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

Abstract

Fracture healing is a regenerative process that involves coordinated responses of many cell types, but characterization of the roles of specific cell populations in this process has been limited. We have identified alpha smooth muscle actin (αSMA) as a marker of a population of mesenchymal progenitor cells in the periosteum that contributes to osteochondral elements during fracture healing. Using a lineage tracing approach, we labeled αSMA-expressing cells, and characterized changes in the periosteal population during the early stages of fracture healing by histology, flow cytometry, and gene expression profiling. In response to fracture, the αSMA-labeled population expanded and began to differentiate toward the osteogenic and chondrogenic lineages. The frequency of mesenchymal progenitor cell markers such as Sca1 and PDGFRα increased after fracture. By 6 days after fracture, genes involved in matrix production and remodeling were elevated. In contrast, genes associated with muscle contraction and Notch signaling were downregulated after fracture. We confirmed that activating Notch signaling in αSMA-labeled cells inhibited differentiation into osteogenic and adipogenic lineages in vitro and ectopic bone formation in vivo. By characterizing changes in a selected αSMA-labeled progenitor cell population during fracture callus formation, we have shown that modulation of Notch signaling may determine osteogenic potential of αSMA-expressing progenitor cells during bone healing.

NOTCH signaling in skeletal progenitors is critical for fracture repair

“…Fracture-repair mechanisms are believed to recapitulate a series of spatiotemporal cellular and signaling events that occur during skeletal development (29,30), suggesting a potential involvement of NOTCH signaling. Evidence that further implicates NOTCH in the general processes of fracture repair has recently emerged, including: (a) an upregulation of some NOTCH components in murine callus tissues during fracture healing (31), (b) a downregulation of NOTCH signaling specifi- cally within certain mouse skeletal progenitors during early fracture repair (32), and (c) evidence that systemic downregulation of NOTCH signaling just prior to fracture prolongs the inflammatory phase and alters fracture healing in mice (33). While these studies have implicated NOTCH in the fracture-repair process, the precise role of NOTCH within specific cell lineages remained unknown.…”

The rational use of animal models in the evaluation of novel bone regenerative therapies

“…Although most studies were conducted with bone marrow derived mesenchymal progenitor cells (MPCs), other tissues have been described to contain osteoprogenitor cells with similar regenerative potential including adipose tissue, muscle, umbilical cord blood, periosteum, dental pulp and periodontal ligament [4,[39][40][41][42][43][44][45]. The multilineage differentiation ability, paracrine effects and immunomodulatory properties of MPCs make them an ideal for tissue engineering and regenerative purposes [5,7,[46][47][48].…”

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

“…Recently, we have published a new high-throughput cryohistological method for assessing several response measures within a given section from mineralized tissue [5][6][7][8][9][10][11][12][13][14] . The process involves stabilizing the cryosection with frozen cryotape, adhering the taped section rigidly to a microscope slide, and conducting several rounds of staining and imaging on each section.…”