Andrea Schwab (1,2), Judith Veldman (1), Nicole Kops (1), Xander den Dekker (1), Kayleigh Hiscock (1), Wilfred van Ijcken (1), Merve Sinram (1, Brigitta Stolze (1), Heiko Richter (1), Jessica Bertrand (2), Eric Farrell (1), Gerjo JVM van Osch (1)
Affiliation(s):
1. Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
2. University Orthopaedic Clinic, Otto-von-Guericke-University Magdeburg, Medical Faculty, Magdeburg, Germany
3. LLS ROWIAK LaserLabSolutions GmbH, Hannover, Germany
The zone of calcified cartilage is the intermediate layer connecting non-calcified articular cartilage to the subchondral bone in adolescence. The tidemark is the wavy line marking the upper boundary of this layer at the transition towards the non-calcified cartilage. The cement line marks the transition towards the subchondral bone. During osteoarthritis (OA), the zone of calcified cartilage advances towards the deep zone of non-calcified cartilage. Thus, the calcification of the deep zone of non-calcified articular cartilage is a pathologic process associated with OA. The limited thickness of 75-250 µm and the low cell density with less than 150 cells per mm2 are the main challenges studying this calcified tissue layer1,2. The aim of this study was to histologically characterize the zone of calcified cartilage and to establish a workflow to isolate this zone for RNA sequencing.
Osteochondral explants were harvested from the medial tibial plateau of OA patients undergoing knee replacement surgery. Tissue was processed for (immune-) histological characterization. The median minimal and maximal thickness of the calcified layer (distance between tidemark and cementline) was quantified on Mallory trichrome stained sections (n=10 measurements per image, n=15 donor).
To isolate the zone of calcified cartilage from fresh osteochondral tissue blocks for phenotypic characterization of the chondrocytes residing in the calcified cartilage layer, the optical coherence tomography guided laser microtome TissueSurgeon was used (n=4 donor). Donor-matched non-calcified cartilage was cut as control tissue. RNA was isolated and the FLASH-seq technology was used for bulk RNA sequencing. Histologically processed tissue from the same donors were used for assessing the OA severity using the OARSI score (grade 0-6) on safranin-orange stained sections.
The zone of calcified cartilage and non-calcified articular cartilage were positive stained for negatively sulfated glycosaminoglycans and collagen type II, but not in the subchondral bone. The minimal and maximal thickness of the calcified layer in tissue from OA patients ranged between 52 µm and 139 µm. We established a workflow to laser cut calcified tissue from fresh osteochondral explants. These samples showed mild signs of OA (OARSI grade 1.5-2.5). We successfully isolated RNA from the zone of calcified cartilage and, for the first time, low yield input RNA from chondrocytes in the zone of calcified cartilage was sequenced. The sequencing resulted in sufficient coverage of the transcriptome with 5000-15000 genes mapped in non-calcified cartilage and the zone of calcified cartilage samples. Using principal component analysis, the zone of calcified cartilage samples clustered together more closely compared to the donor-matched non-calcified cartilage samples. The most upregulated gene in the calcified cartilage was SOST, a negative regulator of bone formation. Chondrogenesis associated genes (ACAN, Col2A1) were less expressed in the zone of calcified cartilage compared to non-calcified cartilage.
The TissueSurgeon guided laser cutting of calcified tissue together with the FLASHseq technology holds promise to study molecular mechanisms at the cartilage-bone interface and to decipher the role of this layer in OA disease progression.
References:
1. Fan X, et al. 2022. doi: 10.1096/fj.202101449R
2. Lane LB, et al. 1980. doi: 10.1302/0301-620X.62B3.7410471