Abstract
There is now considerable experimental data to suggest that inflammatory cells collaborate in the healing of skeletal fractures. In terms of mechanisms that contribute to the recruitment of inflammatory cells to the fracture site, chemokines and their receptors have received considerable attention. Our previous findings have shown that Duffy antigen receptor for chemokines (Darc), the non-classical chemokine receptor that does not signal, but rather acts as a scavenger of chemokines that regulate cell migration, is a negative regulator of peak bone density in mice. Furthermore, because Darc is expressed by inflammatory and endothelial cells, we hypothesized that disruption of Darc action will affect post-fracture inflammation and consequently will affect fracture healing. To test this hypothesis, we evaluated fracture healing in mice with targeted disruption of Darc and corresponding wild type (WT) control mice. We found that fracture callus cartilage formation was significantly greater (33%) at 7 days post-surgery in Darc-KO compared to WT mice. The increased cartilage was associated with greater Collagen (Col) II expression at 3 days post-fracture and Col-X at 7 days post-fracture compared to WT mice, suggesting that Darc deficiency led to early fracture cartilage formation and differentiation. We then compared the expression of cytokine and chemokine genes known to be induced during inflammation. Interleukin (Il)-1β, Il-6, and monocyte chemotactic protein 1 were all down regulated in the fractures derived from Darc-KO mice at one day post-fracture, consistent with an altered inflammatory response. Furthermore, the number of macrophages was significantly reduced around the fractures in Darc-KO compared to WT mice. Based on these data, we concluded that Darc plays a role in modulating the early inflammatory response to bone fracture and subsequent cartilage formation. However, the early cartilage formation was not translated with an early bone formation at the fracture site in Darc-KO compared to WT mice.
Highlights
Fracture healing is a complex process that involves the interaction of soluble mediators, extracellular matrix components, resident cells and infiltrating leukocyte subtypes, which participate differentially in the classically defined three phases of fracture healing: inflammation, bone formation and bone remodeling [1,2]
We found that fracture callus size (Fig. 2A) and fracture cartilage area (Fig. 2B) were significantly greater at 7 days post-surgery in duffy antigen receptor for chemokines (Darc)-KO mice compared to wild type (WT) mice (45% and 33%, respectively, p,0.03)
Since our previous studies have established that bone resorption is reduced in the unfractured bones of the Darc-KO mice [8], we propose that cartilage resorption at the fracture site of Darc-KO mice could be delayed due to reduced recruitment of osteoclast precursors to the fracture calluses in KO mice compared to WT mice
Summary
Fracture healing is a complex process that involves the interaction of soluble mediators, extracellular matrix components, resident cells and infiltrating leukocyte subtypes, which participate differentially in the classically defined three phases of fracture healing: inflammation, bone formation and bone remodeling [1,2]. The role of the duffy antigen receptor for chemokines (Darc), which is known to play an important role in chemokine-regulated leukocyte/neutrophil trafficking during inflammation has not been characterized in fracture healing. Darc is known to bind chemokines that regulate cell trafficking [9] It is highly expressed in erythrocytes as well as vascular endothelial cells [10,11], the cell types that play key role in wound healing process [12,13,14]. Based on the established role of inflammation in fracture healing, and the predicted role of Darc in regulating function of inflammatory chemokines, we proposed that Darc expression plays an important role in post-fracture inflammation and fracture healing. We have used Darc-KO mice and wild type mice to compare femur fracture healing and the postfracture expression levels of inflammatory genes in the two lines of mice
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