Tissue-engineered Vascular Grafts Research Articles

Challenges and Possibilities of Cell-Based Tissue-Engineered Vascular Grafts.

There is urgent demand for biologically compatible vascular grafts for both adult and pediatric patients. The utility of conventional nonbiodegradable materials is limited because of their thrombogenicity and inability to grow, while autologous vascular grafts involve considerable disadvantages, including the invasive procedures required to obtain these healthy vessels from patients and insufficient availability in patients with systemic atherosclerosis. All of these issues could be overcome by tissue-engineered vascular grafts (TEVGs). A large body of evidence has recently emerged in support of TEVG technologies, introducing diverse cell sources (e.g., somatic cells and stem cells) and novel fabrication methods (e.g., scaffold-guided and self-assembled approaches). Before TEVG can be applied in a clinical setting, however, several aspects of the technology must be improved, such as the feasibility of obtaining cells, their biocompatibility and mechanical properties, and the time needed for fabrication, while the safety of supplemented materials, the patency and nonthrombogenicity of TEVGs, their growth potential, and the long-term influence of implanted TEVGs in the body must be assessed. Although recent advances in TEVG fabrication have yielded promising results, more research is needed to achieve the most feasible methods for generating optimal TEVGs. This article reviews multiple aspects of TEVG fabrication, including mechanical requirements, extracellular matrix components, cell sources, and tissue engineering approaches. The potential of periodic hydrostatic pressurization in the production of scaffold-free TEVGs with optimal elasticity and stiffness is also discussed. In the future, the integration of multiple technologies is expected to enable improved TEVG performance.

Cell-assembled extracellular matrix (CAM) sheet production: Translation from using human to large animal cells

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures.

Fibrous scaffold with a tunable nonlinear elasticity

Micro/nanofibers have been widely applied to mimic the fibrillary structure of collagen type I from a bionic point of view, however, the wavy structure of collagen fibers in biological tissues such as arteries and ligaments has hardly been fabricated. In this study, a universal method was developed to fabricate the crimped fibers of different biopolymers by controlling the relaxation of residual stress, allowing the resulting scaffold a stable nonlinear elasticity. The effects of the processing conditions such as pre-strain, temperature of plasticizer, and fixed contraction length on the crimped fiber structure have been investigated. The results suggested that an engineered nonlinear elasticity of the scaffolds can be tailored by tuning the crimped structure. Human umbilical vein endothelial cells (HUVECs) cultured on the mat with crimped fibers showed promoted cell adhesion and cell spreading. Crimped fibers might have a great potential for repairing tendons and soft connective tissue and for constructing of tissue-engineering vascular grafts.

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach-the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis-are discussed in detail.

Silk fibroin vascular graft: a promising tissue-engineered scaffold material for abdominal venous system replacement

No alternative tissue-engineered vascular grafts for the abdominal venous system are reported. The present study focused on the development of new tissue-engineered vascular graft using a silk-based scaffold material for abdominal venous system replacement. A rat vein, the inferior vena cava, was replaced by a silk fibroin (SF, a biocompatible natural insoluble protein present in silk thread), tissue-engineered vascular graft (10 mm long, 3 mm diameter, n = 19, SF group). The 1 and 4 -week patency rates and histologic reactions were compared with those of expanded polytetrafluoroethylene vascular grafts (n = 10, ePTFE group). The patency rate at 1 and 4 weeks after replacement in the SF group was 100.0% and 94.7%, and that in the ePTFE group was 100.0% and 80.0%, respectively. There was no significant difference between groups (p = 0.36). Unlike the ePTFE graft, CD31-positive endothelial cells covered the whole luminal surface of the SF vascular graft at 4 weeks, indicating better endothelialization. SF vascular grafts may be a promising tissue-engineered scaffold material for abdominal venous system replacement.

Human iPS Cell-derived Tissue Engineered Vascular Graft: Recent Advances and Future Directions.

Tissue engineered vascular grafts (TEVGs) generated from human primary cells represent a promising vascular interventional therapy. However, generation and application of these TEVGs may be significantly hindered by the limited accessibility, finite expandability, donor-donor functional variation and immune-incompatibility of primary seed cells from donors. Alternatively, human induced pluripotent stem cells (hiPSCs) offer an infinite source to obtain functional vascular cells in large quantity and comparable quality for TEVG construction. To date, TEVGs (hiPSC-TEVGs) with significant mechanical strength and implantability have been generated using hiPSC-derived seed cells. Despite being in its incipient stage, this emerging field of hiPSC-TEVG research has achieved significant progress and presented promising future potential. Meanwhile, a series of challenges pertaining hiPSC differentiation, vascular tissue engineering technologies and future production and application await to be addressed. Herein, we have composed this review to introduce progress in TEVG generation using hiPSCs, summarize the current major challenges, and encapsulate the future directions of research on hiPSC-based TEVGs. Graphical abstract.

Decellularization of porcine carotid arteries using low-concentration sodium dodecyl sulfate.

The decellularized scaffold is a promising material for producing tissue-engineered vascular grafts (TEVGs) because of its complex, native-like three-dimensional structure and mechanical properties. Sodium dodecyl sulfate (SDS), one of the most commonly used decellularization reagents, appears to be more effective than other detergents for removing cells from dense tissues. The concentrations of SDS used in previous studies and their effects on decellularization are not consistent. In this study, porcine carotid arteries were decellularized using detergent-based protocols using Triton X-100 followed by SDS at different concentrations and exposing time. Cell removal efficiency and composition were evaluated by histological analysis, and DNA and collagen quantification. Ultrastructure, mechanical properties, pore size distribution, and in vivo biocompatibility of decellularized arteries were also evaluated. The DNA content of decellularized scaffolds treated with 0.3% SDS for 72 h or 0.5% SDS for 48 h was significantly less than that treated with 1% SDS for 30 h. There was a significant loss of soluble collagen after treatment with 1% SDS relative to native arteries. The extensive loss of elastin and glycosaminoglycans was observed in decellularized arteries treated with 0.5% SDS or 1% SDS. The basement membrane and biomechanics were also damaged by these two protocols. Moreover, decellularized scaffolds became more porous with many large pores after treatment with 0.3% SDS. Low-concentration SDS could be a suitable choice for artery decellularization. Decellularized porcine carotid arteries, prepared using Triton X-100 followed by 0.3% SDS, may be a promising biological scaffold for TEVGs.

Efficient Differentiation of Human Induced Pluripotent Stem Cells into Endothelial Cells under Xenogeneic-free Conditions for Vascular Tissue Engineering

Tissue engineered vascular grafts (TEVGs) represent a promising therapeutic option for emergency vascular intervention. Although the application of small-diameter TEVGs using patient-specific primary endothelial cells (ECs) to prevent thrombosis and occlusion prior to implantation could be hindered by the long time course required for in vitro endothelialization, human induced pluripotent stem cells (hiPSCs) provide a robust source to derive immunocompatible ECs (hiPSC-ECs) for immediate TEVG endothelialization. To achieve clinical application, hiPSC-ECs should be derived under culture conditions without the use of animal-derived reagents (xenogeneic-free conditions), to avoid unwanted host immune responses from xenogeneic reagents. However, a completely xenogeneic-free method of hiPSC-EC generation has not previously been established. Herein, we substituted animal-derived reagents used in a standard method of xenogeneic hiPSC-EC differentiation with functional counterparts of human origin. As a result, we generated xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) with similar marker expression and function to those of human primary ECs. Furthermore, XF-hiPSC-ECs functionally responded to shear stress with typical cell alignment and gene expression. Finally, we successfully endothelialized decellularized human vessels with XF-hiPSC-ECs in a dynamic bioreactor system. In conclusion, we developed xenogeneic-free conditions for generating functional hiPSC-ECs suitable for vascular tissue engineering, which will further move TEVG therapy toward clinical application.

Cyborg Vessel

Cyborg Vessel

Porcine carotid arteries decellularized with a suitable concentration combination of Triton X-100 and sodium dodecyl sulfate for tissue engineering vascular grafts.

Tissue engineering vascular grafts (TEVGs) constructed by decellularized arteries have the potential to replace autologous blood vessels in bypass surgery for patients with cardiovascular disease. There are various methods of decellularization without a standard protocol. Detergents approaches are simple, and easy control of experimental conditions. Non-ionic detergent Triton X-100 and ionic detergent sodium dodecyl sulfate (SDS) are the most commonly used detergents. In this study, we used Triton X-100 and SDS with different concentrations to decellularize porcine carotid arteries. After that, we investigated the acellular effect and mechanical properties of decellularized arteries to find a promising concentration combination for decellularization. Results showed that any detergents' combination would damage the inherent structure of extracellular matrix, and the destruction increased with the increase of detergents' concentration. We concluded that the decellularization approach of 0.5% Triton X-100 for 24h combined with 0.25% SDS for 72h could help to obtain decellularized arteries with minimum destruction. This protocol may be able to prepare a clinically suitable vascular scaffold for TEVGs.

Xenogeneic-free generation of vascular smooth muscle cells from human induced pluripotent stem cells for vascular tissue engineering

Development of mechanically advanced tissue-engineered vascular grafts (TEVGs) from human induced pluripotent stem cell (hiPSC)-derived vascular smooth muscle cells (hiPSC-VSMCs) offers an innovative approach to replace or bypass diseased blood vessels. To move current hiPSC-TEVGs toward clinical application, it is essential to obtain hiPSC-VSMC-derived tissues under xenogeneic-free conditions, meaning without the use of any animal-derived reagents. Many approaches in VSMC differentiation of hiPSCs have been reported, although a xenogeneic-free method for generating hiPSC-VSMCs suitable for vascular tissue engineering has yet to be established. Based on our previously established standard method of xenogeneic VSMC differentiation, we have replaced all animal-derived reagents with functional counterparts of human origin and successfully derived functional xenogeneic-free hiPSC-VSMCs (XF-hiPSC-VSMCs). Next, our group developed tissue rings via cellular self-assembly from XF-hiPSC-VSMCs, which exhibited comparable mechanical strength to those developed from xenogeneic hiPSC-VSMCs. Moreover, by seeding XF-hiPSC-VSMCs onto biodegradable polyglycolic acid (PGA) scaffolds, we generated engineered vascular tissues presenting effective collagen deposition which were suitable for implantation into an immunodeficient mice model. In conclusion, our xenogeneic-free conditions for generating hiPSC-VSMCs produce cells with the comparable capacity for vascular tissue engineering as standard xenogeneic protocols, thereby moving the hiPSC-TEVG technology one step closer to safe and efficacious clinical translation.

Development of a Semi-Automated, Bulk Seeding Device for Large Animal Model Implantation of Tissue Engineered Vascular Grafts.

Vascular tissue engineering is a field of regenerative medicine that restores tissue function to defective sections of the vascular network by bypass or replacement with a tubular, engineered graft. The tissue engineered vascular graft (TEVG) is comprised of a biodegradable scaffold, often combined with cells to prevent acute thrombosis and initiate scaffold remodeling. Cells are most effectively incorporated into scaffolds using bulk seeding techniques. While our group has been successful in uniform, rapid, bulk cell seeding of scaffolds for TEVG testing in small animals using our well-validated rotational vacuum technology, this approach was not directly translatable to large scaffolds, such as those required for large animal testing or human implants. The objective of this study was to develop and validate a semi-automated cell seeding device that allows for uniform, rapid, bulk seeding of large scaffolds for the fabrication of TEVGs appropriately sized for testing in large animals and eventual translation to humans. Validation of our device revealed successful seeding of cells throughout the length of our tubular scaffolds with homogenous longitudinal and circumferential cell distribution. To demonstrate the utility of this device, we implanted a cell seeded scaffold as a carotid interposition graft in a sheep model for 10 weeks. Graft remodeling was demonstrated upon explant analysis using histological staining and mechanical characterization. We conclude from this work that our semi-automated, rotational vacuum seeding device can successfully seed porous tubular scaffolds suitable for implantation in large animals and provides a platform that can be readily adapted for eventual human use.

Tissue Engineered Vascular Graft Recipient Interleukin 10 Status Is Critical for Preventing Thrombosis.

Tissue engineered vascular grafts (TEVGs) are a promising technology, but are hindered by occlusion. Seeding with bone-marrow derived mononuclear cells (BM-MNCs) mitigates occlusion, yet the precise mechanism remains unclear. Seeded cells disappear quickly and potentially mediate an anti-inflammatory effect through paracrine signaling. Here, a series of reciprocal genetic TEVG implantations plus recombinant protein treatment is reported to investigate what role interleukin-10, an anti-inflammatory cytokine, plays from both host and seeded cells. TEVGs seeded with BM-MNCs from wild-type and IL-10 KO mice, plus unseeded grafts, are implanted into wild-type and IL-10 KO mice. Wild-type mice with unseeded grafts also receive recombinant IL-10. Serial ultrasound evaluates occlusion and TEVGs are harvested at 14 d for immunohistochemical analysis. TEVGs in IL-10 KO mice have significantly higher occlusion incidence compared to wild-type mice attributed to acute (<3 d) thrombosis. Cell seeding rescues TEVGs in IL-10 KO mice comparable to wild-type patency. IL-10 from the host and seeded cells do not significantly influence graft inflammation and macrophage phenotype, yet IL-10 treatment shows interesting biologic effects including decreasing cell proliferation and increasing M2 macrophage polarization. IL-10 from the host is critical for preventing TEVG thrombosis and seeded BM-MNCs exert a significant anti-thrombotic effect in IL-10 KO mice.

Effects of Braiding Parameters on Tissue Engineered Vascular Graft Development.

Tissue engineered vascular grafts (TEVGs) using scaffolds fabricated from braided poly(glycolic acid) (PGA) fibers coated with poly(glycerol sebacate) (PGS) are developed. The approach relies on in vivo tissue engineering by which neotissue forms solely within the body after a scaffold has been implanted. Herein, the impact of altering scaffold braid design and scaffold coating on neotissue formation is investigated. Several combinations of braiding parameters are manufactured and evaluated in a Beige mouse model in the infrarenal abdominal aorta. Animals are followed with 4D ultrasound analysis, and 12 week explanted vessels are evaluated for biaxial mechanical properties as well as histological composition. Results show that scaffold parameters (i.e., braiding angle, braiding density, and presence of a PGS coating) have interdependent effects on the resulting graft performance, namely, alteration of these parameters influences levels of inflammation, extracellular matrix production, graft dilation, neovessel distensibility, and overall survival. Coupling carefully designed in vivo experimentation with regression analysis, critical relationships between the scaffold design and the resulting neotissue that enable induction of favorable cellular and extracellular composition in a controlled manner are uncovered. Such an approach provides a potential for fabricating scaffolds with a broad range of features and the potential to manufacture optimized TEVGs.

Electronic Blood Vessel

Summary Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface.

Improvement of a Novel Small-diameter Tissue-engineered Arterial Graft With Heparin Conjugation

Small diameter (<6 mm), bioabsorbable, arterial, tissue-engineered vascular grafts (TEVGs) remain limited by thromboembolism. The objective of this study was to test whether heparin-eluting (HE) TEVGs prevent early thrombosis in a large animal model. TEVGs were created with an outer poly-ε-caprolactone electrospun nanofiber layer, with a 15-μm average pore size and an inner layer composed of a 50:50 poly(L-lactide-co-ε-caprolactone) copolymer. Adult female sheep (n= 5) underwent bilateral carotid artery interposition grafting, with a control TEVG in 1 carotid artery and an HE TEVG in the contralateral position. Animals were followed for 8 weeks with weekly Duplex ultrasonography to monitor TEVG performance. All sheep survived to the designated endpoint. At 8 weeks all 5 HE TEVGs were patent. Three of 5 control TEVGs had early thrombotic occlusion at <1 week. More than 97% of heparin release occurred within the first 24 hours. Histologic evaluation of the HE TEVG displayed cellularity like a native carotid artery with no evidence of calcification. Significantly fewer platelets adhered to the HE TEVG than to the control TEVG (P < .001). This study suggests HE TEVGs prevent acute graft thrombosis. We hypothesize that the HE properties of the HE TEVG during vascular endothelialization is useful for maintaining TEVG patency. This technique may aid in the translation of small arterial TEVGs to the clinic.

Advancing tissue-engineered vascular grafts via their endothelialization and mechanical conditioning.

Tissue engineering has garnered significant attention for its potential to address the predominant modes of failure of small diameter vascular prostheses, namely mid-graft thrombosis and anastomotic intimal hyperplasia. In this review, we described two main features underpinning the promise of tissue-engineered vascular grafts: the incorporation of an antithrombogenic endothelium, and the generation of a structurally and biomechanically mimetic extracellular matrix. From the early attempts at the in-vitro endothelialization of vascular prostheses in the 1970s through to the ongoing clinical trials of fully tissue-engineered vascular grafts, the historical advancements and unresolved challenges that characterize the current state-of-the-art are summarized in a manner that establishes a guide for the development of an effective vascular prosthesis for small diameter arterial reconstruction. The importance of endothelial cell purity and their arterial specification for the prevention of both diffuse neointimal hyperplasia and the accelerated development of atherosclerotic lesions is delineated. Additionally, the need for an extracellular matrix that recapitulates both the composition and structure of native elastic arteries to facilitate the protracted stability and patency of an engineered vasoactive conduit is described. Finally, the capacity of alternative sources of cells and mechanical conditioning to overcome these technical barriers to the clinical translation of an effective small diameter vascular prosthesis is discussed. In conclusion, this review provides an overview of the historical development of tissue-engineered vascular grafts, highlighting specific areas warranting further research, and commentating on the outlook of a clinically feasible and therapeutically efficacious vascular prosthesis for small diameter arterial reconstruction.

Development and in Vivo Evaluation of a Biodegradable Vascular Graft Reinforced with a Fused PCL Filament

Tissue-engineered vascular grafts developed for cardiovascular surgery must be fully functional after implantation, resistant to all types of kinks, compression and burst pressure. One of the approaches to improve mechanical properties includes the reinforcement with an outer polymeric sheath. This study was aimed at evaluating the mechanical properties of a tissue-engineered vascular graft reinforced with a fused PCL filament and in vivo testing in a carotid artery of sheep within one year.

Fabrication of blood-derived elastogenic vascular grafts using electrospun fibrinogen and polycaprolactone composite scaffolds for paediatric applications.

The development of tissue-engineered vascular grafts (TEVGs) for paediatric applications must consider unique factors associated with this patient cohort. Although the increased elastogenic potential of neonatal cells offers an opportunity to overcome the long-standing challenge of in vitro elastogenesis, neonatal patients have a lower tolerance for autologous tissue harvest and require grafts that exhibit growth potential. The purpose of this study was to apply a multipronged strategy to promote elastogenesis in conjunction with umbilical cord-derived materials in the production of a functional paediatric TEVG. An initial proof-of-concept study was performed to extract fibrinogen from human umbilical cord blood samples and, through electrospinning, to produce a nanofibrous fibrinogen scaffold. This scaffold was seeded with human umbilical artery-derived smooth muscle cells (hUASMCs), and neotissue formation within the scaffold was examined using immunofluorescence microscopy. Subsequently, a polycaprolactone-reinforced porcine blood-derived fibrinogen scaffold (isolated using the same protocol as cord blood fibrinogen) was used to develop a rolled-sheet graft that employed topographical and biochemical guidance cues to promote elastogenesis and cellular orientation. This approach resulted in a TEVG with robust mechanical properties and biomimetic arrangement of extracellular matrix (ECM) with rich expression of elastic fibre-related proteins. The results of this study hold promise for further development of paediatric TEVGs and the exploration of the effects of scaffold microstructure and nanostructure on vascular cell function and ECM production.

A new method for the preparation of three-layer vascular stents: a preliminary study on the preparation of biomimetic three-layer vascular stents using a three-stage electrospun membrane

There is an urgent need to design a tissue-engineered vascular graft that exhibits good biocompatibility and sufficient mechanical strength to repair and facilitate regeneration of defective vascular tissue. It is generally accepted that multi-layer stents can be used to simulate the structure and function of natural blood vessels. Here, we developed a new three-layer tubular graft that is rolled from a single Poly(L-lactide-co-caprolactone) electrospun membrane. We used a new electrospinning technique to place three different structures on a single electrospun membrane such that the stent is comprised of three different layers. The inner layer is dense and suitable for endothelial cell growth, the middle layer is a parallel loose structure suitable for smooth muscle cell growth, and the outer layer is a parallel structure with sparse alternating texture suitable for both smooth muscle cell growth and structural support. The vascular stent has good tensile strength. At the same time, endothelial cells and smooth muscle cells readily proliferate on the material in vitro. In particular, smooth muscle cells grow in parallel on the middle and outer materials. In vivo, all layers of the vascular graft were infiltrated by cells within one week of subcutaneous implantation, indicative of favorable biocompatibility. After a week of subcutaneous implantation, the vascular stent was orthotopically transplanted into the abdominal aorta of Sprague Dawley rats. After ten weeks of transplantation, ultrasound imaging of the abdomen showed vascular patency. The vascular stent was endothelialized, smooth muscle cells readily proliferated, and a large amount of elastic fibers were formed. Therefore, our specially designed tri-layer vascular graft may be of significant benefit in vascular reconstruction.