Transgenic mice exhibit significant increases in empty lacunae and intracortical cavities.

<p>(A) Quantification of empty lacunae in cortical and trabecular bone. (B) Quantification of intracortical cavities.</p

Bone structure in control limb predicts inter- and intra-group variability in adaptation to pressure loading.

<p>Bone structure in control limb is plotted in the x-coordinate, and is indicative of baseline cellular activity in the absence of pressure loading. Relative adaptation is plotted in the y-coordinate, and was found to be negatively correlated with control limb structure independently of genotype and DT dose. Results are shown for (A) trabecular volume fraction, (B) trabecular number, (C) trabecular spacing, (D) cortical thickness, and (E) bone mineral density. Each point represents a single animal (red: WT; blue: Tg; circle: 10 µg/kg DT; square: 50 µg/kg DT; fill: HLS; no fill: Amb). Pearson correlation coefficients and corresponding p-values are shown in the top right of each plot.</p

Systems-Based Identification of Temporal Processing Pathways during Bone Cell Mechanotransduction

<div><p>Bone has long been established to be a highly mechanosensitive tissue. When subjected to mechanical loading, bone exhibits profoundly different anabolic responses depending on the temporal pattern in which the stimulus is applied. This phenomenon has been termed temporal processing, and involves complex signal amplification mechanisms that are largely unidentified. In this study, our goal was to characterize transcriptomic perturbations arising from the insertion of intermittent rest periods (a temporal variation with profound effects on bone anabolism) in osteoblastic cells subjected to fluid flow, and assess the utility of these perturbations to identify signaling pathways that are differentially activated by this temporal variation. At the level of the genome, we found that the common and differential alterations in gene expression arising from the two flow conditions were distributionally distinct, with the differential alterations characterized by many small changes in a large number of genes. Using bioinformatics analysis, we identified distinct up- and down-regulation transcriptomic signatures associated with the insertion of rest intervals, and found that the up-regulation signature was significantly associated with MAPK signaling. Confirming the involvement of the MAPK pathway, we found that the insertion of rest intervals significantly elevated flow-induced p-ERK1/2 levels by enabling a second spike in activity that was not observed in response to continuous flow. Collectively, these studies are the first to characterize distinct transcriptomic perturbations in bone cells subjected to continuous and intermittent stimulation, and directly demonstrate the utility of systems-based transcriptomic analysis to identify novel acute signaling pathways underlying temporal processing in bone cells.</p></div

Experimental setup for pressure loading experiments and measurements of ImP.

<p>(A) Image of a hindlimb suspended mouse subjected to microfluidic pressure loading. The syringe pump consists of a Hamilton syringe (hs) mounted in a computer-controlled loading frame (lf) that actuates the syringe plunger (p). A saline-filled catheter (c) couples the pump to the cannulated mouse (hindlimb suspended via a tail suspension apparatus (tsa)). The catheter is protected from mouse chewing/pulling by an infusion harness (ih). (B) Composite average (± standard error) of intramedullary pressure measurements obtained from four animals in the absence (empty circles) and presence (filled circles) of microfluidic pressure loading. Pressure loading resulted in a 5.1 Hz waveform with a mean peak pressure of ∼70 mmHg.</p

Bone structure and bone mineral density in control limbs.

<p>^gand ^s indicate statistically significant for genotype or suspension, respectively.</p

Bone response at the mid-shaft tibiae from animals subject to loading with or without CsA supplementation.

<p>Illustrative fluorescent images from animals subject to cyclic loading (a) and supplemented with 3.0 mg/kg CsA (b), or subject to rest-inserted loading (c) and supplemented with 0.1 mg/kg CsA (d).</p

Schematic demonstrating two potential mechanisms by which osteocyte ablation may give rise to loss of trabecular bone mass in unpressurized limbs while enhancing pressure loading-induced adaptation.

<p>In the first case (A), osteocyte ablation gives rise to an increase in the number of active osteoclasts, resulting in heightened bone loss in unpressurized limbs. In limbs subjected to pressure loading, the resorptive activity of these active osteoclasts is halted, preserving bone mass. In the second case (B), osteocyte ablation shifts the osteoblastic population to a more quiescent state, resulting in decreased bone mass in unpressurized limbs. However, in pressure-loaded limbs, an enhanced anabolic response occurs due to the newly available pool of quiescent cells activated following exposure to pressure loading-induced IFF.</p

Quantification of canalicular convective velocity from <i>ex vivo</i> measurements of lacunar fluorescence recovery after photobleaching.

<p>(A) Single lacuna immediately prior to (Pre-Bleach) and following photobleaching (Bleach), and the subsequent recovery of fluorescence in the absence (top) and presence (bottom) of pressure loading. Faster recovery can be observed in the presence of pressure loading, indicating convective transport. Color bar on bottom indicates image intensity. (B) Plot of Eq. 1 demonstrating the relationship between convective velocity <i>v_c</i> and recovery rates <i>k</i> and <i>k</i>₀. The red dot corresponds to the canalicular fluid velocity (∼80 µm/s) calculated using the values of <i>k</i> and <i>k</i>₀ obtained in this study.</p

Dose-response relations simulated by the implemented non-linear models.

<p>A single model, which assumed that there is no adaptive benefit of loading supplemented with CsA, could simulate the rp.MS/BS data (a; mean ± s.e.). In contrast, two separate models were required to simulate rp.MAR (b) and rp.BFR/BS (c) induced by cyclic and rest-inserted loading supplemented with CsA. Please note that CsA dosage is plotted on a log-scale.</p

Pressure loading-induced adaptation is enhanced in transgenic mice.

<p>Results are shown for relative changes (defined as the difference between pressure-loaded limb and contralateral limb values) in (A) trabecular volume fraction, (B) trabecular number, (C) trabecular spacing, (D) cortical thickness, and (E) bone mineral density.</p