In polytrauma patients invasive surgeries can potentiate the posttraumatic systemic inflammation thus increasing the risk of multi organ dysfunction. Therefore, fractures are initially treated by external fixators, which later are replaced by intramedullary nails. We showed that a severe trauma impaired the healing of fractures stabilized by external fixation. Here we studied, whether the conversion to an intramedullary nail increases posttraumatic inflammation and leads to further impairment of healing.

44 rats received a femur osteotomy stabilized by an external fixator (FixEx). Half of the rats underwent a thoracic trauma (TXT) at the same time. After 4 days the fixator was replaced by an intramedullary nail (IMN) in half of the rats of each group. The rats were killed after 40 and 47 days. C5a serum levels were measured 0, 6, 24, and 72h after the 1st as well as the 2nd surgery. The calli were evaluated by three-point-bending test, μCT and histomorphometry.

The TXT significantly increased serum C5a levels after the 2nd surgical intervention. After 40 days the switch from FixEx to IMN significantly decreased bending stiffness in rats with and without TXT. After 47 days flexural rigidity in rats subjected to conversion was significantly decreased compared to rats treated only with a FixEx, particularly in combination with TXT.

This study showed that after a severe trauma the conversion of the fixation could provoke a second hit and contribute to delayed fracture healing.

There is evidence that fracture healing is delayed in severely injured patients. We recently demonstrated that a blunt chest trauma, which induced posttraumatic systemic inflammation, considerably impaired fracture healing in rats. Because the complement anaphylatoxin C5a is an important trigger of systemic inflammation, we tested the hypothesis, whether the impairment of fracture healing observed after a severe trauma resulted from systemically activated complement.

16 male Wistar rats received a thoracic trauma and a femur osteotomy stabilized by an external fixator. Immediately and 12 h after the trauma, half of the animals received a C5aR-antagonist to prevent the C5a-dependent systemic inflammation. Control rats received a nonsense peptide, which does not provoke any biological effect. The animals were killed after 35 days and the calli were analyzed by three point bending testing, μCT and histomorphometry. Statistics: Mann-Whitney U test, level of significance to p<0.05.

The treatment with the C5aR-antagonist increased flexural rigidity significantly by 55%, improved bony bridging of the fracture gap and led to a slightly larger and qualitatively improved callus as evaluated by μCT and histological measurements.

This study shows, that the immunomodulation by a C5aR-antagonist significantly reduced the deleterious effects of a thoracic trauma on fracture healing. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma.

There is evidence that fracture healing is impaired in patients with chronic immune disorders the reasons remaining unclear so far. To further elucidate the role of the immune system in bone healing, this study investigated the hypothesis that fracture healing would be considerably disturbed in a mouse model with severe defects of the innate as well as adaptive immune system.

Immune deficient Nod-scidIL2Rγ^null and immune competent BALBcByJ mice were used (12 weeks, male, each n=24). The mice received a femur osteotomy stabilized by an external fixator and were sacrificed at d 21, 28, and 35. The calli were evaluated by three-point-bending testing, μCT and histomorphometry.

The flexural rigidity of the callus did not significantly differ between both genotypes after 21 and 28 days but was significantly lower in Nod-scidIL2Rγ^null mice after 35 days (31%). The maximum moment of inertia was significantly increased after 21 days (by 34%), and the callus cross section area after 21, 28 and 35 days in Nod-scidIL2Rγ^null mice. BV/TV of the callus of Nod-scidIL2Rγ^null mice was significantly decreased after 28 and 35 days (by 32% and 41%). The histological evaluation showed a significantly enhanced amount of cartilage in the fracture gap of Nod-scidIL2Rγ^null mice.

These data indicate an only moderate delay in fracture healing in Nod-scidIL2Rγ^null mice suffering on severe defects in innate and adaptive immune response.

Since osteoimmunology is gaining increasingly interest and evidence for involvement of complement in bone biology was found, the role of complement in bone biology and fracture healing was evaluated.

After characterizing the bone phenotype, a fracture healing experiment with C3- and C5- deficient mice was performed. After osteotomy of the right femur and external fixation, healing was analyzed after 1, 3, 7 and 21 days. Bone characterization revealed a reduced number of osteoclasts in C5-deficient animals with a significantly reduced resorption activity. While bone mineral density was significantly higher in complement-deficient strains, stiffness was significantly reduced. After 21 days of fracture healing, C5-deficient animals showed reduced stiffness and a smaller callus volume compared to controls. Interestingly, C3- more than C5-deficient animals showed reduced bone formation. Altogether, bone phenotype of complement-deficient animals resembles a mild form of osteopetrosis.

This might be due to the resorption defect seen in C5-deficient mice. A reason for the minor involvement of C3-deficient mice compared to the C5-deficient animals could be the cross-talk between the coagulation cascade with side activation of complement factor C5 by thrombin.

These results indicate for the first time an essential role of complement in bone biology and fracture healing. Future studies should focus on the molecular basis of complement involvement and the osteoclastic resorption defect.