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.

Fast-degrading bioresorbable arterial vascular graft with high cellular infiltration inhibits calcification of the graft.

OBJECTIVE: Bioresorbable vascular grafts are biologically active grafts that are entirely reconstituted by host-derived cells through an inflammation-mediated degradation process. Calcification is a detrimental condition that can severely affect graft performance. Therefore, prevention of calcification is of great importance to the success of bioresorbable arterial vascular grafts. The objective of this study was to test whether fast-degrading (FD) bioresorbable arterial grafts with high cellular infiltration will inhibit calcification of grafts. METHODS: We created two versions of bioresorbable arterial vascular grafts, slow-degrading (SD) grafts and FD grafts. Both grafts had the same inner layer composed of a 50:50 poly(l-lactic-co-ε-caprolactone) copolymer scaffold. However, the outer layer of SD grafts was composed of poly(l-lactic acid) nanofiber, whereas the outer layer of FD grafts was composed of a combination of poly(l-lactic acid) and polyglycolic acid nanofiber. Both grafts were implanted in 8- to 10-week-old female mice (n = 15 in the SD group, n = 10 in the FD group) as infrarenal aortic interposition conduits. Animals were observed for 8 weeks. RESULTS: von Kossa staining showed calcification in 7 of 12 grafts in the SD group but zero in the FD group (P < .01, χ2 test). The cell number in the outer layer of FD grafts was significantly higher than in the SD grafts (SD, 0.87 ± 0.65 × 103/mm2; FD, 2.65 ± 1.91 × 103/mm2; P = .02). CONCLUSIONS: The FD bioresorbable arterial vascular graft with high cellular infiltration into the scaffold inhibited calcification of grafts.

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.

Cilostazol, Not Aspirin, Prevents Stenosis of Bioresorbable Vascular Grafts in a Venous Model.

OBJECTIVE: Despite successful translation of bioresorbable vascular grafts for the repair of congenital heart disease, stenosis remains the primary cause of graft failure. In this study, we investigated the efficacy of long-term treatment with the antiplatelet drugs, aspirin and cilostazol, in preventing stenosis and evaluated the effect of these drugs on the acute phase of inflammation and tissue remodeling. APPROACH AND RESULTS: C57BL/6 mice were fed a drug-mixed diet of aspirin, cilostazol, or normal chow during the course of follow-up. Bioresorbable vascular grafts, composed of poly(glycolic acid) mesh sealed with poly(l-lactide-co-ε-caprolactone), were implanted as inferior vena cava interposition conduits and followed up for 2 weeks (n=10 per group) or 24 weeks (n=15 per group). Both aspirin and cilostazol suppressed platelet activation and attachment onto the grafts. On explant at 24 weeks, well-organized neotissue had developed, and cilostazol treatment resulted in 100% graft patency followed by the aspirin (67%) and no-treatment (60%) groups (P<0.05). Wall thickness and smooth muscle cell proliferation in the neotissue of the cilostazol group were decreased when compared with that of the no-treatment group at 24 weeks. In addition, cilostazol was shown to have an anti-inflammatory effect on neotissue at 2 weeks by regulating the recruitment and activation of monocytes. CONCLUSIONS: Cilostazol prevents stenosis of bioresorbable vascular graft in a mouse inferior vena cava implantation model up to 24 weeks and is accompanied by reduction of smooth muscle cell proliferation and acute inflammation.

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.

Dissociation of pulse wave velocity and aortic wall stiffness in diabetic db/db mice: The influence of blood pressure.

Introduction: Vascular stiffness is a predictor of cardiovascular disease and pulse wave velocity (PWV) is the current standard for measuring in vivo vascular stiffness. Mean arterial pressure is the largest confounding variable to PWV; therefore, in this study we aimed to test the hypothesis that increased aortic PWV in type 2 diabetic mice is driven by increased blood pressure rather than vascular biomechanics. Methods and Results: Using a combination of in vivo PWV and ex vivo pressure myography, our data demonstrate no difference in ex vivo passive mechanics, including outer diameter, inner diameter, compliance (Db/db: 0.0094 ± 0.0018 mm2/mmHg vs. db/db: 0.0080 ± 0.0008 mm2/mmHg, p > 0.05 at 100 mmHg), and incremental modulus (Db/db: 801.52 ± 135.87 kPa vs. db/db: 838.12 ± 44.90 kPa, p > 0.05 at 100 mmHg), in normal versus diabetic 16 week old mice. We further report no difference in basal or active aorta biomechanics in normal versus diabetic 16 week old mice. Finally, we show here that the increase in diabetic in vivo aortic pulse wave velocity at baseline was completely abolished when measured at equivalent pharmacologically-modulated blood pressures, indicating that the elevated PWV was attributed to the concomitant increase in blood pressure at baseline, and therefore "stiffness." Conclusions: Together, these animal model data suggest an intimate regulation of blood pressure during collection of pulse wave velocity when determining in vivo vascular stiffness. These data further indicate caution should be exerted when interpreting elevated PWV as the pure marker of vascular stiffness.

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (µCT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. µCT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Imatinib attenuates neotissue formation during vascular remodeling in an arterial bioresorbable vascular graft.

BACKGROUND: Bioresorbable vascular grafts (BVGs) can transform biologically into active blood vessels and represent an alternative to traditional synthetic conduits, which are prone to complications such as infection and thrombosis. Although platelet-derived growth factors and c-Kit positive cells play an important role in smooth muscle cell (SMC) migration and proliferation in vascular injury, atherosclerosis, or allograft, their roles in the vascular remodeling process of an arterial BVG remains unknown. Thus, we assessed the neottisue formation on arterial BVG remodeling by administrating imatinib, which is both a platelet-derived growth factor receptor kinase inhibitor and c-Kit receptor kinase inhibitor, in a murine model. METHODS: BVGs were composed of an inner poly(L-lactic-co-ε-caprolactone) copolymer sponge layer and an outer electrospun poly(L-lactic acid) nanofiber layer, which were implanted into the infrarenal abdominal aortas of C57BL/6 mice. After graft implantation, saline or 100 mg/kg of imatinib was administrated intraperitoneally daily for 2 weeks (n = 20 per group). Five mice in each group were scheduled to be humanely killed at 3 weeks and 15 at 8 weeks, and BVGs were explanted for histologic assessments. RESULTS: Graft patency during the 8-week observational period was not significantly different between groups (control, 86.7% vs imatinib, 80.0%; P > .999). Neotissue formation consisting of endothelialization, smooth muscle proliferation, and deposition of collagen and elastin was not observed in either group at 3 weeks. Similar endothelialization was achieved in both groups at 8 weeks, but thickness and percent area of neotissue formation were significantly higher in the control group than in the imatinib group, (thickness, 30. 1 ± 7.2 µm vs 19.6 ± 4.5 µm [P = .001]; percent area, 9.8 ± 2.7% vs 6.8 ± 1.8% [P = .005]). Furthermore, SMC layer and deposition of collagen and elastin were better organized at 8 weeks in the control group compared with the imatinib group. The thickness of SMC layer and collagen fiber area were significantly greater at 8 weeks in the control group than in the imatinib group (P < .001 and P = .026, respectively). Because there was no difference in the inner diameter of explanted BVGs (831.7 ± 63.4 µm vs 841.8 ± 41.9 µm; P = .689), neotissue formation was thought to advance toward the outer portion of the BVG with degradation of the polymer scaffold. CONCLUSIONS: Imatinib attenuates neotissue formation during vascular remodeling in arterial bioresorbable vascular grafts (BVGs) by inhibiting SMC layer formation and extracellular matrix deposition. CLINICAL RELEVANCE: This study demonstrated that imatinib attenuated neotissue formation during vascular remodeling in arterial Bioresorbable vascular graft (BVG) by inhibiting smooth muscle cell formation and extracellular matrix deposition. In addition, as imatinib did not modify the inner diameter of BVG, neotissue advanced circumferentially toward the outer portion of the neovessel. Currently, BVGs have not yet been clinically applied to the arterial circulation. The results of this study are helpful for the design of BVG that can achieve an optimal balance between polymer degradation and neotissue formation.

Early natural history of neotissue formation in tissue-engineered vascular grafts in a murine model.

Aim: To characterize early events in neotissue formation during the first 2 weeks after vascular scaffold implantation. Materials & methods: Biodegradable polymeric scaffolds were implanted as abdominal inferior vena cava interposition grafts in wild-type mice. Results: All scaffolds explanted at day 1 contained a platelet-rich mural thrombus. Within the first few days, the majority of cell infiltration appeared to be from myeloid cells at the peritoneal surface with modest infiltration along the lumen. Host reaction to the graft was distinct between the scaffold and mural thrombus; the scaffold stimulated an escalating foreign body reaction, whereas the thrombus was quickly remodeled into collagen-rich neotissue. Conclusion: Mural thrombi remodel into neotissue that persistently occludes the lumen of vascular grafts.

Morphologic adaptation of arterial endothelial cells to longitudinal stretch in organ culture.

Arteries in vivo are subjected to large longitudinal stretch, which changes significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on arterial endothelium. The aim of this study was to determine the morphologic adaptation of arterial endothelial cells (ECs) to elevated axial stretch. Porcine carotid arteries were stretched 20% more than their in vivo length while being maintained at physiological pressure and flow rate in an organ culture system. The ECs were elongated with the application of the axial stretch (aspect ratio 2.81+/-0.25 versus 3.65+/-0.38, n=8, p<0.001). The elongation was slightly decreased after three days and the ECs recovered their normal shape after seven days, as measured by the shape index and aspect ratio (0.55+/-0.03 versus 0.56+/-0.04, and 2.93+/-0.28 versus 2.88+/-0.20, respectively, n=5). Cell proliferation was increased in the intima of stretched arteries in three days as compared to control arteries but showed no difference after seven days in organ culture. These results demonstrate that the ECs adapt to axial stretch and maintain their normal shape.

Bone marrow-derived mononuclear cell seeded bioresorbable vascular graft improves acute graft patency by inhibiting thrombus formation via platelet adhesion.

BACKGROUND: Acute thrombosis is a crucial cause of bioresorbable vascular graft (BVG) failure. Bone marrow-derived mononuclear cell (BM-MNC)-seeded BVGs demonstrated high graft patency, however, the effect of seeded BM-MNCs against thrombosis remains to be elucidated. Thus, we evaluated an antithrombotic effect of BM-MNC-seeding and utilized platelet-depletion mouse models to evaluate the contribution of platelets to acute thrombosis of BVGs. METHODS AND RESULTS: BVGs were composed of poly(glycolic acid) mesh sealed with poly(l-lactideco-ε-caprolactone). BM-MNC-seeded BVGs and unseeded BVGs were implanted to wild type C57BL/6 mice (n = 10/group) as inferior vena cava interposition conduits. To evaluate platelet effect on acute thrombosis, c-Mpl-/- mice and Pf4-Cre+; iDTR mice with decreased platelet number were also implanted with unseeded BVGs (n = 10/group). BVG patency was evaluated at 2, 4, and 8 weeks by ultrasound. BM-MNC-seeded BVGs demonstrated a significantly higher patency rate than unseeded BVGs during the acute phase (2-week, 90% vs 30%, p = .020), and patency rates of these grafts were sustained until week 8. Similar to BM-MNC-seeded BVGs, C-Mpl-/- and Pf4-Cre+; iDTR mice also showed favorable graft patency (2-week, 90% and 80%, respectively) during the acute phase. However, the patency rate of Pf4-Cre+; iDTR mice decreased gradually after DTR treatment as platelet number recovered to baseline. An in vitro study revealed BM-MNC-seeding significantly inhibited platelet adhesion to BVGs compared to unseeded BVGs, (1.75 ±â€¯0.45 vs 8.69 ±â€¯0.68 × 103 platelets/mm2, p < .001). CONCLUSIONS: BM-MNC-seeding and the reduction in platelet number prevented BVG thrombosis and improved BVG patency, and those results might be caused by inhibiting platelet adhesion to the BVG.

Tropoelastin inhibits intimal hyperplasia of mouse bioresorbable arterial vascular grafts.

Neointimal hyperplasia, which results from the activation, proliferation and migration of vascular smooth muscle cells (SMCs), is a detrimental condition for vascular stents or vascular grafts that leads to stenosis. Preventing neointimal hyperplasia of vascular grafts is critically important for the success of arterial vascular grafts. We hypothesized that tropoelastin seeding onto the luminal surface of the graft would prevent neointimal hyperplasia through suppressing neointimal smooth muscle cell proliferation. In this study, we investigated the efficacy of tropoelastin seeding in preventing neointimal hyperplasia of bioresorbable arterial vascular grafts. Poly (glycolic acid) (PGA) fiber mesh coated with poly (l-lactic-co-ε-caprolactone) (PLCL) scaffolds reinforced by poly (l-lactic acid) (PLA) nano-fibers were prepared as bioresorbable arterial grafts. Tropoelastin was then seeded onto the luminal surface of the grafts. Tropoelastin significantly reduced the thickness of the intimal layer. This effect was mainly due to a substantial reduction the number of cells that stained positive for SMC (α-SMA) and PCNA in the vessel walls. Mature elastin and collagen type I and III were unchanged with tropoelastin treatment. This study demonstrates that tropoelastin seeding is beneficial in preventing SMC proliferation and neointimal hyperplasia in bioresorbable arterial vascular grafts. STATEMENT OF SIGNIFICANCE: Small resorbable vascular grafts can block due to the over-proliferation of smooth muscle cells in neointimal hyperplasia. We show here that the proliferation of these cells is restricted in this type of graft. This is achieved with a simple dip, non-covalent coating of tropoelastin. It is in principle amendable to other grafts and is therefore an attractive process. This study is particularly significant because: (1) it shows that smooth muscle cell proliferation can be reduced while still accommodating the growth of endothelial cells, (2) small vascular grafts with an internal diameter of less than 1mm are amenable to this process, and (3) this process works for resorbable 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.

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.

Pilot Mouse Study of 1 mm Inner Diameter (ID) Vascular Graft Using Electrospun Poly(ester urea) Nanofibers.

An off-the-shelf, small diameter tissue engineered vascular graft (TEVG) would be transformative to surgeons in multiple subspecialties. Herein, the results of a small diameter (ID ≈ 1 mm) vascular graft constructed from resorbable, amino acid-based poly(ester urea) (PEU) are reported. Electrospun PEU grafts of two different wall thicknesses (type A: 250 µm; type B: 350 µm) are implanted as abdominal infra-renal aortic grafts in a severe combined immune deficient/beige mouse model and evaluated for vessel remodeling over one year. Significantly, the small diameter TEVG does not rupture or lead to acute thrombogenic events during the intervals tested. The pilot TEVG in vivo shows long-term patency and extensive tissue remodeling with type A grafts. Extensive tissue remodeling in type A grafts leads to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells. However, due to slow degradation of the PEU scaffold materials in vivo, the grafts remain after one year. The type B grafts, which have 350 µm thick walls, experience occlusion over the one year interval due to intimal hyperplasia. This study affords significant findings that will guide the design of future generations of small diameter vascular grafts.

Long-Term Functional Efficacy of a Novel Electrospun Poly(Glycerol Sebacate)-Based Arterial Graft in Mice.

Many surgical interventions for cardiovascular disease are limited by the availability of autologous vessels or suboptimal performance of prosthetic materials. Tissue engineered vascular grafts show significant promise, but have yet to achieve clinical efficacy in small caliber (<5 mm) arterial applications. We previously designed cell-free elastomeric grafts containing solvent casted, particulate leached poly(glycerol sebacate) (PGS) that degraded rapidly and promoted neoartery development in a rat model over 3 months. Building on this success but motivated by the need to improve fabrication scale-up potential, we developed a novel method for electrospinning smaller grafts composed of a PGS microfibrous core enveloped by a thin poly(ε-caprolactone) (PCL) outer sheath. Electrospun PGS-PCL composites were implanted as infrarenal aortic interposition grafts in mice and remained patent up to the 12 month endpoint without thrombosis or stenosis. Many grafts experienced a progressive luminal enlargement up to 6 months, however, due largely to degradation of PGS without interstitial replacement by neotissue. Lack of rupture over 12 months confirmed sufficient long-term strength, due primarily to the persistent PCL sheath. Immunohistochemistry further revealed organized contractile smooth muscle cells and neotissue in the inner region of the graft, but a macrophage-driven inflammatory response to the residual polymer in the outer region of the graft that persisted up to 12 months. Overall, the improved surgical handling, long-term functional efficacy, and strength of this new graft strategy are promising, and straightforward modifications of the PGS core should hasten cellular infiltration and associated neotissue development and thereby lead to improved small vessel replacements.

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.

Design and Use of a Novel Bioreactor for Regeneration of Biaxially Stretched Tissue-Engineered Vessels.

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.

Hemodynamic Characterization of a Mouse Model for Investigating the Cellular and Molecular Mechanisms of Neotissue Formation in Tissue-Engineered Heart Valves.

Decellularized allograft heart valves have been used as tissue-engineered heart valve (TEHV) scaffolds with promising results; however, little is known about the cellular mechanisms underlying TEHV neotissue formation. To better understand this phenomenon, we developed a murine model of decellularized pulmonary heart valve transplantation using a hemodynamically unloaded heart transplant model. Furthermore, because the hemodynamics of blood flow through a heart valve may influence morphology and subsequent function, we describe a modified loaded heterotopic heart transplant model that led to an increase in blood flow through the pulmonary valve. We report host cell infiltration and endothelialization of implanted decellularized pulmonary valves (dPV) and provide an experimental approach for the study of TEHVs using mouse models.

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.