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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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1589–1595, 2014.

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

We 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 17b-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 17b-estradiol during the healing period. Fracture healing was evaluated after 8 weeks using pQCT, mCT, 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 17b-estradiol substitution considerably improved bone healing in the HPD-sheep. Our results indicate that fracture healing requires central regulation and that thyroxine T4 and 17b-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

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.

In poly-traumatic patients, second hits are known to potentiate the posttraumatic systemic inflammatory response, thus increasing the risk of multi-organ dysfunction. In accordance with “damage control orthopaedic surgery” principles, fractures are initially treated with external fixators, which are replaced by internal osteosynthesis once the immunological status of the patient is considered stable. Recently, we demonstrated that a severe trauma impaired the healing of fractures stabilized by external fixation during the entire healing period. The question arose, whether switching to intramedullary nailing increases the inflammatory response in terms of a second hit, leading to a further impairment of bone healing. Wistar rats received a femoral osteotomy stabilized by an external fixator. Simultaneously half of the rats underwent an additional thoracic trauma. After 4 days, the external fixator was replaced by an intramedullary nail in half of the rats of the two groups. The inflammatory response was evaluated by measuring serum C5a levels. Fracture healing was determined by three-point-bending, µCT, and histomorphometry. The thoracic trauma significantly increased C5a concentrations 6, 24, and 72 h after the second surgical intervention. After 40 days, conversion to intramedullary nailing considerably decreased the flexural rigidity of the callus, with no significant differences between rats with or without thoracic trauma. After 47 days, flexural rigidity in rats subjected to conversion remained decreased compared to animals solely treated by external fixation, particularly in combination with blunt chest trauma. The results indicate that accumulation of second hits after multiple injuries could lead to aggravation of the fracture healing outcome. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 465–471, 2013

ZusammenfassungMausmodelle werden mehr und mehr für die Frakturheilungsforschung eingesetzt. Es stehen zahlreiche verschiedene Mausstämme zur Verfügung. Zu beachten ist jedoch, dass sich die verschiedenen Mausstämme zum Teil erheblich bezüglich Knochenphänotyp und Heilungscharakteristik unterscheiden. Zudem sind auch in der Maus standardisierte und mechanisch kontrollierte Frakturmodelle obligatorisch, da die mechanischen Bedingungen das Versuchsergebnis erheblich be-einflussen. Die standardisierte Frakturfixation ist in der Maus aufgrund der geringen Skelettgröße eine technische Herausforderung. Inzwischen gibt es jedoch eine Reihe kommerziell verfügbarer Osteosynthesesysteme für das Mäusefemur, mit denen kontrollierte Studien möglich sind. Es können Marknägel, Platten oder Fixateur externe verwendet werden. Alle Systeme haben verschiedene Vor- und Nachteile, die bei der Auswahl des Versuchsdesigns für die jeweilige Fragestellung berücksichtigt werden müssen. Durch diese methodischen Fortschritte werden genetisch veränderte Mausmodelle für die Forschung nutzbar gemacht. Durch Ausschalten, Über- oder sogar ektopische Expression eines einzelnen Gens in der Maus kann nicht nur seine physiologische bzw. pathologische Bedeutung, sondern auch ein möglicher pharmakologischer Effekt überprüft werden. Mausmodelle stellen damit ein sehr wertvolles Werkzeug für die Frakturheilungsforschung dar, wenn auch die Bedeutung der gewonnenen Erkenntnisse im Großtiermodell und im Menschen überprüft werden muss.

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. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:734–739, 2011

The application of autologous mesenchymal stem cells (MSC) for the treatment of bone defects requires two invasive procedures and several weeks of ex vivo cell expansion. To overcome these limitations, the administration of allogeneic MSC may be attractive, because they are anticipated to be immunoprivileged. Because preclinical studies using various animal models are conflicting with respect to the efficacy of allogeneic MSC, we investigated whether autologous and allogeneic human MSC (hMSC) are equally effective in regenerating bone in a humanized mouse model resembling the human immune system. Applying autologous and allogeneic hMSC in critically sized femoral defects, we found that allogeneic hMSC elicited a mild immune response early after implantation, whereas early angiogenic processes were similar in both treatments. At later healing time points, the transplantation of allogeneic hMSC resulted in less bone formation than autologous hMSC, associated with a reduced expression of the osteogenic factor Runx2 and impaired angiogenesis. We found by species-specific staining for collagen-type-1α2 that MSCs of either source did not synthesize new bone matrix, indicating an indirect contribution of transplanted hMSC to bone regeneration. In conclusion, our data suggest that the application of autologous hMSC is superior to that of allogeneic cells for bone defect treatment., publishedVersion

Mesenchymal stem cells (MSC) were shown to support bone regeneration, when they were locally transplanted into poorly healing fractures. The benefit of systemic MSC transplantation is currently less evident. There is consensus that systemically applied MSC are recruited to the site of injury, but it is debated whether they actually support bone formation. Furthermore, the question arises as to whether circulating MSC are recruited only in case of injury or whether they also participate in mechanically induced bone formation. To answer these questions we injected green fluorescent protein (GFP)-labelled MSC into C57BL/6J mice, which were subjected either to a femur osteotomy or to non-invasive mechanical ulna loading to induce bone formation. We detected GFP-labelled MSC in the early (day 10) and late fracture callus (day 21) by immunohistochemistry. Stromal cell-derived factor 1 (SDF-1 or CXCL-12), a key chemokine for stem cell attraction, was strongly expressed by virtually all cells near the osteotomy – indicating that SDF-1 may mediate cell migration to the site of injury. We found no differences in SDF-1 expression between the groups. Micro-computed tomography (µCT) revealed significantly more bone in the callus of the MSC treated mice compared to untreated controls. The bending stiffness of callus was not significantly altered after MSC-application. In contrast, we failed to detect GFP-labelled MSC in the ulna after non-invasive mechanical loading. Histomorphometry and µCT revealed a significant load-induced increase in bone formation; however, no further increase was found after MSC administration. Concluding, our results suggest that systemically administered MSC are recruited and support bone formation only in case of injury but not in mechanically induced bone formation.

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 (mu CT), 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 ER alpha in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERa 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.

Balloon kyphoplasty (BKP) with calcium phosphate cement (CPC) is increasingly being used for spinal surgery in younger patients. In routinely performed follow-up CT scans we observed considerable areas of demineralization in CPC processed vertebrae in several patients. To rule out infections or inflammations histological examinations were planned for these patients. Ten patients (23–54 years; six men) with significant demineralization areas in CT scans after CPC balloon kyphoplasty were selected. Punch biopsies from these areas were taken in local anesthesia using a biopsy needle. One half of the specimen was decalcified and embedded in paraffin, and sections were examined histologically using hematoxylin and eosin, Van Gieson, and trichrome staining. The second half of the specimen was cast directly in methyl methacrylate and sections were examined by Paragon and von Kossa/Safranin staining. Stained slides were viewed under light microscopy. Bone-punch specimens were taken at 17.5 months (mean) after BKP with CPC. In most cases, the cement was well surrounded by newly formed lamellar bone with very tight connections between the cement and new bone. Unmineralized areas were observed sporadically at the cement surface and adjacent to the implant. There were no pronounced signs of inflammation or osteolysis of adjacent bone. No complications were observed during or following patients’ biopsy procedures. CPC demonstrated good biocompatibility and osseointegration in clinical use, with no evidence of inflammation or osteonecrosis. Demineralized areas in CT scans could be a result of remodeling of the cancellous bone in vertebral bodies.

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.

Purpose: It is supposed that the demographic change will lead to an increase in patients with impaired alveolar bone conditions. Large animal models are of particular interest in this context as they are suitable for developing and testing new dental implants. Recently, we demonstrated that surgical hypothalamo–pituitary disconnection (HPD) causes a pronounced low-turnover situation leading to cortical and trabecular bone loss in sheep. In this study, we aimed to investigate the influence of the HPD procedure on the alveolar bone. Methods: Ten adult Merino ewes were randomly assigned to two groups: Control and HPD. After 6 months, we analysed the cortical and trabecular bone of all mandibles by histomorphometry and high-resolution peripheral quantitative computed tomography (HR-pQCT). Results: HPD ewes showed a significant decrease in cortical thickness by ~20%, a significant increase in cortical porosity by ~20% and a significant decrease in bone volume by ~30% in comparison with Control ewes. Conclusion: Our results underline the importance of central regulatory mechanisms of bone turnover. However, further studies are needed to understand these central regulatory elements of bone turnover in detail and to judge the value of the HPD sheep for dental research.

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.

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.

We 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.

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.

The adaptive response of bone to load is dependent on molecular factors, including growth factor signaling, which is involved in the regulation of proliferation, differentiation and function of osteoblasts and osteoclasts. Based on a recent study, which has shown that the deficiency of growth factor midkine (Mdk) in mice at 12 and 18 months of age resulted in increased trabecular bone formation, we hypothesized that mechanically-induced bone remodeling may, at least in part, be dependent on Mdk expression. To investigate this, we loaded the ulnae of Mdk-deficient mice and appropriate wild-type mice at the age of 12 months using the in vivo ulna loading model. Histomorphometric quantification of the periosteal bone demonstrated an increased mineralizing surface, mineral apposition rate, and bone formation rate in ulnae of Mdk-deficient mice compared to wild-type mice in response to loading. Because Mdk has been shown to bind to a complex of receptor-type protein tyrosine phosphatase zeta (Ptprz) and low density lipoprotein receptor-related protein-6 (Lrp-6) together with the α4β1- and α6β1-integrins, we performed in vitro studies using osteoblastic cells, transiently over-expressing Mdk, Wnt-3a, and Ptprz to evaluate whether Mdk has a role in regulating bone formation by modulating Wnt signaling. We observed a negative effect of Mdk on Wnt signaling, the extent of which appeared to be dependent on Ptprz expression. Moreover, we performed in vitro loading studies with osteoblasts treated with recombinant Mdk and observed a negative effect on the expression of Wnt target genes, which play a critical role in osteoblast proliferation. In summary, our data demonstrate that Mdk-deficiency in mice has an anabolic effect on mechanically induced cortical bone formation. This could be due to an improved osteoblast function based on an enhancement of β-catenin-dependent Wnt signaling by both Mdk-deficiency and mechanical loading.

Since osteoimmunology is gaining increasingly interest and evidence for involvement of complement in bone biology was found, the role of complement in bone biology and fracture healing was evaluated. After characterizing the bone phenotype, a fracture healing experiment with C3- and C5- deficient mice was performed. After osteotomy of the right femur and external fixation, healing was analyzed after 1, 3, 7 and 21 days. Bone characterization revealed a reduced number of osteoclasts in C5-deficient animals with a significantly reduced resorption activity. While bone mineral density was significantly higher in complement-deficient strains, stiffness was significantly reduced. After 21 days of fracture healing, C5-deficient animals showed reduced stiffness and a smaller callus volume compared to controls. Interestingly, C3- more than C5-deficient animals showed reduced bone formation. Altogether, bone phenotype of complement-deficient animals resembles a mild form of osteopetrosis. This might be due to the resorption defect seen in C5-deficient mice. A reason for the minor involvement of C3-deficient mice compared to the C5-deficient animals could be the cross-talk between the coagulation cascade with side activation of complement factor C5 by thrombin. These results indicate for the first time an essential role of complement in bone biology and fracture healing. Future studies should focus on the molecular basis of complement involvement and the osteoclastic resorption defect.

It is well accepted that a too flexible fracture fixation delays bone healing compared to a more rigid fixation. There is first evidence, that already the inflammatory phase of fracture healing is influenced by the fixation stability. It has been demonstrated in a sheep study that the inflammatory phase is more pronounced under very flexible fixation possibly contributing to delayed healing [Schmidt-Bleek, 2011]. The aim of this study was to further investigate the effect of the mechanical conditions on the inflammatory response during the early healing phase. We investigated the cellular composition of the callus thereby focussing on polymorphonuclear neutrophils (PMN), macrophages, osteoclasts and the presence of interleukin-6 (IL-6) under controlled biomechanical conditions in a rat osteotomy model.