Jasmin Bäuerle, Carolin Schneider, Agnieszka Halm-Pozniak, Christoph H Lohmann, Jessica Bertrand, Andrea Schwab
Affiliation(s):
Orthopaedic Clinic, Otto-von-guericke-university Magdeburg, Medical Faculty
Background: The main components of cartilage extracellular matrix (ECM) are collagen type II and sulfated glycosaminoglycans. During osteoarthritis (OA) the ECM is degraded and calcium-containing crystals are deposited. Cartilage degeneration is characterized by a loss in sulfated glycosaminoglycans, cartilage fibrillation and collagen type I rich fibrocartilage formation. These changes in the cartilage ECM might play a role in ectopic cartilage calcification during OA. However, the role of matrix proteins in the deposition of calcium-containing crystals in osteoarthritic cartilage is largely unknown. The aim of this study is to study the matrix-associated calcification using a human ex vivo cartilage explant model.
Methodology: Knee explants were taken from patients (n=8) undergoing knee replacement surgery due to OA. The patient cohort was characterized using the Kellgren-Lawrence score (1-4) and the osteoarthritis research society international (OARSI score 0-6) score to evaluate the severity of the patients' OA. The type of calcified crystal deposits (basic calcium phosphate (BCP) and calcium pyrophosphate (CPP)) was examined using Raman spectroscopy.
Explants of full-thickness non-calcified cartilage tissue were collected from the preserved femoral condyles (8 mm in diameter) and divided into four parts. The cells residing in the tissue explants were devitalized by freeze-thawing (four cycles). The devitalized explants were cultured for three weeks to induce cartilage calcification. The basic medium for cartilage explants was based on Dulbecco´s modified eagle medium high glucose, antibiotics (amphotericin B and penicillin/streptomycin), sodium-pyruvate (1mM), ascorbic acid (0.02mM) and dexamethasone (10nM). ß-glycerophosphate (ß-GP) was used as phosphate source to induce calcification. We compared two ß-GP concentrations (10mM vs. 20mM). Additionally, we compared the effect of foetal bovine serum on cartilage calcification. The basic media containing ß-GP was supplemented with either foetal bovine serum (FBS 10%), heat-inactivated FBS (HI-FBS, 10%) or the chemically defined supplement ITS (insulin-transferrin-selenite, 1%). After three weeks of ex vivo culture the samples were stained with safranin orange (S/O) and von-Kossa (VK) to visualize and localize the calcified cartilage in the explants. The VK-positive area was quantified using the ImageJ software and normalized to the total cartilage area. The crystal type of the induced cartilage calcification was characterized using Raman.
Results: The mean Kellgren-Lawrence score for the patient cohort is 3.75. The mean OARSI score of the macroscopic intact cartilage used for the explant culture was 1.5. BCP calcification was prevalent in the explant tissues cultured with FBS and HI-FBS at 10mM and 20mM ß-GP (mean: FBS-10mM: 10.03%, FBS-20mM: 6.161%, HI-FBS-10mM: 9.650%, HI-FBS-20mM: 8.020%). No calcification was found in samples cultured with ITS and in the control media. Media containing FBS and 10mM ß-GP resulted in the most homogenous deposition of BCP compared to the respective control group (p=0.0427). Comparing the two ß-GP concentrations, no differences were apparent (p>0.05). The comparison of the VK positive stained area with the S/O staining showed that the calcification was restricted to the areas rich in sulfated glycosaminoglycans layers.
Conclusion: Our study shows that FBS is required to induce calcification in ex vivo devitalized cartilage tissue. With this model we can study the role of cartilage matrix components in ectopic calcification.