Tissue-Engineered Vessel Graft Research Articles

Challenges and Strategies for Endothelializing Decellularized Small-Diameter Tissue-Engineered Vessel Grafts.

Vascular diseases are the leading cause of ischemic necrosis in tissues and organs, necessitating using vascular grafts to restore blood supply. Currently, small vessels for coronary artery bypass grafts are unavailable in clinical settings. Decellularized small-diameter tissue-engineered vessel grafts (SD-TEVGs) hold significant potential. However, they face challenges, as simple implantation of decellularized SD-TEVGs in animals leads to thrombosis and calcification due to incomplete endothelialization. Consequently, research and development focus has shifted toward enhancing the endothelialization process of decellularized SD-TEVGs. This paper reviews preclinical studies involving decellularized SD-TEVGs, highlighting different strategies and their advantages and disadvantages for achieving rapid endothelialization of these vascular grafts. Methods are analyzed to improve the process while addressing potential shortcomings. This paper aims to contribute to the future commercial viability of decellularized SD-TEVGs.

Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering.

The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling.

Meet the First Authors.

Meet the First Authors.

Binding of Dickkopf-3 to CXCR7 Enhances Vascular Progenitor Cell Migration and Degradable Graft Regeneration.

Vascular progenitor cells play key roles in physiological and pathological vascular remodeling-a process that is crucial for the regeneration of acellular biodegradable scaffolds engineered as vital strategies against the limited availability of healthy autologous vessels for bypass grafting. Therefore, understanding the mechanisms driving vascular progenitor cells recruitment and differentiation could help the development of new strategies to improve tissue-engineered vessel grafts and design drug-targeted therapy for vessel regeneration. In this study, we sought to investigate the role of Dkk3 (dickkopf-3), recently identified as a cytokine promotor of endothelial repair and smooth muscle cell differentiation, on vascular progenitor cells cell migration and vascular regeneration and to identify its functional receptor that remains unknown. Vascular stem/progenitor cells were isolated from murine aortic adventitia and selected for the Sca-1 (stem cell antigen-1) marker. Dkk3 induced the chemotaxis of Sca-1+ cells in vitro in transwell and wound healing assays and ex vivo in the aortic ring assay. Functional studies to identify Dkk3 receptor revealed that overexpression or knockdown of chemokine receptor CXCR7 (C-X-C chemokine receptor type 7) in Sca-1+ cells resulted in alterations in cell migration. Coimmunoprecipitation experiments using Sca-1+ cell extracts treated with Dkk3 showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high-affinity Dkk3-CXCR7 binding with a dissociation constant of 14.14 nmol/L. Binding of CXCR7 by Dkk3 triggered the subsequent activation of ERK1/2 (extracellular signal-regulated kinases 1/2)-, PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)-, Rac1 (Ras-related C3 botulinum toxin substrate 1)-, and RhoA (Ras homolog gene family, member A)-signaling pathways involved in Sca-1+ cell migration. Tissue-engineered vessel grafts were fabricated with or without Dkk3 and implanted to replace the rat abdominal aorta. Dkk3-loaded tissue-engineered vessel grafts showed efficient endothelization and recruitment of vascular progenitor cells, which had acquired characteristics of mature smooth muscle cells. CXCR7 blocking using specific antibodies in this vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodeling. We provide a novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced vascular progenitor migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.

Abstract 479: Dickkopf-3 Binds to CXCR7 of Vascular Progenitor Cells to Enhance Degradable Graft Regeneration

Background: Dickkopf-3 (Dkk3) is a secreted protein that may have a role in vascular diseases by promoting smooth muscle cell (SMC) differentiation and endothelial repair, two processes in which vascular stem/progenitor cells could be involved. However, the effect of Dkk3 on stem/progenitor cell migration and its specific receptor on these cells remain unknown. Methods: Vascular stem/progenitor cells (VPCs) were isolated from murine aortic adventitia and selected for the Sca-1 marker. The chemotactic ability of Dkk3 for Sca-1+ cells was tested in vitro using transwell and wound healing assays and ex vivo by the aortic ring assay. Chemokine receptors identification was carried out using Western blot (WB), siRNA-mediated knockdown, co-Immunoprecipitation (Co-IP) and saturation binding assays. To assess in vivo the role of Dkk3 in recruiting Sca-1+ cells, tissue-engineered vessel grafts, with or without Dkk3, were fabricated and implanted to replace the rat abdominal aorta. Results: We demonstrated that Dkk3 induced the chemotaxis of Sca-1+ cells in vitro , and ex vivo in culture of aortic rings derived from Sca-1-GFP transgenic mice. Flow cytometry and WB analysis revealed that Sca-1+ cells expressed CXCR7, while overexpression or knockdown of CXCR7 resulted in alterations in cell migration. Interestingly, Co-IP experiments showed the physical interaction between DKK3 and CXCR7, and specific saturation binding assays identified a high affinity Dkk3-receptor binding with a dissociation constant of 14.14 nM. Activation of CXCR7 by Dkk3 triggered the ERK1/2, PI3K/AKT, Rac1 and RhoA signalling pathways. Furthermore, when Dkk3-loaded tissue-engineered vessels were grafted to the abdominal artery of rats, the grafts showed efficient endothelization and recruitment of VPCs, which had acquired characteristics of mature SMCs. CXCR7 blocking using specific antibodies in our vessel graft model hampered stem/progenitor cell recruitment into the vessel wall, thus compromising vascular remodelling. Conclusions: We provide novel and solid evidence that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced VPCs migration in vitro and in tissue-engineered vessels, hence harnessing patent grafts resembling native blood vessels.

Contact-free monitoring of vessel graft stiffness - proof of concept as a tool for vascular tissue engineering.

Tissue-engineered vessel grafts have to mimic the biomechanical properties of native blood vessels. Manufacturing processes often condition grafts to adapt them to the target flow conditions. Graft stiffness is influenced by material properties and dimensions and determines graft compliance. This proof-of-concept study evaluated a contact-free method to monitor biomechanical properties without compromising sterility. Forced vibration response analysis was performed on human umbilical vein (HUV) segments mounted in a buffer-filled tubing system. A linear motor and a dynamic signal analyser were used to excite the fluid by white noise (0-200Hz). Vein responses were read out by laser triangulation and analysed by fast Fourier transformation. Modal analysis was performed by monitoring multiple positions of the vessel surface. As an inverse model of graft stiffening during conditioning, HUV were digested proteolytically, and the course of natural frequencies (NFs) was monitored over 120min. Human umbilical vein showed up to five modes with NFs in the range of 5-100Hz. The first natural frequencies of HUV did not alter over time while incubated in buffer (p=0.555), whereas both collagenase (-35%, p=0.0061) and elastase (-45%, p<0.001) treatments caused significant decreases of NF within 120min. Decellularized HUV showed similar results, indicating that changes of the extracellular matrix were responsible for the observed shift in NF. Performing vibration response analysis on vessel grafts is feasible without compromising sterility or integrity of the samples. This technique allows direct measurement of stiffness as an important biomechanical property, obviating the need to monitor surrogate parameters. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanical and seeding properties of human umbilical vein – a potential scaffold for a tissue-engineered vessel graft

Objectives: The mechanical properties and seeding with endothelial cells were investigated in fresh and cryopreserved human umbilical vein. Methods: Human umbilical veins (HUV) were frozen in Euro-Collins/1M DMSO at –1°C/min and stored in liquid nitrogen. Stress-strain relationships of fresh and thawed veins were determined in an uniaxial tension-testing rig. HUV endothelial cells (HUVEC) were seeded onto denuded HUV under static conditions and grown for 3d. Luminal surfaces were analyzed by scanning electron microscopy. Calcein-stained cells were seeded hyperconfluently to determine the cell retention capacity of fresh and cryopreserved veins. Results: The stress-strain relationships of HUV followed a biphasic pattern typical for natural vessels. Neither the failure stress (2.71±0.36 vs. 3.25±0.97 N, n=3) nor the displacement required to achieve failure (9.73±0.9 vs. 7.43±2.07mm, n=3) were altered by cryopreservation. The burst pressure was estimated as approx. 1000mm Hg within the limitations of the uniaxial model. HUVEC seeded onto denuded HUV formed patches (at 9E3 cells per cm2) or an almost confluent endothelium (at 3E4 cells per cm2) within three days. The capacity to retain seeded HUVEC of denuded HUV was not altered by cryopreservation (1.15±0.08E5 vs. 1.26±0.14E5 cells per cm2, n=6). Conclusions: The burst pressure of HUV seems to be sufficiently high for the human arterial circulation and is not altered by cryopreservation. HUVEC can establish a confluent endothelium on denuded HUV. Therefore HUV appears to be a suitable storable scaffold for vascular tissue engineering.

Properties of the Human Umbilical Vein as a Living Scaffold for a Tissue-Engineered Vessel Graft

Umbilical cords are usually discarded after delivery, even though they contain a set of functional vessels. We investigated whether the human umbilical vein (HUV) is suitable as a storable scaffold for the tissue engineering of small-caliber vessel grafts. Isolated HUVs were cryopreserved by freezing or vitrification. The reaction of the vessels to vasoactive compounds and the mechanical properties were determined in an organ bath. Mitochondrial metabolism, release of antithrombotic compounds, and platelet adhesion were measured on the luminal vessel surface. Seeding with endothelial cells was tested on denuded HUVs. The vessels showed a weak response to norepinephrine but were readily contracted by serotonin and by the thromboxane A2 mimetic U46619. Endothelium-dependent vasorelaxation was weak, reaching significance only for histamine. However, the vessels relaxed to sodium nitroprusside, and to acetylcholine if sandwiched with human saphenous vein. Cryopreservation did not change the mechanical properties in the relevant tension range. Vasoconstriction to potassium chloride and serotonin were reduced after freezing (22.9 ± 7.6%, 27.7 ± 10.2%) and after vitrification (2.6 ± 5.8%, 4.3 ± 7.1%). The mitochondrial metabolism was also attenuated after freezing (57.9 ± 25.9%) and after vitrification (21.7 ± 6.7%). Prostacyclin release was elevated after both cryopreservation procedures (4.0-fold, 3.9-fold), whereas there was no significant change in the adhesion of platelets. Denuded HUVs could readily be seeded with isolated endothelial cells before and after freezing. We conclude that HUV is suitable as a storable living scaffold with antithrombogenic properties.

Properties of the Human Umbilical Vein As a Living Scaffold for a Tissue-Engineered Vessel Graft

Properties of the Human Umbilical Vein As a Living Scaffold for a Tissue-Engineered Vessel Graft

Properties of the Human Umbilical Vein as a Living Scaffold for a Tissue-Engineered Vessel Graft

Umbilical cords are usually discarded after delivery, even though they contain a set of functional vessels. We investigated whether the human umbilical vein (HUV) is suitable as a storable scaffold for the tissue engineering of small-caliber vessel grafts. Isolated HUVs were cryopreserved by freezing or vitrification. The reaction of the vessels to vasoactive compounds and the mechanical properties were determined in an organ bath. Mitochondrial metabolism, release of antithrombotic compounds, and platelet adhesion were measured on the luminal vessel surface. Seeding with endothelial cells was tested on denuded HUVs. The vessels showed a weak response to norepinephrine but were readily contracted by serotonin and by the thromboxane A2 mimetic U46619. Endothelium-dependent vasorelaxation was weak, reaching significance only for histamine. However, the vessels relaxed to sodium nitroprusside, and to acetylcholine if sandwiched with human saphenous vein. Cryopreservation did not change the mechanical properties in the relevant tension range. Vasoconstriction to potassium chloride and serotonin were reduced after freezing (22.9+/-7.6%, 27.7+/-10.2%) and after vitrification (2.6+/-5.8%, 4.3+/-7.1%). The mitochondrial metabolism was also attenuated after freezing (57.9+/-25.9%) and after vitrification (21.7+/-6.7%). Prostacyclin release was elevated after both cryopreservation procedures (4.0-fold, 3.9-fold), whereas there was no significant change in the adhesion of platelets. Denuded HUVs could readily be seeded with isolated endothelial cells before and after freezing. We conclude that HUV is suitable as a storable living scaffold with antithrombogenic properties.

Properties of the Human Umbilical Vein As a Living Scaffold for a Tissue-Engineered Vessel Graft

Properties of the Human Umbilical Vein As a Living Scaffold for a Tissue-Engineered Vessel Graft

Effect of cryopreservation on viability and function of human umbilical vein – a potential scaffold for a tissue-engineered vessel graft

Objectives: Human umbilical veins are readily available small-caliber vessels with a non-thrombogenic surface. The vitality and the contractile as well as antithrombogenic function before and after cryopreservation were compared.