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Analysis of αSMA-labeled Progenitor Cell Commitment Identifies Notch Signaling as an Important Pathway in Fracture Healing

• Brya Matthews, University of Connecticut Health Center, Department of Reconstructive Sciences, USA, Danka Grcevic, University of Zagreb, Croatia, Liping Wang, University of Connecticut Health Center, USA, Yusuke Hagiwara, University of Connecticut Health Center, USA, Douglas Adams, University of Connecticut Health Center, USA, Ivo Kalajzic, University of Connecticut Health Center, USA Fracture healing is a complex process that involves coordinated cellular responses, but characterization of specific cell populations in this process has been limited. We identified alpha smooth muscle actin (αSMA) as a marker of periosteal progenitor cells that contribute to fracture healing. To identify and trace cells in periosteum we used αSMA promoter-driven inducible Cre (αSMACreERT2) combined with a Cre-activated fluorescent reporter Ai9 to generate SMA9 mice. Tibial fractures were performed after tamoxifen-induced labeling of cells, and the SMA9+ population was characterized during the early stages of fracture healing by histology, flow cytometry and gene expression profiling. Histology indicated that SMA9+ periosteal cells expanded after fracture, and by day 6 were contributing to chondrocytes and osteoblasts within the callus. Periosteal cells from labeled unfractured bone, or periosteum/fracture callus from fracture day 2 and 6 were isolated for flow cytometry or cell sorting for RNA extraction. Over 95% of SMA9+ cells were negative for hematopoietic and endothelial lineage markers. The frequency of mesenchymal stem cell markers such as Sca1 increased after fracture (9% unfractured SMA9+ cells, 26% at day 6). Gene expression profiling showed that proliferation genes and cytokines/chemokines were upregulated 2 days after fracture. By fracture day 6, genes involved in matrix production and remodeling, including chondrocyte markers Col2a1 and aggrecan, and osteoblast markers alkaline phosphatase and bone sialoprotein were elevated. Genes associated with muscle contraction and Notch signaling were downregulated after fracture. Given that Notch signaling is an important developmental pathway in bone, but its role in fracture healing is undefined, we examined the effect of forced Notch activation in SMA9+ mesenchymal progenitor cells in vitro. Cre-mediated activation of Notch signaling in SMA9+ cells from periosteal or bone marrow stromal cell cultures reduced or blocked differentiation into osteogenic, chondrogenic and adipogenic lineages. This is the first study to characterize a defined population of mesenchymal progenitor cells that actively participate in bone repair. We found that downregulation of Notch signaling may be important for commitment of SMA9+ cells to mature lineages. Further characterization of these cells could potentially reveal mechanisms for recruitment or expansion of progenitor cells to improve bone healing.

progenitor cells; Notch; fracture healing

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