Unraveling the mysteries of the growth plate; An approach in modeling and mapping the process of endochondral ossification without the need for animal sacrifice

Abstract

The musculoskeletal system, fundamental to mobility, is reliant on the intricate organisation of the musculoskeletal tissues including bones, cartilage, tendons, and muscles. The foundation of these tissues is laid during embryonic development through two distinct mechanisms, endochondral and intramembranous ossification. The former entails the differentiation of mesenchymal stem cells into chondrocytes, forming a cartilaginous template that matures into bone. The epiphyseal growth plate, a structure at the end of long bones, regulates longitudinal bone growth through chondrocyte proliferation, differentiation, and ultimately, apoptosis or trans differentiation into osteoblasts. This process separates the growth plate in distinct zones: resting, proliferative, and hypertrophic, each with unique cellular characteristics and matrix composition.

Trauma induced large bone defects that the body itself is unable to repair highlight the need for new treatment strategies. Current strategies to treat large bone defects include grafts. However this approach encounters limitations, which shows the need to explore novel bone regeneration strategies. Studying endochondral ossification, particularly intriguing due to its hypoxia-tolerant nature, presents such a promising strategy. However, this requires a comprehensive understanding of endochondral ossification, a gap that studying the epiphyseal growth plate can help fill.

Animal models are indispensable tools used for growth plate research, even though challenges arise in direct translation of the findings in these models to humans. In vitro models provide controllability, allowing manipulation and large-scale analysis to study endochondral ossification more extensively. Additionally, they have the potential to replace animal models according to 3Rs principle. The ATDC5 cell line is an excellent candidate to model endochondral ossification due to its chondrogenic differentiation potential and easy accessibility and maintenance. Nonetheless, a cell line is limited. The use of primary growth plate cells from larger and more representative animals can enhance the modelling for a better translation to the human process of endochondral ossification. This thesis outlines a comprehensive approach to investigate growth plate biology and endochondral ossification. A 3D in vitro growth plate model is developed using chondrogenic ATDC5 cells to simulate chondrogenic maturation. Subsequently, primary growth plate cells from dogs and pigs are isolated to elevate the model and explore surface markers for identifying resting zone progenitors. Promising markers are identified, which lays a foundation for future research in progenitor cell isolation.

Results demonstrate successful induction of chondrogenic maturation and differentiation in ATDC5 cells and establishment of a 3D model utilizing primary dog growth plate cells. Potential markers, CD146, CD105, CD90, CD44, CD73, and CD271, for identifying progenitor cells are validated for presence and expression in the resting zone. This work provides the foundation for advanced in vitro models, offering insights into endochondral ossification and potential applications in growth disorders, musculoskeletal diseases, and regenerative therapies. As the journey continues, these findings promise to enhance our comprehension of bone formation and contribute to improving global health and well-being.