In Vitro Effects of Hyaluronic Acid on Equine Mesenchymal Stem Cells in Osteoarthritis Research

Mesenchymal stem cells are, due to their potential in cartilage regeneration and their immuno-modulatory functions, of a great interest in osteoarthritis research and therapeutic applications. Osteoarthritis is a painful condition leading to a loss of articular cartilage, inflammation, and changes in synovial fluid composition, such as reduction of hyaluronic acid molecular weight. Hyaluronic acid, a key component of synovial fluid, interacts with cells via its receptor, CD44, and has differing effects on cells depending on its molecular weight. Intra-articular injection of hyaluronic acid, and/or mesenchymal stem cells, is an effective treatment for osteoarthritis. This study investigates how inflammatory conditions and hyaluronic acid molecular weight af-fects equine mesenchymal stem cells. Adipose-derived equine mesenchymal stem cells were treated with TNFα and IL-1β to simulate the onset of inflammatory conditions and simultaneously conditioned with high (> 950 kDa), and low molecular weight (15-40 kDa) hyaluronic acid, to determine if hyaluronic acid modifies effects caused by the cytokines. Effect of hyaluronic acid without cytokine induction was also explored. After six days of conditioning, cell viability was measured and CD44 expression was quantified with flow cytometry and visualised via immunocytofluorescence. Morphology of mesenchymal stem cells was visualised via phase-contrast microscopy. Hyaluronic acid conditioning had either no, or negative effects on viability of cytokine non-induced cells compared to the unconditioned control. Cytokine induction had a positive effect on cell viability compared to the unconditioned control. This effect was enhanced by condition-ing with low molecular weight hyaluronic acid. CD44 was highly expressed across all groups and hyaluronic acid conditioning did not affect CD44 expression in induced and non-induced groups. The presented study brought a novel finding of the effect of hyaluronic acid on cell viability during the onset of inflammatory conditions, which can serve as a basis for further studies.