Publications

The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Models for the study of the physiological processes affecting the BBB, as well as predicting the permeability of therapeutic substances for neurological and neurovascular pathologies, are highly desirable. Existing models, such as Transwell utilizing-models, do not mimic the extracellular environment of the BBB with their stiff, semipermeable, non-biodegradable membranes. To help overcome this, we engineered electrospun membranes from poly L-lactic acid in combination with a nanometric coating of poly(ethyl acrylate) (PEA) that drives fibrillogenesis of fibronectin, facilitating the synergistic presentation of both growth factors and integrin binding sites. Compared to commercial semi-porous membranes, these membranes significantly improve the expression of BBB-related proteins in brain endothelial cells. PEA-coated membranes in combination with different growth factors and extracellular protein coatings reveal nerve growth factor (NGF) and fibroblast growth factor (FGF-2) caused formation of better barriers in vitro. This BBB model offers a robust platform for studying key biochemical factors influencing barrier formation that marries the simplicity of the Transwell model with the highly tunable electrospun PEA-fibronectin membranes. This enables the generation of high-throughput drug permeability models without the need of complicated co-culture conditions. The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Here a simple model of the BBB that allows culture of endothelial cells on growth-factor functionalised membranes is introduced. This novel in vitro model of the BBB offers a robust platform for studying key barriergenic biochemical factors influencing barrier formation without the use of the complicated co-culture conditions. image

JTD Keywords: Bbb, Densit, Differentiation, Ecm, Electrospinning, Endothelial-cell lines, Expression, Fiber diameter, Fibroblast-growth-factor, Growth factors, In vitro mode, In vitro model, Morphology, Permeability, Poly(l-lactic acid), Proteins

Osteoporotic-related fractures are among the leading causes of chronic disease morbidity in Europe and in the US. While a significant percentage of fractures can be repaired naturally, in delayed-union and non-union fractures surgical intervention is necessary for proper bone regeneration. Given the current lack of optimized clinical techniques to adequately address this issue, bone tissue engineering (BTE) strategies focusing on the development of scaffolds for temporarily replacing damaged bone and supporting its regeneration process have been gaining interest. The piezoelectric properties of bone, which have an important role in tissue homeostasis and regeneration, have been frequently neglected in the design of BTE scaffolds. Therefore, in this study, we developed novel hydroxyapatite (HAp)-filled osteoinductive and piezoelectric poly(vinylidene fluoride-co-tetrafluoroethylene) (PVDF-TrFE) nanofibers via electrospinning capable of replicating the tissue's fibrous extracellular matrix (ECM) composition and native piezoelectric properties. The developed PVDF-TrFE/HAp nanofibers had biomimetic collagen fibril-like diameters, as well as enhanced piezoelectric and surface properties, which translated into a better capacity to assist the mineralization process and cell proliferation. The biological cues provided by the HAp nanoparticles enhanced the osteogenic differentiation of seeded human mesenchymal stem/stromal cells (MSCs) as observed by the increased ALP activity, cell-secreted calcium deposition and osteogenic gene expression levels observed for the HAp-containing fibers. Overall, our findings describe the potential of combining PVDF-TrFE and HAp for developing electroactive and osteoinductive nanofibers capable of supporting bone tissue regeneration.© 2023 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis Group.

JTD Keywords: composites, electrospinning, hydroxyapatite, piezoelectricity, promote, pvdf, pvdf-trfe, removal, scaffolds, temperature, Bone tissue engineering, Electrospinning, Electrospun polycaprolactone, Hydroxyapatite, Piezoelectricity, Pvdf-trfe

The incorporation of exogenous lactate into cardiac tissues is a regenerative strategy that is rapidly gaining attention. In this work, two polymeric platforms were designed to achieve a sustained release of lactate, combining immediate and prolonged release profiles. Both platforms contained electrospun poly(lactic acid) (PLA) fibers and an alginate (Alg) hydrogel. In the first platform, named L/K(x)/Alg-PLA, lactate and proteinase K (x mg of enzyme per 1 g of PLA) were directly loaded into the Alg hydrogel, into which PLA fibers were assembled. In the second platform, L/Alg-K(x)/PLA, fibers were produced by electrospinning a proteinase K:PLA solution and, subsequently, assembled within the lactate-loaded hydrogel. After characterizing the chemical, morphological, and mechanical properties of the systems, as well as their cytotoxicity, the release profiles of the two platforms were determined considering different amounts of proteinase K (x = 5.2, 26, and 52 mg of proteinase K per 1 g of PLA), which is known to exhibit a broad cleavage activity. The profiles obtained using L/Alg-K(x)/PLA platforms with x = 26 and 52 were the closest to the criteria that must be met for cardiac tissue regeneration. Finally, the amount of lactate directly loaded in the Alg hydrogel for immediate release and the amount of protein in the electrospinning solution were adapted to achieve a constant lactate release of around 6 mM per day over 1 or 2 weeks. In the optimized bioplatform, in which 6 mM lactate was loaded in the hydrogel, the amount of fibers was increased by a factor of ×3, the amount of enzyme was adjusted to 40 mg per 1 g of PLA, and a daily lactate release of 5.9 ± 2.7 mM over a period of 11 days was achieved. Accordingly, the engineered device fully satisfied the characteristics and requirements for heart tissue regeneration.

JTD Keywords: biodegradable fibers, cardiac tissue regeneration, cell, drug-release, elastic-modulus, electrospinning, heart, nanoindentation, plasma treatment, proteinase, scaffold, stiffness, Alginate, Biodegradable fibers, Cardiac tissue regeneration, Electrospinning, Nanoindentation, Plasma treatment, Proteinase, Skeletal-muscle

JTD Keywords: alignment, bioresorbable stents, cells, design, electrospinning, everolimus, impact, in-vitro, poly(l-lactic-co-e-caprolactone), proliferation, release, sirolimus, Scaffold topography, Solvent-cast direct-writing

Tissue engineering is nowadays a powerful tool to restore damaged tissues and recover their normal functionality. Advantages over other current methods are well established, although a continuous evolution is still necessary to improve the final performance and the range of applications. Trends are nowadays focused on the development of multifunctional scaffolds with hierarchical structures and the capability to render a sustained delivery of bioactive molecules under an appropriate stimulus. Nanocomposites incorporating hydroxyapatite nanoparticles (HAp NPs) have a predominant role in bone tissue regeneration due to their high capacity to enhance osteoinduction, osteoconduction, and osteointegration, as well as their encapsulation efficiency and protection capability of bioactive agents. Selection of appropriated polymeric matrices is fundamental and consequently great efforts have been invested to increase the range of properties of available materials through copolymerization, blending, or combining structures constituted by different materials. Scaffolds can be obtained from different processes that differ in characteristics, such as texture or porosity. Probably, electrospinning has the greater relevance, since the obtained nanofiber membranes have a great similarity with the extracellular matrix and, in addition, they can easily incorporate functional and bioactive compounds. Coaxial and emulsion electrospinning processes appear ideal to generate complex systems able to incorporate highly different agents. The present review is mainly focused on the recent works performed with Hap-loaded scaffolds having at least one structural layer composed of core/shell nanofibers.

JTD Keywords: bone tissue, coaxial electrospinning, composite nanofibers, drug-release behavior, emulsion electrospinning, hydroxyapatite, in-vitro evaluation, mechanical-properties, osteogenic differentiation, pickering emulsions, protein adsorption, structured scaffolds, surface-initiated polymerization, tissue regeneration, Bone tissue, Coaxial electrospinning, Emulsion electrospinning, Hydroxyapatite, Multifunctional scaffolds, Poly(3-hydroxybutyrate) phb patches, Tissue regeneration

The creation of cardiac tissue models for preclinical testing is still a non-solved problem in drug discovery, due to the limitations related to thein vitroreplication of cardiac tissue complexity. Among these limitations, the difficulty of mimicking the functional properties of the myocardium due to the immaturity of the used cells hampers the obtention of reliable results that could be translated into human patients.In vivomodels are the current gold standard to test new treatments, although it is widely acknowledged that the used animals are unable to fully recapitulate human physiology, which often leads to failures during clinical trials. In the present work, we present a microfluidic platform that aims to provide a range of signaling cues to immature cardiac cells to drive them towards an adult phenotype. The device combines topographical electrospun nanofibers with electrical stimulation in a microfabricated system. We validated our platform using a co-culture of neonatal mouse cardiomyocytes and cardiac fibroblasts, showing that it allows us to control the degree of anisotropy of the cardiac tissue inside the microdevice in a cost-effective way. Moreover, a 3D computational model of the electrical field was created and validated to demonstrate that our platform is able to closely match the distribution obtained with the gold standard (planar electrode technology) using inexpensive rod-shaped biocompatible stainless-steel electrodes. The functionality of the electrical stimulation was shown to induce a higher expression of the tight junction protein Cx-43, as well as the upregulation of several key genes involved in conductive and structural cardiac properties. These results validate our platform as a powerful tool for the tissue engineering community due to its low cost, high imaging compatibility, versatility, and high-throughput configuration capabilities.

JTD Keywords: bioreactor, cardiac tissue engineering, cardiomyocytes, electrospinning, fabrication, fibroblasts, heart-on-a-chip, heart-tissue, in vitro models, myocardium, orientation, platform, scaffolds, Cardiac tissue engineering, Electrospinning, Field stimulation, Heart-on-a-chip, In vitro models, Microphysiological system

(Following spinal cord injury, olfactory ensheathing cell (OEC) transplantation is a promising therapeutic approach in promoting functional improvement. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical concentration differences. Here we compare the attachment, morphology, and directionality of an OEC-derived cell line, TEG3 cells, seeded on functionalized nanoscale meshes of Poly(l/dl-lactic acid; PLA) nanofibers. The size of the nanofibers has a strong effect on TEG3 cell adhesion and migration, with the PLA nanofibers having a 950 nm diameter being the ones that show the best results. TEG3 cells are capable of adopting a bipolar morphology on 950 nm fiber surfaces, as well as a highly dynamic behavior in migratory terms. Finally, we observe that functionalized nanofibers, with a chemical concentration increment of SDF-1α/CXCL12, strongly enhance the migratory characteristics of TEG3 cells over inhibitory substrates.

JTD Keywords: cell migration, cxcl12, electrospinning, gradients, pla nanofibers, sdf-1alpha, Cell migration, Cxcl12, Electrospinning, Gradients, Olfactory ensheathing cells, Pla nanofibers, Sdf-1alpha

Nowadays, the main limitation for clinical application of scaffolds is considered to be an insufficient vascularization of the implanted platforms and healing tissues. In our studies, we proposed a novel PLA-based hybrid platform with aligned and random fibrous internal structure and incorporated calcium phosphate (CaP) ormoglass nanoparticles (0, 10, 20 and 30 wt%) as an off-the-shelf method for obtaining scaffolds with pro-angiogenic properties. Complex morphological and physicochemical evaluation of PLA–CaP ormoglass composites was performed before and after in vitro degradation test in SBF solution to assess their biological potential. The degradation process of PLA–CaP ormoglass composites was accompanied by numerous CaP-based precipitations with extended topography and cauliflower-like shape which may enhance bonding of the material with the bone tissue and accelerate the regenerative process. Random fiber orientation was preferable for CaP compounds deposition upon in vitro degradation. CaP compounds precipitated firstly for randomly oriented composite nonwovens with 20 and 30 wt% addition of ormoglass. Moreover, the preliminary bioactivity test has shown that BSA adsorbed to PLA–CaP ormoglass composites (both aligned and randomly oriented) with 20 and 30 wt% of ormoglass nanoparticles which was not observed for pure PLA scaffolds.

JTD Keywords: Calcium phosphate ormoglass, Composites, Degradation, Electrospinning, PLA

Ambroxol is a pharmacological chaperone (PC) for Gaucher disease that increases lysosomal activity of misfolded β-glucocerebrosidase (GCase) while displaying a safe toxicological profile. In this work, different poly(ε-caprolactone) (PCL)-based systems are developed to regulate the sustained release of small polar drugs in physiological environments. For this purpose, ambroxol is selected as test case since the encapsulation and release of PCs using polymeric scaffolds have not been explored yet. More specifically, ambroxol is successfully loaded in electrospun PCL microfibers, which are subsequently coated with additional PCL layers using dip-coating or spin-coating. The time needed to achieve 80% release of loaded ambroxol increases from ≈15 min for uncoated fibrous scaffolds to 3 days and 1 week for dip-coated and spin-coated systems, respectively. Furthermore, it is proven that the released drug maintains its bioactivity, protecting GCase against induced thermal denaturation.

JTD Keywords: Electrospinning, Gaucher's disease, Lysosomal storage disorders, Misfolding diseases, Poly(ε-caprolactone), Polyester, Release regulation

Aim: To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Materials & methods: Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. Results: A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Conclusion: Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.

JTD Keywords: Electrospinning, Fibronectin, Hybrid nanofibers, Osseointegration, PLA, Recombinant protein

The success of scaffold implantation in acellular tissue engineering approaches relies on the ability of the material to interact properly with the biological environment. This behavior mainly depends on the design of the graft surface and, more precisely, on its capacity to biodegrade in a well-defined manner (nature of ions released, surface-to-volume ratio, dissolution profile of this release, rate of material resorption, and preservation of mechanical properties). The assessment of the biological behavior of temporary templates is therefore very important in tissue engineering, especially for composites, which usually exhibit complicated degradation behavior. Here, blended polylactic acid (PLA) calcium phosphate ORMOGLASS (organically modified glass) nanofibrous mats have been incubated up to 4 weeks in physiological simulated conditions, and their morphological, topographical, and chemical changes have been investigated. The results showed that a significant loss of inorganic phase occurred at the beginning of the immersion and the ORMOGLASS maintained a stable composition afterward throughout the degradation period. As a whole, the nanostructured scaffolds underwent fast and heterogeneous degradation. This study reveals that an angiogenic calcium-rich environment can be achieved through fast-degrading ORMOGLASS/PLA blended fibers, which seems to be an excellent alternative for guided bone regeneration.

JTD Keywords: Angiogenesis, Calcium release, Electrospinning, Fast degradation, Nanofibers, ORMOGLASSES

Electrospinning is a method that can be used to efficiently produce scaffolds that mimic the fibrous structure of natural tissue, such as muscle structures or the extracellular matrix of bone. The technique is often used as a way of depositing composites (organic/inorganic materials) to obtain bioactive nanofibers which have the requisite mechanical properties for use in tissue engineering. However, many factors can influence the formation and collection of fibers, including experimental variables such as the parameters of the solution of the electrospun slurry. In this study, we assessed the influence of the polymer concentration, glass content and glass hydrolysis level on the morphology and thickness of fibers produced by electrospinning for a PCL-(Si-Ca-P2) bioactive ormoglass—organically modified glass—blend. Based on previous assays, this combination of materials shows good angiogenic and osteogenic properties, which gives it great potential for use in tissue engineering. The results of our study showed that blend preparation directly affected the features of the resulting fibers, and when the parameters of the blend are precisely controlled, fibers with a regular diameter could be produced fairly easily when 2,2,2-trifluoroethanol was used as a solvent instead of tetrahydrofuran. The diameter of the homogeneous fibers ranged from 360 to 620 nm depending on the experimental conditions used. This demonstrates that experimental optimization of the electrospinning process is crucial in order to obtain a deposit of hybrid nanofibers with a regular shape.

JTD Keywords: Si-based glasses, Ormoglass, Electrospinning, Hybrid materials, Bioactivity, Angiogenesis

Electrospun nanofibrous materials mimicking the architecture of native extracellular matrix (ECM) hold great promise as scaffolds in tissue engineering. In order to optimize the properties of nanofibrous scaffolds it is important to understand the impact of fibres’ organization on cell behaviour. Herein, we investigated the effect of nanofibres (NFs) alignment on human adipose-derived mesenchymal stem cells (hAD-MSCs) adhesion and morphology. Electrospun composite fibrinogen/poly-lactic acid (FNG/PLA) NF scaffolds with same composition and comparable fibre size were fabricated into randomly oriented and aligned configuration and stem cells adhesion was characterized by the meaning of overall cell morphology, actin cytoskeleton organization and expression of molecules, involved in the development of focal adhesion complexes. We found that hAD-MSCs altered their morphology, actin cytoskeleton and cell attachment in accordance with nanofibre orientation while cell spreading, focal adhesions and expression of β1 and αNintegrin receptors were not influenced significantly by fibre orientation. These results confirmed that fibre alignment of scaffold guide cellular arrangement and could be beneficial for stem differentiation and therefore for the successful scaffolds development if its contact guidance coincided with the cell shape and cytoskeletal tension.

JTD Keywords: Electrospinning, Fibrinogen/polylactic acid hybrid nanofibres, Human adipose-derived stem cells

In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass–polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca2+ release analysis, such as stiffness evaluation by AFM, ζ-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.

JTD Keywords: Biological materials, Blood vessels, Calcium, Electrospinning, Glass, Hybrid materials, Silicon oxides, Sol-gel process, Sol-gels, Angiogenesis, Biological characterization, Calcium phosphate glass, Calcium-sensing receptors, Degradation process, Extracellular matrices, Organic/inorganic hybrid materials, ormoglasses, Silicon

In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.

JTD Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning

Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies.

JTD Keywords: Ceramic, Composites, Electrospinning, Nanofi bers, Nanostructured fi bers, Polymer, Tissue regeneration