2nd Bone Fluid Flow Workshop
The City College of New York  9/20/00
Summary

A Second 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 20th, 2000 at the City College of New York (CCNY). The workshop was sponsored by City College’s New York Center for Biomedical Engineering and was organized by Steve Cowin, Shelly Weinbaum, and Susannah Fritton. The program, with abstracts, the list of attendees and other information is located here. There were 80 attendees, 40 from New York State, 28 from out of state including 4 from the west coast and 12 from outside the country (Austria, Bulgaria, Germany, Japan, the Netherlands, Scotland and Switzerland). The greatest measure of success of this workshop, after the high number of attendees, was the fact that others have volunteered to hold the third and fourth bone fluid flow workshops. John Frangos from UCSD will organize the third workshop before the ASBMR meeting in Phoenix in 2001 and Jenneke Klein-Nulend and Theo Smit will organize the fourth workshop in Amsterdam in 2002 to honor Professor Elisabeth Burger of the Oral Cell Biology Department at Vrije Universiteit upon her retirement.

The workshop speakers were Mitch Schaffler (Mount Sinai, New York), Melissa Knothe Tate (ETH-Zurich, AO Research Institute-Davos & Mount Sinai, New York), Mark Otter (SUNY Stony Brook), Elizabeth Burger (Vrije Universiteit, Amsterdam), Theo Smit (Vrije Universiteit, Amsterdam), John Frangos (UCSD), Hank Donahue (Penn State University), Yi-Xian Qin (SUNY Stony Brook), Roland Steck (ETH-Zurich, AO Research Institute-Davos), Nikola Petrov (Bulgarian Academy of Sciences), Shelly Weinbaum (CCNY), Liyun Wang (CCNY), and Lidan You (CCNY).

The contents of the presentations of the thirteen speakers are summarized below.  In addition, the contents of four short communications are described.  The four short communications were given by Chris Jacobs (Penn State University), Colby Swan (University of Iowa), David Denhardt (Rutgers University)& Masaki Noda (Tokyo Medical & Dental University), and Laura McCabe (Michigan State University).



Bone Fatigue and Remodeling in the Development of Stress Fractures
The first technical presentation entitled “Bone fatigue and remodeling in the development of stress fractures” was given by Mitch Schaffler (Mount Sinai, New York). Schaffler began by noting that stress fractures result from repetitive loading. As such they have often been regarded as a mechanical fatigue-driven process.  However, while bone readily sustains fatigue microdamage during the course of repeated loading at the stresses or strains encountered in normal activities, it does not progress to fracture in the time course seen for the development of stress fracture.  This suggests that other mechanisms drive the development of stress fractures.  Histopathological data from humans and racehorses suggest that increased remodeling is a prominent early feature of stress fracture. Early increases in intracortical remodeling were observed experimentally in the rabbit tibial stress fracture model developed in their laboratory. Together these studies suggest a central role for increased intracortical remodeling in the pathogenesis of stress fractures.  Schaffler proposed that the model that best explains the development of stress fracture is that of a biologically (remodeling)-driven damage accumulation system.  In this model, stress fracture occurs as a positive feedback mechanism, wherein increased mechanical usage stimulates bone turnover, which results in focally increased bone remodeling space (porosity) and decreased bone mass.  There is a wide range of factors (low level bone fatigue, altered mechanical loading, injury, cytokines, vascular alterations) that potentially can activate local bone remodeling; all of these can occur in the development of stress fracture.  With continued loading of this focally, transiently osteopenic bone, local stresses would be markedly elevated, leading to accelerated matrix damage and failure.  Fracture is the result of continued repetitive loading superimposed on the decreased bone mass caused by more, and larger, resorption spaces.
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The Role of Interstitial Fluid Flow in the Remodeling Response to Fatigue and Disuse
The second technical presentation entitled “The role of interstitial fluid flow in the remodeling response to fatigue and disuse” was given by Melissa L. Knothe Tate (ETH-Zurich, AO Research Institute-Davos & Mount Sinai, New York). Knothe Tate noted that the ability of bone to regenerate itself in response to dynamic metabolic and structural demands is necessary for the survival of vertebrates. Hence, the structure of mature bone represents a patchwork; the initial construct, resulting from modeling events during growth and development, is interwoven with bone laid down by osteoblasts in areas carved out by osteoclasts during periods of remodeling. The osteocytes, which are located within the mineralized matrix of bone, are presumed to play an important role in “sensing” the mechanical and chemical environment within the tissue. Remodeling events appear to be highly “choreographed,” but the signaling and timing of interactions between osteocytes, osteoclasts and osteoblasts are not clear. Osteotropic agents are likely to mediate remodeling processes; the concentration and distribution of such agents are a function of tissue perfusion as well as diffusive and convective transport conditions prevailing in the tissue. Knothe Tate and her colleagues hypothesize that loss of fluid flow and ensuing compromise to molecular transport and exchange is a mechanism causing loss of cell viability and triggering the remodeling response. Microdamage due to fatigue loading alters interstitial fluid flow and mass transport within bone, reducing the concentration and distribution of osteotropic agents to osteocytes “downstream” from the damage. In the case of disuse, the lack of fluid flow and subsequent deficiency in transport through the tissue causes the osteocytes to fall into a state of deprivation, ultimately resulting in a loss of viability. The hypothesis was discussed in light of ongoing work involving theoretical and experimental models as well as changes in osteocyte integrity observed in association with bone resorption following disuse. A model for mechanochemical transduction in bone was introduced.
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Does Bone Perfusion/Reperfusion Initiate Bone Remodeling and the Stress Fracture Syndrome?
In his talk, Mark Otter (SUNY Stony Brook) addressed the question “Does bone perfusion/reperfusion initiate bone remodeling and the stress fracture syndrome?” Otter noted that stress fractures have been proposed to arise from repetitive activity of training, inducing an accumulation of microfractures in locations of peak strain. However, stress fractures most often occur long before accumulation of material damage could occur; they occur in cortical locations of low, not high, strain; and intracortical osteopenia precedes any evidence of microcracks. Otter proposed that this lesion arises from a focal remodeling response to site-specific changes in bone perfusion during redundant axial loading of appendicular bones. Intramedullary pressures significantly exceeding peak arterial pressure are generated by strenuous exercise and if the exercise is maintained, the bone tissue can suffer from ischemia caused by reduced blood flow into the medullary canal and hence to the inner two-thirds of the cortex. Site specificity is caused by the lack, in certain regions of the cortex, of compensating matrix-consolidation-driven fluid flow, which brings nutrients from the periosteal surface to portions of the cortex. Upon cessation of the exercise, re-flow of fresh blood into the vasculature leads to reperfusion injury, causing an extended no-flow or reduced flow to that portion of the bone most strongly denied perfusion during the exercise. This leads to a cell-stress initiated remodeling which ultimately weakens the bone, predisposing it to fracture.
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Cellular Aspects of Mechanotransduction and Bone Remodeling
Elisabeth Burger (Vrije Universiteit, The Netherlands) spoke on “Cellular aspects of mechanotransduction and bone remodeling.” Burger began by noting that the capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized, but the cellular mechanisms remain poorly understood. She noted that several lines of evidence currently emphasize the role of osteocytes as the professional mechanosensors of bone, and the lacunar-canalicular porosity as the structure that mediates osteocyte mechanosensing. Strain-derived flow of interstitial fluid through this porosity is thought to mechanically activate the osteocytes. The narrowness of the canalicular annulus ensures that even the minute physiological bulk strains in bone produce considerable fluid shear stress over the osteocyte cell “finger.” Extracellular and/or intracellular signaling between activated osteocytes and the osteoclasts/osteoblasts at the bone surface provides a mechanism for strain-regulated modulation of bone mass. However, to fully explain mechanical adaptation, a cellular mechanism must exist whereby not only the density of bone tissue is regulated, but also the alignment of the trabeculae and osteons along the dominant loading directions. This asks for a mechanism that guides the direction in which osteoclastic resorption proceeds during the process of bone remodeling. Finite element analysis of remodeling bone at the microscopic, supracellular scale showed opposite strain levels around the cutting- and closing cone of a (hemi-)osteon loaded in the longitudinal (i.e. dominant) direction. A region of decreased strain appeared in front of the cutting cone of the osteonic tunnel, where osteoclasts are activated to continue resorption. Likewise in a trabecula, the presence of a Howship’s lacuna induced the appearance of decreased strain fields along the trabecular surface in the direction of loading. Around the closing cone of the osteonic tunnel however, where osteoblasts are recruited to refill the gap, elevated strains appeared in the tunnel wall. Elevated strains also appeared at the bottom of the Howship’s lacuna. These results suggest that in remodeling bone, osteocytes, informed by locally reduced strain fields, may guide the osteoclasts to resorb bone in the right direction, thereby ensuring correct alignment of the new (hemi-)osteon. They also may determine, based on locally elevated strain fields, the amount of subsequent bone formation by osteoblasts, and thereby the final density of the remodeled piece of bone. Local regulation of bone metabolism by mechanically informed osteocytes provides therefore a mechanism that explains both aspects of mechanical adaptation, correct bone density and correct bone alignment. As each remodeling cycle is also an adapting cycle, adaptation occurs throughout life.
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Fluid Flow During Osteonic Tunnelling
Theo Smit (Vrije Universiteit, The Netherlands) expanded on the modeling work introduced by Elisabeth Burger with a talk entitled “Fluid flow during osteonic tunneling – a biphasic finite element analysis.” Smit began by noting that he and his colleagues have recently found evidence that BMU-coupling during bone remodeling may be regulated by deformation of the bone matrix under mechanical loading. Further, it is generally assumed that mechanosensing by osteocytes is related to extracellular canalicular fluid flow, which is generated by deformation of the bone matrix under mechanical loading. In an attempt to relate the theory of canalicular fluid flow to BMU coupling, they determined the pattern of fluid flow around a tunneling osteon under axial loading.  They approached the problem with Biot’s theory of poroelasticity and the finite element (FE) method. The tunneling osteon was modeled axisymmetrically as a cylindrical gap with a spherical end, and the bone matrix was described as an isotropic material with a fully saturated lacunar-canalicular porosity of 5.0%. They derived the material properties of the mineralized bone matrix from the isotropic description of cortical bone by Cowin & Sadegh (1976) and the equations for porous materials by Christensen (1979). The bulk modulus of the bone fluid was that of water. An important parameter in the model is the hydraulic permeability k, which varies over several orders of magnitude in the literature. They derived its value from the equation for relaxation time by Rice and Cleary (1976) and the experiments by Otter et al. (1992). Their value of 1.2e-7 was two orders of magnitude smaller than the one derived by Zhang et al. (1998). The value of this parameter was discussed in some detail, and a parameter study was presented.  The FE analysis showed that during a walking cycle (4 km/h) a different fluid flow pattern exists near the cutting cone as compared to the closing cone. Or in other words: the findings of this study suggest that the osteocytes within the bone matrix sense different patterns of mechanical stimulation near sites of osteoclastic and osteoblastic activity. This is compatible with the hypothesis that local patterns of bone fluid flow regulate BMU-coupling.
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All Bone Cells are Mechanosensory Cells
John Frangos (UCSD) spoke to the thesis “All bone cells are mechanosensory cells.” Frangos began by observing that it has been proposed that osteocytes are the single mechanosensory cell type because they are ideally situated to sense mechanical stimulation, such as strain or interstitial fluid flow, as a result of mechanical loading.  He noted that while there is little argument that osteocytes are subject to fluid flow, they are by no means the only bone cell type to experience hydrodynamic forces.  Osteoblasts and bone lining cells are subject to intercellular fluid flow, as are osteoclasts and their precursors.  Consistent with this view, all bone cell types investigated respond to fluid shear stress.  The significant distance between most osteocytes from the appositional and resorption surfaces of bone further suggest that they do not have the dominant role in load-induced remodeling.
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Gap Junctions and Biophysical Regulation of Bone Cell Differentiation
Hank Donahue (Penn State University) addressed the topic “Gap junctions and biophysical regulation of bone cell differentiation.” Donahue began by noting that physical signals, in particular mechanical loading, are clearly important regulators of bone turnover.  Indeed, the structural success of the skeleton is due in large part to the bone's capacity to recognize some aspect of its functional environment as a stimulus for achievement and retention of a structurally adequate morphology.  However, while the skeleton's ability to respond to its mechanical environment is widely accepted, identification of a reasonable mechanism through which a mechanical “load” could be transformed to a signal relevant to the bone cell population has been elusive.  In addition, the downstream response of bone cells to load-induced signals is unclear.  Evidence suggesting that gap junctional intercellular communication (GJIC) contributes to mechanotransduction in bone and, in so doing, contributes to the regulation of bone cell differentiation by biophysical signals was reviewed.  In that context, mechanotransduction is defined as transduction of a load-induced biophysical signal, such as fluid flow, substrate deformation, or electrokinetic effects, to a cell and ultimately throughout a cellular network.  Thus, mechanotransduction would include interactions of extracellular signals with cellular membranes, generation of intracellular second messengers, and the propagation of these messengers, or signals they induce, through a cellular network.  Donahue proposed that gap junctions contribute largely to the propagation of intracellular signals.
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The Relationship between Bone Fluid Flow and Adaptation as Stimulated by Intramedullary Hydraulic Loading
Yi-Xian Qin (SUNY Stony Brook) presented a talk entitled “The relationship between bone fluid flow and adaptation as stimulated by intramedullary hydraulic loading.” Qin began by noting that the motion of intracortical fluid flow, which arises under mechanical loading, has been proposed to be an important mediator for regulating bone mass and morphology. However, mechanical deformation-generated stimulation may only partially examine the mechanism of flow-induced adaptation, because loading of bone results in not only intracortical fluid flow by the sources of matrix deformation and intramedullary (IM) pressure, but also the matrix strain which has been proposed as a key for the remodeling process. Because it has been demonstrated that bone fluid flow and its associated streaming potential product can be significantly influenced by the dynamic IM pressure that can be controlled quantitatively, the hypothesis of fluid-induced bone adaptation was evaluated in an avian ulna model using IM hydraulic loading in the absence of bone matrix strain. The fluid pathways in bone during the loading were discussed. The left ulnae of adult male turkeys were functionally isolated via transverse epiphyseal osteotomies. A specially designed fluid-loading device was firmly attached on bone via a 5-mm hole allowing IM pressure oscillation in the cavity. A sinusoidal fluid pressure was applied to the ulna with the magnitude of 50 mm Hg, 20 Hz, 10 min/day for 4 weeks. With IM pressure generating a spatial fluid pressure gradient distribution through the cortex, fluid loading (n=4) resulted in significant new surface bone formation (12.2±4.2%). In the animal group subject to sham disuse alone (n=4), the cortex showed a decrease in cross-sectional area with 6.1±3.0% reduction compared to the contralateral control.  The results show that low magnitude IM pressure can initiate a spatial fluid flow in bone and thus stimulate a bone adaptive response. This suggests that oscillation of IM pressures may influence the perfusion of bone tissue in many ways, e.g., altering blood supply and enhancing pressure gradients in a variety of fluid channels. IM pressure loading can increase and improve this perfusion process. Moreover, it assumes that there is a fluid pathway directly connected between the marrow cavity and intracortical porous space, e.g., Haversian canal and lacunae-canaliculi, which may play a role in regulating fluid transportation and perfusion in bone. These experiments may yield new insights into the mechanisms, at least at the tissue level, by which bone fluid flow initiates and controls bone morphology.
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Implications of Load-Induced Fluid Flow for Functional Adaptation:  a Combined Theoretical and Experimental Modeling Approach
Roland Steck (ETH-Zurich) described the “Implications of load-induced fluid flow for functional adaptation: a combined theoretical and experimental modeling approach.” Steck and his colleagues hypothesize that load-induced fluid flow through bone enhances transport of substances (i.e. nutrients and osteotropic substances such as signal molecules, molecular factors and hormones) that modulate cellular activities associated with growth, adaptation and repair, thereby providing a mechanochemical transduction mechanism for functional adaptation. In order to start to understand the implications of fluid flow for processes associated with remodeling, they have developed a macroscopic continuum model of the rat tibia in parallel with carrying out molecular tracer experiments using an established four-point-bending model of the rat tibia. They used a two step finite element (FE) approach to calculate different fluid flow parameters on a macroscopic scale. In a first step, bone was modeled as a poroelastic continuum for the calculation of fluid velocities and displacements resulting from the loading schemes of the experimental models. In a second step, these fluid velocities were used in a mass transfer analysis to demonstrate the positive effect of this additional convective flux on the distribution of a simulated tracer within bone cross-sections. The purpose of this presentation was to introduce the theoretical model and to interpret the predictions of the model in light of experimental data from the molecular tracer experiments as well as from parallel experiments in which a functional adaptation response was elicited in response to the hyperphysiological-loading regime.
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An Anatomical Model of Stress Induced Fluid Flow in Osteons
Nikola Petrov (Bulgarian Academy of Sciences) presented “An anatomical model of stress-induced fluid flow in osteons.” Petrov presented a new model for stress-induced fluid flow in bone based upon a triple porosity model. In this model fluid exchange occurs among the Haversian canal, the lacunar-canalicular system and the matrix microporosity. The model represents an extension of the earliest models of Pollack et al. (1984) and Kufahl and Saha (1990) and is an alternative to the Weinbaum et al. (1994) model. The model incorporates a porous boundary in the lacunar-canalicular system, thereby permitting flow between the lacunar-canalicular system and the microporosity. Results for flow in an osteon were presented and used to describe streaming potential data from various authors. Outstanding fit to both "live" and "dead" bone data is achieved along with the explanation of the long standing observation of two time constants.
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Thinking and Feeling Like an Osteocyte in its Mechanical Environment
Shelly Weinbaum (CCNY) spoke to the topic “Thinking and feeling like an osteocyte in its mechanical environment.” Weinbaum began by noting that it is very difficult for people to imagine how an osteocyte senses and responds to its mechanical environment largely because people are unaccustomed to the forces and length scales of the microenvironment in which the osteocyte lives. This is particularly true of the cytoskeletal structure of the osteocyte, its pericellular matrix and the permeability properties of the lacunar-canalicular and mineralized matrix porosities. New insights into the relationship between cellular structure and function were deduced from important new findings with other cells that have structures in common with osteocytes. In particular, Weinbaum addressed the following questions:
1. Why does an osteocyte need a pericellular matrix (glycocalyx)? What are its functions?
    a. structural
    b. hydrodynamic
    c. molecular sieving
     Why does bone not need a lymphatic system?
2. How does this matrix differ from the endothelial glycocalyx in structure and function?
3. Does water pass through the canalicular wall? Will the mineralized matrix porosity allow for water movement if there is a small amount of free water?
4. How does one account for the short and long SGP relaxation times in Pienkowski (1982) and Otter et al. (1992) if there is no fluid interchange between the lacunar-canalicular and mineralized matrix porosities?
5. What changes occur in the lacunar-canalicular porosity when a cell dies and how does this affect SGP relaxation time?
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A Lacunar Mixing Mechanism for Solute Transport in Cyclically Loaded Bone
Liyun Wang (CCNY) described “A lacunar mixing mechanism for solute transport in cyclically loaded bone.” Wang began by noting that a fundamental conundrum in mechanically loaded bone is how, during cyclic loading, there can be net solute (e.g., nutrient, tracer) transport via the lacunar-canalicular porosity when there is no net fluid movement in the canaliculi over a loading cycle. She and her colleagues propose a lacunar mixing mechanism where the fluid space in an osteocytic lacuna facilitates a nearly instantaneous mixing process of bone fluid that creates a difference in tracer concentration between the inward and outward canalicular flow and thus ensures net tracer transport to the osteocytes during cyclic loading, as has been shown experimentally. The sequential spread of the tracer from the osteonal canal to the lacunae is investigated for an osteon experiencing sinusoidal loading.
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A Potential Mechanism for Mechanotransduction in Bone: Fluid flow Induced Strain Amplification on Bone Cells
Lidan You (CCNY) presented a talk entitled “A potential mechanism for mechanotransduction in bone: fluid flow-induced strain amplification on bone cells.” You began by observing that the cell processes of bone cells in their canaliculi provide a greatly simplified and heretofore unexploited model system to explore the effect of fluid drag forces on extracellular matrix, its coupling to the intracellular actin cytoskeleton (IAC) and the strain amplification that results from this coupling. The model also provides a resolution to a fundamental paradox in bone physiology, namely, that the strains applied to whole bone (i.e., tissue level strains) are much smaller (0.04% to 0.3%) than the strains (1% to 10%) that are necessary to cause bone signaling in deformed cell cultures. The model of You and her colleagues shows that (i) it is indeed possible to produce cellular level strains in bone that are more than 100-fold greater than tissue level strains and (ii) that the fluid flow-induced drag forces on the fibers that tether the cell to its surrounding extracellular matrix can be much greater than the fluid shear forces on the cell membrane, the fluid force that has been extensively studied until now. In order to examine the hypothesis of You and her colleagues, a histomorphometric experiment is being carried out to investigate the detailed structure around the osteocytic process.
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The Importance of Flow Reversal in the Response of Bone Cells to Fluid Flow
In the first short communication, Chris Jacobs (Penn State University) addressed the topic “The importance of flow reversal in the response of bone cells to fluid flow.” Jacobs described that loading-induced fluid flow in the lacunar-canalicular system is oscillatory in nature due to the dynamic nature of most physical activities. Thus, it is important the in vitro investigations of loading-induced fluid flow as a physical signal regulating bone cell metabolism also be oscillatory and involve a reversal of flow direction. For example, Jacobs and coworkers have found that GdCl (a putative stretch-activated channel blocker) had no effect on cytosolic calcium mobilization and gene expression in response to oscillatory flow, but does have an effect in the response to steady flow.
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Poroelastic Modeling of Flow at the Haversian and Lacunar Scales
In the second short communication, Colby Swan (University of Iowa) spoke on “Poroelastic modeling of flow at the Haversian and lacunar scales.” Swan described a study in which micromechanical analysis is being used to quantify load-induced stress fields, strain fields, and fluid flows in the Haversian system and in the canalicular-lacunar system. He noted that a significant source of uncertainty in these models is the hydraulic conductivity associated with the canaliculi. With current best estimates of canalicular hydraulic conductivities, Swan's models indicate that fluid flows quite freely in both the Haversian and canalicular-lacunar systems at physiological frequencies (e.g., frequencies below 100 Hz). The fact that fluid flows quite freely at physiological frequencies makes it quite difficult to detect using whole bone viscoelastic spectroscopy.
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Osteopontin and Bone Remodeling
In the third short communication, “Osteopontin and Bone Remodeling,” David Denhardt (Rutgers University) summarized some of what is known about the secreted, phosphorylated sialoglycoprotein, osteopontin, which has the properties of a cytokine but will also promote cell attachment to mineralized matrices. The protein is found in bone and body fluids; it interacts with receptors (integrins, possibly CD44v) to stimulate intracellular signaling pathways that control gene expression and cell behavior. It is chemotactic for macrophages and in some situations aids cell survival, likely by inhibiting apoptosis. Research on OPN-deficient (knock-out) mice has revealed that OPN is required for cell-mediated immunity and for bone remodeling in response to stress.  Masaki Noda (Tokyo Medical & Dental University) described some of the studies in his laboratory on the OPN-deficient mice described by Rittling et al. (JBMR 13:1101, 1998). In contrast to control animals, ovariectomized OPN-deficient mice retain most of their bone mineral. Additionally, the knockout mice are resistant to dis-use osteoporosis, and are much less efficient at resorbing ectopic bone. It appears that in the absence of OPN osteoclast function is impaired.
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Understanding the Mechanisms of Simulated Microgravity Effects on Osteoblast Gene Expression
The fourth short communication was from Laura McCabe (Michigan State University) and was entitled “Understanding the mechanisms of simulated microgravity effects on osteoblast gene expression.” McCabe described modulation of osteoblast phenotype in a rotating cell culture system in which fluid flow is hypothesized to be a component of this responsiveness.
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Steve Cowin and Susannah Fritton, December 20, 2000.