A novel Electrospinning Procedure for the Production of Straight Aligned and Winded Fibers

An electrospinning procedure allowing the spinning of a straight jet of polymer solution was developed. By using proper collector devices, it enables to collect winded and aligned fibers and to prepare polymeric constructs developing along the Z axis. The reported results are expected to provide basic understandings on which parameters are controlling the stability/instability of the process and implement new applications of electrospinning with specific reference to the preparation of well defined three-dimensional structure

Adsorption of the rhNGF Protein on Polypropylene with Different Grades of Copolymerization

The surface properties of drug containers should reduce the adsorption of the drug and avoid packaging surface/drug interactions, especially in the case of biologically-derived products. Here, we developed a multi-technique approach that combined Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM), Contact Angle (CA), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoemission Spectroscopy (XPS) to investigate the interactions of rhNGF on different pharma grade polymeric materials. Polypropylene (PP)/polyethylene (PE) copolymers and PP homopolymers, both as spin-coated films and injected molded samples, were evaluated for their degree of crystallinity and adsorption of protein. Our analyses showed that copolymers are characterized by a lower degree of crystallinity and lower roughness compared to PP homopolymers. In line with this, PP/PE copolymers also show higher contact angle values, indicating a lower surface wettability for the rhNGF solution on copolymers than PP homopolymers. Thus, we demonstrated that the chemical composition of the polymeric material and, in turn, its surface roughness determine the interaction with the protein and identified that copolymers may offer an advantage in terms of protein interaction/adsorption. The combined QCM-D and XPS data indicated that protein adsorption is a self-limiting process that passivates the surface after the deposition of roughly one molecular layer, preventing any further protein adsorption in the long term

Molecular basis of cardiac arrhythmias: genetics of natural variants and electrophysiological investigation of mutant proteins

Channelopathies are diseases caused by deranged functioning of ion channel subunits or the proteins that regulate them. Long QT and Brugada syndrome are included in this group. In particular, long QT syndrome (LQTS) is a familial autosomal dominant disease characterized by prolongation of the QT interval on the surface ECG, syncope, torsade de pointes and sudden cardiac death in young patients. Each type of heritable LQTS (LQTS 1-12) is linked to mutations in a specific gene. Mutations occur more frequently in the cardiac ion channel coding genes (SCN5A, KCNH2,KCNQ1) and ancillary β-subunits (KCNE1 and KCNE2). Differently, BrS is an inherited cardiac disease characterized by ST segment elevation in the right precordial leads (V1 to V3), susceptibility to ventricular tachyarrhythmia and sudden cardiac death, typically during rest or sleep. BrS is inherited as an autosomal dominant trait and its prevalence in Caucasians is 5/1000. The disorder is linked to mutations in the SCN5A gene. Our project was designed to functionally characterize the novel mutations found in genes related to LQTS and BrS to better understand the pathogenesis of pathological phenotypes . To this aim, we firs amplified by PCR all coding exons, 5’ and 3’ UTR of the SCN5A, KCNQ1, KCNH2, KCNE1 and KCNE2 genes and analyzed them by dHPLC and automatic sequencing. The mutants were generated by QuickChange site-directed mutagenesis. Mutants were transiently transfected in mammalian cells for in vitro analysis. We characterized the LQT3 associated p.ΔN1472 mutation that we found in SCN5A gene. The electrophysiological studies demonstrated that the hH1 mutation had a shift in the voltage-dependence of inactivation, a positive shift in the voltage dependence of activation and a slower recovery from inactivation compared to WT channel. Moreover, the persistent current levels were much higher in SCN5A-p.ΔN1472 than in the WT channel. We also studied mutations KCNH2-p.C108Y and KCNQ1-p.R583H. Interestingly, only subjects carrying both mutations manifested severe LQTS. The biophysical studies showed that in the homozygous condition, KCNH2-p.C108Y, led to a non-functional KCNH2 channel, whereas, in the heterozygous condition, mutant KCNH2 had a significantly reduced current density and a negative shift in the voltage dependence of activation compared to the WT. Furthermore, mutant KCNQ1-p.R583H had a significantly reduced tail current density compared to the WT channel, but no significant changes in activating current density and in voltage-dependence of activation. In conclusion, we demonstrate that the SCN5A-p.ΔN1472 and KCNH2-p.C108Y mutants exhibit characteristic biophysical properties causing LQTS; whereas KCNQ1-p.R583H, in combination with KCNE1-WT, does not exhibit striking biophysical defects, but in combination with mutant KCNH2 it results in a more severe phenotype. Our results allow to better understand the pathogenesis of LQTS phenotype and to increase the knowledge of ion channel behavior in the pathological conditions

MICRO-NANOSTRUCTURED BIOCOMPATIBLE POLYMERIC FIBRES IN THE FABRICATION OF BIOACTIVE SCAFFOLDS FOR TISSUE ENGINEERING APPLICATIONS

Tissue Engineering is an interdisciplinary field aiming at the restoration of the function of damaged tissues and organs. The most common tissue engineering approach is based on the use of a biocompatible scaffold onto which cells are seeded, in order to develop a fully or partially functional tissue construct and implant it in the human body, at the defect site. Various scaffolds manufacturing techniques are actually being investigated by the scientifical community and new trends are emerging due to the advancement in manufacturing technology. In particular, a technique called electrospinning is emerging as an interesting methodology for scaffold production, due to the morphological features that electrospun scaffolds possess. Electrospun meshes are in fact 2 or 3D structures which can mimic the nanosized features and the organization of the extracellular matrix of native tissues, providing a favourable environment for cell attachment and proliferation. Since the beginning of this century, an increasing amount of research articles based on electrospinning are being published every year, and new and appealing advances in the electrospinning technology are giving a significant contribution to the field of Tissue Engineering. This PhD program, performed within the European Network of Excellence (NoE) Expertissues, has been devoted to the investigation of electrospinning and the production and characterization of scaffolds for tissue engineering. The PhD fellowship has been carried out among three well internationally recognised laboratories, including: -the Laboratory of Polymeric Materials for Biomedical and Environmental Applications (BIOLab) of the Department of Chemistry and Industrial Chemistry of the University of Pisa (Italy), whose head is Prof. Emo Chiellini (main site of activity) -the Biopolymer Technology Laboratory of the Department of Chemical and Biological Engineering of Chalmers University of Technology in Gothenburg (Sweden) led by Prof. Paul Gatenholm -the Regenerative Medicine Laboratory at the Institute of Health and Biomedical Innovation (IHBI) of Queensland University of Technology in Brisbane (Australia), led by Prof. Dietmar W. Hutmacher Besides the introduction Chapter, reviewing the use of electrospun fibrous meshes in different fields of Tissue Engineering and Drug Delivery, this thesis includes 6 main chapters: Development of Advanced Technologies for Tissue Engineering Applications, describing modification of the standard solution electrospinning apparatus at BioLab in order to optimize the manufacturing process, and the design of a roto-translating collector for the obtainment of electrospun small diameter conduits Biodegradable polymeric micro-nanofibers by electrospinning of polyester/polyether block copolymers, investigating the differences in (solution) electrospun meshes morphology and mechanical properties of block copolymers with different weight ratio and molecular weight of the components Mechanical evaluation of electrospun meshes by uniaxial loading under scanning electron microscopy, which is based on the use of a specific apparatus to visualize fibrous meshes through scanning electron microscopy during uniaxial tensile loading. This chapter is focused on the mechanical loading of meshes produced by solution electrospinning and from 3 different materials, highlighting differences in fibrous network behaviour based on morphological analysis Novel Electrospun Polyurethane/Gelatin Composite Meshes for Vascular Grafts, that discusses a novel (solution) electrospun tubular composite produced at BioLab, made from a biomedical polyurethane (Tecoflex EG-80°) and a biopolymer (gelatin). Particular attention is devoted to the analysis of the mechanical properties in wet conditions and the endothelial cells compatibility of the tubular composite as compared to the conduit constituted by the sole polyurethane Melt Electrospinning and Morphological Characterization of Polycaprolactone and Polycaprolactone Copolymers, which investigates melt electrospinning as a component of electrospinning so far not adequately explored by researchers in the field. The chapter demonstrates how melt electrospinning can succesfully produce uniform Polycaprolactone (PCL) fibrous meshes with average diameter in the micron-size, which could be employed as scaffold in Tissue Engineering. The addition of a PCL copolymer, resulting into smaller diameter fibres as compared to pure PCL, is also discussed. Cellular Characterization of Melt Electrospun Polycaprolactone Meshes, in which human osteoblasts are seeded on PCL melt electrospun scaffolds and cultured up to 19 weeks. Several in-vitro assays are taken into consideration to evaluate cell attachment and proliferation, together with the deposition of mineralized matrix components and production of extracellular matrix proteins by the cells. The seven Chapters constituting the body of this PhD program, in their final form or a modified version, have been already published or are going to be submitted for publication in journals relevant to Biomaterials & Tissue Engineering

The multi-faceted aspects of the complex cardiac Nav1.5 protein in membrane function and pathophysiology

The aim of this mini-review is to draw together the main concepts and findings that have emerged from recent studies of the cardiac channel protein Nav1.5. This complex protein is encoded by the SCN5A gene that, in its mutated form, is implicated in various diseases, particularly channelopathies, specifically at cardiac tissue level. Here we describe the structural, and functional aspects of Nav1.5 including post-translational modifications in normal conditions, and the main human channelopathies in which this protein may be the cause or trigger. Lastly, we also briefly discuss interacting proteins that are relevant for these channel functions in normal and disease conditions

Poly(lactic-co-glycolic acid) electrospun fibrous meshes for the controlled release of retinoic acid

Poly(lactic-co-glycolic acid) (PLGA) meshes loaded with retinoic acid (RA) were prepared by applying the electrospinning technique. The purpose of the present work was to combine the biological effects of RA and the advantages of electrospun meshes to enhancing the mass transfer features of controlled release systems and cell interaction with polymeric scaffolds. The processing conditions for the fabrication of three-dimensional meshes were optimized by studying their influence on mesh morphology. Tensile testing showed that RA loading influenced the meshes mechanical properties by increasing their strength and rigidity. Moreover, the drug release and degradation profiles of the electrospun systems were compared to analogous RA-loaded PLGA films prepared by solvent casting. The results of this study highlight that the electrospun meshes preserved their fibrous structure after 4 months under in vitro physiological conditions and showed a sustained controlled release of the loaded agent in comparison to that observed for cast films. The bioactivity of the loaded RA was investigated on murine preosteoblasts cells by evaluating its influence on cell proliferation and morphology

Electrospun Polymeric Meshes for Application in Tissue Engineering

Electrospun Polymeric Meshes for Application in Tissue Engineerin

Genetic analysis in a family affected by sick sinus syndrome may reduce the sudden death risk in a young aspiring competitive athlete

a CEINGE‐Biotecnologie Avanzate s.c.ar.l., Naples, Italy b Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita di Napoli Federico II, Naples, Italy c Unita Operativa Clinica di Cardiologia, Ospedale dei Colli, Seconda Universita di Napoli, Naples, Italy d Medicina dello Sport, Dipartimento di Scienze Biomediche Avanzate, University of Naples Federico II, Naples, Italy e IRCCS-Fondazione SDN, Naples, Ital

Melt electrospinning of poly(epsilon-caprolactone) scaffolds: Phenomenological observations associated with collection and direct writing

Melt electrospinning and its additive manufacturing analogue, melt electrospinning writing (MEW), are two processes which can produce porous materials for applications where solvent toxicity and accumulation in solution electrospinning are problematic. This study explores the melt electrospinning of poly(ε-caprolactone) (PCL) scaffolds, specifically for applications in tissue engineering. The research described here aims to inform researchers interested in melt electrospinning about technical aspects of the process. This includes rapid fiber characterization using glass microscope slides, allowing influential processing parameters on fiber morphology to be assessed, as well as observed fiber collection phenomena on different collector substrates. The distribution and alignment of melt electrospun PCL fibers can be controlled to a certain degree using patterned collectors to create large numbers of scaffolds with shaped macroporous architectures. However, the buildup of residual charge in the collected fibers limits the achievable thickness of the porous template through such scaffolds. One challenge identified for MEW is the ability to control charge buildup so that fibers can be placed accurately in close proximity, and in many centimeter heights. The scale and size of scaffolds produced using MEW, however, indicate that this emerging process will fill a technological niche in biofabrication