Engineering Vascular Grafts Research Articles

Gelatine and Gelatine/Elastin Nanocomposites for Vascular Grafts: Processing and Characterization

This study involves the preparation, microstructural, physical, mechanical, and biological characterization of novel gelatine and gelatine/elastin gels for their use in the tissue engineering of vascular grafts. Gelatine and gelatine/elastin nanocomposite gels were prepared via a sol-gel process, using soluble gelatine. Gelatine was subsequently cross-linked by leaving the gels in 1% glutaraldehyde. The cross-linking time was optimized by assessing the mass loss of the cross-linked gels in water and examining their mechanical properties in dynamic mechanical tests. Atomic force microscopy (AFM) studies revealed elastin nanodomains, homogeneously distributed and embedded in a bed of gelatine nanofibrils in the 30/70 elastin/gelatine gel. It was concluded that the manufactured nanocomposite gels resembled natural arteries in terms of microstructure and stiffness. The biological characterization involved the culture of rat smooth muscle cells (SMCs) on tubular gelatine and gelatine/ elastin nanocomposite gels, and measurements of the scaffold diameter and the cell density as a function of time.

Regulated Heparin Release using Novel Quantum Dots for Potential Application to Vascular Graft Engineering

This paper presents a new approach in the field of controlled drug delivery systems using a novel quantum dot (QD). We developed a system for polymeric microencapsulated drugs which is conjugated to near infrared (NIR) absorbing quantum dots and tested the feasibility of burst release of a model drug, heparin, from microcapsules triggered by irradiation. We have shown the burst release of heparin from microcapsules can be achieved by irradiation. This system is designed to externally modulate drug release in response to physiological needs by control of the intensity and period of irradiation. These results suggest that QD can be a key component to be used for triggering the release of drugs for various clinical applications. We further investigated the heparin incorporation into and release from decellularized blood vessels for potential application of functionalized heparin for vascular graft engineering.

Intravital fluorescent microscopic evaluation of bacterial cellulose as scaffold for vascular grafts

Although commonly used synthetic vascular grafts perform satisfactorily in large caliber blood vessels, they are prone to thrombosis in small diameter vessels. Therefore, small vessels might benefit from tissue engineered vascular grafts. This study evaluated bacterial cellulose (BC) as a potential biomaterial for biosynthetic blood vessels. We implanted the dorsal skinfold chambers in three groups of Syrian golden hamsters with BC (experimental group), polyglycolic acid, or expanded polytetrafluorethylene (control groups). Following implantation, we used intravital fluorescence microscopy, histology, and immunohistochemistry to analyze the biocompatibility, neovascularization, and incorporation of each material over a time period of 2 weeks. Biocompatibility was good in all groups, as indicated by the absence of leukocyte activation upon implantation. All groups displayed angiogenic response in the host tissue, but that response was highest in the polyglycolic acid group. Histology revealed vascularized granulation tissue surrounding all three biomaterials, with many proliferating cells and a lack of apoptotic cell death 2 weeks after implantation. In conclusion, BC offers good biocompatibility and material incorporation compared with commonly used materials in vascular surgery. Thus, BC represents a promising new biomaterial for tissue engineering of vascular grafts.

Tissue-engineered blood vessels with endothelial nitric oxide synthase activity

Nondegradable synthetic polymer vascular grafts used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. Tissue engineering of vascular grafts with autologous stem cells and biodegradable polymeric materials could solve these problems. The present study is aimed to develop a tissue-engineered vascular graft (TEVG) with functional endothelium using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. TEVGs (internal diameter: 10 mm, length: 40 mm) were fabricated by seeding vascular cells differentiated from BMCs onto PGA/PLCL scaffolds and implanted into the abdominal aorta of bone marrow donor dogs (n = 7). Eight weeks after implantation of the TEVGs, the vascular grafts remained patent. Histological and immunohistochemical analyses of the vascular grafts retrieved at 8 weeks revealed the regeneration of endothelium and smooth muscle and the presence of collagen. Western blot analysis showed that endothelial nitric oxide synthase (eNOS) was expressed in TEVGs comparable to native abdominal aortas. This study demonstrates that vascular grafts with significant eNOS activity can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.

Fibrinogen Concentration Influence on VEGF-Induced Differentiation of Adipose Tissue-Derived Stem Cells into Endothelial Cells

Fibrin is a natural substrate for growth, adhesion, and migration of mature endothelial cells (ECs) and a candidate coating material in approaches to graft endothelialization. Adipose tissue represents an abundant, practical source of donor tissue for stem cells which may be a useful source for engineering of vascular grafts. However, the optimal substrates that promote differentiation of adipose tissue-derived stem cells (ASCs) into ECs remain to be elucidated. In the present study, we investigated whether fibrin can be used as a substratum to support in vitro ECs differentiation of ASCs and whether fibrinogen concentration can be affect on ECs differentiation of ASCs. For determination of phenotypic characteristics of ASCs used in this experiment, we performed flow cytometry analysis. ASCs were plated on fibrin composed of varying concentrations of fibrinogen and induced into ECs differentiation in presence of VEGF. Before inducing into ECs, ASCs did not express any markers of hematopoietic cells (CD34, CD45), ECs (CD31, CD34), and endothelial progenitor cells (CD34, CD133, Flk-1). The degree of ECs differentiation was determined by capillary network formation, ECs-specific gene expression, and F-actin assembly. During the first 12 h after seeding, cells spread randomly, moved and formed small interconnected clusters. These clusters decreased in size and formed a capillary tube at 48 h. During the further incubation in presence of VEGF for 7 days, ASCs expressed mRNA and protein of von Willebrand factor (vWF). The degree of ECs differentiation of ASCs was consistently decreased as fibrinogen concentration increase. Fibrin may be used as biomatrix to promote differentiation of ASCs into ECs for tissue engineering.

In Vitro Models of Angiogenesis

Neovascularization can be categorized into two general processes: vasculogenesis and angiogenesis. Angiogenesis is the formation of new capillaries from pre-existing vessels, requiring growth factor driven recruitment, migration, proliferation, and differentiation of endothelial cells (ECs). Complex cell-cell and cell-extracellular matrix (ECM) interactions contribute to this process, leading finally to a network of tube-like formations of endothelial cells supported by surrounding mural cells. The study of angiogenesis has broad clinical implications in the fields of peripheral and coronary vascular disease, oncology, hematology, wound healing, dermatology, and ophthalmology, among others. As such, novel, clinically relevant models of angiogenesis in vitro are crucial to the understanding of angiogenic processes. We highlight some of the advances made in the development of these models, and discuss the importance of incorporating the three-dimensional cell-matrix and EC-mural cell interactions into these in vitro assays of angiogenesis. This review also discusses our own 3-D angiogenesis assay and some of the in vitro results from our lab as they relate to therapeutic neovascularization and tissue engineering of vascular grafts.

Polyurethane biomaterials for fabricating 3D porous scaffolds and supporting vascular cells

Successful tissue engineering of vascular grafts largely depends on synthetic scaffolds that support the survival, proliferation, and differentiation of seeded cells. To investigate the utility of polyurethanes for vascular tissue engineering, three-dimensional porous polyurethane scaffolds with highly interconnected pore structures were fabricated by a pressure differential/particulate leaching technique. Ammonium chloride and paraffin porogens were prepared to fabricate the scaffolds. Grinding of ammonium chloride resulted in particulates with uniform particle sizes but irregular shapes. Paraffin particulates made by a dispersion method, on the other hand, had spherical shapes and uniform particle sizes. Polyurethane scaffolds fabricated from these particulates had open faced, highly interconnected channels that could allow cellular infiltration and nutrient delivery. Human coronary artery smooth muscle and endothelial cell interactions with polyurethane surfaces revealed these biomaterials to maintain the contractile phenotype of human coronary artery smooth muscle cells and the formation of endothelial monolayers. During longer culture times, surface modification with cell adhesive extracellular matrix (ECM) protein promoted vascular cell proliferation, maintenance of the differentiated phenotype and endothelial monolayer integrity. Our results suggest that these polyurethanes, in conjunction with cell adhesive ECM proteins, could also support vascular cells in three-dimensional bioreactor-based culture conditions.

Processing Methods of Ultrathin Poly(ε-caprolactone) Films for Tissue Engineering Applications

Ultrathin poly(epsilon-caprolactone) (PCL) films were fabricated through biaxially drawn films made from three different methods, namely, spin casting, 2-roll milling, and solution casting. Biaxial drawn spin cast films yield thickness of 1.2 microm which is 9 and 12 times thinner that 2-roll mill and solvent cast films, respectively. The films fabricated were found to exhibit different drawing ratios. 2-roll mill film exhibits the highest drawing ratio of 4 x 4 while spin cast films can only draw up to a ratio of 2 x 2. The morphology of the films, studied using a polarized microscope and atomic force microscope, showed fine fibrillar networks of different thicknesses. Biaxially drawn 2-roll mill and solvent cast films showed thicker fibrils as compared to those for the spin cast films. Such a difference can be attributed to larger spherulites caused by slower cooling rates during melt pressing for both 2-roll mill and solvent cast films and smaller spherulites because of fast cooling during evaporation for spin cast films. Thermal analysis through differential scanning calorimetry revealed a slight increase in the peak-melting temperature after biaxial drawing. A drop in percentage crystallinity was also noted. The result of the water vapor transmission rate (WVTR) was found to be dependent on fabrication techniques that determine the spherulites formation. It was also found that the WVTR was inversely proportional to the thickness of the films. Tensile strength and modulus of the films showed significant improvements after biaxial stretching. By identifying the unique strengths of each individual PCL film produced via different techniques, it is possible to apply to different areas of membrane tissue engineering such as dermatology, ophthalmology, vascular graft engineering, and soft tissue regeneration.

Development of Tissue Engineered Vascular Grafts

Vascular bypass grafting is a commonly performed procedure for ischemic heart disease and peripheral vascular disease. However, approximately one in fourteen patients do not have suitable autologous arteries or veins available for grafting. Synthetic vascular grafts were introduced in the 1960s to overcome these problems, but while they perform adequately in high-flow, large-diameter vessel settings they are generally not suited to low-flow, small-diameter vessels. Tissue engineering is a relatively new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffolds. Because tissue engineering constructs contain living cells, they may have the potential to grow, self-repair, and self-remodel. Therefore, recently there has been much interest in the use of this technique to produce low-flow small-diameter arteries. The latest and most exciting developments in this area involve the use of multipotent stem cells as a cell source for tissue engineering of vascular grafts (both in vivo and in vitro).

Engineering of Vascular Grafts With Genetically Modified Bone Marrow Mesenchymal Stem Cells on Poly (Propylene Carbonate) Graft

Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO(2) at 37 degrees C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency.

A Novel Culture System Shows that Stem Cells Can be Grown in 3D and Under Physiologic Pulsatile Conditions for Tissue Engineering of Vascular Grafts

Currently available vascular grafts have been limited by variable patency rates, material availability, and immunological rejection. The creation of a tissue-engineered vascular graft (TEVG) from autologous stem cells would potentially overcome these limitations. As a first step in creating a completely autologous TEVG, our objective was to develop a novel system for culturing undifferentiated mouse embryonic stem cells (mESC) in a three-dimensional (3D) configuration and under physiological pulsatile flow and pressure conditions. A bioreactor was created to provide pulsatile conditions to a specially modified four-well Labtek Chamber-Slide culture system. Undifferentiated mESC were either suspended in a 3D Matrigel matrix or suspended only in cell-culture media within the culture system. Pulsatile conditions were applied to the suspended cells and visualized by video microscopy. Undifferentiated mESC were successfully embedded in a 3D Matrigel matrix and could withstand physiological pulsatile conditions. Video microscopy demonstrated that the mESC in the 3D matrix were constrained to the wells of the culture system, moved in unison with the applied flows, and were not washed downstream; this was in contrast to the mESC suspended in media alone. Undifferentiated mESC can be grown in 3D and under pulsatile conditions. We will use these results to study the effects of long-term pulsatile conditions on the differentiation of mESC into endothelial cells, smooth muscle cells, and fibroblast cells with the long-term goal of creating a completely autologous TEVG.

A new culture system shows that stem cells can be grown in 3-D and under physiologic pulsatile conditions for tissue engineering of vascular grafts

A new culture system shows that stem cells can be grown in 3-D and under physiologic pulsatile conditions for tissue engineering of vascular grafts

Umbilical Cord Blood Derived Endothelial Progenitor Cells for Tissue Engineering of Vascular Grafts

A substantial limitation regarding present pediatric cardiac surgery is the lack of appropriate materials for the repair of congenital defects. To address this shortcoming, tissue engineering is a scientific field that aims at in vitro fabrication of living autologous grafts with the capacity of growth, repair, and regeneration. Here we focused on tissue engineered vascular grafts using human umbilical cord blood derived endothelial progenitor cells (EPCs), as a noninvasive cell source for pediatric applications. EPCs were isolated from 20 ml fresh human umbilical cord blood by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto three-dimensional (3D) biodegradable vascular scaffolds (porosity > 95%, n = 22). Twenty-four hours after seeding the vascular grafts were positioned into a pulse-duplicator-in vitro system and grown for 48 hours under biomimetic conditions. A second group was grown 6 days statically and an additional 6 days biomimetically. Controls were cultured statically. Analysis of the grafts included immunohistochemistry, histology, and scanning electron microscopy. Preseeding differentiated EPCs indicated constant endothelial phenotypes including acetylated low-density lipoprotein, cluster of differentiation 31, von Willebrand factor, and endothelial nitric oxide synthetase. Seeded EPCs established favorable cell-to-polymer attachment and proliferation into the 3D tubular scaffolds. Both conditioned and static cellular constructs demonstrated positive staining for cluster of differentiation 31, von Willebrand factor, and expression of endothelial nitric oxide synthase. Human umbilical cord derived EPCs indicated exceptional growth characteristics used for tissue engineering of vascular grafts. These cells demonstrated a constant endothelial phenotype and related functional features. Based on these results EPCs seem to be a promising autologous cell source with regard to cardiovascular tissue engineering, particularly for the repair of congenital defects.

Tissue engineering of vascular grafts on xenogenic acellular matrix conduits using endothelial progenitors in human postnatal bone marrow(Cellular & Tissue Engineering)

Tissue engineering of vascular grafts on xenogenic acellular matrix conduits using endothelial progenitors in human postnatal bone marrow(Cellular & Tissue Engineering)

Recent Progress of Vascular Graft Engineering in Japan

The development of a small-diameter vascular graft has long been awaited. This review covers research activities, achievements and progress on vascular engineering in Japan, which was conducted over the last decade. The article includes recently developed experimental scaffolds, biologically active artificial extracellular matrices (ECMs) or non-fouling synthetic coatings, cell sourcing including the autologous vascular cell type, endothelial progenitor cells and genetically-engineered, temporary endothelial-like cells. The discussions were presented from biomechanical, biomaterial, cellular and tissue aspects. Once the mechano-biological and biologically active extracellular milieus are established in a designed vascular graft, the functional, structural and mechanical tissue morphogenesis and adaptation of implanted vascular grafts may proceed with implantation duration, and the spatio-temporal tissue modulations at cytokine, cellular, ECM levels under physiological stress proceed to regenerate vascular tissue architecture. The ultimate solution to a small-diameter vascular graft should be realized by optimal combinations of these factors.

Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.

Endothelial cell delivery for cardiovascular therapy

Obstructive atherosclerotic vascular disease stands as one of the greatest public health threats in the world. While a number of therapies have been developed to combat vascular disease, endothelial cell delivery has emerged as a distinct therapeutic modality. In this article, we will review the anatomy of the normal blood vessel and the biology of the intact endothelium, focusing upon its centrality in vascular biology and control over the components of the vascular response to injury so as to understand better the motivation for a cell-based form of therapy. Our discussion of cell delivery for cardiovascular therapy will be divided into surgical and interventional approaches. We will briefly recount the development of artificial grafts for surgical vascular bypass before turning our attention towards endothelial cell seeded vascular grafts, in which endothelial cells effectively provide local delivery of endogenous endothelial secretory products to maintain prosthetic integrity after surgical implantation. New techniques in tissue and genetic engineering of vascular grafts and whole blood vessels will be presented. Methods for percutaneous interventions will be examined as well. We will evaluate results of endoluminal endothelial cell seeding for treatment of restenosis and gene therapy approaches to enhance endogenous re-endothelialization. Finally, we will examine some innovations in endothelial cell delivery that may lead to the development of endothelial cell implants as a novel therapy for controlling proliferative vascular arteriopathy.

Tissue Engineering of Vascular Grafts: Human Cell Seeding of Decellularised Porcine Matrix

Objectives to develop a biocompatible and mechanically stable vascular graft combining human cells and a xenogenic acellular matrix. Design/Materials decellularised matrix tubes were obtained by enzymatic cell extraction of native porcine aortas. Endothelial cells and myofibroblasts were isolated from human saphenous veins and grown in cell cultures. The inner surface of the tubes was seeded with endothelial cells or myofibroblasts and exposed to pulsatile flow.Results after cell extraction, the absence of cellular components, as well as the maintenance of matrix integrity, was demonstrated by means of light microscopy and scanning electron microscopy. Furthermore, the porcine matrix was successfully seeded with human endothelial cells, which grew to a monolayer under flow conditions. Stable biomechanical properties were achieved at physiological perfusion pressures in vitro.Conclusions cellular components can be extracted from native porcine blood vessels. Vascular grafts can be generated in vitro of animal acellular matrix and human cells.

Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.