dissection of the 5th lumbar vertebra (l5) from the mouse carcass for uniaxial compression testing

Compression Testing of Mouse Vertebra for Determination of Mechanical Strength

Age-related bone changes are widely recognized as problematic due to the increased risk of bone fractures associated with these changes. Bone fractures in humans can lead to chronic pain, reduced mobility, long-term disability, an increased risk of death, and economic burdens1. Common therapies investigated to address the symptoms of age-related bone changes include dietary supplements, hormone treatments, and drugs2,3,4,5,6,7,8,9. Initial investigations of such treatments for human subjects are commonly done using small animal models (e.g., laboratory rats and mice), which possess the two major types of bones found in the human skeleton10. Appendicular long bones, such as the humerus, femur, and tibia, are rich in cortical (i.e., compact) bone, whereas vertebrae are rich in cancellous bone (i.e., woven, spongy, or trabecular bone)4. There is growing knowledge that the mechanisms of bone regulation and signaling pathways differ between cortical bone (e.g., long bone mid-diaphysis) and cancellous bone (e.g., vertebral centrum)2. Because of this, therapies may have differential effects that are bone-specific or even site-specific within the same bone2,3,4.
The application of force to an object (e.g., bone) causes the object to undergo acceleration, deformation, or both, depending on the object&#39;s boundary conditions. When the bone is constrained, an opposite force of equal magnitude resists the acceleration of the bone, and deformation occurs. As the bone sustains deformation, internal resistance called stress is generated, of which there are two basic types: Normal force, in the form of tension or compression, and shear force10. Often, a combination of the basic types of stress is generated, depending on the applied force system10. The strength of a material is its ability to withstand stress without failing. As increasingly larger forces are applied to a material, it eventually undergoes permanent deformation, at which point it is said to have transitioned from an elastic state (i.e., will return to its original shape if the force is removed) to a plastic state (i.e., will not return to its original shape if the force is removed)11. The point at which the transition from an elastic state to a plastic state occurs is called the yield point. As even larger forces are applied to the material beyond the yield point, it increasingly sustains microfractures (i.e., damage) until total fracture occurs; at this point, the material is said to have failed11,12. The fracture of a bone represents a failure at both a structural level and a tissue level10. As an example, the breakage of a vertebral bone happens because not only do multiple trabeculae fail at a structural level, but there&#39;s also a failure of extracellular matrix elements like collagen and hydroxyapatite crystals in an individual trabecula at the tissue level.
The mechanical events leading up to the failure of a material can be measured using a variety of testing methods. Three-point bending is a common method for testing the mechanical properties of long bones from the appendicular skeleton. This method is simple and reproducible, making it the preferred method of biomechanical testing for many researchers13. By lowering a crosshead beam onto the mid-diaphysis of a long bone resting on two lower support beams, this method specifically tests the mechanical properties of the mid-diaphysis region, which is densely organized cortical bone. From load-displacement curves, tensile force effects on elasticity, toughness, force to failure, and the transition from elastic to plastic behavior of bone materials, among other properties, can be determined.
In the second type of bone, referred to as trabecular, spongy, woven, or cancellous bone, bone elements are formed into an array of rods and beams called trabeculae, giving a &#34;spongy&#34; appearance. The main vertebral bodies (i.e., centra) are rich in cancellous bone and are often the sites of age-related compression bone fractures in humans14. Lumbar (i.e., lower back) vertebrae are the largest vertebrae, bear most of the body&#39;s weight, and are the most common site for vertebral fractures15,16. The mechanical properties of vertebral bodies can best be directly assessed using uniaxial compression testing methods since axial compression is the normal force load imposed on vertebral columns in vivo17. Compression of the vertebral bodies in vivo occurs as a result of muscle and ligament contractions, the force of gravity, and ground reaction forces18.
Ex vivo compression testing of small animal vertebra can be difficult due to their small size, irregular shape, and fragility. The shape of vertebral bodies can be estimated as a parallelogram with mild ventral tilt and slight cranial concavity17. This shape presents challenges for achieving uniaxial compression testing ex vivo because, without adequate preparation to the loading surface, compressive forces will be applied to only part of the loading surface, resulting in a &#34;local contact&#34;17,19. This can cause inconsistent results and premature failure19. This is not the case in vivo because the loading surface is surrounded by intervertebral discs at the vertebral joints, which allows the load to be distributed throughout the cranial end plate. The intervertebral disc-cranial end plate complex plays an important role in the application of force throughout the vertebral body and the biomechanics of fracture to the vertebral body14,20. While compression testing is not new to the field of biology, there are limitations in the current methods of mechanical testing of bones. These limitations include the lack of predictor models and simulations for bone mechanics, unique geometric spatial architecture, and even inherent sample-based biological variations21. More importantly, the field is challenged by a lack of standardization between methods and an overall lack of reported methods in the literature22.
There are two methods reported in the literature for the preparation of rodent lumbar vertebrae to achieve uniaxial compression testing: the cutting method and the embedding method17,19,23,24,25,26. The cutting method requires that the vertebral processes, cranial end plate, and caudal end plate are cut from the vertebral body. Pendleton et al.19 have previously reported a detailed method for the use of this method on mouse lumbar vertebrae. This method presents the challenges of achieving perfectly parallel cuts at both the caudal and cranial end plates while also avoiding any damage to the sample. It also has the limitation that the cranial end plate is removed. The cranial end plate contains a dense shell of cortical bone and plays an important role in distributing loads from the intervertebral discs in vivo and is involved in the failure of the bone for in vivo fractures17,20,27. In contrast, the embedding method involves removing the vertebral processes while keeping the cranial end plate of the vertebral body intact. The loading surface is then made approximately horizontal by placing a small amount of bone cement onto the cranial end of the vertebral body. This method has the advantage that it overcomes the technical challenges associated with the cutting method and may better mimic the mechanism of load application and bone failure in vivo due to the preservation of the cranial end plate. This approach has previously been documented in studies involving uniaxial compression testing on rat bones. However, as far as we are aware, it has not been previously documented in the context of smaller mouse lumbar vertebrae17,25,26. The method in question was previously detailed by Chachra et al.25 &#160; and originally used a bone specimen held in between two plates, each with a cylindrical cavity, which was then filled with polymethylmethacrylate (PMMA). The same research group later improved the method where one end is gently sanded (caudal), and the other end has a small spot of bone cement added (cranial)26. This method is an improvement on the previous method because it minimizes the material between the platens and is the focus of this article. Despite the challenges associated with uniaxial vertebral compression testing, it is a method that may provide valuable information regarding the effects of a proposed therapy on bone, especially when paired with three-point bending.
Here, the use of a convertible three-point bending/compression testing machine to allow for easy testing of both long bones and vertebral bodies using a single machine is presented. Furthermore, the use of an embedding method to achieve uniaxial compression testing of mouse lumbar vertebrae is presented. The present study was performed as part of a larger study that aimed to investigate the influences of dietary hempseed supplementation on the properties of skeletal bone in young, growing female C57BL/6 mice5,6. The three-point bending tester was originally constructed by faculty and students in the Engineering Dept. at Colorado State University-Pueblo and used by our research group in three-point bending tests on long bones [rat femur and tibia7 and mouse humerus, femur, and tibia5,6,8,9]. However, its modification and application for use in mouse vertebral body compression testing was&#160;not been explored. The design and construction of the three-point bending machine have been previously described7. This report will focus on methods used to modify the machine for compression testing and to correct for system displacement. Secondly, the embedding method for mouse vertebral body loading surface preparation is described, along with methods for uniaxial compression testing and the analysis of load-displacement data.

Author Spotlight: Enhancing Small Animal Bone Compression Testing for Research

Mouse Lumbar Vertebra Uniaxial Compression Testing with Embedding of the Loading Surface

Our research focuses on mechanical strength testing of small animal bones. Specifically, we aim to present an embedding method for compression testing of lumbar vertebrae. Applications for this research area include a better understanding of aging and dietary influences on vertebral bone mechanical properties.Inherently, the small size of the vertebra is a challenge for compression testing, but the embedding method improves accessibility because the main preparation is to remove only the vertebral processes from the main body, but leaving other structures intact. The small size of mouse vertebra makes them challenging to prepare for compression testing. Our method provides a simpler means of preparing the loading surface for uniaxial compression testing.Furthermore, we present the construction of compression testing machine from a three-point bending machine for easy conversion between the two. Our method allows researchers a simple means of pairing three-point bending and compression testing, which may allow researchers to better discern the effects of pharmacologic therapy, dietary intervention, or other treatments on a cortical-rich versus cancellous-rich bone in a rodent model. We are exploring compression testing for other types of biological specimens.For example, we are developing methods for testing mouse mandibles combined with other bone evaluation techniques. This will enable a more comprehensive approach to studying factors that influence bone properties, such as aging and diet.

Watch this Scientific Journal Video about Dissection of the 5th Lumbar Vertebra L5 from the Mouse Carcass for Uniaxial Compression Testing at JoVE.com

Dissection of the 5th Lumbar Vertebra (L5) from the Mouse Carcass for Uniaxial Compression Testing

To begin, make a small incision on the dorsal midline of the mouse carcass near the base of the tail. Extend the incision across each hind limb and gently pull to remove the pelt from the base of the tail toward the head of the animal. Cut the abdominal wall musculature away until the vertebral column becomes easily visible.Under a dissecting microscope, identify the two sacroiliac joints and locate the cranial end of the sacrum. Using a razor blade or scalpel, make a precise cut to separate the last lumbar vertebrae from the cranial end of the sacrum. Again, cut between the intervertebral space.Remove L six and L five from the vertebral column and set aside L five for analysis. Inspect the vertebra under a dissecting microscope and remove all soft tissues from the bone, including the intervertebral disc using gauze pads and gently with forceps where necessary.

dissection-5th-lumbar-vertebra-l5-from-mouse-carcass-for-uniaxial

Research

JoVE Journal

Biology

243 Views.  Colorado State University-Pueblo. To begin, make a small incision on the dorsal midline of the mouse carcass near the base of the tail. Extend the incision across each hind limb and gently pull to remove the pelt from the base of the tail toward the head of the animal. Cut the abdominal wall musculature away until the vertebral column becomes easily visible.Under a dissecting microscope, identify the two sacroiliac joints and locate the cranial end of the sacrum. Using a razor blade or scalpel, make a precise cut to se...

Video: Dissection of the 5th Lumbar Vertebra (L5) from the Mouse Carcass for Uniaxial Compression Testing

There is increasing awareness that cortical and cancellous bone differ in regulating and responding to pharmaceutical therapies, hormone therapies, and other treatments for age-related bone loss. Three-point bending is a common method used to assess the influence of a treatment on the mid-diaphysis region of long bones, which is rich in cortical bone. Uniaxial compression testing of mouse vertebrae, though capable of assessing bones rich in cancellous bone, is less commonly performed due to technical challenges. Even less commonly performed is the pairing of three-point bending and compression testing to determine how a treatment may influence a long bone's mid-diaphysis region and a vertebral centrum similarly or differently. Here, we describe two procedures to make compression testing of mouse lumbar vertebrae a less challenging method to perform in parallel with three-point bending: first, a procedure to convert a three-point bending machine into a compression testing machine, and second, an embedding method for preparing a mouse lumbar vertebra loading surface.

In this protocol, two approaches are described to make uniaxial compression testing of mouse lumbar vertebrae more attainable. First, the conversion of a three-point bending machine to a compression testing machine is described. Second, an embedding method for preparing the loading surface that uses bone cement is adapted for mouse lumbar vertebrae.

There is increasing awareness that cortical and cancellous bone differ in regulating and responding to pharmaceutical therapies, hormone therapies, and ...

Conversion of a Three-Point Bending Machine to a Compression Testing Machine

To begin unscrew the crosshead beam attached to the load sensor on the three-point bending machine. Screw a self-aligning top platin onto the load sensor with threading identical to the crosshead beam. Turn on the machine at a constant lowering speed to begin compression testing.Using the threaded hex screws, secure the bottom platin to the two lower supports, and tighten them until they are securely fastened. Then to determine machine displacement, lower the top platin onto the bottom platin, until light contact is made with no test material in between. Use digital data collection software to collect load and displacement measurements during the mechanical testing.Continue lowering the top platin onto the bottom platin at a constant speed until forces greater than that produced by all bone samples are reached. Plot the data for system displacement versus applied load, correct the recorded displacement for each data point from mouse vertebra samples obtained in subsequent steps.

Preparing L5 Vertebra Loading Surface for Uniaxial Compression Testing Using the PMMA Bone Cement Embedding Method

To begin, dissect the fifth lumbar vertebra isolated from the frozen mouse carcass. Using a diamond cutoff wheel attached to a rotary tool, cut each pedicle to remove the transverse and spinous processes. Gently sand the coddle end of the vertebrae using fine 120-grit sandpaper to eliminate all intervertebral discs, soft tissue, and irregularities.Then mark the sanded coddle end with a permanent marker for easy identification later. Prepare the PMMA bone cement according to the manufacturer's instructions. Apply a small amount of the semi-soft PMMA bone cement onto the upper side of the unmarked cranial end of the vertebra and rehydrate with saline.While the PMMA is still semi-soft, position the vertebra on the bottom platin with the coddle side facing downwards. Now turn on the machine to engage the drive gears and gradually lower the top platin onto the vertebra and PMMA bone cement complex until it contacts the bone cement. Allow the PMMA bone cement to completely harden while the top platin gently presses down on it.In real time, use digital software designed for data collection of mechanical testing to collect data for load and displacement from the sensors into a spreadsheet. After collecting baseline data for five seconds, applied with a minimal preload force of less than 0.5 newtons. Gradually lower the top platin onto the sample at a constant and predetermined lowering speed, to initiate the compression test.Cease data collection, once a significant reduction in load has been observed indicating material failure. Correct each data point for system displacement, using the equation obtained in previous steps. To create a load displacement curve with the load on the Y axis and the corrected specimen displacement on the X axis.First, create a table by clicking on windows, then selecting new table, followed by do it"Copy the corrected displacement and load data from the raw data spreadsheet into the new table. Then to generate a waveform representing the raw data, click data and select XY pair to waveform. Select corrected displacement data for the X wave and load data for the Y wave, ensure the correct data points are in the number of points box.Name the wave form, and click make wave form. After creating the wave form, visualize the load displacement curve by clicking on windows and selecting new graph. Place the waveform on the Y axis and calculate it on the X axis to generate a graph.Use the cursor tool to mark points or regions of interest on the graph for further analysis.