Implications for chondrogenesis and cartilage repair

Faculty of Health Sciences – Department of Clinical Medicine

Ann Kristin Hansen
A dissertation for the degree of Philosophiae Doctor – December 2017

Osteoarthritis (OA) is characterised by gradual destruction of articular cartilage and leads to painful and dysfunctional knee-, hip- or hand-joints. With the increasing life expectancy and obesity, the prevalence is expected to rise. In the younger population, joint pain and disability can result from local cartilage defects resulting from injury or disease. After the introduction of autologous chondrocyte implantation (ACI) as a treatment option for localised cartilage defects in the late 80’s, cell‑based repair techniques have been extensively explored through clinical trials aiming to improve long‑term clinical outcomes. However, the role of ACI remains that of postponing joint replacement in patients with localised defects, and despite persistent research on cartilage repair strategies, there is no available treatment to halt or reverse the degenerative process of OA once initiated. The focus of this thesis has thus been to gather new basic knowledge on cartilage functions in the context of cell signalling receptors.
In the first study, we have explored the effects of the powerful inflammatory mediator leukotriene B₄ (LTB₄) on human articular chondrocytes based on studies indicating that this mediator could hold a key role in inflammatory joint diseases. When cyclooxygenase (COX) inhibitors are prescribed to reduce inflammation and pain e.g. in patients with OA, there is a shunting from the prostaglandin- to the leukotriene axis with subsequent upregulation of LTB₄. We demonstrated that chondrocytes
express both the high‑affinity (BLT1) and the low‑affinity (BLT2) LTB₄ receptors by immunolabelling and gene expression. By Western blot, we showed that the high‑affinity BLT1 receptor is active. Upon stimulation of chondrocyte cultures by the ligand, we found no effect on biological functions such as release of inflammatory mediators, proliferation, cartilage gene expression or matrix formation. The overall results suggest that the leukotriene axis is not very active in cartilage, and that the role seen in other inflammatory diseases is probably linked to the ability of LTB₄ to recruit neutrophils, a mechanism that is less prominent in osteoarthritis pathology. In the second paper, we investigated the influence of the active hormonal form of vitamin D, 1α,25(OH)₂D₃, on chondrocyte functions and evaluated potential modulating effects on inflammation as suggested by clinical studies showing improved pain scores after vitamin D supplementation. By immunolabelling and gene‑expression, we found that the expression of vitamin D receptor (VDR) in native cartilage is elusive, but that receptor expression increase upon dedifferentiation and during inflammatory conditions. We also demonstrated that 1α‑hydroxylase, the enzyme catalysing the
conversion of 25(OH)D₃ to 1α,25(OH)₂D₃, is expressed in cartilage and that the expression persists through cellular dedifferentiation and redifferentiation. In monolayer cultures the 25(OH)D₃ was converted to 1α,25(OH)₂D₃ in a dose dependent matter, and exposing chondrocytes to both 25(OH)D₃ and 1α,25(OH)2Dincreased their proliferation rate. The proteoglycan genes ACAN and VCAN displayed an inverse expression pattern, and matrix production was diminished in chondrocytes treated with 25(OH)D₃ or 1α,25(OH)₂D₃. The results imply that cartilage can contribute to the increased level
of 1α,25(OH)₂D₃ seen in synovial fluid of OA patients, but 25(OH)D₃ or 1α,25(OH)₂D₃ may only exert effects on chondrocytes upon dedifferentiation or during inflammatory conditions. The third paper aimed at identifying biomarkers of intrinsic chondrogenic potential in chondrocyte cultures established from 17 donors undergoing ACI treatment. Patient‑derived chondrocytes cultures were grouped according to their chondrogenic abilities in scaffold‑free 3D cultures, as evaluated by
the Bern score. The groupwise expression of cell‑surface molecules including integrins, cell adhesion molecules and growth factor receptors were measured using flow cytometry or gene expression. The gene expression of TGF‑β receptor 3 was inversely related to chondrogenic potential, while all other molecules tested had a uniform expression pattern among all donors. A global proteomic profiling of cell‑associated proteins using tandem‑mass‑tag technology pointed at prolyl 4‑hydroxylase, a pivotal enzyme in collagen triple helix formation, as a biomarker potentially linked to chondrogenic potential.