In Vivo Reference Point Indentation Variability in Skeletally Mature Inbred Mice

Abstract

Ex vivo mechanical testing is the gold standard for assessing bone mechanical properties. However, ex vivo assessments preclude longitudinal studies of different interventions to enhance these properties. Reference point indentation (RPI) – a noninvasive probe that cyclically indents a bone surface with a set force and measures resulting displacement – was developed to measure bone mechanical properties in vivo. Studies using RPI have discriminated between patients with previous skeletal fractures and those without, and among dogs given different bone anti‐remodeling drugs. This study assesses variation in RPI outcomes in mice, the most common animal model used to study bone. Twelve 16‐week‐old female C57BL/6 mice were subjected to RPI (3 tests) on the anterior tibia, followed by repeat testing on the contralateral limb 28 days later. A custom MATLAB program derived several outcome parameters from RPI force‐displacement curves: 1st cycle indentation distance (ID‐1st), indentation distance increase (IDI), total indentation distance (TID), 1st cycle unloading slope (US‐1st), and 1st cycle energy dissipation (ED‐1st). Within‐animal variation of the three tests on Day 1 ranged from 12.8 to 33.4%, and from 14.1 to 22.4% on Day 28. Between‐animal variation on Day 1 ranged from 15.9% to 40.9%, and from 14.8% to 33.4% on Day 28. At both time points, within‐ and between‐animals, US‐1st was the least variable parameter and IDI was most variable. All parameters were non‐significantly lower at Day 28 compared to Day 1. These data demonstrate the feasibility of measuring bone mechanical properties longitudinally in mice – an advance for the field of bone biology – and will shape the experimental design of future studies.