Abstract
The purpose of the current study was to establish an in vitro model for osteoarthritis (OA) by co-culture of osteochondral and synovial membrane explants. Osteochondral explants were cultured alone (control-1) or in co-culture with synovial membrane explants (control-2) in standard culture medium or with interleukin-1β (IL1β) and tumor necrosis factor (TNFα) added to the culture medium (OA-model-1 = osteochondral explant; OA-model-2 = osteochondroal-synovial explant). In addition, in OA-model groups a 2-mm partial-thickness defect was created in the centre of the cartilage explant. Changes in the expression of extracellular matrix (ECM) genes (collagen type-1 (Col1), Col2, Col10 and aggrecan) as well as presence and quantity of inflammatory marker genes (IL6, matrix metalloproteinase-1 (MMP1), MMP3, MMP13, a disintegrin and metalloproteinase with-thrombospondin-motif-5 (ADAMTS5) were analysed by immunohistochemistry, qPCR and ELISA. To monitor the activity of classically-activated pro-inflammatory (M1) versus alternatively-activated anti-inflammatory/repair (M2) synovial macrophages, the nitric oxide/urea ratio in the supernatant of osteochondral-synovial explant co-cultures was determined. In both OA-model groups immunohistochemistry and qPCR showed a significantly increased expression of MMPs and IL6 compared to their respective control group. ELISA results confirmed a statistically significant increase in MMP1and MMP3 production over the culturing period. In the osteochondral-synovial explant co-culture OA-model the nitric oxide/urea ratio was increased compared to the control group, indicating a shift toward M1 synovial macrophages. In summary, chemical damage (TNFα, IL1β) in combination with a partial-thickness cartilage defect elicits an inflammatory response similar to naturally occurring OA in osteochondral explants with and without osteochondral-synovial explant co-cultures and OA-model-2 showing a closer approximation of OA due to the additional shift of synovial macrophages toward the pro-inflammatory M1 phenotype.
Highlights
Osteoarthritis (OA), a chronic degenerative joint disease characterized by cartilage breakdown, subchondral bone remodeling and synovial inflammation, is the most common musculoskeletal disorder in humans as well as in horses
There was no difference in overall collagen type-1 (Col1) and Col2 staining over time or between the four groups
There were no significant differences between the 2 control groups with the exception of their Col2 expression, which was significantly higher in control-2 compared to control-1
Summary
Osteoarthritis (OA), a chronic degenerative joint disease characterized by cartilage breakdown, subchondral bone remodeling and synovial inflammation, is the most common musculoskeletal disorder in humans as well as in horses. Secondary to a variety of etiologic factors such as mechanical injury, genetics, ageing, gender and obesity, a common molecular pathway linking biochemical and biomechanical processes leads to the typical pathological progression of OA with an imbalance of cartilage matrix synthesis and degradation and a vicious positive feedback loop involving cartilage breakdown and synovial inflammation [1,2,3,4,5,6,7,8,9,10,11]. The NO/urea ratio reflects the M1/M2 polarization and can be used as a functional readout of their relative proportions [19, 20]
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