gms | German Medical Science

Objectives: Upon activation of the complement system, the terminal complement complex (TCC) is formed by the assembly of complement proteins C5b, C6, C7, C8 and C9 (C5b-9). This pore-forming complex is typically generated on bacterial surfaces, leading to cell lysis to ensure host protection. When present at sublytic concentrations, the TCC exerts immunomodulatory effects. The complement system affects the skeletal system and dysregulation of complement at C5-level contributes to impaired fracture healing. Moreover, the TCC drives osteoarthritis development. Here we investigated the effect of the TCC on bone metabolism and bone regeneration after femur fracture. To this end we used C6-deficient (C6-def) mice, with a reduced TCC formation capacity.

Methods: The bone phenotype of male 17 week-old C6-def and WT mice was analyzed by biomechanical testing, μCT and histological analyses. Dynamic histomorphometry was determined by fluorochrome injections and bone turnover markers and hemolytic activity were analyzed in serum. Primary osteoblasts and osteoclasts were isolated of 8-12 week-old mice and assessed for proliferation and differentiation. Mice aged 12 weeks were subjected to femur fracture stabilized by an external fixator. Cytokine levels in plasma and locally at the fracture site were analyzed 1 day post-fracture. Bone healing properties were assessed 21 days post-fracture using biomechanical testing, μCT and histomorphometry. Statistics: Student's t-test, p≤0.05; n=4-8.

Results and conclusion: C6-def mice displayed a reduced bone mineral density (BMD), reduced numbers of trabeculae and increased numbers of osteoclasts in both femur and spine. Moreover, femora were less stiff and decreased in length, with the growth plate being thinner and showing decreased chondrocyte numbers. Furthermore, the bone formation rate and levels of the bone formation marker P1NP in C6-def serum were reduced. As expected, C6-def serum displayed a significantly reduced hemolytic activity. At day 1 post-fracture, IL-6 and IFN-ϒ levels were significantly enhanced locally at the fracture site, while CXCL1 was increased systemically. Fracture healing at day 21 was impaired in C6-def mice, evident by decreased femur stiffness and an enlarged fracture callus, displaying a decreased quality and decreased BMD. In vitro, C6-def osteoblasts showed reduced differentiation capacities.

Our data show that the complement system, and especially the TCC, needs to be tightly regulated to ensure balanced bone turnover. We show that the absence of the TCC leads to an osteopenic phenotype. Under inflammatory conditions during fracture healing, the presence of the TCC is critical to ensure a regulated early inflammatory response and proper bone regeneration. Moreover, the TCC appears to have direct effects on bone cells, evident by decreased numbers of osteoclasts in vivo and impaired osteoblast differentiation in vitro. Concluding, we showed that TCC-mediated lytic or sublytic effects are necessary for a balance in bone metabolism.