Locking Nail versus Plate Fixation in Calcaneal Fractures: Brief report on a Retrospective Analysis of Treatment Characteristics and Radiographic Correction Potential.

PURPOSE OF THE STUDY Minimal and limited access techniques are gaining increasing interest for the treatment of displaced intra-articular calcaneal fractures. The ideal treatment method is however still debated and largely based on individual case factors and surgeon experience. Aim of this study was thus to compare the treatment characteristics and radiographic correction potential of a locking nail system with a sinus tarsi approach to plate fixation via an extended lateral approach. MATERIAL AND METHODS We retrospectively reviewed 39 cases of patients with calcaneal fractures that received primary fracture treatment for displaced intra-articular calcaneal fractures between July 2017 and March 2020. Patient characteristics, time to surgery, time to discharge, OR time and the correction achieved were analyzed and comparative statistics performed. RESULTS In total 19 patients treated with the locking nail and 20 patients treated with plate fixation were analyzed. Patient age and fracture severity according to the Sanders classification were comparable between the groups. Overall surgical time, as well as the achieved reduction was equal between both groups. Time to surgery, as well as time from surgery to discharge was significantly shorter in the locking nail group. 2 additional soft tissue procedures were necessary in the extended lateral approach group. DISCUSSION AND CONCLUSIONS The results with the locking calcaneus nail and sinus tarsi approach suggest, that similar treatment results can be achieved as with plate osteosynthesis and an extended lateral approach. Soft tissue management, as well as pre- and postoperative timing and discharge management can be improved with the nail. Further controlled trials comparing the longterm outcome between the treatment options are needed. Key words: calcaneus fracture, sinus tarsi approach, calcaneal nail, C-Nail.

Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Erythropoietin does not improve fracture healing in aged mice.

Fracture healing in the elderly is associated with a declined healing potential caused by multiple factors including a delay of vascularization. Erythropoietin (EPO) has been demonstrated to improve vascularization and fracture healing in adult mice. We, therefore, hypothesized that EPO in aged mice also improves fracture healing. For this purpose, EPO was given daily in a femoral fracture model in aged mice and compared to vehicle-treated controls using radiological, biomechanical, histomorphometric and Western blot techniques. Blood analyses revealed significantly higher concentrations of hemoglobin and a higher hematocrit in EPO-treated animals at 14 and 35 days after fracture. Micro-computed tomography (µCT) indicated that the fraction of bone volume/tissue volume within the callus did not differ between the two groups. However, µCT showed a 3-fold increased tissue mineral density (TMD) in the callus of EPO-treated animals compared to controls. The callus TMD of the EPO-treated animals was also 2-fold higher when compared to the TMD of the unfractured contralateral femur. Interestingly, biomechanical analyses revealed a reduced bending stiffness in femurs of EPO-treated animals at day 35. The histomorphometrically analyzed callus size and callus composition did not show significant differences between the study groups. However, Western blot analyses exhibited an increased expression of osteoprotegerin (OPG), but in particular of receptor activator of NF-κB ligand (RANKL) in the callus of the EPO-treated animals. Further histological analyses of the callus tissue showed that this was associated with an increased number of newly formed blood vessels and a higher number of tartrate-resistant acid phosphatase (TRAP)+ cells. Conclusion: In fracture healing of aged mice EPO treatment increases callus TMD as well as OPG and RANKL expression, indicating an accelerated bone turnover when compared to controls. However, EPO does not improve fracture healing in aged mice. The process of fracture healing may be altered by EPO due to a deterioration of the microcirculation caused by the worsened rheological properties of the blood and due to an increased bone fragility caused by the accelerated bone turnover. Thus, EPO may not be used to improve fracture healing in the elderly.

Effects of locally applied adipose tissue-derived microvascular fragments by thermoresponsive hydrogel on bone healing.

Insufficient vascularization is a major cause for the development of non-unions. To overcome this problem, adipose tissue-derived microvascular fragments (MVF) may serve as vascularization units. However, their application into bone defects needs a carrier system. Herein, we analyzed whether this is achieved by a thermoresponsive hydrogel (TRH). MVF were isolated from CD-1 mice and cultivated after incorporation into TRH, while non-incorporated MVF served as controls. Viability of MVF was assessed immunohistochemically over a 7-day period. Moreover, osteotomies were induced in femurs of CD-1 mice. The osteotomy gaps were filled with MVF-loaded TRH (TRH + MVF), unloaded TRH (TRH) or no material (control). Bone healing was evaluated 14 and 35 days postoperatively. MVF incorporated into TRH exhibited less apoptotic cells and showed a stable vessel morphology compared to controls. Micro-computed tomography revealed a reduced bone volume in TRH + MVF femurs. Histomorphometry showed less bone and more fibrous tissue after 35 days in TRH + MVF femurs compared to controls. Accordingly, TRH + MVF femurs exhibited a lower osseous bridging score and a reduced bending stiffness. Histology and Western blot analysis revealed an increased vascularization and CD31 expression, whereas vascular endothelial growth factor (VEGF) expression was reduced in TRH + MVF femurs. Furthermore, the callus of TRH + MVF femurs showed increased receptor activator of NF-κB ligand expression and higher numbers of osteoclasts. These findings indicate that TRH is an appropriate carrier system for MVF. Application of TRH + MVF increases the vascularization of bone defects. However, this impairs bone healing, most likely due to lower VEGF expression during the early course of bone healing. STATEMENT OF SIGNIFICANCE: In the present study we analyzed for the first time the in vivo performance of a thermoresponsive hydrogel (TRH) as a delivery system for bioactive microvascular fragments (MVF). We found that TRH represents an appropriate carrier for MVF as vascularization units and maintains their viability. Application of MVF-loaded TRH impaired bone formation in an established murine model of bone healing, although vascularization was improved. This unexpected outcome was most likely due to a reduced VEGF expression in the early phase bone healing.

[Periprosthetic acetabular fractures in geriatric patients]. / Die periprothetische Azetabulumfraktur des geriatrischen Patienten.

Periprosthetic acetabular fractures in geriatric patients are rare injuries; however, the incidence is increasing because of the current demographic developments. For diagnosis of periprosthetic acetabular fractures, conventional X­ray images are regularly complemented by computed tomography (CT). For exclusion of loosening of the prosthesis more advanced techniques, such as single photon emission CT (SPECT/CT) are applied. In addition to classification of periprosthetic acetabular fractures by the traditional system of Letournel there are several other classification systems, which take into account the etiology of the fracture and the stability of the prosthesis. While, under certain circumstances conservative treatment of periprosthetic acetabular fractures is possible, operative treatment often requires extensive surgical procedures to restore the stability of the acetabulum as a support for the cup of the prosthesis. Besides the traditional techniques of acetabular osteosynthesis, special revision systems, augmentations and allografts are used for the reconstruction of periprosthetic acetabular fractures. To determine a therapeutic regimen patient-specific preconditions as well as fracture pattern and type of prosthesis need to be taken into account. In the literature there are several algorithms, which are aimed at supporting the attending physician in making the correct decision for the treatment of periprosthetic acetabular fractures. In cases of periprosthetic acetabular fractures even experienced surgeons are faced with great challenges. Thus, treatment should be carried out in specialized centers.

[Traumatic hip dislocation in a 6-year-old male child: Seldom but demanding]. / Die Traumatische Hüftgelenkluxation eines 6­jährigen Jungen: Selten, aber anspruchsvoll.

Traumatic hip dislocations in children are not frequent but constitute true emergencies. They require urgent reduction because of the risk of consecutive avascular necrosis of the femoral head. We report a 6-year-old boy with traumatic posterior hip dislocation on a vacation abroad. After closed reduction the day of the accident, a hip spica cast was applied and the patient was transferred home. Once home, X­ray and CT diagnostics were completed by MRI. In future, long-term clinical and radiological investigations for avascular necrosis and growth disorders, as well as thoroughly informing the parents, should be mandatory.

Obesity does not affect the healing of femur fractures in mice.

Obesity is reported to be both protective and deleterious to bone. Lipotoxicity and inflammation might be responsible for bone loss through inhibition of osteoblasts and activation of osteoclasts. However, little is known whether obesity affects the process of fracture healing. Therefore, we studied the effect of high fat diet-induced (HFD) obesity on callus formation and bone remodelling in a closed femur fracture model in mice. Thirty-one mice were fed a diet containing 60kJ% fat (HFD) for a total of 20 weeks before fracture and during the entire postoperative observation period. Control mice (n=31) received a standard diet containing 10kJ% fat. Healing was analyzed using micro-CT, biomechanical, histomorphometrical, immunohistochemical, serum and protein biochemical analysis at 2 and 4 weeks after fracture. HFD-fed mice showed a higher body weight and increased serum concentrations of leptin and interleukin-6 compared to controls. Within the callus tissue Western blot analyses revealed a higher expression of transcription factor peroxisome proliferator-activated receptor y (PPARy) and a reduced expression of runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein (BMP)-4. However, obesity did not affect the expression of BMP-2 and did not influence the receptor activator of nuclear factor κB (RANK)/RANK ligand/osteoprotegerin (OPG) pathway during fracture healing. Although the bones of HFD-fed animals showed an increased number of adipocytes within the bone marrow, HFD did not increase callus adiposity. In addition, radiological and histomorphometric analysis could also not detect significant differences in bone formation between HFD-fed animals and controls. Accordingly, HFD did not affect bending stiffness after 2 and 4 weeks of healing. These findings indicate that obesity does not affect femur fracture healing in mice.

Effect of Stabilization on the Healing Process of Femur Fractures in Aged Mice.

BACKGROUND: The influence of mechanical stability on fracture healing has previously been studied in adult mice, but is poorly understood in aged animals. Therefore, we herein studied the effect of stabilization on the healing process of femur fractures in aged mice. METHODS: Twenty-four 18-month-old CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw. In another 24 18-month-old mice, the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric, and protein expression analyses. RESULTS: After 2 and 5 weeks of healing, the callus of nonstabilized fractures compared to stabilized fractures was significantly larger, containing a significantly smaller amount of osseous tissue and a higher amount of cartilaginous tissue. This was associated with a significantly lower biomechanical stiffness during the early phase of healing. However, during the late phase of fracture healing both nonstabilized and stabilized fractures showed a biomechanical stiffness of ∼40%. Of interest, Western blot analyses of callus tissue demonstrated that the expression of proteins related to angiogenesis, bone formation and remodeling, i.e. VEGF, CYR61, BMP-2, BMP-4, Col-2, Col-10, RANKL, OPG, did not differ between nonstabilized and stabilized fractures. CONCLUSION: Nonstabilized fractures in aged mice show delayed healing and remodeling. This is not caused by an altered protein expression in the callus but rather by the excessive interfragmentary movements.

Characterization of the healing process in non-stabilized and stabilized femur fractures in mice.

BACKGROUND: Although a variety of suitable fracture models for mice exist, in many studies bone healing was still analyzed without fracture stabilization. Because there is little information whether the healing of non-stabilized fractures differs from that of stabilized fractures, we herein studied the healing process of non-stabilized compared to stabilized femur fractures. MATERIALS AND METHODS: Twenty-one CD-1 mice were stabilized after midshaft fracture of the femur with an intramedullary screw allowing micromovements and endochondral healing. In another 22 mice the femur fractures were left unstabilized. Bone healing was studied by radiological, biomechanical, histomorphometric and protein expression analyses. RESULTS: Non-stabilized femur fractures revealed a significantly lower biomechanical stiffness compared to stabilized fractures. During the early phase of fracture healing non-stabilized fractures demonstrated a significantly lower amount of osseous tissue and a higher amount of cartilage tissue. During the late phase of fracture healing both non-stabilized and stabilized fractures showed almost 100 % osseous callus tissue. However, in stabilized fractures remodeling was almost completed with lamellar bone while non-stabilized fractures still showed large callus with great amounts of woven bone, indicating a delay in bone remodeling. Of interest, western blot analyses of callus tissue demonstrated in non-stabilized fractures a significantly reduced expression of vascular endothelial growth factor and a slightly lowered expression of bone morphogenetic protein-2 and collagen-10. CONCLUSION: Non-stabilized femur fractures in mice show a marked delay in bone healing compared to stabilized fractures. Therefore, non-stabilized fracture models may not be used to analyze the mechanisms of normal bone healing.

[Practical courses for students can not only improve the teaching quality but can also increase the attractiveness of orthopaedic and trauma surgery]. / Praktische, berufsrelevante Kurse für Studierende können nicht nur die Lehre verbessern, sondern steigern auch die Attraktivität des Faches Orthopädie/Unfallchirurgie.

BACKGROUND: Students often complain about a lack of teaching of practical skills. This may be a cause for the low attractiveness of surgical disciplines. We therefore established a practical course to improve teaching quality to inspire the students for orthopedic and trauma surgery. METHODS: The platforms of the course included the teaching and acquisition of suture techniques, arthroscopy and osteosynthesis techniques. A total of 119 students participated in 9 courses and performed a detailed evaluation. RESULTS: The main motivation to participate was (i) to acquire practical skills (93 %), (ii) to learn about orthopaedic and trauma surgery (66 %) and (iii) to facilitate decision-making for the occupational choice (21 %). 94 % judged the quality of the course as "excellent", and all 119 participants indicated that they would recommend the course to other students. 43 of 45 students, who had not yet decided on their occupational choice, indicated that the course stimulated them for a career in orthopaedic and trauma surgery. CONCLUSION: This course not only can improve the teaching quality but also can increase the attractiveness of muskuloskeletal surgery.

[Compatibility of science and clinical aspects. Between realism and utopia]. / Vereinbarkeit von Wissenschaft und Klinik. Zwischen Realismus und Utopie.

The working environment for young residents in orthopedic surgery has changed tremendously over the past 10 years. Due to cumulative clinical requirements and increasing demands on work-life balance research activity has become less attractive. Successful incorporation of research into the career of residents is a challenging project for the future. The young forum of the German Association for Orthopedics and Traumatology (DGOU) provides different approaches to enhance the quality of research and to help young orthopedists and trauma surgeons.

Pantoprazole, a proton pump inhibitor, delays fracture healing in mice.

Proton pump inhibitors (PPIs), which are widely used in the treatment of dyspeptic problems, have been shown to reduce osteoclast activity. There is no information, however, on whether PPIs affect fracture healing. We therefore studied the effect of the PPI pantoprazole on callus formation and biomechanics during fracture repair. Bone healing was analyzed in a murine fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analyses at 2 and 5 weeks after fracture. Twenty-one mice received 100 mg/kg body weight pantoprazole i.p. daily. Controls (n = 21) received equivalent amounts of vehicle. In pantoprazole-treated animals biomechanical analysis revealed a significantly reduced bending stiffness at 5 weeks after fracture compared to controls. This was associated with a significantly lower amount of bony tissue within the callus and higher amounts of cartilaginous and fibrous tissue. Western blot analysis showed reduced expression of the bone formation markers bone morphogenetic protein (BMP)-2, BMP-4, and cysteine-rich protein (CYR61). In addition, significantly lower expression of proliferating cell nuclear antigen indicated reduced cell proliferation after pantoprazole treatment. Of interest, the reduced expression of bone formation markers was associated with a significantly diminished expression of RANKL, indicating osteoclast inhibition. Pantoprazole delays fracture healing by affecting both bone formation and bone remodeling.

Increased exercise after stable closed fracture fixation does not affect fracture healing in mice.

PURPOSE: The aim of the present study was to evaluate the systemic biological effect of increased exercise on bone repair after stable fracture fixation. METHODS: Two groups of SKH-1h mice were studied. Animals of the first group (n=36) were housed in cages supplied with a running wheel, while mice of the second group (n=37) were housed in standard cages for control. Using a closed femur fracture model, bone repair was analysed by histomorphometry and biomechanical testing at 2 and 5 weeks. At 2 weeks, we additionally evaluated the expression of the proliferation marker PCNA (proliferating cell nuclear antigen) and the angiogenic and osteogenic growth factor VEGF (vascular endothelial growth factor). To standardise the mechanical conditions in the fracture gap, we used an intramedullary compression screw for stable fracture fixation. RESULTS: Each mouse of the exercise group run a mean total distance of 23.5 km after 2 weeks and 104.3 km after 5 weeks. Histomorphometric analysis of the size and tissue composition of the callus could not reveal significant differences between mice undergoing exercise and controls. Accordingly, biomechanical testing showed a comparable torsional stiffness, peak rotation angle, and load at failure of the healing bones in the two groups. The expression of PCNA and VEGF did also not differ between mice of the exercise group and controls. CONCLUSION: We conclude that increased exercise does not affect bone repair after stable fracture fixation.

Temporal and spatial vascularization patterns of unions and nonunions: role of vascular endothelial growth factor and bone morphogenetic proteins.

BACKGROUND: Failure of fracture-healing with nonunion is a major clinical problem. Angiogenesis is closely linked to bone regeneration, but the role of angiogenesis in nonunion formation remains unclear. Because established nonunions are well vascularized, we hypothesized that lack of vascular endothelial growth factor (VEGF) expression and vascularization during the early time course of fracture-healing determine nonunion formation. METHODS: In seventy-two CD-1 mice, a femoral osteotomy with a gap size of 1.80 mm (nonunion group) or a gap size of 0.25 mm (union group) was created and stabilized by a pin-clip technique. Healing was analyzed after three, seven, fourteen, twenty-one, twenty-eight, and seventy days by micro-computed tomography and histomorphometry. Vascularization was determined in different healing zones by immunohistochemical staining of PECAM-1 (platelet-endothelial cell adhesion molecule). Additional animals were analyzed after seven, fourteen, and twenty-one days with Western blot analysis of VEGF, bone morphogenetic protein (BMP)-2, and BMP-4 expression. RESULTS: Micro-computed tomography and histomorphometry showed complete bone-bridging in the union group, whereas animals in the nonunion group showed atrophic nonunion formation. Vascularization increased from day 3 to day 7 in both groups, with a subsequent decrease after fourteen days. However, overall vascularization did not differ between unions and nonunions over time. It is of interest that vascularization within the endosteal healing zone was even higher in nonunions than in unions after fourteen days. Expression of VEGF was significantly higher in nonunions, while expression of BMP-2 and 4 and proliferating cell nuclear antigen were found significantly reduced compared with unions. CONCLUSIONS: Because vascularization during the early time course of fracture-healing was not impaired despite the failure of bone-healing in nonunions, we rejected our hypothesis and accepted the null hypothesis that nonunion formation is not due to failure of VEGF-mediated angiogenesis. Failure of fracture-healing was associated with a decreased expression of BMP-2 and 4 and a disturbed ratio of angiogenic to osteogenic growth factors, which may be responsible for nonunion. CLINICAL RELEVANCE: Because the intrinsic angiogenic response during nonunion formation was sufficient for adequate vascularization, treatment strategies for nonunions should focus on the stimulation of osteogenesis rather than on the stimulation of angiogenesis.

Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice.

The glycoprotein erythropoietin (EPO) has been demonstrated to stimulate fracture healing. The aim of the present study was to investigate the effect of EPO treatment on bone repair in a femoral segmental defect model. Bone repair was analyzed in mice which were treated by EPO (500IE/kg/d intraperitoneally; n=38) and in mice which received the vehicle for control (n=40). Two and 10 weeks after creating a 1.8mm femoral segmental defect, bone repair was studied by micro-CT, histology, and Western blot analysis. At 10 weeks, micro-CT and histomorphometric analyses showed a significantly higher bridging rate of the bone defects in EPO-treated animals than in controls. This was associated by a significantly higher bone volume within the segmental defects of the EPO-treated animals. At 2 weeks, Western blot analyses revealed a significantly higher expression of vascular endothelial growth factor (VEGF) in EPO-treated animals compared to controls. Accordingly, the number of blood vessels was significantly increased in the EPO group at 2 weeks. At 10 weeks, we found a significantly higher expression of proliferating cell nuclear antigen (PCNA) in EPO-treated animals when compared to controls. Western blot analyses showed no significant differences between the groups in the expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS) and the angiopoietin receptor Tie-2. Immunohistochemistry confirmed the results of the Western blot analyses, demonstrating a significantly higher number of VEGF- and PCNA-positive cells in EPO-treated animals than in controls at 2 and 10 weeks, respectively. We conclude that EPO is capable of stimulating bone formation, cell proliferation and VEGF-mediated angiogenesis in a femoral segmental defect model.

Low dose erythropoietin stimulates bone healing in mice.

Beyond its classical role in regulation of erythropoiesis, erythropoietin (EPO) has been shown to exert protective and regenerative actions in a variety of non-hematopoietic tissues. However, little is known about potential actions in bone regeneration. To analyze fracture healing in mice, a femoral 0.25 mm osteotomy gap was stabilized with a pin-clip technique. Animals were treated with 500 U EPO/kg bw per day or with vehicle only. After 2 and 5 weeks, fracture healing was analyzed biomechanically, radiologically and histologically. Expression of PCNA and NFκB was examined by Western blot analysis. Vascularization was analyzed by immunohistochemical staining of PECAM-1. Circulating endothelial progenitor cells were measured by flow-cytometry. Herein, we demonstrate that EPO-treatment significantly accelerates bone healing in mice. This is indicated by a significantly greater biomechanical stiffness and a higher radiological density of the periosteal callus at 2 and 5 weeks after fracture and stabilization. Histological analysis demonstrated significantly more bone and less cartilage and fibrous tissue in the periosteal callus. Endosteal vascularization was significantly increased in EPO-treated animals when compared to controls. The number of circulating endothelial progenitor cells was significantly greater in EPO-treated animals. The herein shown acceleration of healing by EPO may represent a promising novel treatment strategy for fractures with delayed healing and non-union formation.

Intravital microscopic studies of angiogenesis during bone defect healing in mice calvaria.

PURPOSE: Due to the great availability of specific antibodies, gene-targeted animals and knockout strains, mouse models came into the focus of musculoskeletal research. Herein, we introduce a calvarian defect model in mice that allows the repetitive analysis of blood vessel formation during bone repair by intravital microscopy. METHODS: The right parietal calvaria of 20 adult CD-1 mice were exposed by skin excision. Under continuous irrigation, a circular defect (Ø0.75 mm) was drilled into the calvarium without penetrating the inner cortical shell. A circular glass (Ø12 mm; thickness 0.15 mm) was fixed by two microscrews (M1; length 2mm) to cover the bone defect. Angiogenesis was analysed by intravital microscopy at days 0, 3, 6, 9, 12, 15, 18 and 21. In addition, bone repair was evaluated by histomorphometry at days 3, 6, 9 and 15. Immunohistochemical stainings for the angiogenic growth factor vascular endothelial growth factor (VEGF) and the cell proliferation marker proliferating cell nuclear antigen (PCNA) were performed to assess angiogenic and proliferative activity during healing of the calvarian defect. RESULTS: Histomorphometry showed a typical pattern of intramembranous bone repair. Osseous bridging of the defect was observed at day 9. This was associated with the formation of a neo-periosteum, which covered the new woven bone and contained a dense network of newly formed blood vessels. At day 9, particularly cells of the neo-periosteum showed intense staining for VEGF, whilst PCNA-positive staining was found mainly in osteoblasts. At day 15, the major fraction of fibrous tissue was replaced by bone undergoing extensive remodelling. Intravital microscopy revealed an increase of vascular density between days 3 and 15. Blood vessel diameters showed an increase between days 3 and 9 and a subsequent decrease between days 9 and 21. CONCLUSIONS: The present calvarian defect model provides a powerful tool to evaluate the process of angiogenesis during intramembranous bone repair in mice.

In vivo gait analysis in a mouse femur fracture model.

Although the mouse has become a preferred species for molecular studies on fracture healing, gait analysis after fracture fixation and during bone healing has not yet been performed in mice. Herein, we introduce a novel technique for gait analysis in mice and report the change of motion pattern after fracture and fixation. A standardized femur fracture was stabilized by a common pin. The non-fractured tibia was additionally marked with a pin, allowing continuous analysis of the tibio-femoral angle by digital video-radiography. Dynamic gait analysis was performed at day fourteen after surgery in a radio-opaque running wheel. Fracture fixation resulted in a significantly reduced range and maximum of the tibio-femoral angle compared to non-fractured controls. This was associated with a significantly reduced stride length. Because stride frequency was slightly increased and, thus, stride time diminished, stride velocity was not significantly reduced compared to controls. Thus, our study demonstrates distinct alterations of the gait of mice at 2 weeks after femur fracture and stabilization. Our results support the need of gait analysis in fracture healing studies to assess the animals' well-being.

Rapamycin affects early fracture healing in mice.

BACKGROUND AND PURPOSE: The immunosuppressive drug rapamycin (RAPA) prevents rejection in organ transplantation by inhibiting interleukin-2-stimulated T-cell division. Rapamycin has also been suggested to possess strong anti-angiogenic activities linked to a decrease in production of vascular endothelial growth factor (VEGF). Angiogenesis and VEGF are thought to play a crucial role in fracture healing and as osteoporotic and traumatic fractures are common complications in immunosuppressed, organ transplantation patients, we conducted this study to analyze the effect of rapamycin treatment on bone repair. EXPERIMENTAL APPROACH: We investigated the effect of rapamycin treatment on bone repair in a murine closed femur fracture model using radiological, histomorphometric, immunohistochemical, biomechanical and protein biochemical analyses. KEY RESULTS: X-ray analyses demonstrated that rapamycin treatment inhibits callus formation after two weeks of fracture healing. The radiologically observed lack of callus formation was confirmed by histomorphometric analyses, which revealed a significantly diminished callus size and a reduced amount of bone formation when compared with vehicle-treated controls. Biomechanical testing further demonstrated that rapamycin significantly reduces torsional stiffness of the callus. Interestingly, this effect was associated with decreased vessel formation; a diminished proliferation of osteoblasts, endothelial cells and periosteal cells; and a reduced VEGF expression in hypertrophic chondrocytes. After five weeks treatment, however, the negative impact of rapamycin on fracture healing was overcome. CONCLUSIONS AND IMPLICATIONS: Thus, rapamycin initially delays fracture healing, most probably by inhibiting cell proliferation and neovascularization in the callus. These undesirable effects should be considered when rapamycin is administered to patients sustaining bone fractures.