| dc.description.abstract | Vascular disease remains the leading cause of death globally. Bypass grafting is one of the common surgical interventions to restore the blood flow. Autologous grafts, especially internal thoracic arteries and saphenous veins are the gold standard bypass grafts, but the source can be limited. Synthetic grafts have been extensively developed as alternatives to autografts. Despite the progresses in the past decades, complications, such as thrombosis and intimal hyperplasia formation, persist in synthetic small diameter vascular grafts (<6 mm), leading to their fast occlusion. Polyvinyl alcohol (PVA) is a biocompatible material that has been extensively studied for various biomedical applications. PVA hydrogels prepared by a food grade crosslinker, sodium trimetaphosphate (STMP), were shown to have the potential to for use as small diameter vascular grafts due to its low thrombogenicity and tunable mechanical properties. However, PVA does not support endothelialization due to the lack of cell attaching sites and its hydrophilic nature. Endothelialization on synthetic surface is believed to be crucial to prevent thrombus and intimal hyperplasia formation, and thus affecting the long-term patency. Various approaches including biochemical and biophysical modifications can be employed to improve endothelial cell responses. In this thesis, it is hypothesized that the combination of biochemical and biophysical cues can enhance endothelialization on PVA and therefore improving the patency of PVA vascular grafts. The thesis was set out to investigate the effects of a bioactive sulfated polysaccharide and topographies on endothelial cell responses and develop modification methods to improve endothelialization on PVA hydrogels for small diameter vascular graft application.
Fucoidan is a sulfated polysaccharide, isolated mainly from brown seaweeds. It has been known to have multiple bioactivities, including anticoagulant and antithrombotic, anticancer, and anti-inflammatory activities. In the first part of the thesis, we assessed the effect of fucoidan molecule on hemocompatibility and endothelial cell adhesion. Fucoidan was mixed into PVA solution and co-crosslinked by STMP. The endothelialization and hemocompatibility of fucoidan modified hydrogels were evaluated in vitro, ex vivo and in vivo. The results demonstrated that fucoidan improved endothelial cell adhesion on PVA both in vitro and in vivo without affecting the mechanical properties of PVA. Additionally, in vitro and ex vivo hemocompatibility tests showed that modified PVA-fucoidan hydrogels maintained good hemocompatibility with minimal platelet adhesion and activation and low thrombin generation. The results suggest that fucoidan is a promising molecule that can be used to improve endothelialization without sacrificing hemocompatibility.
Cells are surrounded by a complex microenvironment with geometrically defined structures in vivo. The extracellular environment provided three-dimensional (3D) physical cues in micron and sub-micron scale, that plays an essential role in diverse cell processes. Many studies have demonstrated that micro- and nano-topographies can be used to mimic the natural extracellular environment and thus regulate cell processes and behaviors. PVA hydrogels can be topographically patterned using casting methods. However, topography alone on PVA is not sufficient to support endothelial cell adhesion. In the second part of the thesis, we developed a fucoidan conjugation method that is compatible with topographical patterning. The conjugation was achieved through carbonyldiimidazole (CDI) reaction, and fucoidan was aminated using ethylenediamine (EDA) to facilitate the conjugation. This fucoidan conjugation method improved endothelialization on PVA significantly without affecting the mechanical properties of PVA. It also yielded a surface that has a significantly lower thrombogenicity compared to PVA modified by other fucoidan modification methods. Additionally, this conjugation method can be applied to other material, such as expanded polytetrafluoroethylene (ePTFE), which is the typical material for synthetic vascular grafts. The results in this section showed that the fucoidan can be chemically conjugated on synthetic surfaces to improve endothelial cell responses. More importantly, the study suggested that the methods to present fucoidan on the surfaces can significantly alter the surface chemistry and the bioactivities for endothelial cell responses and hemocompatibility.
In the last part of the thesis, PVA hydrogels was modified with fucoidan and topographies of various dimensions. The synergistic effect of fucoidan and topographies on endothelial cell adhesion and functions as well as hemocompatibility were evaluated. The results showed that the topographies on PVA-fucoidan hydrogels could affect endothelial cell adhesion, monolayer formation, and function, while not affecting the hemocompatibility significantly. Among 6 isotropic and anisotropic topographical patterns evaluated, the 2 μm grating appeared to be the most promising topography and was used for in vivo studies. The in vivo results showed a significantly improvement on the blood flow in the fucoidan-conjugated PVA grafts and fucoidan-conjugated PVA grafts with 2 μm grating. Furthermore, the luminal endothelialization on PVA small vascular grafts was achieved by fucoidan and 2 μm grating modification. Finally, we investigated the potential mechanism underlying the improvement on endothelialization by the modification with fucoidan and topography. The results indicated that fucoidan may function similarly as extracellular matrix (ECM) proteins. Fucoidan provides binding ligands for integrins, β1, ⍺2, ⍺5, ⍺6, and ⍺vβ5, that are actively involved in endothelial cell adhesion to ECM proteins. Vascular endothelial cells formed similar number and size of focal adhesions on fucoidan compared to those on gelatin, although size of focal adhesions was smaller compared to those on laminin. Topographies further affected focal adhesion size and alignment, and 2 μm grating promoted cell migration.
Overall, this thesis has demonstrated the hypothesis of presenting a sulfated polysaccharide and topography as luminal modification to improve performance of PVA small diameter vascular graft. The findings of the research work suggest that both fucoidan and topographical cues can benefit the endothelial cell adhesion and regeneration of endothelial monolayer both in vitro and in vivo, and thus are promising modification strategies for blood contact materials and vascular devices. | en |
