- Email: [email protected]
In vivo gait analysis in a mouse femur fracture model
Journal of Biomechanics 43 (2010) 3240–3243
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Short communication
In vivo gait analysis in a mouse femur fracture model T. Histing a,b,c,n,1, A. Kristen a,c,1, C. Roth d, J.H. Holstein a,b,c, P. Garcia a,b,c, R. Matthys e, M.D. Menger b,c, T. Pohlemann a,c a
Department of Trauma, Hand and Reconstructive Surgery, University of Saarland, D-66421 Homburg/Saar, Germany Institute for Clinical and Experimental Surgery, University of Saarland, D-66421 Homburg/Saar, Germany c CRC Homburg, Collaborative Research Center, AO Foundation, Switzerland d Departemt of Diagnostic and Interventional Neuroradiology, University of Saarland, Homburg/Saar, Germany e AO Development Institute, Davos, Switzerland b
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 24 July 2010
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. & 2010 Elsevier Ltd. All rights reserved.
Keywords: Gait analysis Mouse Femur fracture Fracture stabilization
1. Introduction
2. Material and methods
Gait analysis is a powerful technique to evaluate the motion patterns after surgery. In large animal models, gait analysis has provided a detailed basic understanding of locomotion and represents a common cross-species clinical tool that is sensitive to minor changes associated with disease, injury and rehabilitation. The mouse is a useful model for enhancing our understanding of fracture healing. Due to the increasing availability of specific antibodies and gene-targeted animals, murine models have become preferred tools also in fracture research (Jacenko and Olsen, 1995). However, there are no reports on gait analysis after fracture and stabilization in mice and rats. Herein, we introduce a novel method for gait analysis in mice and report changes to the motion pattern after femur fracture fixation.
2.1. Animals and surgical procedures All animal procedures were performed according to the NIH guidelines, for the use of experimental animals. Female CD-1 mice with a body weight (BW) of 30–40 g were used. Five days before surgery, all mice were given free access to a running wheel to become accustomed to walking in the wheel. Mice were anesthetized by intraperitoneal injection of xylazine (25 mg/kg BW) and ketamine (75 mg/kg BW). A 4 mm medial parapatellar incision was performed at the knee of the right hind leg to dislocate the patella laterally. After drilling a 0.5 mm hole into the intracondylar notch, a tungsten guide wire was inserted retrogradely into the intramedullary canal. Using a 3-point bending device, a closed midshaft femur fracture was produced. A common pin consisting of a 24-gauge injection needle of stainless steel (10 mm length, 0.55 mm diameter) was implanted over the guide wire to stabilize the fracture (n¼ 5). Subsequently, a second pin (5 mm, 0.55 mm) was inserted into the non-fractured tibia. Animals without fracture, which received pins in the femur and the tibia, served as controls (n¼5).
2.2. Radiological analysis
n
Corresponding author at: Department of Trauma, Hand and Reconstructive Surgery, University of Saarland, D-66421 Homburg/Saar, Germany. Tel.: + 49 6841 16 31502; fax: +49 6841 16 31503. E-mail address: [email protected] (T. Histing). 1 These authors contributed equally to this work. 0021-9290/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2010.07.019
After 14 days, gait analysis was performed by a digital video-radiography system (Axiom Artis, Siemens, Erlangen, Germany), using a radio-opaque running wheel. Three series of 600 images each were recorded for analysis. Multiple running cycles were digitized with 30 images/s. Eighteen representative strides per animal were analyzed using Osirix Imaging Software (Osirix Foundation, Geneva, Switzerland) and Image J (W. Rasband, NIH, Bethesda, Maryland, USA). Each stride of the mice was
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243 time-normalized. Cubic-spline interpolation was applied to raw data to obtain 11 images per stride. The normalized data were used to analyze the tibio-femoral angle.
3241
time is the inverse of the stride frequency and represents the time to complete one stride. The stride velocity was defined as stride length n stride frequency. 2.4. Statistics
2.3. Gait parameters Tibio-femoral angles, stride frequency, stride length, stride time and stride velocity were measured. The maximum and minimum tibio-femoral angles were given by the angle between the two implanted pins, representing the longitudinal bone axes of the femur and the tibia. A stride was defined as the first paw contact of the right hind leg with the ground to the following first ground contact of this paw. The stride frequency was calculated as 30 ðimages=secondÞ 30 strides ¼ x ðimages=strideÞ x second
3.1. Tibio-femoral angle
where x represents the number of images per stride. The stride length represents the spatial distance of the sequential contacts of the paw in one stride. The stride
tibio-femoral angle [°]
3. Results
Sham-treated non-fractured control mice showed a maximum tibio-femoral angle of 96.671.21 and a minimum tibio-femoral angle
120
60
100
50
*
80 60 40
tibio-femoral angle [°]
Stride frequency ¼
All data are given as means 7SEM. After proving the assumption for normal distribution and an equal variance, comparison between the experimental groups was performed by an unpaired Student’s t-test. A p-value o 0.05 was considered to indicate significant differences.
20
30 20 10
0
0 control
fracture
80
control
fracture
control
fracture
control
fracture
0.4
60
*
40
0.3 stride time [s]
stride length [mm]
40
20
0.2
0.1
0
0.0 control
fracture
350
5
stride velocity [mm/s]
stride frequency [1/s]
300 4 3 2 1
250 200 150 100 50 0
0 control
fracture
Fig. 1. Quantitative gait analysis, including the maximum tibio-femoral angle (A), the minimum tibio-femoral angle (B), the stride length (C), the stride time (D), the stride frequency (E) and the stride velocity (F). Gait analysis was performed at day 14 after pin stabilization in animals, which underwent femur fracture (white bars) or sham procedure (black bars). Data are given as means7 SEM; *p o 0.05 vs. control.
3242
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243
of 51.471.21 (Fig. 1). The mean of the range of motion, as defined by the tibio-femoral angle during the individual strides was 45.876.31. Fractured femora showed a significant reduction of the maximum tibio-femoral angle to 74.771.21 and a marked reduction of the minimum tibio-femoral angle to 41.671.31 (Fig. 1). This resulted in a significantly decreased range of the tibio-femoral angle during the individual strides (33.177.91). The reduced extension of the knee after fracture and stabilization could be observed over the entire time course of the individual strides (Fig. 2). 3.2. Stride characteristics Non-fractured controls showed stride length of 64.672.4 mm, a stride time of 0.2770.04 s and frequency of 4.170.5 strides/s. This resulted in a stride velocity of 266739 mm/s (Fig. 1). In fractured femora, the stride length was significantly reduced. Because stride frequency was slightly increased and, thus, stride time diminished, stride velocity was not significantly reduced compared to controls (Fig. 1).
4. Discussion
100 90 tibio-femoral angle [°]
We herein introduce a novel method which allows quantitative gait analysis after fracture, fracture stabilization and fracture healing in mice. Using this method, we demonstrate for the first time that femur fracture and stabilization in mice are associated with a lower range and maximum of the tibio-femoral angle, a significantly reduced stride length, but only a slightly diminished stride velocity. The reduced maximum tibio-femoral angle after fracture and stabilization may be the cause for the significantly reduced stride length. Stride velocity is controlled by stride length and stride frequency. After fracture and stabilization, the animals compensate the reduced stride length by a slight increase of stride frequency, resulting in a stride velocity not significantly different from that of controls. Gait analysis has been successfully applied to different animal species, especially to large animals (Muybridge, 1979). To gain deeper insights into the molecular mechanisms of diseases, murine models have become of major interest in biomedical research. This is based on the possibilities of genetic targeting and the wide availability of distinct antibodies. By consequence, gait analysis studies have been introduced in rats to describe normal locomotion (Clarke and Parker, 1986), pain adaptations (Gabriel et al., 2007) and functional deficits after nerve injury (Wang et al., 2008). In mice, only few studies have examined the gait, analyzing characteristics in healthy animals (Clarke and Still, 1999, 2001) as well as functional deficits in arthritic mice (Williams et al., 1993) and SOD1-transgenic mice (Wooley et al., 2005). We herein demonstrate now for the first time distinct alterations in the gait of mice after femur fracture and stabilization. In humans and large animal models, it is well known that fracture healing is strongly influenced by mechanical forces and, thus, the stability of fracture fixation. Depending on the fixation techniques, differences of interfragmentary movements result in different healing rates. Motion at the site of injury induces formation of cartilaginous callus. In contrast, highly stabilized fractures induce intramembranous ossification (Thompson et al., 2002). In sheep, ground reaction forces are strongly related to callus mineralization, and, thus, reflect the recovery of stiffness at the fracture site (Seebeck et al., 2005). The transfer of the fracture model and the gait analysis into the mouse system may now allow the study of the role of mechanical forces, after fracture stabilization, on molecular mechanisms of healing and functional recovery.
80 70 60 50 40 30 subsequent images over the individual stride
Fig. 2. Video-radiographic imaging of a stride (A–C) and quantitative analysis of the tibio-femoral angle during the stride cycle (D). Data are given as means7 SEM for animals after fracture and stabilization (white circles) as well as for sham controls (black circles).
Previous studies have shown that physical activity enhances the callus size and influences the stability of the newly formed bone (Sarmiento et al., 1977). Thus, fracture treatment should aim at an
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243
early recovery of normal physical activity. We have used a conventional pin for fracture stabilization, which does not provide rotational stability (Manigrasso and OConnor, 2004; Histing et al., 2009; Holstein et al., 2008; Garcia et al., 2008). The delayed recovery of normal motion observed is, thus, most probably due to the lack of rotational stability. By roughly assessing the behavioural aspects of the animals, Cheung et al. (2003) and Holstein et al. (2007) reported that mice can resume their normal activity levels within 2–5 days after stable fracture fixation. However, the rough assessment of behavioural aspects of the animals in the present study also showed normal activity levels after 5 days. The fact that gait analysis could demonstrate a markedly affected locomotion still after 14 days indicates that rough inspection of the animals’ behaviour is not appropriate to adequately assess the animals’ well-being. The technique of gait analysis presented herein has also some limitations. Marking of the femur and the tibia by pins was necessary to exactly determine the bone axes by digital videoradiography. Pin implantation induces a minor trauma, which may affect the gait of the animals. In addition, those pins cannot be used in case of plate or an external fixator stabilization. However, this problem can be overcome by using radio-opaque markers placed into the intramedullary cavity or inserted into the cortical bone. Alternatively, a high resolution digital videoradiography system may be used, allowing the determination of the bone axes without radio-opaque markers. In addition, commercially available foils for measurements of ground reaction forces may be incorporated into the ground of the running wheel. During the last few years, a considerable number of implants for fracture stabilization in mice have been developed, including locking nails (Holstein et al., 2007), intramedullary screws (Holstein et al., 2008), locking plates (Histing et al., 2010), pinclip devices (Garcia et al., 2008) and external fixators (Cheung et al., 2003). Our results indicate that further studies are required to characterize the effect of these different osteosynthesis techniques on the mouse gait and motion pattern recovery.
Conflict of interest statement All authors have no affiliation or financial arrangement with an organization or company that has financial interests in the subject matter discussed in the manuscript.
3243
References Cheung, K., Kalurachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K.S., 2003. An external fixed femoral fracture model for mice. Journal of Orthopaedic Research 21, 685–690. Clarke, K.A., Parker, A.J., 1986. A quantitative study of normal locomotion in the rat. Physiology and Behavior 38, 345–351. Clarke, K.A., Still, J., 1999. Gait analysis in mouse. Physiology and Behavior 66, 723–729. Clarke, K.A., Still, J., 2001. Development and consistency of gait in the mouse. Physiology and Behavior 73, 159–164. Gabriel, A.F., Marcus, M.A., Honig, W.M., Walenkamp, G.H., Joosten, E.A., 2007. The CatWalk method: a detailed analysis of behavioral changes after acute inflammatory pain in the rat. Journal of Neuroscience Methods 163, 9–16. Garcia, P., Holstein, J.H., Histing, T., Burkhardt, M., Culemann, U., Pizanis, A., Wirbel, R.J., Pohlemann, T., Menger, M.D., 2008. A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing. Journal of Biomechanics 41, 1689–1696. Histing, T., Holstein, J.H., Garcia, P., Matthys, R., Kristen, A., Claes, L., Menger, M.D., Pohlemann, T., 2009. Ex vivo analysis or rotational stiffness of different osteosynthesis techniques in mouse femur fracture. Journal of Orthopaedic Research 27, 1152–1156. Histing, T., Garcia, P., Matthys, R., Leidinger, M., Holstein, J.H., Kristen, A., Pohlemann, T., Menger, M.D., 2010. An internal locking plate to study intramembranous bone healing in a mouse femur fracture model. Journal of Orthopaedic Research 28, 397–402. Holstein, J.H., Matthys, R., Histing, T., Becker, S.C., Fiedler, M., Garcia, P., Meier, C., Pohlemann, T., Menger, M.D., 2008. Development of a stable closed femoral fracture model in mice. Journal of Surgical Research 153, 71–75. Holstein, J.H., Menger, M.D., Culemann, U., Meier, C., Pohlemann, T., 2007. Development of a locking femur nail for mice. Journal of Biomechanics 40, 215–219. Jacenko, O., Olsen, B.R., 1995. Transgenic mouse models in studies of skeletal disorders. Journal of Rheumatology 43, 39–41. Manigrasso, M.B., OConnor, P.J., 2004. Characterization of closed femur fracture model in mice. Journal of Orthopaedic Trauma 18, 687–695. Muybridge, E., 1979. Human and Animal Locomotion. Dover Publications, New York. Sarmiento, A., Schaeffer, J.F., Beckerman, L., Latta, L.L., Enis, J.E., 1977. Fracture healing in rat femora as affected by functional weight-bearing. Journal of Bone and Joint Surgery (American Volume) 59, 369–375. Seebeck, P., Thompson, M.S., Parwani, A., Taylor, W.R., Schell, H., Duda, G.N., 2005. Gait evaluation: a tool to monitor bone healing? Clinical Biomechanics 20 883–891. Thompson, Z., Miclau, T., Hu, D., Helmes, J.A., 2002. A model for intramembranous ossification during fracture healing. Journal of Orthopaedic Research 20, 1091–1098. Wang, H., Spinner, R.J., Sorenson, E.J., Windebank, A.J., 2008. Measurement of forelimb function by digital video motion analysis in rat nerve transection models. Journal of the Peripheral Nervous System 13, 92–102. Williams, J.M., Zurawski, J., Mikecz, K., Glant, T.T., 1993. Functional assessment of joint use in experimental inflammatory murine arthritis. Journal of Orthopaedic Research 11, 172–180. Wooley, C.M., Sher, R.B., Kale, A., Frankel, W.N., Cox, G.A., Seburn, K.L., 2005. Gait analysis detects early changes in transgenic SOD1 (G93A) mice. Muscle & Nerve 32, 43–50.
Contents lists available at ScienceDirect
Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Short communication
In vivo gait analysis in a mouse femur fracture model T. Histing a,b,c,n,1, A. Kristen a,c,1, C. Roth d, J.H. Holstein a,b,c, P. Garcia a,b,c, R. Matthys e, M.D. Menger b,c, T. Pohlemann a,c a
Department of Trauma, Hand and Reconstructive Surgery, University of Saarland, D-66421 Homburg/Saar, Germany Institute for Clinical and Experimental Surgery, University of Saarland, D-66421 Homburg/Saar, Germany c CRC Homburg, Collaborative Research Center, AO Foundation, Switzerland d Departemt of Diagnostic and Interventional Neuroradiology, University of Saarland, Homburg/Saar, Germany e AO Development Institute, Davos, Switzerland b
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 24 July 2010
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. & 2010 Elsevier Ltd. All rights reserved.
Keywords: Gait analysis Mouse Femur fracture Fracture stabilization
1. Introduction
2. Material and methods
Gait analysis is a powerful technique to evaluate the motion patterns after surgery. In large animal models, gait analysis has provided a detailed basic understanding of locomotion and represents a common cross-species clinical tool that is sensitive to minor changes associated with disease, injury and rehabilitation. The mouse is a useful model for enhancing our understanding of fracture healing. Due to the increasing availability of specific antibodies and gene-targeted animals, murine models have become preferred tools also in fracture research (Jacenko and Olsen, 1995). However, there are no reports on gait analysis after fracture and stabilization in mice and rats. Herein, we introduce a novel method for gait analysis in mice and report changes to the motion pattern after femur fracture fixation.
2.1. Animals and surgical procedures All animal procedures were performed according to the NIH guidelines, for the use of experimental animals. Female CD-1 mice with a body weight (BW) of 30–40 g were used. Five days before surgery, all mice were given free access to a running wheel to become accustomed to walking in the wheel. Mice were anesthetized by intraperitoneal injection of xylazine (25 mg/kg BW) and ketamine (75 mg/kg BW). A 4 mm medial parapatellar incision was performed at the knee of the right hind leg to dislocate the patella laterally. After drilling a 0.5 mm hole into the intracondylar notch, a tungsten guide wire was inserted retrogradely into the intramedullary canal. Using a 3-point bending device, a closed midshaft femur fracture was produced. A common pin consisting of a 24-gauge injection needle of stainless steel (10 mm length, 0.55 mm diameter) was implanted over the guide wire to stabilize the fracture (n¼ 5). Subsequently, a second pin (5 mm, 0.55 mm) was inserted into the non-fractured tibia. Animals without fracture, which received pins in the femur and the tibia, served as controls (n¼5).
2.2. Radiological analysis
n
Corresponding author at: Department of Trauma, Hand and Reconstructive Surgery, University of Saarland, D-66421 Homburg/Saar, Germany. Tel.: + 49 6841 16 31502; fax: +49 6841 16 31503. E-mail address: [email protected] (T. Histing). 1 These authors contributed equally to this work. 0021-9290/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2010.07.019
After 14 days, gait analysis was performed by a digital video-radiography system (Axiom Artis, Siemens, Erlangen, Germany), using a radio-opaque running wheel. Three series of 600 images each were recorded for analysis. Multiple running cycles were digitized with 30 images/s. Eighteen representative strides per animal were analyzed using Osirix Imaging Software (Osirix Foundation, Geneva, Switzerland) and Image J (W. Rasband, NIH, Bethesda, Maryland, USA). Each stride of the mice was
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243 time-normalized. Cubic-spline interpolation was applied to raw data to obtain 11 images per stride. The normalized data were used to analyze the tibio-femoral angle.
3241
time is the inverse of the stride frequency and represents the time to complete one stride. The stride velocity was defined as stride length n stride frequency. 2.4. Statistics
2.3. Gait parameters Tibio-femoral angles, stride frequency, stride length, stride time and stride velocity were measured. The maximum and minimum tibio-femoral angles were given by the angle between the two implanted pins, representing the longitudinal bone axes of the femur and the tibia. A stride was defined as the first paw contact of the right hind leg with the ground to the following first ground contact of this paw. The stride frequency was calculated as 30 ðimages=secondÞ 30 strides ¼ x ðimages=strideÞ x second
3.1. Tibio-femoral angle
where x represents the number of images per stride. The stride length represents the spatial distance of the sequential contacts of the paw in one stride. The stride
tibio-femoral angle [°]
3. Results
Sham-treated non-fractured control mice showed a maximum tibio-femoral angle of 96.671.21 and a minimum tibio-femoral angle
120
60
100
50
*
80 60 40
tibio-femoral angle [°]
Stride frequency ¼
All data are given as means 7SEM. After proving the assumption for normal distribution and an equal variance, comparison between the experimental groups was performed by an unpaired Student’s t-test. A p-value o 0.05 was considered to indicate significant differences.
20
30 20 10
0
0 control
fracture
80
control
fracture
control
fracture
control
fracture
0.4
60
*
40
0.3 stride time [s]
stride length [mm]
40
20
0.2
0.1
0
0.0 control
fracture
350
5
stride velocity [mm/s]
stride frequency [1/s]
300 4 3 2 1
250 200 150 100 50 0
0 control
fracture
Fig. 1. Quantitative gait analysis, including the maximum tibio-femoral angle (A), the minimum tibio-femoral angle (B), the stride length (C), the stride time (D), the stride frequency (E) and the stride velocity (F). Gait analysis was performed at day 14 after pin stabilization in animals, which underwent femur fracture (white bars) or sham procedure (black bars). Data are given as means7 SEM; *p o 0.05 vs. control.
3242
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243
of 51.471.21 (Fig. 1). The mean of the range of motion, as defined by the tibio-femoral angle during the individual strides was 45.876.31. Fractured femora showed a significant reduction of the maximum tibio-femoral angle to 74.771.21 and a marked reduction of the minimum tibio-femoral angle to 41.671.31 (Fig. 1). This resulted in a significantly decreased range of the tibio-femoral angle during the individual strides (33.177.91). The reduced extension of the knee after fracture and stabilization could be observed over the entire time course of the individual strides (Fig. 2). 3.2. Stride characteristics Non-fractured controls showed stride length of 64.672.4 mm, a stride time of 0.2770.04 s and frequency of 4.170.5 strides/s. This resulted in a stride velocity of 266739 mm/s (Fig. 1). In fractured femora, the stride length was significantly reduced. Because stride frequency was slightly increased and, thus, stride time diminished, stride velocity was not significantly reduced compared to controls (Fig. 1).
4. Discussion
100 90 tibio-femoral angle [°]
We herein introduce a novel method which allows quantitative gait analysis after fracture, fracture stabilization and fracture healing in mice. Using this method, we demonstrate for the first time that femur fracture and stabilization in mice are associated with a lower range and maximum of the tibio-femoral angle, a significantly reduced stride length, but only a slightly diminished stride velocity. The reduced maximum tibio-femoral angle after fracture and stabilization may be the cause for the significantly reduced stride length. Stride velocity is controlled by stride length and stride frequency. After fracture and stabilization, the animals compensate the reduced stride length by a slight increase of stride frequency, resulting in a stride velocity not significantly different from that of controls. Gait analysis has been successfully applied to different animal species, especially to large animals (Muybridge, 1979). To gain deeper insights into the molecular mechanisms of diseases, murine models have become of major interest in biomedical research. This is based on the possibilities of genetic targeting and the wide availability of distinct antibodies. By consequence, gait analysis studies have been introduced in rats to describe normal locomotion (Clarke and Parker, 1986), pain adaptations (Gabriel et al., 2007) and functional deficits after nerve injury (Wang et al., 2008). In mice, only few studies have examined the gait, analyzing characteristics in healthy animals (Clarke and Still, 1999, 2001) as well as functional deficits in arthritic mice (Williams et al., 1993) and SOD1-transgenic mice (Wooley et al., 2005). We herein demonstrate now for the first time distinct alterations in the gait of mice after femur fracture and stabilization. In humans and large animal models, it is well known that fracture healing is strongly influenced by mechanical forces and, thus, the stability of fracture fixation. Depending on the fixation techniques, differences of interfragmentary movements result in different healing rates. Motion at the site of injury induces formation of cartilaginous callus. In contrast, highly stabilized fractures induce intramembranous ossification (Thompson et al., 2002). In sheep, ground reaction forces are strongly related to callus mineralization, and, thus, reflect the recovery of stiffness at the fracture site (Seebeck et al., 2005). The transfer of the fracture model and the gait analysis into the mouse system may now allow the study of the role of mechanical forces, after fracture stabilization, on molecular mechanisms of healing and functional recovery.
80 70 60 50 40 30 subsequent images over the individual stride
Fig. 2. Video-radiographic imaging of a stride (A–C) and quantitative analysis of the tibio-femoral angle during the stride cycle (D). Data are given as means7 SEM for animals after fracture and stabilization (white circles) as well as for sham controls (black circles).
Previous studies have shown that physical activity enhances the callus size and influences the stability of the newly formed bone (Sarmiento et al., 1977). Thus, fracture treatment should aim at an
T. Histing et al. / Journal of Biomechanics 43 (2010) 3240–3243
early recovery of normal physical activity. We have used a conventional pin for fracture stabilization, which does not provide rotational stability (Manigrasso and OConnor, 2004; Histing et al., 2009; Holstein et al., 2008; Garcia et al., 2008). The delayed recovery of normal motion observed is, thus, most probably due to the lack of rotational stability. By roughly assessing the behavioural aspects of the animals, Cheung et al. (2003) and Holstein et al. (2007) reported that mice can resume their normal activity levels within 2–5 days after stable fracture fixation. However, the rough assessment of behavioural aspects of the animals in the present study also showed normal activity levels after 5 days. The fact that gait analysis could demonstrate a markedly affected locomotion still after 14 days indicates that rough inspection of the animals’ behaviour is not appropriate to adequately assess the animals’ well-being. The technique of gait analysis presented herein has also some limitations. Marking of the femur and the tibia by pins was necessary to exactly determine the bone axes by digital videoradiography. Pin implantation induces a minor trauma, which may affect the gait of the animals. In addition, those pins cannot be used in case of plate or an external fixator stabilization. However, this problem can be overcome by using radio-opaque markers placed into the intramedullary cavity or inserted into the cortical bone. Alternatively, a high resolution digital videoradiography system may be used, allowing the determination of the bone axes without radio-opaque markers. In addition, commercially available foils for measurements of ground reaction forces may be incorporated into the ground of the running wheel. During the last few years, a considerable number of implants for fracture stabilization in mice have been developed, including locking nails (Holstein et al., 2007), intramedullary screws (Holstein et al., 2008), locking plates (Histing et al., 2010), pinclip devices (Garcia et al., 2008) and external fixators (Cheung et al., 2003). Our results indicate that further studies are required to characterize the effect of these different osteosynthesis techniques on the mouse gait and motion pattern recovery.
Conflict of interest statement All authors have no affiliation or financial arrangement with an organization or company that has financial interests in the subject matter discussed in the manuscript.
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