Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function

In vivo tibial loading of healthy and osteolathrytic mice

Although the in vivo tibial loading model has been used to study the bone formation response of mice to exercise, little emphasis has been placed on the translation of architectural and compositional modifications to changes in mechanical behaviour. The goals of the studies discussed below were to investigate the mechanical response in both healthy and osteolathrytic mice to this loading model and to determine the dose-depended effects of strain level on these properties. In two separately designed studies, strain levels ranging from 1700 to 2400 με were applied to the right tibiae of 8 week old female C57BL/6 mice, while the left tibiae were used as non-loaded control. The first study consisted of loading both PBS- and BAPN-injected mice to 1750 με which resulted in little bone formation but some tissue-level changes in mechanical analyses and an improvement in fatigue-resistance in terms of microdamage accumulation. The second study loaded healthy mice to three strain levels (1700, 2050, and 2400 με). Results indicated that the low end of the strain range did not engender a robust formation response, while the high end of the strain range resulted in a woven bone response in half of the animals in that group. Future studies will focus on the mid-strain level of 2050 με which induced both significant architectural and mechanical improvements

Schematic Representations of Standard Site Geometric Profiles.

<p>A) 1700 με group. B) 2050 με group. C) 2400 με group. There was a dose-dependent increase in cortical parameters with robust periosteal and endocortical formation, especially at the higher strain levels. D) 2400 με group with the addition of those animals that experiences a woven bone response due to loading.</p

Location of mechanical tests and micro-computed tomography (μCT) regions of interest.

<p>The diaphysis of each bone was loaded using 4-point bending with the medial surface in tension. The bottom support points (green) were located 9mm apart and the top loading points (blue) were located 3mm apart. The metaphyseal region used for trabecular analyses began just distal to the growth plate in the proximal metaphysis and extended distally by 12% of the overall bone length. The cortical standard site was located 45% the total bone length from the proximal growth plate. Strain gauges used for calibration were in the region spanning the cortical standard site on the anteromedial surface.</p

Timeline for tibial loading study and waveform profile.

<p>A) The right tibia of each mouse was loaded using the waveform to a set force (8.8N, 10.6N and 12.4N) to elicit a desired periosteal mid-diaphyseal tensile strain level (1700 με, 2050 με and 2400 με) over a 14 day period. B) The loading profile consisted of four haversine waveforms followed by 3 seconds of rest repeated 55 times for a total of 220 cycles of loading per day.</p

Load/Strain Calibration Curve.

<p>This figure demonstrates the linear relationship between applied load and resulting tensile strain at the periosteal mid-diaphysis. For all data points, error bars represent the standard deviation from n = 5 mice.</p

Schematic representations of mechanical testing curves.

<p>A) Representative structural-level force/displacement curves. B) Estimated tissue-level mechanical curves. At 1700 με, there was no mechanical effect noted. Those limbs loaded to 2050 με experienced significant increases in structural- and tissue-level strength and energy dissipation. The 2400 με group also experienced gains. However, when animals that experienced a woven bone response were removed from the analysis, the gains were more modest (especially at the tissue-level) and most failed to reach significance versus the contralateral control limb given the loss in power. For all data points, error bars represent the standard error of the mean (SEM).</p

PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu