The development of in vitro and in vivo models to investigate the therapeutic potential of encapsulated angiogenic and vasculogenic factors

The major focus of this work was to develop a novel porous device to recruit and capture bone marrow endothelial progenitor cells. Vascular endothelial growth factor (VEGF) and granulocyte-monocyte colony stimulating factor (GM-CSF) were encapsulated into biodegradable polymers using phase inversion nanoencapsulation (PIN). Microspheres were less than one micron in diameter and ELISA analysis demonstrated the continuous release of each factor. Maintenance of bioactivity post-encapsulation was confirmed using cell-based bioassays. Two novel cell culture systems showed endothelial cells proliferated and migrated significantly more over time in response to VEGF released from microspheres compared to equivalent unencapsulated VEGF controls, and that the relative bioactivity ranged between 130--140%. At least 50--55% of GM-CSF remained bioactive following encapsulation. Evaluation of these delivery systems in vivo demonstrated their ability to recruit cells into a subcutaneous porous nylon mesh implant. Semi-quantitative immunohistochemical analysis of tissue surrounding implants at 3 and 7 days showed increased vascularization, and analysis of the implant interior revealed evidence of c-Kit+ and VEGF-R2+ cell recruitment that was particular to implants containing VEGF and/or GM-CSF microspheres. A transgenic mouse containing lacz+ bone marrow was created to investigate the origin of recruited cells. Few lacz+ cells were found inside control implants: plain nylon mesh, mesh containing BSA microspheres, or mesh containing VEGF and GM-CSF unencapsulated protein at 1, 2 or 3 weeks. However, implants containing both VEGF and GM-CSF microspheres showed significant lacz+ cell migration into the implant at all time points. Preliminary analysis of implants at 1 week revealed significant lacz+ cell recruitment to implants containing VEGF or GM-CSF microspheres as well. Immunohistochemical analysis of the 3 week implants revealed the majority of recruited cells were positive for CD133, an endothelial progenitor cell marker. Preliminary experiments demonstrated cells captured inside the mesh implant could be removed and cultured ex vivo. It is believed that the potential of specific encapsulated factors to recruit cells of progenitor origin to an ectopic site in vivo was demonstrated for the first time. The therapeutic potential of this system is broad and will be discussed in further detail in the dissertation.