As cytotoxic (CD8(+)) T cells seem to impair shaft fracture healing, we hypothesized that depletion of CD8(+) cells would instead improve healing of cancellous bone. Additionally, we also tested if CD8-depletion would influence the healing of ruptured Achilles tendons. Rats received a single injection of either anti-CD8 antibodies or saline and put through surgery 24 h later. Three different surgical interventions were performed as follows: (1) a drill hole in the proximal tibia with microCT (BV/TV) to assess bone formation; (2) a screw in the proximal tibia with mechanical evaluation (pull-out force) to assess fracture healing; (3) Achilles tendon transection with mechanical evaluation (force-at-failure) to assess tendon healing. Furthermore, CD8-depletion was confirmed with flow cytometry on peripheral blood. Flow cytometric analysis confirmed depletion of CD8(+) cells (p amp;lt; 0.001). Contrary to our hypothesis, depletion of CD8(+) cells reduced the implant pull-out force by 19% (p amp;lt; 0.05) and stiffness by 34% (p amp;lt; 0.01), although the bone formation in the drill holes was the same as in the controls. Tendon healing was unaffected by CD8-depletion. Our results suggest that CD8(+) cells have an important part in cancellous 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 models were similar in cell composition at day 3 and 5. In mass spectrometry there was a large increase in detected proteins at day 3 in the indomethacin exposed cortical model, but otherwise the models were similar. This points to an early and model specific effect of indomethacin. The observed lack of indomethacin-induced effects in cancellous bone healing is in line with the previously noted lack of indomethacin-induced effects on bone weakening. The apparently increased inflammatory activity in the cortical model with indomethacin exposure at day 3 might indicate the healing process to be disturbed and not able to progress from the early proinflammatory state to a more anabolic, anti-inflammatory state.

In paper IV the effect of macrophage depletion on healing of metaphyseal bone was studied. Clodronate was given for depletion at different time points prior to surgery and the pull-out force of a screw or tissue phenotyping of macrophages was performed a varying number of days after surgery. It was noted that metaphyseal bone healing was to a large extent inhibited by macrophage depletion up to two days after surgery, but not if depletion was done more than two days after surgery. Thus, macrophages seem to be most important during the first two days after trauma in cancellous bone healing. 

In summary this thesis provide insight to the natural development of bone healing. The findings emphasise that cancellous and cortical bone healing are different entities with differences in the inflammatory process leading to healing.

Objectives Cortical and cancellous bone healing processes appear to be histologically different. They also respond differently to anti-inflammatory agents. We investigated whether the leucocyte composition on days 3 and 5 after cortical and cancellous injuries to bone was different, and compared changes over time using day 3 as the baseline. Methods Ten-week-old male C56/B16J mice were randomized to either cancellous injury in the proximal tibia or cortical injury in the femoral diaphysis. Regenerating tissues were analyzed with flow cytometry at days 3 and 5, using panels with 15 antibodies for common macrophage and lymphocyte markers. The cellular response from day 3 to 5 was compared in order to identify differences in how cancellous and cortical bone healing develop. Results Between day 3 and 5, the granulocytes increased in the cancellous model, whereas the lymphocytes (T cells, B cells, NK cells) and monocytes (CD11b+, 14/80+, CD206+, CD14+ ) increased in the cortical model. Conclusion These results suggest an acute type of inflammation in cancellous bone healing, and a more chronic inflammation in cortical healing. This might explain, in part, why cancellous healing is faster and more resistant to anti-inflammatory drugs than are diaphyseal fractures.