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Characterizing The Contribution Of Circulating Mesenchymal Cells To Pathologic Wound Healing And Heterotopic Ossification
John Butts, BS, Shawn Loder, BS, Shailesh Agarwal, MD, David Fireman, BS, David Cholok, BS, Michael Chung, MD, Christopher Breuler, BS, John Li, MD, Joseph Habbouche, BS, Arminder Kaura, BS, Kavitha Ranganathan, MD, Hsiao Hsieh, Hsung, Shuli Li, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: Pathologic wound healing after injury represents dysregulation of several cellular components of the physiologic wound niche. Trauma induced heterotopic ossification (tHO) is a highly morbid class of pathologic healing characterized by endochondral formation of de novo osseous lesions in soft tissue. These lesions occur at several anatomic sites and it remains unclear which cell populations form the pre-HO niche. Identification of the specific cells which give rise to tHO is critical to the development of targeted therapeutic options. Similarities between endochondral ossification and tHO suggest a common progenitor, however, it is unclear if this population is entirely local or receives contributions from circulating cells. Here we utilized a parabiotic reporter-based model of tHO to identify and characterize contributions from circulating cells to tHO.
METHODS: A mouse model of parabiosis between wild type mice and reporters carrying the CAG-luc-eGFP L2G85 transgene to examine the presence of circulating (eGFP+) mesenchymal cells at the injury site was generated. These animals carry both luciferase and eGFP reporters allowing for concurrent bioluminescent and histologic analysis of circulating populations. After blood chimerism was confirmed, the wild type parabiotic mouse received a hindlimb Achilles' tenotomy and dorsal burn. Bioluminescence imaging was used to study the timing and localization of circulating cells. Immunofluorescence was performed to identify common inflammatory, vascular, mesenchymal markers and to characterize their source and contribution to each stage of the tHO anlagen.
RESULTS: Early pre-osseous lesions were highly enriched in eGFP+ circulating cells (Fig. 1A,B). These cells primarily co-stained for neutrophil (Ly6G+), macrophage (F4/80+), and trauma-reactive mesenchymal (PDGFRα+) markers consistent with acute inflammation and consequent mesenchymal condensation (Fig. 1B). Vascular (CD31+) and pericyte (CD105+) staining at the wound site demonstrated minimal co-staining. Bioluminescent signals persisted from one- to eight-weeks post-injury diminishing slightly by twelve weeks (Fig. 1A). Samples of developing tHO from this period demonstrated presence of eGFP (+) cells in fibroproliferative, cartilaginous, and stromal/marrow populations consistent with joint contribution to early endochondral ossification (SOX9+; OSX+) and to the development of a mature shared marrow space in the definitive osseous lesion (Fig. 1C).
CONCLUSION: Here we demonstrate the presence of circulating cells at all stages of HO from early-inflammatory populations to the definitive and mature marrow. Importantly, we identify the presence of PDGFRα+ mesenchymal cells in pre-osseous lesions with direct contributions to the endochondral anlagen and (eGFP+/PDGFRα+/SOX9+ or OSX+ cells). These data suggest a contributory role for circulating cells in the pathogenesis of tHO and ultimately identifies a novel therapeutic target, the recruitment of circulating cells, to prevent this disease.


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