Models for Axial Loading of Murine

Long BonesK. Alice Matthews

Jonathon GaliKellen Sakala

Advisors: Dr. Jeffrey Nyman and Dr. Daniel Perrien

Mechanosensitivity of bone is lost with osteoporosis, and understanding the signaling that leads to bone adaptation while under mechanical loading can lead to insight into new targets for therapy.

Bone fractures are costly, but understanding the structural strength of the femoral neck in normal and diseased mice can lead to providing insight into its role in osteoporotic fractures

Motivation

Osteoporosis affects approximately 75 million people in Europe, USA, and Japan

1 in 3 women over the age of 50 will experience osteoporotic fractures as will 1 in 5 men

Exercise has been proven to reduce the risk of osteoporosis , fracture, and fall-related injuries

In the US in 2005, there were 2 million fractures caused by osteoporosis which cost $17 billion

Statistics

1. Design a dynamic tibial-loading system for mice◦ Will aid in studying consequences of deleting the

Sirtuin-1gene in osteocytes. ◦ Sirtuin proteins may play a key role in an organism's

response to stress.◦ Recent studies show they are involved in age related

diseases such as osteoporosis.

2. Design a potting apparatus for loading a murine femur to test femoral neck strength in mice suffering from Perthes disease, a form of osteonecrosis that occurs in the hip.

Objectives

How to design testing fixtures that will allow us to mechanically load a very small bone in a physiological manner

Normal loading while walking is 200-600μ, but μ >1200 needed to stimulate osteogenesis

Problem to Address-Tibia

Manufacture and calibrate an apparatus for dynamic axial loading of tibia of live mice.

Solutions-Tibia

Live mouse on its back

Patella resting in top cup and Calconeum in bottom cup with groove for foot

Tibia Study Tibial loading cups should provide

support during testing and effectively minimize injury/discomfort to mouse.

Effectively transfers mechanical load to murine tibia, causing desired peak tibial strain (1500 μ) in order to examine the effects of Sirtuin-1 on bone growth and bone remodeling.

Performance Criteria

Lanyon, 2008

Created protocol in Instron control software and performing 3 week test on ex-vivo tibia

Fifty trapezoidal wave cycles every 11.5 seconds for ten minutes.

Loading time 1.5 seconds loading from1N to 9N

1500 microstrain (optimal strain for bone growth )

Current Progress-Tibia

Subjected 7 mice to mechanical loading on tibia three times a week

4 KO and 3 Wildtype 2% Isoflorane 98% O2 Sacrificed the 7 mice and

excised the femur, tibia, and spine of each mouse.

Collected blood samples and prepared the OB and OCL cultures and histology

Our design is currently being used in an additional study

Current Progress-Tibia

Have histological tests done on the excised bones

Conduct OB and OCL culture tests Examine the response to mechanical loading

measured by microCT, finite element modeling, and histomorphometric analysis of metaphyseal trabecular and diaphyseal cortical bone

Analyze data and determine whether SIRT1 affects the response to mechanical loading and subsequent bone growth of the tibia

Future Work-Tibia

Loading Comparison

Force will be applied at constant speed until failure (femoral neck fracture). This differs from the loading done in the live mice study where a non-failure force was applied and then released 50 times (to tibia).

Need to be able to test multiple femurs in same orientation

Apparatus will allow the bone to be scanned by micro-computed tomography so that boundary conditions of biomechanical test can be simulated in uCT finite element modeling of femoral neck loading

Couple uCT-FEM predictions with experimental measurements to show how gene affects material properties of bone

Problems to Address-Femoral Head

Femur Potting Apparatus Potted murine femur capable of being

transitioned from loading apparatus to micro-ct tube for imaging and subsequent finite element modeling and analysis.

Performance Criteria

Design of a potting apparatus for testing of multiple femurs in the same orientation that can then be scanned with micro-Ct

Solutions-Femoral Head

Square Block: 1.5 cm X 1.5 cm X 0.5 cmMicro-CT Insertion Tube: 0.39 cm outer diameter, 0.27 cm inner diameter, 0.37 cm tallLoad Cell Attachment Tube: 1 cm diameter, 0.75 cm tall

Jamisa, 1998

Cost Time Materials Available Skills needed to produce the potting

apparatus

Conclusion: SolidWorks Design has been abandoned at this point for a new approach

Drawbacks to SolidWorks Design

Reasons Why:◦ Easily available and plentiful in the biomechanics

lab.◦ Can be easily loaded into the Instron machine.◦ Tube can be cut to fit into microCT scanning tubes◦ Easily place bone into potting mixture in tube

New Solution – Microcentrifuge tubes

3 point loading arm◦ Pros: Reduces error (3-point hold, angle adjustment),

bone protection◦ Cons: Delicate

Tweezer clamp◦ Pros: Stronger hold◦ Cons: Less control of potting angle

Tweezer Clamps vs. 3-point Loading Arm

Decision: 3-Point Loading Arm

Femur Potting Apparatus Timeline Week of March 21: pot test bones Week of March 28: load test bones, perform

microCT Week of April 4: analyze results/process,

deliver protocol for bones of interest Week of April 11: complete project

Future Work

Jamsa T et al. Femoral neck strength of mouse in two loading configurations: Method evaluation and fracture characteristics. Journal of Biomechanics, April 1998.

Leanne Kaye Saxon and Lance Edward Lanyon. Methods in Molecular Biology, Chapter 21: Assessment of the In Vivo Adaptive Response to Mechanical Loading. May 2008

Jamsa T et al. Femoral Neck Is a Sensitive Indicator of Bone Loss in Immobilized Hind Limb of Mouse. Journal of Bone and Mineral Research, November 1999.

Willinghamm et al. Age-related changes in bone structure and strength in female and male BALB/c mice. Calcified Tissue International, June 2010

References