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-an...

Secondary fracture healing processes are strongly influenced by interfragmentary motion. Shear movement is assumed to be more critical than axial movement, however experimental results are controversial. Numerical fracture healing models allow to simulate the fracture healing process with variation of single input parameters and under comparable normalized mechanical conditions. Therefore, a direct comparison of different in vivo scenarios is possible. The aim of this study was to simulate fracture healing under several axial and shear movement scenarios and compare their respective time to heal. We hypothesize that shear movement is always more critical than axial loading. For the presented study, we used a corroborated numerical model for fracture healing in sheep. Numerous variations of the movement amplitude, the fracture gap size and the musculoskeletal loads were simulated for comparable axial compressive and shear load cases. In all simulated cases, axial compressive load had less inhibitory influe...

IntroductionThe fracture healing outcome is often evaluated via ex vivo testing of the fracture callus. However, there is only a small time window, where the callus stiffness is significantly different, i.e. a delayed fracture healing might be undetected if the time point of sacrifice is improper. The aim of this study was to develop an in vivo monitoring concept, which allows determining the fracture callus stiffness in vivo over the whole healing time in rats.HypothesisThe fracture callus stiffness can be monitored by measuring the deformation of the external fixation device during gait analysis at several healing time points.Materials & MethodsThe right femurs of sixteen wistar rats were osteotomized and stabilized with an external fixation device (stiffness 119 N/mm or 32 N/mm). The fixator body was instrumented with a stain gauge to measure the deformation. Gait analysis was performed once per week in a gait wheel equipped with a ground reaction force measuring device.ResultsThe deformation of the fi...

For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. Therefore, standardized specimens were created by selective laser sintering (SLS), mimicking size, directional behavior, and embedding variations of respective rat long bone specimens. For the latter, five different geometries were created which show shifted or tilted specimen alignments. The mechanical tests included three-point bending, four-point bending, cantilever bending, axial compression, constrained torsion, and unconstrained torsion. All three different bending tests showed the same principal behavior. They were highly dependent on the rotational direction of the maximum fracture callus expansion relative to the loading direction (creating experimental errors of more than 60%), however small angular deviations (<15°) were negligible. Differences in the experimental results between the bending tests originate in their respective location of maximal bending moment induction. Compared to four-point bending, three-point bending is easier to apply on small rat and mouse bones under realistic testing conditions and yields robust measurements, provided low variation of the callus shape among the tested specimens. Axial compressive testing was highly sensitive to embedding variations, and therefore cannot be recommended. Although it is experimentally difficult to realize, unconstrained torsion testing was found to be the most robust method, since it was independent of both rotational alignment and embedding uncertainties. Constrained torsional testing showed small errors (up to 16.8%, compared to corresponding alignment under unconstrained torsion) due to a parallel offset between the specimens' axis of gravity and the torsional axis of rotation.

Numerous experimental fracture healing studies are performed on rats, in which different experimental, mechanical parameters are applied, thereby prohibiting direct comparison between each other. Numerical fracture healing simulation models are able to predict courses of fracture healing and offer support for pre-planning animal experiments and for post-hoc comparison between outcomes of different in vivo studies. The aims of this study are to adapt a pre-existing fracture healing simulation algorithm for sheep and humans to the rat, to corroborate it using the data of numerous different rat experiments, and to provide healing predictions for future rat experiments. First, material properties of different tissue types involved were adjusted by comparing experimentally measured callus stiffness to respective simulated values obtained in three finite element (FE) models. This yielded values for Young’s moduli of cortical bone, woven bone, cartilage, and connective tissue of 15,750 MPa, 1,000 MPa, 5 MPa, and 1 MPa, respectively. Next, thresholds in the underlying mechanoregulatory tissue differentiation rules were calibrated by modifying model parameters so that predicted fracture callus stiffness matched experimental data from a study that used rigid and flexible fixators. This resulted in strain thresholds at higher magnitudes than in models for sheep and humans. The resulting numerical model was then used to simulate numerous fracture healing scenarios from literature, showing a considerable mismatch in only 6 of 21 cases. Based on this corroborated model, a fit curve function was derived which predicts the increase of callus stiffness dependent on bodyweight, fixation stiffness, and fracture gap size. By mathematically predicting the time course of the healing process prior to the animal studies, the data presented in this work provides support for planning new fracture healing experiments in rats. Furthermore, it allows one to transfer and compare new in vivo findings to previously performed studies with differing mechanical parameters.

ABSTRACT 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 0.3 g 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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1006–1013, 2014.

ABSTRACTThe outcome of secondary fracture healing processes is strongly influenced by interfragmentary motion. Shear movement is assumed to be more disadvantageous than axial movement, however, experimental results are contradictory. Numerical fracture healing models allow simulation of the fracture healing process with variation of single input parameters and under comparable, normalized mechanical conditions. Thus, a comparison of the influence of different loading directions on the healing process is possible. In this study we simulated fracture healing under several axial compressive, and translational and torsional shear movement scenarios, and compared their respective healing times. Therefore, we used a calibrated numerical model for fracture healing in sheep. Numerous variations of movement amplitudes and musculoskeletal loads were simulated for the three loading directions. Our results show that isolated axial compression was more beneficial for the fracture healing success than both isolated shearing conditions for load and displacement magnitudes which were identical as well as physiological different, and even for strain‐based normalized comparable conditions. Additionally, torsional shear movements had less impeding effects than translational shear movements. Therefore, our findings suggest that osteosynthesis implants can be optimized, in particular, to limit translational interfragmentary shear under musculoskeletal loading. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:865–872, 2014.

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 stiffness of fracture fixation devices together with musculoskeletal loading defines the mechanical environment within a long bone fracture, and can be quantified by the interfragmentary movement. In vivo results suggested that this can have acceleratory or inhibitory influences, depending on direction and magnitude of motion, indicating that some complications in fracture treatment could be avoided by optimizing the fixation stiffness. However, general statements are difficult to make due to the limited number of experimental findings. The aim of this study was therefore to numerically investigate healing outcomes under various combinations of shear and axial fixation stiffness, and to detect the optimal configuration. A calibrated and established numerical model was used to predict fracture healing for numerous combinations of axial and shear fixation stiffness under physiological, superimposed, axial compressive and translational shear loading in sheep. Characteristic maps of healing outcome versus fixation stiffness (axial and shear) were created. The results suggest that delayed healing of 3 mm transversal fracture gaps will occur for highly flexible or very rigid axial fixation, which was corroborated by in vivo findings. The optimal fixation stiffness for ovine long bone fractures was predicted to be 1000-2500 N/mm in the axial and >300 N/mm in the shear direction. In summary, an optimized, moderate axial stiffness together with certain shear stiffness enhances fracture healing processes. The negative influence of one improper stiffness can be compensated by adjustment of the stiffness in the other direction.

The healing process consists of at least three phases: inflammatory, repair, and remodeling phase. Because callus stiffness correlates with the healing phases, it is suitable for evaluating the fracture healing process. Our aim was to develop a method which allows determination of callus stiffness in vivo, the healing time and the duration of the repair phase. The right femurs of 16 Wistar rats were osteotomized and stabilized with either more rigid or more flexible external fixation. Fixator deformation was measured using strain gauges during gait analysis. The strains were recalculated as the callus stiffness over the time course of healing, and the healing phases were identified based on stiffness thresholds. Our hypothesis was that stabilization with more flexible external fixation prolongs the repair phase, therefore resulting in an extended healing time. Confirming our hypothesis, the duration of the repair phase (rigid: approximately 15 days, flexible: approximately 41 days) and the healing time (rigid: approximately 27 days, flexible: approximately 62 days) were significantly longer for more flexible external fixation. Our method allows the quantitative detection of differences in the healing time and duration of the repair phase without multiple time-point sacrifices, which reduces the number of animals in experimental studies. (c) 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1589-1595, 2014.

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. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1845–1850, 2013

Introduction Secondary fracture healing processes are strongly influenced by interfragmentary motion [Pauwels, 1960; Einhorn, 2005; Carter, 1988]. Shear movement is assumed to be more critical than axial movement, however experimental results are controversial [Bishop, 2006; Augat, 2003]. Numerical fracture healing models allow to simulate the fracture healing process with variation of single input parameters and under comparable normalized mechanical conditions. Therefore, a direct comparison of different in vivo scenarios is possible. The aim of this study was to simulate fracture healing under several axial compressive, and shear movement scenarios and compare their respective time to heal. We hypothesize that under comparable normalized displacement magnitudes, shear strain is always more critical than axial loading.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

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.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

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.

ABSTRACTWe recently established a large animal model of osteoporosis in sheep using hypothalamic–pituitary disconnection (HPD). As central regulation is important for bone metabolism, HPD‐sheep develop severe osteoporosis because of low bone turnover. In this study we investigated metaphyseal fracture healing in HPD‐sheep. To elucidate potential pathomechanisms, we included a treatment group receiving thyroxine T4 and 17β‐estradiol. Because clinically osteoporotic fractures often occur in the bone metaphysis, HPD‐sheep and healthy controls received an osteotomy in the distal femoral condyle. Half of the HPD‐sheep were systemically treated with thyroxine T4 and 17β‐estradiol during the healing period. Fracture healing was evaluated after 8 weeks using pQCT, µCT, and histomorphometrical analysis. Bone mineral density (BMD) and bone volume/total volume (BV/TV) were considerably reduced by 30% and 36%, respectively, in the osteotomy gap of the HPD‐sheep compared to healthy sheep. Histomorphometry also revealed a decreased amount of newly formed bone (−29%) and some remaining cartilage in the HPD‐group, suggesting that HPD disturbed fracture healing. Thyroxine T4 and 17β‐estradiol substitution considerably improved bone healing in the HPD‐sheep. Our results indicate that fracture healing requires central regulation and that thyroxine T4 and 17β‐estradiol contribute to the complex pathomechanisms of delayed metaphyseal bone healing in HPD‐sheep. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1851–1857, 2013

Numerical models of secondary fracture healing are based on mechanoregulatory algorithms that use distortional strain alone or in combination with either dilatational strain or fluid velocity as determining stimuli for tissue differentiation and development. Comparison of these algorithms has previously suggested that healing processes under torsional rotational loading can only be properly simulated by considering fluid velocity and deviatoric strain as the regulatory stimuli. We hypothesize that sufficient calibration on uncertain input parameters will enhance our existing model, which uses distortional and dilatational strains as determining stimuli, to properly simulate fracture healing under various loading conditions including also torsional rotation. Therefore, we minimized the difference between numerically simulated and experimentally measured courses of interfragmentary movements of two axial compressive cases and two shear load cases (torsional and translational) by varying several input parameter values within their predefined bounds. The calibrated model was then qualitatively evaluated on the ability to predict physiological changes of spatial and temporal tissue distributions, based on respective in vivo data. Finally, we corroborated the model on five additional axial compressive and one asymmetrical bending load case. We conclude that our model, using distortional and dilatational strains as determining stimuli, is able to simulate fracture-healing processes not only under axial compression and torsional rotation but also under translational shear and asymmetrical bending loading conditions.