Abstract
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.
Highlights
Bone healing is a spatiotemporally regulated regeneration process, ideally leading to complete restoration of the broken bone without fibrous scar formation [1]
In the mouse-osteotomy model used in this study, residuals of cells in the fracture hematoma are visible in Movat’s Pentachrome staining at day 3, while the osteotomy gap is filled with a mixture of bone marrow cells including hematopoietic cells at day 7, and endochondral bone formation occurs between day 14 and day 21 (Figure 1A)
The peripheral borders of the gap and adjacent periosteal regions are populated by cells coexpressing Runx2 and SRY-box transcription factor 9 (Sox9) in the nucleus, indicating their potential for either osteogenic or chondrogenic differentiation (Figure 1B)
Summary
Bone healing is a spatiotemporally regulated regeneration process, ideally leading to complete restoration of the broken bone without fibrous scar formation [1]. Drastic vascular structural plasticity has been shown during bone marrow regeneration between 7 and 21 days by our group using a longitudinal microendoscopic method at cellular resolution [10] Angiogenic factors, such as vascular endothelial growth factor (VEGF) are of great importance in order to induce vascularization. Buettmann et al most recently showed that especially the release of VEGFA by Osterix (Osx)+ osteoprogenitors/pre-osteoblasts is critical for vessel formation during fracture healing [16]. It is well-known that the crosstalk between endothelium and bone cells is essential for the integrity and formation of bone. The presence of type H vessels has been reported at sites of bone regeneration [10, 21], early events inducing the formation of these vessels in those situations have not yet been investigated
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