Engineering biomimetic microenvironment for vascular tissue engineering
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Vascular tissue engineering might provide an alternative solution to treat cardiovascular diseases. Despite the significant progress in the past two decades, there still are issues need to be addressed for successful vascular tissue engineering. Mimicking the microenvironment of the native vascular tissue in vivo is one of the most crucial issues. In this dissertation, I aim to recreate biomimetic microenvironment for fibrin-based tissue engineering vascular tissues. I started by presenting a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context by engineering fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain. In solution the fusion TGF-β1 exhibited similar biological activity as native TGF-β1 as evidenced by activity assays. Immunoprecipitation experiments and protein release experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in the presence of Factor XIII. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells. Subsequently, we investigated the hypothesis that immobilizing TGF-β1 within fibrin hydrogels may act in synergy with cyclic mechanical stimulation to enhance the properties of vascular grafts. To this end, we applied the fusion TGF- β1 protein and a 24-well based bioreactor we developed in which vascular constructs can be mechanically stimulated by distending the silastic mandrel in the middle of each well. TGF-β1 was either conjugated to fibrin or supplied in the culture medium and the fibrin based constructs were cultured statically for a week followed by cyclic distention for another week. The tissues were examined for myogenic differentiation, vascular reactivity, mechanical properties and ECM content. Our results showed that some aspects of vascular function were differentially affected by growth factor presentation vs. pulsatile force application, while others were synergistically enhanced by both. Overall, this two-prong biomimetic approach improved ECM secretion, vascular reactivity and mechanical properties of vascular constructs. These findings may be applied in other tissue engineering applications such as cartilage, tendon or cardiac regeneration where TGF-β1and mechano-stimulation play critical roles. The next section of this dissertation is rooted from the observation that overexpression of a single transcription factor, Nanog, using lentiviral vector can reverse the effects of organismal aging on mesenchymal stem cells (MSCs) proliferation and myogenic differentiation. These results prompted us to hypothesize that delivery of Nanog proteins into MSCs may also overcome the effects of cellular senescence and has more clinical relevance (risks from using lentiviral system: no carcinogenesis or gene integration in to genomic DNA). To this end, I introduced cell penetrating peptide, nona-arginine tage, to Nanog, (named NanogR) and optimized the NanogR purification process. The purified NanogR protein can readily be delivered into 293T cells to promote Nanog transcriptional activity. Subsequently, by applying magnetic forces along with magnetic nanoparticles which can be bound by NanogR, I successfully delivered NanogR proteins to fibroblast cells which NanogR alone cannot efficiently trigger transcriptional activity. By further engineering Nanog proteins (by removing ubiquitination sequence for higher stability and introducing extra nuclear localization signals for swifter nuclear transport, named SVdPNanogR), I also increased the Nanog protein delivery efficiency by up to another 20% comparing to using NanogR. These results suggest that this effective Nanog protein delivery protocol might be applied to reverse MSC senescence replacing the lentiviral Nanog overexpression system used in the previous study or to reprogram cells with other Yamanaka factors.