Track 10. Cel lular and Molecular Mechanics 10.8 Molecular Biomechanics $587

10kPa resulted in 1.1% (±0.13%) in the center of the wells and varied from 0.8% to 1.3% at the borders. Preliminary experiments suggest that human periosteum-derived cells respond to both hydrostatic pressure and stretch. In summary, HyPaS is a valuable tool for studying the cellular response to HP, stretch or PIS. Future studies will determine whether these stimuli activate distinct downstream mechanotransduction pathways.

6770 Mo-Tu, no. 33 (P63) Extracellular matrix and medium serum levels influence the rate of f ibroblast reor ientat ion in response to tensile strain K. Baria 1,2, H. Screen 1 , D. Bader 1 , D. Lee 1 . 1Medical Engineering Division, Department of Engineering, Queen Mary University of London, UK, 2Department of Bioengineering and Robotics, Tohoku University, Sendal, Japan

Cyclic uniaxial tensile strain is known to induce reorientation of fibroblasts [1,2]. Cells sense their mechanical environment primarily through integrin-based cell- matrix adhesions, the nature which depends upon the surrounding extracellular matrix (ECM). There is also evidence of an altered response to mechanical forces in the presence of soluble factors present in serum. Therefore, this study compared the effects of providing substrates functionalised with collagen I or ProNectin F, and media containing three levels of foetal bovine serum (FBS), on the reorientation of dermal fibroblasts in response to uniaxial tensile strain. Human neonatal dermal fibroblasts (Clonetics), plated at 1 104 cells/cm 2 on dumbell shaped membranes coated with collagen I or ProNectin F (Flexcell), were cultured for 2 days. The specimens were mounted into a tensile straining system [3] and provided with media containing 10%, 0.05% or 0% serum (v/v). Specimens were divided into an experimental group, subjected to 5% cyclic tensile strain at 1 Hz for 3, 12 or 24 hours, or an unstrained control group. Samples were imaged using a light microscope with attached digital camera (Nikon). On application of tensile strain, fibroblasts reoriented to an angle between 700 and 850 from the axis of strain. Reorientation on ProNectin F, which was more rapid than on collagen I, was complete within 3 hours in the presence of 10% serum medium. The rate of reorientation on both surfaces was enhanced by the presence of serum. Following reorientation, fibroblasts developed a more elongated and spread morphology. Reorientation was associated with decreased proliferation, and increased total protein and collagen production. In conclusion, the presence of soluble factors and the nature of the insoluble ECM do not alter the ultimate orientation angle of dermal fibroblasts but may modulate the rate of reorientation.

References [1] Berry. Biorheology 2003; 40: 337-345. [2] Neidlinger-Wilke. Journal of Orthopaedic Research 2001; 19: 286-293. [3] Cacou. Medical Engineering & Physics 2000; 22: 327-333.

5964 Mo-Tu, no. 34 (P63) Mechanical loading o f porcine knee joint cartilage activates the Akt/PKB signaling pathway A. Niehoff 1, W. BIoch 2, M. Offermann 2, A. Schmidt 2, J. Dargel 1 , G.-P. BrQggemann 1 . 1Institute of Biomechanics and Orthopaedics, German Sport University of Cologne, Germany, 2Department of Molecular and Ceflular Sport Medicine, German Sport University of Cologne, Germany

Introduction: Previous studies have demonstrated that chondrocytes detect and respond to mechanical loading by altering their metabolism. The objective of this study was to examine the effect of compression on loading-sensitive pAkt/PKB in the porcine knee joint cartilage. Materials and Methods: 15 swines (42±2kg) were used for this study. The patella of the left knee was loaded in compression immediately after sacrifice. Compression was applied with a custom-designed built loading frame for 150s. The swines were randomly divided in 3 groups. The patella of group 1 (n = 5) was loaded with 500N with 12Hz, of group 2 (n=5) with 500N for 1Hz and of group 3 (n =5) statically with 500 N. Full-thickness cartilage plugs from the loaded portion of the patellar and the femoral surfaces were harvested at time point 0s (immediately after knee joint opening, 2±1 min after compression), 300s and 1200s after knee joint opening. The right knee served as control and was treated in the same way as the experimental knee but without loading. Cartilage sections were stained immunohistologically using an anti-pAkt/PKB antibody (Upstate). The staining of pAkt/PKB was semi-quantitatively analyzed using a score system. For statistical analysis a paired Student's t-test for site- matched samples was performed (significance level: p <0.05). Results: The pAkt/PKB was detected throughout the whole cartilage, whereas the strongest activation was found in the radial zone. 300s after mechanical loading pAkt/PKB activation in the lower radial zone was significantly (p < 0.05) lower compared to the cartilage of the unloaded knee. This lower activation was not observed 1200 s after mechanical loading. Furthermore, the activation of pAkt/PKB seems to be related to the loading protocol.

Discussion: Our results demonstrate that mechanical loading of the porcine knee joint cartilage activates the pAkt/PKB signaling pathway in relation to the loading protocol.

7410 Mo-Tu, no. 35 (P63) Effects of PEMFs on the cel lular act iv i ty o f osteoblast-like cells C. Martino 1 , J. Qi 1 , D. Belchenko 1 , V. Ferguson 1 , S. Preiss 2. 1Department of Mechanical Engineering, University of Colorado at Boulder, Boulder, Colorado, USA, 2Department of Endocrinology, University of Colorado Health Science Center, Aurora, CO, USA

Pulsed electromagnetic fields (PEMFs) have been used for treatment of non-union bone fracture healing for more than three decades. It has been experimentally observed that a series of parameters such as pulse shape and frequency should be carefully controlled in order to achieve effective treatments. In this paper, the effects of PEMFs on the cellular activity of SaOS-2 osteoblast-like cells are investigated. PEMFs with different frequencies and different electric field strengths were applied to SaOS-2 cell cultures. The effects of PEMFs on cell proliferation and differentiation were then assessed through direct cell counts, the MTT assay, Alizarin Red Staining, and Alkaline Phosphatase (ALP) activity. These assays were performed following distinc- tive magnetic stimulation protocols. There was a significant increase in cell growth with PEMF exposure over the control group after 1, 2, and 3 days of culture. The ALP activity of SaOS-2 cells was also increased significantly after PEMFs stimulation, although mineral formation did not appear to be increased following 6 days of PEMF exposure. These observations suggest that PEMFs accelerates cellular metabolism and may play a role in the enhancement of SaOS-2 cell differentiation. We are currently pursuing the effect of PEMF on other markers of osteoblastic proliferation and differentiation.

10.8 Molecular Biomechanics

5959 Mo-Tu, no. 36 (P63) Unbinding o f vesicles under flow at a stagnation point S. Chatkaew, M. L6onetti. IRPHE, UMR CNRS 6594, Universities Aix-Marseille I and II, Technopole de Chateau-Gombert, Marseille, France

In the last decade, considerable efforts have been undertaken to measure, model and simulate the motion in Stokes flow of deformable particles such as capsules, vesicles and cells. They are motivated by cosmetic, pharmaceutical and agricultural industries and also by key biological involvements in blood rheology or leukocytes motion. We study the unbinding of a heavy vesicle interacting weakly with a flat substrate under a succion flow induced by a micropipette. The dynamics of unbinding depends mainly on the vesicle size, the flow rate and the contact area between vesicle and substrate, a quantity directly linked to the reduced volume (a measurement of the deflating). The range of applied hydrodynamic forces varies from sub-pN to one hundred picoNewton what covers the buoyancy force of our vesicles. Their detachments appear above a critical flow rate Uc depending on the characteristics of the studied vesicle. Uc varies linearly with the difference of densities and with the power minus two of the distance between the micropipette and the vesicle. At this threshold, the inverse of the rising time vanishes. Rv and Rc are the radii of the vesicle and of the circular contact area. Three kinds of behaviours are observed following if vesicles are quasi-spherical, slightly deflated and deflated. For Rv ~> Rc, Rc(t) varies very slowly during a lag period and after, Rc(t) falls to zero with a finite time. For Rv >Rc, the vesicle shape becomes a truncated egg with a smaller contact radius during a characteristic time. After, we recover the two phases of Rv ~ Rc. For Rv :~ Rc, the shape dynamics is more complex from egg and pear shapes to lipid tubes.

5126 Mo-Tu, no. 37 (P63) Protein- l ip id interactions: correlation of a predictive algorithm for lipid-binding sites with 3D-structural data G. Diez 1 , D.L. Scott 2, W.H. Goldmann 1,2. 1Friedrich-Alexander-University of Erlangen-Nueremberg Center for Medical Physics and Technology, Biophysics Group, Erlangen, Germany, 2Structural Biology Program, Massachusetts General Hospital~Harvard Medical School, Charlestown, MA, USA

Research in our laboratory over the past decade has provided experimental data documenting the interaction of cytoskeletal proteins with lipid interfaces. Fluorescence microscopy, tracking of genetic constructs, and high precision techniques including differential scanning calorimetry, film balance, T-jump, and isothermal titration applied to artifical lipid membranes have shown that do- mains of numerous proteins (e.g. Arp2/3, talin, vinculin, filamin, alpha-actinin, CapZ, huntingtin) are designed to specifically interact with cell membranes. These actin binding and regulatory proteins exhibit functional domains that are partially attracted to the cell membrane and transiently insert into the lipid bilayer. This establishes a membrane scaffold which allows transport and mechanical signaling across cell membranes.

$588 Journal o f Biomechanics 2006, Vol. 39 (Suppl 1) Poster Presentations

We developed an algorithm to predict protein regions that were competent to associate with membrane lipids. The algorithm is based on the identification of hydrophobic and amphipathic amino acid segments while discriminating between surface-seeking and transmembrane configurations. Results from these analyses compared well with the three dimensional consensus structure and the spatially arranged orientation of these protein molecules, indicating alpha-helical binding with lipid membranes. Further efforts to map these sites, including genetic manipulation and/or targeted crystallography, will be needed to define the precise role of these regions in promoting protein-lipid interactions and cell stability. Since the 3D coordinates of the most proteins are incomplete or do not in themselves identify the site of lipid binding and/or association, computational methods are a real alternative.

5369 Mo-Tu, no. 38 (P63) On mathematical model ing o f " in te l l igence"

A.A. Kolpakov. The Novosibirsk State University 324, Novosibirsk, Russia

The recent analysis of "smart" structures [1] predicts the following general properties of the intelligence: 1. A structure demonstrates the existence or absence of the intelligence under

the action of inner factors. 2. There exist at least two level of the intelligence: the high-intelligence and

"if-" instruction intelligence (known as knowledge). In this paper a model of intelligent system is given with no relation to "smart" structures. Denote by s characteristics describing a system and by S the set of all states of the system. We describe evolution of the system from the state s to another state by the equation

(1) 8s = ~ ( s , p ) + 8g(s),

where p is a control parameter, and ~J(S) describes the action of not controlled agents. We consider a partial case of the problem (1) (it allows demonstrate our results):

ds (2) ~ - A s = p ( t ) + g ( s , t ) , s(t0)=s0,

where So is the current state of the system in the current time. The solution of (2) can be written as

+ l "t (3) s(t) = So G ( t - r ) p ( r ) d r + G ( t ) -- K \p// \ + [G(t) + s0]. • 0

Not all s t a tes from S are "comfortable" for the sys tem. We introduce the set Sc -~ S of comfort. To save itself in the set of comfort, the system must control itself in such a way that

(4) s(t) ~ Sc, p ~ P

where P is the set of possible control actions available. We denote the solution of the problem (4),(5) by Pc(S0)-~ P. We are interested not in the analysis of the problems (1)-(4) but in the structure and origin of the set Pc. We demonstrate that for the problem (4),(5) the conclusions (1) and (2) [1] are valid. The following new problems are discussed: a. Decompositions of Pc(S0) into control units different from indicated in (2). b. Many units control problem (protection and exchange of knowledge). c. Production of knowledge from the living experience of the system.

References [1] Kolpakov A.A., Kolpakov A.G. Design of smart beam - an integrated design

procedure. Structural and Multidisciplinary Optimization 2006; 31, N13. [2] Zagoruiko N.G. Applied Methods of Analysis of Data and Knowledge. 1999;

S.L. Sobolev Math. Inst., Novosibirsk.

6078 Mo-Tu, no. 39 (P63) Determining the dissociation kinetics of rol l ingly adherent cells on E-selectin at low shear stress A.M. Wayman 1 , R.P. McEver 2, C. Zhu 1,3. 1Woodruff School of Mechanical Engineering and Joint Emery/Georgia Tech Coulter School of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA, 2 Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 3 and Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

Selectin molecules play a major role in the initial tethering and rolling of leukocytes to vascular surfaces during inflammation and thrombosis. This

rolling behavior is governed by the mechanokinetics of the association and dissociation of two or more selectin-mediated tethers. The dissociation of selectin-mediated bonds has been extensively studied with cells that transiently tether and cannot roll on selectin surfaces. However, the dissociation kinetics for rollingly adherent cells has yet to be determined. Our aim is to use an invertible flow chamber to determine the dissociation kinetics of cells rolling on E-selectin substrates at low shear stresses. By inverting the chamber during the experiment, the detachment of rollingly adherent cells over time can be observed. The semi-log plot of the number of rolling cells bound over time displays great correlation to a linear curve, suggesting that the dissociation kinetics of cells rolling on E-selectin at low forces can be approximated as first order. Using first order analysis, we are able to determine the dissociation kinetics of E-selectin-mediated rolling cells over a low shear stress range. Our analysis quantitatively shows the slower dissociation kinetics of cells rolling on E-selectin compared to transiently tethering cells as well as a minimum shear requirement for rollingly adherent cells on E-selectin.

4622 Mo-Tu, no. 40 (P64) Probing single cell micromechanics in v/vo

D. Wirtz 1'2, B.R. Daniels 1 . 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, USA, 2Howard Hughes Medical Institute graduate training program, Johns Hopkins University, Baltimore, USA

Cells are not directly physically accessible in vive and, therefore, their me- chanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vive assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of the thermally-excited movements of nanoparticles embedded in physiological cytoplasm reflect local viscoelastic properties. As a proof of principle, we probe the spatial variations in the micromechanical properties of the cytoplasm of developing C. elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior- posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of mitotic spindles stems from the asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.

5006 Mo-Tu, no. 41 (P64) Sel f -organized tissue structures for mechanical attachment to artif icial materials K. Takakuda, S. Asoda, T. Arita, H. Koshitomae, '~ Koyama. Institute ef Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan

In a living body, tensile stresses are transmitted between soft and hard tissues via the Sharpey's fibers and strong attachments are realized. On the other hand, since collagen fibers cannot have satisfactory anchors on artificial materials, firm attachments of soft tissues to artificial materials are not yet achieved. Hence we make mesh-like structures on the surfaces of artificial materials for the anchoring of collagen fibers. Test specimens were implanted into subcutaneous tissues of rat's dorsal region. They were retrieved after experimental periods and tensile tests for attachment strength were carried out. Microscopic observations of tissues specimens were also carried out. Consequently, in the case of specific experimental conditions, we found such collagen fibers could be spontaneously induced that penetrated into the mesh structure placed on the surface of the materials, turning around the fibers of the mesh and come back to the tissues. These fibers were suitable for tensile stress transmissions. Such self-organized structures for the anchoring of collagen fibers might be useful for the attachment of soft tissues to artificial materials.

Track 11

Artificial Organs

6000 We-Th, no. 1 (P64) Evaluation of polymyxin B interact ion with endotox ins by molecular model l ing M. Soncini, S. Vesentini, A. Zaupa, G.B. Fiore, A. Redaelli. Department ef Bioengineering, Politecnico di Milano, Milan, Italy

The outer membrane of gram-negative bacteria is mainly constituted by en- dotoxins, which are a trigger of the inflammatory process and crucial in the