Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review

The higher patency rates of cardiovascular implants, including vascular bypass grafts, stents, and heart valves are related to their ability to inhibit thrombosis, intimal hyperplasia, and calcification. In native tissue, the endothelium plays a major role in inhibiting these processes. Various bioengineering research strategies thereby aspire to induce endothelialization of graft surfaces either prior to implantation or by accelerating in situ graft endothelialization. This article reviews potential bioresponsive molecular components that can be incorporated into (and/or released from) biomaterial surfaces to obtain accelerated in situ endothelialization of vascular grafts. These molecules could promote in situ endothelialization by the mobilization of endothelial progenitor cells (EPC) from the bone marrow, encouraging cell-specific adhesion (endothelial cells (EC) and/or EPC) to the graft and, once attached, by controlling the proliferation and differentiation of these cells. EC and EPC interactions with the extracellular matrix continue to be a principal source of inspiration for material biofunctionalization, and therefore, the latest developments in understanding these interactions will be discussed.     

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.     

Induction of EPC homing on biofunctionalized vascular grafts for rapid in vivo self-endothelialization — A review of current strategies

For years intensive research has been done to improve the hemocompatibility of blood contacting vascular devices. Despite the enormous progress in physicochemical surface optimization technologies, the native endothelium still represents the ideal surface for blood contact. Numerous tissue engineering strategies aspired towards the endothelialization of graft surfaces to generate a non-thrombogenic barrier on artificial materials. A paradigm change in surface modification concepts is the in vivo endothelialization of vascular grafts by capturing circulating endothelial progenitor cells (EPCs) directly from the blood stream via biofunctionalized implant materials. Thereby, capture molecules are immobilized on artificial vascular grafts to mimic a pro-homing substrate for EPCs. In this review, different coating strategies for in vivo capturing of EPCs on synthetic implants are discussed. This therapeutic concept opens a new chapter in regenerative medicine by realizing the vision that every patient seeds his implants with his own progenitor cells to make the synthetic grafts unrecognizable for the body's rejection mechanisms.     

An intelligent bioreactor system for the cultivation of a bioartificial vascular graft

Cardiovascular disease is the most common cause of death, accounting for 31% of deaths worldwide. As purely synthetic grafts implicate concomitant anticoagulation and autologous veins are rare, tissue‐engineered vascular grafts are urgently needed. For successful in vitro cultivation of a bioartificial vascular graft, the suitable bioreactor should provide conditions comparable to vasculogenesis in the body. Such a system has been developed and characterized under continuous and pulsatile flow, and a variety of sensors has been integrated into the bioreactor to control parameters such as temperature, pressure up to 500 mbar, glucose up to 4.5 g/L, lactate, oxygen up to 150 mbar, and flow rate. Wireless data transfer (using the ZigBee specification based on the IEEE 802.15.4 standard) and multiple corresponding sensor signal processing platforms have been implemented as well. Ultrasound is used for touchless monitoring of the growing vascular structure as a quality control before implantation (maximally achieved ultrasound resolution 65 μm at 15 MHz). To withstand the harsh conditions of steam sterilization (120°C for 20 min), all electronics were encapsulated. With such a comprehensive physiologically conditioning, sensing, and imaging bioreactor system, all the requirements for a successful cultivation of vascular grafts are available now.     

Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.     

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.     

国产真丝涤纶血管移植物自然内皮化的研究

采用真丝涤纶人造血管行腹主动脉移植,结果显示,术后１０天吻合口管壁有较完整的纤维组织性吻合,并有内皮细胞覆盖,血管内壁有血细胞形成的一薄层血栓覆盖,１０天后开始机化学变成纤维组织,逐渐被内皮细胞覆盖,术后１个月移植血管内形成完整的内皮,但并不完善,３个月血管内皮才被一单层内皮细胞完整覆盖。     

In vivo study of a model tissue-engineered small-diameter vascular bypass graft

Conventionally used vascular grafts such as polyester (Dacron) or expanded polytetrafluoroethylene perform inadequately as small-diameter vascular bypass grafts (SDBGs). SDBGs, which can maintain long-term patency and those that could potentially evolve with the somatic growth, are highly desirable in vascular surgery and thus research into tissue-engineered blood vessels (TEBVs) is of keen interest. A TEBV was developed by seeding endothelial cells onto a collagen matrix that was cross-linked and contracted by smooth muscle cells (SMCs). A polyester graft served as a scaffold. Recovery studies (12 TEBVs and seven controls) were carried out to assess in vivo endothelialization and long-term patency of TEBVs. Hemodynamic observations indicated para-anastomotic turbulences and high shear stress at anastomosis. Recovery studies demonstrated confluent endothelialization, thrombus-free surfaces, and patent TEBVs in all cases. Graft incorporation and neovascularization of the scaffold occurred in both hybrid and control grafts. However, thickened neointima formation occurred in TEBV grafts, which was most likely caused by the rigidity of polyester scaffold. Significant perigraft inflammatory changes could be observed in both TEBVs and control grafts at 1, 4, and 8 weeks. In conclusion, the TEBVs demonstrated satisfactory performance as an infra-renal-aortic graft in a porcine model. The TEBV serves as a promising model and facilitates the development of a TEBV in a clinical setting, potentially with human stem cells and with more biocompatible, biodegradable scaffolds that are mechanically more compliant with natural vessels.     

Tumor Necrosis Factor Improves Vascularization in Osteogenic Grafts Engineered with Human Adipose-Derived Stem/Stromal Cells

The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.     

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity¹. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes². However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia^3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries^5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)⁷ for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.     

Nitric oxide-producing polyurethanes

Thrombus formation and eventual intimal hyperplasia are the leading causes of small-diameter synthetic vascular graft failure. To combat these issues, we have incorporated a diazeniumdiolate-modified nitric oxide (NO)-producing peptide into a polyurethane to improve the thromboresistance of this biocompatible polymer. NO production by polyurethane films occurred for approximately 2 months under physiological conditions, and mechanical properties of the material were suitable for vascular graft applications. Platelet adhesion to NO-releasing polyurethane was dramatically decreased compared to control polyurethane. Furthermore, endothelial cell growth was stimulated in the presence of the NO-releasing polyurethane, while smooth muscle cell growth was greatly inhibited. The ability of this bioactive material to inhibit platelet adhesion and smooth muscle cell proliferation while encouraging endothelialization suggests that this NO-generating polyurethane may be suitable as a candidate material for small-diameter vascular grafts.     

Electrospun tecophilic/gelatin nanofibers with potential for small diameter blood vessel tissue engineering

Tissue engineering techniques particularly using electrospun scaffolds have been intensively used in recent years for the development of small diameter vascular grafts. However, the development of a completely successful scaffold that fulfills multiple requirements to guarantee complete vascular regeneration remains challenging. In this study, a hydrophilic and compliant polyurethane namely Tecophilic (TP) blended with gelatin (gel) at a weight ratio of 70:30 (TP(70)/gel(30)) was electrospun to fabricate a tubular composite scaffold with biomechanical properties closely simulating those of native blood vessels. Hydrophilic properties of the composite scaffold induced non‐thrombogenicity while the incorporation of gelatin molecules within the scaffold greatly improved the capacity of the scaffold to serve as an adhesive substrate for vascular smooth muscle cells (SMCs), in comparison to pure TP. Preservation of the contractile phenotype of SMCs seeded on electrospun TP(70)/gel(30) was yet another promising feature of this scaffold. The nanostructured TP(70)/gel(30) demonstrated potential feasibility toward functioning as a vascular graft. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1165–1180, 2014.     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

Tubular silk scaffolds for small diameter vascular grafts

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.     

Mussel-inspired immobilization of vascular endothelial growth factor (VEGF) for enhanced endothelialization of vascular grafts

Most polymeric vascular prosthetic materials have low patency rate for replacement of small diameter vessels (<5 mm), mainly due to failure to generate healthy endothelium. In this study, we present polydopamine-mediated immobilization of growth factors on the surface of polymeric materials as a versatile tool to modify surface characteristics of vascular grafts potentially for accelerated endothelialization. Polydopamine was deposited on the surface of biocompatible poly(L-lactide-co-ε-caprolactone) (PLCL) elastomer, on which vascular endothelial growth factor (VEGF) was subsequently immobilized by simple dipping. Surface characteristics and composition were investigated by using scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Immobilization of VEGF on the polydopamine-deposited PLCL films was effective (19.8 ± 0.4 and 197.4 ± 19.7 ng/cm(2) for DPv20 and DPv200 films, respectively), and biotin-mediated labeling of immobilized VEGF revealed that the fluorescence intensity increased as a function of the concentration of VEGF solution. The effect of VEGF on adhesion of HUVECs was marginal, which may have been masked by polydopamine layer that also enhanced cell adhesion. However, VEGF-immobilized substrate significantly enhanced proliferation of HUVECs for over 7 days of in vitro culture and also improved their migration. In addition, immobilized VEGF supported robust cell to cell interactions with strong expression of CD 31 marker. The same process was effective for immobilization of basic fibroblast growth factor, demonstrating the robustness of polydopamine layer for secondary ligation of growth factors as a simple and novel surface modification strategy for vascular graft materials.     

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.     

Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip

Neurovascular inflammation is a major contributor to many neurological disorders, but modeling these processes in vitro has proven to be difficult. Here, we microengineered a three-dimensional (3D) model of the human blood-brain barrier (BBB) within a microfluidic chip by creating a cylindrical collagen gel containing a central hollow lumen inside a microchannel, culturing primary human brain microvascular endothelial cells on the gel’s inner surface, and flowing medium through the lumen. Studies were carried out with the engineered microvessel containing endothelium in the presence or absence of either primary human brain pericytes beneath the endothelium or primary human brain astrocytes within the surrounding collagen gel to explore the ability of this simplified model to identify distinct contributions of these supporting cells to the neuroinflammatory response. This human 3D BBB-on-a-chip exhibited barrier permeability similar to that observed in other in vitro BBB models created with non-human cells, and when stimulated with the inflammatory trigger, tumor necrosis factor-alpha (TNF-α), different secretion profiles for granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed depending on the presence of astrocytes or pericytes. Importantly, the levels of these responses detected in the 3D BBB chip were significantly greater than when the same cells were co-cultured in static Transwell plates. Thus, as G-CSF and IL-6 have been reported to play important roles in neuroprotection and neuroactivation in vivo, this 3D BBB chip potentially offers a new method to study human neurovascular function and inflammation in vitro, and to identify physiological contributions of individual cell types.     

Successful development of small diameter tissue-engineering vascular vessels by our novel integrally designed pulsatile perfusion-based bioreactor

Small-diameter (<4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O(2) and CO(2) in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for small-diameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use.     

A Novel Modular Bioreactor to In Vitro Study the Hepatic Sinusoid

We describe a unique, versatile bioreactor consisting of two plates and a modified commercial porous membrane suitable for in vitro analysis of the liver sinusoid. The modular bioreactor allows i) excellent control of the cell seeding process; ii) cell culture under controlled shear stress stimulus, and; iii) individual analysis of each cell type upon completion of the experiment. The advantages of the bioreactor detailed here are derived from the modification of a commercial porous membrane with an elastomeric wall specifically moulded in order to define the cell culture area, to act as a gasket that will fit into the bioreactor, and to provide improved mechanical robustness. The device presented herein has been designed to simulate the in vivo organization of a liver sinusoid and tested by co-culturing endothelial cells (EC) and hepatic stellate cells (HSC). The results show both an optimal morphology of the endothelial cells as well as an improvement in the phenotype of stellate cells, most probably due to paracrine factors released from endothelial cells. This device is proposed as a versatile, easy-to-use co-culture system that can be applied to biomedical research of vascular systems, including the liver.     

Can sites prone to flow induced vascular complications in a-v fistulas be assessed using computational fluid dynamics?

Arterio-venous fistulas (shunts between arteries and veins) are the preferred vascular access for hemodialysis. Despite their superior patency, compared with synthetic tubes and grafts, functional problems and inadequate flow rates are the common complications. Local flow conditions, in particular low and oscillating wall shear stresses (WSS), are central to vascular problems and a robust framework for analyzing flow conditions in vascular structures could provide an understanding of the mechanisms leading to vascular complications, such as stenoses, aneurisms, and thromboses.We hypothesize that a validated computational fluid dynamics (CFD) framework can be used to identify critical fistula configurations with elevated risk of complications. Therefore, the aim of the present study was to develop a CFD framework for analyzing fluid flow in complex vascular structures, such as arterio-venous fistulas validated by comparisons of in vitro volume flows with CFD results and flow fields from ultrasound scans with CFD simulations.Volume flows measured in vitro and CFD data differed quantitatively. However, good relative correlations exist between the data using logarithmic scales. Qualitatively, visual comparisons between ultrasound and CFD images showed good agreement between the two methods. In addition, WSS levels and the oscillatory shear index (OSI) were calculated and visualized on the model surface. The method was successfully validated and the method is deemed suitable for more thorough investigations into the field of vascular complications in a-v fistulas.