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Abstract:
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I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs . Zero -order release can be obtained by conjugating a growth factor with a homobifunctional , amine -reactive , PEG derivative . Growth factors can be entrapped during thrombin -mediated crosslinking and released rapidly . Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and /or disassociation . PDGF -BB was loaded via entrapment while TGF -β1 was sequestered through a combination of physical affinity and conjugation . The affinity of TGF -β1 and fibrinogen had never been previously examined or quantified . I aimed to determine the Ka and Kd between TGF -β1 and fibrinogen through a variety of assays . Binding ELISAs were developed for TGF -β1 and fibronectin , a protein associated with fibrin gels , and TGF -β1 and fibrinogen . However , background was high due to insufficient blocking agents . Other assays explored included western blots , surface plasmon resonance , and radiolabeled TGF -β1 with limited success . The affect of TGF -β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs . With exposure to TGF -β1 , MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF -β1 maintained bioactivity for at least 7 days . Gene expression of MSCs exposed to TGF -β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation . However , confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF -β1 loaded PEGylated fibrin gels . The system developed is highly modular and can be applied to other tissue engineering systems . Furthermore , other growth factors could be incorporated to promote vascular cell differentiation . |