This study investigated mice serum and joint microRNA expression profiles in ageing and osteoarthritis to elucidate the role of microRNAs in the development and progression of disease, and provide biomarkers for ageing and osteoarthritis. Whole joints and serum samples were collected from C57BL6/J male mice and subjected to small RNA sequencing. Groups used included; surgically-induced post-traumatic osteoarthritis, (DMM; 24 months-old); sham surgery (24 months-old); old mice (18 months-old); and young mice (8 months-old). Differentially expressed microRNAs between the four groups were identified and validated using real-time quantitative PCR. MicroRNA differential expression data was used for target prediction and pathway analysis. In joint tissues, miR-140–5p, miR-205–5p, miR-682, miR-208b-3p, miR-499–5p, miR-455–3p and miR-6238 were differentially expressed between young and old groups; miR-146a-5p, miR-3474, miR-615–3p and miR-151–5p were differentially expressed between DMM and Sham groups; and miR-652–3p, miR-23b-3p, miR-708–5p, miR-5099, miR-23a-3p, miR-214–3p, miR-6238 and miR-148–3p between the old and DMM groups. The number of differentially expressed microRNAs in serum was higher, some in common with joint tissues including miR-140–5p and miR-455–3p between young and old groups; and miR-23b-3p, miR-5099 and miR-6238 between old and DMM groups. We confirmed miR-140–5p, miR-499–5p and miR-455–3p expression to be decreased in old mouse joints compared to young, suggesting their potential use as biomarkers of joint ageing in mice. MiR-140–5p, miR-499–5p and miR-455–3p could be used as joint ageing biomarkers in mice. Further research into these specific molecules in human tissues is now warranted to check their potential suitability as human biomarkers of ageing.

SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK-ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with (35)S sulphate during static hyperosmolar loading. MEK-ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition. The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.

To identify the effect of alterations in physical parameters such as oxygen and pH on processes associated with cellular redox balance in osteoarthritic chondrocytes. Human osteoarthritic chondrocytes (HOAC) were isolated from total knee arthroplasty samples and cultured in 3-D alginate beads in four different oxygen tensions (<1%, 2%, 5% and 21% O2), at pH 7.2 and 6.2 and in the presence or absence of 10 ng/ml, interleukin-1β (IL-1β). Cell viability, media glycosaminoglycan (GAG) levels, media nitrate/nitrate levels, active matrix metalloproteinase (MMP)-13 and intracellular adenosine triphosphate (ATPi) were measured over a 96-h time course. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential, intracellular pH and reduced/oxidised glutathione (GSH/GSSG) were additionally measured after 48-h incubation under these experimental conditions. Hypoxia (2% O2) and anoxia (<1% O2), acidosis (pH 6.2) and 10 ng/ml IL-1β reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1β increased nitrite/nitrate release, but increases were moderate at 2% O2 and significantly reduced at <1% O2. ATPi was significantly reduced following hypoxia and anoxia and acidosis. At 48 h cellular ROS levels were increased by acidosis and IL-1β but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1β. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1β. This study shows that oxygen and pH affect elements of the redox system in HOAC including cellular anti-oxidants, mitochondrial membrane potential and ROS levels.