Osteoarthritis (OA) is a major cause of equine lameness, which affects the athletic capacity and welfare of horses across breeds and disciplines. There are no therapies available to the equine practitioner to manipulate the joint environment to slow the destructive process of OA. Though regenerative therapies including mesenchymal stromal cells (MSCs) are often employed with the intent of enhancing the inherent poor healing capacity of articular cartilage, the mechanisms by which MSCs improve clinical symptoms have yet to be elucidated. Further, disadvantages include regulatory burdens, cost, and time delay associated with MSC therapy. Therefore, alternative cell-free regenerative therapies are currently being explored, including extracellular vesicles (EVs).

EVs are small membrane-bound particles that facilitate cell-cell communication. Derived from many cell types, EVs contain diverse cargos, including whole organelles. Recent evidence suggests that intact mitochondria can be transferred from MSCs to injured cells by EVs, or mitovesicles (mitoEVs). This intercellular mitochondrial transfer has been shown to improve recipient cell survival and healing in other tissues but has not been investigated in cartilage. Our group has successfully isolated and characterized mitoEVs produced by MSCs. Furthermore, we have shown that MSCs can donate healthy mitochondria to injured chondrocytes. However, the effects of isolated mitoEVs on chondrocytes have not yet been studied. We hypothesize that isolated mitoEVs will be taken up by stressed chondrocytes, improving their respiratory capacity. Furthermore, we hypothesize that mitoEVs are abundant in equine blood products, which would provide a culture-free source of mitoEVs.

Therefore, our broad objective is to investigate mitoEVs as a potential cell-free and culture-free regenerative therapy. Our specific project aims are 1) to investigate the effects of mitoEVs on chondrocytes and 2) to investigate non-cultured sources of mitoEVs.

We will collect blood and bone marrow from healthy adult horses to obtain plasma, platelet rich plasma (PRP), bone marrow aspirate concentrate (BMAC), and the cell-conditioned media (CCM) of cultured MSCs. Using filtration, mitoEVs will be isolated from each source. To investigate the effect of mitoEVs on chondrocytes (Aim 1) we will culture equine chondrocytes in a validated OA model and treat them with mitoEVs harvested from CCM. Microrespirometry will be used to assess the ability of mitoEVs to rescue chrondrocyte mitochondrial function. To investigate non-cultured sources of mitoEVs (Aim 2), we will use flow cytometry to characterize and compare the subpopulations of mitoEVs obtained from non-cultured blood products (plasma, PRP, BMAC), and further, to compare these to subpopulations from cell-culture (CCM).