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

Stefanowski, Jonathan; Lang, Annemarie; Rauch, Ariana; Aulich, Linus; Köhler, M; Fiedler, Alexander; Buttgereit, Frank; Schmidt‐Bleek, Katharina; Duda, Georg N.; Gaber, Timo; Niesner, Raluca; Hauser, Anja E.

doi:10.3389/fimmu.2019.02588

Cited by 42 publications

(50 citation statements)

References 68 publications

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“…We repeatedly performed imaging in the osteotomy gap over the time course of regeneration, starting at Day 2, up to Day 14 after osteotomy. As shown in Figure and consistent with our observations using immunofluorescence histology , vascularization of the fracture hematoma became detectable between Day 3 and Day 4 in the FOV. Vessels developed sprouts into the nonvascularized tissue (Fig.…”

Section: Resultssupporting

confidence: 91%

“…The flexibility of the system with regard to insertion depth of the lens will allow us to adjust the position of the FOV according to the biological question. As known from histology studies, the callus resolves and becomes replaced by hematopoietic marrow tissue at later phases of regeneration in deeper regions of the bone . Limbostomy will us to analyze this important and fascinating process at cellular resolution.…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, bone regeneration is a process with rapid phases such as the onset of vascularization or the distribution of inflammatory cells . Vascularization in the fracture gap is completed within 24 h after onset.…”

Section: Discussionmentioning

confidence: 99%

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Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

et al. 2020

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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.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

et al. 2020

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“…Macrophages are essential during fracture healing and we have previously reported the crosstalk between macrophages and vessel especially during the early phase (56,57). Since the treatment with both DFO and MIF/DFO resulted in an enhanced and accelerated endochondral ossification ( Fig.…”

Section: Dfo and Mif/dfo Lead To Enhanced Presence Of Macrophages Andmentioning

confidence: 91%

“…Revascularization is crucial for bone regeneration, tightly regulated by the microenvironment and appears in two waves at day 7 and day 21 (17,57). CD31 + endothelial progenitors enter the fracture gap during the initial phase of fracture healing (until day 7) (57).…”

Section: Vessel Formation Is Increased By Hif-stabilizationmentioning

confidence: 99%

HIF-stabilization prevents delayed fracture healing

Lang

Helfmeier

Stefanowski

et al. 2020

Preprint

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AbstractThe initial phase of fracture healing decides on 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, 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 of 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, the application of DFO with or without MIF during the initial healing phase accelerated callus mineralization and vessel formation in a clinically relevant mouse-osteotomy-model in a compromised healing setting. Our findings provide support for a promising preventive strategy towards bone healing disorders in patients with a higher risk due to e.g. delayed neovascularization by accelerating fracture healing using DFO and MIF to stabilize HIF-1α.

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External Mechanical Stability Regulates Hematoma Vascularization in Bone Healing Rather than Endothelial YAP/TAZ Mechanotransduction

Mehl,

Farahani,

Brauer

et al. 2024

Advanced Science

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Bone fracture healing is regulated by mechanobiological cues. Both, extracellular matrix (ECM) deposition and microvascular assembly determine the dynamics of the regenerative processes. Mechanical instability as by inter‐fragmentary shear or compression is known to influence early ECM formation and wound healing. However, it remains unclear how these external cues shape subsequent ECM and microvascular network assembly. As transcriptional coactivators, the mechanotransducers yes‐associated protein 1 (YAP)/transcriptional coactivator with PDZ‐binding motif (TAZ) translate physical cues into downstream signaling events, yet their role in sprouting angiogenesis into the hematoma after injury is unknown. Using bone healing as model system for scar‐free regeneration, the role of endothelial YAP/TAZ in combination with tuning the extrinsic mechanical stability via fracture fixation is investigated. Extrinsically imposed shear across the gap delayed hematoma remodeling and shaped the morphology of early collagen fiber orientations and microvascular networks, suggesting that enhanced shear increased the nutrient exchange in the hematoma. In contrast, endothelial YAP/TAZ deletion has little impact on the overall vascularization of the fracture gap, yet slightly increases the collagen fiber deposition under semi‐rigid fixation. Together, these data provide novel insights into the respective roles of endothelial YAP/TAZ and extrinsic mechanical cues in orchestrating the process of bone regeneration.

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

Cited by 42 publications

References 68 publications

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

HIF-stabilization prevents delayed fracture healing

External Mechanical Stability Regulates Hematoma Vascularization in Bone Healing Rather than Endothelial YAP/TAZ Mechanotransduction

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