Skeletal Adaptation to Intramedullary Pressure-Induced Interstitial Fluid Flow Is Enhanced in Mice Subjected to Targeted Osteocyte Ablation

Interstitial fluid flow (IFF) is a potent regulatory signal in bone. During mechanical loading, IFF is generated through two distinct mechanisms that result in spatially distinct flow profiles: poroelastic interactions within the lacunar-canalicular system, and intramedullary pressurization. While the former generates IFF primarily within the lacunar-canalicular network, the latter generates significant flow at the endosteal surface as well as within the tissue. This gives rise to the intriguing possibility that loading-induced IFF may differentially activate osteocytes or surface-residing cells depending on the generating mechanism, and that sensation of IFF generated via intramedullary pressurization may be mediated by a non-osteocytic bone cell population. To begin to explore this possibility, we used the Dmp1-HBEGF inducible osteocyte ablation mouse model and a microfluidic system for modulating intramedullary pressure (ImP) to assess whether structural adaptation to ImP-driven IFF is altered by partial osteocyte depletion. Canalicular convective velocities during pressurization were estimated through the use of fluorescence recovery after photobleaching and computational modeling. Following osteocyte ablation, transgenic mice exhibited severe losses in bone structure and altered responses to hindlimb suspension in a compartment-specific manner. In pressure-loaded limbs, transgenic mice displayed similar or significantly enhanced structural adaptation to Imp-driven IFF, particularly in the trabecular compartment, despite up to ∼50% of trabecular lacunae being uninhabited following ablation. Interestingly, regression analysis revealed relative gains in bone structure in pressure-loaded limbs were correlated with reductions in bone structure in unpressurized control limbs, suggesting that adaptation to ImP-driven IFF was potentiated by increases in osteoclastic activity and/or reductions in osteoblastic activity incurred independently of pressure loading. Collectively, these studies indicate that structural adaptation to ImP-driven IFF can proceed unimpeded following a significant depletion in osteocytes, consistent with the potential existence of a non-osteocytic bone cell population that senses ImP-driven IFF independently and potentially parallel to osteocytic sensation of poroelasticity-derived IFF

Bone structure and bone mineral density in control limbs.

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

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

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

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

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

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