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

Abstract

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.

Microdamage induced by in vivo Reference Point Indentation in mice is repaired by osteocyte-apoptosis mediated remodeling

“…The IDI parameter has been proposed as the most useful RPI output since it was shown to correlate well with fracture toughness in dogs [6]. In our studies, this was the most variable parameter between animals, which is in agreement with other reports in the literature [17]. It is possible that a size effect of the tissue being tested influences this measurement, which would explain the observed variability in mouse bone, these and other tissue specific effects have been suggested by others [22].…”

“…We first developed a novel method to carry out reproducible RPI tests on living mouse bones. In vivo RPI parameters (ID-1st, CID, TID, IDI, LS, US and ED) have been successfully collected from mouse bones before [17], in a study of their variability in this species. Our method was slightly different and was specifically designed to confirm microcrack formation occurs beneath the indenter tip.…”

True Gold or Pyrite: A Review of Reference Point Indentation for Assessing Bone Mechanical Properties In Vivo

“…Data are plotted as percent difference between baseline and day 28. Figures reproduced with permission from Srisuwananukorn and colleagues and Allen and colleagues …”

Reference point indentation is insufficient for detecting alterations in traditional mechanical properties of bone under common experimental conditions

“…Although several different test settings have been used in the literature, these parameters generally matched those previously published. From the resulting force-displacement curves, we used a custom MATLAB program to calculate the total indentation distance (TID) and the indentation distance increase (IDI), as described previously [5–7]. These parameters were chosen as the focus as they are the most prominently discussed parameters in the RPI literature.…”

“…Reference point indentation (RPI) was developed as a novel method to assess mechanical properties of bone in vivo [2,3]. This technique has been utilized in both patients (for review see [4]) and in several pre-clinical models [5–7], as well as multiple ex vivo studies [8–12] and has demonstrated the ability to separate disparate group means across varying conditions. However, it remains unclear what mechanical and/or morphological aspects of bone dictate changes/differences in RPI-based parameters.…”