טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTimaner Michael
SubjectCo-Culture of Osteogenic and Angiogenic Stem Cells on 3D
Scaffolds for Improved Engraftment in Animal Model
DepartmentDepartment of Medicine
Supervisors Professor Abraham Reznick
Ms. Erella Livne (Deceased)
Full Thesis textFull thesis text - English Version


Abstract

Bone repair is a significant challenge in future medicine. In artificial settings, bone formation is regulated by osteoprogenitor cells and growth factors. It occurs on 3D scaffolds (bone particles) which mimic natural bone structure and which provide the necessary support for osteogenic progenitor cells to proliferate and maintain their differentiated function in the newly formed bone. Additionally, new bone formation and its successful grafting require adequate vascularization. Since the survival of cells in the center of the scaffold is often restricted due to suboptimal oxygenation and nutrition, enhanced vascularization may be used to improve bone cultures for grafting.

The main purpose of this study was to create a biofunctional graft consisting of a co-culture of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) in order to improve vascularization and to enhance bone tissue formation on a 3D scaffold in an animal model.

Human bone marrow derived MSCs are multipotent stem cells, able to differentiate into different mesenchymal tissues. Human peripheral blood derived EPCs originate from the bone marrow and can contribute to formation of blood vessels. Each cell type has been isolated and characterized separately using fluorescent or immunohistochemistry, RNA markers and FACS analysis. Prior to co-culture formation, the osteogenic characteristics of MSCs were validated, as well as the angiogenic potential of EPCs. Based on the extensive proliferation potential of MSCs compared to EPCs and the morphological analysis of co-cultures with different ratios we decided that the optimal ratio between MSCs and EPCs, which preserves both angiogenic and osteogenic capabilities, was 2:1. This ratio was used to study the co-culture effect on vascularization and osteogenesis.

Our results demonstrated that the ability of the co-culture to create bone tissue is equal to the ability of MSCs culture. Furthermore, when supplemented with BMP-2 the co-culture presented even stronger osteogenic potential than MSCs alone. In parallel, the evaluation of vascularization of newly formed bone has revealed that the angiogenic potential of the co-culture is much higher than the potential of MSCs or EPCs cultures. Additionally, we showed that blood vessels inside the co-culture implants are of human origin, based on positive staining results for human CD31 and CD146 around capillaries. Finally, we demonstrated that blood vessels inside the co-culture implants are functional.

In conclusion, our results show that 3D scaffolds containing a co-culture of angiogenic and osteogenic cells can provide better vascularization of the graft and thus enhance new bone formation.