Fracture healing requires proliferation of callus cells at early and mature stages of osteoblast differentiation.

Fractures heal by rapid formation of mineralized callus, a process requiring periosteal cell proliferation and differentiation. Our objective was to dissect the contribution of proliferating osteoblast lineage cells to fracture callus formation. First, mice expressing thymidine kinase (TK) in 3.6Col1a1-lineage cells were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. Immunostaining demonstrated that this approach specifically depleted TK+ proliferating cells in the bony regions of the callus, while sparing other proliferating cells. Single-cell RNA-seq of callus cells revealed that GCV-treated Col1-TK mice had fewer osteoblasts and chondrocytes than controls, with more myofibroblasts and immune cells, consistent with fibrous nonunion. In controls, 15-30% of callus cells expressing the early osteoblast markers osterix (Sp7) and the late marker osteocalcin (Bglap) were in the cell cycle. Next, we targeted proliferating osteolineage cells at different stages of differentiation by crossing Osx-CreERT2, Ocn-Cre and Dmp1-CreERT2 mice with ROSA-TK mice. Following fracture, each Cre;ROSA-TK mouse line exhibited decreased callus bone volume and a shift from callus bone to fibrous tissue. Therefore, during fracture repair, proliferation of callus cells at early and mature stages of osteoblast differentiation is critical to the formation of a mineralized callus that is essential for healing.