Fracture healing requires proliferation of callus cells at early and mature stages of osteoblast differentiation
6 affiliations
- 1Department of Orthopaedic Surgery
- 2Musculoskeletal Research Center
- 3Department of Biomedical Engineering, and
- 4Department of Developmental Biology, Washington University in St. Louis, St. Louis, Missouri, USA.
- 5Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
- 6Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, St. Louis, Missouri, USA.
Abstract
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 marker Osterix (Osx, Sp7) and the late marker osteocalcin (Ocn, 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.
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BibTeX
@article{gould2026fracture,
title = {{Fracture healing requires proliferation of callus cells at early and mature stages of osteoblast differentiation}},
author = {Nicole R. Gould and Andre F. Coello and Jennifer A. McKenzie and Mariam Obaji and Tiandao Li and Katherine R. Hixon and Leyi Chen and Kristen Barwick and Tiffany Lee and Bo Zhang and David Ornitz and Matthew J. Silva},
journal = {JCI Insight},
year = {2026},
month = sep,
doi = {10.1172/jci.insight.196751},
url = {https://doi.org/10.1172/jci.insight.196751},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Fracture healing requires proliferation of callus cells at early and mature stages of osteoblast differentiation
AU - Nicole R. Gould
AU - Andre F. Coello
AU - Jennifer A. McKenzie
AU - Mariam Obaji
AU - Tiandao Li
AU - Katherine R. Hixon
AU - Leyi Chen
AU - Kristen Barwick
AU - Tiffany Lee
AU - Bo Zhang
AU - David Ornitz
AU - Matthew J. Silva
PY - 2026
DA - 2026/09/08
JO - JCI Insight
DO - 10.1172/jci.insight.196751
UR - https://doi.org/10.1172/jci.insight.196751
AB - 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 marker Osterix (Osx, Sp7) and the late marker osteocalcin (Ocn, 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.
SN - 2379-3708
ER -