Ph.D Thesis

Ph.D StudentDado Dekel
SubjectCell-Scaffold Mechanical Interplay within Engineered
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Shulamit Levenberg


Tissue engineering techniques involving cell-embedded scaffolds enables examination of biological processes within a three-dimensional environment. A dynamic mechanical interplay exists between the cell-scaffold components, where cell forces modify the scaffold, while scaffold properties and external forces mediated through the scaffold, directly impact cell behavior. The present study assessed the influence of uniaxial tensile forces applied to cell-seeded constructs on endothelial differentiation and organization, starting with mesodermal differentiation through vessel network formation and organization. Uniaxial static or cyclic stretch was applied to embryonic stem cell-embedded collagen constructs. Cyclic stretching of the cellular constructs led to increased Brachyury expression, characteristic of the primitive streak phase in gastrulation. Further examination of gene expression characteristic of embryonic differentiation and pluripotency under cyclic stretching, revealed changes mostly related to mesodermal processes. We observed the influence of the tensile forces on cell actin fibers orientation and observed correlation between actin fibers organization and cell differentiation with external applied forces. Differentiation to the mesoderm germ layer was mostly influenced by the forces as assessed also by inhibition of non-muscle myosin II.

 In the next stage, uniaxial tensile forces applied to constructs embedded with both endothelial cells and fibroblasts, led to formation of an organized vessel network where static stretching of the scaffold let to parallel orientation with the stretching direction whereas cyclic stretching led to diagonal orientation with the stretching direction. The involvement of forces in the angiogenesis process was also examined through addition of inhibitors showing correlation between the level of cell induced forces and the quality of the resulting network. Implantation of such constructs in mice promoted angiogenesis, as expressed in improved integration of the organized network with the host tissue compared to the control. In addition angiogenic factor secretion under the examined mechanical condition was quantitavely examined showing enhanced secretion of PDGF-ββ with increasing forces suggesting influence on network stability and maturity. In addition, involvement of Ang-2, VEGF and Timp1 and Timp2 in forces induction during angiogenesis was observed. 

Understanding the effects of mechanical forces on stem cell differentiation provides a means of manipulating their differentiation for later use in regenerative medicine applications.  Moreover, regulation of the alignment of vessel networks in vitro can improve tissue integration in vivo.  Taken together, these findings shed light on the involvement of such forces in mesodermal differentiation and vascular network formation.