Microtube embedded hydrogel bioprinting for vascularization of tissue-engineered scaffolds.

Vascular tissue engineering has been considered promising as one of the alternatives for viable artificial tissues and organs. Macro- and microscale hollow tubes fabricated with various techniques have been widely studied to mimic blood vessels. To date, the fabrication of biomimetic capillary vessels with sizes ranging from 1 to 10 µm is still challenging. In this paper, core-sheath microtubes were electrospun to mimic capillary vessels and were embedded in carboxymethyl cellulose/sodium alginate hydrogel for bioprinting. The results showed improved printing fidelity and promoted cell attachment. The tube concentration and tube length both had significant influences on filament size and merging area. Printed groups with higher microtube concentration showed higher microtube density, with filament/nozzle size ratio, and printed/designed grid area ratio closer to 100%. In the in vitro experiments, microtubes were not only compatible with human umbilical vein endothelial cells but also provided microtopographical cues to promote cell proliferation and morphogenesis in three-dimensional space. In summary, the microtubes fabricated by our groups have the potential for the bioprinting of vascularized soft tissue scaffolds.

Developing human tissue engineered arterial constructs to simulate human in vivo thrombus formation.

Thrombus formation is highly dependent upon the physico-chemical environment in which it is triggered. Our ability to understand how thrombus formation is initiated, regulated, and resolved in the human body is dependent upon our ability to replicate the mechanical and biological properties of the arterial wall. Current in vitro thrombosis models principally use reductionist approaches to model the complex biochemical and cellular milieu present in the arterial wall, and so researcher have favored the use of in vivo models. The field of vascular tissue engineering has developed a range of techniques for culturing artificial human arteries for use as vascular grafts. These techniques therefore provide a basis for developing more sophisticated 3D replicas of the arterial wall that can be used in in vitro thrombosis models. In this review, we consider how tissue engineering approaches can be used to generate 3D models of the arterial wall that improve upon current in vivo and in vitro approaches. We consider the current benefits and limitations of reported 3D tissue engineered models and consider what additional evidence is required to validate them as alternatives to current in vivo models.

The basement membrane as a structured surface - role in vascular health and disease.

The basement membrane (BM) is a thin specialized extracellular matrix that functions as a cellular anchorage site, a physical barrier and a signaling hub. While the literature on the biochemical composition and biological activity of the BM is extensive, the central importance of the physical properties of the BM, most notably its mechanical stiffness and topographical features, in regulating cellular function has only recently been recognized. In this Review, we focus on the biophysical attributes of the BM and their influence on cellular behavior. After a brief overview of the biochemical composition, assembly and function of the BM, we describe the mechanical properties and topographical structure of various BMs. We then focus specifically on the vascular BM as a nano- and micro-scale structured surface and review how its architecture can modulate endothelial cell structure and function. Finally, we discuss the pathological ramifications of the biophysical properties of the vascular BM and highlight the potential of mimicking BM topography to improve the design of implantable endovascular devices and advance the burgeoning field of vascular tissue engineering.

An encapsulated fibrin-based bioartificial tissue construct with integrated macrovessels, microchannels, and capillary tubes.

Facilitating sufficient nutrient and oxygen supply in large-scale bioartificial constructs is a critical step in organ bioengineering. Immediate perfusion not only depends on a dense capillary network, but also requires integrated large-diameter vessels that allow vascular anastomoses during implantation. These requirements set high demands for matrix generation as well as for in vitro cultivation techniques and remain mostly unsolved challenges up until today. Additionally, bioartificial constructs must have sufficient biomechanical stability to withstand mechanical stresses during and after implantation. We developed a bioartificial tissue construct with a fibrin matrix containing human umbilical vein endothelial cells and adipose tissue-derived stem cells facilitating capillary-like network formation. This core matrix was surrounded by a dense acellular fibrin capsule providing biomechanical stability. Two fibrin-based macrovessels were integrated on each side of the construct and interconnected via four 1.2 mm thick microchannels penetrating the cellularized core matrix. After 4 days of perfusion in a custom-built bioreactor, homogeneous capillary-like network formation throughout the core matrix was observed. The fibrin capsule stabilized the core matrix and facilitated the generation of a self-supporting construct. Thus, the encapsulated fibrin tissue construct could provide a universal prevascularized matrix for seeding with different cell types in various tissue engineering approaches.

Anti-Inflammatory Fibronectin-AgNP for Regulation of Biological Performance and Endothelial Differentiation Ability of Mesenchymal Stem Cells.

The engineering of vascular regeneration still involves barriers that need to be conquered. In the current study, a novel nanocomposite comprising of fibronectin (denoted as FN) and a small amount of silver nanoparticles (AgNP, ~15.1, ~30.2 or ~75.5 ppm) was developed and its biological function and biocompatibility in Wharton's jelly-derived mesenchymal stem cells (MSCs) and rat models was investigated. The surface morphology as well as chemical composition for pure FN and the FN-AgNP nanocomposites incorporating various amounts of AgNP were firstly characterized by atomic force microscopy (AFM), UV-Visible spectroscopy (UV-Vis), and Fourier-transform infrared spectroscopy (FTIR). Among the nanocomposites, FN-AgNP with 30.2 ppm silver nanoparticles demonstrated the best biocompatibility as assessed through intracellular ROS production, proliferation of MSCs, and monocytes activation. The expression levels of pro-inflammatory cytokines, TNF-α, IL-1ß, and IL-6, were also examined. FN-AgNP 30.2 ppm significantly inhibited pro-inflammatory cytokine expression compared to other materials, indicating superior performance of anti-immune response. Mechanistically, FN-AgNP 30.2 ppm significantly induced greater expression of vascular endothelial growth factor (VEGF) and stromal-cell derived factor-1 alpha (SDF-1α) and promoted the migration of MSCs through matrix metalloproteinase (MMP) signaling pathway. Besides, in vitro and in vivo studies indicated that FN-AgNP 30.2 ppm stimulated greater protein expressions of CD31 and von Willebrand Factor (vWF) as well as facilitated better endothelialization capacity than other materials. Furthermore, the histological tissue examination revealed the lowest capsule formation and collagen deposition in rat subcutaneous implantation of FN-AgNP 30.2 ppm. In conclusion, FN-AgNP nanocomposites may facilitate the migration and proliferation of MSCs, induce endothelial cell differentiation, and attenuate immune response. These finding also suggests that FN-AgNP may be a potential anti-inflammatory surface modification strategy for vascular biomaterials.

The applications of heparin in vascular tissue engineering.

Cardiovascular diseases, among all diseases, are taking the most victims worldwide. Coronary artery occlusion, takes responsibility of about 30% of the yearly global deaths in the world (Heart Disease and Stroke Statistics 2017 At-a-Glance, 2017), raising the need for viable substitutes for cardiovascular tissues. Depending on a number of factors, blocked coronary arteries are now being replaced by autografts or stents. Since the autografts, as the gold standard coronary artery replacements, are not available in adequate quality and quantity, the demand for small diameter vascular substitute comparable to native vessels is rapidly growing. Synthetic grafts have been successfully approved for developing vascular replacements but regarding the special conditions in small-caliber vessels, their use is limited to large-diameter vascular tissue engineering. The major problems associated with the vascular tissue engineered grafts are thrombosis and intimal hyperplasia. Heparin, a negatively charged natural polysaccharide has been used in fabricating vascular grafts since it prevents protein fouling on the surfaces and most importantly, impeding thrombosis. Herein, we focused on heparin, as a multifunctional bioactive molecule that not only serves as an anticoagulant with frequent clinical use but also acts as an anti-inflammatory and angiogenic regulatory substance. We summarized heparin incorporation into stents and grafts and their applicability to restrain restenosis. Also, the applications of heparinzation of biomaterials and heparin mimetic polymers and different approaches invoked to improve heparin bioactivity have been reviewed. We summarized the methods of adding heparin to matrices as they were explained in the literature. We reviewed how heparin influences the biocompatibility of the scaffolds and discussed new advances about using heparin in small-diameter vascular tissue engineering.

Novel Cell-Based and Tissue Engineering Approaches for Induction of Angiogenesis as an Alternative Therapy for Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most frequent microvascular complication of long-term diabetes and the most common cause of blindness, increasing morbidity in the working-age population. The most effective therapies for these complications include laser photocoagulation and anti-vascular endothelial growth factor (VEGF) intravitreal injections. However, laser and anti-VEGF drugs are untenable as a final solution as they fail to address the underlying neurovascular degeneration and ischemia. Regenerative medicine may be a more promising approach, aimed at the repair of blood vessels and reversal of retinal ischemia. Stem cell therapy has introduced a novel way to reverse the underlying ischemia present in microvascular complications in diseases such as diabetes. The present review discusses current treatments, their side effects, and novel cell-based and tissue engineering approaches as a potential alternative therapeutic approach.

Vascular tissue engineering: Fabrication and characterization of acetylsalicylic acid-loaded electrospun scaffolds coated with amniotic membrane lysate.

As the incidence of small-diameter vascular graft (SDVG) occlusion is considerably high, a great amount of research is focused on constructing a more biocompatible graft. The absence of a biocompatible surface in the lumen of the engineered grafts that can support confluent lining with endothelial cells (ECs) can cause thrombosis and graft failure. Blood clot formation is mainly because of the lack of an integrated endothelium. The most effective approach to combat this problem would be using natural extracellular matrix constituents as a mimic of endothelial basement membrane along with applying anticoagulant agents to provide local antithrombotic effects. In this study, we fabricated aligned and random electrospun poly-L-lactic acid (PLLA) scaffolds containing acetylsalicylic acid (ASA) as the anticoagulation agent and surface coated them with amniotic membrane (AM) lysate. Vascular scaffolds were structurally and mechanically characterized and assessed for cyto- and hemocompatibility and their ability to support endothelial differentiation was examined. All the scaffolds showed appropriate tensile strength as expected for vascular grafts. Lack of cytotoxicity, cellular attachment, growth, and infiltration were proved using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scanning electron microscopy. The blood compatibilities of different scaffolds examined by in vitro hemolysis and blood coagulation assays elucidated the excellent hemocompatibility of our novel AM-coated ASA-loaded nanofibers. Drug-loaded scaffolds showed a sustained release profile of ASA in 7 days. AM-coated electrospun PLLA fibers showed enhanced cytocompatibility for human umbilical vein ECs, making a confluent endothelial-like lining. In addition, AM lysate-coated ASA-PLLA-aligned scaffold proved to support endothelial differentiation of Wharton's jelly-derived mesenchymal stem cells. Our results together indicated that AM lysate-coated ASA releasing scaffolds have promising potentials for development of a biocompatible SDVG.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Engineering blood vessels and vascularized tissues: technology trends and potential clinical applications.

Vascular tissue engineering has the potential to make a significant impact on the treatment of a wide variety of medical conditions, including providing in vitro generated vascularized tissue and organ constructs for transplantation. Since the first report on the construction of a biological blood vessel, significant research and technological advances have led to the generation of clinically relevant large and small diameter tissue engineered vascular grafts (TEVGs). However, developing a biocompatible blood-contacting surface is still a major challenge. Researchers are using biomimicry to generate functional vascular grafts and vascular networks. A multi-disciplinary approach is being used that includes biomaterials, cells, pro-angiogenic factors and microfabrication technologies. Techniques to achieve spatiotemporal control of vascularization include use of topographical engineering and controlled-release of growth/pro-angiogenic factors. Use of decellularized natural scaffolds has gained popularity for engineering complex vascularized organs for potential clinical use. Pre-vascularization of constructs prior to implantation has also been shown to enhance its anastomosis after implantation. Host-implant anastomosis is a phenomenon that is still not fully understood. However, it will be a critical factor in determining the in vivo success of a TEVGs or bioengineered organ. Many clinical studies have been conducted using TEVGs, but vascularized tissue/organ constructs are still in the research & development stage. In addition to technical challenges, there are commercialization and regulatory challenges that need to be addressed. In this review we examine recent advances in the field of vascular tissue engineering, with a focus on technology trends, challenges and potential clinical applications.

Vascular Remodeling Process of Heparin-Conjugated Poly(ε-Caprolactone) Scaffold in a Rat Abdominal Aorta Replacement Model.

In the field of vascular graft research, poly-ε-caprolactone (PCL) is used owing to its good mechanical strength and biocompatibility. In this study, PCL scaffold was prepared by electrospinning and surface modification with heparin via hexamethylenediamine. Then the scaffolds were implanted into the infrarenal abdominal aorta of Wistar rats and contrast-enhanced micro-ultrasound was used to monitor the patency of grafts after implantation. These grafts were extracted from the rats at 1, 3, and 6 months for histological analysis, immunofluorescence staining, and scanning electron microscopy observation. Although some grafts experienced aneurysmal change, results showed that all implanted grafts were patent during the course of 6 months and these grafts demonstrated well-organized neotissue with endothelium formation, smooth muscle regeneration, and extracellular matrix formation. Such findings confirm feasibility to create heparin-conjugated scaffolds of next-generation vascular grafts.

Generation of human umbilical cord vein CD146+ perivascular cell origined three-dimensional vascular construct.

Small-diameter vascular grafts are needed for the treatment of coronary artery diseases in the case of limited accessibility of the autologous vessels. Synthetic scaffolds have many disadvantages so in recent years vascular constructs (VCs) made from cellularized natural scaffolds was seen to be very promising but number of studies comprising this area is very limited. In our study, our aim is to generate fully natural triple-layered VC that constitutes all the layers of blood vessel with vascular cells. CD146+ perivascular cells (PCs) were isolated from human umbilical cord vein (HUCV) and differentiated into smooth muscle cells (SMCs) and fibroblasts. They were then combined with collagen type I/elastin/dermatan sulfate and collagen type I/fibrin to form tunica media and tunica adventitia respectively. HUCV endothelial cells (ECs) were seeded on the construct by cell sheet engineering method after fibronectin and heparin coating. Characterization of the VC was performed by immunolabeling, histochemical staining and electron microscopy (SEM and TEM). Differentiated cells were identified by means of immunofluorescent (IF) labeling. SEM and TEM analysis of VCs revealed the presence of three histologic tunicae. Collagen and elastic fibers were observed within the ECM by histochemical staining. The vascular endothelial growth factor receptor expressing ECs in tunica intima; α-SMA expressing SMCs in tunica media and; the tenascin expressing fibroblasts in tunica adventitia were detected by IF labeling. In conclusion, by combining natural scaffolds and vascular cells differentiated from CD146+ PCs, VCs can be generated layer by layer. This study will provide a preliminary blood vessel model for generation of fully natural small-diameter vascular grafts.

Pericytes in Tissue Engineering.

Pericytes have crucial roles in blood-brain barrier function, blood vessel function/stability, angiogenesis, endothelial cell proliferation/differentiation, wound healing, and hematopoietic stem cells maintenance. They can be isolated from fetal and adult tissues and have multipotential differentiation capacity as mesenchymal stem cells (MSCs). All of these properties make pericytes as preferred cells in the field of tissue engineering. Current developments have shown that tissue-engineered three-dimensional (3D) systems including multiple cell layers (or types) and a supporting biological matrix represent the in vivo environment better than those monolayers on plastic dishes. Tissue-engineered models are also more ethical and cheaper systems than animal models. This chapter describes the role of pericytes in tissue engineering for regenerative medicine.

The role of mechanical stimuli in the vascular differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are among the most promising and suitable stem cell types for vascular tissue engineering. Substantial effort has been made to differentiate MSCs towards vascular cell phenotypes, including endothelial cells and smooth muscle cells (SMCs). The microenvironment of vascular cells not only contains biochemical factors that influence differentiation, but also exerts hemodynamic forces, such as shear stress and cyclic strain. Recent evidence has shown that these forces can influence the differentiation of MSCs into endothelial cells or SMCs. In this Commentary, we present the main findings in the area with the aim of summarizing the mechanisms by which shear stress and cyclic strain induce MSC differentiation. We will also discuss the interactions between these mechanical cues and other components of the microenvironment, and highlight how these insights could be used to maintain differentiation.

In vitro remodeling and structural characterization of degradable polymer scaffold-based tissue-engineered vascular grafts using optical coherence tomography.

Non-destructive imaging strategies to monitor long-term cultures is essential for vascular engineering. The goal of this study is to investigate whether optical coherence tomography (OCT) can be a suitable approach to monitor the long-term remodeling process of biodegradable polymeric scaffold-based tissue-engineered vascular grafts (TEVG) after pulsatile stimulation and to observe polymeric scaffold degradation during bioreactor cultivation. In the present study, a perfusion system driven by a ventricular assist device was provided for a three-dimensional culture system as a pulsatile force. We characterized the structural features of wall thickness and polyglycolic acid degradation based on optical signal attenuation using catheter-based OCT. Scanning electron microscopy confirmed morphological changes. Also, polymer degradation and the detection of different types of collagen was visualized after 4 weeks of culture by means of polarized microscopy. Findings on OCT imaging correlated with those on histological examination and revealed the effects of pulsatile stimulation on the development of engineered vessels. This finding demonstrated that real-time imaging with OCT may be a promising tool for monitoring the growth and remodeling characterization of TEVG and provide a basis to promote the ideal and long-term culture of vascular tissue engineering.

A Dynamically Tunable, Bioinspired Micropatterned Surface Regulates Vascular Endothelial and Smooth Muscle Cells Growth at Vascularization.

Regulation of the growth of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) with artificial vascular grafts at vascularization is well-known to regenerate functional blood vessels for treating cardiovascular disease; however, little research has been published on this subject. Here, a novel polymer vascular graft is presented, whose inner surface contains an assembled circular microgroove pattern decorated with a combination of concentric circular microgrooves and radial, straight microgrooves inspired by the orientation of SMCs and ECs in natural tissues. The surface micropatterns can produce dynamically tunable variations via the thermally switched shape memory. The results from the in vitro EC/SMC co-cultures reveal that the surface micropatterns have a great capacity to regulate the specific distribution of ECs/SMCs because the ECs grow along the radial, straight microgrooves and the SMCs grow along concentric circular microgrooves. The in vivo vascularization is further analyzed by implanting the vascular graft in the rabbit carotid artery. Both histological analysis and immunofluorescence staining demonstrate that it is capable of highly effectively capturing ECs and SMCs in the blood and subsequent regeneration of new blood vessels. Therefore, this study opens a new possibility for regenerating neovessels to replace and repair damaged vessels for cardiovascular diseases treatment.

Smooth muscle cells differentiated from reprogrammed embryonic lung fibroblasts through DKK3 signaling are potent for tissue engineering of vascular grafts.

RATIONALE: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. OBJECTIVE: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state. METHODS AND RESULTS: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft. CONCLUSIONS: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

The influence of electrospun scaffold topography on endothelial cell morphology, alignment, and adhesion in response to fluid flow.

Bioengineered vascular grafts provide a promising alternative to autografts for replacing diseased or damaged arteries, but necessitate scaffold designs capable of supporting a confluent endothelium that resists endothelial cell (EC) detachment under fluid flow. To this end, we investigated whether tuning electrospun topography (i.e., fiber diameter and orientation) could impact EC morphology, alignment, and structural protein organization with the goal of forming a confluent and well-adhered endothelium under fluid flow. To test this, a composite polymer blend of poly(ε-caprolactone) (PCL) and type I collagen was electrospun to form scaffolds with controlled fiber diameters ranging from approximately 100-1,200 nm and with varying degrees of fiber alignment. ECs were seeded onto scaffolds, and cell morphology and degree of alignment were quantified using image analysis of fluorescently stained cells. Our results show that ECs form confluent monolayers on electrospun scaffolds, with cell alignment systematically increasing with a larger degree of fiber orientation. Additionally, cells on aligned electrospun scaffolds display thick F-actin bundles parallel to the direction of fiber alignment and strong VE-cadherin expression at cell-cell junctions. Under fluid flow, ECs on highly aligned scaffolds had greater resistance to detachment compared to cells cultured on randomly oriented and semi-aligned scaffolds. These results indicate that scaffolds with aligned topographies may be useful in forming a confluent endothelium with enhanced EC adhesion for vascular tissue engineering applications.

Endothelial differentiation of diabetic adipose-derived stem cells.

BACKGROUND: Diabetic (DM) patients frequently lack autologous vascular tissue required for revascularization procedures and dialysis access creation. We have developed a tissue-engineered graft that uses adipose-derived stem cells (ASC) as endothelial cell substitutes. Here, we compare DM versus nondiabetic (NDM) ASC in terms of isolation efficiency, proliferation, commitment toward endothelial lineage, and seeding onto the luminal surface of a graft. METHODS: ASC were isolated from liposuction specimens of vascular surgery patients. Proliferation was assessed by constructing growth curves over 14 d. ASC were differentiated in endothelial growth medium (EGM2). Endothelial commitment was assessed by measuring endothelial cell-specific gene expression (CD31, von Willebrand factor) and by cord formation on Matrigel. Finally, ASC were seeded onto a vascular scaffold, flow conditioned, and imaged with confocal microscopy. RESULTS: Diabetes did not alter ASC isolation efficiency (224,028 ± 20,231 cells/g adipose for DM (n = 53) versus 259,345 ± 15,441 cells/g adipose for NDM (n = 145; P = 0.21). Growth curves for DM (n = 6) and NDM (n = 6) also appeared similar. After culture in EGM2, upregulation of CD31 and von Willebrand factor message was observed in NDM; these markers were found within the primary cultures of DM but no upregulation was observed after culture in EGM2. Both groups exhibited similar cord formation on Matrigel and retention to vascular scaffolds. CONCLUSIONS: Isolation and proliferation studies suggest that adipose is a promising source of stem cells for tissue engineering in the DM population. The angiogenic potential of DM ASC appears intact; however, differences in acquisition of endothelial cell markers suggest that differentiation may be inhibited or delayed by diabetes.

In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept?

BACKGROUND: Tissue-engineered blood vessels (TEBVs) represent an innovative approach for overcoming reconstructive problems associated with vascular diseases by providing small-caliber vascular grafts. This study aimed to evaluate a novel biomaterial of bacterially synthesized cellulose (BC) as a potential scaffold for small-diameter TEBV. METHODS: Small-diameter blood vessels with a supramolecular fiber network structure consisting of tubular hydrogels from biodesigned cellulose were created using Gluconacetobacter strains and Matrix reservoir technology. BC tubes (length: 100 mm, inner diameter: 4.0-5.0 mm) were applied to replace the carotid arteries of 10 sheep for a period of 3 mo to gain further insights into (a) functional (in vivo) performance, (b) ability of providing a scaffold for the neoformation of a vascular wall and (c) their proinflammatory potential, and the (d) technical feasibility of the procedure. RESULTS: Preoperative analysis revealed a bursting strength of the grafts of approximately 800 mm Hg and suture retention strength of 4-5 N. Postexplantation analysis showed a patency rate of 50% (n = 5) and physiological performance of the patent grafts at 4, 8, and 12 wk postoperatively, compared with native arteries. Histologic analysis revealed a neoformation of a vascular wall-like structure along the BC scaffold consisting of immigrated vascular smooth muscle cells and a homogeneous endothelialization of the inner graft surface without signs of prothrombogenic or inflammatory potential. Scanning electron microscopy revealed a confluent luminal endothelial cell layer and the immigration of vascular smooth muscle cells into the BC matrix. CONCLUSIONS: BC grafts provide a scaffold for the neoformation of a three-layered vascular wall exhibit attractive properties for their use in future TEBV programs for cardiovascular surgery.