Cantilever Bending of Murine Femoral Necks

Summary

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Abstract

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20° relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6° ± 1.5°, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

Introduction

Fracture risk is a serious medical concern associated with osteoporosis. Over 1.5 million fragility fractures are reported each year in the United States alone, with fractures occurring in the hip, specifically the femoral neck, as the leading fracture type¹. It is estimated that 18% of women and 6% of men will experience a femoral neck fracture in their lifetime^2, and the mortality rate at 1 year following the fracture is greater than 20%¹. Therefore, mouse models that allow biomechanical testing of the femoral neck can be suitable for studying fragility fractures. Mouse models also of....

Protocol

Animal studies were approved by The University of Rochester Committee of Animal Resources. The mice used in this study were C57BL/6 males and females ranging from age 24-29 weeks of age. Mice were housed in standard conditions with food and water ad libitum. Upon euthanasia via carbon dioxide inhalation, followed by cervical dislocation, 20 right femurs (10 male and 10 female) were harvested and frozen at -20 °C until tested.

1. Creation of custom 3D printed mounting guides

NOTE: This step might be needed because different strains and genetic phenotypes might have different anatomical....

Results

When potted with the aid of the guide, the femoral shafts were aligned at 21.6° ± 1.5°. While this represents <10% deviation from the intended angle of 20°, the coefficients of variation (COV) of the potting angle across all samples tested were 7.6% and 6.5% for male and female mice, respectively (n = 10 per group) as verified by pre-test planar x-rays (Figure 5). Additionally, the post-testing X-rays should be used to assess the mode in which the samples failed. Fail.......

Discussion

This protocol outlines a reliable cantilever bending test for murine femoral necks. The natural cantilever flexure scenario that occurs at the femoral neck is typically not represented in standard 3- and 4-point bending tests⁵. This testing method is better and more reliably replicates the type of femoral neck fractures experienced by bone fragility patients. The main focus when performing this protocol is eliminating the variability due to inconsistent potting of the femoral shaft. Critically, cl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
¼” x ¼” square aluminum tubing	Grainger	48KU67	Cut to lengths of 1/2" to 1" lengths
1 kN load cell	Instron	2527-130	Any load cell with sub 1 N resolution can be used.
3.5x-45x Zoom Stereo Boom Microscope	Omano	OM2300S-GX4	Microscope used to precisely line up samples with loading platen.
3D printed guides	Custom made		Angled slots at 73.13°, with diameters between 1.9 mm and 2.2 mm
3D printed mount	Custom made		Tapped with M10 threads to fit the mount attachment and with 2 M4 threaded holes adjacent sides to hold the aluminum tubing with sample in place.
Acrylic Base Plate Material Kit	Keystone Industries	921392	Mix 3.5 g of powder with 2 mL of liquid. This will be enough for approximately 8 samples, and will begin to harden quickly.
Amira	ThermoFisher Scientific		Used to compile µCT scans
Biaxial stage	Custom made		Used to center femoral head of sample under the loading platen.
BioMed Amber Resin	formlabs	RS-F2-BMAM-01	Any resin from formlabs could be used for this project.
Bluehill 3	Instron	V3.66	Software used to set up loading protocol and collect load, displacement and time data.
ElectroPuls 10000	Instron	E10000	Mechanical testing system
Faxitron UltraFocus	Faxitron BioOptics	2327A40311	X-ray imaging system
Form 2	formlabs	F2	Used to print the mount and guides
Form 2 Resin Tank LT	formlabs	RT-F2-02	LT Tank was used to be compatible with the BioMed Resin
ImageJ	National Institutes of Health	ImageJ	Used to assess µCT and X-ray images
Laxco iLED Series LED Light Source	ThermoFisher Scientific	AMPSILED30W	Light source used in conjugtion with microscope.
Loading platen	Custom made		This can be any metal rod that is tapered to a diameter of approximately 2.5 mm. We used an M6 screw that was tapered on a lathe.
Mount attachment	Custom made		To secure the 3D printed mount to the load cell. We used a M10/M6 threaded rod
Phosphate Buffer Saline (PBS)	ThermoFisher Scientific	10010031	Need to rehydrate the samples once acrylic base plate material has set.
Plumber's putty	Oatey	31174	Used to seal the end of the aluminum tubing when pouring acrylic base plate material in. Any clay or putty could be used.
PreForm	formlabs	Preform 3.15.2	Formlabs software
Tissue Culture Dish	Corning	353003	Samples can be laid flat in culture dish and covered in PBS to rehydrate.
vivaCT 40	Scanco	µCT 40	Representative set or actual samples can be scanned prior to printing of guides to calculate femoral shaft angle and diameter.