Rna Sequencing of Chondrocytes Isolated From the Calcified Cartilage Layer of Osteoarthritic Osteochondral Tissue

The zone of calcified cartilage connects the non-calcified articular cartilage with the underlying subchondral bone. Mechanisms and stimuli initiating the advancement of the calcified cartilage layer in osteoarthritis (OA) are not yet understood due to the lack of studies on RNA expression on human cells derived from this layer. The challenge in characterizing this interfacial layer are the low cell density with less than 150 cells per mm 2 [PMID:35032407] and its dense and mineralized thin tissue layer ranging between 75 – 250 µm [PMID:7410471]. The aim of this study was to isolate the calcified tissue and sequence the low input RNA to unravel the phenotype of chondrocytes in the zone of calcified cartilage. We established a workflow to specifically isolate the human zone of calcified cartilage from patients (n=4 donor) undergoing knee replacement surgery. The optical coherence tomography guided laser cutting device TissueSurgeon was used to isolate the thin calcified and non-calcified cartilage layer from fresh osteochondral tissue. RNA was isolated from laser cut samples using a standard protocol. FLASH-seq technology was used for RNA sequencing. Differentially expressed gene analysis was performed using DESeq2 (v1.40.2) comparing calcified and non-calcified cartilage harvested from the same osteochondral tissue. After a process of optimisation, we successfully isolated RNA specifically from the layer of calcified cartilage. For the first time, we reliably sequenced low yield input RNA samples from calcified and non-calcified cartilage using the FLASH-seq Bulk approach. The number of genes mapped in both groups ranged between 5000−15000, indicating sufficient coverage of the transcriptome in most of the samples. The calcified samples from all donors seemed to cluster together more closely than the respective non-calcified cartilage samples using principal component analysis. One of the most upregulated genes in calcified cartilage was SOST , a gene associated with Wnt signaling and bone formation. The angiopoietin-Like Protein 4 was one of the most downregulated gene in calcified samples. The chondrogenesis associated genes including COL2A1, ACAN, COMP were expressed in both layers but less in the calcified compare to the non-calcified samples. The established workflow to sequence RNA isolated from the calcified interface hold promise to study molecular mechanisms at the cartilage-bone interface and to decipher the role of this layer in OA disease progression.