The zone of calcified cartilage: A human explant culture model and a proof-of-concept transcriptomic study of 3D laser-cut interface tissue.

OBJECTIVE

The cartilage-bone interface, including the zone of calcified cartilage (ZCC), is a thin layer of calcified tissue with a low cell number, thus making it technically challenging to study. The aim of this study was to develop methods to selectively study the ZCC for gene expression analysis and transcriptomic studies.

DESIGN

We set-up a method to isolate ZCC explants comprising the ZCC and a thin layer of subchondral bone from human osteochondral tissue (n=4 patients) for tissue culture. The cellular response to a pro-inflammatory cytokine cocktail or TGF-ß1 (72 h) was compared to unstimulated explants. Full-thickness non-calcified cartilage (NCC) was used as control group. Next, we have set-up an optical coherence tomography (OCT)-guided 3D-laser microtomy to isolate the calcified ZCC and used FLASH-seq technology (n=4 patients) to characterize the cells in the ZCC compared to donor-matched deep zone of non-calcified cartilage (dNCC).

RESULTS

The culture model revealed metabolically active cells in ZCC explants that responded to inflammatory cytokines and to TGF-ß1. The responses were clearly different from the responses of NCC. With 3D-laser microtomy we isolated sufficient RNA from fresh human ZCC. ZCC had similar expression of cartilage-associated genes, lower expression of angiogenesis-related genes, and higher expression of SOST compared to dNCC.

CONCLUSION

The ZCC explant model holds promise to decipher the role of the cartilage-bone interface in disease progression. OCT-guided 3D-laser microtomy and FLASH-seq are innovative technologies overcoming the limitation of standard approaches to phenotypically characterize chondrocytes in the thin ZCC layer and in other calcified tissues.