Christian Ehrnthaller

The Anaphylatoxin Receptor C5aR Is Present During Fracture Healing in Rats and Mediates Osteoblast Migration In Vitro

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 Our in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone resorbing 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.

Experimental blunt chest trauma impairs fracture healing in rats

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 (µCT 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.

Non-resorbing osteoclasts induce migration and osteogenic differentiation of mesenchymal stem cells†

Osteoclast activity has traditionally been regarded as restricted to bone resorption but there is some evidence that also non‐resorbing osteoclasts might influence osteoblast activity. The aim of the present study was to further investigate the hypothesis of an anabolic function of non‐resorbing osteoclasts by investigating their capability to recruit mesenchymal stem cells (MSC) and to provoke their differentiation toward the osteogenic lineage. Bone‐marrow‐derived human MSC were exposed to conditioned media (CM) derived from non‐resorbing osteoclast cultures, which were generated from human peripheral blood monocytes. Osteogenic marker genes (transcription factor Runx2, bone sialoprotein, alkaline phosphatase (AP), and osteopontin) were significantly increased. Osteogenic differentiation (OD) was also proved by von Kossa and AP staining occurred in the same range as in MSC cultures stimulated with osteogenic supplements. Chemotactic responses of MSC were measured with a modified Boyden chamber assay. CM from osteoclast cultures induced a strong migratory response in MSC, which was greatly reduced in the presence of an anti‐human platelet‐derived growth factor (PDGF) receptor β antibody. Correspondingly, significantly increased PDGF‐BB concentrations were measured in the CM using a PDGF‐BB immunoassay. CM derived from mononuclear cell cultures did not provoke MSC differentiation and had a significantly lower migratory effect on MSC suggesting that the effects were specifically mediated by osteoclasts. In conclusion, it can be suggested that human non‐resorbing osteoclasts induce migration and OD of MSC. While effects on MSC migration might be mainly due to PDGF‐BB, the factors inducing OD remain to be elucidated. J. Cell. Biochem. 109: 347–355, 2010.

Temporary distraction and compression of a diaphyseal osteotomy accelerates bone healing

Mechanical strain during callus distraction is known to stimulate osteogenesis. It is unclear whether this stimulus could be used to enhance the healing of a normal fracture without lengthening the bone. This study tested the hypothesis that a slow temporary distraction and compression of a diaphyseal osteotomy accelerates fracture healing. Fourteen sheep underwent a middiaphyseal osteotomy of the right tibia, stabilized by external fixation. An external fixator allowed either a temporary axial distraction (TD group; n = 6) or a constant fixation (C group; n = 8). Distraction began 7 days postoperatively at a rate of 0.5 mm twice per day for 2 days with subsequent shortening of 1.0 mm twice on the third day. The procedure was repeated four times. Fluorochrome labeling was performed postoperatively. After 8 weeks the sheep were sacrificed and healing was evaluated using densitometric, biomechanical, and histological methods. Bending stiffness of the tibiae after 8 weeks was 58% higher in the TD group than in the C group. The volume of the periosteal callus was significantly (p = 0.05) higher in the TD group (3.9 cm3) than in the C group (2.7 cm3). There was 20% more bone in the fracture gap of the TD group than the C group. There was a significantly higher bone formation rate in the TD group than in the C group. This study demonstrated the feasibility of fracture healing stimulation by the temporary application of distraction and compression.

C5aR-antagonist significantly reduces the deleterious effect of a blunt chest trauma on fracture healing

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.

Complement C3 and C5 Deficiency Affects Fracture Healing

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.

Fracture Healing Is Delayed in Immunodeficient NOD/scid‑IL2Rγcnull Mice.

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγcnull mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγcnull mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγcnull mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγcnull mice. Our results suggest that—under aseptic, uncomplicated conditions—the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγcnull mice.

Early Detection of Junctional Adhesion Molecule-1 (JAM-1) in the Circulation after Experimental and Clinical Polytrauma

Severe tissue trauma-induced systemic inflammation is often accompanied by evident or occult blood-organ barrier dysfunctions, frequently leading to multiple organ dysfunction. However, it is unknown whether specific barrier molecules are shed into the circulation early after trauma as potential indicators of an initial barrier dysfunction. The release of the barrier molecule junctional adhesion molecule-1 (JAM-1) was investigated in plasma of C57BL/6 mice 2 h after experimental mono- and polytrauma as well as in polytrauma patients (ISS ≥ 18) during a 10-day period. Correlation analyses were performed to indicate a linkage between JAM-1 plasma concentrations and organ failure. JAM-1 was systemically detected after experimental trauma in mice with blunt chest trauma as a driving force. Accordingly, JAM-1 was reduced in lung tissue after pulmonary contusion and JAM-1 plasma levels significantly correlated with increased protein levels in the bronchoalveolar lavage as a sign for alveolocapillary barrier dysfunction. Furthermore, JAM-1 was markedly released into the plasma of polytrauma patients as early as 4 h after the trauma insult and significantly correlated with severity of disease and organ dysfunction (APACHE II and SOFA score). The data support an early injury- and time-dependent appearance of the barrier molecule JAM-1 in the circulation indicative of a commencing trauma-induced barrier dysfunction.

Alteration of complement hemolytic activity in different trauma and sepsis models.

Complement activation is involved in various diseases in which innate immunity plays a crucial role. However, its pathophysiological relevance is not clearly understood. Experimental models have been widely used to characterize the role of complement activation under different pathological conditions, such as hypoxemia, ischemia and reperfusion, tissue damage, and polymicrobial invasion. Screening of the complement status and function is, however, strongly dependent on the laboratory-specific techniques being used to sample and measure complement, making it difficult to compare the results found in different laboratories. Therefore, we evaluated complement function by measuring complement hemolytic activity (CH50) in various animal models of isolated ischemia reperfusion (I/R: kidney, liver, gut), hemorrhagic traumatic shock (HTS), endotoxic shock (LPS), and sepsis (CLP). Complement activation was less pronounced in isolated models of ischemia and reperfusion, whereas a strong complement response was observed early after HTS, CLP, and LPS. In summary, CH50 is a well-established, quick, and cost-effective screening method of complement function. However, because we obtained different results in clinically relevant animal models, further differentiation using specific complement factor analysis is necessary.

The role of lesser trochanter fragment in unstable pertrochanteric A2 proximal femur fractures - is refixation of the lesser trochanter worth the effort?

Background: Instability of osteoporotic pertrochanteric fractures is defined by loss of medial/lateral cortical integrity with the posteromedial fragment including the lesser trochanter being pivotal for load distribution. Literature addressing the importance of lesser trochanter refixation is scarce. To clarify the effect of lesser trochanter refixation on primary stability in these fractures, following study was performed. Methods: 21 femora were match‐paired in 3 groups and osteotomized, creating pertrochanteric fractures (AO‐31A2). Group 1 was stabilized with a proximal femoral nail, group 2 with a dynamic hip screw and group 3 with an augmented proximal femoral nail. Each femur was tested non‐destructively at 200 and 400 N with and without refixation of the lesser trochanter (configuration A/B). The overall stiffness and movement of the femoral neck was recorded. Findings: At 200 N, refixation reduced movement of the femoral neck and increased overall stiffness significantly in group 1 and 3. At 400 N, refixation decreased movement of the femoral neck not significantly in all groups (1 = 38%, 2 = 36%, 3 = 43%). The augmented proximal femoral nail after refixation showed the highest stability of all constructs. Interpretation: Refixation of the lesser trochanter may increase the primary stability of pertrochanteric fracture osteosynthesis as all groups showed a higher primary stability. Therefore, refixation should be considered in unstable, osteoporotic fractures. If additional trauma through refixation appears inappropriate, cement augmentation should be performed as it showed only 9% less stability than a non‐augmented proximal femoral nail with refixation of the lesser trochanter. Highlights:Refixation of lesser trochanter should be considered in osteoporotic bone.Lesser trochanter refixation increases primary stability.Highest stability was demonstrated in augmented proximal femoral nails with refixation of the lesser trochanter.