A Population of M2 Macrophages Associated with Bone Formation

Purpose: We had previously published that very early during bone formation we observed a very transient cell that had the phenotype of brown fat and was important in creating a hypoxic microenvironment. This is critical for cartilage formation, the first stage in bone formation. The purpose of the experiments in this manuscript were to determine the phenotype of these "brown fat" cells in more detail. Methods: To determine the phenotype of these cells they were isolated by FACS for the beta 3 adrenergic receptor that we had previously found was a good marker for these cells during bone formation. After isolation, the cells both from HO and from the callus of a fracture were subjected to single cell RNA seq. Results: The results of the single cell RNA seq showed that the cells in HO had many of the phenotypic markers of type 2 macrophages. However, they also retained the markers of brown fat including Ucp1 and Ucp2 as well as many markers of adipogenesis and extreme elevation of mitochondrial proteins. This mixture of proteins is required to burn oxygen in the microenvionment to create hypoxia. The cells made during fracture repair were further along in bone formation because they had other blood cells (B and NK cells) besides these macrophages. However, a small fraction was the same as the macrophages found during HO. Conclusions: The type 2 macrophages made during bone formation had many of the properties of type 2 macrophages in the blood. However, they were also different from macrophages found in blood in that they lacked many of the key markers of blood macrophages. These unique macrophages are made rapidly at the start of bone formation and then disappear four days later probably differentiating to osteoclasts. However, they are critical in determining where the new bone forms as well as its size and shape. scRNA seq in both Heterotopic ossification (HO) and fracture repair. HO and callus were each bar-coded at the single-cell level, which were then combined before preparing the RNA.