Proliferation and Activation of Osterix-Lineage Cells Contribute to Loading-Induced Periosteal Bone Formation in Mice.

Mechanical loading stimulates bone formation. Bone-lining-cell activation and cell proliferation have been implicated in this process. However, the origin of osteoblasts that form bone following mechanical stimulation remains unknown. Our objective was to identity the contributions of activation, differentiation, and proliferation of osteoblast lineage cells to loading-induced periosteal bone formation. Tamoxifen-inducible Osx-Cre-ERT2;Ai9/TdTomato reporter mice (male and female) were aged to young adult (5 months) and middle age (12 months), and were administered tamoxifen for 5 consecutive days to label osterix-lineage cells. Following a 3-week clearance period, mice were subjected to five consecutive bouts of unilateral axial tibial compression. We first confirmed this protocol stimulated an increase in periosteal bone formation that was primarily lamellar apposition. Next, mice received 5-bromo-2'-deoxyuridine (BrdU) in their drinking water daily to label proliferating cells; calcein was given to label active mineralizing surfaces. Tibias were harvested after the fifth loading day and processed for frozen undecalcified histology. The middiaphyseal periosteal surface in the region of peak bone formation was analyzed. Histology revealed both nonloaded and loaded tibias were covered in osterix positive (Osx) cells on the periosteal surface of both 5- and 12-month-old animals. There was a significant increase in the mineralizing surface (calcein) covered with Osx cells in loaded versus control limbs. Furthermore, nearly all of the mineralizing surfaces (>95%) were lined with Osx cells. We also observed approximately 30% of Osx cells were also BrdU, indicating they arose via proliferation. These results show that following mechanical loading, pre-existing cells of the Osx lineage cover the vast majority of surfaces where there is active loading-induced bone formation, and a portion of these cells proliferated in the 5-day loading period. We conclude the initial anabolic response after mechanical loading is based on the activation and proliferation of Osx lineage cells, not the differentiation of progenitor cells. © 2019 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.