Fracture Healing Requires Proliferation of Callus Cells at Early and Mature Stages of Osteoblast Differentiation

To dissect the role of cell proliferation in fracture healing, we asked: How are callus cellular and tissue composition altered when proliferation of osteolineage cells is blocked? Does proliferation of osteolineage cells contribute to callus formation?

First, mice expressing thymidine kinase (TK) driven by the 3.6Col1a1 promoter were treated with ganciclovir (GCV) to ablate proliferating osteolineage cells for 5 or 10 days. 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.