Bone Abstracts

Fracture healing is a multistep process that involves many cell lineages and is still not fully understood. We aimed to identify and characterize population of mesenchymal progenitor cells during its commitment within a fracture callus. To identify and trace cells in periosteum and bone marrow we used αSMA promoter-driven inducible Cre expression (αSMA-CreERT2) combined with a Cre-activated tdTomato reporter (Ai9) to generate αSMACre/Ai9 mice. Tibias, fixed with an intramedullary stainless steel pin, were fractured in 3–4 month old mice treated with tamoxifen the day before and the day of injury. Histological analysis of αSMACre/Ai9 mice indicated an expansion of tomato+ cells with fibroblastic shape in the periosteum proximal and distal to the injury 2 days after fracture. Six days after fracture numerous tomato+, chondrocytes were observed. By day 12 a population of osteoblasts in the fracture were tomato+. Periosteum/soft callus and BM were collected 2 days after tamoxifen treatment (unfractured), and 2 and 6 days after fracture and digested using collagenase/trypsin for cell flow cytometry sorting. RNA was extracted from sorted αSMACre labeled populations (tomato+), amplified, and hybridized to Illumina arrays (n=3).

We observed that Notch signaling components were decreased in tomato+ cells following fracture, including Notch1, 3, 4, Hes1, and Hey1. In order to assess the effect of Notch signaling in progenitor cells, BMSC and periosteal cultures from αSMACre/Ai9 mice with and without the Rosa-NICD transgene were treated with hydroxytamoxifen then sorted to obtain tomato+ cells. In cultures without Notch overactivity cells are capable of differentiation into osteoblast, adipocyte and chondrocyte, however in the presence of Notch activation, chondrogenesis, osteogenesis and adipogenesis are decreased. This is the first study to characterize a population of mesenchymal progenitor cells that actively participate in fracture callus formation. Downregulation of Notch signaling may be important for commitment of the cells to mature lineages.