gms | German Medical Science

Objectives: Syndecan-1 regulates inflammatory responses, controls cell migration during wound healing, and stabilizes endothelial cell-cell junctions [1]. By binding and producing growth factors, it also helps to control endothelial cell proliferation and differentiation [2]. Our previous research indicated that Syndecan-1 has a considerable impact on the development of blood vessels at the cartilage-bone interface and is expressed in chondrocytes at the growth plate and during fracture healing.

Our aim is to establish a 3D spheroid culture method using chondrocytes to study the complex interaction of cells on the chondro-osseous border during enchondral ossification.

Methods: Primary chondrocytes were isolated from the knees of new-born mice (WT/Sdc1-/-) and used to form 10,000 cell number spheroids. We used the ATDC5-chondrocyte cell line as an alternative cell type. Different cell numbers (5,000, 10,000, and 30,000) were used to form spheroids and observed for 7, 14, and 21 days. Syndecan-1, Collagen type 2, and Collagen type X expression were analyzed via quantitative real-time PCR and immunohistochemistry. Alcian blue staining was performed to identify proteoglycan positive areas to prove the formation of extracellular matrix in spheroids.

Results and conclusion: Alcian blue staining showed increasing matrix formation from day 7 to day 14 and proliferative chondrocytes at early time points as well as hypertrophic chondrocyte increasing from day 7 to 14. There was a gradual increase in the size of spheroids from day 7 to day 21. Both WT and Sdc1-/- primary chondrocytes showed increasing mRNA expression of Collagen type 2 and Collagen type X (confirmed by IHC staining) from day 7 to day 21. We could show Syndecan-1 expression especially at the early time point decreasing after day 14. Syndecan-1 deficient spheroids show decreased Collagen 2 and X expression compared to WT. Additionally, ATDC5 cells exhibited a slower progression in chondrogenic differentiation compared to primary chondrocytes from day 14 onwards.

In chondrocyte spheroids, we observed the differentiation of chondrocytes reaching the hypertrophy phase. Primary chondrocytes showed faster development than the ATDC5 cell line. Syndecan-1 was expressed in the early differentiation phase. 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.