Xiaozhou Zhou

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

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.

In situ measurement of transport between subchondral bone and articular cartilage

Subchondral bone and articular cartilage play complementary roles in load bearing of the joints. Although the biomechanical coupling between subchondral bone and articular cartilage is well established, it remains unclear whether direct biochemical communication exists between them. Previously, the calcified cartilage between these two compartments was generally believed to be impermeable to transport of solutes and gases. However, recent studies found that small molecules could penetrate into the calcified cartilage from the subchondral bone. To quantify the real‐time solute transport across the calcified cartilage, we developed a novel imaging method based on fluorescence loss induced by photobleaching (FLIP). Diffusivity of sodium fluorescein (376 Da) was quantified to be 0.07 ± 0.03 and 0.26 ± 0.22 µm2/s between subchondral bone and calcified cartilage and within the calcified cartilage in the murine distal femur, respectively. Electron microscopy revealed that calcified cartilage matrix contained nonmineralized regions (∼22% volume fraction) that are either large patches (53 ± 18 nm) among the mineral deposits or numerous small regions (4.5 ± 0.8 nm) within the mineral deposits, which may serve as transport pathways. These results suggest that there exists a possible direct signaling between subchondral bone and articular cartilage, and they form a functional unit with both mechanical and biochemical interactions, which may play a role in the maintenance and degeneration of the joint.

Quantifying load‐induced solute transport and solute‐matrix interaction within the osteocyte lacunar‐canalicular system

Osteocytes, the most abundant cells in bone, are essential in maintaining tissue homeostasis and orchestrating bones mechanical adaptation. Osteocytes depend upon load‐induced convection within the lacunar‐canalicular system (LCS) to maintain viability and to sense their mechanical environment. Using the fluorescence recovery after photobleaching (FRAP) imaging approach, we previously quantified the convection of a small tracer (sodium fluorescein, 376 Da) in the murine tibial LCS under intermittent cyclic loading. In the present study, we first expanded the investigation of solute transport using a larger tracer (parvalbumin, 12.3 kDa), which is comparable in size to some signaling proteins secreted by osteocytes. Murine tibiae were subjected to sequential FRAP tests under rest‐inserted cyclic loading while the loading magnitude (0, 2.8, or 4.8 N) and frequency (0.5, 1, or 2 Hz) were varied. The characteristic transport rate k and the transport enhancement relative to diffusion (k/k0) were measured under each loading condition, from which the peak solute velocity in the LCS was derived using our LCS transport model. Both the transport enhancement and solute velocity increased with loading magnitude and decreased with loading frequency. Furthermore, the solute‐matrix interaction, quantified in terms of the reflection coefficient through the osteocytic pericellular matrix (PCM), was measured and theoretically modeled. The reflection coefficient of parvalbumin (σ = 0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCMs fiber configurations accounting for the measured interactions were obtained for the first time. The present study provided not only new data on the micro‐fluidic environment experienced by osteocytes in situ but also a powerful quantitative tool for future study of the PCM, the critical interface that controls both outside‐in and inside‐out signaling in osteocytes during normal bone adaptation and in pathological conditions.

Anatomic Variations of the Lacunar-Canalicular System Influence Solute Transport in Bone

Solute transport in the lacunar-canalicular system (LCS) is essential for bone metabolism and mechanotransduction. Using the technique of fluorescence recovery after photobleaching (FRAP) we have been quantifying solute transport in the LCS of murine long bone as a function of loading parameters and molecular size. However, the influence of LCS anatomy, which varies among animal species, bone type and location, age and health condition, is not well understood. In this study, we developed a mathematical model to simulate solute convection in the LCS during a FRAP experiment under a physiological cyclic flow. We found that the transport rate (the reciprocal time constant for refilling the photobleached lacuna) increased linearly with canalicular number and decreased with canalicular length for both diffusion and convection. As a result, the transport enhancement of convection over diffusion was much less sensitive to the variations associated with chick, mouse, rabbit, bovine, dog, horse, and human LCS anatomy, when compared with the rates of diffusion or convection alone. Canalicular density did not affect transport enhancement, while solute size and the lacunar density had more complicated, non-linear effects. This parametric study suggests that solute transport could be altered by varying LCS parameters, and that the anatomical details of the LCS need systemic examination to further understand the etiology of aged and osteoporotic bones.

Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice

Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29‐null mice is due to delayed entry of chondro‐osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro‐computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender‐specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender‐matched controls in three‐point bending tests, and smaller post‐yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29‐deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub‐efficient protein translation increased fracture risk in HIP/RPL29‐null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality.

Deficiency in Perlecan/HSPG2 During Bone Development Enhances Osteogenesis and Decreases Quality of Adult Bone in Mice

Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz–Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.

Investigating the Sieving and Structural Property of the Osteocyte Pericellular Matrix: Experiments and Modeling

Growing evidence shows that osteocytes, the most abundant bone cells, serve as the primary sensory cells that detect external mechanical forces [1], enabling the bone to adapt its mass and structure to meet its environmental requirements and to fulfill its weight bearing functions [2]. Although the cellular and molecular mechanisms of such adaptation phenomena are not fully understood, recent experiments and theoretical models suggest that the pericellular matrix (PCM) filling the tiny gap between the cell membrane and the canalicular matrix wall plays a critical role in the osteocytes’ outside-in signaling process [1]. Weinbaum first hypothesized that a proteoglycan-like fiber matrix, similar to the endothelial glycocalyx, must exist within the PCM to account for the surprisingly long relaxation times of the strain-generated potentials measured in bone [3]. Such a filling matrix was predicted to impose hydraulic resistance, impede fluid pressure relaxation and reduce fluid flow in the tiny lacunar-canalicular pore system in bone, thus protecting the cell membranes from being ruptured under shear. Later electronic microscopic studies confirmed the existence and the proteoglycan nature of the PCM [4]. Previous models using idealized PCM ultrastructure suggested that the hydrodynamic interactions between the PCM and fluid could determine the magnitude of drag forces that deform cytoskeleton via tethered transmembrane components or the focal contacts containing integrins [5,6]. In both scenarios, the PCM is the key to force transmission and strain signal amplification, and responsible for downstream mechanotransduction. In addition, once the mechanically excited osteocytes affect the release of molecular signals such as ATP, NO, PGE2, OPG, RANKL, and sclerostin [2], the PCM, as a molecular sieve and temporary storage, may influence the transport and availability of these bioactive molecules [7]. Therefore, the structural and sieving properties of PCM are important in regulating bone’s mechanotransduction and adaptation. However, due to the small dimensions of the PCM (∼100nm thick) and the difficulty in preserving the PCM in situ, its detailed structure and properties have remained elusive [4]. The objective of this study was to elucidate the sieving property of the PCM in mechanically loaded bone with an innovative imaging approach and to further deduce plausible PCM structures using mathematical modeling.Copyright