Development of a New Device Implementing Standardized Closed Fracture in the Rat

The objective of the study was to produce a user-friendly device for creating standardized close fracture in an experimental animal. We describe a device that takes advantage of the elastic and plastic nature of the bone for producing a closed fracture with reproducible location and structure. Fractures were p roduced by a pendulum-like device applying an external blow directly over the tibiae of female Wistar rats fixed to a platform by dynamic holders. The tibiae were immediately stabilized by plaster fixation that was covered by dental wax. Rad iographically, this method resulted in a highly reproducible b i-cortical transverse fracture. Minimal soft tissue damage was noticed around the fracture site. Creatine phosphokinase activity in the serum and in the gastrocnemius muscle o f the posterior calf compartment was analyzed in order to evaluate the standardization of the soft tissue damage. The device is simple to build and can be modulated and adapted to different sizes of animals. By using this device, a highly reproducible closed fracture system can be implemented with minimal damage to local soft tissues, but still with the local hematoma that is essential for the fracture healing process.


Introduction
Creat ing an anima l model is crucial for understanding the cascade of events occurring follo wing a fracture in a long bone. Several approaches to creating closed fractures that imitate a similar fracture in the hu man long bone have been introduced in the last 20 years. (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) In those methods, the bone to be fractured is rigid ly fixed to the instrument and is injured by a pendulu m, grav ity mass, or a piston effect, creating an ext ramedullary force applied to the desired location that produces a three-point bending effect. The limitat ion of those approaches are that they do not truly reproduce the bio mechanical sequence of events taking p lace when a human long bone is fractured, because the rigid grasp of the bone limits the elastic and plastic characteristics of the bone In an attempt to better imitate the bio mechanical strain produced at the t ime o f a long bone fractu re, we h ave developed a device that creates a closed transverse fracture in a long bone of the rat in which the bone is being held by dynamic ho lders situ ated on a rotat ing p lat form. These holders allo w the bone to bend under the external force applied until the force overco mes the plastic quality o f the bone, resulting in a fracture. Moreover, the magnitude of the force of the blow can be adjusted and controlled. The innovation in our method is in exp loiting the elastic and plastic properties of the long bone in the environ ment of a closed fracture.

Materials and Methods
The device is composed of five basic components ( Fig. 1): (a) a plat form that supports the anima l wh ile the fracture is being produced; (b) a fra me supporting a moveable pendulum and an adjustable stopper that limits the depth of the blow to the bone; (c) a pendulum consisting of a steel pole along which a moveable weight glides and that is used to apply the external blo w. By changing the position of the weight, one can change the mass of the blow; (d) two specially designed dynamic cla mps, each located on a rotating platform that can be adjusted to the size of the limb; and (e) a protractor that enables fixing the pendulum at the angle desired. All co mponents are made of stainless steel.
Sixteen female Wistar rats were rando mly divided into two groups: 10 anima ls served as the experimental group and six as the control group. Animals were anesthetized by intraperitoneal inject ion of sodiu m phenobarbital (4mg/ 100gr body weight, Rhöne Merieu x Limited, Harlow, Essex, UK). (12) The left leg of each of the experimental rats was fixed to the device using the dynamic cla mps that held both ends of the anima l limb. An external b low using an equal swing angle of the pendulum was applied to the tibiae. In both groups, the left limbs were immed iately fixated in a cast covered by dental wax in order to prevent cast nibbling and breakage by the animals. Animals were free to move in their cage with full weight bearing throughout the entire experimental period, and both groups received the same diet. Radio logical and clinical macroscopic assessments were performed. Anima ls were killed 48 hours post-fracture by a high dose of intraperitoneal nembutal. The experimental protocol was approved by the Anima l Care and Use Co mmittee of the Technion Faculty of Medicine The gastrocnemius muscle was removed fro m the left leg of all animals, and creat ine phosphokinase (CPK) act ivity was measured in the muscle and in the serum harvested at the time o f sacrifice.(13) Fracture sites were d issected and their anatomic structure was explored.
Statistical analysis was performed by unpaired t-test. Significance level was set at p < 0.05.

Results
The device and its detailed elements are demonstrated in figure 1. In all animals, a closed fracture of the tib ia was produced. As demonstrated by radiography and proved by anatomical dissection, all fractures were transverse and uniform (Fig. 2). Minima l soft tissue damage was noticed around the fracture site, but no open wounds were observed. CPK activ ity, as anticipated, was significantly higher in the serum of the experimental anima ls than in the serum of the control rats that were not subjected to a fracture (Fig. 3). The values of CPK act ivity in the sera of experimental and control rats are given in Tables 1 and 2. As can be seen, the standard deviation in each group was relatively small. On the other hand, CPK level in the gastrocnemius muscle was reciprocally lower (p<0.0001) in the experimental rats than that in the controls, as a result of the lesion caused by the blow. The results are presented in Fig. 4 and Tables 3 and 4.

Discussion
The production of a standard closed fracture was accomplished using a simp le and rather inexpensive device that can be built in any laboratory. The device enables calculation of both the mo mentum and the total force applied on the limb, simp ly by knowing the weight at the distal pole of the pendulum and the angle to which it was elevated previous to the swing. As in human patients, the structure of the fracture and the extent of the soft tissue damage are related to the loading forces applied by the device.
Most of the methods producing fractures that have been described in the literature until now ma ke use of an open procedure, either by cutting the skin and muscles over the site of the bone to be fractured by cutting, (14) or by creating a closed fracture with devices that do not take in consideration the elastic and plastic properties of the bone. (1,3,4) In the device p resented here, the rotating platform clamps mimic the adjacent joints' movement that takes place at the time of fracture and enable the long bone to explore its mechanical properties. Moreover, internal fixation of the fracture through nailing (11,15,16) has the following disadvantages: a) it necessitates an additional cut; b) reduces the mechanical properties of a long bone, which necessitates an additional cut; c) creates a drainage pathway for the he mato ma initiated by the fracture. The hemato ma is a necessary preliminary step in the cascade of events during the process of fracture healing.
CPK en zy me activity in the serum of the treated animals showed a uniform and statistically significant increase of about 175% when compared with CPK enzy me act ivity in the control rats, as well as a decrease of 28% in the calf muscles. These results demonstrate that using the device of implementing a fracture as described in this paper produces a reproducible fracture pattern, as well as equal and minimal damage to the surrounding soft tissue around the fracture. Moreover, we suggest that the fracture caused by this device and the following healing process present a reliab le reproduction of the physiological healing process of closed fractures in the human patient.
In conclusion, our device is simple to build and might be modulated and adapted to different sizes of anima ls. By using this device, a highly reproducible closed fracture system can be introduced with min ima l damage to local soft tissues, but still with the local he matoma that is essential for the fracture healing process.