Towards autologous endothelialization of 3D bioprinted vascular constructs

Examensarbete för masterexamen

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Type: Examensarbete för masterexamen
Title: Towards autologous endothelialization of 3D bioprinted vascular constructs
Authors: Oskarsdotter, Kristin
Abstract: Vascularization of tissue engineered constructs is a requirement before tissue engineered organs become a reality, and is hindered in part by the inability to mimic the complexity of natural vasculature. A method showing tremendous potential for this application is 3D bioprinting, where cell-laden biomaterials are deposited to form pre-designed constructs. Biomaterials with excellent properties for 3D bioprinting are nanocellulose-based hydrogels. However, these hydrogels generally lack the ability to promote endothelial cell adhesion which is a prerequisite to form vasculature. One very promising approach to circumvent this issue is periodate-mediated bioconjugation of bioactive molecules, such as proteins, onto a nanocellulose surface to promote cell adhesion. This project aimed to develop a bioconjugation protocol for the covalent immobilization of extracellular matrix (ECM) proteins such as laminin, fibronectin and collagen I onto a nanocellulose-based hydrogel surface. The protocol was then applied to 3D bioprinted constructs with vascular architecture to evaluatethe efficiency in promoting endothelial cell adhesion of both human umbilical vein endothelial cells (HUVECs) and autologously sourced cell populations cell populations from stromal vascular fraction (SVF). SVF is a clinically relevant cell system comprised of a heterogenous cell population with large application potential within tissue engineering. Nanocellulose at the surface of a nanocellulose-hydrogel construct was oxidized by topical application of various concentrations of sodium periodate to find optimum treatment conditions. Aldehyde generation was confirmed by X-ray photoelectron spectroscopy (XPS), and the highest aldehyde content without construct disintegration was generated by a periodate concentration of 0.025M for one hour. These conditions were therefore established as optimal. Surfaces were bioconjugated and analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) to confirm protein immobilization. The protocol was then applied to nanocellulose-hydrogel constructs and evaluated in vitro with HUVECs and SVF-derived cells, respectively. Samples were evaluated by live/dead- and fluorescent staining of actin filaments and cell nuclei at day 1, 3 and 7 post-seeding. From analysis, it could be concluded that bioconjugation of ECM proteins can promote endothelial cell adhesion and viability for up to a week in cell culture, and that laminin elicits the most positive cell response of the tested proteins on cell adhesion, viability and overall morphology. In addition, the bioconjugation of laminin could be concluded to facilitate endothelialization by SVF-derived cell populations, although further study is necessary. Although in need of further optimization, the presented result supports that bioconjugation of bioactive molecules can indeed facilitate endothelial cell adhesion of a nanocellulose-based hydrogel materials, which is a prerequisite for vascularization.
Keywords: vascularization;tissue engineering;3D bioprinting;bioconjugation;endothelialization;stromal vascular fraction;nanocellulose;hydrogel
Issue Date: 2020
Publisher: Chalmers tekniska högskola / Institutionen för biologi och bioteknik
Collection:Examensarbeten för masterexamen // Master Theses

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