Maghsoudlou, P; (2015) Tissue engineering using natural acellular matrices. Doctoral thesis , UCL (University College London).

Abstract

Introduction The demand for organ transplantation has rapidly increased worldwide during the past decade. Conventional transplantation of organs from living and/or deceased donors is limited by the mismatch of organ availability and demand, as well as by the requirement of life-long immunosupression. The field of tissue engineering (TE) offers a therapeutic alternative. The two main components of a tissue-engineered organ are the cells and the scaffold these cells are seeded onto. While synthetic materials can be used for the production of simple structures, only decellularised matrices can be used to reproduce complex organs. Decellularised scaffolds are derived from animal or human organs that have been treated to remove cells and immunogenic material, resulting in natural scaffolds that maintain their architecture of origin. Decellularised scaffolds developed so far are either far too simple or use harsh decellularisation chemicals that damage the extracellular matrix (ECM) thus limiting successful in vivo applications. Methods Intestinal (rat and human), oesophageal (rat and rabbit), pulmonary (rat and sheep) and hepatic (rat) tissues were decellularised using dynamic intravascular infusion of gentle decellularisation chemicals (i.e. deioinised water, sodium deoxycholate, DNase). Scaffolds were characterized to assess cell removal, maintenance of microarchitecture, ECM components, mechanical properties, growth factors, and biocompatibility. Oesophageal scaffolds were stored using four different storage protocols to determine the optimal preservation technique, with follow-up timepoints of up to 6 months. Cells were cultured and seeded to repopulate the intestinal epithelium (i.e. amniotic fluid stem cells [AFSC], organoid units [OU], crypt organoids [CO]), oesophageal muscle (i.e. mesangioblasts [MABs]), and liver parenchyma (i.e. hepatoblastoma cell line [HepG2], induced pluripotent stem cells [iPS]). Intestinal and oesophageal scaffolds were implanted in the omental and 4 subcutaneous compartments of mice to assess neovascularization, cell survival and differentiation following in vitro seeding. Results Intravascular infusion of gentle decellularisation chemicals allowed cell removal while preserving microarchitecture, ECM components, mechanical properties, and growth factors in all scaffolds. The use of chemicals such EDTA, sodium dodecyl sulphate (SDS), and Triton X-100 (TX100) led to destruction in microarchitecture and loss of mechanical properties. Intermittent chemical infusion, mimicking inspiration and expiration, improved microarchitecture preservation in pulmonary scaffolds. Oesophageal scaffold storage in liquid nitrogen vapour, following slow cooling in medium and dimethyl sulfoxide (DMSO) preserved microarchitecture in the most optimal manner for up to 6 months. Seeding with AFSC led to intestinal scaffold repopulation but did not allow epithelial differentiation. Trans-differentiation of AFSC towards an endodermal phenotype was achieved by blockade of the TGFβ pathway and promotion of E-Cadherin expression. Seeding with OU led to intestinal epithelium formation in vivo but not in vitro. Seeding with CO and modulation of the Wnt pathway allowed the formation of intestinal epithelium in vitro. Seeding with HepG2 and iPS allowed the repopulation of hepatic parenchyma. Omental transplantation led to neovascularization and host cell migration. Conclusions Decellularised scaffolds that retain original tissue characteristics can be produced efficiently using gentle chemical and infusion methodologies. Seeding with a cell source that is sufficiently proliferative and can be differentiated in vitro allows the formation of tissue-engineered constructs.

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