Confluent Endothelial Layer Research Articles

Protein‐Engineered Elastin Fibers as Building Blocks for The Textile‐Based Assembly of Tissue Equivalents

AbstractNative tissues feature unique hierarchical designs, in which fiber units are arranged from the bottom up in anisotropic patterns. The processing of biomaterials into fibers, followed by their textile‐like assembly into complex patterns, is therefore a promising avenue to engineer native‐like tissue replacements. Here it is shown for the first time the fabrication of meter‐long hydrogel fibers prepared from engineered elastin using a microinjection system and exploiting the catalyst‐free click chemistry. Given their similarity to native elastin, the fabricated elastin‐like fibers achieved excellent stretching (500%) and recoiling performance. Moreover, the fabrication scheme is compatible with the implementation of a salt‐leaching gas‐foaming approach, resulting in highly porous elastin‐like fibers (the first of their kind). From the translation perspective, the fibers can be autoclaved, which allows for sterilization and long‐term storage. Human umbilical vein endothelial cells cultured on autoclaved fibers produced a confluent endothelial layer lining the fiber surface, in which the cells became aligned in response to physiological fiber stretching. It is also shown that these functional fibers can be assembled by weaving, braiding and knitting, with various spatial patterns. Overall, the elastin‐like fibers can be used as building blocks for the reconstruction of functional tissues using the principles of textile technology.

Successful endothelial monolayer formation on melt electrowritten scaffolds under dynamic conditions to mimic tunica intima

The lack of transplantable tissues and organs as well as the limitations of synthetic implants highlight the need for tissue-engineered constructs to obtain safe, long-lasting, and limitless tissue replacements. Scaffolds for cardiovascular applications, such as for a tissue-engineered vascular graft (TEVG), are thus highly required. For TEVGs, tubular scaffolds should support the formation of confluent endothelial layers in particular under dynamic conditions to prevent thrombosis and maintain hemostasis. For that purpose, a porous and highly diffusible scaffold structure is necessary to allow optimal cell adhesion as well as oxygen and nutrient exchange with the surrounding tissue. Here, we present a three-dimensional-printed scaffold made by a combination of fused deposition modeling (FDM) and melt electrowriting (MEW) out of polycaprolactone that enables monolayer formation and alignment of endothelial cells in the direction of medium flow under a shear stress of up to 10 dyn cm-2. Pore size and coating with human fibrin were optimized to enable confluent endothelial layers on the printed scaffold structures. Cell orientation and shape analysis showed a characteristic alignment and elongation of the tested endothelial cells with the direction of flow after dynamic cultivation. In contrast, melt electrospun scaffolds based on the same CAD design under comparable printing and cultivation conditions were not sufficient to form gapless cell layers. Thus, the new scaffold fabricated by MEW/FDM approach appears most suitable for TEVGs as a template for the innermost vascular wall layer, the tunica intima.

In Situ Endothelialization of Free-Form 3D Network of Interconnected Tubular Channels via Interfacial Coacervation by Aqueous-in-Aqueous Embedded Bioprinting.

The challenge of bioprinting vascularized tissues is structure retention and in situ endothelialization. The issue is addressed by adopting an aqueous-in-aqueous 3D embedded bioprinting strategy, in which the interfacial coacervation of the cyto-mimic aqueous two-phase systems (ATPS) are employed for maintaining the suspending liquid architectures, and serving as filamentous scaffolds for cell attachment and growth. By incorporating endothelial cells in the ink phase of ATPS, tubular lumens enclosed by coacervated complexes of polylysine (PLL) and oxidized bacteria celluloses (oxBC) can be cellularized with a confluent endothelial layer, without any help of adhesive peptides. By applying PLL/oxBC ATPS for embedded bioprinting, free-form 3D vascular networks with in situ endothelialization of interconnected tubular lumens are achieved. This simple approach is a one-step process without any sacrificed templates and post-treatments. The resultant functional vessel networks with arbitrary complexity are suspended in liquid medium and can be conveniently handled, opening new routes for the in vitro production of thick vascularized tissues for pathological research, regeneration therapy and animal-free drug development.

Breast adenocarcinoma-derived exosomes lower first-contact de-adhesion strength of adenocarcinoma cells to brain endothelial layer

Despite of advances in modern therapeutics, one of the most feared complications of cancer are brain metastases, which often cause life impairing profound neurological symptoms and premature death. Breast adenocarcinoma is among the leading “sources” of brain metastases. Since the central nervous system lacks a classical lymphatic circulation, invading metastatic cells can reach the brain parenchyma only through haematogenous routes and must breach the blood-brain barrier (BBB). The key step before the transmigration of metastatic cells through the highly regulated interface of the BBB is the establishment of firm adhesion between the tumor cell and the cerebral endothelial layer. Using atomic force microscopy, as a high resolution force spectrograph, direct measurements of intercellular interactions was performed between living adenocarcinoma cells and a confluent endothelial layer pre-treated with carcinoma cell-derived exosomes. By immobilization of a living adenocarcinoma cell to an atomic force microscope’s cantilever, intercellular de-adhesions were directly measured by single cell force spectroscopy (SCFS) at quasi-physiological conditions. De-adhesion dynamics and strength was characterized by several different calculated parameters, involving aspects of both membrane and cell surface related factors. Our results indicate that de-adhesion strength was lower in case of exosome pre-treated endothelial cells as compared to non-treated controls. Breast adenocarcinoma-derived exosomes have direct effect on de-adhesion pattern of brain endothelium.

Frugal Approach toward Developing a Biomimetic, Microfluidic Network-on-a-Chip for In Vitro Analysis of Microvascular Physiology.

Several disease conditions, such as cancer metastasis and atherosclerosis, are deeply connected with the complex biophysical phenomena taking place in the complicated architecture of the tiny blood vessels in human circulatory systems. Traditionally, these diseases have been probed by devising various animal models, which are otherwise constrained by ethical considerations as well as limited predictive capabilities. Development of an engineered network-on-a-chip, which replicates not only the functional aspects of the blood-carrying microvessels of human bodies, but also its geometrical complexity and hierarchical microstructure, is therefore central to the evaluation of organ-assist devices and disease models for therapeutic assessment. Overcoming the constraints of reported resource-intensive fabrication techniques, here, we report a facile, simple yet niche combination of surface engineering and microfabrication strategy to devise a highly ordered hierarchical microtubular network embedded within a polydimethylsiloxane (PDMS) slab for dynamic cell culture on a chip, with a vision of addressing the exclusive aspects of the vascular transport processes under medically relevant paradigms. The design consists of hierarchical complexity ranging from capillaries (∼80 μm) to large arteries (∼390 μm) and a simultaneous tuning of the interfacial material chemistry. The fluid flow behavior is characterized numerically within the hierarchical network, and a confluent endothelial layer is realized on the inner wall of microfluidic device. We further explore the efficacy of the device as a vascular deposition assay of circulatory tumor cells (MG-63 osteosarcoma cells) present in whole blood. The proposed paradigm of mimicking an in vitro vascular network in a low-cost paradigm holds further potential for probing cellular dynamics as well as offering critical insights into various vascular transport processes.

A Three-Dimensional Microfluidic Device for Monitoring Cancer and Chemotherapy-Associated Platelet Activation.

Platelet activation and the risk of thrombosis are increased in cancer patients, especially after chemotherapy. Our previous studies indicated that chemotherapy-induced platelet activation is largely due to endothelial cell damage. Thus, simple in vitro tests, such as aggregometry, are not desirable tests to predict platelet responsiveness to different chemotherapeutic agents because other contributory factors, such as tumor cells, endothelial cells, and the flow rate of platelets, also contribute to the formation of cancer-associated thrombosis. Therefore, developing a platelet detection system, which includes all possible risk parameters, is necessary. In the present study, we described a microengineered microfluidic system that contained a drug concentration generator, cancer cell culture chip, and three-dimensional (3D) circular microvascular model covered with a confluent endothelial layer and perfused with human platelets at a stable flow rate. Doxorubicin was injected through two injection sites. Endothelial cell injury was evaluated by counting, cell cytoskeleton observation, and the level of IACM1 and ET-1 in endothelial cells or a culture medium. Prestained platelets were perfused into the artificial blood vessel, and platelet-endothelial cell adhesion was measured. We found that (i) MCF7 cell-released factors had a cytotoxicity effect on both endothelial cells and platelets. (ii) We confirmed that doxorubicin-induced platelet activation was endothelial cell-dependent. (iii) A lower dosage of doxorubicin (0–2.0 μM) induced platelet activation, while a higher dosage of doxorubicin (2.0–4.0 μM) led to platelet death. Our findings indicated that platelet-endothelial cell adhesion could be used as a diagnostic marker of platelet activation, providing a simple and rapid detective way to predict platelet responsiveness before or during chemotherapy.

Void-free 3D Bioprinting for In-situ Endothelialization and Microfluidic Perfusion.

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. We address both of these issues by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer-by-layer alongside a matrix bioink to establish void-free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well-defined 3D network of interconnected tubular channels. This void-free 3D printing (VF-3DP) approach circumvents the traditional concerns of structural collapse, deformation and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered "unprintable". By pre-loading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in-situ endothelialization method can be used to produce endothelium with a far greater uniformity than can be achieved using the conventional post-seeding approach. This VF-3DP approach can also be extended beyond tissue fabrication and towards customized hydrogel-based microfluidics and self-supported perfusable hydrogel constructs.

Long-term nitric oxide release for rapid endothelialization in expanded polytetrafluoroethylene small-diameter artificial blood vessel grafts

Without a complete and confluent endothelial layer covering the luminal surface, expanded polytetrafluoroethylene (ePTFE) small-diameter (<6 mm) artificial blood vessels would fail in vivo due to thrombosis and restenosis. In order to promote rapid endothelialization and anticoagulation, we modified the surface of ePTFE by effectively grafting functional biomolecules, such as nitric oxide (NO) catalyst selenocystamine (SeCA) and vascular endothelial growth factor (VEGF), via dopamine coating. Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) results showed the successful incorporation of SeCA and VEGF. The release profile of NO, a cell-signaling molecule that regulates cell behavior and platelet aggregation, lasted beyond 35 days and could be controlled by the ratio of SeCA to dopamine. The biological results in terms of cell culture and antithrombogenic properties showed that the surface functionalization strategies promoted human coronary artery endothelial cell (HCAEC) growth and proliferation while preventing platelet adhesion and the formation of thrombi in vitro. A complete endothelial cell monolayer on ePTFE was found to form by day 10. This simple and effective surface functionalization method is beneficial for the eventual clinical application of small-diameter ePTFE blood vessel grafts.

Void-free 3D Bioprinting for In-situ Endothelialization and Microfluidic Perfusion.

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. We address both of these issues by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer-by-layer alongside a matrix bioink to establish void-free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well-defined 3D network of interconnected tubular channels. This void-free 3D printing (VF-3DP) approach circumvents the traditional concerns of structural collapse, deformation and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered "unprintable". By pre-loading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in-situ endothelialization method can be used to produce endothelium with a far greater uniformity than can be achieved using the conventional post-seeding approach. This VF-3DP approach can also be extended beyond tissue fabrication and towards customized hydrogel-based microfluidics and self-supported perfusable hydrogel constructs.

Layer-by-layer biofabrication of coronary covered stents with clickable elastin-like recombinamers

Coronary artery disease is the leading cause of death around the world. Endovascular stenting is the preferred treatment option to restore blood flow in the coronary arteries due to the lower perioperative morbidity when compared with more invasive treatment options. However, stent failure is still a major clinical problem, and further technological solutions are required to improve the performance of current stents. Here, we developed coronary stents covered with elastin-like recombinamers (ELRs) by exploiting a layer-by-layer technique combined with catalyst-free click chemistry. The resulting ELR-covered stents were intact after an in vitro simulated implantation procedure by balloon dilatation, which evidenced the elastic performance of the membrane. Additionally, the stents were mechanically stable under high flow conditions, which is in agreement with the covalent and stable nature of the click chemistry crosslinking strategy exploited during the ELR-membrane manufacturing and the successful embedding of the stent. Minimal platelet adhesion was detected after blood exposure in a Chandler loop as shown by scanning electron microscopy. The seeding of human endothelial progenitor cells (EPCs) on the ELR-membranes resulted in a confluent endothelial layer. These results prove the potential of this strategy to develop an advanced generation of coronary stents, with a stable and bioactive elastin-like membrane to exclude the atherosclerotic plaque from the blood stream or to seal coronary perforations and aneurysms, while providing a luminal surface with minimal platelet adhesion and favouring endothelialization.

Polyurethane End-Capped by Tetramethylpyrazine-Nitrone for Promoting Endothelialization Under Oxidative Stress.

Thrombus and restenosis are two main factors that cause the failure of vascular implants. Constructing a functional and confluent layer of endothelial cells (ECs) is considered an ideal method to prevent these problems. However, oxidative stress induced by the disease and implantation can damage ECs and hinder the endothelialization of implants. Thus, developing biomaterials that can protect ECs adhesion and proliferation from oxidative stress is urgently needed for the rapid endothelialization of vascular implants. In this work, a novel polyurethane (PU-TBN) is synthesized by employing tetramethylpyrazine-nitrone (TBN) as end-group to endow polymers with dual functions of antioxidant activity and promoting endothelialization. Common PU without TBN is also prepared to be control. Compared to PU, PU-TBN significantly promotes human umbilical vein endothelial cells (HUVECs) adhesion and proliferation, where cells spread well and a confluent endothelial layer is formed. PU-TBN also shows obvious free radical scavenging activity, and thus effectively attenuates oxidative stress to protect HUVECs from oxidative apoptosis. Moreover, PU-TBN exhibits enhanced antiplatelets effect, excellent biocompatibility, and similar mechanical properties to PU. These characteristics can endow PU-TBN with great potential to be used as vascular implants or coatings of other materials for rapid endothelialization under complex oxidative stress environment.

Direct writing alginate bioink inside pre-polymers of hydrogels to create patterned vascular networks

We describe a strategy to fabricate a hydrogel-based microvascular construct by direct writing alginate bioink inside the viscous pre-polymer of hydrogels, which acts as a support bath. As the print needle translates through the polymers, the extruded alginate instantaneously forms calcium alginate hydrogel (Ca-Alg) templates deposited within the bath. This phase change allows the formed templates to be anchored within the pre-polymers, while maintaining their structure. After the printing process, the pre-polymers are solidified to form a mechanically robust hydrogel. Finally, a hydrogel construct with embedded microchannels is generated by liquefying and removing the Ca-Alg templates. Using this method, not only the alginate ink alone can be directly printed within the engineered constructs, but also the size and shape of the formed microchannels are controllable. Furthermore, a confluent endothelial layer for the generation of vascular networks can be constructed by adhering and proliferating endothelial cells on the channel linings. This strategy demonstrates a promising technique for rapid construction of in vitro vasculatures, which would provide a versatile platform for a wide array of applications such as tissue engineering, organ-on-a-chip and drug screening.

Regulation of endothelial migration and proliferation by ephrin-A1

Endothelial migration and proliferation are fundamental processes in angiogenesis and wound healing of injured or inflamed vessels. The present study aimed to investigate the regulation of the Eph/ephrin-system during endothelial proliferation and the impact of the ligand ephrin-A1 on proliferation and migration of human umbilical venous (HUVEC) and arterial endothelial cells (HUAEC).Endothelial cells that underwent contact inhibition showed a massive induction of ephrin-A1. In contrast, an injury to a confluent endothelial layer, associated with induction of migration and proliferation, showed reduced ephrin-A1 levels. In addition, reducing ephrin-A1 expression by siRNA led to increased proliferation, whereas the overexpression of ephrin-A1 led to decreased proliferative activity. Due to the fact that wound healing is a combination of proliferation and migration, migration was investigated in detail. First, classical wound-healing assays showed increased wound closure in both ephrin-A1 silenced and overexpressing cells. Live-cell imaging enlightened the underlying differences. Silencing of ephrin-A1 led to a faster but more disorientated migration. In contrast, ephrin-A1 overexpression did not influence velocity of the cells, but the migration was more directed in comparison to the controls. Additional analysis of EphA2-silenced cells showed similar results in terms of proliferation and migration compared to ephrin-A1 silenced cells. Detailed analysis of EphA2 phosphorylation on ligand-dependent phospho-site (Y588) and autonomous activation site (S897) revealed a distinct phosphorylation pattern. Furthermore, the endothelial cells ceased to migrate when they came in contact with an ephrin-A1 coated surface. Using a baculoviral-mediated expression system, ephrin-A1 silencing and overexpression was shown to modulate the formation of focal adhesions. This implicates that ephrin-A1 is involved in changes of the actin cytoskeleton which explains the alterations in migratory actions, at least in part.In conclusion, ephrin-A1 expression is regulated by cellular density and is itself a critical determinant of endothelial proliferation. According to current knowledge, ephrin-A1 seems to be remarkably involved in elementary processes of endothelial migration like cellular polarization, migratory direction and speed. These data support the notion that ephrin-A1 plays a pivotal role in basal mechanisms of re-endothelialization.

Strategies and Techniques to Enhance theIn SituEndothelialization of Small-Diameter Biodegradable Polymeric Vascular Grafts

Due to the lack of success in small-diameter (<6 mm) prosthetic vascular grafts, a variety of strategies have evolved utilizing a tissue-engineering approach. Much of this work has focused on enhancing the endothelialization of these grafts. A healthy, confluent endothelial layer provides dynamic control over homeo-stasis, influencing and preventing thrombosis and smooth muscle cell proliferation that can lead to intimal hyperplasia. Strategies to improve endothelialization of biodegradable polymeric grafts have encompassed both chemical and physical modifications to graft surfaces, many focusing on the recruitment of endothelial and endothelial progenitor cells. This review aims to provide a compilation of current and developing strategies that utilize in situ endothelialization to improve vascular graft outcomes, providing a context for the future directions of vascular tissue-engineering strategies that do not require preprocedural cell seeding.

The influence of endothelial cells on the ECM composition of 3D engineered cardiovascular constructs

Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach to develop viable alternatives for autologous vascular grafts. Development of a functional, adherent, shear resisting endothelial cell (EC) layer is one of the major issues limiting the successful application of these tissue engineered grafts. The goal of the present study was to create a confluent EC layer on a rectangular 3D cardiovascular construct using human venous cells and to determine the influence of this layer on the extracellular matrix composition and mechanical properties of the constructs. Rectangular cardiovascular constructs were created by seeding myofibroblasts (MFs) on poly(glycolic acid) poly-4-hydroxybutyrate scaffolds using fibrin gel. After 3 or 4 weeks, ECs were seeded and co-cultured using EGM-2 medium for 2 or 1 week, respectively. A confluent EC layer could be created and maintained for up to 2 weeks. The EGM-2 medium lowered the collagen production by MFs, resulting in weaker constructs, especially in the 2 week cultured constructs. Co-culturing with ECs slightly reduced the collagen content, but had no additional affect on the mechanical performance. A confluent endothelial layer was created on 3D human cardiovascular constructs. The layer was co-cultured for 1 and 2 weeks. Although, the collagen production of the MFs was slightly lowered, co-culturing ECs for 1 week results in constructs with good mechanical properties and a confluent EC layer.

Development of a novel endothelial cell‐seeded endovascular stent for intracranial aneurysm therapy

The metallic stent has been widely used in endovascular treatment of intracranial aneurysms and arterial stenosis. Endothelialization at the neck of the aneurysm or stenotic lesion after stent deployment plays a pivotal role in preventing aneurysm recurrence, as well as local thrombus formation and restenosis. To deliver autologous endothelial cells and to promote the endothelialization on the luminal wall of the parent artery, we established an endothelial cell-seeded intracranial stent device. Endothelial cells were isolated from canine jugular vein and identified by FACS assay and immunohistochemistry. We demonstrated that the seeded endothelial cells formed a confluent endothelial layer on the stent's surface. After being brushed with 100 dyne/cm(2) of shear stress, we found that this endothelial layer remained intact for at least 48 h on the heparinized polymer coated stent, rather than the poly-lactic-acid coated stent (p < 0.05). The results suggest that an autologous endothelial cell-seeded stent may be a feasible and optimal tool for endothelial delivery during stenting and may overcome some limitations of the traditional bare stent in the treatment of intracranial aneurysms and arterial stenosis.

Addressing thrombogenicity in vascular graft construction

Thrombosis is a major cause of poor patency in synthetic vascular grafts for small diameter vessel (< 6 mm) bypass. Arteries have a host of structural mechanisms by which they prevent triggering of platelet activation and the clotting cascade. Many of these are present in vascular endothelial cells. These mechanisms act together with perpetual feedback at different levels, providing a constantly fine-tuned non-thrombogenic environment. The arterial wall anatomy also serves to promote thrombosis as a healing mechanism when it has been severely injured. Surface modification of synthetic graft surfaces to attenuate the coagulation cascade has reduced thrombosis levels and improved patency in vitro and in animal models. Success in this endeavor is critically dependent on the methods used to modify the surface. Platelets adhere to positively charged surfaces due to their own negative charge. They also preferentially attach to hydrophobic surfaces. Therefore synthetic graft development is concerned with hydrophilic materials with negative surface charge. However, fibrinogen has both hydrophilic and hydrophobic binding sites-amphiphilic materials reduce its adhesion and subsequent platelet activation. The self-endothelializing synthetic graft is an attractive proposition as a confluent endothelial layer incorporates many of the anti-thrombogenic properties of arteries. Surface modification to promote this has shown good results in animal models. The difficulties experienced in achieving spontaneous endothelialisation in humans have lead to the investigation of pre-implantation in vitro endothelial cell seeding. These approaches ultimately aim to result in novel synthetic grafts which are anti-thrombogenic and hence suitable for coronary and distal infrainguinal bypass.

Functional tissue-engineered blood vessels from bone marrow progenitor cells

Stem cells have significant potential for development of cell-based therapeutics for cardiovascular tissue regeneration. We developed a novel method for isolating smooth muscle cells (SMC) from ovine bone marrow using a tissue-specific promoter and fluorescence-activated cell sorting. As compared to vascular SMC, bone marrow-derived smooth muscle progenitor cells (BM-SMPC) exhibited similar morphology, showed higher proliferation potential and expressed several SMC markers including alpha-actin, calponin, myosin heavy chain, smoothelin, caldesmon and SM22. When embedded in fibrin hydrogels, BM-SMPC contracted the matrix and displayed receptor- and non-receptor-mediated contractility, indicating that BM-SMPC can generate force in response to vasoreactive agonists. We also prepared tissue-engineered blood vessels from BM-SMPC and BM-derived endothelial cells and implanted them into the jugular veins of lambs. As early as five weeks post-implantation, grafted tissues displayed a confluent endothelial layer overlaying the medial layer in which BM-SMPC were aligned circumferentially and synthesized significant amounts of collagen. In contrast to previous results with vascular SMC, BM-SMPC synthesized high amounts of elastin that was organized in a fibrillar network very similar to that of native vessels. Our results suggest that BM-SMPC may be useful in studying SMC differentiation and have high potential for development of cell therapies for the treatment of cardiovascular disease.

The effect of high-density-lipoprotein on thrombus formation on and endothelial cell attachement to biomaterial surfaces

Cardiovascular implants such as vascular grafts fail frequently because they lack genuine blood-compatibility. The blood-contacting surface should simultaneously prevent thrombus formation and promote formation of a confluent endothelial cell layer, to achieve sustained haemostasis. Contact activation and endothelialization are known to be determined by the plasma proteins which adsorb onto virtually all synthetic surfaces almost immediately upon contact with blood. A common approach in blood-compatibility research is, therefore, to use hydrophilic biomaterials, which are sometimes claimed to be “protein-repellent”. We report here that, for synthetic polymeric surfaces, hydrophilicity is by no means synonymous to protein-repellency. We discovered that significant amounts of proteins, especially high-density lipoprotein, adsorb to hydrophilic surfaces. Pre-incubation of hydrophilic synthetic surfaces with high-density lipoprotein provides a blood–biomaterial interface, which inhibits thrombin generation and subsequent thrombus formation, and also accommodates overgrowth with a confluent endothelial layer. This approach may open the way to truly functional small-caliber arterial prostheses, and may also be relevant to cardiovascular tissue engineering in which de novo vascular tissues are cultured on or within a biomaterial scaffold.