Abstract
Introduction
Cartilage comprises chondrocytes and extracellular matrix. The matrix contains different collagens, proteoglycans, and growth factors produced by chondroprogenitor cells that differentiate from proliferating to hypertrophic chondrocytes. In vitro chondrocyte growth is challenging due to differences in behaviour between 2D and 3D cultures. Our aim is to establish a murine 3D spheroid culture method using chondrocytes to study the complex interaction of cells on the chondro-osseous border during enchondral ossification.
Method
Primary chondrocytes were isolated from the knee of WT new-born mice and used to form 10,000 cell number spheroids. We used the ATDC5-chondrocyte cell line as an alternative cell type. Spheroids were observed for 7, 14, and 21 days before embedding in paraffin for slicing. Alcian blue staining was performed to identify proteoglycan positive areas to prove the formation of extracellular matrix in spheroids. Collagen type 2, and Collagen type X expression were analyzed via quantitative real-time PCR and immunohistochemistry.
Result
Alcian blue staining showed increasing matrix formation from day 7 to day 14 and proliferative chondrocytes at early time points. Both cell types showed increasing mRNA expression of Collagen type 2 from day 7 to day 21. Collagen type X positive staining starting from day 14 on confirmed the development of hypertrophic stage of chondrocytes. ATDC5 cells exhibited a slower progression in chondrogenic differentiation compared to primary chondrocytes.
Conclusion
In chondrocyte spheroids, we observed proceeding differentiation of chondrocytes reaching hypertrophic phase. Primary chondrocytes showed faster development than ATDC5 cell line. Overall, spheroid culture of chondrocytes could be a good basis to study the interaction of different cells types of the chondro-osseous border by combination of chondrocytes with e.g., endothelial cells and osteoblasts within the spheroid. Those organoid cultures might also help to reduce animal experiments in the future, by mimicking complex regeneration procedures like bone growth or fracture healing.
DFG(German Research Foundation)
