Toshiharu Shinoka

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

Late-term results of tissue-engineered vascular grafts in humans

OBJECTIVEnThe development of a tissue-engineered vascular graft with the ability to grow and remodel holds promise for advancing cardiac surgery. In 2001, we began a human trial evaluating these grafts in patients with single ventricle physiology. We report the late clinical and radiologic surveillance of a patient cohort that underwent implantation of tissue-engineered vascular grafts as extracardiac cavopulmonary conduits.nnnMETHODSnAutologous bone marrow was obtained and the mononuclear cell component was collected. Mononuclear cells were seeded onto a biodegradable scaffold composed of polyglycolic acid and epsilon-caprolactone/L-lactide and implanted as extracardiac cavopulmonary conduits in patients with single ventricle physiology. Patients were followed up by postoperative clinic visits and by telephone. Additionally, ultrasonography, angiography, computed tomography, and magnetic resonance imaging were used for postoperative graft surveillance.nnnRESULTSnTwenty-five grafts were implanted (median patient age, 5.5 years). There was no graft-related mortality (mean follow-up, 5.8 years). There was no evidence of aneurysm formation, graft rupture, graft infection, or ectopic calcification. One patient had a partial mural thrombosis that was successfully treated with warfarin. Four patients had graft stenosis and underwent successful percutaneous angioplasty.nnnCONCLUSIONnTissue-engineered vascular grafts can be used as conduits in patients with single ventricle physiology. Graft stenosis is the primary mode of graft failure. Further follow-up and investigation for the mechanism of stenosis are warranted.

Vascular tissue engineering: towards the next generation vascular grafts.

The application of tissue engineering technology to cardiovascular surgery holds great promise for improving outcomes in patients with cardiovascular diseases. Currently used synthetic vascular grafts have several limitations including thrombogenicity, increased risk of infection, and lack of growth potential. We have completed the first clinical trial evaluating the feasibility of using tissue engineered vascular grafts (TEVG) created by seeding autologous bone marrow-derived mononuclear cells (BM-MNC) onto biodegradable tubular scaffolds. Despite an excellent safety profile, data from the clinical trial suggest that the primary graft related complication of the TEVG is stenosis, affecting approximately 16% of grafts within the first seven years after implantation. Continued investigation into the cellular and molecular mechanisms underlying vascular neotissue formation will improve our basic understanding and provide insights that will enable the rationale design of second generation TEVG.

A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts

The primary graft‐related complication during the first clinical trial evaluating the use of tissue‐engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11b‐diphtheria toxin‐receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P= 0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.—Hibino, N., Yi, T., Duncan, D. R., Rathore, A., Dean, E., Naito, Y., Dardik, A., Kyriakides, T., Madri, J., Pober, J. S., Shinoka, T., Breuer, C. K. A critical role for macrophages in neovessel formation and the development of stenosis in tissue‐engineered vascular grafts. FASEB J. 25, 4253–4263 (2011). www.fasebj.org

Tissue engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model

Introduction:The development of a living, autologous vascular graft with the ability to grow holds great promise for advancing the field of pediatric cardiothoracic surgery. Objective:To evaluate the growth potential of a tissue-engineered vascular graft (TEVG) in a juvenile animal model. Methods:Polyglycolic acid nonwoven mesh tubes (3-cm length, 1.3-cm id; Concordia Fibers) coated with a 10% copolymer solution of 50:50 l-lactide and &egr;-caprolactone were statically seeded with 1 × 106 cells/cm2 autologous bone marrow derived mononuclear cells. Eight TEVGs (7 seeded, 1 unseeded control) were implanted as inferior vena cava (IVC) interposition grafts in juvenile lambs. Subjects underwent bimonthly magnetic resonance angiography (Siemens 1.5 T) with vascular image analysis (www.BioimageSuite.org). One of 7-seeded grafts was explanted after 1 month and all others were explanted 6 months after implantation. Neotissue was characterized using qualitative histologic and immunohistochemical staining and quantitative biochemical analysis. Results:All grafts explanted at 6 months were patent and increased in volume as measured by difference in pixel summation in magnetic resonance angiography at 1 month and 6 months. The volume of seeded TEVGs at explant averaged 126.9% ± 9.9% of their volume at 1 month. Magnetic resonance imaging demonstrated no evidence of aneurysmal dilation. TEVG resembled the native IVC histologically and had comparable collagen (157.9 ± 26.4 &mgr;g/mg), elastin (186.9 ± 16.7 &mgr;g/mg), and glycosaminoglycan (9.7 ± 0.8 &mgr;g/mg) contents. Immunohistochemical staining and Western blot analysis showed that Ephrin-B4, a determinant of normal venous development, was acquired in the seeded grafts 6 months after implantation. Conclusions:TEVGs demonstrate evidence of growth and venous development when implanted in the IVC of a juvenile lamb model.

Concise Review: Tissue-Engineered Vascular Grafts for Cardiac Surgery: Past, Present, and Future

In surgical repair for heart or vascular disease, it is often necessary to implant conduits or correct tissue defects. The most commonly used graft materials to date are (a) artificial grafts; (b) autologous tissues, such as pericardium and saphenous vein; (c) allografts; and (d) xenografts. However, none of these four options offer growth potential, and all are associated with varying levels of thrombogenicity and susceptibility to infection. The lack of growth potential of these four options is particularly important in pediatric cardiac surgery, where patients will often outgrow their vascular grafts and require additional operations. Thus, developing a material with sufficient durability and growth potential that will function as the child grows older will eliminate the need for reoperation and significantly reduce morbidity and mortality of some types of congenital heart defects. Vascular tissue engineering is a relatively new field that has undergone enormous growth over the last decade. The goal of vascular tissue engineering is to produce neovessels and neo‐organ tissue from autologous cells using a biodegradable polymer as a scaffold. The most important advantage of tissue‐engineered implants is that these tissues can grow, remodel, rebuild, and respond to injury. Once the seeded autologous cells have deposited an extracellular matrix and the original scaffold is biodegraded, the tissue resembles and behaves as native tissue. When tissue‐engineered vascular grafts are eventually put to use in the clinical arena, the quality of life in patients after surgery will be drastically improved.

Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel

We developed a tissue‐engineered vascular graft composed of biodegradable scaffold seeded with autologous bone marrow‐derived mononuclear cells (BMMCs) that is currently in clinical trial and developed analogous mouse models to study mechanisms of neovessel formation. We previously reported that seeded human BMMCs were rapidly lost after implantation into immunodeficient mice as host macrophages invaded the graft. As a consequence, the resulting neovessel was entirely of host cell origin. Here, we investigate the source of neotissue cells in syngeneic BMMC‐seeded grafts, implanted into immunocompetent mouse recipients. We again find that seeded BMMCs are lost, declining to 0.02% at 14 d, concomitant with host macrophage invasion. In addition, we demonstrate using sex‐mismatched chimeric hosts that bone marrow is not a significant source of endothelial or smooth muscle cells that comprise the neovessel. Furthermore, using composite grafts formed from seeded scaffold anastomosed to sex‐mismatched natural vessel segments, we demonstrate that the adjacent vessel wall is the principal source of these endothelial and smooth muscle cells, forming 93% of proximal neotissue. These findings have important implications regarding fundamental mechanisms underlying neotissue formation; in this setting, the tissue‐engineered construct functions by mobilizing the bodys innate healing capabilities to “regenerate” neotissue from preexisting committed tissue cells.— Hibino, N., Villalona, G., Pietris, N., Duncan, D. R., Schoffner, A., Roh, J. D., Yi, T., Dobrucki, L. W., Mejias, D., Sawh‐Martinez, R., Harrington, J. K., Sinusas, A., Krause, D. S., Kyriakides, T., Saltzman, W. M., Pober, J. S., Shinoka, T., Breuer, C. K. Tissue‐engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel. FASEB J. 25, 2731‐2739 (2011). www.fasebj.org

Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts

OBJECTIVEnThe development of a living, tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiovascular surgery. However, the ultimate source and time needed to procure these cells remain problematic. Induced puripotent stem (iPS) cells have recently been developed and have the potential for creating a pluripotent cell line from a patients own somatic cells. In the present study, we evaluated the use of a sheet created from iPS cell-derived vascular cells as a potential source for the construction of TEVG.nnnMETHODSnMale mouse iPS cells were differentiated into embryoid bodies using the hanging-drop method. Cell differentiation was confirmed by a decrease in the proportion of SSEA-1-positive cells over time using fluorescence-activated cell sorting. The expression of endothelial cell and smooth muscle cell markers was detected using real-time polymerase chain reaction (PCR). The differentiated iPS cell sheet was made using temperature-responsive dishes and then seeded onto a biodegradable scaffold composed of polyglycolic acid-poly-l-lactide and poly(l-lactide-co-ε-caprolactone) with a diameter of 0.8 mm. These scaffolds were implanted as interposition grafts in the inferior vena cava of female severe combined immunodeficiency/beige mice (nxa0=xa015). Graft function was serially monitored using ultrasonography. The grafts were analyzed at 1, 4, and 10 weeks with histologic examination and immunohistochemistry. The behavior of seeded differentiated iPS cells was tracked using Y-chromosome fluorescent in situ hybridization and SRY real-time PCR.nnnRESULTSnAll mice survived without thrombosis, aneurysm formation, graft rupture, or calcification. PCR evaluation of iPS cell sheets inxa0vitro demonstrated increased expression of endothelial cell markers. Histologic evaluation of the grafts demonstrated endothelialization with von Willebrand factor and an inner layer with smooth muscle actin- and calponin-positive cells at 10 weeks. The number of seeded differentiated iPS cells was found to decrease over time using real-time PCR (42.2% at 1 week, 10.4% at 4 weeks, 9.8% at 10 weeks). A fraction of the iPS cells were found to be Y-chromosome fluorescent positive at 1 week. No iPS cells were found to co-localize with von Willebrand factor or smooth muscle actin-positive cells at 10 weeks.nnnCONCLUSIONSnDifferentiated iPS cells offer an alternative cell source for constructing TEVG. Seeded iPS cells exerted a paracrine effect to induce neotissue formation in the acute phase and were reduced in number by apoptosis at later time points. Sheet seeding of our TEVG represents a viable mode of iPS cell delivery over time.

Tissue-engineered vascular grafts: does cell seeding matter?

PURPOSEnUse of tissue-engineered vascular grafts (TEVGs) in the repair of congenital heart defects provides growth and remodeling potential. Little is known about the mechanisms involved in neovessel formation. We sought to define the role of seeded monocytes derived from bone marrow mononuclear cells (BM-MNCs) on neovessel formation.nnnMETHODSnSmall diameter biodegradable tubular scaffolds were constructed. Scaffolds were seeded with the entire population of BM-MNC (n = 15), BM-MNC excluding monocytes (n = 15), or only monocytes (n = 15) and implanted as infrarenal inferior vena cava (IVC) interposition grafts into severe combined immunodeficiency/bg mice. Grafts were evaluated at 1 week, 10 weeks, or 6 months via ultrasonography and microcomputed tomography, as well as by histologic and immunohistochemical techniques.nnnRESULTSnAll grafts remained patent without stenosis or aneurysm formation. Neovessels contained a luminal endothelial lining surrounded by concentric smooth muscle cell layer and collagen similar to that seen in the native mouse IVC. Graft diameters differed significantly between those scaffolds seeded with only monocytes (1.022 +/- 0.155 mm) and those seeded without monocytes (0.771 +/- 0.121 mm; P = .021) at 6 months.nnnCONCLUSIONSnMonocytes may play a role in maintaining graft patency. Incorporation of such findings into the development of second-generation TEVGs will promote graft patency and success.

Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation

Background: The extracellular matrix (ECM) is a critical determinant of neovessel integrity. Materials and Methods: Thirty-six (polyglycolic acid + polycaprolactone and poly lactic acid) tissue-engineered vascular grafts seeded with syngeneic bone marrow mononuclear cells were implanted as inferior vena cava interposition grafts in C57BL/6 mice. Specimens were characterized using immunohistochemical staining and qPCR for representative ECM components in addition to matrix metalloproteinases (MMPs). Total collagen, elastin, and glycosaminoglycan (GAG) contents were determined. MMP activity was measured using zymography. Results: Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks. Conclusions: ECM production during neovessel formation is characterized by early ECM deposition followed by extensive remodeling.