Andrea Schwab, Christoph Lohmann, Jessica Bertrand
Affiliation(s):
Department of Orthopaedics, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
Background: Ectopic mineralization is one hallmark of osteoarthritis mainly found as mineral deposits on the superficial layer of articular cartilage. In healthy patients, cartilage calcification is restricted to the zone of calcified cartilage, a thin (75 – 250 µm) interfacial layer connecting the articular cartilage and the subchondral bone. The expansion of this layer towards the articular cartilage is characterized in associated with osteoarthritis. This expansion might be a result of cartilage mineralization in the deep zone. So far, there are no predictive ex vivo models on cartilage mineralization to study mechanisms and inhibitors of cartilage calcification.
Objectives: In this study we compared the effect of different phosphate sources (β-glycerophosphate: βGP, adenosine triphosphate: ATP) on cartilage calcification in a human ex vivo explant model. Further, we investigated the influence of extracellular matrix proteoglycan content on mineral deposition.
Methodology: Non-calcified cartilage explants were harvested from macroscopically intact condyles of patients (n>5 donors) undergoing knee replacement. Full thickness cartilage explants (8mm diameter) were divided in four pieces and cultured overnight in chondrogenic media (DMEM high glucose (HG), 10% FBS, sodium pyruvate and antibiotics (Pen/Strep) to equilibrate. Hereafter, explants were cultured for one week in calcification media (chondrogenic media supplemented with dexamethasone and ascorbic-acid-2-phosphate). Either βGP (20mM) or ATP (1mM) or a combination of both was used to induce explant calcification.
To study the effect of proteoglycans on cartilage calcification, explants (n≥4 donors) were treated with Pronase or Hyaluronidase (1mg/ml, 1h, 37°C), and cultured them as described before. The distribution of proteoglycans and calcium crystal deposition in the extracellular matrix of explants was analyzed using Safranin-O and von Kossa staining (day 0 and 7). For image quantification, positive stained area was normalized to the respective day 0 sample.
Findings: Proteoglycan content varied in the zones of articular cartilage with an increase from superficial zone towards the deep zone. The one week culture in presence of phosphates in the culture media did not affect the proteoglycan content based on Safranin-O stainings. The von Kossa staining showed a spontaneous calcification in the control media (44±95 fold relative to day 0) and βGP supplemented media (25±42 fold). Due to donor variation, the calcifying capacity and the response to the phosphate stimuli varied between the patients.
We detected no calcium cyrstals in the superficial layer whereas in the middle and deep zone a stronger von Kossa staining was observed, indicating a zonality of calcification propensity. Further, calcium staining was restricted to the cytoplasm and nuclei of chondrocytes in all conditions with only few donors showing a pericellular staining. We observed no differences between the different phosphate sources supplemented to the culture media.
The enzymatic pre-treatment of explants with hyaluronidase and Pronase removed all proteoglycans from the cartilage matrix. Interestingly, image quantification of von Kossa stained sections nearly abolished calcification increase (<10 fold) in all groups after 7 days of incubation in calcification medium.
Significance The results of this study indicate that extracellular matrix proteoglycans directly influence the capability of chondrocytes to deposit calcium crystals.