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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Immunol. | doi: 10.3389/fimmu.2019.02588

Spatial distribution of macrophages during callus formation and maturation reveals close crosstalk between macrophages and newly forming vessels

 Jonathan Stefanowski1, 2,  Annemarie Lang1, 2, Ariana Rauch1, 2, Linus Aulich1, 2, Markus Köhler2,  Alexander F. Fiedler2,  Frank Buttgereit1, 2, 3,  Katharina Schmidt-Bleek1, 3, 4,  Georg N. Duda4,  Timo Gaber1, 2, 3,  Raluca A. Niesner2, 5 and  Anja E. Hauser1, 6*
  • 1Charité Medical University of Berlin, Germany
  • 2Deutsches Rheuma-Forschungszentrum (DRFZ), Germany
  • 3Charité Medical University of Berlin, Germany
  • 4Julius Wolff Institute for Biomechanics and Musculoskeletal Regeneration, Charité Medical University of Berlin, Germany
  • 5Department of Veterinary Medicine, Free University of Berlin, Germany
  • 6A Leibniz Institute, Deutsches Rheuma-Forschungszentrum (DRFZ), Germany

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization.
In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration – the expansion phase (d1 - d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14 - d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2, Osx) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the facture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization.

Keywords: Bone Regeneration, macrophage, endothelial cell, H-type vessel, intravital microscopy, limb, CX3CR1 myeloid cells

Received: 30 Jul 2019; Accepted: 18 Oct 2019.

Copyright: © 2019 Stefanowski, Lang, Rauch, Aulich, Köhler, Fiedler, Buttgereit, Schmidt-Bleek, Duda, Gaber, Niesner and Hauser. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Anja E. Hauser, Charité Medical University of Berlin, Berlin, 10117, Baden-Wurttemberg, Germany,