An improved murine femur fracture device for bone healing studies

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Abstract

Murine models are commonly used to investigate bone healing and test new treatments before human trials. Our objective was to design an improved murine femur fracture device and determine optimal mass and velocity settings for maximal likelihood of transverse fracture. Fracture reproducibility was maximized using an adjustable kinetic energy level, a novel mouse positioning system and an electromagnet striker release assembly. Sixty wild-type mice of 8–12-week-old male and female with a weight of 26.4±6.1 g were subjected to an experimental postmortem fracture in the left and right femur (n=120) using variable kinetic energy inputs. A best-fit prediction equation for transverse fracture was developed using multivariate linear regression. Transverse fracture was shown to correlate most highly with kinetic energy with a maximum likelihood at mv2=292 where m is mass (g) and v is velocity (m/s). Model validation with a group of 134 anesthetized C57BL/6 mice resulted in a favorable transverse fracture rate of 85.8%. Simple modifications to existing fracture devices can improve accuracy and reproducibility. The results may assist researchers studying the effects of genetic modifications and novel treatments on boney healing in murine femur fracture models. Maintaining kinetic energy parameters within suggested ranges may also aid in ensuring accuracy and reproducibility.

Introduction

Debilitating skeletal bone fractures occur over five million times each year in the United States alone (Holstein et al., 2007b). The increasing life expectancy of the population creates an ever-increasing risk of fracture. To study bone healing in a controlled manner, pre-clinical small animal models have been developed and utilized extensively. The original fracture models from the 1970s and 1980s used the rat (Bonnarens and Einhorn, 1984; Jackson et al., 1970), and its popularity has extended to present-day studies (Schmidmaier et al., 2004). More recently, mouse models have sparked great interest due to their well mapped genome and ability to delete or mutate specific genes (Baldik et al., 2005; Cheung et al., 2003; Hiltunen et al., 1993), which affords the opportunity to observe the effect of an individual gene in the healing process (Manigrasso and O’Connor, 2004).

Multiple bones have been studied in these models, with the most prevalent being the femur and tibia. Although the mouse tibia is easier to access than the femur, the tibia is not an ideal model for fracture studies because of its curved major axis that complicates mechanical testing and the minimal local soft tissue surrounding the bone (Holstein et al., 2007a; Manigrasso and O’Connor, 2004). Furthermore, the proximity of the fibula to the tibia may change healing rate if it accidentally fractures, which can occur at rates up to 30% (Thompson et al., 2002). For these reasons, the transgenic mouse femur is an exciting fracture model for modern orthopedic research.

The most widely utilized small animal fracture device is the Bonnarens and Einhorn (1984) rat tibia fracture device. Its simple gravity-driven three-point bending design is easy to construct, operate, and maintain. However, the device as originally described has several shortcomings for use with transgenic mice. First, while it is relatively simple to position the tibia of a rat centered between the two anvils, positioning the femur of a mouse is much more difficult due to the intra-torso location and small size. Another disadvantage of the device is the use of the reset spring, which experiences fatigue and may not create a reproducible fracture over time. Perhaps the most significant device limitation has been the absence of a systematic study to determine the ideal parameters for creating a transverse fracture in the mouse femur using a gravity-driven fracture device (Carmouche et al., 2005; Taguchi et al., 2005; Thompson et al., 2002). Taken together, these issues may result in undesirable fractures, which can be particularly detrimental when using transgenic mice that are typically difficult to obtain.

The goal of this work is to develop a device and method to improve quality and reproducibility of experimental fractures in a murine femur model. Using the results of this study, researchers will be able to more efficiently study the effects of genetic modifications and novel treatments on boney healing in murine femur fracture models.

Section snippets

Theoretical considerations

Based on physics fundamentals, fracture mechanics, and bone biomechanics, the following four device parameters were considered: impact mass, impact velocity, “gap” between two fracture anvils in three-point bending and “depth” that the striker is permitted to traverse past the top skin surface of the mouse femur.

The impact mass and velocity are related in the equation of kinetic energy, EK=12mv2. The effect of kinetic energy load on cortical bone has been directly related to fracture type in

Results

We found that the device performs in a reproducible manner, is simple to operate with a single user, and is capable of rapid adjustment. The shaft does not visually reverberate during impact of the thumbwheel and stop-block. Further, the cylindrical linear bearings eliminate rotation except when significant manual torque is applied.

The weight of the mice used to derive the prediction model ranged from 18 to 41 g with a mean of 26.4±6.1 g. A histogram of mouse weight is shown in Fig. 3. An example

Discussion

The specific objective of this study was to adapt the popular Bonnarens and Einhorn (1984) gravity-driven fracture device for use with the mouse model and to improve on its functionality in three areas: femur positioning, impact velocity consistency, and development of the ideal energy inputs for transverse fracture. Our results demonstrate that the device and method developed for this study produce reproducible transverse fractures of the mouse femur when the mass and velocity are selected

Conflict of interest statement

We, the authors hereby declare that individually and as a collective we have not acquired any personal, professional or financial conflicts of interest related to the work. To the best of our knowledge the work was completed with the highest level of objectivity and interpretations were free from bias. The results and conclusions of this work were reached knowing that their outcome would not have any personal, professional or financial effects on the authors or the institutions at which they

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1

Also at: Department of Orthopedic Surgery, University of Massachusetts Memorial Hospital, 55 Lake Avenue North, Worcester, MA 01655, USA.

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