Mouse Lumbar Vertebra Uniaxial Compression Testing with Embedding of the Loading Surface
December 1st, 2023
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.
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 dimension, or other treatments on a cortical bridge 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. To begin, unscrew the crosshead beam attached to the load sensor on the three-point bending machine.
Screw a self-aligning top platen 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 platen to the two lower supports and tighten them until they are securely fastened.
Then to determine machine displacement, lower the top platen onto the bottom platen 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 platen onto the bottom platen 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. 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 vertebra from the cranial end of the sacrum. Again, cut between the intervertebral space, remove L6 and L5 from the vertebral column and set aside L5 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.
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 caudal end of the vertebra using fine 120-grit sandpaper to eliminate all intervertebral discs, soft tissue, and irregularities.
Then mark the sanded caudal 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 platen with the caudal side facing downwards. Now turn on the machine to engage the drive gears and gradually lower the top platen 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 platen 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 for five seconds, applied with a minimal preload force of less than 0.5 Newtons, gradually lower the top platinum 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 waveform and click Make Waveform. After creating the waveform, 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.
Overview
This study addresses the challenge of mechanical strength testing of mouse lumbar vertebrae by presenting an embedding method and the conversion of a three-point bending machine for uniaxial compression testing. The research aims to improve the understanding of factors influencing vertebral bone mechanical properties, such as aging and diet.
Key Study Components
Research Area
- Mechanical strength testing of small animal bones
- Effects of aging and diet on bone properties
- Innovative methods for biological specimen testing
Background
- The small size of mouse vertebrae poses challenges for standard compression testing.
- Traditional methods often require extensive preparation that can damage the sample.
- Finding efficient methods can enhance comparative studies across different testing scenarios.
Methods Used
- Embedding method for preparing vertebrae using PMMA bone cement
- Conversion of a three-point bending machine to a compression testing machine
- Digital data collection for load and displacement measurements
Main Results
- A simpler means of preparing lumbar vertebrae for testing has been developed.
- The method enables comparative analysis between bending and compression testing conditions.
- Data collection techniques were validated to ensure accurate mechanical assessments.
Conclusions
- This study showcases effective modifications to mechanical testing methods for small bones.
- The findings are significant for researchers exploring pharmacologic and dietary effects on bone health.
Frequently Asked Questions
Executive Industry Relevance
Reliable mechanical testing of both cortical and cancellous bone is critical for evaluating the effects of pharmaceutical and dietary interventions on skeletal integrity in preclinical models. The described embedding and machine conversion methods enable parallel, quantitative assessment of vertebral and long bone strength, supporting more predictive and translationally relevant bone research. This dual-modality approach enhances confidence in target validation and mechanistic de-risking for bone-active therapeutics.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct comparison of treatment effects on cortical versus cancellous bone compartments.
- Supports mechanistic de-risking by clarifying bone-type specific responses to interventions.
- Improves predictive confidence in preclinical bone strength endpoints.
Screening & Assay Development
- Facilitates preparation of standardized vertebral samples for reproducible compression testing.
- Allows conversion of existing three-point bending systems for dual-mode mechanical assays.
- Generates quantitative load-displacement data suitable for compound screening workflows.
Translational & Preclinical Research
- Aligns preclinical bone strength measurements with disease-relevant endpoints for osteoporosis and bone loss models.
- Enables continuity from discovery through preclinical validation by supporting both bending and compression modalities.
- Supports risk-adjusted advancement decisions by providing robust, comparative mechanical data.
Pipeline & Workflow Integration
This method integrates into the discovery-to-preclinical continuum by enabling both cortical and cancellous bone strength testing within the same experimental pipeline.
- Discovery Biology: Supports hypothesis testing on bone compartment-specific drug effects and dietary influences.
- Screening: Provides assay-ready, reproducible vertebral samples for quantitative mechanical evaluation.
- Analytics: Delivers load-displacement curves and failure thresholds for comparative analysis across conditions.
- Translational Research: Bridges discovery findings to preclinical models of bone fragility and therapeutic response.
- Enterprise Reuse: Offers a scalable, adaptable workflow for diverse bone strength studies in rodent models.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in bone-targeted R&D.
- Operational Value: Standardizes sample preparation and testing, improving reproducibility and throughput.
- Strategic Value: Enables more informed go/no-go decisions and reduces late-stage biological risk for bone therapies.
- Portfolio Impact: Supports risk-adjusted prioritization of bone-active candidates based on robust mechanical endpoints.
Implementation Considerations
- Requires expertise in small animal dissection and mechanical testing protocols.
- Needs access to three-point bending machines and digital data acquisition systems.
- Demands cross-team standardization of sample preparation and data analysis workflows.
- Adaptable to other small bone models with appropriate modifications.
- Sample size and preparation precision are critical for reliable, reproducible results.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:36
Conversion of a Three‐Point Bending Machine to a Compression Testing Machine
3:00
Dissection of the 5th Lumbar Vertebra (L5) from the Mouse Carcass for Uniaxial Compression Testing
4:11
Preparing L5 Vertebra Loading Surface for Uniaxial Compression Testing Using the PMMA Bone Cement Embedding Method
