IntroductionEquine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10β1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling.MethodsAdipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics.ResultsA total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs.DiscussionTo date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.
Equine osteoarthritis is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterised by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilises their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin aplha 10 beta 1-selected mesenchymal stem cell treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Adipose tissue derived, integrin aplha 10 beta 1-MSCs were injected into the osteoarthritis afflicted joint after 18 days post surgery. Sixty-nine synovial fluid samples were collected via aseptic arthrocentesis at day 0, 18, 21, 28, 35, and 70. Synovial fluid was hyaluronidase treated and extracellular vesicles isolated using differential ultracentrifugation. Extracellular vesicles were characterised using the Exoview human tetraspanin chip. Extracellular vesicle concentration, surface marker identification, fluorescent microscopy and tetraspanin colocalization analysis was undertaken, in conjunction with nanoparticle tracking analysis. For proteomics, extracellular vesicle pellets were suspended in urea lysis buffer. Samples were reduced, alkylated and digested on SP3 beads with trypsin/LysC. A data independent acquisition mode was utilised for nano liquid chromatography tandem mass spectrometry analysis on a Triple TOF 6600 mass spectrometer. A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR≤ 0.05) between control and osteoarthritis treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p=0.023), complement activation (classical pathway) (p=0.023), and collagen containing extracellular matrix (p=0.034). To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis and an improved phenotype.
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