Although mouse models are crucial in orthopedic studies, no study has explored the bone healing process in mice through continuous imaging. Thus, we optimized a mouse model for femoral defect by continually monitoring the bone healing process of different‐sized femoral defects. We observe bone healing images and quantify the trabecular bone value up to 1 month after surgery. In the 0.3‐mm group, significant bone bridge formation and spongy bone trabeculae were found on the day 14 after the defect operation. The bone healed on day 21 with the formation of external callus and internal callus structures and transformed to a remodeled compact bone on day 28. In the 1‐mm group, bone bridge formation occurred on day 21 after surgery. Moreover, bone healing was observed on day 28 with the formation of external callus and internal callus structures. Mice in the 1.5‐mm group were prone to fracture during feeding after bone defect surgery. The fluorescent staining results of osterix and cathepsin K also revealed that the bone formation process in the 0.3‐mm group was faster than that in the 1‐mm group. This study optimized a bone healing mouse model that can be applied for developing new strategies for accelerating bone healing. Support or Funding Information During the study period, the authors received funding support from the Ministry of Science and Technology, Taiwan (grant numbers MOST 107‐2314‐B‐182A‐036‐MY3 and MOST 108‐2320‐B‐182A‐020 ‐MY3), and the Chang Gung Memorial Hospital (grant numbers CRRPG3H1311, CRRPG3H1312, CRRPG3H1313, CMRPG3F2011, CMRPG3F2012, CMRPG3F2013). The funders had no role in study design, data collection, data interpretation and analysis, decision to publish, or preparation of the manuscript. Continuous observation of the bone healing process in the 0.3‐mm bone defect group of the mouse femoral bone. Images of 3D micro‐computed tomography (CT) angiography as well as hematoxylin and eosin (H&E)‐stained and Safranin O (SFO)‐stained histopathological slides. Immunofluorescence staining of osterix and cathepsin K indicates osteoblast and osteoclast signals, respectively.
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