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Hibino N.,Johns Hopkins Hospital | Best C.A.,Tissue Engineering Program and Surgical Research | Engle A.,George Washington University | Ghimbovschi S.,George Washington University | And 6 more authors.
Tissue Engineering - Part A | Year: 2016

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. © Copyright 2016, Mary Ann Liebert, Inc. 2016.

Melchiorri A.J.,University of Maryland University College | Hibino N.,Tissue Engineering Program and Surgical Research | Yi T.,Nationwide Childrens Hospital | Lee Y.U.,Nationwide Childrens Hospital | And 5 more authors.
Biomacromolecules | Year: 2015

Surface modification of biodegradable vascular grafts is an important strategy to improve the in situ endothelialization of tissue engineered vascular grafts (TEVGs) and prevent major complications associated with current synthetic grafts. Important strategies for improving endothelialization include increasing endothelial cell mobilization and increased endothelial cell capture through biofunctionalization of TEVGs. The objective of this study was to assess two biofunctionalization strategies for improving endothelialization of biodegradable polyester vascular grafts. These techniques consisted of cross-linking heparin to graft surfaces to immobilize vascular endothelial growth factor (VEGF) or antibodies against CD34 (anti-CD34Ab). To this end, heparin, VEGF, and anti-CD34Ab attachment and quantification assays confirmed the efficacy of the modification strategy. Cell attachment and proliferation on these groups were compared to unmodified grafts in vitro and in vivo. To assess in vivo graft functionality, the grafts were implanted as inferior vena cava interpositional conduits in mice. Modified vascular grafts displayed increased endothelial cell attachment and activity in vivo, according to microscopy techniques, histological results, and eNOS expression. Inner lumen diameter of the modified grafts was also better maintained than controls. Overall, while both functionalized grafts outperformed the unmodified control, grafts modified with anti-CD34Ab appeared to yield the most improved results compared to VEGF-loaded grafts. © 2014 American Chemical Society.

Lee Y.U.,Tissue Engineering Program and Surgical Research | Yi T.,Tissue Engineering Program and Surgical Research | Tara S.,Tissue Engineering Program and Surgical Research | Lee A.Y.,Tissue Engineering Program and Surgical Research | And 3 more authors.
Journal of visualized experiments : JoVE | Year: 2014

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with ε-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (>90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

Lee Y.U.,Tissue Engineering Program and Surgical Research | Naito Y.,Texas Childrens Hospital | Kurobe H.,Tissue Engineering Program and Surgical Research | Breuer C.K.,Tissue Engineering Program and Surgical Research | Humphrey J.D.,Yale University
Journal of Biomechanics | Year: 2013

Multiple murine models have proven useful in studying the natural history of neovessel development in the tissue engineering of vascular grafts. Nevertheless, to better understand longitudinal changes in the biomechanics of such neovessels, we must first quantify native tissue structure and properties. In this paper, we present the first biaxial mechanical data for, and nonlinear constitutive modeling of, &QJ;the inferior vena cava from two models used in tissue engineering: wild-type C57BL/6 and immunodeficient CB-17 SCID/bg mice. Results show that inferior vena cava from the latter are significantly stiffer in the circumferential direction, both materially (as assessed by a stored energy function) and structurally (as assessed by the compliance), despite a lower intramural content of fibrillar collagen and similar wall thickness. Quantifying the natural history of neovessel development in different hosts could lead to increased insight into the mechanisms by which cells fashion and maintain extracellular matrix in order to match best the host stiffness while ensuring sufficient vascular integrity. © 2013.

Huang A.H.,Yale University | Lee Y.-U.,Tissue Engineering Program and Surgical Research | Calle E.A.,Yale University | Boyle M.,Yale University | And 3 more authors.
Tissue Engineering - Part C: Methods | Year: 2015

Conventional bioreactors are used to enhance extracellular matrix (ECM) production and mechanical strength of tissue-engineered vessels (TEVs) by applying circumferential strain, which is uniaxial stretching. However, the resulting TEVs still suffer from inadequate mechanical properties, where rupture strengths and compliance values are still very different from native arteries. The biomechanical milieu of native arteries consists of both circumferential and axial loading. Therefore, to better simulate the physiological stresses acting on native arteries, we built a novel bioreactor system to enable biaxial stretching of engineered arteries during culture. This new bioreactor system allows for independent control of circumferential and axial stretching parameters, such as displacement and beat rate. The assembly and setup processes for this biaxial bioreactor system are reliable with a success rate greater than 75% for completion of long-term sterile culture. This bioreactor also supports side-by-side assessments of TEVs that are cultured under three types of mechanical conditions (static, uniaxial, and biaxial), all within the same biochemical environment. Using this bioreactor, we examined the impact of biaxial stretching on arterial wall remodeling of TEVs. Biaxial TEVs developed the greatest wall thickness compared with static and uniaxial TEVs. Unlike uniaxial loading, biaxial loading led to undulated collagen fibers that are commonly found in native arteries. More importantly, the biaxial TEVs developed the most mature elastin in the ECM, both qualitatively and quantitatively. The presence of mature extracellular elastin along with the undulated collagen fibers may contribute to the observed vascular compliance in the biaxial TEVs. The current work shows that biaxial stretching is a novel and promising means to improve TEV generation. Furthermore, this novel system allows us to optimize biomechanical conditioning by unraveling the interrelationships among the applied mechanical stress, the resulting ECM properties, and the mechanics of TEVs. © 2015, Mary Ann Liebert, Inc.

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