Tissue Engineering Of Small-diameter Blood Vessels Research Articles

Preparation and Characterization of a Novel Tissue-Engineered Blood Vessel of dECM/CS/Gel Bilayer Scaffolds

At present, decellularized extracellular matrix (dECM) has become a popular research material worldwide due to its good biocompatibility in the research of small-diameter tissue engineering blood vessels (diameter ≤6mm). In this study, the cells of porcine coronary artery were removed by acellular method to obtain dECM, which was coated with chitosan (CS)/gelatin (Gel) mixed hydrogels with different concentrations to bionic the natural blood vessel’s multilayer structure. The mechanical properties, microstructure, and biocompatibility of scaffolds were characterized. The experiment result showed that the bilayer scaffolds with different proportions were successfully constructed by this method, and the outer layers and inner layers of scaffolds were closely connected, and the pores were evenly distributed. The critical tension and elastic modulus of the five scaffolds showed no significant difference, and all scaffolds had good biocompatibility.

Klotho functionalization on vascular graft for improved patency and endothelialization

The Klotho (KL) gene is related to aging. In this study, SKL (secreted KL) and heparin were cross-linked to the acellular small intestinal submucosa (SIS). Based on this, tissue-engineered bioactive small blood vessels were constructed. The goal of this study was to determine whether the release of SKL could improve the patency of small-diameter tissue-engineered blood vessels (TEVs) through promoting cell adhesion. The recombinant human SKL protein was generated from HEK293 cells with overexpression of SKL. Then the SIS membrane was cross-linked with heparin and SKL respectively, to prepare heparin group and SKL group artificial vascular grafts. SKL treatment promoted endothelial cells proliferation and upregulated the levels of Focal adhesion kinase (FAK) phosphorylation and Ras homolog gene family, member A (RhoA). SKL effectively enhanced the endothelial cells adhesion on the SIS membrane. In vivo evaluation of SKL modified SIS grafts in rabbits exhibited increased patency rate, endothelialization, and smooth muscle regeneration. In this study, SKL-modified SIS grafts can effectively improve patency of small-diameter TEVs through enhancing cell adhesion, and it is expected to exhibit an important effect in the construction of substitutes for coronary artery bypass grafting.

Quantitative Evaluation of Mechanical Stimulation for Tissue-Engineered Blood Vessels.

Functional small-diameter tissue-engineered blood vessels (TEBVs) have been developed in silico using biodegradable polymeric scaffolds under pulsatile perfusion. Accurate simulation of physiological mechanical stimulations in vitro is a crucial factor in vascular engineering. However, little is known about the patterns of mechanical stimulation on silicone tubes. This study aimed to determine the optimal mechanical conditions required for inducing circumferential deformations in silicone tubes during in vitro vascular development under pulsatile perfusion. For this purpose, we established a data acquisition (DAQ) system with a laser micrometer and pressure transducers to evaluate changes in the diameter of silicone tubes in response to pulsatile flow and validated the results on cultured TEBVs. The established DAQ system showed satisfactory reproducibility for measuring diameter variation in the in silico model. Furthermore, the hardness and thickness of the silicone tubes affected the mechanical conditioning in the three-dimensional culture system under different working pressures, frequencies, and circumferential deformations. We demonstrated a simple and reliable approach to quantify the circumferential strain and deformations to ensure optimal mechanical stimulation of the cultured TEBVs under pulsatile perfusion. Based on the results, we were able to dynamically culture dense cellularized small-diameter TEBVs. This study highlights the importance of mechanical stimulation in vascular tissue engineering. Impact statement This study demonstrated a direct and noncontact data acquisition system for quantifying the strain on the supporting silicone medium during three-dimensional tissue-engineered blood vessel culture, which can help optimize the mechanical parameters for vascular tissue engineering.

Bio-composites reinforced with unique coral collagen fibers: Towards biomimetic-based small diameter vascular grafts

Cardiovascular Diseases (CVDs) are the leading cause of death worldwide. Approximately 31% of all global deaths are caused by CVDs, of which 42% are attributable to coronary artery disease (CAD). CAD is characterized by a narrowing of arteries that restricts the normal blood flow. Over time, surgical intervention is required in severe cases of occlusions and includes implantation of autologous vessels. Today synthetic grafts are used successfully as replacements for blood vessels with a diameter larger than 6 mm. However, they often fail as small-diameter blood vessel replacements.This study introduces a new biocomposite material system consisting of unique and long (cm-scale) collagen fibers derived from soft corals embedded within an alginate hydrogel matrix. The new biocomposite layers were used to fabricate grafts, towards developing a new class of tissue-engineered small-diameter blood vessels. These constructs consisted of both circumferentially and longitudinally oriented collagen fibers. The mechanical properties of the grafts were investigated via a new experimental setup constructed in our lab for this purpose, which applied internal pressure levels of 0–300 mmHg. Similar to native coronary arteries, the biocomposite tubes demonstrated a compliance of 4.88 ± 0.99%/100 mmHg for a physiologic pressure range of 80–120 mmHg. Furthermore, a numerical finite element simulation model is proposed to generate the overall mechanical response of the construct. It is composed of axial and circumferential fibers embedded within the continuum alginate elements. Good prediction is demonstrated when compared with the measured pressure-strain response. Moreover, we examined biocompatibility and cell growth on the collagen fibers. Fibroblast cells proliferated during the experiment that lasted for 32 days and showed aligned configuration with the collagen fiber orientation.The novelty of this study is manifested in the use of naturally derived coral-based long collagen fibers for the development of a new class of tissue-engineered grafts. The proposed novel biocomposite graft demonstrated both mechanical and biological compatibility and can be further developed for small-diameter blood-vessel replacement.

Design and characterization of small-diameter tissue-engineered blood vessels constructed by electrospun polyurethane-core and gelatin-shell coaxial fiber

ABSTRACT Substitution or bypass is the most effective treatment for vascular occlusive diseases. The demand for artificial blood vessels has seen an unprecedented rise due to the limited supply of autologous blood vessels. Tissue engineering is the best approach to provide artificial blood vessels. In this study, a new type of small-diameter artificial blood vessel with good mechanical and biological properties was designed by using electrospinning coaxial fibers. Four groups of coaxial fibers vascular membranes having polyurethane/gelatin core-shell structure were cross-linked by the EDC-NHS system and characterized. The core-shell structure of the coaxial vascular fibers was observed by transmission electron microscope. After the crosslinking, the stress and elastic modulus increased and the elongation decreased, burst pressure of 0.11 group reached the maximum (2844.55 ± 272.65 mmHg) after cross-linking, which acted as the experimental group. Masson staining identified blue-stained ring or elliptical gelatin ingredients in the vascular wall. The cell number in the vascular wall of the coaxial group was found in muscle embedding experiment significantly higher than that of the non-coaxial group at all time points(p < 0.001). Our results showed that the coaxial vascular graft with the ratio of 0.2:0.11 had better mechanical properties (burst pressure reached 2844.55 ± 272.65 mmHg); Meanwhile its biological properties were also outstanding, which was beneficial to cell entry and offered good vascular remodeling performance. Polyurethane (PU); Gelatin (Gel); Polycaprolactone (PCL); polylactic acid (PLA);1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC); N-Hydroxy succinimide (NHS); 4-Morpholine-ethane-sulfonic (MES); phosphate buffered saline (PBS); fetal calf serum (FCS); Minimum Essential Medium (MEM); Dimethyl sulfoxide (DMSO); hematoxylin-eosin (HE).

Preparation of PU/Fibrin Vascular Scaffold with Good Biomechanical Properties and Evaluation of Its Performance in vitro and in vivo.

PurposeThe development of tissue-engineered blood vessels provides a new source of donors for coronary artery bypass grafting and peripheral blood vessel transplantation. Fibrin fiber has good biocompatibility and is an ideal tissue engineering vascular scaffold, but its mechanical property needs improvement.MethodsWe mixed polyurethane (PU) and fibrin to prepare the PU/fibrin vascular scaffolds by using electrospinning technology in order to enhance the mechanical properties of fibrin scaffold. We investigated the morphological, mechanical strength, hydrophilicity, degradation, blood and cell compatibility of PU/fibrin (0:100), PU/fibrin (5:95), PU/fibrin (15:85) and PU/fibrin (25:75) vascular scaffolds. Based on the results in vitro, PU/fibrin (15:85) was selected for transplantation in vivo to repair vascular defects, and the extracellular matrix formation, vascular remodeling, and immune response were evaluated.ResultsThe results indicated that the fiber diameter of the PU/fibrin (15:85) scaffold was about 712nm. With the increase of PU content, the mechanical strength of the composite scaffolds increased, however, the degradation rate decreased gradually. The PU/fibrin scaffold showed good hydrophilicity and hemocompatibility. PU/fibrin (15:85) vascular scaffold could promote the adhesion and proliferation of mesenchymal stromal cells (MSCs). Quantitative RT-PCR experimental results showed that the expression of collagen, survivin and vimentin genes in PU/fibrin (15:85) was higher than that in PU/fibrin (25:75). The results in vivo indicated the mechanical properties and compliance of PU/fibrin grafts could meet clinical requirements and the proportion of thrombosis or occlusion was significantly lower. The graft showed strong vasomotor response, and the smooth muscle cells, endothelial cells, and ECM deposition of the neoartery were comparable to that of native artery after 3 months. At 3 months, the amount of macrophages in PU/fibrin grafts was significantly lower, and the secretion of pro-inflammatory and anti-inflammatory cytokines decreased.ConclusionPU/fibrin (15:85) vascular scaffolds had great potential to be used as small-diameter tissue engineering blood vessels.

In Vivo Performance of Decellularized Vascular Grafts: A Review Article.

Due to poor vessel quality in patients with cardiovascular diseases, there has been an increased demand for small-diameter tissue-engineered blood vessels that can be used as replacement grafts in bypass surgery. Decellularization techniques to minimize cellular inflammation have been applied in tissue engineering research for the development of small-diameter vascular grafts. The biocompatibility of allogenic or xenogenic decellularized matrices has been evaluated in vitro and in vivo. Both short-term and long-term preclinical studies are crucial for evaluation of the in vivo performance of decellularized vascular grafts. This review offers insight into the various preclinical studies that have been performed using decellularized vascular grafts. Different strategies, such as surface-modified, recellularized, or hybrid vascular grafts, used to improve neoendothelialization and vascular wall remodeling, are also highlighted. This review provides information on the current status and the future development of decellularized vascular grafts.

Innovative Tissue-Engineered and Synthetic Vascular Graft Models for the Treatment of PAD in Small-Diameter Arteries

Current treatment of peripheral arterial disease (PAD) incorporates a variety of methods that aim to restore normal vascular flow in the peripheral arteries. Treatment options to prevent complications such as chronic critical limb ischemia often include autografts, percutaneous transluminal angioplasty, stents, or synthetic grafts. In small-diameter arteries, such as those located in the lower leg, most of these treatment options often seek to control symptoms instead or trying to restore complete vascularization through the artery. There are design niches to come up with better alternatives to treat long-term complications derived from PAD in the lower limbs. To meet this goal, more research is still needed to dedicate to the treatment of PAD in small-diameter arteries. This review will provide design rationale and results on (a) major clinical breakthroughs in the development of small-diameter tissue-engineered blood vessels (TEBVs) and (b) innovative research involving TEBVs and synthetic vascular grafts. This review will also describe the main characteristics of the TEBV and synthetic vascular graft designs presented in these research groups to provide a basis for future research related to the treatment of PAD in small-diameter arteries. Excess fatty deposits within arteries can lead to occlusion in the small arteries below the knee. When this happens, there can be complications involving the loss of movement of the leg and foot. Methods primarily designed to treat larger arteries are commonly used to treat these complications. However, several issues may arise with these methods that often lead to additional treatment costs to patients. New techniques aimed specifically at replacing occluded arteries have been developed in the last few years. The reasoning behind these techniques and the studies that examined them are described in this review.

Construction of Antithrombotic Tissue-Engineered Blood Vessel via Reduced Graphene Oxide Based Dual-Enzyme Biomimetic Cascade.

Thrombosis is one of the biggest obstacles in the clinical application of small-diameter tissue-engineered blood vessels (TEBVs). The implantation of an unmodified TEBV will lead to platelet aggregation and further activation of the coagulation cascade, in which the high concentration of adenosine diphosphate (ADP) that is released by platelets plays an important role. Inspired by the phenomenon that endothelial cells continuously generate endogenous antiplatelet substances via enzymatic reactions, we designed a reduced graphene oxide (RGO) based dual-enzyme biomimetic cascade to successively convert ADP into adenosine monophosphate (AMP) and AMP into adenosine. We used RGO as a support and bound apyrase and 5'-nucleotidase (5'-NT) on the surface of RGO through covalent bonds, and then, we modified the surface of the collagen-coated decellularized vascular matrix with the RGO-enzyme complexes, in which RGO functions as a platform with a large open surface area and minimal diffusion barriers for substrates/products to integrate two catalytic systems for cascading reactions. The experimental results demonstrate that the two enzymes can synergistically catalyze procoagulant ADP into anticoagulant AMP and adenosine successively under physiological conditions, thus reducing the concentration of ADP. AMP and adenosine can weaken or even reverse the platelet aggregation induced by ADP, thereby inhibiting thrombosis. Adenosine can also accelerate the endothelialization of TEBVs by regulating cellular energy metabolism and optimizing the microenvironment, thus ensuring the antithrombotic function and patency of TEBVs even after the RGO-enzyme complex loses its activity.

Netrin-1 Promotes Inflammation Resolution to Achieve Endothelialization of Small-Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ.

The transplant of small‐diameter tissue engineering blood vessels (small‐diameter TEBVs) (<6 mm) in vascular replacement therapy often fails because of early onset thrombosis and long‐standing chronic inflammation. The specific inflammation state involved in small‐diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small‐diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin‐1) is found present in endothelial cells of natural blood vessels and has anti‐inflammatory effects. This work generates netrin‐1‐modified small‐diameter TEBVs by using layer‐by‐layer self‐assembly to resolve the inflammation. The results show that netrin‐1 reprograms macrophages (MΦ) to assume an anti‐inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small‐diameter TEBVs modified by netrin‐1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small‐diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ‐derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

Determining the optimal protocol for preparing an acellular scaffold of tissue engineered small-diameter blood vessels.

Although detergent-based decellularization protocols have been widely used to obtain a natural extracellular matrix (ECM) scaffold in tissue engineering, some key challenges still exist. To achieve an optimum natural decellularized scaffold for the construction of tissue-engineered small-diameter blood vessels (TEBV), porcine carotid arteries (PCAs) were decellularized by combining sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) and Triton X-100 (Triton) in different concentrations. Tissue samples were processed and their histological, biochemical and biomechanical characteristics were investigated. Results showed that only two methods 0.5% (SDS + SDC) and 1% (SDS + SDC) could completely remove of the cellular contents and preserve the native ECM architecture. Furthermore, 1% (SDS + SDC) based methods acquire preferable porosity and suitable mechanical strength. Residual Triton in the ECM scaffold holds intensive cytotoxity. In conclusion, 1%(SDS + SDC) based method can obtain a superior PCAs scaffold for the construction of TEBV. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 619-631, 2018.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

Maintaining Moderate Platelet Aggregation and Improving Metabolism of Endothelial Progenitor Cells Increase the Patency Rate of Tissue-Engineered Blood Vessels.

Small-diameter tissue-engineered blood vessels (TEBVs) have been associated with low, long-term patency rates primarily because of acute thrombosis in early stages and an inability to achieve early endothelialization. Platelets and endothelial progenitor cells (EPCs) play a key role in these processes. A nano delayed-release 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR)-bound TEBV was implanted in rat carotid arteries for 3 months. AICAR-bound TEBVs had a high patency rate compared with control TEBVs after 3 months. We found that AICAR maintained moderate platelet aggregation in vivo. In vitro data indicated that AICAR inhibits the release of 5-hydroxytryptamine and thromboxane A2 in activating platelets to reduce platelet aggregation. Then, we confirmed that AICAR strengthens the EPC energy state, which results in earlier endothelialization. The homing, migration, and paracrine function of EPCs were enhanced by AICAR in vitro. Besides, AICAR can contribute to the migration of endothelial cells near the anastomosis. The cellularization of TEBVs at different time points was observed too. In conclusion, our study suggests that the application of nanodelivery material containing AICAR can effectively improve small-diameter TEBVs by maintaining moderate platelet aggregation and improving metabolism of EPCs.

Late-outgrowth endothelial progenitors from patients with coronary artery disease: Endothelialization of confluent stromal cell layers

Patients with coronary artery disease (CAD) are the primary candidates to receive small-diameter tissue-engineered blood vessels (TEBVs). Peripheral blood derived endothelial progenitor cells (EPCs) from CAD patients (CAD EPCs) represent a minimally invasive source of autologous cells for TEBV endothelialization. We have previously shown that human CAD EPCs are highly proliferative and express many of the hallmarks of mature and healthy endothelial cells; however, their behavior on stromal cells that comprise the media of TEBVs has not yet been evaluated. Primary CAD EPCs or control human aortic endothelial cells (HAECs) were seeded over confluent, quiescent layers of human smooth muscle cells (SMCs) using a direct co-culture model. The percent coverage, adhesion strength, alignment under flow and generation of flow-induced nitric oxide of the seeded CAD EPCs were compared to that of HAECs. The integrin-binding profile of CAD EPCs was also evaluated over a layer of confluent, quiescent SMCs. Direct comparison of our CAD EPC results to analogous co-culture studies with cord blood EPCs show that both types of blood-derived EPCs are viable options for endothelialization of TEBVs.

Gradient nanofibrous chitosan/poly ɛ-caprolactone scaffolds as extracellular microenvironments for vascular tissue engineering

One of the major challenges of tissue-engineered small-diameter blood vessels is restenosis caused by thrombopoiesis. The goal of this study was to develop a 3D gradient heparinized nanofibrous scaffold, aiding endothelial cells lined on the lumen of blood vessel to prevent thrombosis. The vertical graded chitosan/poly ɛ-caprolactone (CS/PCL) nanofibrous vessel scaffolds were fabricated with chitosan and PCL by sequential quantity grading co-electrospinning. To mimic the natural blood vessel microenvironment, we used heparinization and immobilization of vascular endothelial growth factor (VEGF) in the gradient CS/PCL. The quantity of heparinized chitosan nanofibers increased gradually from the tunica adventitia to the lumen surfaces in the gradient CS/PCL wall of tissue engineered vessel. More heparin reacted to chitosan nanofiber in gradient CS/PCL than in uniform CS/PCL nanofibrous scaffolds. Antithrombogenic properties of the scaffolds were enhanced by the heparinization of these scaffolds, as shown by activated partial thromboplastin time and platelet adhesion assay. Compared to the uniform CS/PCL scaffold, the release of VEGF from the gradient CS/PCL was more stable and sustained, and the burst release of VEGF was reduced approximately 42.5% within the initial 12 h. The adhesion and proliferation of human umbilical vein endothelial cells (HUVEC) were enhanced on the gradient CS/PCL scaffold. Furthermore, HUVEC grew and formed an entire monolayer on the top side of the gradient CS/PCL scaffold. Therefore, use of vertical gradient heparinized CS/PCL nanofibrous scaffolds could provide an approach to create small-diameter blood vessel grafts with innate properties of mammalian vessels of anticoagulation and rapid induction of re-endothelialization.

The use of BDNF to enhance the patency rate of small-diameter tissue-engineered blood vessels through stem cell homing mechanisms

The patency rate of small-diameter tissue-engineered blood vessels is the determinant for their application in coronary artery bypass grafting. The coronary artery is innervated by vagus nerves. The origin of vagus nerves is rich in brain-derived neurotrophic factors (BDNF). We have investigated whether BDNF could improve the patency rate of small-diameter tissue-engineered blood vessels through promoting stem cell homing and paracrine activity. In vitro, we isolated early and late endothelial progenitor cells (EPCs) and found BDNF could promote single clone formation and paracrine function of EPCs, and could also induce the proliferation, migration and differentiation of late EPCs. BDNF could enhance the capturing of EPCs in parallel-plate flow chamber. Flow cytometric analysis and laser-scanning confocal microscope showed BDNF could enhance the mobilization and homing of C57BL/6 mouse EPCs after wire injury. Based on it, BDNF was coupled to the lumen surface of the blood vessel matrix material incubated with collagen through SPDP to construct BDNF-modified small-diameter tissue-engineered blood vessel. The blood vessel patency rate was significantly higher than that of control group 8 weeks after implantation in rats and the endothelialization level was superior to control. These results demonstrate that BDNF can effectively improve patency of small-diameter tissue-engineered blood vessels through stem cell homing and paracrine, and it is expected to play an important role in the construction of substitutes for coronary artery bypass grafting.

Intravital Molecular Imaging of Small-Diameter Tissue-Engineered Vascular Grafts in Mice: A Feasibility Study

Creating functional small-diameter tissue-engineered blood vessels has not been successful to date. Moreover, the processes underlying the in vivo remodeling of these grafts and the fate of cells seeded onto scaffolds remain unclear. Here we addressed these unmet scientific needs by using intravital molecular imaging to monitor the development of tissue-engineered vascular grafts (TEVG) implanted in mouse carotid artery. Green fluorescent protein-labeled human bone marrow-derived mesenchymal stem cells and cord blood-derived endothelial progenitor cells were seeded on polyglycolic acid-poly-L-lactic acid scaffolds to construct small-caliber TEVG that were subsequently implanted in the carotid artery position of nude mice (n = 9). Mice were injected with near-infrared agents and imaged using intravital fluorescence microscope at 0, 7, and 35 days to validate in vivo the TEVG remodeling capability (Prosense680; VisEn, Woburn, MA) and patency (Angiosense750; VisEn). Imaging coregistered strong proteolytic activity and blood flow through anastomoses at both 7 and 35 days postimplantation. In addition, image analyses showed green fluorescent protein signal produced from mesenchymal stem cell up to 35 days postimplantation. Comprehensive correlative histopathological analyses corroborated intravital imaging findings. Multispectral imaging offers simultaneous characterization of in vivo remodeling enzyme activity, functionality, and cell fate of viable small-caliber TEVG.

Three-Dimensional Microchannels in Biodegradable Polymeric Films for Control Orientation and Phenotype of Vascular Smooth Muscle Cells

The poor mechanical strength and vasoactivity of current small-diameter tissue engineered blood vessels (TEBVs) remain unsolved problems. Given the plasticity of smooth muscle cells (SMCs), 1 of the main limitations of current scaffolding techniques is the difficulty in controlling SMC phenotype shifts in vitro. A synthetic phenotype allows the cells to rapidly proliferate and produce extracellular matrix (ECM), whereas a shift to contractile phenotype with organized ECM ultimately provides a functional blood vessel. In this study, 3D deep (65 microm) and wide microchannels separated by high-aspect ratio (8) microwalls were successfully ultraviolet (UV) microembossed using a liquid UV polymerizable biodegradable macromer (poly(epsilon-caprolactone-r-L-lactide-r-glycolide) diacrylate) and the in vitro guidance effects of varying channel width (40-160 microm) on SMCs were verified. The results show that SMCs cultured in the wider microchannels (80-160 microm wide) switch from fibroblast morphology and random orientation to spindle-shaped morphology, and align along the direction of the microchannel nearing confluence achieved with similar cell density to unpatterned film. Further, an enhanced expression of smooth muscle alpha-actin of SMCs grown on micropatterns was found nearing confluence, which demonstrates a phenotype shift to a more contractile phenotype. These films are flexible and can be folded into tubular and lamellar structures for tissue engineering of small-diameter TEBVs as well as other organs such as esophagus or intestine. These results suggest that these micropatterned synthetic biodegradable scaffolds may be useful for guiding SMCs to grow into functional, small-diameter vascular grafts.

Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo.

Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases.