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Identification and Characterization of a Mesenchymal Progenitor Cell Population Involved in Fracture Healing

Introduction: Bone fractures are common injuries that are associated with significant morbidity and cost, particularly if they heal poorly. Fracture healing is a complex, multistep process that involves many cell lineages and is still not fully understood. Understanding the sources of progenitor cells involved in natural healing, and the mechanisms governing their recruitment and expansion is important for the development of better therapeutic strategies. Studies of gene expression in whole fracture callus have shown that around 50% of transcripts in the mouse genome are altered during the process of healing and do not distinguish contributions from different cell lineages. We have shown that smooth muscle alpha actin (αSMA) is a marker of progenitor cells that expand rapidly following fracture, and show significant contribution to callus formation. We aimed to identify and characterize this population of mesenchymal progenitor cells during its commitment to mature elements within a fracture callus. Methods: All animal procedures were IACUC approved by UConn Health Center. To identify and trace cells in periosteum and bone marrow (BM) we used αSMA promoter-driven inducible Cre expression (αSMA-CreERT2) combined with a Cre-activated TdTomato reporter Ai9 to generate αSMACre/Ai9 mice. To track differentiation into mature lineages we have utilized triple transgenic mice with an osteoblast/osteocyte specific GFP-reporter (Col2.3GFP) or a chondrocyte-specific reporter (Col2A1Cyan). 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 was performed on frozen sections of fixed and decalcified fractures at various timepoints. Periosteum/soft callus and BM were collected 2 days after tamoxifen treatment (unfractured), and 2 and 6 days after fracture. Periosteum was digested using collagenase/trypsin to obtain a single cell suspension for cell sorting or flow cytometry (FACS) analysis. RNA was extracted from sorted αSMACre labeled populations (tomato+), amplified, and hybridized to Illumina MouseRef-8 arrays (n=3 per group). Following the observation that components of the Notch signaling pathway decreased after fracture we crossed αSMACre mice with a transgenic model expressing a Cre-mediated active Notch1 (Rosa-NICD). αSMACre/Rosa-NICD/Ai9 mice and their αSMACre/Ai9 littermates were used to assess the effects of Notch pathway activation in bone marrow stromal cell (BMSC) and periosteal cell cultures in vitro. Results: Histological analysis of fractures in 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+, Col2A1cyan+ chondrocytes were observed. By day 12 a population of osteoblasts (indicated by concomitant Col2.3GFP expression) in the fracture were tomato+. Tomato+ osteoblasts remained in the remodeled cortical bone 40 days after fracture indicating that the αSMA-expressing cells present in the periosteum prior to injury represent a population of osteochondroprogenitors that contribute to the healing process. FACS analysis indicated that these cells represented 2.4% of periosteal cells in unfractured bone, 3.7% of cells 2 days after fracture and 35.5% of cells at Day 6. In the bone marrow of the same samples tomato+ cells comprise 0.05%, 0.2% and 1.5% of cells. Gene expression analysis of tomato+ periosteal cells revealed that two days after fracture many genes associated with mitosis or immune response (e.g. chemokines Cxcl2 and Ccl7) were upregulated compared to cells in unfractured bones. By day 6, upregulated genes were associated with bone and cartilage, with >50-fold increases in aggrecan and Col2a1, and elevated bone sialoprotein and osterix (Fig 1). Numerous downregulated genes were associated with vascular and muscle development, including the expected decrease in αSMA expression. Notch signaling components were decreased in tomato+ cells following fracture, including Notch1, 3, 4, Hes1 and Hey1. These changes were confirmed by real time PCR (Fig 1). FACS analysis indicated that the majority of tomato+ cells in periosteum before and after fracture are CD45- (>95%), and 30-40% express Sca1 and PDGFRβ, markers of progenitor cells. In contrast, ~80% of tomato+ cells in BM are CD45+, and this proportion increases after fracture. In addition, microarray analysis revealed over 3000 genes differentially regulated between BM and periosteal cell populations in intact bones. In order to assess the effect of Notch signaling in αSMA-expressing 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 these cells are capable of differentiation into osteoblast, adipocyte and chondrocyte lineages, however in the presence of Notch activation, chondrogenesis was blocked, and osteogenesis and adipogenesis decreased in both cultures. The periosteal cells overexpressing Notch also showed increased proliferation. Discussion: We have characterized αSMA as a marker of mesenchymal progenitor cells in the periosteum that make a major contribution to fracture repair. After fracture, proliferation is stimulated in αSMA-labeled periosteal progenitor cells followed by differentiation into chondrocytes and osteoblasts. Downregulation of Notch signaling may be important for commitment of the cells to mature lineages. We are also able to label cells within the bone marrow that can contribute to bone formation using this marker, however the gene expression and cell surface marker profiles of these two cell populations are very different suggesting that we are labeling different cell populations within the bone tissue. Significance: This is the first study to identity and characterize a defined population of mesenchymal progenitor cells that actively participate in fracture callus formation and bone repair. We have analyzed this population using gene expression and cell surface marker analysis prior to and during fracture. Further characterization of this population of cells has the potential to identify means by which we can recruit or expand progenitor cells that could improve fracture healing.

Bone Fracture; Bone Biology; Chondrocytes

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