Electrospun scaffolds for tissue engineering of vascular grafts

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications.     

Fabrication of small-diameter polyurethane vascular grafts with microporous structure

 Microporous polyurethane vascular grafts with a diameter of 3 mm were fabricated by a spray phase inversion technique (SPIT). The microporous structure and hydraulic permeability of the grafts were regulated by changing the fabrication conditions. The maximum hydraulic permeability of 26 ml/min/cm², which was obtained in this series of grafts, satisfied the target value of about 10–40 ml/min/cm², which our previous studies suggested would lead to satisfactory graft patency. Further investigation is, however, needed to optimize the microporous structure and hydraulic permeability of the grafts. Received: October 10, 2000 / Accepted: February 4, 2002     

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20–30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4β1 ligand, REDV. Six grafts were transplanted in the femoral–femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.     

Usefulness of polyurethane for small-caliber vascular prostheses in comparison with autologous vein graft

 For long-term patency of small-caliber vascular prostheses, antithrombogenicity and microporous structure are very important. We have developed a new technique to give a microporous structure to a polyurethane vascular prosthesis that has favorable antithrombogenicity. A solution of tetrahydrofuran/dimethylformamide (1 : 1) containing 13 wt% of segmented polyurethane (PTMG + MDI) and calcium carbonate (mean particle size, 8 μm) was dipcoated on a glass mandrel 3 mm in diameter and placed into distilled water. After the glass mandrel was removed, the polyurethane tube was placed into hydrochloric acid, and a microporous polyurethane vascular prosthesis was produced. Prostheses made in this fashion, and autologous jugular vein grafts were implanted into the femoral artery and the carotid artery of mongrel dogs. Patency was recognized on the arteriogram and duplex scanning (ultrasonography), and the removed grafts were inspected macroscopically and microscopically. This prosthesis was similar in elasticity to a vein graft. Patency was defined 8 weeks after implantation, and this prosthesis showed less intimal hyperplasia than the autologous vein graft. The new polyurethane prosthesis might be useful for small-caliber vascular reconstruction. Received: December 18, 2000 / Accepted: January 28, 2002     

Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates

The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).     

Ultrathin-wall vascular grafts for endovascular surgery

 The purposes of this study were to develop ultrathin-wall grafts suitable for stent-graft procedures and to examine the feasibility of the grafts from the physical point of view. We fabricated ultrathin-wall grafts with a wall thickness of 42 to 137 μm, using 50 denier polyester threads consisting of 72 polyester monofilaments. We studied the following physical properties of the ultrathin wall: the surface structure of the grafts, the longitudinal tensile strength of the grafts, the water permeability of the grafts, and the size of introducers through which the stent grafts can pass. In ultrathin-wall grafts with a wall thickness of 75 μm or more, a regular surface structure with zero planimetric porosity was recognized. In those with a thickness less than 64 μm, the porosity increased as the wall thickness decreased. The longitudinal tensile strengths of the 75 μm and 64 μm grafts were 13.1 ± 0.9 and 9.5 ± 0.9 kg, respectively. The water permeability of the 75-μm grafts was 380 ml/min, and that of the thinner grafts increased as the wall thickness decreased. Stents with a diameter of 40 mm covered with the ultrathin-wall grafts could pass through introducers with an inner diameter of 18 French. We conclude that the newly developed ultrathin-wall grafts are physiologically suitable for endovascular surgery. Received: December 4, 2000 / Accepted: August 13, 2001     

Tailored design of electrospun composite nanofibers with staged release of multiple angiogenic growth factors for chronic wound healing

The objective of this research study is to develop a collagen (Col) and hyaluronic acid (HA) inter-stacking nanofibrous skin equivalent substitute with the programmable release of multiple angiogenic growth factors (vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and endothelial growth factor (EGF)) either directly embedded in the nanofibers or encapsulated in the gelatin nanoparticles (GNs) by electrospinning technology. The delivery of EGF and bFGF in the early stage is expected to accelerate epithelialization and vasculature sprouting, while the release of PDGF and VEGF in the late stage is with the aim of inducing blood vessels maturation. The physiochemical characterizations indicate that the Col–HA–GN nanofibrous membrane possesses mechanical properties similar to human native skin. The design of a particle-in-fiber structure allows growth factors for slow controlled release up to 1 month. Cultured on biodegradable Col–HA membrane with four kinds of growth factors (Col–HA w/4GF), endothelial cells not only increase in growth rate but also form a better network with a thread-like tubular structure. The therapeutic effect of Col–HA w/4GF membrane on streptozotocin (STZ)-induced diabetic rats reveals an accelerated wound closure rate, together with elevated collagen deposition and enhanced maturation of vessels, as revealed by Masson’s trichrome stain and immunohistochemical analysis, respectively. From the above, the electrospun Col–HA–GN composite nanofibrous skin substitute with a stage-wise release pattern of multiple angiogenic factors could be a promising bioengineered construct for chronic wound healing in skin tissue regeneration.     

Fabrication of highly interconnected porous silk fibroin scaffolds for potential use as vascular grafts

Silk fibroin (SF) scaffolds have been designed and fabricated for multiple organ engineering owing to SF’s remarkable mechanical property, excellent biocompatibility and biodegradability, as well as its low immunogenicity. In this study, an easy-to-adopt and mild approach based on a modified freeze-drying method was developed to fabricate a highly interconnected porous SF scaffold. The physical properties of the SF scaffold, including pore morphology, pore size, porosity and compressive modulus, could be adjusted by the amount of ethanol added, the freezing temperature and the concentration of SF. Fourier transform infrared spectroscopy illustrated that treatment of the lyophilized scaffolds with 90% methanol led to a structure transition of SF from silk I (random coil) to silk II (beta-sheet), which stabilized the SF scaffolds in water. We also incorporated heparin during fabrication to obtain a heparin-loaded scaffold which possessed excellent anticoagulant property. The heparin that was incorporated into the SF scaffolds could be released in a sustain manner for approximately 7 days, inhibiting the proliferation of human smooth muscle cells within the scaffold in vitro while promoting neovascularization in vivo. We therefore propose that the SF porous scaffold fabricated here may be an attractive candidate for use as a potential vascular graft for implantation based on its high porosity, excellent blood compatibility and mild fabrication process.     

Development of sutureless vascular connecting system for easy implantation of small-caliber artificial grafts

A novel sutureless vascular connecting system, an assembly with a delivery rod, an introducing sheath, and a connecting device, was developed for easy implantation of small-caliber vascular grafts less than 2 mm in internal diameter. A microporous stainless tube (length 2 mm, external diameter 1.6 mm, wall thickness 65 µm, pore diameter 400 µm, pore-to-pore distance 500 µm) was designed to serve as a connecting device. The feasibility of the system was tested using two types of preliminary animal experiments. One animal model consisted of graft implantation into the rat abdominal aorta (1.5 mm in diameter). The connecting device was inserted into the proximal and distal ends of the aorta through the introducing sheath by pushing the delivery rod with the connecting device placed over it. Subsequently, the aortic segments were inserted into both ends of model grafts made of segmented polyurethane (1.8 mm in internal diameter) and were fixed with banding silk threads from the exterior. The procedure was completed within 20 min without requiring specialized microsurgery techniques. Blood leakage and obstruction did not occur. The second model consisted of an end-to-end anastomosis between rabbit common carotid arteries (2 mm in diameter), which was performed within several minutes of blood flow interruption. Scanning electron microscopy demonstrated that the luminal surface of the device was fully covered with endothelial cells (ECs) after 1 week as a result of transluminal ingrowth of native ECs through the micropores in the device. This endothelialization may prevent early thrombus-induced occlusion. This simple and “easy-to-learn” technique will promote the development of small-caliber arterial grafts, and furthermore, it may have potential for clinical application.     

Mechanical analysis of a prototype of small diameter vascular prosthesis: numerical simulations

This paper concerns a mechanical analysis of a prototype of a small diameter vascular prosthesis made of a fibre reinforcement silicone material. The theoretical approach is carried out for a neoHookean strain energy function augmented with unidirectional reinforcing that is characterized by a single additional constitutive parameter for strength of reinforcement. Numerical simulations based on a finite element model compare the compliance of different grafts and predict the degree of the compliance mismatch in an anastomosis between native artery and vascular prosthesis. Furthermore, specific applied strains on the prototype, viewed as arising surgical manipulation and implying telescopic shear have been simulated. Thus, for different fibre reinforcements, the stress gradient through the wall of the tubular structure is evaluated.     

Achieving the ideal properties for vascular bypass grafts using a tissue engineered approach: a review

The multiple demands placed on small calibre cardiovascular bypass grafts have meant that a synthetic prosthesis with good long-term patency has not been developed. A tissue-engineered graft could fulfil the ideal characteristics present in an artery. However, the great disadvantage of such a conduit is the time necessary for maturation leading to unacceptable delays once the decision to intervene surgically has been made. This maturation process is essential to produce a graft which can withstand haemodynamic stress. Once implanted, the tissue-engineered graft can contract in response to immediate haemodynamic conditions and remodel in the long term. We review the latest tissue engineering approaches used to give the favourable properties of mechanical strength, arterial compliance, low thrombogenicity, long-term resistance towards biodegradation as well as technological advances which shorten the time required for production of an implantable graft.     

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by l-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo.     

Modified n-HA/PA66 scaffolds with chitosan coating for bone tissue engineering: cell stimulation and drug release

The dipping-drying procedure and cross-linking method were used to make drug-loaded chitosan (CS) coating on nano-hydroxyapatite/polyamide66 (nHA/PA66) composite porous scaffold, endowing the scaffold controlled drug release functionality. The prefabricated scaffold was immersed into an aqueous drug/CS solution in a vacuum condition and then crosslinked by vanillin. The structure, porosity, composition, compressive strength, swelling ratio, drug release and cytocompatibility of the pristine and coating scaffolds were investigated. After coating, the scaffold porosity and pore interconnection were slightly decreased. Cytocompatibility performance was observed through an in vitro experiment based on cell attachment and the MTT assay by MG63 cells which revealed positive cell viability and increasing proliferation over the 11-day period in vitro. The drug could effectively release from the coated scaffold in a controlled fashion and the release rate was sustained for a long period and highly dependent on coating swelling, suggesting the possibility of a controlled drug release. Our results demonstrate that the scaffold with drug-loaded crosslinked CS coating can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to be a promising high performance biomaterial in bone tissue engineering.     

Heparinized PCL/keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation

Abstract

Heparins are capable of improving blood compatibility, enhancing HUVEC viability, while inhibiting HUASMC proliferation. Combination of biodegradable poly(ε-caprolactone) (PCL) with keratin and heparins would provide an anticoagulant and endothelialization supporting environment for vascular tissue engineering. Herein, PCL and keratin were first coelectrospun and then covalently conjugated with heparins. The resulting mats were surface-characterized by ATR-FTIR, SEM, WCA, and XPS. Cell viability data showed that the heparinized PCL/keratin mats could motivate the adhesion and growth of HUVEC, while inhibit HUASMC proliferation. In addition, these mats could prolong blood clotting time and reduce platelet adhesion as well as no erythrolysis. Interestingly, these mats could catalyze the NO donor in blood to release NO, which could enhance endothelial cell growth, while decrease smooth muscle cell proliferation and platelet adhesion. In summary, the heparinized mats would be a good candidate as a scaffold for vascular tissue engineering. This study is novel in that we prepared a type of heparinized tissue scaffold that could catalyze the NO donor to release NO to regulate endothelialization without angiogenesis and thrombus formation.