Combination of electrospinning with other techniques for the fabrication of 3D polymeric and composite nanofibrous scaffolds with improved cellular interactions.

The development of three-dimensional (3D) scaffolds with physical and chemical topological cues at the macro-, micro-, and nanometer scale is urgently needed for successful tissue engineering applications. 3D scaffolds can be manufactured by a wide variety of techniques. Electrospinning technology has emerged as a powerful manufacturing technique to produce non-woven nanofibrous scaffolds with very interesting features for tissue engineering products. However, electrospun scaffolds have some inherent limitations that compromise the regeneration of thick and complex tissues. By integrating electrospinning and other fabrication technologies, multifunctional 3D fibrous assemblies with micro/nanotopographical features can be created. The proper combination of techniques leads to materials with nano and macro-structure, allowing an improvement in the biological performance of tissue-engineered constructs. In this review, we focus on the most relevant strategies to produce electrospun polymer/composite scaffolds with 3D architecture. A detailed description of procedures involving physical and chemical agents to create structures with large pores and 3D fiber assemblies is introduced. Finally, characterization and biological assays including in vitro and in vivo studies of structures intended for the regeneration of functional tissues are briefly presented and discussed.

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.

Multiscale constitutive model with progressive recruitment for nanofibrous scaffolds.

Biomedical applications need tailor-made scaffolds that exhibit biomimetic mechanical properties. In this context, electrospinning has emerged as a technique with promising features for their production. However, the electrospun scaffolds mechanical behavior as a function of the microstructure and nanofiber properties is still poorly understood. Besides, multiscale constitutive modeling appears as a powerful design tool, not only able to characterize electrospun structures, but also to determine the fiber properties and scaffold microstructure that would achieve the objective response. With focus in this last aspect, we developed a multiscale constitutive model for nanofibrous structures that takes into account the material constitutive properties, scaffold microstructure, and nanofiber progressive recruitment. A statistical approach of the nanofibers tortuosity with a modified Gaussian distribution was adopted, which allowed for reproducing the scaffolds macroscopic nonlinear mechanical behavior. It was observed that such behavior arises even if the nanofibers response is considered as mechanically linear. Experimental data from pressure vs. diameter inflation tests of electrospun tubular scaffolds was used to validate the model. In addition, the influence of the microstructural parameters upon the macroscopic constitutive behavior was studied. Finally, the model parameters were adjusted to obtain a vascular graft able to reproduce the mechanical response of a target natural tissue. The current study presents a step towards understanding, characterizing, and optimizing the mechanical properties of nanofibrous biomaterials.

Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (L-lactic acid) scaffolds for potential use in cardiac regeneration.

Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiber-aligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.

Effect of poly (l-lactic acid) scaffolds seeded with aligned diaphragmatic myoblasts overexpressing connexin-43 on infarct size and ventricular function in sheep with acute coronary occlusion.

Diaphragmatic myoblasts (DM) are stem cells of the diaphragm, a muscle displaying high resistance to stress and exhaustion. We hypothesized that DM modified to overexpress connexin-43 (cx43), seeded on aligned poly (l-lactic acid) (PLLA) sheets would decrease infarct size and improve ventricular function in sheep with acute myocardial infarction (AMI). Sheep with AMI received PLLA sheets without DM (PLLA group), sheets with DM (PLLA-DM group), sheets with DM overexpressing cx43 (PLLA-DMcx43) or no treatment (control group, n = 6 per group). Infarct size (cardiac magnetic resonance) decreased ∼25% in PLLA-DMcx43 [from 8.2 ± 0.6 ml (day 2) to 6.5 ± 0.7 ml (day 45), p < .01, ANOVA-Bonferroni] but not in the other groups. Ejection fraction (EF%) (echocardiography) at 3 days post-AMI fell significantly in all groups. At 45 days, PLLA-DM y PLLA-DMcx43 recovered their EF% to pre-AMI values (PLLA-DM: 61.1 ± 0.5% vs. 58.9 ± 3.3%, p = NS; PLLA-DMcx43: 64.6 ± 2.9% vs. 56.9 ± 2.4%, p = NS), but not in control (56.8 ± 2.0% vs. 43.8 ± 1.1%, p < .01) and PLLA (65.7 ± 2.1% vs. 56.6 ± 4.8%, p < .01). Capillary density was higher (p < .05) in PLLA-DMcx43 group than in the remaining groups. In conclusion, PLLA-DMcx43 reduces infarct size in sheep with AMI. PLLA-DMcx43 and PLLA-DM improve ventricular function similarly. Given its safety and feasibility, this novel approach may prove beneficial in the clinic.

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.

Similarities of arterial collagen pressure-diameter relationship in ovine femoral arteries and PLLA vascular grafts.

INTRODUCTION: In-vivo implanted vascular grafts fail due to the mechanical mismatch between the native vessel and the implant. The biomechanical characterization of native vessels provides valuable information towards the development of synthetic grafts. MATERIALS AND METHODS: Five samples of electrospun nanofibrous poly(L-lactic acid)(PLLA) tubular structures were subjected to physiological pulsating pressure using an experimental setup. Four ovine femoral arteries were also tested in the experimental setup under the same conditions. Instantaneous diameter and pressure signals were obtained using gold standard techniques, in order to estimate the dynamic pressure-strain elastic modulus (E(Pε)) of both native vessels and grafts. RESULTS: Synthetic grafts showed a significant increase of E(Pε) (10.57±0.97 to 17.63±2.61 10(6) dyn/cm(2)) when pressure was increased from a range of 50-90 mmHg (elastin-response range) to a range of 100-130 mmHg (collagen-response range). Furthermore, femoral arteries also exhibited a significant increase of EPε (1.66±0.30 to 15.76±4.78 10(6) dyn/cm(2)) with the same pressure variation, showing that both native vessels and synthetic grafts have a similar behavior in the collagen-acting range. CONCLUSION: The mechanical behavior of PLLA vascular grafts was characterized In vitro. However, the procedure can be easily extrapolated to In vivo experiences in conscious and chronically instrumented animals.