Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Flynn, Lauren E

Abstract

Adipose tissue engineering holds promise for the development of therapeutic strategies for subcutaneous adipose tissue regeneration to treat defects resulting from congenital birth defects, invasive surgical procedures and traumatic injuries. Decellularized adipose tissue (DAT) scaffolds represent a potential off-the-shelf tissue substitute for volume augmentation. Seeding the DAT with adipose-derived stromal cells (ASCs) has been shown to enhance adipose tissue regeneration in immunocompetent animals in vivo. Although promising, this strategy is limited by low cell attachment on the DAT. As such, this thesis focused on the development of bioreactor strategies to enhance the capacity of human ASCs to stimulate angiogenesis and adipogenesis within the DAT.

Culturing human ASCs on the DAT scaffolds within a perfusion bioreactor under hypoxia (2% O2) promoted ASC expansion, and altered cell phenotype, upregulating the expression of hypoxia inducible factor 1-alpha (HIF-1a), inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-a) in the ASCs in the peripheral regions of the DAT. Further, bioreactor culture modulated the expression of pro-angiogenic and immunomodulatory paracrine factors, suggesting that the capacity of the ASCs to stimulate regeneration may have been altered by shear stress stimulation. In vivo testing in athymic nude mice demonstrated that angiogenesis and adipogenesis within the DAT were markedly enhanced when the ASCs were cultured for 14 days within the perfusion bioreactor under 2% O2 prior to implantation as compared to bioreactor culture under 20% O2, as well as static-cultured, freshly-seeded and unseeded controls. Analysis of host cell infiltration indicated that there was increased CD31+ endothelial cell recruitment and potentially adipogenic progenitor cell recruitment into the DAT implants, as well as a shift towards a more pro-regenerative macrophage response, in the 2% O2 bioreactor group relative to static cultured controls.

Building from this work, a novel scalable rocking bioreactor platform was explored as an expansion and preconditioning system for ASCs. Preliminary studies indicated that culturing ASCs on DAT coatings within the rocking bioreactor enhanced the expression of the pro-angiogenic and immunomodulatory markers CD146 and iNOS. Collectively, this work supports that dynamic culture systems can be applied to enhance the pro-regenerative potential of ASC-seeded DAT bioscaffolds.

Summary for Lay Audience

The adipose (fat) tissue underneath the skin has limited ability for self-healing. Damage or loss of these tissues resulting from conditions including trauma, burns, or congenital birth defects can lead to anxiety and post-traumatic stress disorder (PTSD). Current therapeutic strategies have numerous limitations and there is a need for new approaches that promote the stable regeneration of adipose tissue for volume augmentation applications in plastic and reconstructive surgery.

To address this challenge, human adipose tissue discarded as surgical waste was processed to extract cellular components that would cause an immune response and produce protein-based decellularized adipose tissue (DAT) scaffolds. In combination with adipose-derived stromal cells (ASCs), a population of regenerative cells found in fat, the DAT was shown to promote the regeneration of fat tissues. Building from this work, the current thesis primarily focused on investigating whether culturing the ASCs on the DAT within a perfusion bioreactor could enhance their capacity to stimulate blood vessel and adipose tissue formation. In-depth biological characterization indicated that human ASC function was altered by culturing within the bioreactor under low oxygen tension. More specifically, these cells produced a range of proteins that could stimulate tissue regeneration. Testing in a pre-clinical mouse model demonstrated that culturing the ASCs on the DAT scaffolds using the established perfusion bioreactor strategy was effective at promoting adipose tissue regeneration within the DAT. These effects were mediated by enhancing the infiltration of host cells that contributed to the formation of new blood vessels and fat, as well as by promoting a more pro-regenerative host immune response.

Although promising, it may be challenging to scale up the perfusion bioreactor for clinical applications in humans where a large number of cells are needed. As such, the development of a more scalable rocking bioreactor was explored as an alternative to the perfusion bioreactor. Initial results showed that this system could also promote the expression of positive markers of the regenerative capacity of ASCs. Overall, the work in this thesis represents an advancement towards a clinically translatable strategy to support the stable and predictable regeneration of the fat tissue layer below the skin.

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