ENGINEERING OF CLINICAL-SCALE, IN VITRO VASCULARIZED BONE TISSUE FOR IMPLANTATION

Tissue engineering has been a rapidly expanding field dedicated to regeneration of tissue. The field has focused on application through combinations of 3 key components: cells, signals, and scaffolds. One ambitious combination of all three is the desire to engineer functional tissues in vitro to meet the clinical-demand of organ replacement. While major advances have been made, a critical obstacle that has yet to be overcome is the need to grow large volumes of complex 3D tissue. In this proposal, this issue is addresed in two ways: the use of a perfusion bioreactor system to culture 3D scaffolds to enhance mass transport, and engineering of a vascular network withing the scaffold for rapid perfusion once implanted in vivo. This thesis aims to address both aspects for bone tissue engineering by engineering pre-vascularized, mineralizing scaffolds that can be scaled up to clinically-relevant volumes by using a tubular perfusion bioreactor system (TPS). To address this, 3 aims were addressed. First, 3D culture of endothelial colony forming cells (ECFCs), a clinically-relevant cell population, was demonstrated utilizing fibrin gels within the TPS. The TPS allowed for viable culture of ECFCs within fibrin bead scaffold up to 1 week without a reduction in cell amount or genomic quality of the cells. Second, a co-culture model of angiogenesis utilizing ECFCs and mesenchymal stem cells (MSCs) was demonstrated to reproducibly form pre-formed vessel networks within a mineralizing fibrin scaffold. Data shows that MSC suspension concentration and fibrinogen concentration modulate the angiogenic response. Mineralization is demonstrated without the use of osteogenic media utilizing shear stress within the TPS. Finally, functionality of the pre-formed vessels is demonstrated following implantation to a SCID mouse model. Engineered human vessels showed anastasmosis to the host vasculature, with evidence of interconnected host and human vessel networks as well as formation of hybrid vessels. Additionally, evidence of mineralization within the scaffolds is maintained in TPS-cultured samples. In demonstrating these aims, future work should focus on fortifying the scaffold material to enable addressing implantation and persistence of clinically-relevant tissue volumes. In conclusion, pre-vascularization within bioreactor-cultured scaffolds represents a promising solution for future tissue engineering application.Ph.D. in Biomedical Engineering, May 201