Skeletal fractures are one of the most common traumatic injuries. While the majority of fractures go on to heal, the incidence of fractures progressing to delayed- or non-union remains a significant concern. Fracture healing and vascularization are closely linked through angiogenic-osteogenic coupling, which regulates the biology of bone development, homeostasis, and repair. Enhancing vascularization by disruption of anti-angiogenic pathways could be a potential therapeutic to improve fracture healing. Thrombospondins (TSPs) are an important class of anti-angiogenic molecules that are produced by mesenchymal cells in the fracture callus. While implicated in regulation of angiogenesis, the TSP interactome is complex and includes over 83 unique ligands. A dominant anti-angiogenic mechanism of TSP1 and TSP2 is believed to be through interaction with cluster of differentiation 47 (CD47), a ubiquitously expressed integrin-associated penta-transmembrane protein that also plays an important role in regulation of cell cycle and homeostasis of the immune system. The purpose of this research is to define the spatiotemporal expression of TSP2 during fracture healing, and to describe disruption of the TSP-CD47 axis in skeletal fractures. A new method to generate physiologically relevant fractures and improve experimental rigor is also described. TSP2 spatiotemporal expression in skeletal fractures was studied using TSP2 green fluorescent protein reporter (TSP2-rep) mice. TSP2 is expressed in the periosteum of uninjured diaphyseal bone. Expression of TSP2 decreases immediately after fracture and then increases in undifferentiated regions of the early fracture callus. As mesenchymal stem cells (MSCs) differentiate to become chondrocytes, TSP2 expression is lost. Immunophenotyping by flow cytometry confirms that the majority of TSP2 expressing cells are MSCs. In vitro assays demonstrate all adherent and colony forming cells express TSP2, but that the non-adherent cells are TSP2 negative. The effect of disrupting the TSP-CD47 axis on fracture healing was studied using TSP1-null, TSP2-null, CD47-null, and wildtype (WT) mice. Disruption of the TSP-CD47 axis has a variable effect on callus morphology depending on whether TSP1, TSP2, or CD47 is absent. TSP2-null mice show accelerated intramembranous bone formation. TSP1-null and CD47-null mice show an opposite callus phenotype of reduced bone volume and bone mineral content relative to TSP2-null mice. To focus on the downstream receptor rather than the thrombospondin molecule, disruption of CD47 was studied in the context of ischemic fracture healing using CD47-null mice and mice treated with a CD47 disrupting morpholino. Loss of CD47 limits recovery of perfusion after ischemia relative to WT mice. CD47-null and morpholino-treated mice show reduced ischemic callus bone volume and bone mineral content. Mesenchymal stem cells (MSC) from CD47-null mice exhibit decreased cellular proliferation and colony formation. Loss of CD47 also downregulates expression of stem cell genes and limits entry into S phase of the cell cycle. The TSP-CD47 axis is a potent regulator of cell homeostasis and has potential for therapeutic development; however, because of cell and context specificity, therapeutics should be cautiously developed and the potential for diverse off-target effects considered.
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