# Studying the formation of the zone of calcified cartilage in bovine cartilage explants ex vivo

## TL;DR

This study introduces an ex vivo model to investigate how calcified cartilage forms in bovine joints, revealing distinct calcification patterns between top and bottom cartilage layers.

## Contribution

The paper presents the first ex vivo model to study zone of calcified cartilage formation and its regulation by top and bottom cartilage layers.

## Key findings

- A distinct calcified cartilage layer formed in explants, resembling the zone of calcified cartilage in mature tissue.
- Bottom cartilage layers calcified with viable chondrocytes, while top layers calcified only when devitalized.
- Top layers inhibited calcification in bottom layers and expressed higher FGF18, PTHLH, and MGP.

## Abstract

The zone of calcified cartilage (ZCC) connects non-calcified articular cartilage to the subchondral bone, acting as transitional layer. Regeneration of this layer is key for cartilage repair but remains a challenge. Knowledge on the formation of this layer during development is limited. This study describes the use of an ex vivo explant culture model to investigate the formation of the ZCC.

Explants were harvested from immature bovine metacarpophalangeal joints and cultured in the presence of β-glycerophosphate for 3 weeks as osteochondral explants, full-thickness cartilage or divided in top and bottom cartilage layers. To investigate cell-driven vs matrix-dependent calcification, explants were devitalized. Calcification was analysed using calcium uptake, micro-computed tomography, gene expression analysis, and histological stainings.

A distinct area of calcified cartilage formed in the explants ex vivo. This layer showed similar characteristics to the ZCC in mature bovine tissue. Viable chondrocytes in bottom layers actively contributed to cartilage calcification, while calcification in top layers was only present in devitalized top layer explants. Top layers inhibited cartilage calcification in bottom layers and expressed higher levels of FGF18, PTHLH and MGP, while the bottom layers expressed more ALPL, COL10A1 and IHH.

We present the first ex vivo model allowing to study and modulate cartilage calcification and the formation of the ZCC. We demonstrated an inherent zone-specific calcification pattern within the cartilage explants. This model allows future studies investigating mechanisms of ZCC formation in cartilage repair procedures, and the role of the top layer in pathological cartilage calcifications and potential interventions.