In vivo reference point indentation measurement variability in skeletally mature inbred mice

Reference point indentation (RPI) was developed to measure material-level mechanical properties of bone in vivo. Studies using RPI in vivo have discriminated between human subjects with previous skeletal fractures and those without and among dogs given different anti-remodeling drugs. Recently, this technology was extended to rats, providing the first in vivo data for rodents. The goal of the present study was to perform in vivo RPI measurements in mice, the most common animal model used to study bone. Twelve 16-week-old female C57BL/6 mice were subjected to RPI (three tests) on the anterior tibia, followed by a repeat test session on the contralateral limb 28 days later. A custom MATLAB program was used to derive several outcome parameters from RPI force-displacement curves: first cycle indentation distance (ID-1st), ID increase (IDI), total ID (TID), first cycle unloading slope (US-1st) and first cycle energy dissipation (ED-1st). Data within an individual were averaged across the three tests for each time point. Within-animal variation of all RPI parameters 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 11.4% to 22.8% and from 7.5% to 24.7% 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 nonsignificantly lower at day 28 compared with day 1. These data are important to demonstrate the feasibility of collecting bone material property data longitudinally in mice and will inform the design of future studies in terms of statistical power and appropriate sample size considerations.