RESUMO
CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.
RESUMO
Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin-2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony-forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing.
