Fabrication and characterization of bioresorbable, electroactive and highly regular nanomodulated cell interfaces.

Biomaterial-based implantable scaffolds capable of promoting physical and functional reconnection of injured spinal cord and nerves represent the latest frontier in neural tissue engineering. Here, we report the fabrication and characterization of self-standing, biocompatible and bioresorbable substrates endowed with both controlled nanotopography and electroactivity, intended for the design of transient implantable scaffolds for neural tissue engineering. In particular, we obtain conductive and nano-modulated poly(D,L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) free-standing films by simply iterating a replica moulding process and coating the polymer with a thin layer of poly(3,4-ethylendioxythiophene) polystyrene sulfonate. The capability of the substrates to retain both surface patterning and electrical properties when exposed to a liquid environment has been evaluated by atomic force microscopy, electrochemical impedance spectroscopy and thermal characterizations. In particular, we show that PLA-based films maintain their surface nano-modulation for up to three weeks of exposure to a liquid environment, a time sufficient for promoting axonal anisotropic sprouting and growth during neuronal cell differentiation. In conclusion, the developed substrates represent a novel and easily-tunable platform to design bioresorbable implantable devices featuring both topographic and electrical cues.

Controlled Release of H2S from Biomimetic Silk Fibroin-PLGA Multilayer Electrospun Scaffolds.

The possibility of incorporating H2S slow-release donors inside biomimetic scaffolds can pave the way to new approaches in the field of tissue regeneration and anti-inflammatory treatment. In the present work, GYY4137, an easy-to-handle commercially available Lawesson's reagent derivative, has been successfully incorporated inside biomimetic silk fibroin-based electrospun scaffolds. Due to the instability of GYY4137 in the solvent needed to prepare silk fibroin solutions (formic acid), the electrospinning of the donor together with the silk fibroin turned out to be impossible. Therefore, a multilayer structure was realized, consisting of a PLGA mat containing GYY4137 sandwiched between two silk fibroin nanofibrous layers. Before their use in the multilayer scaffold, the silk fibroin mats were treated in ethanol to induce crystalline phase formation, which conferred water-resistance and biomimetic properties. The morphological, thermal, and chemical properties of the obtained scaffolds were thoroughly characterized by SEM, TGA, DSC, FTIR, and WAXD. Multilayer devices showing two different concentrations of the H2S donor, i.e., 2 and 5% w/w with respect to the weight of PLGA, were analyzed to study their H2S release and biological properties, and the results were compared with those of the sample not containing GYY4137. The H2S release analysis was carried out according to an "ad-hoc" designed procedure based on a validated high-performance liquid chromatography method. The proposed analytical approach demonstrated the slow-release kinetics of H2S from the multilayer scaffolds and its tunability by acting on the donor's concentration inside the PLGA nanofibers. Finally, the devices were tested in biological assays using bone marrow-derived mesenchymal stromal cells showing the capacity to support cell spreading throughout the scaffold and prevent cytotoxicity effects in serum starvation conditions. The resulting devices can be exploited for applications in the tissue engineering field since they combine the advantages of controlled H2S release kinetics and the biomimetic properties of silk fibroin nanofibers.

Reusable Cavitand-Based Electrospun Membranes for the Removal of Polycyclic Aromatic Hydrocarbons from Water.

The removal of toxic and carcinogenic polycyclic aromatic hydrocarbons (PAHs) from water is one of the most intractable environmental problems nowadays, because of their resistance to remediation. This work introduces a highly efficient, regenerable membrane for the removal of PAHs from water, featuring excellent filter performance and pH-driven release, thanks to the integration of a cavitand receptor in electrospun polyacrylonitrile (PAN) fibers. The role of the cavitand receptor is to act as molecular gripper for the uptake/release of PAHs. To this purpose, the deep cavity cavitand BenzoQxCav is designed and synthetized and its molecular structure is elucidated via X-Ray diffraction. The removal efficiency of the new adsorbent material toward the 16 priority PAHs is demonstrated via GC-MS analyses at ng L-1 concentration. A removal efficiency in the 32%, to 99% range is obtained. The regeneration of the membrane is performed by exploiting the pH-driven conformational switching of the cavitand between the vase form, where the PAHs uptake takes place, to the kite one, where the PAHs release occurs. The absorbance and regeneration capability of the membrane are successfully tested in four uptake/release cycles and the morphological stability.

Thermoactive Smart Electrospun Nanofibers.

The recent burst of research on smart materials is a clear evidence of the growing interest of the scientific community, industry, and society in the field. The exploitation of the great potential of stimuli-responsive materials for sensing, actuation, logic, and control applications is favored and supported by new manufacturing technologies, such as electrospinning, that allows to endow smart materials with micro- and nanostructuration, thus opening up additional and unprecedented prospects. In this wide and lively scenario, this article systematically reviews the current advances in the development of thermoactive electrospun fibers and textiles, sorting them, according to their response to the thermal stimulus. Hence, several platforms including thermoresponsive systems, shape memory polymers, thermo-optically responsive systems, phase change materials, thermoelectric materials, and pyroelectric materials, are described and critically discussed. The difference in active species and outputs of the aforementioned categories is highlighted, evidencing the transversal nature of temperature stimulus. Moreover, the potential of novel thermoactive materials are pointed out, revealing how their development could take to utmost interesting achievements.

A Modular Composite Device of Poly(Ethylene Oxide)/Poly(Butylene Terephthalate) (PEOT/PBT) Nanofibers and Gelatin as a Dual Drug Delivery System for Local Therapy of Soft Tissue Tumors.

In the clinical management of solid tumors, the possibility to successfully couple the regeneration of injured tissues with the elimination of residual tumor cells left after surgery could open doors to new therapeutic strategies. In this work, we present a composite hydrogel-electrospun nanofiber scaffold, showing a modular architecture for the delivery of two pharmaceutics with distinct release profiles, that is potentially suitable for local therapy and post-surgical treatment of solid soft tumors. The composite was obtained by coupling gelatin hydrogels to poly(ethylene oxide)/poly(butylene terephthalate) block copolymer nanofibers. Results of the scaffolds' characterization, together with the analysis of gelatin and drug release kinetics, displayed the possibility to modulate the device architecture to control the release kinetics of the drugs, also providing evidence of their activity. In vitro analyses were also performed using a human epithelioid sarcoma cell line. Furthermore, publicly available expression datasets were interrogated. Confocal imaging showcased the nontoxicity of these devices in vitro. ELISA assays confirmed a modulation of IL-10 inflammation-related cytokine supporting the role of this device in tissue repair. In silico analysis confirmed the role of IL-10 in solid tumors including 262 patients affected by sarcoma as a negative prognostic marker for overall survival. In conclusion, the developed modular composite device may provide a key-enabling technology for the treatment of soft tissue sarcoma.

Ether-Oxygen Containing Electrospun Microfibrous and Sub-Microfibrous Scaffolds Based on Poly(butylene 1,4-cyclohexanedicarboxylate) for Skeletal Muscle Tissue Engineering.

We report the study of novel biodegradable electrospun scaffolds from poly(butylene 1,4-cyclohexandicarboxylate-co-triethylene cyclohexanedicarboxylate) (P(BCE-co-TECE)) as support for in vitro and in vivo muscle tissue regeneration. We demonstrate that chemical composition, i.e., the amount of TECE co-units (constituted of polyethylene glycol-like moieties), and fibre morphology, i.e., aligned microfibrous or sub-microfibrous scaffolds, are crucial in determining the material biocompatibility. Indeed, the presence of ether linkages influences surface wettability, mechanical properties, hydrolytic degradation rate, and density of cell anchoring points of the studied materials. On the other hand, electrospun scaffolds improve cell adhesion, proliferation, and differentiation by favouring cell alignment along fibre direction (fibre morphology), also allowing for better cell infiltration and oxygen and nutrient diffusion (fibre size). Overall, C2C12 myogenic cells highly differentiated into mature myotubes when cultured on microfibres realised with the copolymer richest in TECE co-units (micro-P73 mat). Lastly, when transplanted in the tibialis anterior muscles of healthy, injured, or dystrophic mice, micro-P73 mat appeared highly vascularised, colonised by murine cells and perfectly integrated with host muscles, thus confirming the suitability of P(BCE-co-TECE) scaffolds as substrates for skeletal muscle tissue engineering.

Thermal Annealing to Modulate the Shape Memory Behavior of a Biobased and Biocompatible Triblock Copolymer Scaffold in the Human Body Temperature Range.

A biodegradable and biocompatible electrospun scaffold with shape memory behavior in the physiological temperature range is here presented. It was obtained starting from a specifically designed, biobased PLLA-based triblock copolymer, where the central block is poly(propylene azelate-co-propylene sebacate) (P(PAz60PSeb40)) random copolymer. Shape memory properties are determined by the contemporary presence of the low melting crystals of the P(PAz60PSeb40) block, acting as switching segment, and of the high melting crystal phase of PLLA blocks, acting as physical network. It is demonstrated that a straightforward annealing process applied to the crystal phase of the switching element gives the possibility to tune the shape recovery temperature from about 25 to 50 °C, without the need of varying the copolymer's chemical structure. The thermal annealing approach here presented can be thus considered a powerful strategy for "ad hoc" programming the same material for applications requiring different recovery temperatures. Fibroblast culture experiments demonstrated scaffold biocompatibility.

The role of 3D microenvironmental organization in MCF-7 epithelial-mesenchymal transition after 7 culture days.

We present a multi-technique study on in vitro epithelial-mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(l-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, ß-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, ß-catenin and vimentin) and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and ß-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-ß2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold.

Comparative performance of collagen nanofibers electrospun from different solvents and stabilized by different crosslinkers.

Collagen electrospun scaffolds well reproduce the structure of the extracellular matrix (ECM) of natural tissues by coupling high biomimetism of the biological material with the fibrous morphology of the protein. Structural properties of collagen electrospun fibers are still a debated subject and there are conflicting reports in the literature addressing the presence of ultrastructure of collagen in electrospun fibers. In this work collagen type I was successfully electrospun from two different solvents, trifluoroethanol (TFE) and dilute acetic acid (AcOH). Characterization of collagen fibers was performed by means of SEM, ATR-IR, Circular Dichroism and WAXD. We demonstrated that collagen fibers contained a very low amount of triple helix with respect to pristine collagen (18 and 16% in fibers electrospun from AcOH and TFE, respectively) and that triple helix denaturation occurred during polymer dissolution. Collagen scaffolds were crosslinked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a commonly employed crosslinker for electrospun collagen, and 1,4-butanediol diglycidyl ether (BDDGE), that was tested for the first time in this work as crosslinking agent for collagen in the form of electrospun fibers. We demonstrated that BDDGE successfully crosslinked collagen and preserved at the same time the scaffold fibrous morphology, while scaffolds crosslinked with EDC completely lost their porous structure. Mesenchymal stem cell experiments demonstrated that collagen scaffolds crosslinked with BDDGE are biocompatible and support cell attachment.

Nanovascularization of polymer matrix: generation of nanochannels and nanotubes by sacrificial electrospun fibers.

Several methods for creating vascular structures, made of either discrete or interconnected channels have been developed. The currently employed methods enable the formation of channels with diameters in the millimetric and micrometric scale. However, the formation of an interconnected three-dimensional (3D) vasculature by using a rapid and scalable process is a challenge and largely limits the fields of applicability of these innovative materials. Here, we propose the use of electrospun nonwoven mats as sacrificial fibers to easily generate 3D macroscale vascularized composites containing interconnected networks with channels and tubes having submicrometric and nanometric diameters. The novel approach has the potentialities to give rise to a novel generation of composites potentially displaying new and enhanced functionalities thanks to the nanoscale features of the cavities.

Peptide direct growth on poly(acrylic acid)/poly(vinyl alcohol) electrospun fibers coated with branched poly(ethylenimine): A solid-phase approach for scaffolds biofunctionalization.

Due to their resemblance to the fibrillar structure of the extracellular matrix, electrospun nanofibrous meshes are currently used as porous and mechanically stable scaffolds for cell culture. In this study, we propose an innovative methodology for growing peptide sequences directly onto the surface of electrospun nanofibers. To achieve this, electrospun fibers were produced from a poly(acrylic acid)/poly(vinyl alcohol) blend that was thermally crosslinked and subjected to a covalent coating of branched poly(ethylenimine). The exposed amino functionalities on the fiber surface were then used for the direct solid-phase synthesis of the RGD peptide sequence. In contrast to established strategies, mainly involving the grafting of pre-synthesized peptides onto the polymer chains before electrospinning or onto the nanofibers surface, this method allows for the concurrent synthesis and anchoring of peptides to the substrate, with potential applications in combinatorial chemistry. The incorporation of this integrin-binding motive significantly enhanced the nanofibers' ability to capture human cervical carcinoma (HeLa) cells, selected as a proof of concept to assess the functionalities of the developed material.

Microporous electrospun nonwovens combined with green solvents for the selective peel-off of thin coatings from painting surfaces.

Over the last few decades, significant research efforts have been devoted to developing new cleaning systems aimed at preserving cultural heritage. One of the main objectives is to selectively remove aged or undesirable coatings from painted surfaces while preventing the cleaning solvent from permeating and engaging with the pictorial layers. In this work, we propose the use of electrospun polyamide 6,6 nonwovens in conjunction with a green solvent (dimethyl carbonate). By adjusting the electrospinning parameters, we produced three distinct nonwovens with varying average fiber diameters, ranging from 0.4 µm to 2 µm. These samples were characterized and tested for their efficacy in removing dammar varnish from painted surfaces. In particular, the cleaning process was monitored using macroscale PL (photoluminescence) imaging in real-time, while post-application examination of the mats was performed through scanning electron microscopy. The solvent evaporation rate from the different nonwovens was evaluated using gravimetric analysis and Proton Transfer Reaction- Time-of-Flight. It was observed that the application of the nonwovens with small or intermediate pore sizes for the removal of the terpenic varnish resulted in the swollen resin being absorbed into the mats, showcasing a peel-off effect. Thus, this protocol eliminates the need for further potentially detrimental removal procedures involving cotton swabs. The experimental data suggests that the peel-off effect relates to the microporosity of the mats, which enhances the capillary rise of the swollen varnish. Furthermore, the application of these systems to historical paintings underwent preliminary validation using a real painting from the 20th century.

Carbon dot/polylactic acid nanofibrous membranes for solar-mediated oil absorption/separation: Performance, environmental sustainability, ecotoxicity and reusability.

Carbon dots (CDs) are promising photothermal nanoparticles that can be utilized in environmental treatments. They exhibit favorable physicochemical properties, including low toxicity, physical and chemical stability, photo-dependant reversible behaviour, and environmentally friendly synthesis using benign building blocks. Here, we synthesized innovative CDs/polylactic acid (PLA) electrospun composite membranes for evaluating the removal of hydrophobic compounds like long-chain hydrocarbons or oils in biphasic mixtures with water. The ultimate goal was to develop innovative and sustainable solar-heated oil absorbents. Specifically, we fabricated PLA membranes with varying CD contents, characterized their morphology, thermal, and mechanical properties, and assessed the environmental impact of membrane production according to ISO 14040 and 14044 standards in a preliminary "cradle-to-gate" life cycle assessment study. Solar radiation experiments demonstrated that the CDs/PLA composites exhibited greater uptake of hydrophobic compounds compared to pure PLA membranes, ascribable to the CDs-induced photothermal effect. The adsorption and regeneration capacity of the new CDs/PLA membrane was demonstrated through multiple uptake/release cycles. Ecotoxicity analyses confirmed the safety profile of the new adsorbent system towards freshwater microalgae, further emphasizing its potential as an environmentally friendly solution for the removal of hydrophobic compounds in water treatment processes.

Advantages of surface-initiated ATRP (SI-ATRP) for the functionalization of electrospun materials.

Surface-initiated atom transfer radical polymerization (SI-ATRP) is successfully applied to electrospun constructs of poly(L-lactide). ATRP macroinitiators are adsorbed through polyelectrolyte complexation following the introduction of negative charges on the polyester surface through its blending with a six-armed carboxy-terminated oligolactide. SI-ATRP of glycerol monomethacrylate (GMMA) or 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) allows then to grow surface films with controllable thickness, and in this way also to control the wetting and interactions of the construct.

Electrospun fibers coated with nanostructured biomimetic hydroxyapatite: A new platform for regeneration at the bone interfaces.

Reconstruction of gradient organic/inorganic tissues is a challenging task in orthopaedics. Indeed, to mimic tissue characteristics and stimulate bone regeneration at the interface, it is necessary to reproduce both the mineral and organic components of the tissue ECM, as well as the micro/nano-fibrous morphology. To address this goal, we propose here novel biomimetic patches obtained by the combination of electrospinning and nanostructured bone apatite. In particular, we deposited apatite on the electrospun fibers by Ionized Jet Deposition, a plasma-assisted technique that allows conformal deposition and the preservation in the coating of the target's stoichiometry. The damage to the substrate and fibrous morphology is a polymer-dependent aspect, that can be avoided by properly selecting the substrate composition and deposition parameters. In fact, all the tested polymers (poly(l-lactide), poly(D,l-lactide-co-glycolide, poly(ε-caprolactone), collagen) were effectively coated, and the morphological and thermal characterization revealed that poly(ε-caprolactone) suffered the least damage. The coating of collagen fibers, on the other hand, destroyed the fiber morphology and it could only be performed when collagen is blended with a more resistant synthetic polymer in the nanofibers. Due to the biomimetic composition and multiscale morphology from micro to nano, the poly(ε-caprolactone)-collagen biomimetic patches coated with bone apatite supported MSCs adhesion, patch colonization and early differentiation, while allowing optimal viability. The biomimetic coating allowed better scaffold colonization, promoting cell spreading on the fibers.

A high-sensitivity long-lifetime phosphorescent RIE additive to probe free volume-related phenomena in polymers.

The photophysical behaviour of phosphorescent rigidification-induced emission (RIE) dyes is highly affected by their micro- and nanoenvironment. The lifetime measure of RIE dyes dispersed in polymers represents an effective approach to gain valuable information on polymer free volume and thus develop materials potentially able to self-monitor physical ageing and mechanical stresses.

Electrospun Poly(vinyl alcohol)-Based Conductive Semi-interpenetrating Polymer Network Fibrous Hydrogel: A Toolbox for Optimal Cross-Linking.

Cross-linking of poly(vinyl alcohol) (PVA) creates a three-dimensional network by bonding adjacent polymer chains. The cross-linked structure, upon immersion in water, turns into a hydrogel, which exhibits unique absorption properties due to the presence of hydrophilic groups within the PVA polymer chains and, simultaneously, ceases to be soluble in water. The properties of PVA can be adjusted by chemical modification or blending with other substances, such as polymers, e.g., conductive poly[3-(potassium-5-butanoate)thiophene-2,5-diyl] (P3KBT). In this work, PVA-based conductive semi-interpenetrating polymer networks (semi-IPNs) are successfully fabricated. The systems are obtained as a result of electrospinning of PVA/P3KBT precursor solutions with different polymer concentrations and then cross-linking using "green", environmentally safe methods. One approach consists of thermal treatment (H), while the second approach combines stabilization with ethanol and heating (E). The comprehensive characterization allows to evaluate the correlation between the cross-linking methods and properties of nanofibrous hydrogels. While both methods are successful, the cross-linking density is higher in the thermally cross-linked samples, resulting in lower conductivity and swelling ratio compared to the E-treated samples. Moreover, the H-cross-linked systems have better mechanical properties-lower stiffness and greater tensile strength. All the tested systems are biocompatible, and interestingly, due to the presence of P3KBT, they show photoresponsivity to solar radiation generated by the simulator. The results indicate that both methods of PVA cross-linking are highly effective and can be applied to a specific system depending on the target, e.g., biomedical or electronic applications.

Nanocomposite electrospun fibers of poly(ε-caprolactone)/bioactive glass with shape memory properties.

Electrospun fibers of shape memory triethoxysilane-terminated poly(epsilon-caprolactone) (PCL-TES) loaded with bioactive glasses (BG) are here presented. Unloaded PCL-TES, as well as PCL/BG nanocomposite fibers, are also considered for comparison. It is proposed that hydrolysis and condensation reactions take place between triethoxysilane groups of the polymer and the silanol groups at the BG particle surface, thus generating additional crosslinking points with respect to those present in the PCL-TES system. The as-spun PCL-TES/BG fibers display excellent shape memory properties, in terms of shape fixity and shape recovery ratios, without the need of a thermal crosslinking treatment. BG particles confer in vitro bioactivity to PCL-based nanocomposite fibers and favor the precipitation of hydroxycarbonate apatite on the fiber surface. Preliminary cytocompatibility tests demonstrate that the addition of BG particles to PCL-based polymer does not inhibit ST-2 cell viability. This novel approach of using bioactive glasses not only for their biological properties, but also for the enhancement of shape memory properties of PCL-based polymers, widens the versatility and suitability of the obtained composite fibers for a huge portfolio of biomedical applications.

Effect of electrospun fiber diameter and alignment on macrophage activation and secretion of proinflammatory cytokines and chemokines.

Macrophage activation can be modulated by biomaterial topography according to the biological scale (micrometric and nanometric range). In this study, we investigated the effect of fiber diameter and fiber alignment of electrospun poly(L-lactic) (PLLA) scaffolds on macrophage RAW 264.7 activation and secretion of proinflammatory cytokines and chemokines at 24 h and 7 days. Macrophages were cultured on four different types of fibrous PLLA scaffold (aligned microfibers, aligned nanofibers, random microfibers, and random nanofibers) and on PLLA film (used as a reference). Substrate topography was found to influence the immune response activated by macrophages, especially in the early inflammation stage. Secretion of proinflammatory molecules by macrophage cells was chiefly dependent on fiber diameter. In particular, nanofibrous PLLA scaffolds minimized the inflammatory response when compared with films and microfibrous scaffolds. The histological evaluation demonstrated a higher number of foreign body giant cells on the PLLA film than on the micro- and nanofibrous scaffolds. In summary, our results indicate that the diameter of electrospun PLLA fibers, rather than fiber alignment, plays a relevant role in influencing in vitro macrophage activation and secretion of proinflammatory molecules.

Elastomeric electrospun scaffolds of poly(L-lactide-co-trimethylene carbonate) for myocardial tissue engineering.

In myocardial tissue engineering the use of synthetically bioengineered flexible patches implanted in the infarcted area is considered one of the promising strategy for cardiac repair. In this work the potentialities of a biomimetic electrospun scaffold made of a commercial copolymer of (L)-lactic acid with trimethylene carbonate (P(L)LA-co-TMC) are investigated in comparison to electrospun poly(L)lactic acid. The P(L)LA-co-TMC scaffold used in this work is a glassy rigid material at room temperature while it is a rubbery soft material at 37 °C. Mechanical characterization results (tensile stress-strain and creep-recovery measurements) show that at 37 °C electrospun P(L)LA-co-TMC displays an elastic modulus of around 20 MPa and the ability to completely recover up to 10% of deformation. Cell culture experiments show that P(L)LA-co-TMC scaffold promotes cardiomyocyte proliferation and efficiently preserve cell morphology, without hampering expression of sarcomeric alpha actinin marker, thus demonstrating its potentialities as synthetic biomaterial for myocardial tissue engineering.