Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

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 fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow

Ergründung der Mechanismen der Knochenregeneration mit Hilfe longitudinaler intravitaler Multiphotonen-Endomikroskopie

Bone regeneration is a complex process that involves the interaction between cells of the vascular network, the immune system, and mesenchymal cells. The underlying mechanisms and dynamics are not fully understood. To allow a visual analysis of these mechanisms during regeneration and over time, a system for longitudinal intravital multiphoton microendoscopy of the murine bone (LIMB) was developed. This dissertation aims to 1) describe the immunophysiological reaction to the LIMB system, 2) to adapt the system for longitudinal microendoscopy in osteotomies (Limbostomy), and 3) to elucidate the role of mononuclear phagocytes in the process of angiogenesis during healing. The implantation of the LIMB system resulted in a transient immunophysiological reaction accompanied by the expression of the extracellular matrix marker Laminin, an accumulation of CD45+ lymphocytes, and the presence of CD31hiEmcnhi (type H) endothelium at the site of injury. All reactions cleared after 28 days without impairment of bone vessel perfusion or hematopoiesis. Longitudinal analysis of vasculature using vascular fluorescent dye revealed vascular plasticity in the bone marrow during homeostasis. A modular design of the LIMB system for longitudinal intravital microendoscopy of murine osteotomies, termed Limbostomy, was developed. It is specified by an improved field of view and image quality, as well as adaptability of the endoscope tissue penetration depth. Using transgenic fluorescent reporter mice for the endothelial cell promotor Cadherin, two phases of vascularization were observed after osteotomy. In the first phase, neovascularization started between day 3 and 4 post-osteotomy and was completed within 24 hours. Activated endothelial cells that penetrated the initial fracture hematoma were derived from an existing vascular network. In the second phase, the endothelial network continuously remodeled, while the bony callus was built and restructured. Using immunofluorescent histology, type H endothelium was found to be the predominant vasculature and co-localized with F4/80hi macrophages during healing. In the osteotomy gap, the majority of cells seven days post-osteotomy were CX3CR1+Gr1-F4/80+ macrophages. Using Limbostomy, the increase of CX3CR1+ cells was observed to be initiated 2 – 3 days post-osteotomy. CX3CR1+ cells preceded vascularization and were present throughout the regeneration process. In summary, LIMB reveals vascular dynamics during homeostasis. In addition, the new LIMB approach was further developed to visualize spatiotemporal organization during bone regeneration. The findings imply that niches in the bone marrow are subject to continued tissue changes and that CX3CR1+ mononuclear phagocytes play a role in promoting vascularization and endothelial remodeling.Die Knochenregeneration ist ein komplexer Prozess mit enger Interaktion von Zellen des Gefäßnetzes, des Immunsystems und mesenchymaler Zellen. Die dem Prozess zugrundeliegenden Mechanismen und die Dynamiken sind noch nicht vollständig verstanden. Methoden, die die Analyse dieser Mechanismen während der Heilung und über die Zeit visualisieren, sind nicht verfügbar. Daher wird ein System für longitudinale intravitale Multi-Photonen-Mikroendoskopie des murinen Knochens (longitudinal intravital multiphoton endomicroscopy in the murine bone marrow, LIMB) entwickelt. Diese Arbeit zielt darauf ab, 1) die immunologische Reaktion auf das LIMB-System zu beschreiben, 2) das System für die longitudinale Mikroendoskopie bei Osteotomien (Limbostomy) anzupassen und 3) die Rolle mononukleärer Phagozyten im Prozess der Angiogenese während der Heilung aufzuklären. Die Implantation des LIMB-Systems führte zu einer transienten immunphysiologischen Reaktion, die von der Expression des extrazellulären Matrixmarkers Laminin, einer Akkumulation von CD45+ Lymphozyten und der Anwesenheit von CD31hiEmcnhi (Typ H) Endothel am Ort der Knochenverletzung begleitet wurde. Alle Reaktionen verschwanden nach 28 Tagen, ohne bleibende Beeinträchtigung der Knochengefäßperfusion oder Hämatopoese. Die Analyse des Gefäßsystems mit Hilfe eines vaskulären Fluoreszenzfarbstoffs zeigte vaskuläre Plastizität im Knochenmark während der Homöostase über die Zeit. Ein adaptiertes LIMB-System mit modularem Design, Limbostomie benannt, wurde mit verbessertem Sichtfeld und Bildqualität, sowie Anpassungsfähigkeit in der Eindringtiefe des Endoskops entwickelt. Mit transgenen Fluoreszenz-Reportermäusen unter dem Endothelzellpromotor Cadherin wurden nach der Osteotomie zwei Phasen der Vaskularisierung beobachtet. Eine initiale Neovaskularisierung, die zwischen Tag 3 und 4 nach der Osteotomie einsetzt und innerhalb von 24 h abgeschlossen war. Aktivierte Endothelzellen, die in das initiale Frakturhämatom eindrangen, stammten aus dem bestehenden Gefäßnetz. Das Endothelnetzwerk wurde in einer zweiten Phase kontinuierlich umgestaltet, während Knochenkallus auf- und umgebaut wurde. Mit Hilfe der Immunfluoreszenz-Histologie wurde festgestellt, dass das vorherrschende Gefäßsystem einen Typ-H Endothelphänotyp aufweiste und während der Heilung mit F4/80hi Makrophagen kolokalisierte. Am siebten Tag nach der Osteotomie waren die meisten Zellen in dem Osteotomiespalt CX3CR1+Gr1-F4/80+ Makrophagen. Mit der Limbostomie-Methode wurde beobachtet, dass die Zunahme der CX3CR1+ Zellen 2 – 3 Tage nach der Osteotomie einsetzte. CX3CR1+ Zellen gingen der Vaskularisierung voraus und waren während des gesamten Regenerationsprozesses vorhanden. Zusammenfassend lässt sich sagen, dass die LIMB-Methode vaskuläre Plastizität während der Homöostase offenbarte. Außerdem wurde hier der neue, technische Ansatz der LIMB-Methode erweitert, um die Visualisierung der räumlichen und zeitlichen Organisation während der Knochenregeneration zu ermöglichen. Die Ergebnisse implizieren, dass Nischen im Knochenmark kontinuierlichen Gewebeveränderungen unterworfen sind und dass mononukleäre CX3CR1+ Phagozyten an der Vaskularisierung und dem Umbau des Gefäßnetzwerks beteiligt sind

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

At present, it is not clear how memory B lymphocytes are maintained over time, and whetheronly as circulating cells or also residing in particular tissues. Here we describe distinctpopulations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bonemarrow, identified according to individual transcriptional signature and B cell receptorrepertoire. A population of marginal zone-like cells is located exclusively in the spleen, while apopulation of quiescent Bsm is found only in the bone marrow. Three further residentpopulations, present in spleen and bone marrow, represent transitional and follicular B cellsand B1 cells, respectively. A population representing 10-20% of spleen and bone marrowmemory B cells is the only one qualifying as circulating. In the bone marrow, all cells individuallydock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasmacells, are void of activation, proliferation and mobility.Peer Reviewe