Devid Maniglio

Genipin-Modified Silk-Fibroin Nanometric Nets

Nanometric silk-fibroin nets were fabricated by electrospinning from regenerated Bombyx mori silk-fibroin (SF)/formic acid solutions with the addition of genipin (GE), 2, 15 and 24 h after the solution preparation. After spinning, the pure fibroin nanofibers were water soluble and needed a further stabilization process, whereas the reaction of fibroin with genipin resulted in water-insoluble fibroin nets due to conformational changes induced in the fibroin by the genipin. SFGE nanofibers presented diameters ranging from 140 to 590 nm and were generally thinner than pure SF nanofibers. The secondary structure of SF into SFGE nanofibers showed the presence of a beta-sheet conformation together with beta-turn intermediates (turns and bends). The approach described in this paper provides an alternative method of designing SF nanofibers that are already water insoluble, without any stability post-treatment needed.

Biohybrid nanofiber constructs with anisotropic biomechanical properties.

Synthetic implant materials often lack of the anisotropic mechanical properties and cell-interactive surface which are shown by natural tissues. For example, engineered vascular grafts need to be developed to address the mechanical and biological problems associated with the graft materials. This study has demonstrated a double-electrospinning fabrication process to produce a poly(ε-caprolactone)-fibroin multilayer composite which shows well-integrated nanofibrous structure, endothelial-conducive surface and anisotropic mechanical property, suitable as engineered vascular constructs. Electrospinning parameters such as voltage, solution concentration, feed rate, and relative humidity were optimized to obtain defect-free, uniform nanofibers. To mimic the different mechanical properties of natural vessels in the circumferential and longitudinal directions, a rotating cylinder was used as collector, resulting in the production of constructs with anisotropic properties. The combination of the collector shape and the collector rotation allows us to produce a tubular structure with tunable anisotropic mechanical properties. Fourier transform infrared spectroscopy, differential scanning calorimetry, and uniaxial tensile tests were used to characterize the electrospun constructs. Cell cultures with primary endothelial cells demonstrated that cells showed spread morphology and strong adhesion on fibroin richer surfaces. The platform for producing robust multilayer scaffolds with intermixing nanofiber structure, tunable anisotropy ratio, and surface with specific compositions may hold great potential in tissue engineering applications.

Surface properties and blood compatibility of commercially available diamond‐like carbon coatings for cardiovascular devices

The aim of this study was to determine the relationships between the surface properties and blood compatibility of in-use diamond-like carbon (DLC) coatings for cardiovascular components. Commercially available DLC films were characterized with respect to surface topography and wettability, protein adsorption from human plasma, and platelets adhesion/activation. Fibrinogen (Fng) and human serum albumin (HSA) adsorbed onto the sample surfaces were in particular quantified as two of the main proteins involved in blood compatibility. A low tendency of platelets to spread and form aggregates onto the DLC-coated surfaces has been described and related to a low Fng-to-HSA adsorption ratio. This study provides evidence that the rapid and tenacious binding of albumin molecules to DLC materials tends to passivate the surfaces and to inhibit Fng adsorption, thus imparting thromboresistance to the carbon coatings by rendering the surfaces less adhesive and activating for platelets. Albumin preferential adsorption was ascribed to high chemical heterogeneity of the DLC sample surfaces. The DLC films tested present a favorable behavior as regards blood compatibility with respect to platelet thrombus formation by reason of their surface properties.

Silk Fibroin Processing and Thrombogenic Responses

Silkworm-derived fibroin, which constitutes the core of the silk filament, is an attractive protein–polymer for biomedical applications. Fibroin can also be processed into a variety of 2-D and 3-D formats to match morphological and structural features to specific applications. The focus of the present research was to correlate the structure of silk fibroin-derived biomaterials with plasma protein adsorption, platelet activation and inflammatory cell (THP-1 cell line) adhesion and activation. The amino-acid composition of the two types of silk studied influenced the crystallinity of the films, hydrophobicity, surface roughness and biological interactions. Protein adsorption was lower on samples with the higher crystallinity and hydrophobicity, in particular the chemotactic factors (C3a, C5a, C3b), while other proteins such as fibrinogen were comparable in terms of adsorption. As a consequence, platelets and immune cells responded differently to the various films obtained by following different processing protocols and stabilized by different methods (methanol or water vapour) in terms of their adherence, activation, and the secretion of inflammatory mediators by monocytes. The data presented here demonstrate that bioactivity can be influenced by changing the chemistry, such as the source of silk protein, or by the specific process used in the preparation of the materials used to assess biological responses.

Electrodeposition of Silk Fibroin on Metal Substrates

Silk fibroin, one of the most promising natural materials for tissue engineering, has positive interactions with the biological environment, particularly in the field of bone and cartilage regeneration. A new approach was developed to create hydrogels from water-based fibroin solutions by applying an electric field to effect protein migration and coagulation at the anode (Aluminium or Ti6Al4V alloy) of an electrochemical cell. The process was easily controlled by the voltage applied to the electrodes (3, 10, and 30 V), solution concentration (1%, 2%, 2.6% w/v), time (up to 100 s) and electrode distance (1—6 mm). The hydrogel thickness can be increased up to 60 μm and, depending on processing conditions, porous coatings or compact films can be obtained. The ability of electrodeposited fibroin hydrogels to coat metal objects with complex shape and surface morphology, together with the acclaimed properties of fibroin, makes it a promising technique to enhance the osteointegration of dental or orthopedic prostheses.

Microencapsulation of cells in alginate through an electrohydrodynamic process

The encapsulation of living cells within a semi-permeable matrix is an attractive process for transplanting nonautologous cells by limiting the interaction with the host immune system. The electrohydrodynamic process is a low-cost and high-throughput system to encapsulate cells by means of a static potential. We evaluated the use of this system for cell entrapment by assessing and then manufacturing capsules that had the best dimensions. The effect of different cell densities on the beads was determined to set up the basic parameters of the encapsulation system. The cell viability inside the beads and as a function of release time was observed for their biological response.

Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance.

Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.

An electrohydrodynamic bioprinter for alginate hydrogels containing living cells.

In this work we present a bioprinting technique that exploits the electrohydrodynamic process to obtain a jet of liquid alginate beads containing cells. A printer is used to microfabricate hydrogels block by block following a bottom-up approach. Alginate beads constitute the building blocks of the microfabricated structures. The beads are placed at predefined position on a target substrate made of calcium-enriched gelatin, where they crosslink upon contact without the need of further postprocessing. The printed sample can be easily removed from the substrate at physiological temperature. Three-dimensional printing is accomplished by the deposition of multiple layers of hydrogel. We have investigated the parameters influencing the process, the compatibility of the printing procedure with cells, and their survival after printing.

Folding and Assembly of Fibroin Driven by an AC Electric Field : Effects on Film Properties

Silk fibroin from Bombyx mori is a high-molecular-weight protein, largely employed in the biomaterials field. Several parameters can affect the folding and assembly of fibroin heavy and light chains. The present work has shown that anisotropic and water-stable films are produced when fibroin solution is cast under an alternating electric field (AC). The treatment can affect the mechanical, thermal and surface properties of fibroin films. These effects have been related to the alignment of molecular dipoles and the formation of oriented supramolecular assemblies. Cell response is affected by this novel processing: MRC5 fibroblasts, cultured on anisotropic fibroin films, preferentially spread parallel to the field direction 6 h after seeding. [Figure: see text].

Blood compatibility of polymers derived from natural materials

Several polymers derived from natural materials are effective for tissue engineering or drug delivery applications, due to specific properties, such as biocompatibility, biodegradability, and structural activity. Their blood compatibility needs to be carefully evaluated to avoid thrombosis and other material-related adverse events in the hematic environment. We compared the surface properties and blood compatibility of protein and polysaccharide polymers, including fibroin, gelatin, and chitosan. Both fibroin and chitosan showed good hemocompatibility, with low platelet adhesion and spreading. Chitosan induced strong interactions with plasma proteins, especially with albumin. It was hypothesized that surface passivation by albumin inhibited the adsorption of other procoagulant and proadhesive proteins on chitosan and fibroin films, which limited platelet spreading. However, the significant and rapid polymer swelling encouraged protein entrapment within the soft, gelatin films, inducing higher platelet adhesion and activation. Thrombin generation assay confirmed the higher blood compatibility of chitosan and fibroin with regard to clotting.