Ph.D Thesis

Ph.D StudentKoffler Jacob
SubjectSkeletal Muscle Tissue Engineering: Integration of
Engineered Vascularized Graft
DepartmentDepartment of Biotechnology
Supervisor PROF. Shulamit Levenberg


Traumatic events such as burns, involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use.  Engineered vascularized grafts provide an alternative for this need. Our aims were to investigate the degree of pre-vascularization and myogenesis required for improved implantation prospects. In addition, we explored two approaches to increase graft vascularity: tissue fabrication using a large blood vessel and angiogenic gene therapy. This research describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. Our grafts were composed of HUVEC, HFF and, C2C12, seeded on SIS bioscaffold. SIS is a resorbable, acellular bioscaffold, composed of extracellular matrix proteins derived from the jejunum of pigs, and has been shown to be completely replaced by the host within 90 days. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semi-organized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle.  We suggest a “relay” approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration. The vascularized graft was further prefabricated in vivo using the femoral vessels in mice. It showed increased blood perfusion and a complex network of blood vessels, relatively to empty or myoblasts graft. Thus, we have created a vascularized tissue that includes a vascular axis, which is a flap. This flap was transferred to reconstruct a defect in the abdominal wall and was viable one day post transfer, even without skin coverage. We further improved vascularity in the graft by increasing the angiogenic response using adeno vectors. We show that among VEGF/HIF/PDGF vectors, PDGF was the only one to induce improved perfusion coupled with increased vessel diameter that sustained for two months post implantation.