Real-time measurement of solute transport within the lacunar-canalicular system of mechanically loaded bone: direct evidence for load-induced fluid flow

Abstract

Since proposed by Piekarski and Munro in 1977, load-induced fluid flow through the bone lacunar-canalicular system (LCS) has been accepted as critical for bone metabolism, mechanotransduction, and adaptation. However, direct unequivocal observation and quantification of load-induced fluid and solute convection through the LCS have been lacking due to technical difficulties. Using a novel experimental approach based on fluorescence recovery after photobleaching (FRAP) and synchronized mechanical loading and imaging, we successfully quantified the diffusive and convective transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in the bone LCS of adult male C57BL/6J mice. We demonstrated that cyclic end-compression of the mouse tibia with a moderate loading magnitude (-3 N peak load or 400 µε surface strain at 0.5 Hz) and a 4-second rest/imaging window inserted between adjacent load cycles significantly enhanced (+31%) the transport of sodium fluorescein through the LCS compared with diffusion alone. Using an anatomically based three-compartment transport model, the peak canalicular fluid velocity in the loaded bone was predicted (60 µm/s), and the resulting peak shear stress at the osteocyte process membrane was estimated (∼5 Pa). This study convincingly demonstrated the presence of load-induced convection in mechanically loaded bone. The combined experimental and mathematical approach presented herein represents an important advance in quantifying the microfluidic environment experienced by osteocytes in situ and provides a foundation for further studying the mechanisms by which mechanical stimulation modulates osteocytic cellular responses, which will inform basic bone biology, clinical understanding of osteoporosis and bone loss, and the rational engineering of their treatments.

Fig. 1

Experimental setup for the mechanical loading of a murine tibia and FRAP imaging of the load-induced solute transport/fluid flow inside the bone LCS. (A) The integrated system consisted of an electromagnetic loading device and an inverted confocal microscope. (B) Axial end loading of the intact murine tibia and the FRAP imaging region, where a tensile strain was induced due to a combination of bending and compression. (C) Synchronization of mechanical loading and FRAP imaging of murine tibiae. Top row: A representative trace of the applied load. Thick lines indicate the periods when the loading device was operated in the load control mode. Middle row: The corresponding trace of the actuator position. The loading device was switched to the displacement control mode (thick lines) during the resting periods when images were acquired between loading cycles. Bottom row: The time sequence for executing the three phases (prebleach, photobleaching, and recovery) of the FRAP experiment as well as the imaging trigger signals (indicated by arrows) sent from the loading device to the confocal microscope.

Fig. 2

A three-compartment LCS transport model (modified from Zhou et al., 2008). See text for a description of the model parameters; their values are listed in Table 1.

Fig. 3

A representative pair of FRAP experiments with sodium fluorescein (376 Da) in a murine tibia subjected to cyclically loaded (peak load of 3 N at 0.5 Hz with a 4-second resting/imaging period between two cycles) or nonloaded paired tests. (A) Prebleach image showing a cluster of osteocyte lacunae chosen for FRAP imaging, including the target (outlined in yellow) and surrounding lacunae, along with a reference lacuna (outlined in white) for autofading correction. (B) The time courses of fluorescence recovery within the same photobleached lacuna under loaded or nonloaded conditions. (C) Normalized fluorescence intensities I_n(t') = [I(t') – I_b]/(I_∞ – I_b) of the paired FRAP trials were fit with a nonlinear regression in the form of y = 1 – e^–t/τ. The fluorescence recovery time constant τ_diff and τ_load (dashed vertical lines) were 65 and 43 seconds (r² = 0.97 and 0.93), respectively. (D) Transport rates (k_diff = 0.017/s and k_load = 0.024/s) were calculated from the slopes of the fitting lines of y = ln[1 – I_n(t')] versus time (r² = 0.99, p < .0001 for both). A steeper slope indicated a faster solute transport rate. For this pair of loaded/nonloaded FRAP trials, a transport enhancement k_load/k_diff of 1.4 was found.

Fig. 4

Simulated transport enhancement (k_diff/k_load) as a function of the peak LCS fluid velocity in loaded murine tibia. A power relationship was found between the peak flow velocity and transport enhancement (k_load/k_diff = 1.0 + 3.7 × 10⁻⁴ × u^1.65, the hatched curve, r² = 0.99). A range of flow velocities (24 to 84 µm/s, the shaded region) corresponded to the experimentally observed transport enhancements (k_load/k_diff = 1.31 ± 0.24).