Tissue-engineered Vascular Grafts Research Articles

Insight on the endothelialization of small silk-based tissue-engineered vascular grafts.

Along with an increased incidence of cardiovascular diseases, there is a strong need for small-diameter vascular grafts. Silk has been investigated as a biomaterial to develop such grafts thanks to different processing options. Endothelialization was shown to be extremely important to ensure graft patency and there is ongoing research on the development and behavior of endothelial cells on vascular tissue-engineered scaffolds. This article reviews the endothelialization of silk-based scaffolds processed throughout the years as silk non-woven nets, films, gel spun, electrospun, or woven scaffolds. Encouraging results were reported with these scaffolds both in vitro and in vivo when implanted in small- to middle-sized animals. The use of coatings and heparin or sulfur to enhance, respectively, cell adhesion and scaffold hemocompatibility is further presented. Bioreactors also showed their interest to improve cell adhesion and thus promoting in vitro pre-endothelialization of grafts even though they are still not systematically used. Finally, the importance of the animal models used to study the right mechanism of endothelialization is discussed.

NON-INVASIVE MOLECULAR IMAGING OF INFLAMMATION IN TISSUE-ENGINEERED VASCULAR GRAFTS USING 18F-FDG PET/CT

Quantitative in vivo imaging to track combined functional and structural remodeling of tissue-engineered vascular grafts (TEVGs) has not been demonstrated. This study used functional positron emission tomography (PET) and anatomical x-ray computed tomography angiography (CTA) imaging to non-

Generating strong vascular grafts

Vascular Biology When treating injury or vascular disease, autologous or synthetic grafts are useful, but supplies are limited and often suboptimal. Robust tissue-engineered vascular grafts (TEVGs), particularly ones that can withstand mechanical stress, are highly desirable. Luo et al. generated vascular smooth muscle cells from human induced pluripotent stem cells and cultured them on biodegradable polyglycolic acid scaffolds exposed to pulsatile radial stress. When implanted into rats, these TEVGs showed high mechanical strength with no dilation, elongation, or wall thickening. Moreover, the TEVGs associated with limited thrombosis and showed no teratoma formation. Further optimization and engineering to limit immune rejection should provide cells and tissues suitable for treating injury or dysfunction of the cardiovascular system. Cell Stem Cell 26 , 251 (2020).

Design and comprehensive assessment of a biomimetic tri-layer tubular scaffold via biodegradable polymers for vascular tissue engineering applications.

Considering the structural complexity of the native artery wall and the limitations of current treatment strategies, developing a biomimetic tri-layer tissue-engineered vascular graft is a major developmental direction of vascular tissue regeneration. Biodegradable polymers exhibit adequate mechanical characteristics and feasible operability, showing potential prospects in the construction of tissue engineering scaffold. Herein, we present a bio-inspired tri-layer tubular graft using biodegradable polymers to simulate natural vascular architecture. The inner layer made of polycaprolactone (PCL) nanofiber possesses high tensile strength and contributed to endothelial cell adhesion and proliferation. The middle layer consisted of poly(lactic-co-glycolide) (PLGA) with a three-dimensional porous structure is appropriate for vascular smooth muscle cells (SMCs) penetration. The polyurethane (PU) was selected to be the outer layer, aiming to hold the entire tubular structure, suggesting superior mechanical properties and ideal biocompatibility. Adhesion between independent layers is achieved by thermal crosslinking. The compliance, burst pressure and suture retention force of the tubular scaffold were 2.50±1.60%, 2737.73±583.41mmHg and 13.06±1.89N, respectively. The in vivo study of subcutaneous implantation for 8weeks demonstrated the biomimetic tri-layer vascular graft could maintain intimal integrity, cell infiltration, collagen deposition and scaffold biodegradation. Overall, the biomimetic tri-layer vascular graft promises to be a potential candidate for vascular replacement and regeneration.

Human iPSCs Stretch to Improve Tissue-Engineered Vascular Grafts.

Human induced pluripotent stem cells (hiPSCs) provide a potentially unlimited cell source for producing autologous tissue-engineered vascular grafts (TEVGs), which currently suffer from low mechanical strength. In this issue of Cell Stem Cell, Luo etal. (2020) describe optimized culture media and a mechanical stretching regimen to produce hiPSC-derived TEVGs with mechanical behavior similar to that of natural vessels.

Human textiles: A cell-synthesized yarn as a truly “bio” material for tissue engineering applications

In the field of tissue engineering, many groups have come to rely on the extracellular matrix produced by cells as the scaffold that provides structure and strength to the engineered tissue. We have previously shown that sheets of Cell-Assembled extracellular Matrix (CAM), which are entirely biological yet robust, can be mass-produced for clinical applications using normal, adult, human fibroblasts. In this article, we demonstrate that CAM yarns can be generated with a range of physical and mechanical properties. We show that this material can be used as a simple suture to close a wound or can be assembled into fully biological, human, tissue-engineered vascular grafts (TEVGs) that have high mechanical strength and are implantable. By combining this truly “bio” material with a textile-based assembly, this original tissue engineering approach is highly versatile and can produce a variety of strong human textiles that can be readily integrated in the body. Statement of SignificanceYarn of synthetic biomaterials have been turned into textiles for decades because braiding, knitting and weaving machines can mass-produce medical devices with a wide range of shapes and mechanical properties. Here, we show that robust, completely biological, and human yarn can be produced by normal cells in vitro. This yarn can be used as a simple suture material or to produce the first human textiles. For example, we produced a woven tissue-engineered vascular grafts with burst pressure, suture retention strength and transmural permeability that surpassed clinical requirements. This novel strategy holds the promise of a next generation of medical textiles that will be mechanically strong without any foreign scaffolding, and will have the ability to truly integrate into the host's body.

Local Delivery of Dual MicroRNAs in Trilayered Electrospun Grafts for Vascular Regeneration.

Globally growing problems related to cardiovascular diseases lead to a considerable need for synthetic vascular grafts. For small-caliber vascular prosthesis, it remains essential to fulfill rapid endothelialization, inhibit intimal hyperplasia, and prevent calcification for keeping patency. To modulate vascular regeneration, herein, we developed a bioactive trilayered tissue-engineered vascular graft encapsulating both microRNA-126 and microRNA-145 in the fibrous inner and middle layers, respectively. In vitro cell activities demonstrated that the trilayered electrospun membranes had significant biological advantages in enhanced growth and intracellular nitric oxide production of vascular endothelial cells, modulation of phenotypes of vascular smooth muscle cells (SMCs), and restraint of calcium deposition through fast-releasing microRNA-126 and slow-releasing microRNA-145. Histological and immunofluorescent analyses of in vivo implantation in a rat abdominal aorta interposition model suggested that the dual-microRNA-loading trilayered electrospun graft exerted a positive effect on accelerating endothelialization, improving contractile SMC regeneration, and promoting normal extracellular matrix formation. Meanwhile, the local bioactivity of microRNA-126 and microRNA-145 in the trilayered vascular graft could regulate inflammation and depress calcification possibly by facilitating transformation of macrophages into the anti-inflammatory M2 phenotype. These findings indicated that the trilayered electrospun graft by local delivery of dual microRNAs could be possibly used as a bioactive substitute for replacement of artificial small-caliber blood vessels.

Bioresorbable silk grafts for small diameter vascular tissue engineering applications: In vitro and in vivo functional analysis

The success of tissue-engineered vascular graft (TEVG) predominantly relies on the selection of a suitable biomaterial and graft design. Natural biopolymer silk has shown great promise for various tissue-engineering applications. This study is the first to investigate Indian endemic non-mulberry silk (Antheraea assama-AA) – which inherits naturally superior mechanical and biological traits (e.g., RGD motifs) compared to Bombyx mori-BM silk, for TEVG applications. We designed bi-layered biomimetic small diameter AA-BM silk TEVGs adopting a new fabrication methodology. The inner layer showed ideally sized (~40 µm) pores with interconnectivity to allow cellular infiltration, and an outer dense electrospun layer that confers mechanical resilience. Biodegradation of silk TEVGs into amino acids as resorbable byproducts corroborates their in vivo remodeling ability. Following our previous reports, we surgically implanted human adipose tissue-derived stromal vascular fraction (SVF) seeded silk TEVGs in Lewis rats as abdominal aortic interposition grafts for 8 weeks. Adequate suture retention strength (0.45 ± 0.1 N) without any blood seepage post-implantation substantiate the grafts’ viability. AA silk-based TEVGs showed superior animal survival and graft patency compared to BM silk TEVGs. Histological analysis revealed neo-tissue formation, host cell infiltration and graft remodeling in terms of extracellular matrix turnover. Altogether, this study demonstrates promising aspects of AA silk TEVGs for vascular tissue engineering applications. Statement of significanceClinical ‘off the shelf’ implementation of tissue-engineered vascular grafts (TEVGs) remains a challenge. Achieving optimal blood vessel regeneration requires the use of bioresorbable materials having suitable degradation rates while producing minimal or no toxic byproducts. Host cell recruitment and preventing acute thrombosis are other pre-requisites for successful graft remodeling. In this study, for the first time we explored the use of naturally derived Indian endemic non-mulberry Antheraea assama silk in combination with Bombyx mori silk for TEVG applications by adopting a new biomimetic approach. Our bi-layered silk TEVGs were optimally porous, mechanically resilient and biodegradable. In vivo implantation in rat aorta showed long-term patency and graft remodeling by host cell infiltration and extracellular matrix deposition corroborating their clinical feasibility.

Tissue-Engineered Vascular Grafts with Advanced Mechanical Strength from Human iPSCs.

Vascular smooth muscle cells (VSMCs) can be derived in large numbers from human induced pluripotent stem cells (hiPSCs) for producing tissue-engineered vascular grafts (TEVGs). However, hiPSC-derived TEVGs are hampered by low mechanical strength and significant radial dilation after implantation. Here, we report generation of hiPSC-derived TEVGs with mechanical strength comparable to native vessels used in arterial bypass grafts by utilizing biodegradable scaffolds, incremental pulsatile stretching, and optimal culture conditions. Following implantation into a rat aortic model, hiPSC-derived TEVGs show excellent patency without luminal dilation and effectively maintain mechanical and contractile function. This study provides a foundation for future production of non-immunogenic, cellularized hiPSC-derived TEVGs composed of allogenic vascular cells, potentially serving needs to a considerable number of patients whose dysfunctional vascular cells preclude TEVG generation via other methods.

Potential of mesenchymal stem cells for bioengineered blood vessels in comparison with other eligible cell sources.

Application of stem cells in tissue engineering has proved to be effective in many cases due to great proliferation and differentiation potentials as well as possible paracrine effects of these cells. Human mesenchymal stem cells (MSCs) are recognized as a valuable source for vascular tissue engineering, which requires endothelial and perivascular cells. The goal of this review is to survey the potential of MSCs for engineering functional blood vessels in comparison with other cell types including bone marrow mononuclear cells, endothelial precursor cells, differentiated adult autologous smooth muscle cells, autologous endothelial cells, embryonic stem cells, and induced pluripotent stem cells. In conclusion, MSCs represent a preference in making autologous tissue-engineered vascular grafts (TEVGs) as well as off-the-shelf TEVGs for emergency vascular surgery cases.

Corrugated nanofiber tissue-engineered vascular graft to prevent kinking for arteriovenous shunts in an ovine model

ObjectiveProsthetic grafts are often needed in open vascular procedures. However, the smaller diameter prosthetic grafts (<6 mm) have low patency and often result in complications from infection. Tissue-engineered vascular grafts (TEVGs) are a promising replacement for small diameter prosthetic grafts. TEVGs start as a biodegradable scaffold to promote autologous cell proliferation and functional neotissue regeneration. Owing to the limitations of graft materials; however, most TEVGs are rigid and easily kinked when implanted in limited spaces, which precludes clinical application. We have developed a novel corrugated nanofiber graft to prevent kinking. MethodsTEVGs with corrugated walls (5-mm internal diameter by 10 cm length) were created by electrospinning a blend of poly-ε-caprolactone and poly(L-lactide-co-caprolactone). The biodegradable grafts were then implanted between the carotid artery and the external jugular vein in a U-shape using an ovine model. TEVGs were implanted on both the left and right side of a sheep (n = 4, grafts = 8). The grafts were explanted 1 month after implantation and inspected with mechanical and histologic analyses. Graft patency was confirmed by measuring graft diameter and blood flow velocity using ultrasound, which was performed on day 4 and every following week after implantation. ResultsAll sheep survived postoperatively except for one sheep that died of acute heart failure 2 weeks after implantation. The graft patency rate was 87.5% (seven grafts out of eight) with one graft becoming occluded in the early phase after implantation. There was no significant kinking of the grafts. Overall, endothelial cells were observed in the grafts 1 month after the surgeries without graft rupture, calcification, or aneurysmal change. ConclusionsOur novel corrugated nanofiber vascular graft displayed neotissue formation without kinking in large animal model.

Synergistic Effect of Inducible Factors on the Differentiation of Human Adipose-Derived Stem Cells to Vascular Cells

The use of cells derived from the patient’s tissue is an attractive approach to develop autologous tissue-engineered vascular graft (TEVG) to bypass the diseased blood vessel. Multipotent human adipose-derived mesenchymal stem cells (hADMSCs) have the potential to differentiate into both smooth muscle cells (SMCs) and endothelial cells (ECs). However, the disease conditions affecting the growth and differentiation potential of cardiovascular disease (CVD) patient’s MSC (pADMSCs) is a hindrancee. This study aimed to derive both endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) from pADMSCs using a specific niche comprising fibrin and other inducers. pADMSCs were seeded on SMC-specific/EC-specific fibrin matrix coated on tissue culture polystyrene (TCPS) surface. In the third passage, pADMSCs were exposed to GFs for a specific period to induce pADMSCs to SMPCs. For inducing hADMSCs to EPCs, cells were exposed to GFs, followed by hypoxia. The EPCs were seeded in the lumen of a scaffold and applied shear stress to induce into ECs. The relative gene expressions at different stages were assessed by mRNA quantification and at the protein level by immunocytochemistry of final differentiated cells. A consistent matrix and GF dependent protocol facilitated derivation of SMPCs/SMCs with early, mid, and late markers. Sequential action by GF, hypoxia (HO), and shear stress (SS) differentiated pADMSCs to EPCs/ECS. The study established that patient-specific ECs and SMCs may be derived from ADMSCs for the construction of intimal and medial layers of TEVG aiming to fabricate autologous blood vessels.

Fabrication of PCL/keratin composite scaffolds for vascular tissue engineering with catalytic generation of nitric oxide potential.

Tissue-engineered vascular grafts (TEVGs) have been proposed as a promising approach to fulfill the need for small-diameter blood vessel substitutes. However, common failure caused by thrombosis and neointimal proliferation after implantation has restricted their use in the clinic. Herein, a NO-generating scaffold for vascular tissue engineering was developed by coelectrospinning poly(ε-caprolactone) (PCL) with keratin. The morphology and surface chemical composition were characterized via SEM, ATR-FTIR spectroscopy and XPS. The biocomposite scaffold selectively enhanced the adhesion and growth of endothelial cells (ECs) while suppressing the proliferation of smooth muscle cells (SMCs) in the presence of GSH and GSNO due to the catalytic generation of NO. In addition, these mats displayed excellent blood compatibility by prolonging the blood-clotting time. In summary, these NO-generating PCL/keratin scaffolds have potential applications in vascular tissue engineering with rapid endothelialization and reduced SMC proliferation.

Is it Possible to Create Readily Available Tissue-Engineered Vascular Grafts Without Using Cells?

Introduction - In modern vascular surgery remains the problem of small-diameter vascular grafts. Autologous or synthetic vascular grafts are used routinely for arterial bypass in patients with cardiovascular disease. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. Currently, there are a number of techniques to receipt such artificial vessel, some of which are already in clinical trials. The method of using acellular biodegradable scaffold suggests that host cells can regenerate new vascular wall in situ, thats allows to overcome the scalability issue and make the grafts available off-the-shelf. The aim of the study is a comprehensive assessment of bioresorbable prosthesis from L-polylactide (PLA), and determining the possibility of its use as acellular vascular graft available off-the-shelf. Methods - Tubular grafts were obtained by the electrospinning method, (1.2 mm inner diameter) on the basis of nonwoven nano- and microfiber biodegradable polymer PLA. Mechanical characteristics such as tensile strength ,Young’s modulus E, and tensile deformation at break ofthe samples were determined using a UTS 10 universaltesting machine (Germany). Porosity parameters were determined by mercury porosimetry and BET method. Cytology analyses were performed with nanofibersfrom PLA and culture of mesenchymal stem cellsobtained from adipose tissue (ASCs). To assess the degree of cell adhesion toPLA grafts investigated materials were placed insquare-hole board (Nunc, USA). Investigation of barrier propertiesof tubular grafts were carried out on a developed laboratory setup. Obtained grafts were implanted in the abdominal aorta of rats (n=18). Follow-up was 16 months. The material was subjected to histological examination, electron microscopy, immunohistochemistry CD 31 +. After 14 months implantation angiography was performed (n=1). Results - During the operations and by the results of the investigation physical and mechanical properties it has been shown that obtained grafts are suitable for sewing in the bloodstream. For grafts from L-polylactide the pore volume was 78% and the surface area per gram of polymer 3.6 m2 (S BET).Investigation of adhesion and proliferation of mesenchymal stem cells derived from adipose tissue of rats showed safety and biocompatibility of used materials. All grafts formed neointima, with no signs of hyperplasia in the areas of anastomoses. Patency for PLA grafts 88% (n = 18). After 16 months (n=3) histology shows total resorption of PLA fibers, new vascular wall consist of connecctive tissue with endotelial lining, but all zone of reconstruction was presented by aneurysm. Conclusion - Small diameter vascular prostheses with high porosity obtained by electrospinning from PLA have sufficient exploitation mechanical properties. In in vivo experiments proved its safety, biocompatibility. Received patency rates are satisfactory for grafts with small diameter. Low mechanical properties of the connective tissue formed in the area of reconstruction have led to the formation of aneurysms. To sum up, the need for the introduction of cells in the wall of the graft in vitro before implantation is not in doubt.

Influence of Mesenchymal Stem Cells Seeding on Morphology of Tissue-Engineered Vascular Graft Based on Poly (L-lactide) Scaffold

Introduction - The problem of small-diameter vascular grafts still exists in modern vascular surgery. Autologous or synthetic vascular grafts are used routinely for arterial bypass in patients with cardiovascular diseases. However, some patients either lack suitable autologous tissue or cannot receive synthetic grafts. Such patients could benefit from a vascular graft produced by tissue engineering. The aim of the study is to evaluate the effects of adipose-derived mesenchymal stem cells (AD-MSC) on the formation of tissue-engineered vascular graft (TEVG). Methods - Tubular scaffolds 1.1 mm inner diameter were obtained by the electrospinning method on the basis of nano- and microfibers of poly(L-lactide) (PLLA). AD-MSC were seeded on the scaffolds by the developed filtration method. The subsequent culturing for 14 days was carried out in the constructed flow bioreactor. Cell distribution in the scaffold wall was assessed by the fluorescence microscopy. Obtained grafts were implanted into rat aorta: group 1 without AD-MSC (n=36) with follow-up till 16 months, group 2 with seeded AD-MSC (n=36) with follow-up till 16 months. The material was subjected to histological examination, electron microscopy, immunohistochemistry CD 31+, aSMA and morphometric analysis (cell counting, neointima and neoadventia thickness). To track AD-MSC in vivo PKH-26 labeling and subsequent fluorescence microscopy were performed in 2, 7 and 14 days. Results - Filtration seeding and subsequent cultivation in the flow bioreactor led to a uniform distribution of AD-MSC in the scaffold wall. Graft patency in the first group was 86%, in the second 96%. All grafts formed neointima, with no signs of hyperplasia in the area of anastomoses. The total resorption of PLLA fibers in group 1 was observed by histological examination, new vascular wall consisted of endothelial lining and connective tissue without smooth muscle cells (SMC); in all cases aneurysm formation was detected. Total cells number of implant wall was more in group 2, including SMC that formed neomedia. Also the outer connective tissue layer was much thicker. Histology identified the uniform formation of new tissues in group 2 with no signs of aneurysm. Conclusion - AD-MSC seeding leads to the formation of TEVG, morphologically similar to the natural structure of the vessels. The study was supported by Russian Science Foundation (project 14-33-00003)

Long-term Functioning Aneurysmal Free Tissue-engineered Vascular Graft Based on Composite Bi-layered (PLLA/FPL) Scaffold

Long-term Functioning Aneurysmal Free Tissue-engineered Vascular Graft Based on Composite Bi-layered (PLLA/FPL) Scaffold

Effectiveness of Using Endothelial Colony-Forming Cells for Creating Tissue-Engineered Vascular Grafts Under in vitro Conditions

Effectiveness of Using Endothelial Colony-Forming Cells for Creating Tissue-Engineered Vascular Grafts Under in vitro Conditions

Stiffness of the Aligned Fibers Affects Structural and Functional Integrity of the Oriented Endothelial Cells

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, stiffness influence of the substrate/scaffold with anisotropic topography on ECs with in vivo like oriented morphology has rarely been attempted. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68~2141.72 MPa) resembling that of the healthy and diseased subendothelial matrix were used to investigate the effects of aligned fiber stiffness on EC attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs capable of providing topographical cues is a crucial endothelium-protective microenvironmental factor by maintaining the long-standing, stable and quiescent endothelium with in vivo like orientation and strong cell-cell junctions. Stiffer AFSs exacerbated disruption of endothelium integrity, occurrence of endothelial-to-mesenchymal transition (EndMT), and inflammation-induced activation in endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels.

Resveratrol-loaded polyurethane nanofibrous scaffold: viability of endothelial and smooth muscle cells

Acellular small-caliber tissue-engineered vascular grafts (SCTEVGs) have low patency rate due to complications including thrombosis and intimal hyperplasia. Rapid endothelialization, antithrombosis and antiproliferation approaches are suitable for dispelling these complications. Nevertheless, common antithrombosis and antiproliferation techniques are usually incompatible with rapid endothelialization on vascular grafts. To overcome these obstacles, we developed nanofibrous polyurethane scaffolds loaded with resveratrol drug, which is a natural compound extracted from plants and shows multifaceted effects in cardiovascular protection. It was found that the tensile strength and Young’s modulus in modified scaffolds were significantly increased by resveratrol loading into membranes. The tensile strengths and breaking strains of resveratrol-loaded scaffolds were close to that of native vessels. The resveratrol release profile from the nanofibrous scaffolds occurred in a sustained manner. The anti-thrombogenicity of resveratrol-loaded nanofibers increased compared to polyurethane alone, with the result that prolonged human blood clotting time and lower hemolysis were detected on these scaffolds. The viability of human umbilical vein endothelial cells and smooth muscle cells on resveratrol-loaded scaffolds was evaluated. Our findings demonstrated that resveratrol-loaded nanofibers resulted in not only appropriate antithrombotic properties, but the formation of a monolayer of endothelial cells on the scaffold surface and lower smooth muscle cell growth. These resveratrol-loaded nanofibers are suggested as potential scaffolds for SCTEVGs.

Strategies in cell‐free tissue‐engineered vascular grafts

Cardiovascular diseases (CVD) remain the leading cause of morbidity and mortality in the world, among which coronary artery diseases (CAD) are the most common type of CVD. Coronary artery bypass grafting (CABG) using autologous vein and artery grafts is the typical surgical intervention for CAD patients. However, for patients whose autologous grafts are not available, there are no appropriate substitutes for vascular grafts. Investigation of tissue-engineered vascular graft (TEVG) has persisted over decades with significant advancement, utilizing different types of biomaterials. In the past two decades, a great number of studies based on cell-seeding strategies were reported. However, limitations of cell-based strategies made clinical application difficult. With the understanding of stem cells and tissue remodeling process, strategies without cell-seeding emerged as potential methods to achieve in situ regeneration. A cell-free graft may recruit host cells and guide their participation in vascular remodeling. The grafts modified by bio-active molecules showed good results in promoting in situ regeneration and exhibited potential to make the vascular grafts off-the-shelf. In this review, the strategies for cell-free TEVG manufacturing were discussed, including the materials for fabricating TEVGs, the methods of functionalization to promote in situ regeneration, the challenges researchers faced in TEVG investigation, and finally the prospects in TEVG design.