Development of in vitro endothelialized drug-eluting stent using human peripheral blood-derived endothelial progenitor cells.

We propose in vitro endothelialization of drug-eluting stents (DES) to overcome late stent thrombosis by directly introducing late-outgrowth human endothelial progenitor cells (EPCs) at the target site utilizing abluminal DES. Isolated EPCs were confirmed as late-outgrowth EPCs by flow cytometric analysis. Abluminally paclitaxel-loaded stents were seeded with different cell concentrations and durations to determine optimal seeding conditions, in both uncrimped and crimped configurations. The seeding yield was determined by evaluating the percent coverage of the stent struts' area. The EPC-seeded DES were exposed to arterial shear stress to evaluate the effect of high shear stress on EPCs. To investigate how much paclitaxel elutes during the seeding procedure, a pharmacokinetic analysis was performed. Finally, to validate the proof of concept, EPC-seeded DES were placed on a fibrin matrix with and without smooth muscle cells (SMCs) and cultured for 3 days under perfusion. The seeding procedure resulted in 47% and 26% coverage of the stent surface in uncrimped and crimped conditions, respectively. After the optimal seeding, almost 99% of drug was still available. When EPC-seeded DES were placed on a fibrin matrix and cultured for 3 days, the EPCs confluently covered the stent surface and spread to the surrounding fibrin gel. When EPC-seeded DES were placed on SMC-containing fibrin layers, cells in contact with the struts died. EPCs can be successfully seeded onto DES without losing drug-eluting capability, and EPCs exhibit sufficient proliferative ability. EPC-seeded DES may combine early re-endothelialization ability with the antirestenotic effectiveness of DES.

Small Caliber Compliant Vascular Grafts Based on Elastin-Like Recombinamers for in situ Tissue Engineering.

Vascular disease is a leading cause of death worldwide, but surgical options are restricted by the limited availability of autologous vessels, and the suboptimal performance of prosthetic vascular grafts. This is especially evident for coronary artery by-pass grafts, whose small caliber is associated with a high occlusion propensity. Despite the potential of tissue-engineered grafts, compliance mismatch, dilatation, thrombus formation, and the lack of functional elastin are still major limitations leading to graft failure. This calls for advanced materials and fabrication schemes to achieve improved control on the grafts' properties and performance. Here, bioinspired materials and technical textile components are combined to create biohybrid cell-free implants for endogenous tissue regeneration. Clickable elastin-like recombinamers are processed to form an open macroporous 3D architecture to favor cell ingrowth, while being endowed with the non-thrombogenicity and the elastic behavior of the native elastin. The textile components (i.e., warp-knitted and electrospun meshes) are designed to confer suture retention, long-term structural stability, burst strength, and compliance. Notably, by controlling the electrospun layer's thickness, the compliance can be modulated over a wide range of values encompassing those of native vessels. The grafts support cell ingrowth, extracellular matrix deposition and endothelium development in vitro. Overall, the fabrication strategy results in promising off-the-shelf hemocompatible vascular implants for in situ tissue engineering by addressing the known limitations of bioartificial vessel substitutes.

MR and PET-CT monitoring of tissue-engineered vascular grafts in the ovine carotid artery.

The modification of biomaterials to comply with clinically employed monitoring techniques is a promising strategy to support clinical translation in regenerative medicine. Here, multimodal imaging of tissue-engineered vascular grafts (TEVG) was enabled by functionalizing the textile scaffold with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The resulting MR-imageable grafts (iTEVG) were monitored non-invasively throughout their whole life-cycle, from initial quality control to longitudinal functional evaluation in an ovine model for up to 8 weeks. Crucial features such as the complete embedding of the textile mesh in the developing tissue and the grafts' structural stability were assessed in vitro using 1T-, 3T- and 7T-MRI scanners. In vivo, the grafts were imaged by 3T-MRI and PET-CT. Contrary to unlabeled constructs, iTEVG could be delineated from native arteries and precisely localized by MRI. USPIO labeling neither induced calcifications, nor negatively affected their remodeling with respect to tissue-specific extracellular matrix composition and endothelialization. Functionality was confirmed by MR-angiography. 18F-FDG uptake (assessed via PET-CT) indicated only transient post-surgical inflammation. In conclusion, USPIO-labeling enables accurate localization of TEVG and opens up opportunities for multimodal imaging approaches to assess transplant acceptance and function. Thereby, it can support clinical decision-making on the need for further pharmacological or surgical interventions.

Biologically Inspired Scaffolds for Heart Valve Tissue Engineering via Melt Electrowriting.

Heart valves are characterized to be highly flexible yet tough, and exhibit complex deformation characteristics such as nonlinearity, anisotropy, and viscoelasticity, which are, at best, only partially recapitulated in scaffolds for heart valve tissue engineering (HVTE). These biomechanical features are dictated by the structural properties and microarchitecture of the major tissue constituents, in particular collagen fibers. In this study, the unique capabilities of melt electrowriting (MEW) are exploited to create functional scaffolds with highly controlled fibrous microarchitectures mimicking the wavy nature of the collagen fibers and their load-dependent recruitment. Scaffolds with precisely-defined serpentine architectures reproduce the J-shaped strain stiffening, anisotropic and viscoelastic behavior of native heart valve leaflets, as demonstrated by quasistatic and dynamic mechanical characterization. They also support the growth of human vascular smooth muscle cells seeded both directly or encapsulated in fibrin, and promote the deposition of valvular extracellular matrix components. Finally, proof-of-principle MEW trileaflet valves display excellent acute hydrodynamic performance under aortic physiological conditions in a custom-made flow loop. The convergence of MEW and a biomimetic design approach enables a new paradigm for the manufacturing of scaffolds with highly controlled microarchitectures, biocompatibility, and stringent nonlinear and anisotropic mechanical properties required for HVTE.

Bio-Based Covered Stents: The Potential of Biologically Derived Membranes.

IMPACT STATEMENT: The use of bio-based materials (i.e., biologically derived materials that have either a biological origin, including engineered tissues, or a bio-inspired chemical composition) offers the potential to obtain covered stents (CS) with superior performance with respect to the currently available ones, which employ synthetic materials. This will advance and expand the clinical applicability of CS not only in the cardiovascular field but also for the treatment of other target areas such as segments of the respiratory, gastrointestinal, biliary, and urinary tracts.

Combining Catalyst-Free Click Chemistry with Coaxial Electrospinning to Obtain Long-Term, Water-Stable, Bioactive Elastin-Like Fibers for Tissue Engineering Applications.

Elastic fibers are a fundamental requirement for tissue-engineered equivalents of physiologically elastic native tissues. Here, a simple one-step electrospinning approach is developed, combining i) catalyst-free click chemistry, ii) coaxial electrospinning, and iii) recombinant elastin-like polymers as a relevant class of biomaterials. Water-stable elastin-like fibers are obtained without the use of cross-linking agents, catalysts, or harmful organic solvents. The fibers can be directly exposed to an aqueous environment at physiological temperature and their morphology maintained for at least 3 months. The bioactivity of the fibers is demonstrated with human vascular cells and the potential of the process for vascular tissue engineering is shown by fabricating small-diameter tubular fibrous scaffolds. Moreover, highly porous fluffy 3D constructs are obtained without the use of specially designed collectors or sacrificial materials, further supporting their applicability in the biomedical field. Ultimately, the strategy that is developed here may be applied to other click systems, contributing to expanding their potential in medical technology.

Macroporous click-elastin-like hydrogels for tissue engineering applications.

Elastin is a key extracellular matrix (ECM) protein that imparts functional elasticity to tissues and therefore an attractive candidate for bioengineering materials. Genetically engineered elastin-like recombinamers (ELRs) maintain inherent properties of the natural elastin (e.g. elastic behavior, bioactivity, low thrombogenicity, inverse temperature transition) while featuring precisely controlled composition, the possibility for biofunctionalization and non-animal origin. Recently the chemical modification of ELRs to enable their crosslinking via a catalyst-free click chemistry reaction, has further widened their applicability for tissue engineering. Despite these outstanding properties, the generation of macroporous click-ELR scaffolds with controlled, interconnected porosity has remained elusive so far. This significantly limits the potential of these materials as the porosity has a crucial role on cell infiltration, proliferation and ECM formation. In this study we propose a strategy to overcome this issue by adapting the salt leaching/gas foaming technique to click-ELRs. As result, macroporous hydrogels with tuned pore size and mechanical properties in the range of many native tissues were reproducibly obtained as demonstrated by rheological measurements and quantitative analysis of fluorescence, scanning electron and two-photon microscopy images. Additionally, the appropriate size and interconnectivity of the pores enabled smooth muscle cells to migrate into the click-ELR scaffolds and deposit extracellular matrix. The macroporous structure together with the elastic performance and bioactive character of ELRs, the specificity and non-toxic character of the catalyst-free click-chemistry reaction, make these scaffolds promising candidates for applications in tissue regeneration. This work expands the potential use of ELRs and click chemistry systems in general in different biomedical fields.

VascuTrainer: A Mobile and Disposable Bioreactor System for the Conditioning of Tissue-Engineered Vascular Grafts.

In vitro tissue engineering of vascular grafts requires dynamic conditioning in a bioreactor system for in vitro tissue maturation and remodeling to receive a mechanically adequate and hemocompatible implant. The goal of the current work was to develop a bioreactor system for the conditioning of vascular grafts which is (i) able to create a wide range of flow, pressure and frequency conditions, including physiological ones; (ii) compact and easy to assemble; (iii) transportable; (iv) disposable. The system is driven by a small centrifugal pump controlled via a custom-made control unit, which can also be operated on batteries to allow for autonomous transportation. To show the potential of the newly developed bioreactor system small-caliber vascular composite grafts (n = 5, internal diameter = 3 mm, length = 12.5 cm) were fabricated using a fibrin scaffold embedding human umbilical artery smooth muscle cells and a polyvinylidene fluoride warp-knitted macroporous mesh. Subsequently, the vascular grafts were endothelialized and mounted in the bioreactor system for conditioning. The conditioning parameters remained within the predefined range over the complete conditioning period and during operation on batteries as tested for up to 25 h. Fabrication and pre-conditioning under arterial pressure and shear stress conditions resulted in robust and hemocompatible tissue-engineered vascular grafts. Analysis of immunohistochemical stainings against extracellular matrix and cell-specific proteins revealed collagen I and collagen III deposition. The luminal surface was confluently covered with endothelial cells. The developed bioreactor system showed cytocompatibility and pH, pO2, pCO2, glucose and lactate stayed constant. Sterility was maintained during the complete fabrication process of the vascular grafts. The potential of a versatile and mobile system and its functionality by conditioning tissue-engineered vascular grafts under physiological pressure and flow conditions could be demonstrated.

In Vitro Quantification of Luminal Denudation After Crimping and Balloon Dilatation of Endothelialized Covered Stents.

PURPOSE: Covered stents have been demonstrated to reduce restenosis; however, the membrane's limited biocompatibility can still lead to thrombus formation. To obtain optimal surface hemocompatibility, endothelialization of the luminal surface has been proposed. However, the effect of delivery procedures, such as crimping and balloon dilatation, on the endothelial layer has not been quantified. This study investigated the impact of such procedures on endothelialized covered stents in vitro. METHODS: Using an injection molding technique, bare metal stents were covered with fibrin subsequently, endothelialized and conditioned in a bioreactor under arterial pressure (80-120 mmHg) and shear stress (1 Pa). For each set of experiments, three covered stents were prepared, one being subjected to crimping alone, one to crimping followed by balloon dilatation and one serving as control. The experiment was repeated three times. The endothelial coverage was quantified by scanning electron microscopy (SEM). The functionality of the endothelium after exposure to platelet-rich plasma was evaluated by immunohistochemistry and SEM. RESULTS: The mean endothelial coverage of control, crimped, crimped and balloon-dilated stents was 87.6, 80.1 and 52.1%, respectively, indicating that endothelial cells detached significantly not after crimping (P = 0.465) but following balloon dilatation (P < 0.001). The cells present on the stent's surface, either after crimping or crimping followed by balloon dilatation, expressed eNOS and CD31 and exhibited no platelet adhesion. CONCLUSION: The simulated delivery procedure resulted in the retention of viable cells on more than half of the luminal surface. The main damage to the layer occurred during balloon dilatation.

Tissue-Engineered Fibrin-Based Heart Valve with Bio-Inspired Textile Reinforcement.

The mechanical properties of tissue-engineered heart valves still need to be improved to enable their implantation in the systemic circulation. The aim of this study is to develop a tissue-engineered valve for the aortic position - the BioTexValve - by exploiting a bio-inspired composite textile scaffold to confer native-like mechanical strength and anisotropy to the leaflets. This is achieved by multifilament fibers arranged similarly to the collagen bundles in the native aortic leaflet, fixed by a thin electrospun layer directly deposited on the pattern. The textile-based leaflets are positioned into a 3D mould where the components to form a fibrin gel containing human vascular smooth muscle cells are introduced. Upon fibrin polymerization, a complete valve is obtained. After 21 d of maturation by static and dynamic stimulation in a custom-made bioreactor, the valve shows excellent functionality under aortic pressure and flow conditions, as demonstrated by hydrodynamic tests performed according to ISO standards in a mock circulation system. The leaflets possess remarkable burst strength (1086 mmHg) while remaining pliable; pronounced extracellular matrix production is revealed by immunohistochemistry and biochemical assay. This study demonstrates the potential of bio-inspired textile-reinforcement for the fabrication of functional tissue-engineered heart valves for the aortic position.

Bioengineered vascular constructs as living models for in vitro cardiovascular research.

Cardiovascular diseases represent the most common cause of morbidity and mortality worldwide. In this review, we explore the potential of bioengineered vascular constructs as living models for in vitro cardiovascular research to advance the current knowledge of pathophysiological processes and support the development of clinical therapies. Bioengineered vascular constructs capable of recapitulating the cellular and mechanical environment of native vessels represent a valuable platform to study cellular interactions and signaling cascades, test drugs and medical devices under (patho)physiological conditions, with the additional potential benefit of reducing the number of animals required for preclinical testing.

Elastin-like recombinamer-covered stents: Towards a fully biocompatible and non-thrombogenic device for cardiovascular diseases.

We explored the use of recently developed gels obtained by the catalyst free click reaction of elastin-like recombinamers (ELRs) to fabricate a new class of covered stents. The approach consists in embedding bare metal stents in the ELR gels by injection molding, followed by endothelialization under dynamic pressure and flow conditions in a bioreactor. The mechanical properties of the gels could be easily tuned by choosing the adequate concentration of the ELR components and their biofunctionality could be tailored by inserting specific sequences (RGD and REDV). The ELR-covered stents exhibited mechanical stability under high flow conditions and could undergo crimping and deployment without damage. The presence of RGD in the ELR used to cover the stent supported full endothelialization in less than 2weeks in vitro. Minimal platelet adhesion and fibrin adsorption were detected after exposure to blood, as shown by immunostaining and scanning electron microscopy. These results prove the potential of this approach towards a new and more effective generation of covered stents which exclude the atherosclerotic plaque from the blood stream and have high biocompatibility, physiological hemocompatibility and reduced response of the immune system.

USPIO-labeled textile materials for non-invasive MR imaging of tissue-engineered vascular grafts.

Non-invasive imaging might assist in the clinical translation of tissue-engineered vascular grafts (TEVG). It can e.g. be used to facilitate the implantation of TEVG, to longitudinally monitor their localization and function, and to provide non-invasive and quantitative feedback on their remodeling and resorption. We here incorporated ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles into polyvinylidene fluoride (PVDF)-based textile fibers, and used them to prepare imageable tissue-engineered vascular grafts (iTEVG). The USPIO-labeled scaffold materials were molded with a mixture of fibrin, fibroblasts and smooth muscle cells, and then endothelialized in a bioreactor under physiological flow conditions. The resulting grafts could be sensitively detected using T1-, T2- and T2*-weighted MRI, both during bioreactor cultivation and upon surgical implantation into sheep, in which they were used as an arteriovenous shunt between the carotid artery and the jugular vein. In vivo, the iTEVG were shown to be biocompatible and functional. Post-mortem ex vivo analyses provided evidence for efficient endothelialization and for endogenous neo-vascularization within the biohybrid vessel wall. These findings show that labeling polymer-based textile materials with MR contrast agents is straightforward and safe, and they indicate that such theranostic tissue engineering approaches might be highly useful for improving the production, performance, personalization and translation of biohybrid vascular grafts.