Fracture Hematoma Micro-Architecture Plays a Crucial Role in Determining Bone Healing Outcomes

The fracture hematoma that forms between the broken fragments of bone serves as a natural fibrin scaffold. However, there is no data regarding the differences between the micro-architectural and biological properties of hematomas formed in normally healing, delayed healing, and non-healing bone defects. Mimicking these three conditions in the rat femur, we demonstrate clear differences in fibrin clot morphology, which directly affect the gene expression pattern. Specifically, RNA-sequencing reveals that the expression of essential osteogenic genes in normally healing defects are significantly up-regulated, whereas in delayed and non-healing defects they are down-regulated. Surprisingly, there were no substantial differences between delayed and non-healing defects. Most importantly, this study demonstrates that the healing outcome has already been determined at the earliest stage of bone healing. These findings could be used to develop biomaterial scaffolds mimicking the micro-architectural properties of normally healing fracture hematoma as a treatment strategy for bone defects. The hypothesis of this study was that the micro-architectural properties of the initially formed hematoma has a significant effect on the regulation of the biological process at the fracture site, which ultimately determines the outcome of bone healing. Three different healing models were investigated - normally healing, delayed healing, and non-healing defects. The results demonstrated that the intrinsic micro-architectural properties of hematomas between those groups varied distinctly in terms of fiber diameter, porosity, and density of the formed fibrin network. Those differences influenced biological responses, as evidenced by a higher expression of osteogenic genes in normally healing compared to delayed and non-healing defects, and the failed activation of BMP-2 in non-healing defects. More importantly, our results demonstrate healing outcomes are already determined at the initial (hematoma) stage of bone healing. Overall design: Three different healing models were investigated - normally healing, delayed healing, and non-healing defects. PreOP group also examined. 3 Samples/Group: Experimental Group 1: Normal Healing (0.5 mm defect) Experimental Group 2: Delayed Healing (1.5 mm defect) Experimental Group 3: Nonunion (5.0 mm defect) Reference Sample: preOP (healthy bone piece removed at surgery from 5.0 mm defects)