Fracture hematoma micro-architecture influences transcriptional profile and plays a crucial role in determining bone healing outcomes

Abstract

The hematoma that forms between broken fragments of bone serves as a natural fibrin scaffold, and its removal from the defect site delays bone healing. The hypothesis of this study is that the microarchitectural and mechanical properties of the initially formed hematoma has a significant effect on the regulation of the biological process, which ultimately determines the outcome of bone healing. To mimic three healing conditions in the rat femur (normal, delayed, and non-healing bone defects), three different defect sizes of 0.5, 1.5, and 5.0 mm, are respectively used. The analysis of 3-day-old hematomas demonstrates clear differences in fibrin clot micro-architecture in terms of fiber diameter, fiber density, and porosity of the formed fibrin network, which result in different mechanical properties (stiffness) of the hematoma in each model. Those differences directly affect the biological processes involved. Specifically, RNA-sequencing reveals almost 700 differentially expressed genes between normally healing and non-healing defects, including significantly up-regulated essential osteogenic genes in normally healing defects, also differences in immune cell populations, activated osteogenic transcriptional regulators as well as potential novel marker genes. Most importantly, this study demonstrates that the healing outcome has already been determined during the hematoma phase of bone healing, three days post-surgery.

Keywords

Fracture hematoma, Bone healing, Large bones defects, Delayed bone healing, Nonunion, micro-architectural properties & Gene expression profile.