Abstract
Fractures in horses–whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.
Highlights
Fractures in horses are often fatal, extremely expensive to treat, and in certain cases an injury leading to euthanasia [1, 2]
mesenchymal stromal cells (MSCs) play a fundamental role in the initial phase of fracture healing
MSCs that fulfill these criteria were utilized to establish the equine in vitro fracture hematoma (FH) model, consisting of peripheral blood and MSCs.After cultivation for three passages, the MSCs adhered to the plastic surface and showed their typical fibroblastoid morphology (Fig 1A)
Summary
Fractures in horses are often fatal, extremely expensive to treat, and in certain cases an injury leading to euthanasia [1, 2]. Equine fractures and their subsequent treatment are of great economic interest [3], especially for horses deployed in racing. Similar to treatment in humans, current strategies towards long bone fractures in horses focus on stabilization of the fracture site by means of screws or nails [8, 9]. Cell therapy with either mesenchymal stromal cells [13, 14] or osteoprogenitor cells [15] is of upcoming interest especially for fractures that cannot be stabilized due to the location (fetlock, coffin bone). Bone healing is generally considered to be delayed [16] and contradictory to phylogenetically lower developed animals, the bone quality is diminished after trauma [17]
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