Ronny Bindl

Small animal bone healing models: Standards, tips, and pitfalls results of a consensus meeting

Small animal fracture models have gained increasing interest in fracture healing studies. To achieve standardized and defined study conditions, various variables must be carefully controlled when designing fracture healing experiments in mice or rats. The strain, age and sex of the animals may influence the process of fracture healing. Furthermore, the choice of the fracture fixation technique depends on the questions addressed, whereby intra- and extramedullary implants as well as open and closed surgical approaches may be considered. During the last few years, a variety of different, highly sophisticated implants for fracture fixation in small animals have been developed. Rigid fixation with locking plates or external fixators results in predominantly intramembranous healing in both mice and rats. Locking plates, external fixators, intramedullary screws, the locking nail and the pin-clip device allow different degrees of stability resulting in various amounts of endochondral and intramembranous healing. The use of common pins that do not provide rotational and axial stability during fracture stabilization should be discouraged in the future. Analyses should include at least biomechanical and histological evaluations, even if the focus of the study is directed towards the elucidation of molecular mechanisms of fracture healing using the largely available spectrum of antibodies and gene-targeted animals to study molecular mechanisms of fracture healing. This review discusses distinct requirements for the experimental setups as well as the advantages and pitfalls of the different fixation techniques in rats and mice.

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.

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.

Systemic inflammation induced by a thoracic trauma alters the cellular composition of the early fracture callus.

BACKGROUND We recently demonstrated that a blunt chest trauma, a strong inducer of the posttraumatic systemic inflammatory response and one of the most critical injuries in polytrauma patients, significantly delayed fracture healing in rats, possibly by the interaction of the systemic inflammation with early regeneration processes locally at the fracture site. The underlying cellular mechanisms, however, have as yet remained unknown. Therefore, the aim of this study was to analyze the cellular and morphologic composition of the early fracture callus after a blunt chest trauma. METHODS Rats received an osteotomy of the right femur stabilized by an external fixator in combination with a blunt chest trauma or not. The animals were killed after 3, 7, and 35 days, and the fracture calli were analyzed histologically for new tissue formation, polymorphonuclear leucocytes, macrophages, osteoclasts, and the presence of the proinflammatory cytokine interleukin 6. RESULTS The blunt chest trauma considerably increased the number of polymorphonuclear leucocytes in the callus by Day 3 compared with animals with isolated fractures. The number of macrophages was significantly reduced by the thoracic trauma at Days 3 and 7. The number of osteoclasts was not changed at any postoperative time point. After 3 days, the blunt chest trauma led to a significantly stronger interleukin 6 staining within the periosteal callus in zones of intramembranous ossification. During the time of cortical bridging at Day 35, the amount of newly formed bone was significantly decreased after blunt chest trauma. CONCLUSION 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.

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.

The Wnt Serpentine Receptor Frizzled-9 Regulates New Bone Formation in Fracture Healing

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9 −/− mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures.

Distinct Frequency Dependent Effects of Whole-Body Vibration on Non-Fractured Bone and Fracture Healing in Mice

Low‐magnitude high‐frequency vibration (LMHFV) provokes anabolic effects in non‐fractured bone; however, in fracture healing, inconsistent results were reported and optimum vibration conditions remain unidentified. Here, we investigated frequency dependent effects of LMHFV on fracture healing. Twelve‐week‐old, female C57BL/6 mice received a femur osteotomy stabilized using an external fixator. The mice received whole‐body vibrations (20 min/day) with 0.3g peak‐to‐peak acceleration and a frequency of either 35 or 45 Hz. After 10 and 21 days, the osteotomized femurs and intact bones (contra‐lateral femurs, lumbar spine) were evaluated using bending‐testing, µ‐computed tomography, and histomorphometry. In non‐fractured trabecular bone, vibration with 35 Hz significantly increased the relative amount of bone (+28%) and the trabecular number (+29%), whereas cortical bone was not influenced. LMHFV with 45 Hz failed to provoke anabolic effects in trabecular or cortical bone. Fracture healing was not significantly influenced by whole‐body vibration with 35 Hz, whereas 45 Hz significantly reduced bone formation (−64%) and flexural rigidity (−34%) of the callus. Although the exact mechanisms remain open, our results suggest that small vibration setting changes could considerably influence LMHFV effects on bone formation in remodeling and repair, and even disrupt fracture healing, implicating caution when treating patients with impaired fracture healing.

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.

Systemic treatment with the sphingosine‐1‐phosphate analog FTY720 does not improve fracture healing in mice

Sphingosine‐1‐phosphate (S1P) has recently been recognized as a crucial coupling molecule of osteoclast and osteoblast activity provoking osteoanabolic effects. Targeting S1P receptors could, therefore, be a potential strategy to support bone formation in osteopenic diseases or in fracture repair. Here we investigated whether systemic treatment with the S1P analog FTY720 (Fingolimod) could improve fracture healing. Twelve‐week‐old, female C57BL/6 mice received an osteotomy of the femur, which was stabilized using an external fixator. The mice received a daily subcutaneous injection of either FTY720 (6 mg/kg) or vehicle from the third postoperative day. Fracture healing was evaluated after 10 and 21 days using biomechanical testing, µ‐computed tomography, and histomorphometry. Because FTY720 is supposed to influence osteoclast recruitment, osteoclasts were identified in the fracture callus by staining for tartrate resistant acid phosphatase (TRAP). There were no significant differences in callus mechanical properties, tissue composition and osteoclast number between the groups, suggesting that systemically applied FTY720 did not influence bone regeneration in this model of regular fracture healing. Even if further studies should test the potency of FTY720 under unfavorable healing conditions, we conclude that the effect of systemically applied FTY720 on fracture healing might be inferior compared to other anabolic treatments.

Exposure to 100% Oxygen Abolishes the Impairment of Fracture Healing after Thoracic Trauma

In polytrauma patients a thoracic trauma is one of the most critical injuries and an important trigger of post-traumatic inflammation. About 50% of patients with thoracic trauma are additionally affected by bone fractures. The risk for fracture malunion is considerably increased in such patients, the pathomechanisms being poorly understood. Thoracic trauma causes regional alveolar hypoxia and, subsequently, hypoxemia, which in turn triggers local and systemic inflammation. Therefore, we aimed to unravel the role of oxygen in impaired bone regeneration after thoracic trauma. We hypothesized that short-term breathing of 100% oxygen in the early post-traumatic phase ameliorates inflammation and improves bone regeneration. Mice underwent a femur osteotomy alone or combined with blunt chest trauma 100% oxygen was administered immediately after trauma for two separate 3 hour intervals. Arterial blood gas tensions, microcirculatory perfusion and oxygenation were assessed at 3, 9 and 24 hours after injury. Inflammatory cytokines and markers of oxidative/nitrosative stress were measured in plasma, lung and fracture hematoma. Bone healing was assessed on day 7, 14 and 21. Thoracic trauma induced pulmonary and systemic inflammation and impaired bone healing. Short-term exposure to 100% oxygen in the acute post-traumatic phase significantly attenuated systemic and local inflammatory responses and improved fracture healing without provoking toxic side effects, suggesting that hyperoxia could induce anti-inflammatory and pro-regenerative effects after severe injury. These results suggest that breathing of 100% oxygen in the acute post-traumatic phase might reduce the risk of poorly healing fractures in severely injured patients.