Inflammation in Cancellous and Cortical Bone Healing

Fractures in humans most commonly occur near the joints, in the metaphyseal bone area mainly consisting of cancellous bone. Despite this, mainly cortical fractures, located in the diaphyseal bone area, have been studied in experimental models of bone healing. It is known from previous studies that the diaphyseal fracture is sensitive to anti-inflammatory treatment, while metaphyseal bone healing is more resistant. The aim of this thesis is to study the inflammatory response to bone trauma in cancellous and cortical bone. A flow cytometric method was established for the purpose of examining the cellular composition of the inflammatory process in models of bone healing In paper I the cellular composition of metaphyseal bone healing was studied with flow cytometry. The proximal tibia was traumatized and then studied at day 1, 3, 5 and 10 afterwards and compared to healthy mice. The contralateral proximal tibia was also studied at the same time points to delineate the trauma site specific inflammation. A few changes could be noted that seemed specific to the trauma site in macrophage phenotype development. However, the cellular composition was similar at the trauma site and in the contralateral proximal tibia. This notion of a general skeletal response was confirmed with analysis of the humerus at day 5. In paper II a model of cortical bone healing apt for flow cytometry was developed and compared to cancellous bone healing. A furrow was milled along the femoral cortex and the healing bone tissue analyzed. The earliest time point that enough cells were present for flow cytometry was day 3. The cortical and cancellous model of bone healing was compared at day 3 and 5 to study how they evolve in comparison to each other. It was noted that they were similar in cellular composition at day 3, but had diverged at day 5. The cancellous model increased in neutrophilic granulocytes, whereas the cortical model increased in lymphocytes. In paper III the cancellous and cortical model were compared under experimental intervention of indomethacin. It is known that indomethacin leads to weakened biomechanical properties in cortical bone healing, but not in cancellous bone healing. The effect on cellular composition with indomethacin was studied with flow cytometry and the extracellular protein profile in the healing bone tissue with mass spectrometry. Unexpectedly, inflammatory monocytes were increased in the cortical model at day 3 with indomethacin, but otherwise the model