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  • Bone Fluid Flow Workshop Summary

    Summary of a Workshop on Bone Fluid Flow

    A Bone Fluid Flow Workshop with the objective of summarizing the
    state of the research on bone fluid flow and its role in the bone
    tissue mechanosensory system was held on September 8th, 1997 at the
    City College of the City University of New York (CCNY). The workshop
    was sponsored by City College's Center for Biomedical Engineering and
    was organized by Steve Cowin, Shelly Weinbaum, and Susannah Fritton.
    More than 50 people attended the workshop, and the speakers were Shelly
    Weinbaum (Center for Biomedical Engineering, CCNY), Yi-Xian Qin
    (Musculo-Skeletal Research Lab, SUNY Stony Brook), Steve Doty
    (Analytical Microscopy Core, Hospital for Special Surgery), Melissa
    Knothe Tate (University and Swiss Federal Institute of
    Technology-Zurich, AO Research Institute-Davos), Eric Nauman
    (Orthopaedic Biomechanics Lab, UC Berkeley), Dajun Zhang (Center for
    Biomedical Engineering, CCNY), P.J. Kelly (Orthopedics, Mayo Clinic,
    Retired), Todd McAllister (Bioengineering, UCSD), Jenneke Klein-Nulend
    (Dept. of Oral Cell Biology, Free University, Amsterdam), Sol Pollack
    (Bioengineering, Penn) and Elisabeth Burger (Dept. of Oral Cell
    Biology, Free University, Amsterdam). The contents of the presentations
    of the eleven speakers are summarized in the following eleven

    The first technical presentation was an Overview of Bone Fluid
    Flow by Shelly Weinbaum from CCNY. The overview addressed fluid movement
    in both the lacunar-canalicular porosity and blood flow in bone. A
    review of bone blood flow was accompanied by the suggestion that the
    periosteum acts as a high pressure membrane maintaining the mean pore
    pressure in bone at about 50 mm of Hg. The possibility of fluid pores
    within the mineralized matrix was examined and the arguments for and
    against the mineralized matrix or the lacunar-canalicular porosity being
    the site of the strain generated potential were presented. The role and
    characterization of the gel-like matrix structure in the fluid annulus
    surrounding the osteocytic processes was discussed and its importance
    in the electromechanical coupling between the fluid flow in the annulus
    and intracellular currents explored. The role of this matrix in
    modulating the fluid shear stresses on osteocytes induced by mechanical
    loading was analyzed and recent experiments investigating the
    intracellular chemical response of bone cells to shear stress in tissue
    culture were summarized.

    Yi-Xian Qin, presently a post-doc from the Musculo-Skeletal
    Research Laboratory at SUNY Stony Brook, spoke on The Interdependence
    of Intracortical Fluid Flow and Loading Frequency, and Their Regulatory
    Role in Bone Adaptation. The speaker noted that there exists an
    increasing body of analytical and experimental evidence which
    demonstrates that fluid flow may be an important mediator of bone cell
    activity. He also observed that perturbation of this flow, via changes
    in functional activity, may ultimately prove the key influence in the
    plasticity of the skeleton. Should fluid flow control bone cell
    activity, then loading regimens which are osteogenic should generate a
    distribution of intracortical fluid pressure which correlates to the
    remodeling response. In the experiments reported by Dr. Qin the
    potential role of fluid flow in adaptive responses in bone was
    investigated through the use of an in vivo animal model in which the
    mechanical loading environment can be controlled, the adaptive response
    quantified, and the fluid flow estimated through the resultant streaming
    potentials and numerical modeling. The influence of surface leakage
    boundary conditions was discussed and evaluated in the context of
    analytical one-dimensional models. A strong correlation was found
    between transcortical fluid flow and streaming potentials in two
    distinct loading cases, with a strong dependence on the loading
    frequency. The results suggested that intracortical fluid flow is a
    product of both matrix strain gradients and intramedullary pressure, the
    latter arising primarily through volumetric changes in the marrow
    cavity. These two distinct sources for remodeling stimuli may explain,
    at least in part, the differential remodeling responses observed as a
    function of strain frequency.

    Steve Doty from the Hospital for Special Surgery in New York
    gave a talk entitled Morphologic and Tracer Studies of the Flow
    Through Bone. Dr. Doty reported on a morphologic approach to
    understanding flow through bone that was executed by using in-vivo
    tracers of different size and chemistry. Vascular transport of
    microperoxidase, native ferritin and tetracycline was described as they
    flow from the blood vessels to surrounding osteocytes within compact
    bone. He also considered the escape of these tracers from the vascular-
    osteocyte system into the surrounding bone matrix. A comparison was
    made between tracer flow through lamellar bone (rat) and Haversian
    systems (rabbit). This information is being collected to aid in the
    description of the flow path in bone and the size of "pores" in this
    path which might regulate the flow rate. Dr. Doty also reported on a 3-
    dimensional morphologic analysis whose objective was to describe the
    vascular and osteocyte relationships and the osteocyte and their
    canalicular systems. Methacrylate or resin casts of these structures
    were made from compact bone and following polymerization, the bone is
    etched away from the plastic to provide a 3-dimensional structure.
    Scanning electron micrographs are being taken and relevant dimensions
    and sizes of the cell structures will be collected. These electron
    micrographs were considered by the audience to be quite striking and
    revealing, although the results were consistent with the present state
    of knowledge of these nano-scale structures.

    Melissa Knothe Tate from Zurich and Davos reported on her computational
    modelling and animal experiments in a presentation entitled Measurement
    of Load-Induced Fluid Flow as a Function of Mechanical Loading
    Parameters. She began with the observation that, due to the
    inaccessibility of the minute spaces through which load-induced fluid
    flow is believed to occur, it is inherently difficult to measure
    directly load-induced fluid displacements using experimental methods.
    She then reported on theoretical and newly developed experimental models
    designed to elucidate interstitial fluid flow and its role in processes
    associated with growth and functional adaptation. Data from her mass
    transport and fluid displacement finite element models, ex vivo model of
    the sheep forelimb, in vitro model of small, cylindrical compact bone
    specimens and in vivo model of the rat tibia show significant
    enhancement of molecular transport resulting from mechanical loading of
    the poroelastic, fluid-filled bone tissue. In addition, observed
    patterns of fluid flow and tracer transport were delineated as a
    function of mechanical parameters (e.g. strain magnitude, number of
    cycles, strain rate) as well as tracer molecule size. Finally, she
    observed that, if specific tracer distributions caused by deformation
    induced fluid flow can be related to cellular activity associated with
    remodeling processes, the mechanisms for functional adaptation within
    the context of Wolff's Law would have to be expanded to include effects
    of load-induced fluid flow.

    Eric Nauman, presently a graduate student working with Tony
    Keaveny at Berkeley, presented their joint work: The Dependence of
    Inter-Trabecular Permeability on Volume Fraction and Trabecular
    Orientation. This research concerns the pressure gradients in the marrow
    and interstitial fluid created by the mechanical loading of trabecular
    bone which deforms the trabecular matrix. The resulting fluid flow
    exerts shear stresses on the bone lining cells and may stimulate
    remodeling. In addition, fluid flow plays an important role in the
    integration of bone grafts and the hydraulic stiffening of trabecular
    bone during impact loading. A fundamental parameter for characterizing
    the flow through the inter-trabecular pores is the permeability. The
    wide range of experimental values in the literature indicates that this
    aspect of trabecular fluid flow is not well understood. Thus, there is a
    need for development of a theoretical model that can be used to
    interpret existing data. This model could also be used with poroelastic
    models of trabecular bone to determine the physiological range of fluid
    shear stresses exerted on the bone lining cells. The goal of this work
    is to develop a simple cellular solid model that describes the
    dependence of inter-trabecular permeability on volume fraction and
    orientation. The model will then be validated by comparison with
    experimentally obtained inter-trabecular permeabilities for a range of
    anatomic sites.

    Dajun Zhang, presently a post-doc in the Center for Biomedical
    Engineering at CCNY, gave a talk entitled Modeling Electrical Signal
    Transmission in the Bone Cell Network. Dr. Zhang noted that it is now
    generally accepted that the weak strain generated electrical potentials
    (SGPs) in wet bone are dominantly caused by the streaming potentials
    established by strain-induced bone fluid flow. He reported his
    calculation of the intracellular potential and current induced by the
    load-driven streaming potentials within a representative osteocytic
    process along the radius of a typical osteon. The streaming potential is
    derived based on poroelasticity theory and electrokinetic theory and the
    intracellular electrical response is evaluated through the cable theory.
    Particularly, his results demonstrated that the SGP-induced variations
    in the transmembrane potential at bone lining cells located along the
    wall of the Haversian canal behave as a high-pass, low-pass filter with
    respect to loading frequency. This strong frequency selectivity suggests
    that intermediate-frequency (15 - 30 Hz), low-amplitude mechanical
    loading, such as those contributed by muscle tone, may be important to
    bone maintenance and remodeling.

    Pat Kelly, Emeritus Professor of Orthopedics at the Mayo
    Clinic, spoke on the topic Fluid Flow and Bone Formation. Dr. Kelly
    described a venous tourniquet model in the canine that was employed to
    study fluid flow, bone formation and pressure effects across the
    capillary barrier (Kelly et al., Clinical Orthopedics and Related
    Research, Vol. 254, 1990). The channels for fluid flow were
    demonstrated in the presentation. Experiments on weight bearing and
    non weight bearing canine tibiae show that less bone appears in non
    weight bearing tibial defects than in weight bearing tibial defects.
    Studies in the same model reveal that the interstitial fluid space (ISF)
    is less on the non weight bearing side than on the weight bearing side.
    The hypothesis offered is that less function results in decreased bone
    formation because of a decrease in capillary filtration and a decrease
    in perfusion of the osteoblast with important solutes that are needed
    for osteoblastic activity; alternative explanations were offered.

    Todd McAllister, presently a graduate student working with
    John Frangos in Bioengineering at UCSD, presented their joint work:
    Characteristics of Flow-Induced Nitric Oxide Release in Osteoblasts. In
    their background remarks the authors noted that transcortical
    interstitial fluid flow has been shown to be a potent stimulus for
    osteogenic autocrine/paracrine factors. Previously the authors have
    demonstrated that fluid flow-induced shear stress stimulates nitric
    oxide (NO) and prostaglandin E2 (PGE2) release in cultured osteoblasts.
    The purpose of the current study was to identify the role of calcium and
    G-proteins in this flow-mediated signal transduction. Flow-induced NO
    release in osteoblasts demonstrated a biphasic response, with an initial
    burst (8.2 nmols/mg/hr) followed by a steady and sustained production
    (2.2 nmols/mg/hr). Treatment with GDPbS (900 uM) or quin 2/AM (30uM)
    inhibits this initial response, but does not significantly attenuate
    sustained production. G-protein activation with GTPgS (300-900 uM)
    stimulated a dose dependent and sustained release. Calcium ionophore
    (1uM) stimulated an initial burst, but no sustained production. Taken
    together, these data suggest that flow-induced NO production in
    osteoblasts is regulated by two distinct mechanisms. Transients in
    shear activate a G-protein and calcium dependent pathway, while steady
    flow activates a calcium independent pathway.

    Jenneke Klein-Nulend from the Dental School ACTA-Vrije Universiteit in
    Amsterdam spoke on the topic Osteocyte Mechanosensitivity and
    Prostaglandins. As a background and motivation, Dr. Klein-Nulend
    observed that mechanical stress produces flow of interstitial fluid in
    the bone lacunar-canalicular network along the surface of osteocytes and
    lining cells. This flow has been postulated to provide the physiological
    signal for bone cell adaptive responses in vivo. Over the last few
    years, Dr. Klein-Nulend and colleagues have examined this theory
    experimentally, using among others cell cultures of isolated osteocytes
    from embryonic chicken calvariae. Their work has shown that bone cells,
    in particular osteocytes, are extremely sensitive to mechanical stress,
    and that osteocytes are much more sensitive to fluid flow than to
    hydrostatic compression. The response of bone cells in culture of fluid
    flow includes prostaglandin synthesis and induction of expression of
    prostaglandin G/H synthase (PGHS-2 or inducible cyclooxygenase, COX-2),
    an enzyme that mediates the induction of bone formation by mechanical
    loading in vivo. This response was also observed in the presence of very
    low serum concentration (0.1%). Disruption of the actin-cytoskeleton
    abolishes the response to stress, suggesting that the cytoskeleton is
    involved in cellular mechanotransduction. The data reported support the
    hypothesis that stress on bone causes fluid flow in the
    lacunar-canalicular system, which stimulates osteocytes to produce
    prostaglandins that induce an osteogenic response.

    Sol Pollack from Bioengineering at Penn gave a talk entitled
    Fluid Flow Effects on Osteoblast Intracellular Calcium Concentration.
    Professor Pollack began by noting that investigations of cellular
    interactions with their local physical environment aim to elucidate the
    mechanism by which physical forces are transduced into cytostolic and
    nuclear events that ultimately determine the state and function of the
    cell. Efforts in his laboratory have detailed a distinct dose-response
    interaction involving fluid flow induced shear stress amplitude and an
    increase in intracellular calcium concentration, [Ca2+], in primary
    cultured osteoblast-like cells. The amplitude of the calcium response
    was significantly increased by the presence of serum. By the use of
    appropriate blockers they have identified that it is the inositol
    phospholipid pathway that leads to the intracellular calcium
    mobilization from the endoplasmic reticulum. However in the presence of
    serum the flow transduction is dependent on pertussis toxin sensitive
    G-proteins while the serum free transduction is not. Furthermore, the
    amplitude of the calcium response in the absence of serum is reduced by
    passing the primary cells, is non-existent in cloned and transformed
    osteoblasts, is blocked by Gadolinium suggesting the involvement of
    stretch receptors and is significantly reduced when calcium is
    eliminated from the perfusate. Combined with the observed increase in
    the calcium amplitude with serum concentration, Professor Pollack labels
    the serum free mechanism "mechano-transduction" and the mechanism with
    serum as a "mass transport" mechanism. Arguments for both were

    Elisabeth Burger from the Dental School ACTA-Vrije Universiteit in
    Amsterdam spoke on the topic Bone Cell Mechanosensitivity and
    Osteoporosis. Professor Burger related the day's discussions to clinical
    problems. She noted that recent studies address the issue of bone cell
    mechanosensitivity in relation to the emerging awareness that
    mechanical disuse may be an important determinant of bone weakness
    as in osteoporosis. It is known that bone metabolism is changed in
    osteoporotic (OP) patients but a relationship with abnormal
    mechanosensitivity of bone tissue is unknown. As a first step to test
    the hypothesis that a low bone cell mechanosensitivity may predispose an
    individual for osteoporosis in later life, she compared the in vitro
    response to stress of bone cells from OP patients with cells from age-
    matched controls. Primary bone cell cultures from iliac bone biopsies of
    9 OP patients (3 male, 6 females, 47-72y) and 6 controls (4 males, 2
    females, 44-77y) were mechanically stressed for 1 h by pulsating fluid
    flow (PFF, 0.7*0.03 Pa at 5 Hz, peak stress rate 12 Pa/sec). Both OP
    and CO cells increased their release of prostaglandin E2 (PGE2) and
    nitric oxide (NO) during 1 h PFF-treatment, in agreement with earlier
    findings in mouse and chicken bone cells. However, at 24 h after 1 h
    PFF treatment, the release of PGE2 was still enhanced by more than
    two-fold in the CO cell cultures, but not in the OP cultures. As PGE2
    is likely involved in the transduction of mechanical signals, these data
    suggest that the long-term response of osteoporotic bone to mechanical
    stress may be changed. She speculated that a disease-related abnormality
    in the mechanosensitivity of bone cells may be involved in the
    pathogenesis of osteoporosis.

    Steve Cowin and Susannah Fritton, September 25th, 1997.