Harry Hields, Eithne Comerford, Emily Clarke, Maddie Jones, James Anderson

University of Liverpool, Liverpool, United Kingdom

Objectives

Canine cruciate ligament disease (CCLD) is the leading cause of pelvic limb lameness in dogs and is strongly associated with osteoarthritis. Current treatments often restrict activity or involve significant financial cost and surgical risk. Bovine milk-derived extracellular vesicles (BMEVs) have demonstrated anti-inflammatory and anti-catabolic effects in cartilage models; however, their role in ligament disease has not been investigated. This study aimed to investigate whether BMEVs modulate gene markers associated with CCLD using an in vitro model to assess their potential as a novel, cost effective CCLD therapeutic.

Methods

BMEVs were isolated from pasteurised skimmed bovine milk (1 ml) using size exclusion chromatography and characterised by nanoparticle tracking analysis. Ruptured canine cruciate ligamentocytes (n=6) were isolated from ruptured cruciate ligaments obtained as clinical waste during stifle surgeries with owner consent and ethical approval. Cells were treated with BMEVs alone or either prior or following cytokine (interleukin-1β and interleukin-6) stimulation. Relative gene expression was assessed using qRT-PCR. Data normality was assessed using a Shapiro-Wilk test, followed by mixed-effects analysis or Friedman tests where appropriate, with p<0.05 considered significant following Dunn’s multiple comparison adjustment.

Results

Pre-treatment of ligamentocytes with BMEVs (mean size 219.3 nm and concentration 3.91×10¹⁰ particles/ml) demonstrated a downregulation of SOX9 (p=0.02) and IL-6 (p=0.01) genes compared to the vehicle only group.

Statement: Impact/ Clinical Significance

BMEVs modulated baseline gene expression in diseased ligamentocytes and prevented cytokine-induced dysregulation. These findings suggest BMEVs may attenuate inflammatory and degenerative pathways involved in CCLD progression and warrant further investigation as a potential non-invasive therapeutic strategy for the management of CCLD.