You are invited to attend the interactive and informative Workshop on Fluid Flow in Bone.  First Annual Meeting to be held in conjunction with the ASBMR 25th Annual meeting in Minneapolis, Minnesota.

Physiology and Biology of Fluid Flow in Bone - Full Program w/Session Abstracts

Physiology and Biology of Fluid Flow in Bone - Session Abstracts

7:15 P.M.

Introduction - The Physiology and Biology of Fluid Flow in Bone Program

John A. Frangos, PhD.

La Jolla Bioengineering Institute

La Jolla, California, USA

7:40 P.M.

Blood Circulation Consideration for Modeling Bone Fluid Flow

Howard Winet, PhD.

Department of Orthopedic Surgery

UCLA Orthopedic Hospital

University of Southern California

Los Angeles, California, USA

Session Abstract
Tissue engineered scaffolds require vascularization to 1) enhance nutrient exchange and 2) provide cells needed to build new tissue.  Cell-seeded scaffolds—bioreactors--require rapid penetration of vessels or enhanced fluid percolation to keep their contents alive until normal nu­trient exchange can be established.  Accordingly, scaffolds implanted in bone for purposes of stimulating osteogenesis must be at least angio- and osteo- conductive.  The ultimate scaffold will be angio- and osteo- inductive as well.  Growth factors/polypeptide cytokines are usually employed as growth inducers.   They are ineffective, however, if their release rate is not con­trolled to match the state of “readiness” of target cells.
 

Bone and endothelial cells are highly sensitive to another inductive stimulus, fluid flow.  In cortical bone water occupies about 13% of the space[Morris #2714] and molecules at least as large as ferritin percolate through the matrix via solven drag from osteon to osteon at rates well in excess of diffusion[Dillaman #2879;Dillaman #525;Kelly #1084;Knothe Tate #3168;Knothe Tate #3168;Knothe Tate #3168;Li #1231;Montgomery #1412].  Control of endothelial cell activity by blood flow-generated fluid shear stress is well established[Frangos #3552].  Bone cell re­sponse is similar[Reich #2582;Klein-Nuland #3555;Johnson #2579;Hillsley #2425]—as would be expected given the high incidence of redundancy in evolved tissues and the common ancestry of the two cell types in mesenchymal stem cells—but with important differences.  First, osteocytes and their processes are surrounded by relatively thin fluid annuli in the lacunar and cana­licular compartments.  Second, there is some evidence for an influence of streaming potentials on bone cells[Chen #2486;MacGinitie #1280;MacGinitie #3130;Otter #1524;Otter #1525;Otter #1526;Otter #3549;Pollack #3633;Walsh #2156;Walsh #2157].  In any case, the need for con­vective flow over the surface of bone cells, whether stimulatory for mechanoreceptors or elec­trokinetic receptors, is supported by an increasing volume of data.  No measures of this flow in vivo have been obtained, but in vitro experiments indicate a shear stress stimulation threshold producing 1% µε[Burger #3632] and a streaming potential threshold of 10µV[Hung #3860].

8:05 P.M.

The Role of Extracellular Fluid in Bone Mechanotransduction

Charles Turner, PhD.

Department of Orthopedic Surgery
Biomechanics and Biomaterials Research Center

Indiana University School of Medicine

Indianapolis, Indiana, USA

Session Abstract
Mechanical loading can elicit an anabolic response in bone tissue, yet bone is very sensitive to the way loading is applied.  Dynamic loading induces new bone formation, whereas static loads can inhibit bone formation.  Adding rest periods between dynamic loading cycles (or bouts) greatly enhances the anabolic response.  Many of these effects appear to be due to the role extracellular fluid plays in mechanotransduction.  It is known that the osteogenic response is almost proportional to the dynamic energy dissipation in the bone tissue.  Energy dissipation in turn is largely due to fluid-solid interactions within the tissue.  Furthermore, bone loss associated with disuse follows distinct regional patterns that resemble hydrostatic fluid pressure changes in the bone tissue.  In cell culture, osteoblasts are far more sensitive to fluid flow than they are to membrane stretching suggesting that bone tissue deformations, which tend to be small due to bone’s stiffness, are insufficient to induce cellular responses unless accompanied by extracellular fluid forces on the cell membrane.  Several theories have been proposed to explain how fluid forces initiate biochemical events within a bone cell.  These theories will be reviewed along with the data from numerous studies of skeletal mechanotransduction. 

8:30 P.M.

Coffee Break

8:40 P.M.

Osteocytes and Mechanosensing

Lynda Bonewald, PhD.

Department of Oral Biology and Biological Science

University of Missouri School of Dentistry

Kansas City, Missouri, USA

Session Abstract
LF Bonewald, J. Feng, J. Gluhak-Heinrich, SE Harris, JX Jiang, D. Nicolella, D. Pavlin, M. Schaffler, E. Sprague.

Our team of investigators is studying cells in bone that function as a team-osteocytes.  Our hypothesis is that the osteocyte in response to cell deformation generated by biophysical forces results in biochemical responses that modify osteocyte function and communication.  Our approach is to use fluid flow shear stress (FFSS) on in vitro cell lines and primary cells to generate data to be tested and validated using in vivo loading models.
 

A protein that is highly expressed and highly activated in osteocytes in response to mechanical loading is Dentin Matrix Protein 1 (DMP1).  DMP1 mRNA expression is increased in osteocytes, but not osteoblasts, in response to mechanical loading in the mouse tooth movement model.  In the rat fatigue loading model, DMP1 expression is greatly expressed in osteocytes on both the tension and compression sides.  To identify the cis regulatory regions of the DMP1 gene, a construct was prepared containing the -9624 to +1996 region driving GFP that was stably transfected into MLO-Y4 osteocyte-like cells.  GFP expression is dramatically enhanced upon application of FFSS, therefore efforts are underway to correlate fluorescence intensity with cellular deformation.  Mice lacking this gene will be used to determine the function of DMP1 in osteocytes.
 

MLO-Y4 cells respond to FFSS with an increase in gap junction function and production of signaling molecules.  Autocrine production of PGE₂ is responsible for elongation of dendritic projections and branching and for increased gap junction protein expression such as connexin 43.  These effects are mediated through the EP₂ receptor. Both primary osteocytes and the osteocyte-like cell line MLO-Y4 express large amounts of Cx43, yet only the tips of the dendritic processes are in contact, therefore another function for this molecule must exist.  The production of PGE₂ by MLO-Y4 cells lacking physical contact in response to FFSS is inhibited by hemichannel blockers.  Therefore, we hypothesize that these cells use hemichannels to exchange information between the cell and its extracellular compartment.  Correlations exist between cellular deformation, dendrite extension, and PGE₂ production in response to FFSS. 
  

Time and space only permit highlights of the team approach to the study of osteocyte function.  Other topics to be briefly highlighted include osteocyte support of osteoclastic bone resorption, osteoblast differentiation into osteocytes, osteocyte specific markers, and genes in osteocytes regulated by mechanical strain. 

9:05 P.M.

Cytoskeleton-Integrin-Matrix Interactions in Mechanotransduction

Frederick Pavalko, PhD.

Department of Cellular Physiology

Indiana University School of Medicine

Indianapolis, Indiana, USA

Session Abstract
Osteoblasts respond to mechanical stimuli such as fluid shear stress, but the mechanisms by which mechanical signals are transduced from the extracellular environment, across the membrane, and into the cell remain poorly understood. Specialized sites of cytoskeletal-integrin-extracellular matrix interaction, referred to as focal adhesions, have been suggested to function as mechanosensors in osteoblasts. To investigate the role of focal adhesions in the sensation and transmission of mechanical signals we limited focal adhesion formation in MC3T3 osteoblasts and then measured cyclooxygenase-2 (Cox-2) protein expression and prostaglandin-E₂ (PGE₂) release in response to fluid shear stress. We found that fluid shear stress-induced Cox-2 protein expression and PGE₂  release were inhibited in osteoblasts when reduced focal adhesion were reduced compared to cells with robust focal adhesions.
 

Methods: To enhance focal adhesion formation, MC3T3-E1 osteoblasts were cultured on fibronectin coated slides in the absence of serum. To limit focal adhesion formation we either cultured cells on bovine serum albumin (BSA) coated slides or treated cells cultured on fibronectin coated slides with Arg-Gly-Asp-Ser (RGDS) peptides to competitively inhibit integrin-fibronectin binding. Osteoblasts were then subjected to 1.5 or 5 hours of laminar fluid shear stress (10 dynes/cm²) followed by analysis of Cox-2 protein levels and release of PGE₂.
 

Results and Conclusions: Limiting focal adhesion formation by either culturing osteoblasts on BSA or by treating osteoblasts cultured on fibronectin with RGDS peptides inhibited fluid shear stress-induced Cox-2 protein levels and PGE₂ release compared to cells cultured on fibronectin, which formed robust focal adhesions. Also, in response to fluid shear stress focal adhesions reorganized into fibrillar adhesions in cells cultured on fibronectin. These results suggest tha formation of focal and fibrillar adhesions play a role in fluid shear stress-induced prostaglandin metabolism and that focal adhesions may function as mechanosensors in MC3T3 osteoblasts.

9:25 P.M.

Concluding Discussion

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