Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Anatomy and Cell Biology

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Flynn, Lauren E.

2nd Supervisor

Séguin, Cheryle A.

Co-Supervisor

Abstract

Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a novel detergent-free protocol was developed for decellularizing bovine NP tissues that was effective at removing cellular content while preserving key ECM constituents including collagens and glycosaminoglycans. Culture studies showed that 2-D coatings derived from the DNP could support cell attachment but did not maintain or rescue the phenotype of primary bovine NP cells, which dedifferentiated when serially passaged in monolayer culture on tissue culture plastics. Similarly, the incorporation of DNP particles within methacrylated chondroitin sulphate hydrogels as a 3-D culture platform was insufficient to maintain or rescue the bovine NP cell phenotype based on gene expression patterns.

Summary for Lay Audience

Low back pain is the leading cause of disability worldwide. While the cause of back pain is not well understood, intervertebral disc (IVD) degeneration is thought to be a major contributor. IVD degeneration is thought to originate in the central region of the IVD, which is a gel-like tissue termed the nucleus pulposus (NP). Current therapies primarily focus on pain management rather than targeting the underlying degenerative condition. Surgical approaches often result in the degeneration of adjacent IVDs due to altered spine biomechanics. The lack of disease-modifying treatments has motivated the investigation of biomaterial-based therapies in order to regenerate the NP and restore mechanical function. In particular, extracellular matrix (ECM)-derived bioscaffolds present a promising approach for promoting tissue regeneration. The ECM is unique to each tissue and consists of proteins and sugars that provide tissue structure and biological cues that can guide cellular behaviour. Tissue decellularization, a process aimed at removing the cellular content of a tissue, while preserving the cell-instructive ECM, can be applied to develop ECM-derived bioscaffolds capable of guiding cellular behaviour for use in cell culture and delivery platforms. The current study developed a detergent-free decellularization protocol that effectively extracted immunogenic cellular components from the tissue while preserving pro-regenerative ECM components such as collagens and glycosaminoglycans. Following validation of the decellularization protocol and the characterization of the decellularized NP (DNP), the DNP was applied to generate novel 2-D coatings and 3-D hydrogel bioscaffold platforms. As a first step towards testing the potential of these culture platforms, the behaviour of bovine NP cells grown on the 2-D DNP coatings or within the 3-D hydrogels containing DNP was assessed. The bioscaffolds were shown to support bovine NP cell attachment and viability but the incorporation of the tissue-specific ECM was insufficient to prevent undesired changes in NP-associated gene expression that are observed when the cells are cultured in 2-D on tissue plastics. Overall, these studies developed novel DNP-derived bioscaffolds that should be further investigated as cell culture or delivery platforms for applications in IVD regeneration.

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