Spatio-temporal characterization of compositional and cellular properties in the murine fracture callus

Advances in the understanding of the physiological process of bone healing and its impairment (i.e., nonunion) require appropriate models and precise standardization. The femoral osteotomy and external fixation mouse model meets these requirements, but a comprehensive spatio-temporal characterization of callus mineralization along the bone healing process has not been available. Here, we differentiated into three regions within the healing bone and examined eight time points post-osteotomy. We were able to demonstrate a gradual increase in callus mineralization alongside endochondral ossification. We further demonstrated a temporary increase in vascularization, followed by increased modeling and remodeling activity of the bone. This was evidenced by a transient high abundance of type-H vessels, increased bone mineralization heterogeneity, and a strong increase in osteocyte numbers. Notably, at the end of the fracture healing cascade, demineralization occurred in the cortical bone adjacent to the fracture callus, suggesting a considerable risk of refracture in the vicinity of the former fracture. In summary, our results provide insight into the progression of callus mineralization at the microstructural, cellular and bone quality levels, providing a reference for the quantification of respective parameters at multiple length scales, which can be used for future studies in this field. STATEMENT OF SIGNIFICANCE: Our data provide the first comprehensive view of murine fracture healing through the multimodal analysis of callus tissue focusing on mineralization, cellular bone turnover, and nanomechanics. The results obtained from several time points (7, 9, 11, 13, 15, 17, 21 and 28 days) and the inclusion of different regions in and around the healing bone allowed us to provide evidence for the transient involvement of mechanosensitive osteocytes as well as the mobilization of calcium from the unfractured bone. Our data also provide scientists with useful multidimensional reference values when interpreting data on fracture healing in mice.