Transmural macrophage migration into an arterial bioresorbable vascular graft promotes inflammatory-mediated response and collagen deposition for vascular remodeling.

Macrophages are the primary cell type orchestrating bioresorbable vascular graft (BVG) remodeling and infiltrate from three sources: the adjacent native vessel, circulating blood, and transmural migration from outer surface of the graft. To elucidate the kinetics of macrophage infiltration into the BVG, we fabricated two different bilayer arterial BVGs consisting of a macroporous sponge layer and a microporous electrospun (ES) layer. The Outer ES graft was designed to reduce transmural cell infiltration from the outer surface and the Inner ES graft was designed to reduce cell infiltration from the circulation. These BVGs were implanted in mice as infrarenal abdominal aorta grafts and extracted at 1, 4, and 8 weeks (n = 5, 10, and 10 per group, respectively) for evaluation. Cell migration into BVGs was higher in the Inner ES graft than in the Outer ES graft. For Inner ES grafts, the majority of macrophage largely expressed a pro-inflammatory M1 phenotype but gradually changed to tissue-remodeling M2 macrophages. In contrast, in Outer ES grafts macrophages primarily maintained an M1 phenotype. The luminal surface endothelialized faster in the Inner ES graft; however, the smooth muscle cell layer was thicker in the Outer ES graft. Collagen fibers were more abundant and matured faster in the Inner ES graft than that in the Outer ES graft. In conclusion, compared to macrophages infiltrating from the circulating blood, transmural macrophages from outside promote the acute inflammatory-mediated response for vascular remodeling and subsequent collagen deposition within BVGs. STATEMENT OF SIGNIFICANCE: To elucidate the kinetics of macrophage infiltration into the bioresorbable vascular graft (BVG), two different bilayer arterial BVGs were implanted in mice as infrarenal abdominal aorta grafts. Cell migration into BVGs was higher in the inner electrospun graft which cells mainly infiltrate from outer surface than in the outer electrospun graft which cells mainly infiltrate from the circulating blood. In the inner electrospun grafts, the majority of macrophages changed from the M1 phenotype to the M2 phenotype, however, outer electrospun grafts maintained the M1 phenotype. Collagen fibers matured faster in the Inner electrospun graft. Compared to macrophages infiltrating from the circulating blood, transmural macrophages from outside promote the acute inflammatory-mediated response for vascular remodeling and subsequent collagen deposition within BVGs.

Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

We previously developed a tissue-engineered vascular graft (TEVG) made by seeding autologous cells onto a biodegradable tubular scaffold, in an attempt to create a living vascular graft with growth potential for use in children undergoing congenital heart surgery. Results of our clinical trial showed that the TEVG possesses growth capacity but that its widespread clinical use is not yet advisable due to the high incidence of TEVG stenosis. In animal models, TEVG stenosis is caused by increased monocytic cell recruitment and its classic ("M1") activation. Here, we report on the source and regulation of these monocytes. TEVGs were implanted in wild-type, CCR2 knockout ( Ccr2-/-), splenectomized, and spleen graft recipient mice. We found that bone marrow-derived Ly6C+hi monocytes released from sequestration by the spleen are the source of mononuclear cells infiltrating the TEVG during the acute phase of neovessel formation. Furthermore, short-term administration of losartan (0.6 g/L, 2 wk), an angiotensin II type 1 receptor antagonist, significantly reduced the macrophage populations (Ly6C+/-/F480+) in the scaffolds and improved long-term patency in TEVGs. Notably, the combined effect of bone marrow-derived mononuclear cell seeding with short-term losartan treatment completely prevented the development of TEVG stenosis. Our results provide support for pharmacologic treatment with losartan as a strategy to modulate monocyte infiltration into the grafts and thus prevent TEVG stenosis.-Ruiz-Rosado, J. D. D., Lee, Y.-U., Mahler, N., Yi, T., Robledo-Avila, F., Martinez-Saucedo, D., Lee, A. Y., Shoji, T., Heuer, E., Yates, A. R., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

Novel Bioresorbable Vascular Graft With Sponge-Type Scaffold as a Small-Diameter Arterial Graft.

BACKGROUND: Current commercialized small-diameter arterial grafts have not shown clinical effectiveness due to their poor patency rates. The present study evaluated the feasibility of an arterial bioresorbable vascular graft, which has a porous sponge-type scaffold, as a small-diameter arterial conduit. METHODS: The grafts were constructed by a 50:50 poly (1-lactic-co-ε-caprolactone) copolymer (PLCL) scaffold reinforced by a poly (1-lactic acid) (PLA) nanofiber. The pore size of the PLCL scaffold was adjusted to a small size (12.8 ± 1.85 µm) or a large size (28.5 ± 5.25 µm). We compared the difference in cellular infiltration, followed by tissue remodeling, between the groups. The grafts were implanted in 8- to 10-week-old female mice (n = 15 in each group) as infrarenal aortic interposition conduits. Animals were monitored for 8 weeks and euthanized to evaluate neotissue formation. RESULTS: No aneurysmal change or graft rupture was observed in either group. Histologic assessment demonstrated favorable cell infiltration into scaffolds, neointimal formation with endothelialization, smooth muscle cell proliferation, and elastin deposition in both groups. No significant difference was observed between the groups. Immunohistochemical characterization with anti-F4/80 antibody demonstrated that macrophage infiltration into the grafts occurred in both groups. Staining for M1 and M2, which are the two major macrophage phenotypes, showed no significant difference between groups. CONCLUSIONS: Our novel bioresorbable vascular grafts showed well-organized neointimal formation in the high-pressure arterial circulation environment. The large-pore scaffold did not improve cellular infiltration and neotissue formation compared with the small-pore scaffold.

TGF-ß receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation.

Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-ß receptor 1 (TGF-ßR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-ßR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-ßR1 inhibitor systemic treatment for the first 2 wk. The TGF-ßR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-ß to LPS/IFN-γ-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-α, IL-12, and IL-6; this effect was blocked by TGF-ßR1 inhibition (P < 0.0001). These findings suggest that the TGF-ß signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-ßR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-ß receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation.

Rational design of an improved tissue-engineered vascular graft: determining the optimal cell dose and incubation time.

AIM: We investigated the effect of cell seeding dose and incubation time on tissue-engineered vascular graft (TEVG) patency. MATERIALS & METHODS: Various doses of bone marrow-derived mononuclear cells (BM-MNCs) were seeded onto TEVGs, incubated for 0 or 12 h, and implanted in C57BL/6 mice. Different doses of human BM-MNCs were seeded onto TEVGs and measured for cell attachment. RESULTS: The incubation time showed no significant effect on TEVG patency. However, TEVG patency was significantly increased in a dose-dependent manner. In the human graft, more bone marrow used for seeding resulted in increased cell attachment in a dose-dependent manner. CONCLUSION: Increasing the BM-MNC dose and reducing incubation time is a viable strategy for improving the performance and utility of the graft.

Comparison of a closed system to a standard open technique for preparing tissue-engineered vascular grafts.

We developed a prototype for a closed apparatus for assembling tissue-engineered vascular grafts (TEVGs) with the goal of creating a simple operator-independent method for making TEVGs to optimize safety and enable widespread application of this technology. The TEVG is made by seeding autologous bone marrow-derived mononuclear cells onto a biodegradable tubular scaffold and is the first man-made vascular graft to be successfully used in humans. A critical barrier, which has prevented the widespread clinical adoption of the TEVG, is that cell isolation, scaffold seeding, and incubation are performed using an open method. To reduce the risk of contamination, the TEVG is assembled in a clean room. Clean rooms are expensive to build, complex to operate, and are not available in most hospitals. In this investigation, we used an ovine model to compare the safety and efficacy of TEVGs created using either a standard density centrifugation-based open method or the new filter-based closed system. We demonstrated no graft-related complications and maintenance of growth capacity in TEVGs created using the closed apparatus. In addition, the use of the closed system reduced the amount of time needed to assemble the TEVG by ∼ 50%. Adaptation of similar methodologies may facilitate the safe translation and the widespread use of other tissue engineering technologies.

Mechanical instability of normal and aneurysmal arteries.

Tortuous arteries associated with aneurysms have been observed in aged patients with atherosclerosis and hypertension. However, the underlying mechanism is poorly understood. The objective of this study was to determine the effect of aneurysms on arterial buckling instability and the effect of buckling on aneurysm wall stress. We investigated the mechanical buckling and post-buckling behavior of normal and aneurysmal carotid arteries and aorta's using computational simulations and experimental measurements to elucidate the interrelationship between artery buckling and aneurysms. Buckling tests were done in porcine carotid arteries with small aneurysms created using elastase treatment. Parametric studies were done for model aneurysms with orthotropic nonlinear elastic walls using finite element simulations. Our results demonstrated that arteries buckled at a critical buckling pressure and the post-buckling deflection increased nonlinearly with increasing pressure. The presence of an aneurysm can reduce the critical buckling pressure of arteries, although the effect depends on the aneurysm's dimensions. Buckled aneurysms demonstrated a higher peak wall stress compared to unbuckled aneurysms under the same lumen pressure. We conclude that aneurysmal arteries are vulnerable to mechanical buckling and mechanical buckling could lead to high stresses in the aneurysm wall. Buckling could be a possible mechanism for the development of tortuous aneurysmal arteries such as in the Loeys-Dietz syndrome.

Transplantation of pulmonary valve using a mouse model of heterotopic heart transplantation.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient's heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

Implantation of inferior vena cava interposition graft in mouse model.

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.

Regenerative implants for cardiovascular tissue engineering.

A fundamental problem that affects the field of cardiovascular surgery is the paucity of autologous tissue available for surgical reconstructive procedures. Although the best results are obtained when an individual's own tissues are used for surgical repair, this is often not possible as a result of pathology of autologous tissues or lack of a compatible replacement source from the body. The use of prosthetics is a popular solution to overcome shortage of autologous tissue, but implantation of these devices comes with an array of additional problems and complications related to biocompatibility. Transplantation offers another option that is widely used but complicated by problems related to rejection and donor organ scarcity. The field of tissue engineering represents a promising new option for replacement surgical procedures. Throughout the years, intensive interdisciplinary, translational research into cardiovascular regenerative implants has been undertaken in an effort to improve surgical outcome and better quality of life for patients with cardiovascular defects. Vascular, valvular, and heart tissue repair are the focus of these efforts. Implants for these neotissues can be divided into 2 groups: biologic and synthetic. These materials are used to facilitate the delivery of cells or drugs to diseased, damaged, or absent tissue. Furthermore, they can function as a tissue-forming device used to enhance the body's own repair mechanisms. Various preclinical studies and clinical trials using these advances have shown that tissue-engineered materials are a viable option for surgical repair, but require refinement if they are going to reach their clinical potential. With the growth and accomplishments this field has already achieved, meeting those goals in the future should be attainable.

Effects of elastin degradation and surrounding matrix support on artery stability.

Tortuous arteries are often associated with aging, hypertension, atherosclerosis, and degenerative vascular diseases, but the mechanisms are poorly understood. Our recent theoretical analysis suggested that mechanical instability (buckling) may lead to tortuous blood vessels. The objectives of this study were to determine the critical pressure of artery buckling and the effects of elastin degradation and surrounding matrix support on the mechanical stability of arteries. The mechanical properties and critical buckling pressures, at which arteries become unstable and deform into tortuous shapes, were determined for a group of five normal arteries using pressurized inflation and buckling tests. Another group of nine porcine arteries were treated with elastase (8 U/ml), and the mechanical stiffness and critical pressure were obtained before and after treatment. The effect of surrounding tissue support was simulated using a gelatin gel. The critical pressures of the five normal arteries were 9.52 kPa (SD 1.53) and 17.10 kPa (SD 5.11) at axial stretch ratios of 1.3 and 1.5, respectively, while model predicted critical pressures were 10.11 kPa (SD 3.12) and 17.86 kPa (SD 5.21), respectively. Elastase treatment significantly reduced the critical buckling pressure (P < 0.01). Arteries with surrounding matrix support buckled into multiple waves at a higher critical pressure. We concluded that artery buckling under luminal pressure can be predicted by a buckling equation. Elastin degradation weakens the arterial wall and reduces the critical pressure, which thus leads to tortuous vessels. These results shed light on the mechanisms of the development of tortuous vessels due to elastin deficiency.

Effects of Geometric Variations on the Buckling of Arteries.

Arteries often demonstrate geometric variations such as elliptic and eccentric cross sections, stenosis, and tapering along the longitudinal axis. Effects of these variations on the mechanical stability of the arterial wall have not been investigated. The objective of this study was to determine the buckling behavior of arteries with elliptic, eccentric, stenotic, and tapered cross sections. The arterial wall was modeled as a homogenous anisotropic nonlinear material. Finite element analysis was used to simulate the buckling process of these arteries under lumen pressure and axial stretch. Our results demonstrated that arteries with an oval cross section buckled in the short axis direction at lower critical pressures compared to circular arteries. Eccentric cross-sections, stenosis, and tapering also decreased the critical pressure. Stenosis led to dramatic pressure variations along the vessel and reduced the buckling pressure. In addition, tapering shifted the buckling deformation profile of the artery towards the distal end. We conclude that geometric variations reduce the critical pressure of arteries and thus make the arteries more prone to mechanical instability than circular cylindrical arteries. These results improve our understanding of the mechanical behavior of arteries.

A Nonlinear Thin-Wall Model for Vein Buckling.

Tortuous or twisted veins are often seen in the retina, cerebrum, and legs (varicose veins) of one-third of the aged population, but the underlying mechanisms are poorly understood. While the collapse of veins under external pressure has been well documented, the bent buckling of long vein segments has not been studied. The objectives of this study were to develop a biomechanical model of vein buckling under internal pressure and to predict the critical pressure. Veins were modeled as thin-walled nonlinear elastic tubes with the Fung exponential strain energy function. Our results demonstrated that veins buckle due to high blood pressure or low axial tension. High axial tension stabilized veins under internal pressure. Our buckling model estimated the critical pressure accurately compared to the experimental measurements. The buckling equation provides a useful tool for studying the development of tortuous veins.