Tissue-engineered Vascular Grafts Research Articles

Composite vascular scaffold combining electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves structure

While the field of tissue engineered vascular grafts has greatly advanced, many inadequacies still exist. Successfully developed scaffolds require mechanical and structural properties that match native vessels and optimal microenvironments that foster cell integration, adhesion and growth. We have developed a small diameter, three-layered composite vascular scaffold which consists of electrospun fibers and physically-crosslinked hydrogel with copper wire-induced grooves by combining the electrospinning and dip-coating methods. Scaffold morphology and mechanics were assessed, quantified and compared to native vessels. Scaffolds were seeded with Human Umbilical Vein Endothelial Cells (HUVECs), cultured in vitro for 3 days and were evaluated for cell viability and morphology. The results showed that composite scaffolds had adjustable mechanical strength and favorable biocompatibility, which is important in the future clinical application of Tissue-engineered vascular grafts (TEVGs).

Promotion of extracellular matrix deposition by human vascular smooth muscle cells and monocytes within a biodegradable polyurethane scaffold

Event Abstract Back to Event Promotion of extracellular matrix deposition by human vascular smooth muscle cells and monocytes within a biodegradable polyurethane scaffold Xiaoqing Zhang1, Kyle Battiston1, Rosalind Labow2, Craig Simmons1, 3, 4 and Paul Santerre1, 4 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Ottawa, Department of Biochemistry, Microbiology and Immunology, Canada 3 University of Toronto, Department of Mechanical and Industrial Engineering, Canada 4 University of Toronto, Faculty of Dentistry, Canada Introduction: Tissue engineered vascular grafts (TEVGs) have shown the potential to replace diseased arteries[1]. In developing TEVGs, it is desirable to incorporate contractile vascular smooth muscle cells (VSMCs) into biomaterial scaffolds and stimulate extracellular matrix (ECM) production allowing for fast maturation of tissue. It is also desired to have a polymeric scaffold which provides immunomodulatory capability early after implantation[2]. Monocytes are a predominant cell type at an implant site and play an important role in wound healing[3]. VSMC-monocyte co-culture on an immunomodulatory polyurethane (D-PHI) scaffold has previously demonstrated enhanced migration and proliferation of VSMCs, as well as a reduced pro-inflammatory monocytic state[4],[5]. The objective of this study was to evaluate the effect of varying VSMC:monocytes ratios on regulating ECM production in co-culture with D-PHI. Materials and Methods: D-PHI scaffolds were prepared as previously described[6]. Monocytes isolated from human peripheral blood (University of Toronto ethics approval #22203)[5] and human coronary artery VSMCs (Lonza CC-2583) (300k) were co-seeded in 50:50 RPMI:DMEM medium at VSMC:monocyte ratios of 1:2 (600k monocytes), 1:1 (300k monocytes), 2:1 (150k monocytes), 4:1 (75k monocytes), 8:1 (37.5k monocytes) and 1:0 (0 monocytes) on each D-PHI scaffold (6 mm diameter, 1 mm thickness) and maintained in culture for 4 weeks. Collagen production was assessed by hydroxyproline (OH-Pro) (accounts for 10% of total collagen[5]) and elastin expression was assessed by immunostaining with elastin-positive areas analyzed by ImageJ. Enzyme-linked immunosorbent assay (ELISA) was used to quantify insulin growth factor 1 (IGF-1, which promotes elastin production) and matrix metallo-proteinase 2 (MMP2, which degrades ECM proteins). Data were analyzed by analysis of variance (SPSS 17.0) using Tukey test for pair-wise comparisons. Statistical significance was reported for p < 0.05. Results and Discussion: Total OH-Pro quantification data (Fig. 1A) showed that collagen content was highest with 2:1, 4:1 and 8:1 VSMC:monocytes ratios, when compared to the previously reported co-culture ratio of 1:2 [5]. 2:1 and 8:1 ratios promoted more collagen deposition when compared to the 1:0 ratio. Immunostaining for elastin (Fig. 1B) demonstrated significantly more elastin on the scaffold for 2:1 and 4:1 vs. 1:2 and 1:0 VSMC:monocyte ratios. IGF-1 release was significantly higher in co-culture (1:2, 2:1 and 4:1 ratios) (Fig. 2A), however, a 1:2 co-culture ratio produced more MMP2 than other conditions (Fig. 2B) . Conclusion: This study demonstrates that the co-culture of VSMCs with a modest number of monocytes promotes ECM accumulation. The reduced ECM synthesis observed at the 1:0 and 1:2 co-culture ratios may be caused by lower matrix producing cytokine (IGF-1) (1:0 condition) and higher matrix degrading protein (MMP2) (1:2 condition) released respectively, in those conditions. Canadian Institutes of Health Research (CIHR) operating grant #230762.

Pericyte-based collagen scaffold for vascular tissue engineering

Event Abstract Back to Event Pericyte-based collagen scaffold for vascular tissue engineering Betül Çelebi-Saltik1, 2 and Beyza Gökçinar-Yagci1, 2 1 Hacettepe University, Department of Stem Cell Sciences, Graduate School of Health Sciences, Türkiye 2 Hacettepe University, Center for Stem Cell Research and Development, Türkiye Pericytes are a perivascular cell population embedded on the capillary wall in a shared basement membrane with the endothelium. During vasculogenesis and angiogenesis, endothelial cells secrete and/or present cell bound molecular cues that stimulate pericytes to proliferate, migrate and attach to nascent capillary tubes. It has been previously developed cell sheet engineering using temperature-responsive culture dishes in order to avoid traditional tissue engineering approaches. In this study our aim is to isolate human umbilical cord vein pericytes then differentiate into smooth muscle cells and fibroblasts then co-culture these cells on 3D collagen scaffold that will be used for generating tissue engineering vascular grafts. Pericytes were enzymatically isolated from human umbilical cord vein and were purified by using MACs cell separation with CD146 microbeads. They were cultured in fibroblast growth medium and smooth muscle growth medium for 21 days on poly(N-isopropylacrylamide) to obtain cell sheets. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibroblast markers; Tenascin-C and Collagen type I, smooth muscle cell markers; Caldesmon, Calponin and Alpha smooth muscle actin). Collagen gel was prepared with human collagen type I (3mg/mL) + 10X DMEM (adjustment pH of mixture to 7.2–7.6 using sterile 0.1 M NaOH). Collagen gel was covered by cell sheets (sandwich system) and vascular graft was incubated in 37oC for 7 days (Figure 1). The results indicated that perivascular cells could differentiate into fibroblast and smooth muscle and formed vascular graft with collagen gel. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support. The Scientific and Technological Research Council of Turkey (Project Number: 113S815) Keywords: Tissue Engineering, cell, 3D scaffold, tissue niche Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in mesenchymal and hematopoietic stem cell biology Citation: Çelebi-Saltik B and Gökçinar-Yagci B (2016). Pericyte-based collagen scaffold for vascular tissue engineering. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02836 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Betül Çelebi-Saltik Beyza Gökçinar-Yagci Google Scholar Betül Çelebi-Saltik Beyza Gökçinar-Yagci PubMed Betül Çelebi-Saltik Beyza Gökçinar-Yagci Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells

Event Abstract Back to Event Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells Beyza Gökçinar-Yagci1, 2 and Betül Çelebi-Saltik1, 2 1 Hacettepe University, Department of Stem Cell Sciences,Graduate School of Health Sciences, Türkiye 2 Hacettepe University, Center for Stem Cell Research and Development, Türkiye Pericytes are perivascular cells that surround endothelial cells of capillaries, microvessels and larger vessels; arteries and veins. They have important roles in vascular development (vasculogenesis and angiogenesis), stability, maturation and remodeling, regulation of vessel diameter and blood flow, blood pressure control, contractility and tone of vascular smooth muscle cells. The cell sheet engineering is based on the capability of cells to secrete and organize their own extracellular matrix (ECM). In this study our aim is to isolate human umbilical cord vein pericytes (CD146+ cells) then differentiate into smooth muscle cells and fibroblasts that will be used for generating tissue engineering vascular grafts and also reduce the time required to produce a tissue-engineered vascular graft. Human perivascular cells were isolated from umbilical cord vein and pericytes were purified by using MACs cell separation with CD146 microbeads. Perivascular cells were cultured in FGM-2, SMCGM-2 and EGM-2 medium for 21 days to investigate fibroblast, smooth muscle cell and endothelial cell differentiation. Cell differentiation was confirmed by immunofluorescence staining and flow cytometry analysis (fibroblast markers; Tenascin-C and Collagen type I, smooth muscle cell markers; Caldesmon and alpha smooth muscle actin, endothelial cell markers; VEGFR1, VEGFR2 and CD31). Next, a tissue-engineered vascular graft was fabricated by rolling the perivascular cells derived endothelial cell, smooth muscle cell and fibroblast sheets (Rapid cell sheet detachment from Poly(N-isopropylacrylamide)) around a mandrel. Different human ECM molecules such as collagen type I and IV, fibrin, elastin, fibronectin and glycosaminoglycans were used among the vascular layers to increase the mechanical properties of the graft. The results indicated that perivascular cells could differentiate into fibroblast, smooth muscle, endothelial cells and formed tubular vascular graft with ECM proteins. As a conclusion, differentiated pericytes offer an alternative cell source for constructing tissue engineered vascular graft. The authors wish to thank The Scientific and Technological Research Council of Turkey (Project Number: 113S815) for their financial support. The Scientific and Technological Research Council of Turkey (Project Number: 113S815) Keywords: Extracellular Matrix, Regenerative Medicine, 3D scaffold, tissue niche Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Gökçinar-Yagci B and Çelebi-Saltik B (2016). Engineered small diameter vascular grafts by cell sheet engineering with human umbilical cord vein perivascular cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01061 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Beyza Gökçinar-Yagci Betül Çelebi-Saltik Google Beyza Gökçinar-Yagci Betül Çelebi-Saltik Google Scholar Beyza Gökçinar-Yagci Betül Çelebi-Saltik PubMed Beyza Gökçinar-Yagci Betül Çelebi-Saltik Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Investigation of cell seeding and culture conditions for vascular smooth muscle cell culture on a degradable polyurethane scaffold

Event Abstract Back to Event Investigation of cell seeding and culture conditions for vascular smooth muscle cell culture on a degradable polyurethane scaffold Xiaoqing Zhang1, Kyle Battiston1, Craig Simmons1, 2, 3 and Paul Santerre1, 3 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Department of Mechanical and Industrial Engineering, Canada 3 University of Toronto, Faculty of Dentistry, Canada Introduction: The development of tissue engineered vascular grafts (TEVGs) requires large quantities of uniformly distributed vascular smooth muscle cells (VSMCs) in biomaterial scaffolds, VSMC proliferation, and induction of extracellular matrix (ECM) production[1]. A biodegradable polyurethane scaffold (D-PHI) has been shown to support an anti-inflammatory phenotype for monocytes, which are one of the first cells to migrate to an implant site and provide vital signalling for wound healing and new tissue regeneration[2]. Vitamin C (Vc) derivatives ( ascorbic acid (AA) and sodium ascorbate (SA)), have been shown to stimulate VSMC growth and ECM production, and modulate cytokine-release from monocytes[3]-[6]. The objective of the current study was to investigate cell seeding strategies on D-PHI, and to use the identified conditions to compare AA and SA with respect to supporting ECM production and maintaining a desired VSMC phenotype. Materials and Methods: D-PHI scaffolds were prepared using a previously established protocol[7]. Human coronary artery SMC (Lonza CC-2583) seeding optimization was performed on D-PHI disk-formed scaffolds (6 mm diameter, 1 mm thickness) to find the maximum number of cells that could be seeded as a function of time, frequency of seeding, and the seeding volume. Seeded scaffolds were analyzed for total DNA and stained by hematoxylin and eosin (H&E) for histology. 300,000 (300k) VSMCs were seeded onto each disk scaffold and treated with 50 µM, 100µM or 200µM AA or SA for 1 week and characterized for DNA and ECM production (glycosaminoglycans (GAG), collagen and elastin). Data were analyzed by analysis of variance (SPSS 17.0). Statistical significance was reported for p < 0.05. Results and Discussion: The highest scaffold seeding efficiency was achieved with 75k or 150k VSMCs (Fig 1A). However, scaffolds seeded with 75k up to 300k VSMCs all demonstrated good cell infiltration into the material (Fig 1B). 300k VSMCs was selected to be used to study the optimal loading volume despite having lower seeding efficiency since it achieved the greatest number of loaded cells with good infiltration. Of the loading volumes tested, 8.3 µl allowed maximum cell uptake into the porous scaffolds (Fig 1C). Given that lower cell concentrations seeded with greater efficiency, a two-stage seeding protocol with 150k cells/stage, loaded over 48 hr using the optimal volume of 8.3µl, was assessed (Fig. 1). The final seeding efficiency of this latter approach was close to 85% (Fig 1D). AA and SA comparative data are reported in Fig. 2 and show that SA outperformed AA in inducing GAG (50 µM, 100µM and 200µM) and elastin (100µM and 200µM) production. Conclusion: This study has identified important seeding conditions that improved cellularity in D-PHI scaffolds. When used to compare AA vs SA on DNA and ECM protein production it was found that SA outperformed AA. These conditions will be used for future TEVG development in co-culture with human monocytes. Canadian Institutes of Health Research operating grant #230762.

Preliminary In Vivo Evaluation of a Hybrid Armored Vascular Graft Combining Electrospinning and Additive Manufacturing Techniques.

In this study, we tested in vivo effectiveness of a previously developed poly-l-lactide/poly-ε-caprolactone armored vascular graft releasing heparin. This bioprosthesis was designed in order to overcome the main drawbacks of tissue-engineered vascular grafts, mainly concerning poor mechanical properties, thrombogenicity, and endothelialization. The bioprosthesis was successfully implanted in an aortic vascular reconstruction model in rabbits. All grafts implanted were patent at four weeks postoperatively and have been adequately populated by endogenous cells without signs of thrombosis or structural failure and with no need of antiplatelet therapy. The results of this preliminary study might warrant for further larger controlled in vivo studies to further confirm these findings.

Novel Association of miR-451 with the Incidence of TEVG Stenosis in a Murine Model.

The development of a tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiac surgery. Despite the successful translation of this technology, previous reports identify the primary mode of graft failure as stenosis secondary to intimal hyperplasia. MicroRNAs (miRNAs) regulate gene expression by interfering with mRNA function and recent research has suggested miRNA as a potential therapeutic target. The role of miRNAs in TEVGs during neotissue formation is currently unknown. In this study, we investigated if miRNAs regulate the inhibition of graft stenosis. Biodegradable PGA-P(LA/CL) scaffolds were implanted as inferior vena cava interposition grafts in a murine model (n = 14). Mice were sacrificed 14 days following implantation and TEVGs were harvested for histological analysis and miRNA profiling using Affymetrix miRNA arrays. Graft diameters were measured histologically, and the largest grafts (patent group) and smallest grafts (stenosed group) were profiled (n = 4 for each group). Cell population in each graft was analyzed with immunohistochemistry using antismooth muscle actin (SMA) and antimacrophage (F4/80) antibodies. The graft diameter was significantly greater in the patent group (0.63 ± 0.06 mm) than in the stenosed group (0.17 ± 0.06 mm) (p < 0.01). Cell proliferation was significantly greater in the stenosed grafts than in patent grafts (p < 0.01: SMA [187 ± 11 vs. 77 ± 8 cells] vs. p = 0.025: F4/80 [245 ± 23 vs. 187 ± 11 cells]). MiRNA array of 1416 genes showed that in stenosed grafts, mir-451, mir-338, and mir-466 were downregulated and mir-154 was upregulated. Mir-451 exhibited the greatest difference in expression between stenosed and patent grafts by -3.1-fold. Significant negative correlation was found between the expression of mir-451 and cell proliferation (SMA: r = -0.86, p = 0.003; F4/80: r = -0.89, p = 0.001). Our data, along with previous evidence that mir-451 regulates tumor suppressor genes, suggest that downregulation of mir-451 promotes acute proliferation of macrophages and smooth muscle cells, thereby inducing TEVG stenosis. Adequate expression of mir-451 may be critical for improving TEVG patency.

Computationally Optimizing the Compliance of a Biopolymer Based Tissue Engineered Vascular Graft.

Coronary heart disease is a leading cause of death among Americans for which coronary artery bypass graft (CABG) surgery is a standard surgical treatment. The success of CABG surgery is impaired by a compliance mismatch between vascular grafts and native vessels. Tissue engineered vascular grafts (TEVGs) have the potential to be compliance matched and thereby reduce the risk of graft failure. Glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen constructs were fabricated and mechanically tested in a previous study by our research group at 2, 8, and 24 hrs of GLUT vapor exposure. The current study details a computational method that was developed to predict the material properties of our constructs for crosslinking times between 2 and 24 hrs by interpolating the 2, 8, and 24 hrs crosslinking time data. matlab and abaqus were used to determine the optimal combination of fabrication parameters to produce a compliance matched construct. The validity of the method was tested by creating a 16-hr crosslinked construct of 130 μm thickness and comparing its compliance to that predicted by the optimization algorithm. The predicted compliance of the 16-hr construct was 0.00059 mm Hg-1 while the experimentally determined compliance was 0.00065 mm Hg-1, a relative difference of 9.2%. Prior data in our laboratory has shown the compliance of the left anterior descending porcine coronary (LADC) artery to be 0.00071 ± 0.0003 mm Hg-1. Our optimization algorithm predicts that a 258-μm-thick construct that is GLUT vapor crosslinked for 8.1 hrs would match LADC compliance. This result is consistent with our previous work demonstrating that an 8-hr GLUT vapor crosslinked construct produces a compliance that is not significantly different from a porcine coronary LADC.

Biomechanical Comparison of Glutaraldehyde-Crosslinked Gelatin Fibrinogen Electrospun Scaffolds to Porcine Coronary Arteries

Cardiovascular disease (CVD) is the leading cause of death for Americans. As coronary artery bypass graft surgery (CABG) remains a mainstay of therapy for CVD and native vein grafts are limited by issues of supply and lifespan, an effective readily available tissue-engineered vascular graft (TEVG) for use in CABG would provide drastic improvements in patient care. Biomechanical mismatch between vascular grafts and native vasculature has been shown to be the major cause of graft failure, and therefore, there is need for compliance-matched biocompatible TEVGs for clinical implantation. The current study investigates the biaxial mechanical characterization of acellular electrospun glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen cylindrical constructs, using a custom-made microbiaxial optomechanical device (MOD). Constructs crosslinked for 2, 8, and 24 hrs are compared to mechanically characterized porcine left anterior descending coronary (LADC) artery. The mechanical response data were used for constitutive modeling using a modified Fung strain energy equation. The results showed that constructs crosslinked for 2 and 8 hrs exhibited circumferential and axial tangential moduli (ATM) similar to that of the LADC. Furthermore, the 8-hrs experimental group was the only one to compliance-match the LADC, with compliance values of 0.0006±0.00018 mm Hg-1 and 0.00071±0.00027 mm Hg-1, respectively. The results of this study show the feasibility of meeting mechanical specifications expected of native arteries through manipulating GLUT vapor crosslinking time. The comprehensive mechanical characterization of cylindrical biopolymer constructs in this study is an important first step to successfully develop a biopolymer compliance-matched TEVG.

Abstract 14338: Fast Degrading Tissue-Engineered Vascular Graft With High Cellular Infiltration Inhibits Calcification of the Graft

Introduction: Tissue-engineered vascular grafts (TEVGs) are biologically active grafts which are entirely reconstituted by host-derived cells over an inflammation-mediated degradation process. Calcification of the graft is a detrimental condition that can severely impact graft performance. Therefore, preventing graft calcification is of great importance to success of arterial TEVGs. Hypothesis: We assessed the hypothesis that the fast degrading TEVGs with high cellular infiltration into the scaffold could inhibit calcification of the grafts. Methods: We created two versions of aortic TEVGs: 1) Slow Degrading (SD) group and 2) Fast Degrading (FD) group. Both groups had the same inner layer composed of a 50:50 poly (l-lactic-co- ε -caprolactone) copolymer (PLCL) scaffold. However, the outer layer of the SD group was composed of a poly (l-lactic acid) (PLA) nano-fiber whereas the outer layer of the FD group was composed of a combination of PLA and polyglycolic acid (PGA) nano-fiber. The PGA in the outer layer of the FD group will degrade around 2-4 weeks after implantation, which will let more inflammatory cells infiltrate the scaffold than in the SD group. Both TEVGs were implanted in 8-10 week old female mice (n = 10 in each group) as infra-renal aortic interposition conduits. Animals were followed for 8 weeks. Results: In the SD group, acute thrombosis occurred in two mice. Von Kossa staining showed calcification of the scaffold in 6 out of 8 grafts in the SD group, but none in the FD group (Fig. A). The cell number in the outer layer of the FD group was significantly higher than in the SD group (2.65 ± 1.91 x 10 3 /mm 2 vs. 0.87 ± 0.65 x 10 3 /mm 2 , FD group and SD group, respectively, p =0.02); no significant difference was observed in the inner layer (1.80 ± 0.53 x 10 3 /mm 2 vs. 1.75 ± 0.60 x 10 3 /mm 2 , FD group and SD group, respectively, p =0.85) (Fig. A, B). Conclusions: In conclusion the fast degrading TEVG with high cellular infiltration inhibited calcification of the grafts.

Hybrid small-diameter vascular grafts: Anti-expansion effect of electrospun poly ε-caprolactone on heparin-coated decellularized matrices

Small-diameter vascular grafts (SDVGs) (D < 6 mm) are increasingly needed in clinical settings for cardiovascular disease, including coronary artery and peripheral vascular pathologies. Vessels made from synthetic polymers have shortcomings such as thrombosis, intimal hyperplasia, calcification, chronic inflammation and no growth potential. Decellularized xenografts are commonly used as a tissue-engineering substitute for vascular reconstructive procedures. Although acellular allogeneic vascular grafts have good histocompatibility and antithrombotic properties, the decellularization process may damage the biomechanics and accelerate the elastin deformation and degradation, finally resulting in vascular graft expansion and even aneurysm formation. Here, to address these problems, we combine synthetic polymers with natural decellularized small-diameter vessels to fabricate hybrid tissue-engineered vascular grafts (HTEV). The donor aortic vessels were decellularized with a combination of different detergents and dehydrated under a vacuum freeze-drying process. Polycaprolactone (PCL) nanofibers were electrospun (ES) outside the acellular aortic vascular grafts to strengthen the decellularized matrix. The intimal surfaces of the hybrid small-diameter vascular grafts were coated with heparin before the allograft transplantation. Histopathology and scanning electron microscope revealed that the media of the decellularized vessels were severely injured. Mechanical testing of scaffolds showed that ES-PCL significantly enhanced the biomechanics of decellularized vessels. Vascular ultrasound and micro-CT angiography showed that all grafts after implantation in a rat model were satisfactorily patent for up to 6 weeks. ES-PCL successfully prevented the occurrence of vasodilation and aneurysm formation after transplantation and reduced the cell inflammatory infiltration. In conclusion, the HTEV with perfect histocompatibility and biomechanics provide a facile and useful technique for the development of SDVGs.

Novel method for the generation of tissue-engineered vascular grafts based on a highly compacted fibrin matrix

The generation of tissue-engineered blood vessel substitutes remains an ongoing challenge for cardiovascular tissue engineering. Full biocompatibility and immediate availability have emerged as central issues for clinical use. To address these issues, we developed a technique that allows the generation of highly stable tubular fibrin segments. The process is based on the compaction of fibrin in a custom-made high-speed rotation mold. In an automated process, fibrin is precipitated from plasma by means of the Vivostat® system. Following application to the rotating mold, the fibrin was compacted by centrifugal force and excess fluid was pressed out. This compaction results in increasing cross-links between the fibrin fibrils and a corresponding significant increase of biomechanical stability up to a burst strength of 230mm of mercury. The molding process allows for a simultaneous seeding procedure. In a first in vivo evaluation in a sheep model, segments of the carotid artery were replaced by tissue-engineered vascular grafts, generated immediately prior to implantation (n=6). Following subjection to the body’s remodeling mechanisms, the segments showed a high structural similarity to a native artery after explantation at 6months. Thus, this technique may represent a powerful tool for the generation of biomechanically stable vascular grafts immediately prior to implantation. Statement of significanceFibrin has previously been shown to be suitable as a matrix for the seeding of different celltypes and for that reason was widely used as scaffold in different fields of tissue engineering. Nevertheless, fibrin’s lack of stability has strongly limited its application. Our study describes a novel moulding technique for the generation of a highly compacted fibrin matrix. Using this approach, it was possible to optimize the engineering process of tubular fibrin segments to provide bioartificial vascular grafts within one hour with sufficient stability for immediate implantation in the arterial system. Thus, this technique may represent a powerful tool to get closer to the ultimate aim of an optimal bioartificial vascular graft.

Current Status of Cardiovascular Tissue Engineering

The development of vascular and heart valve surgeries have contributed to improve outcomes in patients with cardiovascu- lar disease. However, there are drawbacks, such as risk of infection and lack of growth potential. Tissue engineered vascular graft (TEVG) and tissue engineered heart valve (TEHV) hold great promise to address these drawbacks as the ideal TEVG and TEHV is easily implanted, biocompatible, non thrombogenic, durable, degradable, and ultimately remodels into native- like tissue. In general, the TEVG and/or TEHV concept consists of scaffold, cells for scaffold seeding, and a subsequent remodeling process driven by cell accumulation and proliferation, and/or biochemical and mechanical signaling. Despite rapid progress in the field over the past decade, small-diameter arterial TEVG and TEHV have not been translated into clinical applications successfully. To successfully utilize TEVGs and TEHVs clinically, further elucidation of the mecha- nisms for TEVG and TEHV remodeling and further translational research outcome evaluations will be required.

The Tissue-Engineered Vascular Graft-Past, Present, and Future.

Cardiovascular disease is the leading cause of death worldwide, with this trend predicted to continue for the foreseeable future. Common disorders are associated with the stenosis or occlusion of blood vessels. The preferred treatment for the long-term revascularization of occluded vessels is surgery utilizing vascular grafts, such as coronary artery bypass grafting and peripheral artery bypass grafting. Currently, autologous vessels such as the saphenous vein and internal thoracic artery represent the gold standard grafts for small-diameter vessels (<6 mm), outperforming synthetic alternatives. However, these vessels are of limited availability, require invasive harvest, and are often unsuitable for use. To address this, the development of a tissue-engineered vascular graft (TEVG) has been rigorously pursued. This article reviews the current state of the art of TEVGs. The various approaches being explored to generate TEVGs are described, including scaffold-based methods (using synthetic and natural polymers), the use of decellularized natural matrices, and tissue self-assembly processes, with the results of various in vivo studies, including clinical trials, highlighted. A discussion of the key areas for further investigation, including graft cell source, mechanical properties, hemodynamics, integration, and assessment in animal models, is then presented.

The Current Status of Tissue-Engineered Vascular Grafts.

Tissue-engineered vascular grafts (TEVGs) are currently being developed to overcome the limitations and complications associated with traditional synthetic grafts. This article aims to review the current status of research into the production and use of tissue-engineered grafts. TEVGs have a number of theoretical advantages over synthetic grafts. The results of animal and human studies have been promising, but more work must be done before TEVGs can replace traditional grafts.

Bio-inspired micropatterned hydrogel to direct and deconstruct hierarchical processing of geometry-force signals by human mesenchymal stem cells during smooth muscle cell differentiation

Micropatterned biomaterial-based hydrogel platforms allow the recapitulation of in vivo-like microstructural and biochemical features that are critical physiological regulators of stem cell development. Herein, we report the use of muscle mimicking geometries patterned on polyacrylamide hydrogels as an effective strategy to induce smooth muscle cell (SMC) differentiation of human mesenchymal stem cells (hMSCs). hMSCs were systemically coerced to elongate with varying aspect ratios (AR) (that is, 1:1, 5:1, 10:1 and 15:1) at a fixed projection area of ~7000 μm2. The results showed engineered cellular anisotropy with an intermediate AR 5:1 and AR 10:1, promoting the expression of alpha smooth muscle actin (α-SMA) and enhancement of contractile output. Further mechanistic studies indicated that a threshold cell traction force of ~3.5 μN was required for SMC differentiation. Beyond the critical cytoskeleton tension, hMSCs respond to higher intracellular architectural cues such as the stress fiber (SF) alignment, SF subtype expression and diphosphorylated myosin regulatory light-chain activity to promote the expression and incorporation of α-SMA to the SF scaffold. These findings underscore the importance of exploiting biomimetic geometrical cues as an effective strategy to guide hMSC differentiation and are expected to guide the rational design of advanced tissue-engineered vascular grafts. Muscle-mimicking geometries patterned on hydrogel facilitate the acquisition of a smooth-muscle-cell-like phenotype from human stem cells. Adult mesenchymal stem cells (MSCs) are a valuable source of cells for regenerative medicine. Now, researchers in Singapore and China have examined the complex interdependence of engineered cell shape and human transforming growth factor beta-1 treatment to direct smooth muscle cells differentiation of MSCs. They discovered that geometries resembling those of human smooth muscle cells provide the optimal shape for the expression and recruitment of the mechano-sensitive protein α-smooth muscle actin to the filamentous actin cytoskeleton. Based on these results, the researchers proposed an elegant model to describe the decision-making process that human MSCs take during differentiation into smooth muscle cells. The findings highlight the importance of using biomimetic cues for guiding differentiation of human MSCs. Inspired by the intrinsic morphology of smooth muscle cells (SMCs), a micropatterned hydrogel is developed to direct and define the boundary conditions for efficient SMC differentiation of human mesenchymal stem cells (hMSCs). The results show that in conjunction with TGF-β1 treatment, muscle-mimicking shapes with intermediate aspect ratios ranging from 5:1 to 10:1 exert the strongest pro-SMC differentiation effects in a structural–contractile force-dependent manner. These findings are expected to provide critical insights and design rules for vascular-related engineered tissue grafts.

Pericytes: Properties, Functions and Applications in Tissue Engineering.

Mesenchymal stem cells (MSCs) are one of the most studied adult stem cells and in recent years. They have become attractive agents/cell source for cellular therapy and regenerative medicine applications. During investigations about their origin, researchers hypothesized that perivascular regions are the common anatomical regions where MSCs come from and perivascular cells like pericytes (PCs) (Rouget cells, mural cells) are in vivo counterparts of MSCs. Beside capillaries and microvessels as their most common locations, PCs are also found in large vessels (arteries and veins). They can be isolated from several tissues and organs particularly from retina and brain. There are different approaches about their isolation, characterization and culture but there has been no common protocol yet because of the lack of defined PC-specific marker. They make special contact with endothelial cells in the basement membrane and have very important functions in several tissues and organs. They participate in vascular development, stabilization, maturation, and remodeling, blood pressure control, endothelial cell proliferation and differentiation, contractility of vascular smooth muscle cells, wound healing, vasculogenesis and angiogenesis, long-term maintenance of hematopoietic stem cells in bone marrow niche. Their multipotential differentiation capacity and participation in many events in the body make PCs preferred cells in tissue engineering applications including 3D blood-brain barrier models, skeletal muscle constructs, bone tissue engineering and tissue-engineered vascular grafts.

Development of small diameter nanofiber tissue engineered arterial grafts.

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.

Tissue-engineered vascular grafts

There is no doubt that the introduction of synthetic materials was the prerequisite for success of vascular surgery. Biological inertness, durability, eases of sterilization and modeling of synthetic vascular grafts contributed to their widespread use as in aortic and great vessels. However, analysis of the accumulated clinical experience in using of synthetic grafts showed that fascination with them was gradually replaced by cautious attitude, and sometimes by refusing, because in the presence of well-known advantages, synthetic grafts are prone to thrombosis and the development of infection. Thereby, it takes place searching of anticoagulant and antibiotic therapy schemes, and the ways of creation of such grafts which will minimize the risk of thrombus formation and the development of infectious complications. Not without reason one of such ways includes tissue engineering, which allows to create substitute for biological tissues and organs using the principles and methods of engineering and biology. Tissue engineering vascular grafts (TIVG), created on the basis of natural acellular allogeneic or xenogeneic vascular matrices and populated with patient cells, so personalized, are thought to be biocompatible, athrombogenic, and deprived of any deficiencies of synthetic grafts. Being biocompatible products, they will be able to grow and will be suitable not only for adults but also for children with cardiovascular defects. However, a number of questions related to the search of optimal conditions for obtaining TIVG remain open.

Biomechanical diversity despite mechanobiological stability in tissue engineered vascular grafts two years post-implantation.

Recent advances in vascular tissue engineering have enabled a paradigm shift from ensuring short-term graft survival to focusing on long-term stability and growth potential. We present the first experimental-computational study of a tissue-engineered vascular graft (TEVG) effectively over the full lifespan of the recipient. We show that grafts implanted within the venous circulation of mice remained patent over 2 years without thrombus, stenosis, or aneurysmal dilatation. Moreover, the gross appearance and mechanical properties of the grafts evolved to be similar to the host vein within 24 weeks, with mean neovessel geometry and properties remaining unchanged thereafter despite a continued turnover of extracellular matrix. Biomechanical diversity manifested after 24 weeks, however, via two subsets of grafts despite all procedures being the same. Computational modeling and associated immunohistological analyses suggested that this diversity likely resulted from a differential ratio of collagen types I and III, with lower I to III ratios promoting grafts having a compliance similar to the native vein. We submit that TEVGs can exhibit the desired long-term mechanobiological stability; hence, we must now focus on evaluating growth potential and optimizing scaffold properties to achieve compliance matching throughout neovessel development.