Biomedical Engineering - Theses
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ItemFabrication of transplantable vascularised tissue-engineered constructs( 2024-01)Tissue engineering aims to fabricate functional tissues to overcome the organ shortage crisis. However, one of the major challenges is to ensure adequate vascularization of the artificial scaffolds that host the cells. Without proper blood supply, the cells will suffer from hypoxia and nutrient deprivation, leading to cell death or dysfunction. The vascular invasion from the host tissue is often insufficient and slow, resulting in necrosis of the core region of the implanted tissue. Therefore, pre-vascularization of the tissue before implantation is essential for most tissues to provide immediate and uniform oxygen and nutrient delivery to all cells. Although current pre-vascularization techniques can preserve cell viability in vitro, they often fail to achieve mechanical stability and integration with the host vasculature in vivo. In this study, a novel solution is presented to the vascularization challenge that allows the creation and transplantation of large, functional vascularized engineered tissues. Our technique involves vascularizing a cell-laden hydrogel by inducing angiogenesis from a suturable tissue-engineered vascular graft (TEVG). The TEVG could be directly connected and perfused by the host circulation upon implantation, ensuring the survival and function of the cells in the hydrogel. Electrospinning is a widely used technique for creating TEVGs. These fibrous structures possess high porosity, allowing them to mimic the mechanical properties of native blood vessels (NBVs). Silk fibroin (SF) stands out as a popular biomaterial for electrospinning TEVGs due to its exceptional strength and biocompatibility. However, a key limitation of SF is its stiffness, which significantly deviates from the elasticity of natural blood vessels. This discrepancy is crucial for the long-term success of NBV replacement strategies. The first study in this thesis investigated the potential of incorporating glycerol into SF to enhance its elasticity. The impact of glycerol on both the mechanical and biological properties of the resulting TEVGs was evaluated. It was found that glycerol addition effectively yielded TEVGs with mechanical properties closely resembling those of NBVs. Moreover, this modification preserved SF's inherent ability to facilitate the growth of endothelial cells (ECs) and smooth muscle cells (SMCs), crucial components of the vascular system. Notably, after just 5 days of culture, the TEVGs exhibited a well-established endothelial monolayer lining the lumen, demonstrating their potential for functional vascular tissue regeneration. The findings of this study hold significant promise for the development of improved TEVGs with mechanical properties closely mimicking NBVs. The addition of glycerol to SF emerged as a successful strategy to address the critical issue of elasticity, paving the way for more functional and durable TEVGs for future clinical applications. The EC axial alignment and the SMC circumferential alignment are essential for functional NBVs. Nevertheless, accomplishing this alignment in TEVGs is still a challenging task. In the second study, A low-cost technique was developed to align ECs axially and SMCs circumferentially in TEVGs within hours. The TEVGs consisted of a layer of electrospun polycaprolactone (PCL) and a layer of cast gelatin methacryloyl (GelMA). A technique of freezing-induced alignment was used to partially align the electrospun fibres along the axial direction, which significantly improved the EC axial alignment. Furthermore, SMCs cultured in a GelMA layer with moderate stiffness around a PCL tube adapted to the tube curvature, leading to their circumferential alignment. The TEVGs also exhibited mechanical properties like NBVs, which could facilitate future applications. This method enables a major advancement in tissue engineering, as it allows the TEVG fabrication with suitable mechanical properties that resemble the structural features of key NBV cells in a few hours using a straightforward and scalable technique. Vascularization is a major challenge for the clinical application of tissue engineering. In the third study, a novel solution was developed to the vascularization challenge in tissue engineering that allows the creation and transplantation of large, functional vascularized engineered tissues. The technique involves vascularizing a cell-laden hydrogel by inducing angiogenesis from a suturable TEVG. The TEVG is made from electrospun PCL with micron-scale pores. The graft is surrounded with a layer of cell-laden photo-crosslinked GelMA and the lumen is lined with ECs. Angiogenesis occurs through the pores in the graft, forming a hydrogel with an extensive vascular network that is connected to an implantable TEVG. The constructs are suturable and have mechanical properties comparable to native vessels. The results demonstrate that the ECs form vascular networks in the GelMA only when the gel is soft (4.3 kPa). The engineered tissue size and vascularization increase by adding multiple TEVGs into one construct. These findings address the longstanding problem of fabricating suturable pre-vascularized tissues for tissue engineering.