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Title: An investigation of the effect of surface functionalisation and electrical stimulation on the thrombogenicity of PCL-based materials
Authors: Xiong, Gordon Minru
Keywords: DRNTU::Engineering::Materials::Biomaterials
Issue Date: 2016
Source: Xiong, G. M. (2016). An investigation of the effect of surface functionalisation and electrical stimulation on the thrombogenicity of PCL-based materials. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: Thrombosis in small-diameter synthetic vascular grafts is a recurring issue that does not yet have an effective clinical solution, despite decades of development in anti-thrombotic materials. One of the most widely-accepted reasons for thrombosis in small-diameter constructs is the slow rate of blood flow, which accentuates the importance of material surface chemistry in preventing thrombosis in such synthetic graft designs. Material surface chemistry influences not only the biological processes occurring at the blood-material interface, such as protein adsorption and platelet activation, but it is also purported to influence the behaviour and thrombogenicity of endothelial cells seeded on the material surface. Hence, designing materials to prevent thrombosis requires a multi-angled approach to analyse both the material and cellular aspects of thrombogenicity. In addition, novel approaches to control thrombosis will have to be proposed. In separate studies, this thesis examines the role of material surface chemistry in mediating cell thrombogenicity and the hypothesised the use of electrical stimulation to regulate thrombosis. PCL substrates were first functionalised for endothelial cell growth. Solvent cast PCL (scPCL) was successfully functionalised with poly(glycidyl methacrylate) [P(GMA)] polymer brushes using surface-initiated atom transfer radical polymerisation (ATRP). The epoxy functional groups in P(GMA) then facilitated the conjugation of gelatin. The chemical intermediates at every reaction step were verified by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The amount of gelatin conjugated was found to correlate with increasing ATRP time, indicating a tunable reaction. Increased hydrophilicity and the presence of amine signatures in FTIR and XPS indicated the successful immobilisation of gelatin. Next, in order to develop a conductive vascular graft scaffold, electrospun PCL (ePCL) was coated with polypyrrole (PPy), a conducting polymer, through oxidative template polymerisation. As heparin is a well-established anti-thrombotic agent, it was used as a dopant of PPy. Thus, a novel PPy-heparin-coated ePCL (ePCL/PPyHEP) scaffold was fabricated. XPS and toluidine blue assay confirmed the doping of heparin in the PPy nanocoating. ePCL/PPyHEP scaffolds exhibited better bulk electrical conductivity and electrochemical properties than undoped PPy-coated ePCL (ePCL/PPy) and chloride-doped PPy-coated ePCL (ePCL/PPyCl) scaffolds. An increasing amount of heparin resulted in the increase of bulk conductivity. Finally, the synthesis of PPy-based nanoparticles as a conductive nanofiller on potential graft substrate material was investigated using heparin as a steric stabiliser during dispersion polymerisation. Sizing of the PPy nanoparticles was performed by dynamic light scattering (DLS) and it was found that heparin could be immobilised onto the nanoparticle surface. Heparin-immobilised PPy (PPy-HEP) nanoparticles exhibited better colloidal stability than conventional polyvinyl alcohol (PVA)-stabilised PPy nanoparticles. The PPy-HEP nanoparticles were utilised for the binding of vascular endothelial growth factor (VEGF). Gelatin-functionalised scPCL substrates supported excellent endothelial cell (EC) coverage, and resulted in downregulated expression of thrombogenic markers von Willebrand Factor (vWF) and matrix metalloproteinase-2 (MMP-2). ECs on gelatinfunctionalised scPCL substrates also demonstrated better nitric oxide production than control gelatin-coated coverslips, indicating the effect of other material properties in governing EC phenotype. The ePCL/PPyHEP scaffolds prolonged blood clotting responses when evaluated by human plasma, but promoted platelet activation that as marked by P-selectin upregulation. However, further application of 10 μA, 100 Hz AC sine current in customised cell array chambers reduced platelet activation. The AC current did not significantly affect the adsorption of fibrinogen onto all the ePCL/PPy and ePCL/PPyHEP scaffolds, but significantly reduced the adhesion of macrophages onto the scaffolds. PPy-HEP nanoparticles were demonstrated as conductive nanofillers in an insulative polymer matrix, and when conjugated with VEGF, demonstrated the retention of bioactivity and pro-angiogenic properties of VEGF by the Matrigel assay. By integrating these multidisciplinary studies together, this thesis has shown the feasibility of designing a multifunctional graft material comprising of ATRP-functionalised PCL substrates dispersed with PPy-HEP nanoparticles that will function as VEGF carriers. Such a hybrid material will also allow the transmission of an AC stimulation current to control thrombosis.
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