Ph.D Thesis


Ph.D StudentBen-Shaul Shahar
SubjectIn Vivo Integration Dynamics of Engineered
Vascularized Constructs
DepartmentDepartment of Biotechnology
Supervisor PROF. Shulamit Levenberg


Abstract

Graft vascularization remains one of the most critical challenges still facing tissue engineering experts, particularly in their attempt to create large and thick tissues. In vitro prevascularization of engineered tissues has been suggested to promote rapid anastomosis between the graft and host vasculatures. We hypothesized that in vitro vessel maturation in transplantable grafts accelerates vessel integration and perfusion, and prevents thrombotic events in the grafts. Human endothelial cells and fibroblasts cultured on three-dimensional, biodegradable scaffolds for 1 or 14 days, formed vasculature with different degrees of maturation. The scaffolds were implanted into immunodeficient nude mice and monitored via a dorsal skinfold window chamber. The 14-day in vitro culture period proved sufficient to obtain vessel maturation within the engineered tissue, which promoted host vessel penetration into the graft, graft-host vessel anastomosis and graft perfusion. Moreover, the presence of the mature vessels prevented clot accumulation within the grafts. These observations were not made in constructs cultured for only one day before implantation. Elevated expression levels of the coagulation factors, von Willebrand factor and tissue factor, was demonstrated in constructs bearing immature vasculature compared to mature vasculature.

To test the importance of vessel maturation in a clinically relevant model we tested it in islet transplantation model. The moderate success of islets revascularization upon implantation has been attributed to the a-vascular nature of the islets, thus we created a new prevascularized platform enabling us to seed isolated murine pancreatic islets on construct bearing mature vasculature and visualize its revascularization in vivo. Host vessel penetration and islet perfusion were demonstrated two days post-implantation. The implantation of the prevascularized pancreatic tissue demonstrated a decrease in blood glucose levels of stz- induced diabetic mice. These results demonstrate the importance of establishing a stable, mature vasculature in engineered tissues before implantation to promote anastomosis with the host and accelerate graft perfusion and functionality.

Macrophages are major players in vascularization and exist on a spectrum of functionally diverse phenotypes, from pro-inflammatory (M1) to anti-inflammatory (M2). Studies suggest that sequential M1 and M2 macrophages are needed for normal wound healing. To investigate macrophages role in vessel development, we created a new platform enabling us introduce macrophages in a two- step in vitro sequential seeding procedure. M0 macrophages addition to endothelial and supporting cells co- culture inhibited vessel network formation. However, sequential seeding of M1 macrophages at day 3 and M2a, at day 6 of seeding promoted the sprouting and development of the engineered vasculature. To investigate macrophages role in graft- host vascular integration we implanted the 14-days prevascularized constructs in a murine subcutaneous implantation model. We demonstrated macrophages infiltration into the graft already a day post- implantation and interaction with both the implanted- engineered vasculature and the host vessels. Moreover, in the absence of macrophage, both host vessel penetration and graft perfusion were significantly decreased compared to implantation of constructs in the present of macrophages. The results emphasize the important role of macrophages in graft- host vascular integration, suggesting that harnessing the host macrophages may be considered when designing prevascularized constructs for implantation purposes.