First Bone Fluid Flow Workshop

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. The speakers were Shelly Weinbaum (CCNY), Yi-Xian Qin (SUNY Stony Brook), Steve Doty (Hospital for Special Surgery), Melissa Knothe Tate (ETH- Zurich, AO Research Institute- Davos), Eric Nauman (Orthopaedic Biomechanics Lab., UC Berkeley), Dajun Zhang (CCNY), P.J. Kelly (Orthopedics, Mayo Clinic, Retired), Todd McAllister (Bioengineering, UCSD), Jenneke Klein-Nulend (Free University, Amsterdam), Sol Pollack (Bioengineering, Penn) and Elizabeth Burger (Free University, Amsterdam). The contents of the presentations of the eleven speakers are summarized in the following paragraphs.

The first technical presentation was an Overview of Bone Fluid Flow by Shelly Weinbaum. 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 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 work 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 flows using experimental methods. She then reported on theoretical and experimental methods developed to elucidate the role of interstitial fluid flow within the relatively impermeable tissue of compact bone. She reported that data from her theoretical (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. The 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. 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 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 also 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 ACTA-Vrije Universiteit in Amsterdam spoke on the topic: Osteocyte Mechanosensitivity and Prostaglandins. As background and motivation, Dr. Klein-Nulend observed that bone cells, in particular osteocytes, are extremely sensitive to mechanical stress, a quality that is probably linked to the process of mechanical adaptation (Wolff’s Law). She observed that mechanical stress produces flow of interstitial fluid in the bone lacunar-canalicular network along the surface of osteocytes and lining cells, and is likely the physiological signal for bone cell adaptive responses in vivo. Her previous work has shown osteocytes to be particularly sensitive to fluid flow, and less sensitive to hydrostatic compression. The response of bone cells in culture to fluid flow includes prostaglandin synthesis and expression of inducible 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. 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 we 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 discussed.

Elizabeth Burger from the 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.

—Summary by Steve Cowin and Susannah Fritton, September 15, 1997