NEOVASCULARIZAnON WITHIN POROUS PEG HYDROGELS
Development of engineered tissues of clinically relevant size requires the ability to control vascularization within biomaterial scaffolds. Poly(ethylene glycol) (PEG) hydrogels have been extensively investigated for use as synthetic scaffolds to support engineered tissue formation. The goal of this work described was to develop techniques that can be used to enhance vascularized tissue formation in PEG-based hydrogels. In the first part of the study a technique was developed to generate porous PEG hydrogels using a salt leaching technique. This technique was then used to examine the role of pore size on vascularization and tissue remodeling in porous PEG hydrogel in vitro and in vitro. Both in vitro and in vivo studies showed that vessel invasion was pore size dependent. In addition, a thin layer of inflammatory tissue was observed between PEG hydrogel and blood vessels that formed within the gels. This layer suggested that inflammatory cells, not vascular cells, interacted with the surface of the material. This suggests that peptides covalently incorporated within PEG may not directly interact with endothelial cells (ECs) following implantation. The porous PEG hydrogels were very stable in vitro and in vivo and did not exhibit any signs of degradation. Hydrogels used in tissue engineering need to exhibit controlled degradation. In order to address the stability of PEG hydrogels, porous hydrogels were rendered using degradable PEG-co-(L-Lactic acid) diacrylate PEG-PLLA-DA. This polymer is degraded via hydrolysis of the PLLA chains. The porous PEG-PLLA-DA hydrogels were generated by solvent casting, photopolymerization, and particulate leaching. The influence of polymer conditions on the architecture, degradation, and mechanical properties of the hydrogels were investigated in vitro. The hydrogels were found to exhibit autofluorescence that allowed for the unique ability to nondestructively image hydrogel structure under fully swelled conditions using confocal microscopy. Initial pore size was a function of particulate size and independent of polymer concentration. Interestingly, pore size remained stable though out the study, and was not a function of degradation. In addition, degradation time of porous PEG-LLA-DA hydrogels was influenced by polymer concentration. Compressive modulus was a function of polymer concentration and pore size and decreased during hydrogel degradation. The incorporation of cell adhesion sequences into the hydrogel showed that they can support cell adhesion with morphology varying with pore size. This technique could be used to tailor porous biodegradable scaffolds for tissue engineering applications. In the final portion of this thesis a poly-lysine (PLL) molecule was synthesized in order to allow clustering of adhesion sequences in PEG hydrogels. Clusters of peptide sequences have been shown to enhance cell interactions with substrate surfaces. The sequence was synthesized and purified by high performance liquid chromatography (HPLC) and characterized by mass spectrometry. The side chains of the PLL molecule was used to attach peptide sequences. Cysteine contained within the PLL allowed incorporation into the PEG hydrogel by mixed mode polymerization. Cells were observed to adhere to hydrogels containing the RGD clusters and not to the control gels. The results presented here describe various techniques that can be used to optimize the design of polymer scaffolds for tissue engineering. In addition, the data provide insight into the process of vascularization in porous hydrogels and the influence of synthesis conditions and degradation on properties of porous hydrogels. Future studies should investigate the optimization of these material techniques for control of neovascularization within PEG hydrogels for tissue engineering applications.