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 µɛ 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. © 2011 American Society for Bone and Mineral Research.
Highlights
In vivo tracer perfusion studies have demonstrated the positive correlations between mechanical loading and the penetration of tracers within the bone lacunar-canaliculi system (LCS).[18,19,20] these studies provide only static snapshots of tracer localization, lack the temporal dynamics of fluid and solute transport processes, and often are prone to histologic artifacts that could confound the interpretation of results.[22]. Measurements of stress-generated streaming potentials[23,24,25] have provided more direct evidence of macroscopic fluid movement in loaded bone, but the physical size of the probing electrodes limits these studies to exposed surfaces and carries a low spatial resolution beyond the canalicular level
We pooled the data from both groups and found that the applied mechanical loading induced a þ31% increase in the transport of sodium fluorescein over diffusion, which was significant when compared with a theoretical kload/kdiff value of 1.0 using both a one-sample t test and a Wilcoxon signed-rank test ( p < .0001)
Over the last 30 years, load-induced fluid flow within the bone LCS has emerged as an important transport enhancement mechanism between the vasculature and osteocytes as well as a potent mechanical stimulation to bone cells
Summary
Osteocytes, the most numerous cells in bone, form a cellular network embedded within the mineralized matrix and are ideally situated to regulate the homeostasis and mechanical adaptation of bone.[1,2] The viability and function of osteocytes require an adequate supply of nutrients (eg, glucose), disposal of waste products (eg, lactic acid), and exchange of endocrine, paracrine, and autocrine regulatory signals (eg, sex hormones, nitric oxide, prostaglandins, cytokines, and growth factors).(1,2) simple diffusion of these molecules may not be sufficient for maintaining cell viability and function[3] because [1] osteocytes are embedded in a largely impermeable matrix,(4) [2] solute transport is restricted to the narrow pericellular annular fluid space surrounding the osteocyte cell body and processes (gap < 1 mm),(5) whereas the cell-to-cell spacing is relatively long ($30 mm),(6) and [3] some osteocytes are found at great distances from the vascular supply (up to 200 to 300 mm).(7) As a potential solution to this problem, Pierkarski and Munro, in their seminal 1977 paper, proposed that load-induced fluid flow within the bone lacunar-canaliculi system (LCS) serves as the primary transport mechanism operating between the blood supply and osteocytes.[3]. To overcome the challenges of quantifying transport dynamics at the bone’s cellular level, we recently developed a novel in situ imaging approach based on fluorescence recovery after photobleaching (FRAP).(6) By irreversibly photobleaching exogenously injected tracer molecules within individual osteocyte lacunae and recording their subsequent fluorescence recovery, we were the first to directly quantify the diffusion of various molecules within the intact bone LCS in the absence of applied load.[6,26] The feasibility of using FRAP to measure loadinduced convection was investigated further in our multiscaled modeling study,(16) where a poroelastic model of an intact murine tibia subjected to cyclic intermittent end compression was combined with a microscopic three-compartment LCS model to simulate hypothetical FRAP experiments under various loading parameters (such as peak load, loading period, resting period, and tracer size). The goal of this study was to quantitatively measure the dynamic process of solute convection and fluid flow within the LCS of bones subjected to physiologically relevant mechanical loading using the novel FRAP approach
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