Addition of shock wave therapy to nail dynamization increases the chance of long-bone non-union healing.

BACKGROUND: Long-bone non-unions after intramedullary nailing can be treated by nail dynamization or focused high-energy extracorporal shock wave therapy (fESWT). The objective of this study was to assess the effect of the combination therapy of nail dynamization and fESWT on long-bone non-unions. MATERIALS AND METHODS: 49 patients with long-bone non-unions (femur and tibia) after nailing were treated with nail dynamization (group D, n = 15), fESWT (group S, n = 17) or nail dynamization in addition to fESWT (group DS, n = 17). Patients were followed up for 6 months retrospectively. Furthermore, age, sex, Non-Union Scoring System (NUSS) score, time intervals from primary and last surgery until intervention and smoking status were analysed for their correlations to bone union. RESULTS: Union rates were 60% for group D, 64.7% for group S and 88.2% for group DS, with a significant difference between group D and DS (p = 0.024). Successful treatment was correlated with high age (OR 1.131; 95% CI 1.009-1.268; p = 0.034), female gender (OR 0.009; 95% CI 0.000-0.89; p = 0.039), low NUSS score (OR 0.839; 95% CI 0.717-0.081; p = 0.028) and negative smoking status (OR 86.018; 95% CI 3.051-2425.038; p = 0.009). CONCLUSIONS: Data from the present study indicate that the combination therapy of nail dynamization and fESWT leads to a higher union rate than dynamization or fESWT alone. LEVEL OF EVIDENCE: Level 3.

Effect of Bone Morphogenetic Protein-2 in the Treatment of Long Bone Non-Unions.

BACKGROUND: Delayed fracture healing continues to cause significant patient morbidity and an economic burden to society. Biological stimulation of non-unions includes application of recombinant bone morphogenetic protein-2 (rhBMP-2). However, rhBMP-2 use continues to be a matter of controversy as literature shows scarce evidence for treatment effectiveness. QUESTIONS: The objective of this study was to evaluate the effectiveness of rhBMP-2 treatment on long bone non-unions measuring union rate and time to union. Furthermore, we assess risk factors for treatment failure. METHODS AND PATIENTS: A total of 91 patients with non-unions of long bones were treated with rhBMP-2 (n = 72) or standard care without BMP (n = 19) at our institution. Patient characteristics, comorbidities, nicotine consumption, and complications were recorded. Bone healing was assessed by plane X-rays and clinical examination. Patients were followed up with for 24 months. RESULTS: Overall, there was significantly faster bone healing after rhBMP-2 application compared to the no-BMP group (p < 0.001; HR = 2.78; 95% CI 1.4-5.6). Union rates differed significantly between rhBMP-2 compared to the no-BMP group (89% vs. 47%; p < 0.001). At the humerus, there was neither a significantly higher union rate in the rhBMP-2 (83%) compared to the no-BMP group (50%) (p = 0.26; n = 12) nor a faster bone healing with a median time of 9 months in both groups (HR = 2.01; 95% CI 0.49-8.61; p = 0.315). The 33 femora treated using rhBMP-2 healed significantly faster than 9 femora in the no-BMP group (HR = 2.93; 95% CI 1.00-8.4; p = 0.023) with significant differences in union rate with 85% and 44%, respectively (p = 0.022). Regarding tibia non-unions, 25 out of 27 (93%) healed with a median of 9 months after rhBMP-2 application with no significant difference in the no-BMP group (33%) in time to union (p = 0.097) but a significantly higher union rate (p = 0.039). There was no effect of comorbidities, age, sex, soft tissue damage, or nicotine use on time to union, union rate, or secondary interventions. CONCLUSION: Consistent with the literature, overall, significantly higher union rates with reduced time to union were achieved after rhBMP-2 application. Femoral and tibial non-unions in particular seem to profit from rhBMP-2 application.

Trends in arthrodeses and total joint replacements in Foot and Ankle surgery in Germany during the past decade-Back to the fusion?

BACKGROUND: The goal of this study was to analyze trends in Foot and Ankle surgery in Germany during the past ten years. METHODS: Data of the German Federal Statistical Office from 2008 until 2018 were screened. We focused on the analysis of fusions and total joint replacements. RESULTS: The total amount of the surgeries increased (+39.5%). Especially fusions like arthrodesis of the ankle (+31%) and of the MTP-1 joint (+77%) were on the rise. In 2017 60% of ankle arthrodesis were conducted in men, while 69% of MTP-1 arthrodesis were performed in women. The amount of joint replacements showed a decline during the last decade, in TAR (-39%) and in the MTP-1 (-48%). Regarding the patients age while undergoing a TAR, we observed a shift of the peak age group from 65 to 70 years in 2008 towards 55-60 years in 2017. CONCLUSION: Our study represents actual health care reality in Germany and shows a constant increase of ankle arthritis surgeries in the last decade, while there was a massive shift regarding the ratio of TAR in favour of fusion procedures.

Dose-dependent effect of parathyroid hormone on fracture healing and bone formation in mice.

BACKGROUND: Parathyroid hormone (PTH) is the only clinically approved osteoanabolic drug for osteoporosis treatment. However, PTH is not established for the treatment of fracture healing, and doses of PTH diverge significantly between different studies. We hypothesized that the effect of PTH on promoting fracture healing and bone formation is dose dependent. MATERIALS AND METHODS: In vivo, mice were treated with PTH (10, 40, and 200 µg/kg) in a closed femoral fracture model. Fracture healing was analyzed after 4 weeks. The fourth lumbar vertebra was analyzed to assess systemic effects. In addition, osteoblasts from calvaria of mice were treated in vitro with PTH doses of 10-5-50 nM, and their differentiation was analyzed after 26 days. RESULTS: In vivo, PTH dose-dependently stimulated bone formation in the fracture callus and the vertebral body. However, PTH treatment did not increase biomechanical stiffness of the fractured femora in a dose-dependent manner. The increased bone formation in the 200 µg/kg group was associated with a depletion of osteoclasts, indicating diminished bone remodeling. Of interest, in vitro, we observed diminished mineralization with the highest doses of PTH in osteoblast cultures. CONCLUSIONS: PTH dose-dependently stimulates bone formation in vivo. However, during fracture healing, this did not result in a dose-dependent increase of the mechanical stiffness of the fracture callus. Taken together, our in vivo and in vitro data indicate that the dose-dependent effects of PTH during fracture healing are based on the actions on multiple cell types, thereby influencing not only bone formation but also osteoclastic callus remodeling.

Mechanical and biological characterization of alkaline substituted orthophosphate bone substitutes containing meta- and diphosphates.

Despite the growing knowledge on the mechanisms of fracture healing, bone defects often do not heal in a timely manner. Clinically, tricalcium phosphate (TCP) bone substitutes are used to fill bone defects and promote bone healing. However, the degradation rate of these implants is often too slow for sufficient bone replacement. The use of calcium phosphate material with the crystalline phase Ca10[K/Na](PO4)7 containing different amounts of di- and metaphosphates may overcome this problem, because these materials show an accelerated degradation. Therefore, we generated alkaline substituted Ca-P scaffolds with different amounts of ortho-, di- and metaphosphates. The degradation of these materials was analyzed in TRIS-HCl buffer solution in vitro. Moreover, we measured the compressive strength and porosity of the scaffolds by micro-CT analysis. The biocompatibility of the scaffolds was evaluated in vivo in the mouse dorsal skinfold chamber by means of intravital fluorescence microscopy and histology. We found that higher amounts of incorporated di- and metaphosphates increase the degradation rate and compressive strength of the scaffolds without inducing a stronger leukocytic inflammatory host tissue reaction after implantation. Histological analyses confirmed the good biocompatibility of the scaffolds containing di- and metaphosphates. In summary, this study demonstrates that the compressive strength and degradation rate of Ca-P scaffolds can be improved by incorporation of di- and metaphosphates without affecting their good biocompatibility. Hence, this material modification may be particularly beneficial for the treatment of metaphyseal bone defects in weight bearing locations.

Trends and incidences of ligament-surgeries and osteotomies of the knee: an analysis of German inpatient records 2005-2013.

INTRODUCTION: In Germany most surgical procedures of the ligaments of the knee and correcting osteotomies of the knees are performed within an inpatient setting. The purpose of this study was to analyze the available data to assess epidemiological trends and incidences in inpatient knee joint surgery. MATERIALS AND METHODS: Approximately 154 million anonymized DRG inpatient records were collected by the German Federal Statistical Office between 2005 and 2013. These data were screened and analyzed for knee joint surgical ligamental procedures and osteotomies. RESULTS: Anterior cruciate ligament reconstruction (ACLR) or repair had a high incidence (46 per 100,000 persons/year). In addition, the most frequent grafts included hamstring tendon (90%) and the patellar tendon (6.3%) autografts (2013). Peak age group for ACLR was 15-20 years (14.5%) for female and 20-25 years (23.6%) for male patients, while there was a second peak in the age group 45-50 years (13.8%) for female patients. Male patients had a higher risk for receiving ACLR (RR 1.96; 95% CI 1.92-2.00) and for receiving valgizing high tibial osteotomy (RR 2.43; 95% CI 2.27-2.61). Females had a higher risk for receiving varizing distal femur osteotomy (RR 1.89; 95% CI 1.58-2.26). CONCLUSION: There are growing trends for joint- and activity-preserving procedure in the middle-aged patients. The knowledge of the trends and incidences related to these procedures may help to validate and discuss the results of clinical and register studies.

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

Endostatin inhibits Callus remodeling during fracture healing in mice.

Information on the impact of endogenous anti-angiogenic factors on bone repair is limited. The hypothesis of the present study was endostatin, an endogenous inhibitor of angiogenesis, disturbs fracture healing. We evaluated this hypothesis in a closed femoral fracture model studying two groups of mice, one that was treated by a daily injection of 10 µg recombinant endostatin subcutaneously (n = 38) and a second one that received the vehicle for control (n = 37). Histomorphometric analysis showed a significantly increased callus formation in endostatin-treated animals at 2 and 5 weeks post-fracture. This was associated with a significantly higher callus tissue fraction of cartilage and fibrous tissue at 2 weeks and a significantly higher fraction of bone at 5 weeks post-fracture. Biomechanical testing revealed a significantly higher torsional stiffness in the endostatin group at 2 weeks. For both groups, we could demonstrate the expression of the endostatin receptor unit integrin alpha5 in endothelial cells, osteoblasts, osteoclasts, and chondrocytes at 2 weeks. Immunohistochemical fluorescence staining of CD31 showed a lower number of blood vessels in endostatin-treated animals compared to controls. The results of the present study indicate endostatin promotes soft callus formation but inhibits callus remodeling during fracture healing most probably by an inhibition of angiogenesis.

Delayed fracture healing in aged senescence-accelerated P6 mice.

BACKGROUND: Osteoporosis is characterized by poor bone quality. However, it is still controversially discussed whether osteoporosis compromises fracture healing. Herein, we studied whether the course of healing of a femur fracture is affected by osteoporosis or age. METHODS: Using the senescence-accelerated osteoporotic mouse, strain P6 (SAMP6), and a closed femur fracture model, we studied the process of fracture healing in 5- and 10-month-old animals, including biomechanical, histomorphometric, and protein biochemical analysis. RESULTS: In five-month-old osteoporotic SAMP6 mice, bending stiffness, callus size, and callus tissue distribution as well as the concentrations of the bone formation marker osteocalcin and the bone resorption markers tartrate-resistant acid phosphatase form 5b (TRAP) and deoxypyridinoline (DPD) did not differ from that of non-osteoporotic, senescence-resistant, strain 1 (SAMR1) controls. In contrast, femur fractures in 10-month-old SAMP6 mice showed a significantly reduced bending stiffness and an increased callus size compared to fractures in age-matched SAMR1 controls. This indicates a delayed fracture healing in advanced age SAMP6 mice. The delay of fracture healing was associated with higher concentrations of TRAP and DPD. Significant differences in osteocalcin concentrations were not found between SAMP6 animals and SAMR1 controls. CONCLUSION: In conclusion, the present study indicates that fracture healing in osteoporotic SAMP6 mice is not affected in five-month-old animals, but delayed in animals with an age of 10 months. This is most probably due to the increased osteoclast activity in advanced age SAMP6 animals.

Reduction and fixation capabilities of different plate designs for pubic symphysis disruption: a biomechanical comparison.

BACKGROUND: Typical stabilisation of pelvic open book injuries consists in plate fixation of the symphysis, leading to many different plate designs and procedures that have evolved. However, implant loosening and development of chronic instability are still evident and represent major complications after plate fixation of the symphysis. The aim of this study was to analyse reduction and fixation capabilities of different classical plate techniques with dynamic compression (DC), prebending or modern interlocking screws. METHODS: Compression injuries (OTA B1.1) were simulated on synthetic composite pelvises. Sensor films placed in the disrupted symphysis allowed assessment of reduction and compression forces, as well as contact characteristics by implants at defined time points under static non loaded conditions. The commercially available steel plates used in our study differed in curved design, prebending and DC- or locking screw capabilities, as narrow large fragment (4.5) or small fragment plates (3.5). RESULTS: DC procedure clearly increased the compressive force in the symphysis and improved the reduction by enhanced contact areas. These effects were preserved to the end of the experiments only when the plates were prebended (10°). Anatomically contoured and prebended 3.5 plates had a similar effect, but the contact area was even more pronounced. Best results were observed using the "3.5 symphyseal plate" with DC-effect medially and locking screws laterally. Purely interlocking screw plates by themselves allowed an optimal contact area, yet failed to preserve the initial compressive reduction force. CONCLUSIONS: The experimental results suggest a biomechanical advantage in using prebended plates for symphysis fixation compared to non-bended plates. Best results with regard to compression and increased contact area can be achieved by anatomically contoured plates with combined DC and locking screw capabilities. These findings are of special interest in pelvic surgery for choosing the right implant in severe displacements, obese patients and symphysiodesis techniques.

Excess dietary methionine does not affect fracture healing in mice.

BACKGROUND: An elevated serum concentration of homocysteine (hyperhomocysteinemia) has been shown to disturb fracture healing. As the essential amino acid, methionine, is a precursor of homocysteine, we aimed to investigate whether excess methionine intake affects bone repair. MATERIAL/METHODS: We analyzed bone repair in 2 groups of mice. One group was fed a methionine-rich diet (n=13), and the second group received an equicaloric control diet without methionine supplementation (n=12). Using a closed femoral fracture model, bone repair was analyzed by histomorphometry and biomechanical testing at 4 weeks after fracture. Blood was sampled to measure serum concentrations of homocysteine, the bone formation marker osteocalcin, and the bone resorption marker collagen I C-terminal crosslaps RESULTS: Serum concentrations of homocysteine were significantly higher in the methionine group than in the control group, while serum markers of bone turnover did not differ significantly between the 2 groups. Histomorphometry revealed no significant differences in size and tissue composition of the callus between animals fed the methionine-enriched diet and those receiving the control diet. Accordingly, animals of the 2 groups showed a comparable bending stiffness of the healing bones. CONCLUSIONS: We conclude that excess methionine intake causes hyperhomocysteinemia, but does not affect fracture healing in mice.

A new model to analyze metaphyseal bone healing in mice.

BACKGROUND: Despite the increasing clinical problems with metaphyseal fractures, most experimental studies investigate the healing of diaphyseal fractures. Although the mouse would be the preferable species to study the molecular and genetic aspects of metaphyseal fracture healing, a murine model does not exist yet. Using a special locking plate system, we herein introduce a new model, which allows the analysis of metaphyseal bone healing in mice. METHODS: In 24 CD-1 mice the distal metaphysis of the femur was osteotomized. After stabilization with the locking plate, bone repair was analyzed radiologically, biomechanically, and histologically after 2 (n=12) and 5 wk (n=12). Additionally, the stiffness of the bone-implant construct was tested biomechanically ex vivo. RESULTS: The torsional stiffness of the bone-implant construct was low compared with nonfractured control femora (0.23 ± 0.1 Nmm/°versus 1.78 ± 0.15 Nmm/°, P<0.05). The cause of failure was a pullout of the distal screw. At 2 wk after stabilization, radiological analysis showed that most bones were partly bridged. At 5 wk, all bones showed radiological union. Accordingly, biomechanical analyses revealed a significantly higher torsional stiffness after 5 wk compared with that after 2 wk. Successful healing was indicated by a torsional stiffness of 90% of the contralateral control femora. Histological analyses showed new woven bone bridging the osteotomy without external callus formation and in absence of any cartilaginous tissue, indicating intramembranous healing. CONCLUSION: With the model introduced herein we report, for the first time, successful metaphyseal bone repair in mice. The model may be used to obtain deeper insights into the molecular mechanisms of metaphyseal fracture healing.

Balloon tibioplasty: a useful tool for reduction of tibial plateau depression fractures.

Reduction of the articular surface in displaced tibial plateau fractures is still challenging and may result in joint incongruence, leading to posttraumatic arthrosis. Conventional techniques use bone tamps and similar instruments, which can increase the surgical trauma due to their size. "Balloon tibioplasty" is a novel minimally invasive technique for the reduction of depressed tibial plateau fractures. We successfully applied an inflatable balloon, commercially available from kyphoplasty, to elevate the depressed articular fragments. This technique allowed for reduction of the depressed tibial plateau fragment without classic fenestration of the tibia, thereby minimizing surgical trauma. Furthermore, under fluoroscopic control, optimal centering of the expanding tibioplasty balloon allows a widespread and continuously increasing reduction force to the fracture area. After fluoroscopy or arthroscopic confirmation of reduction of the articular surface, the cavity resulting from tibioplasty was filled with ceramic bone cement through small incisions and fractures were fixed with a small fragment locking T-plate (3.5 mm). Balloon tibioplasty was applied in 5 patients with displaced tibial plateau fractures (OTA type B2/3). No intra- or postoperative complications were observed. This new technique may be a useful tool to facilitate the reduction of select depressed tibial fractures in the future.

Melatonin impairs fracture healing by suppressing RANKL-mediated bone remodeling.

BACKGROUND: Melatonin, the major pineal hormone, is known to regulate distinct physiologic processes. Previous studies have suggested that it supports skeletal growth and bone formation, most probably by inhibiting bone resorption. There is no information, however, whether melatonin affects fracture healing. We therefore studied in a mouse femur fracture model the influence of melatonin on callus formation and biomechanics during fracture healing. METHODS AND MATERIALS: Thirty CD-1 mice received 50 mg/kg body weight melatonin i.p. daily during the entire 2-wk or 5-wk observation period. Controls (n = 30) received equivalent amounts of vehicle. Bone healing was studied by radiological, biomechanical, histomorphometrical, and protein biochemical analyses at 2 and 5 wk after fracture. RESULTS: Biomechanical analysis at 2 wk after fracture healing showed a significantly lower bending stiffness in melatonin-treated animals compared with controls. A slightly higher amount of cartilage tissue and a significantly larger callus size indicated a delayed remodeling process after melatonin treatment. Western blot analysis showed a significantly reduced expression of receptor activator of nuclear factor-κB ligand (RANKL) and collagen I after melatonin treatment. The reduced expression of RANKL was associated with a diminished number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts within the callus of the newly formed bone. CONCLUSIONS: Because bone resorption is an essential requirement for adequate remodeling during fracture healing, we conclude that melatonin impairs fracture healing by suppressing bone resorption through down-regulation of RANKL-mediated osteoclast activation.

Atypical extensive extratemporal hyperpneumatization of the skull base including the cervical spine: case report and review of the literature.

STUDY DESIGN: Case report and clinical discussion. OBJECTIVE: To describe a rare case of hyperpneumatization of the skull base including the cervical spine with the atlas bone. SUMMARY OF BACKGROUND DATA: Initial imaging studies of physically traumatized patients consist of conventional radiographs. An atypical radiolucency is often misdiagnosed as a primary malignancy or a secondary osteolytic metastasis. Further imaging studies may reveal an underlying atypical hyperpneumatization as a very rare benign differential diagnosis. Pathophysiologically, embryological developmental anomalies as well as an elevated pressure to the middle and inner ear are discussed. METHODS: We present a symptomatic 40-year-old man with conventional radiographs, after computed tomography and magnetic resonance imaging examinations. RESULTS: Imaging studies reveal an uncommon radiolucency of the skull base including the atlas bone, free air beneath the mastoid bone, stylomastoid foramen, epidural air adjacent to the atlas bone, and surrounding soft-tissue emphysema. CONCLUSION: Atypical radiolucency may represent a very rare benign hyperpneumatization of the skull base, which may include the craniocervical junction. Because of microfractures of the thinned and consecutive, less stable bones, this also can lead to free air and soft-tissue emphysema, which has not been described previously. Special care should be taken to identify epidural free air because of a possible communication of the epidural space with the external environment. Harmful activities and especially high-speed trauma could result in fractures of the cervical spine due to decreased stability of the hyperpneumatized bones.

Increased osteoblast and osteoclast activity in female senescence-accelerated, osteoporotic SAMP6 mice during fracture healing.

BACKGROUND: Previous studies have shown that fracture healing depends on gender and that in females, ovariectomy-induced osteoporosis impairs the healing process. There is no information, however, whether the alteration of fracture healing in osteoporosis also depends on gender. MATERIALS AND METHODS: Therefore, we herein studied fracture healing in female and male senescence-accelerated osteoporotic mice, strain P6 (SAMP6), including biomechanical, histomorphometric, and protein biochemical analysis. RESULTS: Bending stiffness was reduced in male and female SAMP6 mice compared with senescence-resistant strain 1 (SAMR1) controls. This was associated with elevated serum concentrations of tartrate-resistent acid phosphatase form 5b (TRAP) in both female and male SAMP6 mice. Callus size, however, was significantly larger in female SAMP6 mice compared with male SAMP6 mice and female SAMR1 controls. This indicates a delayed remodeling process in female SAMP6 mice. The delay of callus remodeling in female SAMP6 mice was associated with a significantly higher osteoprotegerin (OPG) callus tissue expression and increased serum concentrations of osteocalcin (OC) and deoxypyridinoline (DPD), indicating elevated osteoblast and osteoclast activities. CONCLUSION: The present study shows that remodeling during fracture healing in female, but not in male, SAMP6 mice is delayed, most probably due to an increased osteoblast and osteoclast activity.

High bone concentrations of homocysteine are associated with altered bone morphology in humans.

Accumulation of homocysteine and S-adenosylhomocysteine in bone has been shown to be associated with reduced bone quality in rats. The aim of the present study was to investigate whether high bone concentrations of homocysteine and S-adenosylhomocysteine as well as a low methylation capacity are related to an impaired bone morphology in humans. Concentrations of homocysteine and its precursors S-adenosylhomocysteine and S-adenosylmethionine were measured in femoral bone samples of eighty-two males and females (age 71 (SD 8) years) who underwent elective hip arthroplasty. Cancellous bone structure was analysed by histomorphometry. In addition, blood was sampled to measure serum concentrations of homocysteine. Results of bone and serum analyses were grouped for individuals with high or low bone concentrations of homocysteine, S-adenosylhomocysteine and S-adenosylmethionine, as well as for individuals with a high or a low methylation capacity, which is indicated by a low or a high S-adenosylhomocysteine:S-adenosylmethionine ratio (n 41, each). Histomorphometry showed a higher trabecular separation and a lower trabecular thickness, trabecular number and trabecular area in individuals with high bone concentrations of homocysteine and S-adenosylhomocysteine compared with individuals with low bone concentrations of homocysteine and S-adenosylhomocysteine. There was no association between the S-adenosylhomocysteine:S-adenosylmethionine ratio and bone morphology. It was found that 48 % of bone homocysteine was bound to the collagen of the extracellular bone matrix. Blood analyses demonstrated a significant correlation between serum and bone homocysteine. The results of the present study indicate an association between altered bone morphology and elevated bone concentrations of homocysteine and S-adenosylhomocysteine, but not between altered bone morphology and methylation capacity.

Exercise enhances angiogenesis during bone defect healing in mice.

The aim of the present study was to investigate the effect of exercise on angiogenesis during bone defect healing in mice. We evaluated angiogenesis during cranial bone defect healing by intravital fluorescence microscopy (IVM) at days 0-21. To characterize the type of bone repair, we performed additional histomorphometric analyses at days 3-15. IVM was conducted in mice, which were housed in cages supplied with running wheels (exercise group; n=7) and compared to IVM results of mice, which were housed in cages without running wheels (controls; n=7). In the exercise group, we additionally performed correlation analyses between results of the IVM and the running distance. IVM showed an accelerated decrease of bone defect area in the exercise group compared to the control group. This was associated with a significantly higher blood vessel diameter in animals undergoing exercise at days 9 and 12 and a significant correlation between running distance and blood vessel density at day 9 (r = 0.74). Histomorphometry showed osseous bridging of the defect at day 9. The newly woven bone was covered by a neo-periosteum containing those blood vessels, which were visible by IVM. We conclude that exercise accelerates bone defect healing and stimulates angiogenesis during bore repair.

Sildenafil accelerates fracture healing in mice.

Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phospodiesterase-5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro-angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro-angiogenic and osteogenic cysteine-rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61-associated pathway.

The LockingMouseNail--a new implant for standardized stable osteosynthesis in mice.

BACKGROUND: Mouse models are of increasing interest to study cellular and molecular mechanisms during fracture healing. However, unlike in large animals and in humans, stable fixation of fractures has been difficult due to the small size of the mouse. METHODS: Based on µCT-scans of a mouse femur, we developed a new intramedullary implant system comparable to a human locking nail. We analyzed fracture healing with osteotomy gap sizes of 0.00, 0.25, and 2.00 mm, which were stabilized with the LockingMouseNail. RESULTS: Femora with a gap size of 0.00 mm and 0.25 mm showed complete fracture healing after 5 wk. Femora showed a secondary bone healing pattern with induction of a small periosteal callus. In contrast, femora with a gap size of 2.00 mm showed sparse periosteal callus formation and a lack of bone bridging even after 10 wk, indicating atrophic non-union. CONCLUSION: The LockingMouseNail allows standardized fixation of mouse femur fractures and also stabilization of segmental defects. By introducing different gap sizes, the healing process can be influenced, ranging from normal fracture healing to atrophic non-union formation. Therefore, the model may ideally be suited to study molecular mechanisms of normal fracture healing, delayed healing, and non-union formation. It may additionally allow studying biological properties and effectiveness of different bone substitutes in stabilized segmental defects.