טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFreiman Alina
SubjectEngineering Tissue Flap Vascularized by Adipose-Derived
ECs and MSCs for Deep Tissue Reconstruction
Applications
DepartmentDepartment of Biotechnology
Supervisors Professor Shulamit Levenberg
Dr. Dana Egozi


Abstract

There is a constant clinical need of strategies for proper reconstruction of various soft-tissue-defects occurring as a consequence of deep burns, tumor removal or trauma. These defects involve a significant tissue loss, requiring surgical reconstruction by means of autologous muscle and vessel flaps. The main drawbacks of autologous flaps are the long surgery duration, donor-site morbidity after tissue harvest, and their inadequate availability. Engineering vascularized tissues with functional blood supply provides an alternative for this need. Therefore, the goal of the study was to investigate whether incorporation of large and functional blood vessels with vascularized engineered graft will induce sprouting of host vessels, thus enhancing its vascularization and eventually serve as a pedicle flap to treat full-thickness abdominal wall defect as well as exposed bone and tendon of an ankle. The potential of using autologous cells for self-reconstruction applications is clinically expanding. Human adipose-derived mesenchymal stem cells (MSCs) and microvascular endothelial cells (HAMECs) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co-culturing human adipose derived cell-combination (MSCs and HAMECs) on porous biodegradable three dimensional (3D) scaffolds in vitro, following implantation in vivo, will undergo anastomosis with the host tissue and will serve as a viable vascularized tissue to treat different full thickness defects. The study was divided into two major parts: (1) in vitro investigation of optimal cell-combination to induce angiogenesis and vessel-like network formation in 3D scaffolds. Cells were immunofluorescently stained within the scaffolds at different time points post-seeding. Various analyses were performed to determine vessel length, complexity and degree of maturity. (2) In vivo analysis of graft and flap. HAMECs:MSCs combination was seeded on poly L-lactic acid (PLLA) / poly lactic-co-glycolyc acid (PLGA) constructs. 1 week post culturing, the graft was implanted around femoral artery and vein of a rat. In order to confirm that sprouting will occur only from the main vessels, the graft was isolated from the surrounding tissue. Grafts were analyzed in different time points for various vascularization properties to determine blood flux, degree of vessel maturity and functionality, total number of vessels, average vessel area and human-implanted-cells survival to determine the best time point for flap transfer. Two flap applications were studied over time and flaps were analyzed to study the vascularization over time. Our findings provide experimental evidence that adipose-derived specific combination can induce successful graft vascularization and integration, crucial for flap survival.