Cartilage heals poorly after injury, which can lead to irreversible joint disease and catastrophic fractures in racehorses. Therefore, regenerative therapies including mesenchymal stromal cells (MSCs) are often used clinically to promote healing. However, the mechanisms underlying the positive effects of cell therapies are unclear, and the most effective strategies to stimulate repair of orthopedic tissues have yet to be determined.

Extracellular vesicles (EVs) are small, membrane-bound structures derived from many cell types, and are known to play critical roles in cell-cell communication. EVs contain diverse cargoes, including proteins, micro- RNAs, and even whole mitochondria (so-called ‘mitovesicles’). EV cargos can be taken up by target cells, affecting a wide array of cell functions. The role of EVs in pathologic signaling (i.e. disease pathogenesis) has long been recognized. More recently, MSC-derived EVs have been investigated for their anti-inflammatory and pro-healing properties in human medicine. However little information is available regarding EVs in veterinary medicine. Furthermore, mitovesicles have not been investigated as possible regenerative orthobiologics.

The broad objective is to investigate mitochondria-containing extracellular vesicles (mitovesicles, mitoEVs) as potential regenerative therapeutics, as well as biomarkers of joint injury in the horse.

Our overarching hypotheses are that mitoEVs containing healthy mitochondria, such as those derived from MSCs, can rescue injured cells and promote tissue repair. Furthermore, mitoEVs containing dysfunctional mitochondria are released into synovial fluid after joint injury, representing a potential early biomarker. Finally, unhealthy mitovesicles can enhance cartilage repair mechanisms by stimulating the release of healthy mitovesicles by MSCs.

In Aim 1, we will determine if mitoEVs isolated from MSCs can improve mitochondrial function, prevent cell death and stimulate pro-healing mechanisms in injured chondrocytes. In Aim 2, we will characterize EVs from joint fluid of horses with and without injury, to determine if there is a difference in function of mitochondrial cargo within mitoEVs between healthy and injured joints. Finally, we will investigate if there is crosstalk between these two processes; we will determine if dysfunctional mitoEVs can enhance healthy mitoEV release by MSCs.

Relevance to equine health and racing: Understanding the role of mitoEVs in the pathologic events after joint injury may lead to new biomarkers, and help identify horses requiring targeted therapy or modified training programs. These studies will also provide a foundation for developing MSC mitoEVs as a new cell-free regenerative therapy in horses. These studies will provide data in direct support of a larger NIH grant proposal.