml) and Th (final 6-12.5-25-50 U/ml). The scaffolds were maintained for 1 hour at 37?C, 5% CO2 before cell seeding. The ultrastructure of fibrin was investigated by scanning electron microscopy (SEM), cryogenic SEM (CRYO-SEM) and atomic force microscopy (AFM) that allow the hydratating analysis of the sample, to evaluate fibre diameter and density. EPC were obtained from peripheral blood of healthy donors and cultured for 1 week on fibrin at the concentration of 1x106 cell/ml in endothelial growth medium. EPC seeded on fibronectin were used as control. Metabolic cell activity on the different scaffolds was assessed after 7 and 14 days by WST1 while cell viability by confocal microscopy (Calcein AM incorporation). Results: Fibrin polymerization rate ranged between 17 and 68 seconds and increased at higher Fb or Th concentrations. Both AFM and SEM analysis revealed a nanometric fibrous structure, with a decrease in fiber diameter with higher fibrinogen concentrations (4.5 mg/ml: 166?4 nm. vs. 36 mg/ml: 119?3 nm, p<0.005, n=5). Different concentrations of Th didn\u27t affect fibre diameter and density. CRYO-SEM suggested a reticulate structure with mesh-size up to 10?m. WST1 assay showed that EPC metabolic activity was better with lower fibrinogen concentrations (4.5 mg/ml: 0.890?0.134 a.u. vs. 36 mg/ml 0.234?0.046 a.u., p<0.05, n=5), while Th had no significant effect. Calcein staining demonstrated that EPC were viable at 14 days and even organised in cluster. Conclusions: Fibrin combines important properties of an ideal biological scaffold, like the nanometric structure, important for the growth and migration of cells. Fibrin is also an ideal scaffold for EPC but the ratio between fibrinogen and thrombin is important for cell viability

Instructional Message Design: Theory, Research, and Practice (Volume 2)

Message design is all around us, from the presentations we see in meetings and classes, to the instructions that come with our latest tech gadgets, to multi-million-dollar training simulations. In short, instructional message design is the real-world application of instructional and learning theories to design the tools and technologies used to communicate and effectively convey information. This field of study pulls from many applied sciences including cognitive psychology, industrial design, graphic design, instructional design, information technology, and human performance technology to name just a few. In this book we visit several foundational theories that guide our research, look at different real-world applications, and begin to discuss directions for future best practice. For instance, cognitive load and multimedia learning theories provide best practice, virtual reality and simulations are only a few of the multitude of applications. Special needs learners and designing for online, e-learning, and web conferencing are only some of many applied areas where effective message design can improve outcomes. Studying effective instructional message design tools and techniques has and will continue to be a critical aspect of the overall instructional design process. Hopefully, this book will serve as an introduction to these topics and inspire your curiosity to explore further!https://digitalcommons.odu.edu/distancelearning_books/1003/thumbnail.jp

Instructional Message Design: Theory, Research, and Practice (Volume 2)

Message design is all around us, from the presentations we see in meetings and classes, to the instructions that come with our latest tech gadgets, to multi-million-dollar training simulations. In short, instructional message design is the real-world application of instructional and learning theories to design the tools and technologies used to communicate and effectively convey information. This field of study pulls from many applied sciences including cognitive psychology, industrial design, graphic design, instructional design, information technology, and human performance technology to name just a few. In this book we will visit several foundational theories that guide our research, look at different real-world applications, and begin to discuss directions for future best practice. For instance, cognitive load and multimedia learning theories provide best practice, virtual reality and simulations are only a few of the multitude of applications. Special needs learners and designing for online, e-learning, and web conferencing are only some of many applied areas where effective message design can improve outcomes. Studying effective instructional message design tools and techniques has and will continue to be a critical aspect of the overall instructional design process. Hopefully, this book will serve as an introduction to these topics and inspire your curiosity to explore further

Da Accettanti a Setantí: il processo di integrazione di una famiglia

Gli uomini d’affari toscani che giunsero a Barcellona nel corso del XV secolo vennero accolti dalla comunità dei cittadini in maniera differenziata a seconda dei casi e delle circostanze. Le ragioni della partenza dalla madrepatria, lo status sociale, così come la concezione della propria permanenza nella città comitale influenzavano l’immagine che l’universitas e le istituzioni avevano dei gruppi e dei singoli, favorendone l’ospitalità o il rigetto. Le strategie di integrazione economica, sociale e politica delle singole famiglie modulavano in maniera fondamentale la percezione dei nuovi gruppi che, acquisita la cittadinanza ufficiale per i propri componenti, giungevano a far dimenticare l’origine per godere a pieno titolo dei diritti riservati ai cittadini di Barcellona e ai sudditi della Corona d’Aragona. In questa sede prenderemo in esame il processo di integrazione e naturalizzazione di una famiglia di origine lucchese nella società barcellonese del XV secolo. Il passaggio da straniero a cittadino era un processo articolato e fortemente dipendente dal mutare delle condizioni dell’individuo e dei percorsi familiari. Per alcuni membri di questa famiglia avvenne attraverso un iter complesso fatto di strategie patrimoniali e matrimoniali intrecciate con un’attenta gestione delle relazioni col potere, che si concluse con un completo inserimento economico, sociale e politico: gli Accettanti, il cui cognome venne presto catalanizzato in Setantí, divennero una delle famiglie principali della nobiltà catalana.Peer reviewe