Development of a novel 3D culture system to understand bone-cartilage crosstalk in osteoarthritis

Hughes, David James

Abstract

Osteoarthritis (OA) is a chronic degenerative disease of the articulated joint that affects approximately 10% of men and 18% of women over the age of 60 globally. There is no cure for OA, and current treatments aim to attenuate the symptoms e.g., non-steroidal anti-inflammatories are used to manage pain, and in severe cases, surgical intervention may be necessary. There is currently no validated in vitro model of OA, which limits understanding of the disease and makes the development of new therapies more challenging.
Animal models of OA do exist; however, scientists are now expected to adhere to the “3Rs” principle of animal experimentation: reduce, replace, and refine. The development of a physiological in vitro model of OA would reduce the need for animal experimentation and improve the consistency of results obtained using in vitro and in vivo models.Development of a typical two-dimensional (2D) in vitro model of OA is challenging as the key cell types, in particular cartilage cells, often lose their phenotype. Additionally, in vitro models often focus on only one cell type (predominantly chondrocytes in the case of OA) which does not give any indication of crosstalk between cartilage and bone cells (osteoblasts) that becomes dysregulated in articulated joints during OA progression, a phenomenon which has been recognised in more recent OA research.
With the development of three-dimensional (3D) cell culture technologies, such as self-assembling supramolecular hydrogels, there is an opportunity to create a physiological in vitro model of OA using these new materials; 3D models have been shown to maintain cellular phenotypes and provide opportunities for the co-culture of multiple cell types and for the creation of direct cellular interfaces.
The research described here aimed to develop a 3D hydrogel co-culture model of OA by determining the most appropriate cell types and the optimal hydrogel conditions to recapitulate the articulated joint under both physiological and OA pathological conditions. Firstly, the most appropriate cell types were identified by analysis of the expression of genes and proteins associated with healthy and diseased chondrocytes and osteoblasts using RT-qPCR and immunoblotting techniques. The optimum hydrogel conditions for growing these cells were determined using RT-qPCR, immunoblotting, immunostaining, and dye-based fluorescent staining, across a variety of hydrogel culture conditions, including hydrogels of varying stiffnesses and composition.
Finally, autophagy, a cellular degradation pathway that plays a key role in OA, and is a druggable target, was manipulated in vitro using the thiopurine Azathioprine, a potential novel drug for the treatment of OA.

Citation

Hughes, D. J. Development of a novel 3D culture system to understand bone-cartilage crosstalk in osteoarthritis. (Thesis). Edinburgh Napier University

• Thesis Type: Thesis
• Deposit Date: Sep 2, 2024
• Publicly Available Date: Sep 2, 2024
• DOI: https://doi.org/10.17869/enu.2024.3789809
• Award Date: Jul 3, 2024