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Biomechanics

Biomechanics - Poster Session D

Poster D9 - Microindentation Testing Analysis of Rat Cortical Bones Exposed to Low Dose Proton Radiation

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  As NASA continues to send astronauts into space, there is a heightened concern surrounding the damaging effects of space radiation on their health. Research endeavors have extensively documented the consequences of space travel on bone loss [1]. When traveling into space, astronauts are outside the protection of Earth’s magnetic field and are thus exposed to space radiation. The effects of high dose space radiation on skeletal deterioration have been vastly explored, yet there is limited information on how low dose space radiation specifically affects the material properties of bones. Microindentation testing provides insight into the viscoelastic material properties of bone at the tissue level. The purpose of this study is to determine the effects of acute exposure to proton radiation on the material properties of rat cortical bones at short (7 and 30 day) and long (90 and 180 day) time points after radiation exposure.

Materials and

Methods:  In this study, hind-limb bones were collected from male and female behaviorally trained Long Evans rats that were acutely exposed to proton radiation (50cGy; 25 MeV/n and 1000 MeV/n) at Brookhaven National Lab with corresponding sham controls. Right hind limb bones were dissected and cleaned of superficial soft tissues after the rats were euthanized at 7, 30, 90 and 180 days post exposure. Bones were stored in 70% ethanol and transported back to TCNJ for further biomechanical analysis. This is an ongoing study; additional animal samples are currently undergoing collection.

Right femur bones were dried then embedded into epoxy. Using a diamond low speed circular saw, bones were cut to the height of lesser trochanter, creating a flat surface on the cross section of the femur (Figure 2). All epoxy pucks were then polished to create a smooth surface for indentation testing. Light microscopy was utilized to identify areas on the bone with high porosity for avoidance during testing. Before testing, the bones were soaked in saline for at least 24 hours.

A 150 µm radius, ruby spherical indentation tip was used for material testing of the bones. An indentation test was performed, to a depth of 10 µm below the surface of the bone and held for 45 seconds. Four indents were performed per bone, one at each anatomic location; medial, lateral, posterior, and anterior. Post indentation testing, the Keyence VHX-S770E Digital Microscope was utilized to visualize the bone and residual plastic deformation from indentation.

Results, Conclusions, and Discussions: 

Results:
Exposure to 50 cGy proton radiation did not have any statistically significant effects on the instantaneous modulus of the cortical bone. Preliminary data shows no statistical variability in the elastic moduli of female rats post acute proton radiation. Significant differentiation in relaxed and instantaneous elastic modulus was observed amongst the four regions of the bone that underwent indentation testing; anterior-lateral, lateral-medial, anterior-posterior, and medial-posterior p < 0.05. Within the posterior region of the cortical bone, a statistically significant increase was found in the instantaneous elastic modulus of male rats exposed to acute proton radiation, between the 7 and 180 day timepoints p< 0.05. This increase is shown in Figure 1. Analysis of the female cohort is on-going as the specimens are still pending collection.

Discussion:
The differentiation amongst the bone regions was likely caused by the indentation testing performed into pores and osteons, unidentifiable by the standard light microscope. Usage of a Keyence VHX microscope allowed for visualization of crack propagation in bone, seen in Figure 2, which is consistent with expected crack propagation behavior; perpendicular to the osteons or pores [2]. The osteoblast damage as a result of radiation exposure is likely causing the decreased modulus, observed in Figure 2. This decrease in instantaneous elastic modulus post irradiation, was likely due to a change in the biological mechanisms attributed with bone growth, specifically, osteoblast damage [3].
Nanoindentation, performed at a smaller length scale, could address variability observed in microindentation. Future work aims to utilize nanoindentation testing to create modulus maps across rat cortical bone, which can provide insight into a better understanding of the risk of bone fracture due to radiation, specifically amongst regions of the bone.

Conclusion:
Changes in the elastic moduli amongst bone regions was likely due to pores, unidentifiable by standard light microscope prior to indentation testing. Differences based on the anatomical location of individual indentation tests were observed, specifically within the posterior region of male cortical bone. Future work may conclude that micropores result in a decreased elastic modulus.

Acknowledgements (Optional):  

References:
[1] Genah S. et al (2021) Int J Mol Sci 22, 4585.
[2] Fang, H., Deng, Z., Liu, J., Chen, S., Deng, Z., & Li, W. (2022). The Mechanism of Bone Remodeling After Bone Aging. Clinical interventions in aging.
[3] Thomas, P. K., Sullivan et al (2021) Calcified tissue international.