Exploring the role of primary cilia in growth plate function and mechanoadaptation by combining high resolution tissue imaging and complex in vitro models

Thesis

Primary cilia are essential organelles in skeletogenesis, with key roles in chondrogenesis, osteogenesis and limb patterning. The micrometre-scale size and delicate structure of primary cilia poses a significant challenges to their in vivo analysis, particularly, for example, within the matrix rich, dense but also dynamic environment of the growth plate. In this project, I developed and applied a high-resolution 3D imaging and analysis pipeline to characterise primary cilia organisation in the postnatal murine growth plate. Using endogenous fluorescent reporters and semi-automated image analysis, I quantitatively mapped cilia prevalence, orientation, length and basal body positioning across thousands of chondrocytes from multiple anatomical regions. These data revealed spatial patterns in cilia organisation, particularly in the resting zone of the middle of the growth plate, implying local micro-environmental influences.

To investigate the functional relevance of these patterns, I examined how genetic deletion of Ift88 and mechanical unloading affect ciliary structure and growth plate architecture. Both Ift88 deletion and limb offloading resulted in tibial growth plate shortening accompanied by subtle reductions in percentage ciliation in the hypertrophic zone. These did not arise with changes in cilia length or orientation, raising questions about the relationship between ciliary morphology and function. My findings, combined with other studies in the literature, suggest that primary cilia may integrate spatial and mechanical cues in a context-dependent manner and highlight the need for further research into the molecular mechanisms linking cilia structure to tissue-level outcomes.

I then described the further development of an in vitro osteochondral model, incorporating a BMP-2 gradient and mechanical loading to assess cilia-mediated responses in a controlled bioengineered context. Adipose-derived murine mesenchymal stem cells and costal chondrocytes were used as a chondroprogenitor source in order to exploit the genetic tools available in mouse lines, such as conditional Ift88 deletion. Although differentiation of mouse MSCs proved difficult, the system demonstrated the feasibility of generating a spatially patterned growth factor gradient in vitro and applying mechanical loading, offering a promising framework for further refinement.

Together, this work combines in vivo and in vitro approaches to advance our understanding of primary cilia in skeletal development and provides methodological platforms for future research in cartilage biology mechanotransduction and ciliopathies.

Authors: Johnson, T

• DOI: 10.5287/ora-zbqdoqpe1
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: in vitro model; primary cilia; growth plate; bone; cartilage