Optimal fracture treatment requires knowledge of the complex physiological process of bone healing. The course of bone healing is mainly influenced by fracture fixation stability (biomechanics) and the blood supply to the healing site (revascularization after trauma). The repair process proceeds via a characteristic sequence of events, described as the inflammatory, repair and remodeling phases. An inflammatory reaction involving immune cells and molecular factors is activated immediately in response to tissue damage and is thought to initiate the repair cascade. Immune cells also have a major role in the repair phase, exhibiting important crosstalk with bone cells. After bony bridging of the fragments, a slow remodeling process eventually leads to the reconstitution of the original bone structure. Systemic inflammation, as observed in patients with rheumatoid arthritis, diabetes mellitus, multiple trauma or sepsis, can increase fracture healing time and the rate of complications, including non-unions. In addition, evidence suggests that insufficient biomechanical conditions within the fracture zone can influence early local inflammation and impair bone healing. In this Review, we discuss the main factors that influence fracture healing, with particular emphasis on the role of inflammation.

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, MSC and osteoclasts. qRT-PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane-bound regulatory proteins CD46, CD55, and CD59 were expressed in human mesenchymal stem cells, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)-6 and IL-8 from osteoblasts. However, co-stimulation with the proinflammatory cytokine IL-1β significantly induced IL-6 and IL-8 expression as well as the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro-inflammatory environment as well as osteoblast-osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage-colony stimulating factor (M-CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro-inflammatory environment, for example during bone healing or in inflammatory bone disorders.

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In poly-traumatic patients a blunt chest trauma is an important trigger of the posttraumatic systemic inflammatory response. There is clinical evidence that fracture healing is delayed in such patients, however, experimental data are lacking. Therefore, we investigated the influence of a thoracic trauma on fracture healing in a rat model. Male Wistar rats received either a blunt chest trauma combined with a femur osteotomy or an isolated osteotomy. A more rigid or a more flexible external fixator was used for fracture stabilization to analyze whether the thoracic trauma influences regular healing and mechanically induced delayed bone healing differently. The blunt chest trauma induced a significant increase of IL-6 serum levels after 6 and 24 h, suggesting the induction of a systemic inflammation, whereas the isolated fracture had no effect. Under a more rigid fixation the thoracic trauma considerably impaired fracture healing after 35 days, reflected by a significantly reduced flexural rigidity (three-point-bending test), as well as a significantly diminished callus volume, moment of inertia, and relative bone surface (mCT analysis). In confirming the clinical evidence, this study reports for the first time that a blunt chest trauma considerably impaired bone healing, possibly via the interaction of the induced systemic inflammation with local inflammatory processes. ß

Background There is evidence that complement components regulate cytokine production in osteoblastic cells, induce cell migration in mesenchymal stem cells, and play a regulatory role in normal enchondral bone formation. We proved the hypothesis that complement might be involved in bone healing after fracture. Methods We investigated the expression of the key anaphylatoxin receptor C5aR during fracture healing in rats by immunostaining after 1, 3, 7, 14, and 28 days. C5aR expression was additionally analyzed in human mesenchymal stem cells (hMSC) during osteogenic differentiation, in human primary osteoblasts, and osteoclasts by reverse transcriptase polymerase chain reaction and immunostaining. Receptor functionality was proven by the migratory response of cells to C5a in a Boyden chamber. Results C5aR was expressed in a distinct spatial and temporal pattern in the fracture callus by differentiated osteoblast, chondroblast-like cells in cartilaginous regions, and osteoclasts. In vitro C5aR was expressed by osteoblasts, osteoclasts, and hMSC undergoing osteogenic differentiation. C5aR was barely expressed by undifferentiated hMSC but was significantly induced after osteogenic differentiation. C5aR activation by C5a induced strong chemotactic activity in osteoblasts, and in hMSC, which had undergone osteogenic differentiation, being abolished by a specific C5aR antagonist. In hMSC, C5a induced less migration reflecting their low level of C5aR expression. Conclusions Out in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone rcsorbing osteoclasts. It is suggested that C5aR might play a regulatory role in fracture healing in intramembranous and in enchondral ossification, one possible function being the regulation of cell recruitment.

Our results suggest that the systemic posttraumatic inflammation induced by a thoracic trauma disturbed the inflammatory balance during the early healing stage by altering the recruitment of inflammatory cells and cytokine expression locally at the fracture site and thus impaired fracture healing. These findings provide new insights in the pathomechanisms of impaired fracture healing in patients experiencing severe trauma.

Confirming clinical evidence, we recently demonstrated that a blunt chest trauma considerably impaired fracture healing in rats, possibly via the interaction of posttraumatic systemic inflammation with local healing processes, the underlying mechanisms being unknown. An important trigger of systemic inflammation is the complement system, with the potent anaphylatoxin C5a. Therefore, we investigated whether the impairment of fracture healing by a severe trauma resulted from systemically activated complement. Rats received a blunt chest trauma and a femur osteotomy stabilized with an external fixator. To inhibit the C5a dependent posttraumatic systemic inflammation, half of the rats received a C5aR-antagonist intravenously immediately and 12 h after the thoracic trauma. Compared to the controls (control peptide), the treatment with the C5aR-antagonist led to a significantly increased flexural rigidity (three-point-bending test), an improved bony bridging of the fracture gap, and a slightly larger and qualitatively improved callus (μCT, histomorphometry) after 35 days. In conclusion, immunomodulation by a C5aR-antagonist could abolish the deleterious effects of a thoracic trauma on fracture healing, possibly by influencing the function of inflammatory and bone cells locally at the fracture site. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3-/- and C5-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3-/-: -25%, p=0.02; C5-/-: -20% p=0.052) and newly formed bone (C3-/-: -38%, p=0.01; C5-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.