MIF does only marginally enhance the pro-regenerative capacities of DFO in a mouse-osteotomy-model of compromised bone healing conditions.

The initial phase of fracture healing is crucial for the success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis or smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy for fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer - deferoxamine (DFO) - and a less known HIF-enhancer - macrophage migration inhibitory factor (MIF) - to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, only the application of DFO without MIF during the initial healing phase increased callus mineralization and vessel formation in a preclinical mouse-osteotomy-model modified to display a compromised healing. Although we did not find a synergistically effect of MIF when added to DFO, our findings provide additional support for a preventive strategy towards bone healing disorders in patients with a higher risk by accelerating fracture healing using DFO to stabilize HIF-1α.

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 whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

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. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.

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.

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.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease.

Specific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with predefined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.