Tissue-engineered blood vessels with endothelial nitric oxide synthase activity

Successful construction of a small-diameter bioartificial vascular graft remains a great challenge. This study reports on novel tissue engineering vascular grafts (TEVGs) constructed by endothelial progenitor cells and heparin-coated decellularized vessels (DV). The DVs were fabricated from canine carotid arteries with observable depletion of cellular components. After heparin coating, the scaffolds possessed excellent antithrombogeneity. Canine endothelial progenitor cells harvested from peripheral blood were expanded and seeded onto heparin-coated DVs and cocultured in a custom-made bioreactor to construct TEVGs. Thereafter, the TEVGs were implanted into the carotid arteries of cell-donor dogs. After 3 months of implantation, the luminal surfaces of TEVGs exhibited complete endothelium regeneration, however, only a few disorderly cells and thrombosis overlaid the luminal surfaces of control DVs grafts, and immunofluorescent staining showed that the seeded cells existed in the luminal sides and the medial parts of the explanted TEVGs and partially contributed to the endothelium formation. Specifically, TEVGs exhibited significantly smaller hyperplastic neointima area compared with the DVs, not only at midportion (0.64 ± 0.08 vs. 2.13 ± 0.12 mm(2) , p < 0.001), but also at anastomotic sites (proximal sites, 1.03 ± 0.09 vs. 3.02 ± 0.16 mm(2), p < 0.001; distal sites, 1.84 ± 0.15 vs. 3.35 ± 0.21 mm(2), p < 0.001). Moreover, TEVGs had a significantly higher patency rate than the DVs after 3 months of implantation (19/20 vs. 12/20, p < 0.01). Overall, this study provided a new strategy to develop small-diameter TEVGs with excellent biocompatibility and high patency rate.

Introduction: Cardiovascular diseases such as coronary heart disease often necessitate the surgical repair using conduits. Although autografts still remain the gold standard, the inconvenience of harvesting and/or insufficient availability in patients with atherosclerotic disease has given impetus to look into alternative sources for vascular grafts.Areas covered: There are four main techniques to produce tissue-engineered vascular grafts (TEVGs): i) biodegradable synthetic scaffolds; ii) gel-based scaffolds; iii) decellularised scaffolds and iv) self-assembled cell-sheet–based techniques. The first three techniques can be grouped together as scaffold-guided approach as it involves the use of a construct to function as a supportive framework for the vascular graft. The most significant advantages of TEVGs are that it possesses the ability to grow, remodel and respond to environmental factors. Cell sources for TEVGs include mature somatic cells, stem cells, adult progenitor cells and pluripotent stem cells.Expert opinion: TEVG holds great promise with advances in nanotechnology, coupled with important refinements in tissue engineering and decellularisation techniques. This will undoubtedly be an important milestone for cardiovascular medicine when it is eventually translated to clinical use.

First long-term preclinical evaluation of woven vascular grafts made from human cell-assembled extracellular matrix.

An exploratory study on the preparation and evaluation of a “same-day” adipose stem cell–based tissue-engineered vascular graft

Electronic Blood Vessel

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.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (n = 5/group) as abdominal aortic interposition conduits. The grafts were harvested at 1 month to evaluate patency, mechanical properties, vascular neotissue formation, and the expression of ECM cross-linking enzymes. All TEVGs were patent without any aneurysmal formation at 1 month. ECM area, TG2-positive area, and LOX-positive area were significantly greater in fast-degrading TEVGs compared to slow-degrading TEVGs, with significantly less remaining scaffold. The mechanical properties of fast-degrading TEVGs were similar to that of native aorta, as demonstrated by strain-stress curve. In conclusion, at 1 month, fast-degrading TEVGs had rapid and well-organized ECM with greater TG2 and LOX expression and native-like mechanical properties, compared to slow-degrading TEVGs. Impact statement Around 1.4 million patients in the United States require arterial prostheses each year due to cardiovascular diseases. Current synthetic vascular grafts suffer from increased risk of infection, thrombosis, a lack of endothelialization, and compliance mismatch to the native vasculature. Tissue-engineered vascular graft (TEVGs) presented in this study exhibited tunable biodegradation profiles by controlling the polymer ratio of polydioxanone/poly(L-lactide-co-caprolactone). One month after implantation, the fast-degrading TEVGs exhibited mechanical properties similar to that of native aorta, formation of endothelium, and well-organized extracellular matrix (ECM) with increased expression of tissue transglutaminase and lysyl oxidases, which are critical to the ECM remodeling process.

Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model. Vascular grafts fabricated from biodegradable scaffolds (ID 18+/-l mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21 days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for < or = 100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography-angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries. This study provides evidence of growth in living, functional pulmonary arteries engineered from vascular cells in a full growth animal model.

Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells

The objective of this study is to investigate if bone marrow-derived cells (BMCs) regenerate vascular tissues and improve patency in tissue-engineered small-diameter (internal diameter = 3 mm) vascular grafts. BMCs have demonstrated the ability to differentiate into endothelial-like cells and vascular smooth muscle-like cells and may offer an alternative cell source for vascular tissue engineering. Thus, we tissue-engineered small-diameter vascular grafts with BMCs and decellularized arteries. Canine BMCs were differentiated in vitro into smooth muscle alpha-actin/smooth muscle myosin heavy-chain-positive cells and von Willebrand factor/CD31-positive cells and seeded onto decellularized canine carotid arteries (internal diameter = 3 mm). The seeded grafts were implanted in cell donor dogs. The vascular-tissue regeneration and graft patency were investigated with immunohistochemistry and angiography, respectively. The vascular grafts seeded with BMCs remained patent for up to 8 weeks in the canine carotid artery interposition model, whereas nonseeded grafts occluded within 2 weeks. Within 8 weeks after implantation, the vascular grafts showed regeneration of the 3 elements of artery (endothelium, media, and adventitia). BMCs labeled with a fluorescent dye prior to implantation were detected in the retrieved vascular grafts, indicating that the BMCs participated in the vascular tissue regeneration. Here we show that BMCs have the potential to regenerate vascular tissues and improve patency in tissue-engineered small-diameter vascular grafts. This is the first report of a small-diameter neovessel engineered with BMCs as a cell source.

To accelerate the fabrication of thick-walled autologous tissue-engineered vascular grafts (TEVGs), a novel biotube inserting technique was developed. After 2 weeks of subcutaneous embedding in rabbits, silicone rods (diameter, 3 mm; length, 35 mm) became encapsulated in connective tissues. Single-layered biotubes were obtained after removing the silicone rods. One silicone rod encapsulated in tissues was inserted into a single-layered biotube to form two layers of autologous tubular tissues. Three layers of autologous tubular tissues were also obtained by applying the same technique. Following a 2-week re-embedding procedure, two layers or three layers of autologous tubular tissues were integrated to form two-layered or three-layered TEVGs. Both wall thickness and burst pressure of three-layered TEVGs were significantly higher than those of two-layered and single-layered TEVGs (P < 0.05). The two-layered TEVGs could be applied as small-caliber vascular grafts, and three-layered TEVGs could be applied as medium- or large-caliber vascular grafts.

Pathologies related to the cardiovascular system are the leading causes of death worldwide. One of the main treatments is conventional surgery with autologous transplants. Although donor grafts are often unavailable, tissue-engineered vascular grafts (TEVGs) show promise for clinical treatments. A systematic review of the recent scientific literature was performed using PubMed (Medline) and Web of Science databases to provide an overview of the state-of-the-art in TEVG development. The use of TEVG in human patients remains quite restricted owing to the presence of vascular stenosis, existence of thrombi, and poor graft patency. A total of 92 original articles involving human patients and animal models were analyzed. A meta-analysis of the influence of the vascular graft diameter on the occurrence of thrombosis and graft patency was performed for the different models analyzed. Although there is no ideal animal model for TEVG research, the murine model is the most extensively used. Hybrid grafting, electrospinning, and cell seeding are currently the most promising technologies. The results showed that there is a tendency for thrombosis and non-patency in small-diameter grafts. TEVGs are under constant development, and research is oriented towards the search for safe devices.

The development of a living, autologous vascular graft with the ability to grow holds great promise for advancing the field of pediatric cardiothoracic surgery. To evaluate the growth potential of a tissue-engineered vascular graft (TEVG) in a juvenile animal model. Polyglycolic acid nonwoven mesh tubes (3-cm length, 1.3-cm id; Concordia Fibers) coated with a 10% copolymer solution of 50:50 L-lactide and epsilon-caprolactone were statically seeded with 1 x 10 cells/cm autologous bone marrow derived mononuclear cells. Eight TEVGs (7 seeded, 1 unseeded control) were implanted as inferior vena cava (IVC) interposition grafts in juvenile lambs. Subjects underwent bimonthly magnetic resonance angiography (Siemens 1.5 T) with vascular image analysis (www.BioimageSuite.org). One of 7-seeded grafts was explanted after 1 month and all others were explanted 6 months after implantation. Neotissue was characterized using qualitative histologic and immunohistochemical staining and quantitative biochemical analysis. All grafts explanted at 6 months were patent and increased in volume as measured by difference in pixel summation in magnetic resonance angiography at 1 month and 6 months. The volume of seeded TEVGs at explant averaged 126.9% +/- 9.9% of their volume at 1 month. Magnetic resonance imaging demonstrated no evidence of aneurysmal dilation. TEVG resembled the native IVC histologically and had comparable collagen (157.9 +/- 26.4 microg/mg), elastin (186.9 +/- 16.7 microg/mg), and glycosaminoglycan (9.7 +/- 0.8 microg/mg) contents. Immunohistochemical staining and Western blot analysis showed that Ephrin-B4, a determinant of normal venous development, was acquired in the seeded grafts 6 months after implantation. TEVGs demonstrate evidence of growth and venous development when implanted in the IVC of a juvenile lamb model.

An off-the-shelf, small diameter tissue engineered vascular graft (TEVG) would be transformative to surgeons in multiple subspecialties. Herein, the results of a small diameter (ID ≈ 1 mm) vascular graft constructed from resorbable, amino acid-based poly(ester urea) (PEU) are reported. Electrospun PEU grafts of two different wall thicknesses (type A: 250 μm; type B: 350 μm) are implanted as abdominal infra-renal aortic grafts in a severe combined immune deficient/beige mouse model and evaluated for vessel remodeling over one year. Significantly, the small diameter TEVG does not rupture or lead to acute thrombogenic events during the intervals tested. The pilot TEVG in vivo shows long-term patency and extensive tissue remodeling with type A grafts. Extensive tissue remodeling in type A grafts leads to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells. However, due to slow degradation of the PEU scaffold materials in vivo, the grafts remain after one year. The type B grafts, which have 350 μm thick walls, experience occlusion over the one year interval due to intimal hyperplasia. This study affords significant findings that will guide the design of future generations of small diameter vascular grafts.

The advent of single-cell RNA sequencing (scRNA-Seq) has brought with it the ability to gain greater insights into the cellular composition of tissues and heterogeneity in gene expression within specific cell types. For tissue-engineered blood vessels, this is particularly impactful to better understand how neotissue forms and remodels into tissue resembling a native vessel. A notable challenge, however, is the ability to separate cells from synthetic biomaterials to generate high-quality single-cell suspensions to interrogate the cellular composition of our tissue-engineered vascular grafts (TEVGs) during active remodeling in situ. We present here a simple, commercially available approach to separate cells within our TEVG from the residual scaffold for downstream use in a scRNA-Seq workflow. Utilizing this method, we identified the cell populations comprising explanted TEVGs and compared these with results from immunohistochemical analysis. The process began with explanted TEVGs undergoing traditional mechanical and enzymatic dissociation to separate cells from scaffold and extracellular matrix proteins. Magnetically labeled antibodies targeting murine origin cells were incubated with enzymatic digests of TEVGs containing cells and scaffold debris in suspension allowing for separation by utilizing a magnetic separator column. Single-cell suspensions were processed through 10 × Genomics and data were analyzed utilizing R to generate cell clusters. Expression data provided new insights into a diverse composition of phenotypically unique subclusters within the fibroblast, macrophage, smooth muscle cell, and endothelial cell populations contributing to the early neotissue remodeling stages of TEVGs. These populations were correlated qualitatively and quantitatively with immunohistochemistry highlighting for the first time the potential of scRNA-Seq to provide exquisite detail into the host cellular response to an implanted TEVG. These results additionally demonstrate magnetic cell isolation is an effective method for generating high-quality cell suspensions for scRNA-Seq. While this method was utilized for our group's TEVGs, it has broader applications to other implantable materials that use biodegradable synthetic materials as part of scaffold composition. Impact statement Single-cell RNA sequencing is an evolving technology with the ability to provide detailed information on the cellular composition of remodeling biomaterials in vivo. This present work details an effective approach for separating nondegraded biomaterials from cells for downstream RNA-sequencing analysis. We applied this method to implanted tissue-engineered vascular grafts and for the first time describe the cellular composition of the remodeling graft at a single-cell gene expression level. While this method was effective in our scaffold, it has broad applicability to other implanted biomaterials that necessitate separation of cell from residual scaffold materials for single-cell RNA sequencing.