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 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.
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'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'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 "spongy" 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'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 "local contact"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 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 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.
All experiments and protocols were conducted in compliance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and received approval from the Colorado State University-Pueblo Institutional Animal Care and Use Committee (Protocol Number: 000-000A-021). Detailed procedures for animal care have been previously described5,6. The mice were obtained at three weeks of age as part of a broader study aimed at investigating the effects of a hempseed-supplemented diet on young, growing female C57BL/6 mice (see Table of Materials). From 5 to 29 weeks of age, the mice were raised on one of three diets: control (0% hempseed), 50 g/kg (5%) hempseed, or 150 g/kg (15%) hempseed, with eight mice per group5,6. Throughout the study, mice had ad libitum access to their respective diets and water, were pair-housed in polycarbonate cages, and maintained on a 12 h light:12 h dark cycle (with lights on from 06:00 to 18:00 h). The mice's weight and health were assessed weekly, and all mice successfully completed the study without developing any adverse health conditions. At twenty-nine weeks of age, the mice were deeply anesthetized using isoflurane gas and euthanized via cervical dislocation5,6. A midline incision was made on the ventral surface from the sternum to the tail, and all intrathoracic, peritoneal, and retroperitoneal organs were removed from the carcasses. The eviscerated carcasses were preserved in 0.9% sodium chloride solution at -70 °C until the time of bone dissection for vertebra testing, which occurred approximately one year later.
1. Conversion of a three-point bending machine to a compression testing machine
2. Correcting for the displacement of the compression testing machine
3. Dissection of the 5 th lumbar vertebra (L5) from the mouse carcass
4. Preparing L5 vertebra loading surface for uniaxial compression testing using PMMA bone cement embedding method
5. Analysis of load-displacement curves for L5 vertebra uniaxial compression tests
With this step-by-step protocol that uses embedding of the L5 loading surface and a convertible three-point bending machine/compression testing machine, it is possible to perform compression testing on mouse lumbar vertebra for inter-group comparisons. A total of twenty-four mouse L5 vertebrae were prepared using the embedding method. Three of the samples, however, were damaged during the removal of the vertebral processes using a diamond cutoff wheel on a rotary tool and, thus, were not tested. Given this, the mechanical properties listed were successfully obtained from twenty-one of twenty-four samples using the embedding method. Specimens were visually inspected after each test, and the PMMA cap sustained no damage in any of the tests. As noted, the mice used in the present study were part of a larger study aiming to determine the effects of dietary hempseed on the bones of young and growing C57BL/6 female mice. Descriptive statistics of five commonly reported whole-bone mechanical properties are offered in Table 1. The load-displacement curves for all twenty-one samples are provided in Figure 7.
Figure 1: The conversion of a three-point bending machine to a compression testing machine. (A) The machine fully equipped to operate as a three-point bending machine with the displacement sensor and load sensor indicated (white arrows). (B) The machine after the crosshead beam has been removed. (C) The machine after a self-aligning top platen has been placed where the crosshead beam was previously placed. (D) The lower support beams with holes drilled into them. (E) The stainless-steel bottom platen with four threaded holes tapped into it, and a screw partially screwed into one of the holes. The other two holes not seen in the photo are on the opposite side. (F) The lower support beams with the bottom platen attached to them by four hex screws. Please click here to view a larger version of this figure.
Figure 2: An example system displacement (Δxmachine) vs. load plot fitted with a linear (A), logarithmic (B), second-order polynomial (C), and third-order polynomial (D) regression. In this example, the third-order polynomial provides the best fit per R2 value, and its regression is used as the system displacement correction factor. Images represent example data to demonstrate regression fitting and will need to be obtained by researchers for individual machines. Please click here to view a larger version of this figure.
Figure 3: Mouse lumbar vertebral column. A mouse lumbar vertebral column under a dissecting microscope before L6 was removed (A), and after L6 had been removed, leaving L5 attached (B). L5 will be subsequently removed and prepared for compression testing. The white colored bands are the intervertebral discs which were dissected and removed. Please click here to view a larger version of this figure.
Figure 4: Anatomy of L5 vertebra. A representative mouse L5 vertebra in cranial, caudal, dorsal, and ventral views under a dissecting microscope. Important dimensions for the vertebral body include height, dorsoventral width, and lateral width, as shown by the colored lines. The black dashed lines show approximately where cuts are to be made to remove the vertebral processes. Please click here to view a larger version of this figure.
Figure 5: Hardening period of PMMA bone cement. An example L5 vertebra with PMMA bone cement (green) placed on the cranial endplate and the top platen lowered onto the PMMA bone cement + bone complex. Once PMMA bone cement has fully hardened, the compression test will begin. The top platen will be further lowered until failure of the material is observed. Please click here to view a larger version of this figure.
Figure 6: Mouse vertebral bone compression test load-displacement curve and data analysis. Cursor A marks the start of the compression test. Cursor B marks the point of material failure. Cursor C marks the start of the linear elastic region, whereas cursor D marks the end (i.e., the yield point). The area shaded in light gray is the linear elastic region, where the material will return to its original shape if the load is removed. The area shaded dark gray is the plastic region, where the material has undergone permanent deformity and will not return to its original shape if the load is removed. Please click here to view a larger version of this figure.
Figure 7: Load-displacement curves for all twenty-one bone samples. Patterns varied between bones. In general, the greatest variability was in post-yield displacement, with a few (n = 5) of the bones having a relatively small post-yield displacement and others (n = 16) having a relatively large post-yield displacement. Please click here to view a larger version of this figure.
Group | Work-to-Failure (N*mm) | Maximum Load (N) | Stiffness (N/mm) | Yield Load (N) | Post-Yield Displacement (mm) |
CON (n = 7) | 13.43 ± 2.44 A,B | 37.93 ± 3.28 | 109.14 ± 11.86 | 22.68 ± 2.04 | 0.34 ± 0.06 |
5HS (n = 8) | 12.12 ± 1.23 A | 33.62 ± 2.43 | 99.70 ± 16.62 | 20.88 ± 2.69 | 0.38 ± 0.08 |
15HS (n = 6) | 19.55 ± 2.13 B | 41.82 ± 1.85 | 134.58 ± 19.73 | 28.07 ± 3.20 | 0.51 ± 0.07 |
Combined Groups (n = 21) | 14.68 ± 1.27 | 37.40 ± 1.63 | 121.82 ± 9.43 | 23.54 ± 1.60 | 0.40 ± 0.04 |
Table 1: Representative values for commonly reported whole-bone mechanical properties obtained using the loading surface preparation embedding method. Values were obtained using all protocols detailed in the present study. Thus, the values represent those that can be obtained using the methods described here. Values are means ± SEM. Groups represent C57BL/6 female mice fed a diet enriched with whole hempseed at concentrations of 0% (CON), 50 g/kg (5%) (5HS), or 150 g/kg (15%) (15HS) from ages 5-29 weeks. For one of the parameters (work-to-failure), it appears that the diet influenced the values per a one-way ANOVA (p < 0.05). Values sharing the same letter superscript are not significantly different (p > 0.05), while values with different letter superscripts are significantly different (p < 0.05), per Tukey-Kramer post hoc analysis.
Supplementary File 1: Example code to obtain whole-bone mechanical properties. Please click here to download this File.
The goal of the present study was to describe the construction of a convertible three-point bending machine/compression testing machine, as well as the use of a PMMA bone cement embedding method for the preparation of mouse lumbar vertebrae samples before uniaxial compression testing. Descriptive statistics were obtained and reported for the bone samples, which will be useful for comparison in future studies. Some of the most commonly reported whole-bone mechanical properties were analyzed in the present study. However, it's worth noting that there are several additional whole-bone and tissue-level mechanical properties that were not investigated here.
It remains unclear how the mechanical properties obtained from samples prepared using the embedding method compare to those prepared using the cutting method for mouse lumbar vertebrae. Schumancher17 previously assessed the mechanical properties of rat vertebrae prepared using the two different methods and found that vertebrae prepared using the embedding method had significantly lower stiffness, higher yield displacement, and higher yield strain than samples prepared using the cutting method. Further characterization is needed to understand how the vertebral mechanical properties of mice or other animal models compare when measured using the two different methods of loading surface preparation. It's expected that some parameters differ between vertebrae prepared using different methods, given that the embedding method adds material to the sample but preserves the end plate, which is an important structure in vertebral fractures in vivo17,27. The addition of bone cement to the cranial end adds height to the sample, whereas cutting the end plates removes height, altering the aspect ratio and thereby changing mechanical properties like stiffness. Furthermore, although PMMA is stiffer than vertebral cancellous bone, it's possible that the PMMA undergoes displacement, and the extent of this displacement needs further characterization. Additionally, it's unclear how the results obtained from either the embedding method or cutting method compare to predictions of bone parameters using finite element analysis for mouse vertebrae or how the results vary under different conditions (e.g., lowering speed, different vertebral levels, PMMA compositions). Nonetheless, because all specimens are prepared in a similar manner, this method is appropriate and allows for an easy and cost-effective means of making comparisons between treatment groups in a single study where samples are prepared and tested under similar conditions.
Regarding specimen preparation before compression testing, it's essential to prepare samples in a reproducible manner. One possible limitation of the method described in the present study is the use of a rotary tool to remove the vertebral processes. Another method to remove the vertebral processes of mouse lumbar vertebrae has been described by Pendleton et al.19, which may allow for more consistent sample preparation. Furthermore, inconsistencies may arise from the application of PMMA bone cement. Therefore, it's important to apply the bone cement consistently in terms of volume, placement, and hardening time. However, the embedding method may provide a simpler means of achieving consistent sample preparation compared to the cutting method, as it can be challenging to achieve perfectly even, parallel cuts consistently between all samples due to their small size and fragility. Future studies will be needed to assess the precision of results obtained from samples prepared using the embedding vs. cutting method.
As mentioned, further characterization and investigation of the embedding method for specimen preparation of mouse lumbar vertebrae before uniaxial compression testing are needed. Nonetheless, this study demonstrates that such a method can be employed, provides a detailed description of the proposed method, and offers descriptive statistics of the parameters measured from samples prepared using the method. This protocol is valuable to the field due to the current lack of available methodology. Moreover, this method may better mimic the mechanism by which in vivo vertebral fractures occur compared to other methods17,27. The method also has the advantage of overcoming the technical difficulties associated with other currently reported methods, making uniaxial compression testing more feasible in bone research. This is particularly significant because drugs, diets, or other interventions may influence cortical-rich bones (e.g., long bone mid-diaphysis) and trabecular-rich bones (e.g., vertebral bodies) differently, yet three-point bending is the predominant method to assess the mechanical properties of bones13. The combination of three-point bending and uniaxial compression testing can become even more easily achievable through the use of a convertible three-point bending/compression testing machine. Thus, the present study proposes two possible means of making the assessment of both cortical-rich and trabecular-rich bone in the same study more available to researchers, potentially leading to a better understanding of how a given treatment affects different bone types between experimental groups.
The authors have nothing to disclose.
We are grateful for the significant efforts that the Colorado State University-Pueblo Dept. of Engineering provided in constructing the three-point bending machine and its modification to a convertible three-point bending/compression testing machine. We are especially thankful to Mr. Paul Wallace, machine shop coordinator, for his efforts in planning and carrying out the construction and modification of the machine. Expertise and feedback from Dr. Bahaa Ansaf (Colorado State University-Pueblo, Dept. of Engineering) and Dr. Franziska Sandmeier (Colorado State University-Pueblo, Dept. of Biology) also significantly contributed to this project. The Institute of Cannabis Research Grant at Colorado State University-Pueblo funded the larger project that this experiment was a part of and allowed for the purchase of the mice, reagents, and some of the equipment used.
120-Grit Sand Paper | N/A | N/A | For removal of caudal end plate soft tissues and irregularities |
24-bit Load Cell Interface | LoadStar Sensors, Freemont, California, USA | DQ-1000 | To connect load and displacement sensors to personal coputer |
Base Mouse Diet | Dyets, Inc, Bethlehem, PA, USA | AIN-93G | Diet the mice were fed, without added hempseed |
Diamond Cutoff Wheel w/ Rotary Tool | Dremel US, Mt. Prospect, Illinois, USA | F0130200AK | To remove vertebral proccesses |
Displacement Sensor | Mitutoyo, Aurora, Illinois, USA | ID-S112EX | Displacement sensor with 0.001 mm resolution and 0.00305 mm accuracy |
External Variable Voltage Power Source | Extech Instruments, Nashua, New Hampshire, USA | 382213 | To provide power to compression testing machine |
Female C57BL/6 Mice | Charles River Laboratories, Wilmington, Massachusetts, USA | 027 (Strain Code) | Mouse model used in present study |
Hempseed | Natera, Pitt Meadows, Canada | 670834012199 | Hempseed added to Base Mouse Diet |
Igor Pro Software (Version 8.04) | Wave Metrics, Portland, Oregon, USA | N/A | Sofware used for load-displacement curve analysis |
iLoad Mini Force Sensor | LoadStar Sensors, Freemont, California, USA | MFM-010-050-S | Load (force) sensor with 1.0% accuracy |
Isotonic (0.9%) Saline Solution | N/A | N/A | To keep bone sampels hydrated |
Leica EZ4 W Miscoscope | Leica Microsystems, Wetzlar, Germany | NC1601884 | For bone dissections and vertebral process removal |
Microsoft Excel Software | Microsoft Corporation, Redmond, Washington, USA | N/A | For data transfer from SensorVue software |
PALACOS R Bone Cement | Hareus Medical, Wehreim, Germany | 00-1112-140-01 | PMMA bone cement for embedding of the loading surface |
Personal Computer | N/A | N/A | For data recording (see 24-bit Load Cell Interface, SensorVue Software, Microsoft Excel Software) and analysis (see Igor Pro Software) |
SensorVue Software | LoadStar Sensors, Freemont, California, USA | N/A | Software used for real-time data collection during compression testing |
Small Animal Dissecting Kit | N/A | N/A | Dissecting scissors, forceps, scalpel, blades, pins, gauze pads |
Stainless Steel Top Platen (Self-Alligning) and Bottom Platen Pair | N/A | N/A | Constructed by Colorado State University-Pueblo Dept. of Engineering |
Three-Point Bending Machine | N/A | N/A | Constructed by Colorado State University-Pueblo Dept. of Engineering. Refer to Sarper et al. (2014) for further details regarding construction |