Advances in the Establishment of Dened Mouse Models
for the Study of Fracture Healing and Bone Regeneration J. H. Holstein, MD,* P. Garcia, MD,* T. Histing, MD,* A. Kristen, MD,* C. Scheuer, PhD, M. D. Menger, MD, and T. Pohlemann, MD* Summary: The availability of a broad spectrum of antibodies and gene-targeted animals caused an increasing interest in mouse models for the study of molecular mechanisms of fracture healing and bone regeneration. In most murine fracture models, the tibia or the femur is fractured using a 3-point bending device (closed models) or is osteotomized using an open surgical approach (open models). For fracture studies in mice, the tibia has to be considered less appropriate compared with the femur because the stabilization of the fracture is more difcult due to its triangular, distally declining caliber and its bowed longitudinal axis. Biomechanical factors critically inuence the bone healing process. Thus, the use of stable osteosynthesis techniques is also of interest in murine fracture models. To achieve stable xation, several biomechanically standardized implants have recently been introduced, including a locking nail and an intra- medullary compression screw. Other implants, such as a pin-clip, an external xator, and a locking plate, additionally allow the stabilization of fractures with distinct gap sizes. This enables the study of healing of critical size defects and nonunions. The use of these implants further allows a rigid xation of fractures in bridle bones, which is essential for fracture studies in animals suffering from metabolic bone diseases like osteoporosis. In general, the analysis of bone healing in these models includes different imaging techniques and histologic, immunohistochemical, biomechanical, and molecular methods. To evaluate the impact of different osteosynthesis techniques on physical activity and rehabilitation, gait analysis may additionally be performed. By this, the gait of the animals can be visualized and quantitatively analyzed using modied running wheels and dynamic high-resolution radiography systems. Taken together, a variety of different murine femur fracture models have become available, providing dened biomechanical conditions for fracture research. The use of these mouse models may now allow studying the inuence of fracture stabilization techniques on molecular mecha- nisms of bone healing. Key Words: animal model, bone repair, mice, fracture stabilization (J Orthop Trauma 2009;23:S31S38) THE VALUE OF MURINE FRACTURE MODELS Although in vitro studies and clinical trials provide adequate techniques and instruments to improve our knowl- edge on bone repair, animal studies still represent an essential tool to analyze the biology of fracture healing. 1 Accordingly, numerous mammalian species, ranging from the mouse to the horse, have been introduced as models to study fracture healing. 28 Nonetheless, differences in the anatomy and metabolism of animals compared with humans must be con- sidered in the experimental setup and in the interpretation of experimental results. In addition, the transfer of the experi- mental data to the clinical situation must be interpreted in light of these limitations. Because of a more primitive bone structure without a haversian system, small rodents, like mice and rats, are thought to be less appropriate for bone healing studies compared with larger animals. In contrast to larger rodents and other mammalians, small rodents use resorption cavities for bone remodeling during fracture healing, and this process of remodeling has been shown similar to the haversian remodeling in larger animals. 1 There are several reasons why small animal models and in particular mouse models have become of increasing interest for fracture healing studies. The expenses for purchase, breeding, and holding of mice are relatively low. Compared with larger animals, the space necessary for holding and the time necessary for bone healing are reduced. Thus, the implementation of larger groups in the experimental study design is more feasible. Finally and of utmost importance, a broad spectrum of antibodies and gene-targeted animals is available for mice, allowing mechanistic studies on molecular mechanisms of bone healing and regeneration. 911 Thus, the murine model is considered as the ideal species for bone healing and regeneration studies. The establishment of standardized fracture models in mice requires sophisticated surgical skills and ne technical developments. For biomechanical testing, highly sensitive testing devices are necessary to detect minor differences in the biomechanical properties of the fragile mouse bones. In addition, the accurate positioning and xation of the small mouse bones in the testing machine is demanding and represents a prerequisite because minor deviations in the testing setup may signicantly affect the test results. Previous studies have shown that the age of the animals has a critical impact on fracture healing. 12 Therefore, animals with an age of completed bone growth should be used to mimic fracture healing in adults. In humans, the ratio of age at Accepted for publication January 27, 2009. From the *Department of Trauma, Hand and Reconstructive Surgery; Institute for Clinical & Experimental Surgery; and Collaborative Research Center AO Foundation, University of Saarland, Homburg/Saar, Germany. Disclosure: The authors report no conicts of interest. Reprints: J. H. Holstein, MD, Department of Trauma, Hand and Reconstructive Surgery, University of Saarland, Homburg/Saar D-66421, Germany (e-mail: joerg.holstein@uks.eu). Copyright 2009 by Lippincott Williams & Wilkins J Orthop Trauma
Volume 23, Number 5 Supplement, May/June 2009 www.jorthotrauma.com | S31 growth plate closure and life expectancy is about 20%. This ratio is quite comparable to that of mice, whereas it markedly differs from that of other species of established models such as rats (30%), rabbits, dogs, and sheep (5%10%). 13 ANATOMIC AND SURGICAL CONSIDERATIONS IN MURINE FRACTURE MODELS The small size of mice is challenging for the de- velopment of a fracture model. Therefore, particularly large long bones, such as the femur and the tibia, are thought to be most appropriate for studies on fracture healing. 6,14 Nonethe- less, previous studies have also used the rib, the radius, the ulna, the mandible, and the calvaria to investigate bone repair. 1520 However, these bones are unfavorable for bio- mechanical testing due to their small size and their anatomic conguration. The tibia fracture model, which was rst described by Hiltunen et al 6 in 1993, is supposed to be the most established murine fracture model. However, due to the distally declining caliber of the tibia, slightly different fracture sites in closed models may result in markedly different callus sizes, which limits both standardization and comparability. The triangular conguration and the bowed longitudinal axis further affords a more sophisticated design for implants, guaranteeing stable xation. In addition, the biomechanical test accuracy is limited due to the irregular shape of bone (Fig. 1). Although the tibia is relatively easy to fracture because of its thin soft tissue cover, this anatomic condition is disadvantageous when analyzing the role of soft tissue in bone repair. The last issue that has to be considered when using tibia fracture models is the inuence of the bula. In closed tibial midshaft fractures, the bula may also break, which results in either 2 different calluses or 1 combined callus (Fig. 2). To avoid a fracture of the bula, the tibia has to be broken very distally and closely to the metaphysis, which, however, is not feasible in a standardized fashion in closed models. FIGURE 1. The mouse tibia with bula (A) and the mouse femur (B). Note the triangular, distally declining caliber and the bended longitudinal axis of the tibia. In contrast to the tibia, the mouse femur is a tubular bone with a relatively consistent inner and outer diameter. FIGURE 2. X-ray of a mouse tibia at day 28 after closed fracture and stabilization with an intramedullary pin (Hiltunen et al 6 ). Note the combined callus of the tibia and the distal bula. A more proximal fracture of the tibia may create even 2 calluses, one of the tibia and another of the bula. To avoid a fracture of the bula, the tibia has to be broken very distally and closely to the metaphysis. Because an exact positioning of the fracture is not possible in closed tibia fracture models, the callus formation is highly variable and not predictable. S32 | www.jorthotrauma.com q 2009 Lippincott Williams & Wilkins Holstein et al J Orthop Trauma
Volume 23, Number 5 Supplement, May/June 2009 In contrast to the tibia, the mouse femur is a tubular bone (length about 15 mm; Fig. 1) with a relatively consistent inner and outer diameter (outer diameter about 1.5 mm). Accord- ingly, different fracture sites within the diaphyseal part of the bone show comparable callus responses. Because of the straight longitudinal axis, standardized fracture stabilization is easier in the femur than in the tibia. Unlike the tibia, the femur is capable of rotation testing, and also 3- and 4-point bending tests provide more consistent results in the femur than in the tibia. As mentioned above, the relatively bulky muscle cover of the femur is challenging when creating a closed fracture. But on the other hand, the muscle cover is an important element to investigate the role of soft tissue during bone repair. Thus, in our opinion, the femur has to be considered more appropriate for fracture studies compared with the tibia. To analyze bone repair, both closed and open fracture models have been used. Closed femur fracture models in mice are based on the model described by Manigrasso and OConnor, 14 which is a modication of the rat femur fracture model reported by Bonnarens and Einhorn. 3 A standardized transverse fracture pattern associated with minor soft tissue trauma is accomplished in these models by a 3-point bending device. Using a parapatellar approach, the patella is dislocated to insert the intramedullary implant retrogradely at the intercondylar notch. The advantage of closed models is that the parapatellar approach and the closed fracture production cause only a minor soft tissue trauma. However, the intramedullary implant might affect the endosteum and the bone marrow. In open models, the femur is fractured or osteotomized visibly using a lateral approach. 21,22 Thereby, the entire lateral muscle layer has to be split to expose the full length of the femur from the greater trochanter to the lateral femoral condyle. In fact, the open surgery procedure produces a major soft tissue trauma, which may have a critical impact on the vascularization of the femur. In contrast to closed models, which use intramedullary implants, open models use extra- medullary implants, which preserve the endosteum and the bone marrow. However, care has to be taken during the surgical procedure to avoid damage to the periosteum. THE RELEVANCE OF BIOMECHANICALLY STANDARDIZED OSTEOSYNTHESIS IN MURINE FEMUR FRACTURE MODELS Because of the lack of stable xation techniques, most of the former femur fracture studies in mice were conducted with an unstable intramedullary pin xation 14,23,24 or even without fracture stabilization. 2527 However, the course of fracture healing has been demonstrated to depend on the stability of the osteosynthesis technique. 2831 In large animal models, it has been shown that interfragmentary movements lead to an increased formation of brocartilage, which is associated with a signicantly decreased bone formation. 30,32 Thereby, bone mineral density and bending rigidity are strongly reduced in fractures that experience shear compared with those that are exposed to axial compression. 28 It has been further demon- strated that interfragmentary motion disturbs angiogenesis. 30,33 In fact, the expression of angiogenic and osteogenic cytokines is affected by the biomechanical environment in the fracture gap. 33,34 Murine studies have illustrated that mechanical fac- tors during fracture healing alter the chronology of chondro- genic and osteogenic cytokine induction, the differentiation of mesenchymal cells, and the size and tissue composition of the callus. 22,26 Standardized osteosynthesis techniques that guarantee dened biomechanical and biologic conditions for fracture research are a matter of course in large animal models. 35 In contrast, it is commonly argued that murine fracture models do not require biomechanical standardization because the pre- dominance of research using mouse models has focused on the molecular aspects of fracture healing. However, as outlined above, the cellular and molecular mechanisms of fracture healing are critically affected by the mechanical environment in the fracture gap. EXPERIMENTAL FIXATION TECHNIQUES TO STABILIZE FEMUR FRACTURES IN MICE Several femoral fracture models have been developed in mice. 14,21,22,3638 Additional models have been introduced for the analysis of bone repair in segmental bone defects. 39,40 Due to the recent advances in osteosynthesis in mice, standardized stabilization may also become feasible in osteoporotic bones. 41 Intramedullary Pin The intramedullary pin provides a simple but unstable closed fracture stabilization (Fig. 3A). 14 Using a medial para- patellar incision, the patella is dislocated to ream the intra- medullary canal at the intercondylar notch. A stainless steel wire (diameter 0.25 mm) is applied for intramedullary fracture stabilization. Migration of the pin is prevented by an additional wedge (length 2 mm and diameter 0.3 mm) at the distal end of the intramedullary canal. The diaphyseal fracture is produced by a 3-point bending device after the insertion of the implant. Intramedullary Locking Nail To overcome the lack of rotational stability observed with pin stabilization, a locking nail has been developed (Fig. 3B). 36 The nail system consists of a modied injection needle (diameter 0.55 mm) and a tungsten guide wire (diameter 0.1 mm). Rotation stability is accomplished by attening the proximal and distal ends of the needle. Using the same surgical approach as described for the pin stabilization, the guide wire is inserted into the intramedullary canal. After the production of a closed diaphyseal fracture, the locking nail is pushed over the guide wire. The guide wire is then removed. Due to the application of the guide wire, it is possible to produce the fracture without prenailing the bone. This avoids alterations to the implant and also more closely mimics the clinical setting. Intramedullary Compression Screw Recently, an intramedullary compression screw (length 18 mm and diameter 0.5 mm) has been engineered (Fig. 3C). 37 It provides not only rotational but also axial stability after osteosynthesis of closed femoral fractures in mice. The technique uses also a guide wire to allow the production of the fracture before insertion of the implant. Rotational and axial stability after fracture xation is achieved by means of q 2009 Lippincott Williams & Wilkins www.jorthotrauma.com | S33 J Orthop Trauma
Volume 23, Number 5 Supplement, May/June 2009 Murine Fracture Models interfragmentary compression through the screw, which consists of a proximal cone-shaped head and a distal thread. According to a pilot study in senescence accelerated mice, strain P6 (SAMP 6), osteoporotic bones can be stabilized by the use of the intramedullary compression screw (Holstein JH, Histing T, Garcia P et al, unpublished data). External Fixator The application of an external xator also allows stable fracture xation (Fig. 3D). 21 In addition, segmental bone defects might be stabilized by an external xator. As an external stabilization device, the xator preserves the endosteum and the bone marrow. However, a traumatizing surgery with a signicant injury of the soft tissue is necessary to insert the xator pins (diameter 0.3 mm) and to fracture the bone. Furthermore, the bulky design of the external xator might restrict the physiologic activity and gait of the animals. Pin-clip Device The pin-clip device represents an open model using an intramedullary pin in combination with an extramedullary clip for fracture xation (Fig. 3E). 22,39 Of interest, the pin-clip technique permits also the stabilization of segmental bone defects. In accordance, it is possible to create different gap sizes and thereby to affect the healing process resulting in critical size defects or even nonunions. Compared with the bulky external xator, this internal stabilization device causes no obvious alteration of the animals gait. In contrast to the external xator, however, the intramedullary pin may affect the endosteal bone repair. Also, the insertion of the pin-clip device requires a traumatizing surgical approach. Locking Plate The locking plate is a third implant that enables a stable fracture xation in the mouse femur but does require open surgery (Fig. 3F). 38,40 The plate is xed to the bone by 4 interlocking screws. The plate is designed to minimize the implant to bone contact area, avoiding major damage to the periosteum. Nonetheless, the plate xation may affect the external callus formation at the implant site. Instead of a rigid plate, a exible plate can be used in which the medial part is replaced by 2 elastic splinting wires. These 2 plate models allow either stable fracture xation or standardized exible stabilization of the mouse femur. Comparable to the pin-clip device, the plate also permits the stabilization of different gap sizes. As a common drawback of open fracture models, the insertion of the locking plate is also associated with a traumatizing surgical approach, which affects the soft tissue cover of the bone. As the intramedullary compression screw, the locking plate can also be used in osteoporotic bones (Histing T, Holstein JH, Garcia P et al, unpublished data). BIOMECHANICAL EX VIVO TESTING OF OSTEOSYNTHESIS DEVICES To evaluate the biomechanical properties of different osteosynthesis devices, biomechanical ex vivo testings have been performed. For these analyses, murine cadaver femora are osteotomized and stabilized by the implant of interest. Using highly sensitive testing devices, the rotational or axial stiffness of the osteosynthesis devices can be analyzed. Of interest, these analyses revealed highly signicant differences in rotation stiffness of the different devices. The locking plate showed a rotational stiffness, which was comparable to that of FIGURE 3. X-rays of fractured mouse femurs after stabilization with (A) an intramedullary pin (Manigrasso and OConnor 14 ), (B) an intramedullary locking nail (Holstein et al 36 ), (C) an intramedullary compression screw (Holstein et al 37 ), (D) an external xator (Cheung et al 21 ), (E) a pin-clip (Garcia et al 22 ; Garcia et al 39 ), and (F) a locking plate (Grongroft et al 40 ; Matthys and Perren 38 ). X-rays in gures B, C, and F were taken at the day of fracture and those in gure A, D, and E were taken at day 7, day 21, or 35 after fracture. Figures AC represent closed femur fracture models and gures DF show open models. S34 | www.jorthotrauma.com q 2009 Lippincott Williams & Wilkins Holstein et al J Orthop Trauma
Volume 23, Number 5 Supplement, May/June 2009 unfractured control femora. In contrast, the conventional pin failed to provide any rotationally stable fracture xation. 22,37 METHODS FOR THE ANALYSIS OF BONE REPAIR IN MURINE FEMUR FRACTURE Imaging Techniques To study femur and tibia fracture healing in mice, the animals have in most cases to be killed at different time points after fracture. This has to be done to resect and prepare the bones for further radiologic, histologic, biomechanical, cyto- logic, and molecular analyses. 14,42 However, beside these post- mortem analyses, noninvasive imaging techniques have been introduced during the last years to evaluate bone repair, including micro-positron emission tomography and micro- magnetic resonance imaging. 43,44 In addition, a variety of molecular imaging techniques, such as bioluminescence, near- infrared uorescence, and nuclear and magnetic resonance imaging, have been applied for noninvasive real-time studies on gene expression, protein degradation, cell migration, and cell death in living animals. 45 Nevertheless, the most established imaging techniques to study murine fracture healing are high-resolution radiog- raphy and 2-dimensional and 3-dimensional microcomputer tomography (CT). Using conventional x-ray techniques, the size and radiologic density of the fracture callus can be analyzed in living animals and in resected bones. 22,39,46 Although micro-CT scans can be applied also in vivo, ex vivo micro-CT scans reveal a signicantly higher resolution than in vivo scans. 47,48 Common parameters that are evaluated by micro-CT are tissue mineral density, total callus volume, and bone volume fraction of the callus. 24,49 By the postmortem injection of a chromate-based contrast agent, the vasculature of the callus can also be visualized and quantitatively assessed by 3-dimensional micro-CT techniques. 50 Histologic Analysis The quantitative histologic analysis of the fracture callus (histomorphometry) has been proven to be an important parameter to assess bone repair. 51 Thereby, the American Society of Bone and Mineral Research has provided recom- mendations regarding a standardized nomenclature, appro- priate indices for assessment, and methodologic approaches for histomorphometric analyses. 52 In addition to histomor- phometric studies, immunohistochemical analyses allow the in situ detection of different proteins like cytokines and cell markers within the fracture callus. 46,53 Because the callus is a heterogeneous, 3-dimensional structure comprising several different types of tissue, it is a great challenge to accurately analyze the different tissue areas using 2-dimensional histo- logic sections. Therefore, it is crucial to dene representative, standardized longitudinal or transverse bone sections that allow a reproducible calculation of the size and tissue com- position of the callus. 51 From the technical aspect, decalcied and undecalcied bone sections are used for a variety of different staining methods. 51 In general, the implant has to be removed; however, when applying the sawing and grinding technique, the implant can also be left in place. 54 Biomechanical Analysis Three-point and 4-point bending tests and torsion tests have proven to be applicable for biomechanical studies on bone repair in murine femur fracture. 40,46,55 In addition, axial testing, which has been reported for the ex vivo analysis of osteosynthesis devices, might be additionally appropriate for the evaluation of bone repair. 37 As mentioned above, the small size of the murine femur is a great challenge for biomechanical tests because an inaccurate, nonstandardized xation of the bones in the testing machine critically affects the testing results. As a matter of course, highly sensitive testing devices are required for biomechanical tests in mouse bones. Using nondestructive 3-point and 4-point testing devices, usually the bending stiffness of the healed bone is assessed from load displacement curves. When applying destructive devices, the ultimate load at failure of the bone is determined. 55 In accordance, torsion stiffness and ultimate torque and angle at failure can be quantitatively analyzed in torsion testing procedures. 46 In general, the results of the biomechanical analysis of the healing bone are expressed as percentage of that of the contralateral intact bone to account for individual differences of the animals. 46,55 Cytologic and Molecular Analysis Beside the new exciting eld of molecular imaging, conventional techniques are available to investigate cellular and molecular aspects of bone repair. Numerous proteins like cytokines, cell metabolism markers, and cell surface proteins are detectable in situ by immunohistochemical methods. 46,53 Results of immunohistochemical assessments can be sup- ported by semiquantitative protein analyses using biochemical methods such as Western blotting and enzyme-linked immu- nosorbent assay techniques. 46,53 In situ hybridization studies provide further information on the corresponding messenger RNA expression in the different cells. 56,57 Also, the assessment of the in situ messenger RNA expression can be additionally supported by semiquantitative techniques such as Northern blot analysis and reverse transcriptionpolymerase chain reaction (RT-PCR). 50,57 Furthermore, cell counting methods like FACS analysis have been reported to be effective to evaluate the effect of different bone marrow cells on bone repair. 58 Cell apoptosis can be assessed by in situ labeling of nuclear DNA fragments using the TdT-mediated duTP-biotin nick end labeling (TUNEL) technique. 59 Finally, cells of the fracture callus can be harvested for further cell culture studies. 60 METHODS TO EVALUATE THE IMPACT OF OSTEOSYNTHESIS ON ANIMAL REHABILITATION As indicated above, bone repair is dependent on the local mechanical environment within the fracture gap. 61 Although mechanical loading during the rst days after fracture (stage of inammation) seems to disturb bone healing, controlled loading stimulates endochondral ossication and bone remodeling during the later stages of healing. 62,63 Of interest, in 1977, Sarmiento et al 64 demonstrated that weight bearing is capable of accelerating bone repair in rats. The animals activity and capability of weight bearing, which might be affected by the q 2009 Lippincott Williams & Wilkins www.jorthotrauma.com | S35 J Orthop Trauma
Volume 23, Number 5 Supplement, May/June 2009 Murine Fracture Models osteosynthesis technique used, has therefore always to be addressed when analyzing bone repair. In large animal models, gait analysis has proven to be a powerful tool for monitoring the course of fracture healing. Thereby, a signicant corre- lation between the stiffness of the osteosynthesis technique, the mineralization of the callus, and the amount of weight bearing of the animals could be demonstrated. 65 First gait analyses in mice were reported in 1974. 66 Using simultaneous video and reaction force analyses, Clarke and Still provided a great review of the murine gait. 42,67 The gait of mice has been further analyzed using treadmills and high-speed video cameras combined with cineuoroscopic equipment. 68 In addition, treadmills were used for the evaluation of the animals running speed. 69 To assess locomotor alterations in arthritic mice, paw print pattern analysis was included in the gait analysis. 70 Recently, we developed a novel approach for gait analysis during murine fracture healing (Fig. 4). Using modied radiolucent running wheels, we were able to visualize the articulating bones of mice by dynamic high-resolution radiography. Thereby, we analyzed characteristic gait parameters such as the tibiofe- moral angle of the fractured limb. Of interest, we observed signicant alterations of the mouse gait when using different osteosynthesis devices for fracture stabilization (Histing et al., unpublished data). In fact, we are convinced that this analysis method represents an additional powerful tool to evaluate healing and rehabilitation of murine femur fractures. SUMMARY AND RECOMMENDATIONS Taken together, a variety of different murine femur fracture models have recently been introduced. These models allow distinct evaluation of the molecular aspects of bone repair. Due to its anatomy, the mouse femur is more appropriate for fracture studies compared with the mouse tibia. Depending on the study, open or closed fracture models with external or internal osteosynthesis devices can be used. The standardization of the experimental setup is a precondition for every animal study and should also include the fracture stabilization. Therefore, biomechanical parameters of fracture stabilization have to be assessed not only in large animals but also in mouse models. A large number of analytical methods, ranging from conventional histology and biomechanics to novel molecular imaging techniques, can be used to evaluate bone repair in mice. In addition, new methods for gait analyses are applicable, which can be either used as a further approach to indirectly assess the fracture healing process or to evaluate the impact of different osteosynthesis devices on physical activity and rehabilitation of the animals. In conclusion, current murine femur fracture models provide standardized conditions with regard to the fracture creation, the character of soft tissue trauma, and the osteosynthesis technique for fracture xation. So far, the mouse has been of interest particularly to analyze molecular and genetic aspects of bone repair. Due to the great progress in the standardization of murine fracture, however, mouse models have become also a powerful tool for research on general aspects of bone repair. REFERENCES 1. Nunamaker DM. Experimental models of fracture repair. Clin Orthop Relat Res. 1998;355:S56S65. 2. Ashhurst DE, Hogg J, Perren SM. A method for making reproducible experimental fractures of the rabbit tibia. Injury. 1982;14:236242. 3. Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res. 1984;2:97101. 4. Cheal EJ, Mansmann KA, DiGioia AM, et al. Role of interfragmentary strain in fracture healing: ovine model of a healing osteotomy. J Orthop Res. 1991;9:131142. 5. Davy DT, Connolly JF. The biomechanical behavior of healing canine radii and ribs. J Biomech. 1982;15:235247. 6. Hiltunen A, Vuorio E, Aro HT. A standardized experimental fracture in the mouse tibia. J Orthop Res. 1993;11:305312. 7. Nunamaker DM, Richardson DW, Butterveck DM, et al. A new external skeletal xation device that allows immediate full weightbearing: application in the horse. Vet Surg. 1986;15:13451355. FIGURE 4. Running wheels for gait analysis in murine fracture models. The physical activity of the animals can be quantita- tively analyzed by using rev counters (A). Using modied radiolucent running wheels, it is possible to visualize the articulating bones of mice by dynamic high-resolution radiography (B). S36 | www.jorthotrauma.com q 2009 Lippincott Williams & Wilkins Holstein et al J Orthop Trauma
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