In situ measurement of solute transport in the bone lacunar-canalicular system.
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC1187997 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 µ? 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.
| S-EPMC3179346 | biostudies-literature
Project description:Osteocytes, the most abundant cells in bone, are essential in maintaining tissue homeostasis and orchestrating bone's 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 (? = 0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCM's 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.
| S-EPMC3593787 | biostudies-literature
Project description:The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both "outside-in" (mechanosensing) and "inside-out" (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (?100?nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (-35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.
| S-EPMC3962519 | biostudies-literature
Project description:In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.
| S-EPMC4773178 | biostudies-literature
Project description:The lacunar-canalicular network (LCN) of bone contains osteocytes and their dendritic extensions, which allow for intercellular communication, and are believed to serve as the mechanosensors that coordinate the processes of bone modeling and remodeling. Imbalances in remodeling, for example, are linked to bone disease, including fragility associated with aging. We have reported that there is a reduction in scale for one component of the LCN, osteocyte lacunar volume, across the human lifespan in females. In the present study, we explore the hypothesis that canalicular porosity also declines with age. To visualize the LCN and to determine how its components are altered with aging, we examined samples from young (age: 20-23 y; n = 5) and aged (age: 70-86 y; n = 6) healthy women donors utilizing a fluorescent labelling technique in combination with confocal laser scanning microscopy. A large cross-sectional area of cortical bone spanning the endosteal to periosteal surfaces from the anterior proximal femoral shaft was examined in order to account for potential trans-cortical variation in the LCN. Overall, we found that LCN areal fraction was reduced by 40.6% in the samples from aged women. This reduction was due, in part, to a reduction in lacunar density (21.4% decline in lacunae number per given area of bone), but much more so due to a 44.6% decline in canalicular areal fraction. While the areal fraction of larger vascular canals was higher in endosteal vs. periosteal regions for both age groups, no regional differences were observed in the areal fractions of the LCN and its components for either age group. Our data indicate that the LCN is diminished in aged women, and is largely due to a decline in the canalicular areal fraction, and that, unlike vascular canal porosity, this diminished LCN is uniform across the cortex.
| S-EPMC5517690 | biostudies-literature
Project description: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 microm(2)/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 (approximately 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.
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Project description:Myeloma bone disease (MBD) affects ~90% of multiple myeloma patients, but current treatment options are suboptimal. Therefore, to successfully develop new therapies or optimize current ones, we must improve our fundamental knowledge of how myeloma affects bone microstructure and function. Here, we have investigated the osteocyte lacuno-canalicular network (LCN) in MBD, as bone porosity affects bone quality and resilience. We used the syngeneic 5TGM1-C57BL-Kalwrij and the xenograft U266-NSG models at end stage and compared them to healthy controls (naïve). Micro-computed tomography (μCT) and histomorphometry indicated the 5TGM1 and U266 models developed mild and extensive MBD, respectively, with the U266 model producing large osteolytic lesions. High-resolution synchrotron micro-CT (SR-μCT) revealed significant osteocyte lacunae changes in U266 bones but not 5TGM1, with a reduction in lacunae number and sphericity, and an increase in lacunae volume compared with naïve. Canalicular length, visualized using histological Ploton silver staining, appeared significantly shorter in 5TGM1 and U266 bones compared with naïve. Canalicular area as a proportion of the bone was also decreased by 24.2% in the U266 model. We observed significant upregulation of genes implicated in peri-lacunar remodeling (PLR), but immunohistochemistry confirmed that the osteocyte-specific protein sclerostin, a known driver of PLR, was unchanged between MBD and naïve bones. In summary, we have demonstrated evidence of PLR and altered organization of the osteocyte LCN in MBD mouse models. The next step would be to further understand the drivers and implications of PLR in MBD, and whether treatments to manipulate PLR and the LCN may improve patient outcomes.
| S-EPMC11299509 | biostudies-literature
Project description:ObjectiveTo establish a method for investigating the permeability of calcified cartilage zone (CCZ) and to observe solute transport between articular cartilage (AC) and subchondral bone (SB) through intact CCZ in vivo.DesignWe developed a novel fixing device combined with un-decalcified fluorescence observation method to address the permeability of CCZ in live mice. Twenty-four Balb/c female mice aged 1 to 8 months were used to observe the development of CCZ. Eighty-four Balb/c female mice (aged 1 or 6 months) with mature or immature CCZ of distal femur were used to investigate the permeability of intact CCZ in vivo. Diffusivity of rhodamine B (476 Da) and tetramethyl-rhodamine isothicyanate-dextran (TRITC-Dextran, 20 kDa) was tested from AC to SB in 0 minutes, 1 minute, 15 minutes, 30 minutes, 1 hour, and 2 hours. None diffused knee joints (0 minutes) served as blank control, while in vitro immersion of distal femurs in rhodamine B or TRITC-Dextran for 72 hours served as positive control.ResultsCCZ was well developed in 6-month mice. Both tracers penetrated immature CCZ down to SB in less than 1 hour in live mice, while the diffusion of both tracers decreased rapidly at tidemark in all testing time points.ConclusionCurrent study provided direct evidence of blocking effect of CCZ in solute transportation during short diffusion period in live animal, indicating the important role of CCZ in joint development and microenvironment maintenance.
| S-EPMC8804871 | biostudies-literature
Project description:Extremity reconstruction surgery is increasingly performed rather than amputation for patients with large-segment pathologic bone loss. Debate persists as to the optimal void filler for this "limb salvage" surgery, whether metal or allograft bone. Clinicians focus on optimizing important functional gains for patients, and the risk of devastating implant infection has been thought to be similar regardless of implant material. Recent insights into infection pathophysiology are challenging this equipoise, however, with both basic science data suggesting a novel mechanism of infection of Staphylococcus aureus (the most common infecting agent) into the host lacunar-canaliculi network, and also clinical data revealing a higher rate of infection of allograft over metal. The current translational study was therefore developed to bridge the gap between these insights in a longitudinal murine model of infection of allograft bone and metal. Real-time Staphylococci infection characteristics were quantified in cortical bone vs metal, and both microarchitecture of host implant and presence of host immune response were assessed. An orders-of-magnitude higher bacterial burden was established in cortical allograft bone over both metal and cancellous bone. The establishment of immune-evading microabscesses was confirmed in both cortical allograft haversian canal and the submicron canaliculi network in an additional model of mouse femur bone infection. These study results reveal a mechanism by which Staphylococci evasion of host immunity is possible, contributing to elevated risks of infection in cortical bone. The presence of this local infection reservoir imparts massive clinical implications that may alter the current paradigm of osteomyelitis and bulk allograft infection treatment.
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Project description:The glymphatic system (GS) hypothesis states that advective driven cerebrospinal fluid (CSF) influx from the perivascular spaces into the interstitial fluid space rapidly transport solutes and clear waste from brain. However, the presence of advection in neuropil is contested and solutes are claimed to be transported by diffusion only. To address this controversy, we implemented a regularized version of the optimal mass transport (rOMT) problem, wherein the advection/diffusion equation is the only a priori assumption required. rOMT analysis with a Lagrangian perspective of GS transport revealed that solute speed was faster in CSF compared to grey and white matter. Further, rOMT analysis also demonstrated 2-fold differences in regional solute speed within the brain. Collectively, these results imply that advective transport dominates in CSF while diffusion and advection both contribute to GS transport in parenchyma. In a rat model of cerebral small vessel disease (cSVD), solute transport in the perivascular spaces (PVS) and PVS-to-tissue transfer was slower compared to normal rats. Thus, the analytical framework of rOMT provides novel insights in the local dynamics of GS transport that may have implications for neurodegenerative diseases. Future studies should apply the rOMT analysis approach to confirm GS transport reductions in humans with cSVD.
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