Recent Progress and Trends in the Development of Electrospun and 3D Printed Polymeric-Based Materials to Overcome Antimicrobial Resistance (AMR).

Antimicrobial resistance (AMR) developed by microorganisms is considered one of the most critical public health issues worldwide. This problem is affecting the lives of millions of people and needs to be addressed promptly. Mainly, antibiotics are the substances that contribute to AMR in various strains of bacteria and other microorganisms, leading to infectious diseases that cannot be effectively treated. To avoid the use of antibiotics and similar drugs, several approaches have gained attention in the fields of materials science and engineering as well as pharmaceutics over the past five years. Our focus lies on the design and manufacture of polymeric-based materials capable of incorporating antimicrobial agents excluding the aforementioned substances. In this sense, two of the emerging techniques for materials fabrication, namely, electrospinning and 3D printing, have gained significant attraction. In this article, we provide a summary of the most important findings that contribute to the development of antimicrobial systems using these technologies to incorporate various types of nanomaterials, organic molecules, or natural compounds with the required property. Furthermore, we discuss and consider the challenges that lie ahead in this research field for the coming years.

Novel Poly(ester urethane urea)/Polydioxanone Blends: Electrospun Fibrous Meshes and Films.

In this work, we report the electrospinning and mechano-morphological characterizations of scaffolds based on blends of a novel poly(ester urethane urea) (PHH) and poly(dioxanone) (PDO). At the optimized electrospinning conditions, PHH, PDO and blend PHH/PDO in Hexafluroisopropanol (HFIP) solution yielded bead-free non-woven random nanofibers with high porosity and diameter in the range of hundreds of nanometers. The structural, morphological, and biomechanical properties were investigated using Differential Scanning Calorimetry, Scanning Electron Microscopy, Atomic Force Microscopy, and tensile tests. The blended scaffold showed an elastic modulus (~5 MPa) with a combination of the ultimate tensile strength (2 ± 0.5 MPa), and maximum elongation (150% ± 44%) in hydrated conditions, which are comparable to the materials currently being used for soft tissue applications such as skin, native arteries, and cardiac muscles applications. This demonstrates the feasibility of an electrospun PHH/PDO blend for cardiac patches or vascular graft applications that mimic the nanoscale structure and mechanical properties of native tissue.

Evaluation of human umbilical vein endothelial cells growth onto heparin-modified electrospun vascular grafts.

One of the main challenges of cardiovascular tissue engineering is the development of bioresorbable and compliant small-diameter vascular grafts (SDVG) for patients where autologous grafts are not an option. In this work, electrospun bilayered bioresorbable SDVG based on blends of poly(L-lactic acid) (PLLA) and segmented polyurethane (PHD) were prepared and evaluated. The inner layer of these SDVG was surface-modified with heparin, following a methodology involving PHD urethane functional groups. Heparin was selected as anticoagulant agent, and also due to its ability to promote human umbilical vein endothelial cells (HUVECs) growth and to inhibit smooth muscle cells over-proliferation, main cause of neointimal hyperplasia and restenosis. Immobilized heparin was quantified and changes in SDVG microstructure were investigated through SEM. Tensile properties of the heparin-functionalized SDVG resembled those of saphenous vein. Vascular grafts were seeded with HUVECs and cultured on a flow-perfusion bioreactor to analyze the effect of heparin on graft endothelization under simulated physiological-like conditions. The analysis of endothelial cells attachment and gene expression (Real-Time PCR) pointed out that the surface functionalization with heparin successfully promoted a stable and functional endothelial cell layer.

Surface-modified bioresorbable electrospun scaffolds for improving hemocompatibility of vascular grafts.

The replacement of small-diameter vessels is one of the main challenges in tissue engineering. Moreover, the surface modification of small-diameter vascular grafts (SDVG) is a key factor in the success of the therapy due to their increased thrombogenicity and infection susceptibility caused by the lack of a functional endothelium. In this work, electrospun scaffolds were prepared from blends of poly(L-lactic acid) (PLLA) and segmented polyurethane (PHD) with a composition designed to perform as SDVG inner layer. The scaffolds were then successfully surface-modified with heparin following two different strategies that rely on grafting of heparin to either PLLA or PHD functional groups. Both strategies afforded high heparin density, being higher for urethane methodology. The functionalized scaffolds did not cause hemolysis and inhibited platelet adhesion to a large extent. However, lysozyme/heparin-functionalized scaffolds obtained through urethane methodology achieved the highest platelet attachment inhibition. The increase in hydrophilicity and water absorption of the surface-functionalized nanostructures favored adhesion and proliferation of human adipose-derived stem cells. Heparinized surfaces conjugated with lysozyme presented microbial hydrolysis activity dependent on heparin content. Overall, a better performance obtained for urethane-modified scaffold, added to the fact that no chain scission is involved in urethane methodology, makes the latter the best choice for surface modification of PLLA/PHD 50/50 electrospun scaffolds. Scaffolds functionalized by this route may perform as advanced components of SDVG suitable for vascular tissue engineering, exhibiting biomimetic behavior, avoiding thrombi formation and providing antimicrobial features.

Mechanical behavior of bilayered small-diameter nanofibrous structures as biomimetic vascular grafts.

To these days, the production of a small diameter vascular graft (<6mm) with an appropriate and permanent response is still challenging. The mismatch in the grafts mechanical properties is one of the principal causes of failure, therefore their complete mechanical characterization is fundamental. In this work the mechanical response of electrospun bilayered small-diameter vascular grafts made of two different bioresorbable synthetic polymers, segmented poly(ester urethane) and poly(L-lactic acid), that mimic the biomechanical characteristics of elastin and collagen is investigated. A J-shaped response when subjected to internal pressure was observed as a cause of the nanofibrous layered structure, and the materials used. Compliance values were in the order of natural coronary arteries and very close to the bypass gold standard-saphenous vein. The suture retention strength and burst pressure values were also in the range of natural vessels. Therefore, the bilayered vascular grafts presented here are very promising for future application as small-diameter vessel replacements.

High pressure assessment of bilayered electrospun vascular grafts by means of an Electroforce Biodynamic System®.

INTRODUCTION: Tissue engineering offers the possibility of developing a biological substitute material in vitro with the inherent properties required in vivo. However, the inadequate performance in vascular replacement of small diameter vascular grafts (VG) reduces considerably the current alternatives in this field. In this study, a bilayered tubular VG was produced, where its mechanical response was tested at high pressure ranges and compared to a native femoral artery. MATERIALS AND METHOD: The VG was obtained using sequential electrospinning technique, by means of two blends of Poly(L-lactic acid) and segmented poly(ester urethane). Mechanical testing was performed in a biodynamic system and the pressure-strain relationship was used to determine the elastic modulus. RESULTS: Elastic modulus assessed value of femoral artery at a high pressure range (33.02×106 dyn/cm(2)) was founded to be 36% the magnitude of VG modulus (91.47×106 dyn/cm(2)) at the same interval. CONCLUSION: A new circulating mock in combination with scan laser micrometry have been employed for the mechanical evaluation of bioresorbable bilayered VGs. At same pressure levels, graft elasticity showed a purely "collagenic" behavior with respect to a femoral artery response.

Structural characterization of electrospun micro/nanofibrous scaffolds by liquid extrusion porosimetry: a comparison with other techniques.

Poly(ε-caprolactone) micro/nanofibrous scaffolds obtained by electrospinning technique from polymer solutions were characterized in terms of fiber diameter (as measured by scanning electron microscopy-SEM), pore size and its distribution (as measured by liquid extrusion porosimetry), and porosity (as determined by gravimetric measurement, liquid intrusion method, SEM image analysis and liquid extrusion porosimetry - LEP). Nonwoven micro/nanofibrous scaffolds were formed by uniform bead-free fibers with mean diameters in the range of 0.4 to 7 µm. The results indicate that pore size and pore size distribution are strongly associated to fiber diameter. Porosity results were analyzed taking into account the accuracy and limitations of each method. LEP resulted as the most suitable technique for measuring through-pore diameter and porosity. In order to compare empirical data of pore size from LEP, a theoretical multiplanar model for stochastic fiber networks was applied. The results predicted by the model were in good agreement with the experimental data provided by LEP for mean diameters higher than 1 µm. The present study shows the potential of LEP as a valuable instrumental technique for characterizing the porous structure of electrospun fibrous scaffolds.

Evaluation of in vitro cytotoxic activity of mono-PEGylated StAP3 (Solanum tuberosum aspartic protease 3) forms.

StAP3 is a plant aspartic protease with cytotoxic activity toward a broad spectrum of pathogens, including potato and human pathogen microorganisms, and cancer cells, but not against human T cells, human red blood cells or plant cells. For this reason, StAP3 could be a promising and potential drug candidate for future therapies. In this work, the improvement of the performance of StAP3 was achieved by means of a modification with PEG. The separation of a mono-PEGylated StAP3 fraction was easily performed by gel filtration chromatography. The mono-PEGylated StAP3 fraction was studied in terms of in vitro antimicrobial activity, exhibiting higher antimicrobial activity against Fusarium solani spores and Bacillus cereus, but slightly lower activity against Escherichia coli than native protein. Such increase in antifungal activity has not been reported previously for a PEGylated plant protein. In addition, PEGylation did not affect the selective cytotoxicity of StAP3, since no hemolytic activity was observed.

Electrospinning of novel biodegradable poly(ester urethane)s and poly(ester urethane urea)s for soft tissue-engineering applications.

The development of biomimetic highly-porous scaffolds is essential for successful tissue engineering. Segmented poly(ester urethane)s and poly(ester urethane urea)s have been infrequently used for the fabrication of electrospun nanofibrous tissues, which is surprising because these polymers represent a very large variety of materials with tailored properties. This study reports the preparation of new electrospun elastomeric polyurethane scaffolds. Two novel segmented polyurethanes (SPU), synthesized from poly(epsilon-caprolactone) diol, 1,6-hexamethylene diisocyanate, and diester-diphenol or diurea-diol chain extenders, were used (Caracciolo et al. in J Mater Sci Mater Med 20:145-155, 2009). The spinnability and the morphology of the electrospun SPU scaffolds were investigated and discussed. The electrospinning parameters such as solution properties (polymer concentration and solvent) and processing parameters (applied electric field, needle to collector distance and solution flow rate) were optimized to achieve smooth, uniform bead-free fibers with diameter (~700 nm) mimicking the protein fibers of native extracellular matrix (ECM). The obtained elastomeric polyurethane scaffolds could be appropriate for soft tissue-engineering applications.