Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

Area of Science:

• Bone biology
• Regenerative medicine
• Skeletal healing

Background:

• Fracture healing relies on periosteal cell proliferation to generate osteoblasts.
• The contribution of osteoblast proliferation to callus formation remains incompletely understood.

Purpose of the Study:

• To investigate the impact of blocking osteolineage cell proliferation on fracture callus composition.
• To determine if mature osteoblasts contribute to callus formation through proliferation.

Main Methods:

• Utilized herpes simplex virus-thymidine kinase (HSV-TK) and ganciclovir (GCV) to ablate proliferating cells in Col1-TK mice.
• Employed single-cell RNA-seq to analyze callus cell composition.
• Generated transgenic mouse lines (Osx-CreERT2, Ocn-Cre, Dmp1-CreERT2 crossed with ROSA-TK) to target proliferating osteoblasts at different differentiation stages.

Main Results:

• GCV treatment resulted in fewer osteoblasts and chondrocytes, with increased myofibroblasts and immune cells, indicating fibrous nonunion.
• In control calluses, 15-30% of cells expressing early (OSX) and late (OCN) osteoblast markers were actively cycling.
• Targeting proliferating osteoblasts in Cre;ROSA-TK mouse lines led to impaired radiographic healing, reduced callus bone volume, and a shift towards fibrous tissue.

Conclusions:

• Osteoblasts, previously considered terminally differentiated, actively proliferate following fracture.
• Osteoblast proliferation is crucial for the formation of mineralized callus, essential for successful fracture healing.