Zone of Calcified Cartilage – a Natural Boarder in the Osteochondral Unit to Control Diffusion of Molecules between Articular Cartilage and Subchondral Bone
Cell Biology
Final Report Abstract
Osteoarthritis (OA) is the commonest joint disease worldwide with more than 20% of the German society suffering from this painful and disabling disease. To intervene the progression of this disease, there is urgent need to better understand the mechanisms at molecular, cell and tissue level of the osteochondral unit, especially the zone of calcified cartilage (ZCC). The ZCC connects the deep zone of non-calcified articular cartilage to the subchondral bone. However, little is known about the role of the ZCC in cartilage homeostasis and its role in OA progression. The aim of this project was to increase our understanding of the role of the ZCC in OA disease and to characterize the phenotype of the cells residing in this thin interfacial layer. We showed that chondrocytes in OA cartilage tissue can take up neutral TRITC-dextran molecules. The size of the dextran molecules was chosen to mimic the size of morphogens and growth factors associated with cartilage homeostasis and hypertrophy. The diffusion of the molecules was limited due to the dense collagen and proteoglycan rich matrix components in the extracellular matrix of the explants. We found a deeper diffusion of the smaller 40kDa TRITC-dextran molecules compared to the 155kDa TRITC-dextran molecules. However, the diffusion was limited to the boundaries of the explant. To study the phenotype of the cells residing in the ZCC we established a workflow to harvest the ZCC from fresh human OA osteochondral tissue. We successfully isolated RNA from this thin layer using the laser microtome TissueSurgeon (collaboration with LLS Hannover) and, for the first time, sequenced the low input RNA (collaboration with Genomics core facility at guest institute). To study the response of the cells in the ZCC to osteoarthritic factors (TGFβ1, a pro-inflammatory cytokine mix), I set up an ex vivo calcified tissue explant culture model comprising the ZCC with a low amount of subchondral bone attached. Cells residing in the calcified tissue explants distinguished from chondrocytes in the non-calcified cartilage explants by a higher expression in hypertrophy and bone associated genes (COL10A1, COL1A1, SPP1, RUNX2). The cartilage associated genes COL2A1 and ACAN were less expressed compared to the non-calcified cartilage explants. Cells in the calcified explants responded to cytokines by an increase in MMP13 and IL6. The stimulation with TGFβ resulted in an increase in the hypertrophy associated gene COL10A1 compared to the non-stimulated control. The characterization of the human ZCC and the response of the cells in the calcified explants to OA associated factors provide a unique approach to deeper understand the mechanism related to cartilage calcification and chondrocyte hypertrophy in OA. The workflow to sequence RNA from this thin and mineralized layer allows for an advanced and deeper characterization of the chondrocytes in the ZCC. This knowledge will open the development of novel approaches to delay OA progression and cartilage degeneration.
Publications
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GRC on Cartilage Biology and Pathology 2023 – Poster “Changes in the human calcified cartilage during osteoarthritis”
Schwab, Andrea
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International Society for Ectopic Calcification (INTEC) Annual Meeting 2023 – Oral “An ex vivo human calcified tissue explant model to study the response of cells in the cartilage-bone interface to cytokines and TGF-β”
Schwab, Andrea
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Loose Bodies Found in the Human Intra-Articular Space Showed Characteristics Similar to Endochondral Bone Formation. CARTILAGE, 15(4), 353-362.
Schwab, Andrea; Pap, Thomas; Krenn, Veit; Rüther, Wolfgang; Lohmann, Christoph & Bertrand, Jessica
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OARSI World Congress 2024 – Poster “A human calcified tissue explant model to decipher the role of the zone of calcified cartilage in osteoarthritis”
Schwab, Andrea
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European Orthopaedic Research Society (EORS) annual meeting 2025 – Oral (accepted) “RNA-sequencing of chondrocytes isolated from the calcified cartilage layer of osteoarthritic osteochondral tissue”
Schwab, Andrea
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Gordon Research Conference (GRC) on Cartilage Biology and Pathology 2025 – Poster (accepted) “Unravel the phenotype of chondrocytes in the zone of calcified cartilage and their response to osteoarthritic factors”
Schwab, Andrea
