$444 Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)

with DiO, regardless of medium. The mean fractional dimension (Db) of these networks was 1.80±0.036. The F-actin cytoskeleton was apparent in cell bodies in all media, but canalicular processes stained positive only in non- isotonic environments. The mean Db for the phalloidin labeled hypotonic treated sections (1.74 ± 0.053) was significantly different from that of the iso- tonic treated sections (1.51 ±0.058), p<0.01, and the DiO labeled networks, p<0.05. This suggests such measures are useful for establishing baseline quantifiers of connectivity, and evaluating network changes. Time lapse DAF- FM imaging demonstrated persistent intercellular transport within the section. These models provide an unprecedented toolset for mechanobiological study of osteocytes, and provide the underpinnings for a comprehensive understand- ing in the osteocyte's role in the regulation of bone function.

6650 Tu, 16:15-16:30 (P25) Microstructure of cort ical bone around the foramen in bovine lumbar vertebra B. Giri, S. Tadano, M. Todoh, K. Fujisaki. Division ef Human Mechanical Systems and Design, Graduate School of Engineering, Hokkaido University, Sapporo, Japan

Naturally occurring holes in bone, i.e. foramina, not detected as regions of crack initiation or growth, are present in considerable number in the lumbar vertebra. Mechanical characteristics of bone depend on mineral content, i.e. hydroxyapatite crystals (HAp) and collagen fibers alignment; c-crystallographic axis of the crystals being preferentially oriented parallel to the collagen fibers. In the current work, we examined the crystal orientation and mineral distribution around the foramen to explore the reason of foramen not being a cause of failure. From the diffracted intensity profile obtained by latest X-ray diffraction method, the orientations of HAp crystals were determined distinctly. The mineral fractions were measured from the fraction of X-rays absorbed by the specimen. The lumbar vertebrae are additionally subjected to the external complex loads. So, the orientations of crystals (collagen fibers) are expected to be different from the case of long bone (e.g. femur). In this regard, for the first time, we examined the orientation of crystals around the foramen of the vertebra by not just observing the diffracted intensity pattern as a whole, but by calculating the actual intensity values at different angles for more specific results. The tangential orientation of the crystals revealed around the foramen edge, irrespective of the shape and axis of the vertebra, was considered as the main reinforcing parameter for the foramen analogous to the fiber reinforced hole. Unlike reported by the previous work, a slight variation in the mineral distribution of the specimen with no specific tendency was observed, hence, was not considered as the strengthening parameter for the foramen. As another novel discovery, a thicker cortical region was found existing near the surface around the top edge of the foramen. This cortical part was extended within the cancellous mass covering the foramen from inside in the form of a thin cylindrical wall consisting of circumferentially oriented crystals. The existence of these structures has additionally made the foramen a damage resistant.

5543 Tu, 16:30-16:45 (P26) Multiscale analysis of different driving effects governing fluid movement in cort ical bones: A coupled viewpoint for the mechanotransduction T. Lemaire, S. Naili, A. R6mond. Laboratoire de Biom6canique et Biomat6riaux Ost6o-Articulaires, Universit6 Paris Val de Marne, Cr6teil, France

Fluid movement within cortical bone at the lacuno-canalicular scale is known to play an important role in these tissues remodeling. Indeed interactions between fluid and osteocytes are key elements of the mechanotransduction of bone adaptation. These multiphysical interactions consist in mechanical, electrical and biochemical signals. Nevertheless, models often adopt a simplified point of view and all of these multiphysical phenomena are not always included in interstitial fluid movement description. That is why this work uses a complete electro-chemo-hydraulical coupled mod- eling suitable to analyse fluid movement in the submicronic pores of cortical bone. The study is not only derived at the lacuno-canalicular porosity but also in the micropores of the collagen-apatite matrix. The periodic homogenization is combined to an efficient change of variables. It provides a modified Darcy law including osmotic and electro-osmotic driving mechanisms for the flow in addition to the classical Poiseuille term. This model permits us to propose innovative arguments concerning the role of transverse glycocalyx filaments located in the canaliculi. Indeed, these microelements not only reduce the flow in the canaliculi but also increase electro-chemical couplings effects. Moreover, numerical simulations derived from this model provide new evidences of the role of lacuno-canalicular in bone's remodelling. It especially enables us to account for electric field stimulation on bone cells and their consequence on bone adaptation.

Oral Presentations

7162 Tu, 16:45-17:00 (P26) Full-f ield character isat ion o f mechanical strain in the Flexercell and stimulation optimization study M. Thompson 1 , C.-E. Ott 2, S. Ahrens 2, U. Kornak 2, S. Mundlos 3, G. Duda 1 . 1 Center for Musculoskeletal Surgery, Charit6 - Universit~tsmedizin Berlin, Germany, 2Institute for Medical Genetics, Charit~ - Universit~tsmedizin Bedin, Germany, 3 Max-Planck-lnstitute for Molecular Genetics, Berlin.

Introduction: Mechanical stimulation of cells using the Flexercell device has become a well established method. However, no full-field mechanical characterisation of the strains to which the cells are exposed is available and further no systematic optimisation study for all available parameters has been performed. Such studies are vital to enable results from widely differing stimulation protocols to be compared and to confirm the homogeneity of the mechanical stimulus. Materials and Methods: We analysed the mechanical behaviour of Flexercell membranes with high resolution photography and Vic2D Digital Image Cor- relation (DIC) software. DIC analysis of images of unstrained and nominal strains of 1-10% was performed in un-used and used Flexercell wells. The systematic optimisation study varied strain level, frequency and duration to identify a protocol giving maximal response of MC3T3-E1 cells to mechanical loading. Expression of c-Fos and Ptgs2 (COX2) was analysed by quantitative PCR relative to GapDH and Actb as endogenous controls at different time points. In addition we performed Western blot analysis to assess Erk and Akt activity. Results: In two independent experiments per stimulation protocol we showed an up to 200-fold up-regulation of c-Fos as well as an up to 50-fold enhanced mRNA-Expression of Ptgs2 compared to non-stimulated wells. The cellular response was confirmed by western blot analysis showing increased levels of p-Akt and p-Erk. Additionally, similar results were obtained when we stimu- lated mouse osteoblasts under the same conditions. DIC strain maps of the Flexercell membrane confirmed the homogeneity of the biaxial strain field, and showed linear correspondence with nominal applied strains. Discussion: The homogeneity of the applied strain field and the linearity with the nominal value showed that the Flexercell device is an appropriate tool for the reproducible application of mechanical loading in vitro. Quantitative PCR and western blot analysis showed strong gene regulation changing over time with mechanical stimulation.

6167 Tu, 17:00-17:15 (P26) Characterizing mechanical behavior of cells and soft tissues by indentation: Potential and limitations N.K. Simha 1 , R. Namani 2, S. Chiravarambath 1,3, J.L. Lewis 1 , P.H. Leo 3. 1Biomechanics Lab, Department of Orthopaedic Surgery, University of Minnesota, Minneapolis, MN, USA, 2Department of Mechanical Engineering, University of Miami, Coral Gables, FL, USA, 3Department of Aerospace Engineering and Mechanics, University of Minnesota, Minneapolis, MN, USA

Indentation is being increasingly used to study the mechanical behavior of cells, cell-protein bonds as well as native and engineered soft tissues; multiple parameters are required to describe such behavior, which typically involves nonlinear elastic and/or time-dependent responses. The primary measurement in indentation is the tip displacement and corresponding indent force (elastic test) or indent force vs. time at fixed displacement (stress relaxation). The critical issue is how many parameters can be extracted, and with what accuracy, from such measurements. The goal of this paper is to determine the sensitivity of indentation tests to nonlinear elastic and poroviscoelastic parameters. Indentation of isotropic, homogeneous, tissue layers lying on a substrate is modeled in ABAQUS, and parameters are extracted by minimizing an error between the FEM predicted and benchmark data using Simplex minimization. First, we focus on nonlinear elastic properties, and consider two parameter Mooney-Rivlin, Polynomial, and Exponential models. Our inverse FEM method extracts both nonlinear elastic parameters with excellent accuracy (errors less than 1.5%) from ideal benchmark data. However, when benchmark data includes only a 5% noise, accuracy of extracted parameters decreases considerably. Results suggest that indentation data from a single tip (i) can extract only one parameter, a generalized elastic modulus, with good accuracy, but (ii) cannot distinguish between the three models if material behavior is not known a priori. Second, we fix the elastic parameters, and consider poroviscoelastic (perme- ability, constant spectrum relaxation) models. The accuracy of the parameters extracted for benchmark data (with and without noise) is assessed to determine the sensitivity of the indentation stress-relaxation test to the permeability and viscoelastic parameters. Based on these results, the potential and limitations of indentation for characterizing mechanical behavior of cells and soft tissues are discussed.