טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPerry Luba
SubjectEffect of Cell Composition on Integration of Transplanted
Vascularized Engineered Muscle Tissue
DepartmentDepartment of Biotechnology
Supervisor Professor Shulamit Levenberg


Abstract


In vitro prevascularization of engineered tissue constructs promises to enhance their clinical applicability. We hypothesize that adult endothelial cells (ECs), isolated from limb veins of elderly patients, bear the vasculogenic properties required to form vascular networks in vitro that can later integrate with the host vasculature upon implantation.  Here, we show that adult ECs formed vessel networks that were more developed and complex than those formed by human umbilical vein endothelial cells (HUVEC) seeded with various supporting cells on three dimensional (3D) biodegradable polymer scaffolds. In parallel, secreted levels of key proangiogenic cytokines were significantly higher in adult EC-bearing scaffolds as compared to HUVEC scaffolds. As a proof of concept for applicability of this model, adult ECs were co-seeded with human myoblasts as well as supporting cells and successfully formed a branched network, which were surrounded by aligned human myotubes. The vascularized engineered muscle tissue implanted into an abdominal wall defect in immunodeficient mice, remained viable and anastomosed with the host vasculature within 9 days of implantation. Functional "chimeric" blood vessels and various types of anastomosis were observed. These findings provide strong evidence of the applicability of adult ECs in construction of clinically relevant autologous vascularized tissue. Since rapid vascularization is critical for transplanted tissue survival, we next attempted to shorten and optimize the host vascularization process. Here, we found that seeding a multicellular culture of ANGPT1 expressing adult ECs (EC ANGPT1) with VEGF expressing adult SMC (SMC VEGF) and myoblasts on 3D polymer scaffolds promoted a more rapid in vitro vessel-like networks formation and accelerated host neovascularization of the transplant upon implantation.


These results may have profound implications for autologous construct engineering intended for transplantation, by accelerating and improving the host vascularization process and the integration of the various tissue constructs. However, the problem with these studies and many others in the field of tissue engineering is that they utilize immune-deficient animal models or administrate immunosuppressive therapy to study the integration and survival of human engineered tissues. Since the immune system plays a major role in transplant's integration and vascularization within the host, the obtained results may not necessarily reflect the actual process that will take place upon an autologous transplantation into humans. Here, we constructed a fully murine vascularized engineered muscle and transplanted it as an isograft to immuno-intact CD1 mice and as an allograft to immuno-deficient NUDE mice. We hypothesized that the presence of immune system will have an effect on the integration and on the vascularization processes of the transplant. We found that at days 14 and 18 post transplantation, more functional vessels were detected in the isograft transplantation into immuno-intact mice compared to allograft transplantation into immuno-deficient mice.  We also found that co culture of ECs with SCs promoted vascularization and myogenesis and improved integration compared to control scaffolds in both immuno-intact and immuno-deficient mice. These results can elucidate the transplantation process and might bring us one step closer to transplantation of autologous engineered tissues in the clinic in order to overcome autologous flap and donor organ shortage.