Tina Histing

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

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

Inhibition of angiotensin‐converting enzyme stimulates fracture healing and periosteal callus formation – role of a local renin‐angiotensin system

Background and purpose:  The renin‐angiotensin system (RAS) regulates blood pressure and electrolyte homeostasis. In addition, ‘local’ tissue‐specific RAS have been identified, regulating regeneration, cell growth, apoptosis, inflammation and angiogenesis. Although components of the RAS are expressed in osteoblasts and osteoclasts, a local RAS in bone has not yet been described and there is no information on whether the RAS is involved in fracture healing. Therefore, we studied the expression and function of the key RAS component, angiotensin‐converting enzyme (ACE), during fracture healing.

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.

Development of a stable closed femoral fracture model in mice.

BACKGROUND Mice have become of increasing interest as experimental model for fracture studies. Due to their small size, most studies use simple pins for fracture stabilization, although insufficient rigidity of fixation critically affects fracture healing. Herein, we studied whether longitudinal fracture compression by an intramedullary screw represents a standardized, stable osteosynthesis technique in mice, and whether it may accelerate fracture healing. MATERIALS AND METHODS A micro-screw (MouseScrew) was constructed, allowing closed fracture stabilization without traumatizing surgery. Fracture stabilization was achieved by longitudinal compression, which was confirmed by biomechanical testing of osteotomized cadaver femora. Bone repair was analyzed histomorphometrically at 2 and 5 wk after surgery. RESULTS Ex vivo analyses showed a significantly increased rotational and axial stiffness after screw stabilization (n = 8 each) compared with stabilization techniques using a conventional pin (n = 8 each) or a locking nail (n = 8 each). In the in vivo setting, 2 wk of screw stabilization (n = 8) demonstrated a significantly decreased fibrous tissue formation and an increased cartilage production compared with fractures stabilized by the locking nail (n = 8). After 5 wk callus consisted exclusively of bone in all animals studied without differences between the two stabilization techniques (n = 8 each). CONCLUSIONS Because prolonged fibrous tissue formation indicates delayed fracture healing, we conclude that the increased stability of the fracture by the use of our newly developed MouseScrew accelerates initial bone repair. Further, this fracture model may represent an ideal tool to study bone repair in mice under conditions of stable fixation.

A new technique for internal fixation of femoral fractures in mice: Impact of stability on fracture healing

Mouse models are of increasing interest to study the molecular aspects of fracture healing. Because biomechanical factors greatly influence the healing process, stable fixation of the fracture is of interest also in mouse models. Unlike in large animals, however, there is a lack of mouse models which provide stable osteosynthesis. The purpose of this study was therefore to develop a technique for a more stable fixation of femoral fractures in mice and to analyze the impact of stability on the process of fracture healing. The new technique introduced herein includes an intramedullary pin and an extramedullary metallic clip. Ex vivo biomechanical analysis revealed a significantly higher implant stiffness of our pin-clip technique when compared with previously described intramedullary fixation techniques. In vivo, we studied the course of healing after the more stable fixation with our pin-clip technique and compared the results with that observed after unstable fixation with the pin-clip technique after cutting the clip. After 2 and 5 weeks of fracture healing radiological analysis demonstrated that the more stable fixation with the pin-clip technique results in a significantly higher union rate compared to the unstable fixation. Torsional stiffness at 5 weeks was almost 3-fold of that measured after unstable fixation. Histomorphological analysis further showed that fractures stabilized with the pin-clip technique healed with a smaller periosteal callus area, an increased fraction of bone and a reduced amount of fibrous tissue. Of interest, the pin-clip fixation showed reliable union after 5 weeks, whereas the unstable pin fixation did not regularly achieve adequate fracture healing. In conclusion, we introduce a novel, easily applicable internal osteosynthesis technique in mice, which provides rotational stability after femoral fracture fixation. We further show that a more stable osteosynthesis significantly improves the process of fracture healing also in mice.

Rapamycin affects early fracture healing in mice

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.

An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.

In most murine fracture models, the femur is stabilized by an intramedullary implant and heals predominantly through endochondral ossification. The aim of the present study was to establish a mouse model in which fractures heal intramembranously. Femur fractures of 16 SKH‐mice were stabilized by an internal locking plate. Femur fractures of another 16 animals were stabilized by an intramedullary screw. Bone repair was analyzed by radiographic, biomechanical, and histological methods. At 2 weeks, histological analysis showed a significantly smaller callus diameter and callus area after locking plate fixation. Cartilage formation within the callus could only be observed after screw fixation, but not after fracture stabilization with the locking plate. Radiological and biomechanical analysis after 2 and 5 weeks showed a significantly improved healing and a higher bending stiffness of fractures stabilized by the locking plate. Fractures stabilized by the locking plate healed exclusively by intramembranous ossification, which is most probably a result of the anatomical reduction and stable fixation. The fractures that healed by intramembranous ossification showed an increased stiffness compared to fractures that healed by endochondral ossification. This model may be used to study molecular mechanisms of intramembranous bone healing.

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.

Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture

The various molecular mechanisms of cell regeneration and tissue healing can best be studied in mouse models with the availability of a wide range of monoclonal antibodies and gene‐targeted animals. The influence of the mechanical stability of individual stabilization techniques on the molecular mechanisms of fracture healing has not been completely elucidated yet. Although during recent years several osteosynthesis techniques have been introduced in mouse fracture models, no comparative study on fracture stabilization is available yet. We therefore analyzed herein in a standardized ex vivo setup the rotational stiffness of seven different osteosynthesis techniques using osteotomized right cadaver femora of CD‐1 mice. Uninjured femora without osteotomy served as controls. Femur stabilization with a locking plate or an external fixator resulted in a rotational stiffness almost similar to the intact femur. The use of a “pin‐clip” device, a “locking nail,” a “mouse nail,” or an “intramedullary screw” produced a lower torsional stiffness, which, however, was still significantly higher than that achieved with the widely applied conventional pin. By the use of the presented data a more specific choice of stabilization technique will be possible according to the various questions concerning molecular aspects in fracture healing.

Low serum folate and vitamin B-6 are associated with an altered cancellous bone structure in humans.

BACKGROUND Several clinical trials have reported B vitamins to be associated with osteoporosis. OBJECTIVE Our objective was to investigate whether low serum B vitamins are associated with altered structural and biomechanical properties of human bone. DESIGN Femoral heads of 94 men and women who underwent hip arthroplasty were analyzed by using dual-energy X-ray absorptiometry (DXA), biomechanical testing (indentation method), and histomorphometry. In addition, blood was collected to measure serum concentrations of homocysteine, folate, vitamin B-6, vitamin B-12, the bone formation marker osteocalcin, and the bone resorption marker tartrate-resistant acid phosphatase (TRAP). Measurement outcomes were grouped according to subjects with high and low serum concentrations, respectively, of folate, vitamin B-6, and vitamin B-12 (n = 47 for each group). RESULTS Histomorphometric analysis showed a significantly lower trabecular thickness and trabecular area in subjects with low serum folate concentrations than in those with high serum folate concentrations and a significantly lower trabecular number in subjects with low serum vitamin B-6 concentrations than in those with high serum vitamin B-6 concentrations. In contrast, we found a comparable trabecular structure in subjects with high and low serum vitamin B-12 concentrations. DXA and biomechanical testing did not show significant differences between subjects with high and low serum B vitamin concentrations. Osteocalcin was significantly lowered in subjects with a low serum B vitamin concentration, whereas there was no association between serum B vitamins and TRAP. CONCLUSION The results of the present study indicate that low serum folate and vitamin B-6 concentrations, but not low serum vitamin B-12 concentrations, are associated with an altered morphology of human bone.