Evaluating Differences in Elastic Modulus of Regenerated and Uninjured Mouse Digit Bone through microCT Density-Elasticity Calculation and Nanoindentation Testing

Abstract

Bone is an essential, healing structure in vertebrates that ensures daily function. However, the regenerative capacity of bone declines with age, compromising quality of life in the elderly and increasing cost of care. Here, for the first time, the elasticity of regenerated bone in a mouse digit amputation model is evaluated in order to better investigate biomechanics of skeletal regeneration. Amputation of the distal one third of the digit (third phalangeal element P3) results in de novo regeneration of the digit, where analyzing the structural quality of this regenerated bone is a challenging task due to its small scale and triangular shape. To date, the evaluation of structural quality of the P3 bone has primarily focused on mineral density and bone architecture. This work describes an image-processing based method for assessment of elasticity in the whole P3 bone by using microcomputed tomography-generated mineral density data to calculate spatially discrete elastic modulus values across the entire P3 bone volume. Further, we validate this method through comparison to nanoindentation-measured values for elastic modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower elastic modulus compared to the uninjured digit, with a similar trend for experimental hardness values. This method will be impactful in predicting and evaluating the regenerative outcomes of potential treatments and heightens the utility of the P3 regenerative model.

Age-Dependent Changes in Bone Architecture, Patterning, and Biomechanics During Skeletal Regeneration

“…By using this processing method, we found that age increases the elasticity of the distal tip of the unamputated digit, while the process of regeneration predictably decreases elasticity and hardness. Surprisingly, we found that both calculated elasticity and direct measurements of hardness show no significant difference between the 6-month-old ( Hoffseth K. et al, 2021 ) and 18-month-old mice after regeneration. These biomechanical similarities are in contrast with age-dependent differences seen in the bone architecture of young and aged mice.…”

“…Calculation of elastic modulus values utilized a processing pipeline starting with BMD as measured by μCT and using established density-elasticity relationships ( Knowles et al, 2016 ) as detailed in Hoffseth K. et al (2021) using 6-month-old mice. Mineral density values were calculated as previously described ( Hoffseth K. F. et al, 2021 ) by averaging grayscale pixel intensity using L3-sized voxels ( L = 3 pixels) for each representative data point through iterative operation over the digit image stack, reducing computation time without avoiding loss of digit characteristics.…”

“…Mineral density values were calculated as previously described ( Hoffseth K. F. et al, 2021 ) by averaging grayscale pixel intensity using L3-sized voxels ( L = 3 pixels) for each representative data point through iterative operation over the digit image stack, reducing computation time without avoiding loss of digit characteristics. We utilized our predictive processing method ( Hoffseth K. et al, 2021 ) to calculate elasticity values for the entire P3 bone in unamputated digits (all cortical bone) and day 42 regenerated digits (both cortical and trabecular bone). As previously described ( Hoffseth K. et al, 2021 ), the density-modulus relationship equation below was used to calculate values of elasticity that were calculated from μCT measured volumetric BMD values using quantitative computed tomography ( Knowles et al, 2016 ).…”

“…We utilized our predictive processing method ( Hoffseth K. et al, 2021 ) to calculate elasticity values for the entire P3 bone in unamputated digits (all cortical bone) and day 42 regenerated digits (both cortical and trabecular bone). As previously described ( Hoffseth K. et al, 2021 ), the density-modulus relationship equation below was used to calculate values of elasticity that were calculated from μCT measured volumetric BMD values using quantitative computed tomography ( Knowles et al, 2016 ).…”

“…To overcome these limitations and explore the biomechanical properties of skeletal regeneration, we utilized both nanoindentation and micro-computed tomography (microCT) image-processing methods for the assessment of elasticity in the whole P3 bone ( Hoffseth K. et al, 2021 ) and evaluated the impact of aging on bone elasticity after regeneration. In this study, we showed that the elasticity measurements calculated from our processing method are able to detect age-dependent differences in elasticity that small samples of Young’s modulus values, generated from directly measured reduced modulus values from nanoindentation, are not able to detect ( Hoffseth K. et al, 2021 ).…”