Abstract

Bone healing depends of a transient inflammatory response, involving selective migration of leukocytes under the control of chemokine system. CCR2 has been regarded as an essential receptor for macrophage recruitment to inflammation and healing sites, but its role in the intramembranous bone healing on craniofacial region remains unknown. Therefore, we investigated the role of CCR2 on F4/80+ cells migration and its consequences to the intramembranous healing outcome. C57BL/6 wild-type (WT) and CCR2KO mice were subjected to upper right incisor extraction, followed by micro-computed tomography, histological, immunological, and molecular analysis along experimental periods. CCR2 was associated with F4/80+ cells influx to the intramembranous bone healing in WT mice, and CCR2+ cells presented a kinetics similar to F4/80+ and CCR5+ cells. By contrast, F4/80+ and CCR5+ cells were significantly reduced in CCR2KO mice. The absence of CCR2 did not cause major microscopic changes in healing parameters, while molecular analysis demonstrated differential genes expression of several molecules between CCR2KO and WT mice. The mRNA expression of TGFB1, RUNX2, and mesenchymal stem cells markers (CXCL12, CD106, OCT4, NANOG, and CD146) was decreased in CCR2KO mice, while IL6, CXCR1, RANKL, and ECM markers (MMP1, 2, 9, and Col1a2) were significantly increased in different periods. Finally, immunofluorescence and FACS revealed that F4/80+ cells are positive for both CCR2 and CCR5, suggesting that CCR5 may account for the remaining migration of the F4/80+ cells in CCR2KO mice. In summary, these results indicate that CCR2+ cells play a primary role in F4/80+ cells migration along healing in intramembranous bones, but its deficiency does not critically impact healing outcome.

Highlights

  • The recruitment of circulating blood monocytes and its transition into macrophages at injured tissues are essential steps of inflammatory immune response and healing processes [1,2,3]

  • At 0 day time point, there was a peak of Ly6g-Gr1+ cells (Figure 1I) observed in the blood clot formed post-extraction in WT and CCR2KO mice, with a significant decrease from 7 to 21 days in C57Bl/6 mice, and with no differences observed among different time points in CCR2KO mice

  • A different kinetics for Ly6g-Gr1+ and CD68+ cells infiltration was observed in CCR2KO mice compared with WT, with a slight higher number of Ly6g-Gr1+ and CD68+ cells in CCR2KO mice compared with WT at 21 days

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Summary

Introduction

The recruitment of circulating blood monocytes and its transition into macrophages at injured tissues are essential steps of inflammatory immune response and healing processes [1,2,3]. Macrophages comprise a heterogeneous myeloid cell lineage that participate directly or indirectly in tissue healing by playing a number of functions, such as removing debris and dead cells after injury, as well as producing a large range of growth factors, immunological molecules, and proteolytic enzymes [3,4,5]. While these macrophages beneficial contributions to tissue healing are well defined in soft tissue healing [1], soft- and mineralized-tissues substantially differ in their healing processes and outcomes [3]. Macrophages depletion in mice significantly suppresses woven bone deposition and mineralization during bone fracture healing in endochondral bones [15]

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