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Cell and Tissue Engineering - Posters

Poster Presentations 259 DIFFERENTIAL EFFECTS OF GROWTH FACTORS ON TISSUE ENGINEERING OF CARTILAGE A Sieminski, T Blunk, L Freed, R Langer, G Vunjak-Novakovic Dept. of Chemical Engineering, MIT, Cambridge, MA.

262 TISSUE ENGINEERING OF LIGAMENTS V. Lin, J. McKean, and K - L Paul Sung Departments of Bioengineering and Orthopaedics University of California - San Diego, La Jolla, CA, 92093-0412

In order to tissue-engineer cartilage for potential clinical use, primary bovine articular chondrocytes were cultivated on biodegradable, porous, three-dimensional scaffolds (polyglycolic acid; 5 mm diameter, 2 mm height) [1]. To improve tissue quality, we investigated the effects of various growth factors known to increase proliferation or extraceliular matrix production of articular chondrocytes. Cells were cultured on scaffolds for four weeks in mixed six-well plates with growth factors supplemented to the culture medium. IGF-I, TGF-beta, and IL-4 increased construct weight and absolute amounts of major matrix components, GAG and collagen, in a dose-dependent manner. Even more important for tissue quality, the relative fractions of GAG and collagen (based on wet weight) in the tissues were increased. In contrast, both PDGF and bFGF were detrimental in our system. We also investigated the possible modulation of the growth factor effects by different bioreactors using IGF-I (100 ng/ml). The rotating bioreactor [1] proved to be especially favorable for IGF-I treated tissues by considerably increasing the GAG fraction and wet weight. [1] Freed and Vunjak-Novakovic, in The Biomedical Engineering Handbook (ed Bronzino) 1778-1796 (CRC Press, Boca Raton, 1995).

Tissue engineering offers the possibility of replacing damaged human ligaments, especially the poorly-healing anterior cruciate ligament (ACL) by engineered ligament tissues. We are attempting the construction of ligament tissues by culturing biodegradable polymeric biomaterial scaffolds seeded with ACL fibroblast cells. We have seeded cells on polylactic acid (PLA) and collagen biomaterials. We found that the rate of cell incorporation into either biomaterial was 90%. After 2-3 weeks of in vitro culturing, the scaffolds were populated throughout by a robust population of cells, most of which were elongated in shape as they should in vivo. Furthermore, the cells seeded into the collagen matrix appeared to have caused the contraction of the matrix, suggesting that the cells interact constructively with the collagen biomaterial. Thus it appears that these seeded scaffolds are suitable and ready for continued culturing or for immediate implantation into the patient. Work is now underway for the continued culturing of seeded scaffolds, both in vitro and in vivo using the nude mouse as the animal model. Mechanical and biochemical assays will also be performed on scaffolds cultured for varying periods of time.

260 ACTIVATION OF MONOCYTES AND GRANULOCYTES IN RAT VEIN GRAFFS S. Q. Liu, M. Glucksberg, A. Mok, J. Grotberg, M. Smith, M. Katada Dept. of Biomedical Engineering, Northwestern Univ., Evanston, IL A vein graft is subjected to the influence of eddy blood flow due to graft-host diameter mismatch as well as to the influence of a suddenly increasect tensile stress aue to exposure to arterial blood pressure. This study was designed to examine the influence of these mechanical factors on the interaction between the monocytes / granulocytes and the endothelium, which was assessed by measuiing the density of the cells attached to the vein graft endothelium by using anti-CD 11 b/c, an antibody specific to the monocyNs and granulocytes. A vein graft was constructea by interposing a jugular vein into the abdominal aorta of the rat. The density of the monocytes and granulocytes attached to the endothelium increased durimz the early pe.riod from several minutes to days and then decreased gradually toward me normal level. The cell density at the proximal region, where apparent eddy flows were found, was higher than that at the middle and distal regions, where no app.arent eddy flows were found. When the eddy flows were eliminated ani:t the tensile stress was relieved by applying an intestinal sheath to the vein graft,_the density of the activated monocytes and granulocytes decreased. slgnitlcantly in all observed areas compared with that of tbe unmodulatecl vein grafts, and no apparent differences were found from the proximal to the distal anastomosis. These results suggested that disturbed fluid shear stress due to eddy flow and increased tensile stress aue to arterial blood pressure might modulate the structure and function of the vein graft endothelial cells, causing activation of the monocytes and granulocytcs.

263 MICROSTRUCTURE-BIAXIAL MECHANICAL RESPONSE OF PORCINE INTESTINAL SUBMUCOSA Michael S. Sacks, University of Miami

261 ASCORBATE AFFECTS THE MORPHOLOGY AND MAGNITUDE OF COLLAGEN GEL CONTRACTION BY SMOOTH MUSCLE CELLS L L Hilenski and R.M. Nerem Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0363

264 MECHANICAL PROPERTIES OF A SELF-ASSEMBLING OLIGOPEPTIDE BIOMATERIAL E.J. Leon, N. Verma, S. Zhang, D.A. Lanffenburger, and R.D. Kamm Center for Biomedical Engineering, M.I.T., Cambridge, MA 02139

One approach to fabrication of an artificial vascular graft is to cast aortic smooth muscle cells into a reconstituted 3-D collagen gel, a design that partially simulates in vivo geometry of the arterial media. In studying various parameters which affect gel architecture, we found that contraction kinetics and morphology of the contracted gels could be altered by addition of L-ascorbate, a modifier of collagen fiber organization and stimulant of biosynthetic activity. Porcine aortic smooth muscle cells contract floating matrices reproducibly and uniformly into dense tissue-like equivalents. Histological examination of 3-week control gels reveals cells with varying phenotypes in three locations: embedded within the gel, often in discrete channels; on the surface in monolayers; and on the surface in multicellular spherical nodules (150-250 I.tm in diameter) with acellular cores containing lipids and fibrillar material. Adding ascorbate at concentrations of 25, 50, 100 and 200 gg/ml results in an initial dosedependent decrease in contraction rates. Although ascorbate-treated gels contract more slowly and less uniformly than controls, they ultimately attain a smaller size than controls and do not form nodules in the monolayer on the gel surface. Understanding factors which affect gel contraction and organization will aid in the design and evaluation of vascular grafts.

Porcine intestinal submucosa (PIS) has been shown to be a successful biomaterial when used as vascular replacement without chemical pretreatment. However, line is known about the nature of the remodeling process from a formal biomechanics perspective. We conducted the following "baseline" study to quantify PIS microstrucure and mechanical behavior using small angle light scattering (SALS) to quantify collagen fiber architecture and biaxial mechanical testing to characterize PPIS aniostropic mechanical behavior. SALS results indicated that PIS collagen fibers were well oriented along the longitudinal axis. PIS specimens chosen from multiple regions along the porcine small intestine showed no signficant regional variations. Biaxial mechancal results demonstrated that PIS is signflcantly anisotropic, with the circumferential direction experiencing 25% greater strain than the longitudinal direction under equi-biaxial stress of 1 MPa. PIS also demonstrated a complex mechanical coupling behavior that suggests microstructural interactions at the fiber level. Interestingly, biaxial stress-relaxation indicated little directional dependence. This study presents the first known biaxial mechanical analysis of PIS, and will provide the basic information for ongoing remodeling studies.

We have studied a new biomaterial derived from a class of ionic selfcomplementary oligopeptides. These short (8-32 amino acid residue) peptides self-assemble in aqueous salt solution into 3-dimensional matrices capable of favorable interactions with cells, and offer promise for useful design based on rational changes in sequence. Here we investigate mechanical properties, combining experimental and theoretical approaches, of one particular example of these peptide materials, EFK8. The static elastic modulus was measured using an apparatus designed to allow sample fabrication and mechanical testing in the same system with the sample in aqueous solution. The material microstructure was examined by SEM and the measurements interpreted with the aid of a model for cellular solids. Values for the elastic modulus increased from 1.59 to t4.7 kPa for peptide concentrations increasing from 2.7 to 10 mg/ml respectively. SEMS showed the microstructure to consist of a homogeneous lattice with fiber thickness of 10-30 nm independent of peptide concentration, but with fiber density increasing with peptide concentration. This behavior is consistent with scaling predictions from the model, and yields an estimate of the individual fiber elastic modulus in the range of 1-20 MPa. These results provide guidance for the improvement of the mechanical properties of these new materials.

Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

265 PEPTIDE-AMPHIPHE.,ES: A NOVEL STRATEGY FOR BIOACTIVE MATERIALS DESIGN Y.-C. Yu, M. Tin'ell and G. B. Fields University of Minnesota, BMEC, Depts. of Lab Medicine & Pathology and Chemical Engineering & Materials Science, Minneapolis, MN. Peptides containing the GVKGDKGNPGWPGAP sequence from the triple-helical region of type IV collagen, which is recognized by melanoma cells, were covalently coupled with single and double chain lipid tails by solid-phase peptide synthesis. The structures of peptides and peptideamphiphiles were studied by circular dichroism and NMR spectroscopies. The peptide alone forms a triple-helix of low thermal stability in solution. When coupled with lipid tails, the peptide-amphiphiles form thermally stable triple-helices. The melting temperatures of the triple-helices increase with the length of the lipid tails. The peptide-amphiphiles form stable monolayers at the air-water interface and can be deposited on surfaces. Melanoma cells adhere to and spread on this surface. REFERENCE Y.-C. Yu, P. Bemdt, M. Tirrell, and G. B. Fields J. Am. Chem. Soc. 1996, 118, 12515-12520

266 PENTOSANPOLYSULFATE BIOCOMPATIBILE COATING P Zupkas, P Stein, J Svahn, A Torri, and C L Parsons VA Medical Center, Urology Research, San Diego, CA Biocompatibility of devices in the urinary tract was investigated by surface coating silicone strips with pentosanpolysulfate (PPS). Study objective was to develop a method for reducing encrustation and bacterial adherence to the silicone surface, improving the safety and function of devices used in the urinary tract. Encrustation was investigated using two groups of ten rabbits undergoing bladder resection with concurrent placement of PPS coated and uncoated silicone strips. After six weeks, all rabbits were sacrificed and the bladders removed. Uncoated silicone strips were completely encrusted in phosphate salts, average weight 3 2 g. PPS coated silicone strips were free of encrustation except in areas were the suture had broken the silicone strip surface during placement, average weight 1. I g Bacterial adherence was investigated using PPS coated and uncoated silicone strips incubated three days in three strains of radiolabeled E. Coli after two hours of radiolabeling with C14-HCO3. Tests showed the adherence of all bacteria strains was 5-15 fold greater for uncoated strips versus coated strips for all strains of bacteria

267 ENHANCEMENT OF PEROXYNITRITE BREAKDOWN BY TITANIUM AND TITANIUM DIOXIDE R. Suzuki and J.A. Frangos Dept. of Bioengineering, University of California, San Diego, La Jolla CA Titanium has been used as an implant material due its biocompatible properties. We are investigating the hypothesis that this biocompatibility involves interaction of a mediator of the inflammatory response, peroxynitrite, with the surface layer of titanium oxide. Peroxynitrite is a highly reactive and unstable compound which is produced in vivo by the reaction of nitric oxide with superoxide. We investigated if titanium oxides affect the stability of peroxynitrite by promoting its breakdown. Peroxynitrite levels can be measured by its absorbance at 302 nm. At pH=13.2, we found a 100% increase in the rate of degradation of peroxynitrite in the presence of titanium particles. Peroxynitrite is capable of nitrating 4-hydroxyphenolacetic acid (4-HPA). The nitrated form of 4HPA can be measured by its absorbance at 432nm. 3-morpholinosydnonimine (SIN-1), a nitric oxide donor, has been shown to produce superoxide during its breakdown resulting in the formation of peroxynitrite. At physiological pH (7.4), a solution of 5raM 4-HPA was exposed to 5raM SIN-1 in the presence of titanium dioxide powder. After one day there was 42% less nitrated 4-HPA in the solution containing titanium dioxide compared to controls. These experiments suggest that titanium is capable of enhancing the breakdown of the inflammatory compound peroxynitrite which may account for the metal's biocompatible properties.

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268 MEASURING THE KINETIC RATES OF INTERACTIONS OF SURFACEBOUND RECEPTORS AND LIGANDS Scott Cbesla and Cheng Zhu Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA Kinetic rate constants of adhesive receptors for ligands and their dependence on molecular structure are essential to a wide variety of biological phenomena. While binding of soluble ligand to cell surface receptors can be measured via many conventional methods, the only existing technique for determining the kinetic rate constants of surfacebound adhesion molecules is that of the flow chamber. We developed a novel micropipet method to determine these constants by measuring the relationship between adhesion probability and contact duration. A monvalent biomolecular binding model was supported by kinetic data from two CD16 (Fc7 receptor III) isoforms anchored to the Chinese hamster ovary cell via either a transmembrane polypeptide chain or a glycosyl phosphatidyl inositol moeity interacting with two immunoglobulin (Ig) G ligands of either human or rabbit origin. The membrane anchor of the receptor was found to directly influence the forward, but not the reverse, rate constant. In addition, the effect of ligand orientation on the kinetic rate constants was examined by coating the human red blood cell (RBC) either with specific rabbit anti-human RBC IgG to obtain oriented Fc sites of CD16 binding or with non-specific rabbit IgG via chromum chloride coupling to obtain randomly oriented Fc sites. The forward, but not the reverse, rate constant was found to depend on the ligand coating method. These data provide new insight to the structure-function relationship of molecular interactions.

269 A MODEL STUDY OF THE IMPACT OF ADHERENT LEUKOCYTES ON VENULAR BLOOD FLOW G.B. Chapman and G.R. Cokelet University of Rochester, Depts. of Biochemistry & Biophysics and Pharmacology & Physiology, Rochester, NY 14642 The blood flow drag on multiple leukocytes adherent on a venule wall, and the impact of the leukocytes on the blood flow resistance were determined by solving the Navier-Stokes equations with a commercial finite-volume solution package (CFD Res. Corp., Huntsville, AL) for flow of a Newtonian fluid past spheres adherent to the wall of a cylindrical vessel. For two interacting spheres, when the spheres are on opposite sides of the vessel axis, the drag force on one of the spheres (Fs) is greater than that for a single isolated sphere (Fo) and a function of sphere separation distance (ds) and the sphere to vessel diameter ratio (d/D). When the second sphere is directly downstream from the first, Fs/Fo < 1.0, depending on d s and d/D. For 3 or more interacting spheres in a line Fs/Fo for the interior spheres is less than that for the end spheres, but not very sensitive to the spatial arrangement of the interior spheres. The resistance to flow in a vessel is calculated from the pressure drop (AP) across the spheres and varies with d/D and the relative radial positions of the spheres. There is little increase in AP for interacting spheres when an adherent sphere is added directly downstream from other spheres compared to when a sphere is added on the opposite side of the vessel. Supported by NIH HL PO1 18208

270 MODELING THE MIGRATION AND PROLIFERATION OF ENDOTHELIAL CELLS WITH CHANGING SHAPE AND SIZE Aparna Ambani l, K. Zygourakis 2 and P. Markenscoff 1 1 Dept. of Electrical and Computer Engineering, University of Houston. 2 Dept. of Bioengineering, Rice University, Houston, TX. Due to the multitude of parameters affecting the dynamics of cellular systems, the development of engineered tissues will be greatly facilitated by mathematical models that can predict cellular movement and tissue growth. This study focuses on analyzing the opposing effects of contact inhibition and migration on the proliferation rates of endothelial cells (EC). A new model based on the concept of cellular automata is developed to accurately describe the dynamics of migrating and proliferating EC. Each EC can now occupy several adjacent sites on the computational grid and its shape varies with time as the cell changes directions, collides with other cells and divides. Changes in the direction of cell movement (caused by cell-cell collisions or intracellular events) involve the formation of a leading lamella pointing in the new direction. The new model is a significant extension of earlier models developed by our group. Simulation results show that cell proliferation rates are significantly affected by the speed of locomotion, the persistence of cell movement and the elongation of migrating ceils. Also, the cell shape and cell density affect the population-average speed of migration. Model predictions agree well with the results of cell growth experiments.

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Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

271 f i l e MOLECULAR BINDING AND RUPTURE CHARACTERISTICS BETWEEN FC GAMMA RECEPTOR Ill (CDI6) AND ITS L1GANDS Scott Chesla and Cheng Zhu Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332 Specific interaction (binding) between cell surface receptors is essential for living organisms to sense and adapt to their environment Fc gamma receptor III (CD16) is natively expressed in two membrane anchor isoforms on leukocyte subsets and represents an excellent experimental model to closely examine the binding and detachment tbrce characteristics of a biologically relevant receptor upon interaction with its natural ligands. Using an extension of Evans' micropipet technique, we determined both the debonding force characteristics and binding kinetics of CD16 (expressed on CHO cells) to its ligands (coated on red blood cells) while both were membrane anchored Extracting this 2-D information requires a probabilistic formulation of the debonding process (master equation) and a theoretically based relation between the reverse rate constant and applied separation force For the latter, we tested both Bell's exponential growth and Evans' power law ibrmulations. We have determined that the membrane anchor of CDI6 directly effects the binding affinity and on-rate only when both molecules are membrane anchored In addition, the master equation was found to closely match our experimental data: however, neither reverse rate constant-force relationship provided a good representation of our data over its entire range. (Supported by NSF grant No BCS 9350370 and NIH grant No. R29 A138282)

272 QUANTIFICATION OF RECEPTOR EXTRACTION PROBABILITY Scott E. Chesla, Bryan T. Marshall and Cheng Zhu Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA Recently, there has been an increasing interest in measuring the interaction forces between membrane bound cell adhesion receptors and their substrate or membrane bound ligands. Nineteen years ago, George Bell estimated the force required to break a receptor-ligand bond and that required to uproot the receptor from the cell membrane to be of the same order of magnitude. The interpretation of the force data therefore requires the knowledge of detachment mode, i.e., via adhesive mechanism if the receptor-ligand bond is dissociated or via cohesive mechanism if the receptor-membrane anchor is disrupted. Previously, we developed a simple micropipet assay to measure the force and kinetics FcT receptor III (CD16) binding to its ligands. We noted that in a series of repeated adhesion tests either stable or declining running adhesion score averages might be observed depending on ligand or receptor membrane anchor type; and we suggested the latter as an indication for receptor uprooting. Here, we report a statistical analysis of the running adhesion frequency to enable a quantifiable measurement of the extraction probability. The basic idea is that, since the force to break a bond is a random variable, whether the detachment of a particular crossbridge results in receptor extraction also is stochastic. Nevertheless, a defined probability of such an event exists and reflects the relative strengths of the bond and membrane anchors. By correlating the extraction probability with various types of receptor-ligand bonds and membrane anchors, we hope to provide both insight into the stmcture-fucntion relationship of the molecules and basis for the development of a single cell assay for cytoskeletal linkage of integral proteins.

273 SUPPORTED BIOACT1VE L A N G M U I R - B L O D G E T r BILAYERS Y. Doff and M. Tirrell Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455 Supported bioactive membranes have many industrial applications such as scaffolding for artificial tissue and for immobilizing proteins. We have created supported membranes using novel peptide amphiphiles containing a peptide sequence, called peptide IVH1, covalently linked to two hydrocarbon chains. This peptide is present in type IV collagen and is known to promote adhesion of melanoma cells. Bilayer membranes, containing the bioactive peptide amphiphile mixed with a biologically inert amphiphile, were deposited on hydrophobic substrates using the Langmuir-Blodgett deposition technique. Atomic Force Microscopy micrographs of such layers show unique star shaped patterns whose structure depends on experimental variables, such as temperature, and the properties of the amphiphile, such as tail length. Cell adhesion assays using M4 melanoma cells show that cells selectively adhere and spread on layers containing the peptide amphiphile. Further experiments are intended to study how surface morphology and composition affect the cell behavior on these layers.

274 ,\ PRELIMINARY STUDY OF NON-ADHESIVE STATE OF LFA-I ~T SINGLE BOND LEVEL * Jing Zhang, Yah Jun Zeng and * Feng Yuan Zhuang * Beijing Polytechnic University ; * China-Japan Friendship Institute of Clinical Medical Sciences For single LFA-1/ICAM-1 bond, little is known in quantitive terms about the adhesion properties such as adhesion probability and bond strength in the non-adhesive(or resting) state of LFA-1 A biophysical approach was employed to study this issue in vitro, including the micromanipulation technique, the chromium chloride method and the single bond theory. Our preliminary result showed that the probability for non-adhesive LFA-1 to bind to ICAM-1 is low, less than 21 5 %, and this probability is closely related to ICAM-I's density on cell surfaces. It also showed that the bond formed by resting LFA-1 and ICAM-1 is weak, with a strength less than 20pN.The low adhesion probability and weak bond strength provide direct evidence to the "low avidity" of LFA-1 in its resting state which observed macroscopically; and the role of this non-adhesive state of LFA-1 suggests an important self-protecting mechanism for leukocyte

275 RECOMBINANT

ADHESIN PROTEIN INHIBITS ADHESION TO COLLAGEN UNDER DYNAMIC CONDITIONS. Nehal Mohamed and Julia M. Ross University of Maryland Baltimore County, Department of Chemical and Biochemical Engineering, Baltimore, MD 21250.

STAPHYLOCOCCUS

AUREUS

Staphylococcus aureus is one of the most common etiological agents

of bacterial arthritis and acute osteomyelitis, and has been shown to bind collagen under static conditions. However, since many infections caused by S. aureus are acquired hematogenously, their interactions with extracellular matrix proteins under dynamic conditions are important. Receptor-mediated adhesion of S. aureus with collagen II under dynamic conditions was studied in vitro using video microscopy coupled with digital image processing. It was found that adhesion follows first order kinetics and that the adhesion rate is shear stress dependent. Pre-incubation of the collagen matrix with a recombinant form of the collagen receptor blocked bacterial adhesion up to 95%. These results suggest the possible therapeutic use of the recombinant protein in preventing infection. The effect of collagen receptor-specific antibodies on adhesion is cmTently being studied.

276 STAPHYLOCOCCUS EPIDERMIDIS ADHESION TO AN IN VITRO MODEL OF HOST MODIFIED SURFACES UNDER HYDRODYNAMIC FLOW CONDITIONS J.M. Higashi*, J.S. Temenoff* and R.E. Marchant*> Depts. of Biomedical Engineering* and Macromolecular Science*~, Case Western Reserve University, Cleveland, OH

Infection is a serious problem associated with the use of blood contact devices because the only effective treatment is surgical replacement. We have studied adhesion of S. epidermidis to plasma proteins and platelets adsorbed on a model hydrophobic biomaterial surface. A dose-response curve of S. epi adhesion to plasma proteins from 10E7-10E9 bac/mL showed that S.tepi adhesion resembled a multilayer adsorption isotherm. Increasing shear stress decreased bacterial adhesion to adsorbed plasma proteins significantly up to 36 dynes/cm2, after which there were no further reductions in adhesion. This suggests that the adhesion of S.$epi to adsorbed plasma proteins at low shear is weak, indicating that a critical time window exists during which these bacteria could be dislodged before they become integrated into a strongly adherent biofilm. Preliminary results suggest that at low shear stress (0-2 dynes/cm2), the adhesion of the bacteria is greater to the plasma proteins than to surfaceadherent platelets, while at high shear stress there is greater adhesion to the platelet-adsorbed surface.

Cell and Tissue E n g i n e e r i n g - Posters

277 ALLOGRAFTING 1N THE BODY WALL OF A SEA CUCUMBER: IS REAPPROXIMATION AN A-CELLULAR PHENOMENON? G. K. Szulgit and R. E. Shadwick Scripps Institution of Oceanography, UCSD, La Jolla, CA The collagenous connective tissues of echinoderms are structurally similar in many ways to those of vertebrates. Likewise, they possess cellular wound responses that are analogous to those of vertebrate tissues. While these cellular responses are probably essential for proper wound healing in holothurians (sea cucumbers), we have found that an astonishing level of adhesion between two wound surfaces can be rapidly achieved without cellular activity. Specifically, we found that dissected pieces of body wall from Porastichopus parvimensis will adhere to each other whether the cells in the tissues are intact or destroyed. Lapshear tests show that breaking strains of adhered tissues increase over time, reaching approximately 0.5 KPa after 24 hours of contact. Furthermore, body walt at{ografts are successfully incorporated into live animals without any external pressures, sutures, or artificial gels to keep them in place. Dislodging the grafts from the animals required shear strains of approximately 14 KPa after 24 hours of contact. We suspect that the adhesive ability of the body wall plays a key role in the initiation of this grafting.

278 COMPUTER SIMULATION OF TIME DEPENDENT RED BLOOD CELL SHAPE CHANGES T. Leibner, H. Henning*, AL. Li and G.M. Artmann Cell Biophysics, Aachen Univ.of Applied Sciences, *Dept. Bioeng. and Medical Informatics, Ilmenau University of Technology, Germany Red blood cells (RBCs) attached as monolayers to a cover slip in a flow chamber respond time dependently with shape changes to stepwise modifications of buffer composition, shear forces, etc. These shape changes were recorded photometrically* following an automated experimental protocol defining the exposure of the RBCs to the modifications chosen (time intervals, concentrations, etc.). To derive system properties from the signal, as characteristic time constants and amplitudes, a structural model based on system theory has been developed and numerically fitted to the experimental data. As an example stomatocytes were induced via flushing (1Pa shear force lasting 10 s) Chlorpromazine containing buffer into the flow chamber (wash-in). After a flow stop of 30 min. CP-free buffer was administered (lPa/10s) and the CP wash-out was observed. From this, RBC stiffness and relaxation time, wash-in time of CP, shape reversal time (in presence of CP) and CP-wash out time were obtained. This data analysis principle is extendible to similar experiments on cells with processes overlapping in time and on-line data acquisition.*Artmann, Biorh., 32(5)1995, 33(3)1996

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280 A DEVICE TO IMPOSE PRESCRIBED HOMOGENOUS STRAINS ON CULTURED CELL MONOLAYERS CM Waters, MR Glucksberg, EP Lautenschlager, KE Healy, U Savla, CW Lee, RM Van Matre, ILl Warp Northwestern University, Biomedical Engineering, Anesthesiology and Dental Biological Materials Departments, Evanston and Chicago, ]L Most existing devices designed to impose known oscillatory stretch on cultured cell monolayers have features that limit their usefulness in our laboratory. We have therefore developed, and now report, a system designed to impose large-strain, homogeneous, stretch on a multi-well surface-treated elastomer (Dow Coming Silastic T-2) substrate plated with pulmonary epithelial cells. Additional design considerations were the ability to use this device with a permeable substrate, the ability to vary the degree of strain isotropy and the ability to control the frequency, strain rate, duty cycle and magnitude of the oscillations. The pneumatically driven mechanism is compact enough to fit inside an incubator and consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle Stretch parameters are set by computer-controlled electronic valves connecting a 100 psi compressed air source to the pneumatic cylinders. Pilot studies show that this device provides an effective way to study the influence of large (>10%) biaxial stretch on pulmonary epithelial cell monolayers. Support: Whitaker Foundation, NSF

281 A DEV1CE FOR THE APPLICATION OF EQU1BIAXIAL STRAIN TO CULTURED BOVINE BLADDER SMOOTH MUSCLE CELLS N. Peter Davis and Robert S. Cargill II George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, 30332 Lar e mechanical strain is a major feature of normal urinary bladder fungtion. Pathologies such as outlet obstruction change the strain field within the bladder and profound changes in bladder morphology result at both the tissue anal cellular levels. We present here a device developed to study the effects of mechanical strain on fetal bovine bladder smooth muscle cells (BBSM) in vitro. This device generates a uniform equibiaxial strain field by deflecting a circular 0.200 mm thick elastomeric membrane (mixture of Sylgard'~ 184 and 186, Dow Coming) over a stationary Cylindrical form. Cells adherent to the surface of the membrane are strained as the membrane is strained. Membrane strain versus deflection over the form is a slightly nonlinear function with a maximum achievable strain of 57%. Video m~ages of ink dots on the membrane were analyzed with NIH Image and a finite approximation to the Greens strain tensor was calculated from triads of points with a Matlab code to characterize the strain field. Results show no significant difference between strains along the two video axes confirming equibiaxial strain conditions BBSM cells have qualitatively b~en shown to adhere to static membranes and to remain attached to membranes deflected at a rate of 0.064 mm/s. These results demonstrate the efficacy of this device for studying the in vitro response of BBSM to quantified mechanical strain.

279 OXIDATIVE STRESS INCREASES RED BLOOD CELL(RBC) ADHESION TO BOVINE AORTIC ENDOTHELIAL CELLS(BAEC) Elena A. Gorbatenkova, Gerhard M. Artmann Cell Biophysics, Aachen University of Appl. Sciences, Ginsterweg 1, D-52428 Juelich, Germany

282 A DEVICE TO BIAXIALLY STRESS BIOMATERIALS IN CULTURE S.B. Mitchell, JL. Garbini, P K Schuessler, and JE. Sanders University of Washington Engineering Biomaterials (UWEB) Program, Department of Bioengineering, University of Washington, Seattle, WA

Active oxygen species (AOS), generated in vivo by neutrophil and monocyte-macrophage activation, promote RBC- BAEC adhesion. We studied the effect of H202, hypochlorous acid (HOCI) and HOCImodified blood lipoproteins on BAEC-RBC adhesion. BAECmonolayers were cultured on a microscope slide, activated with AOS and placed in a parallel flow chamber. Human RBCs were added, al-lowed to settle and form attachments. The RBC detachment was quanti-fied at 0.1 Pa wall shear stress. Compared to control, 100gM H202 in-creased the RBC adhesion to 200%; 25, 50, 100 g.M HOC1 to 240, 500, 270%, respectively. 50 and 250gM HOCl-modified blood lipoproteins elevated the adhesion to 200 and 450%, respectively. The enhanced ad-hesion was in parallel with an increased molar cholesterol/phospholipid ratio in the BAECs. The results suggest that AOS at physiological concentrations either directly or due to lipoprotein modification modulate BAEC-RBC adhesion.

Mechanical stresses at the interface of a biomaterial and natural tissue can have an important impact on the acceptance and performance of the implant. The long-term goal of this research is to investigate the effects of mechanical stress on cytokine release, fibrous ingrowth, and cell density in biomaterials intended for skin or cardiothoracic applications. Within this project, a system to apply stresses was designed. The system was a closed-loop displacement control system that could apply any reference cyclic waveform within a 19 Hz bandwidth in one direcron while the orthogonal direction was maintained at a constant displacement. Up to ten specimens, each in its own media chamber and maintained in a sterile environment, could be loaded simultaneous. Closed-loop control systems were also designed to control temperature of the media to within 1~ C and [CO2] to within 5%. The device, its performance, and preliminary test results with skin fibroblasts in a blow-molded fiber porous biomaterial will be presented

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Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

283 A STRAIN DEVICE IMPOSING DYNAMIC AND UNIFORM EQUIBIAXIAL STRAIN ON CULTURED CELLS M. Sotoudeh, S. Usami, J. Shyy, and S. Chien UCSD, Dept. of Bioengineering, San Diego, CA, 92037.

286 MORPHOLOGICAL CHANGES IN ENDOTHELIAL CELLS OF EXCISED AORTIC SEGMENTS UNDER SHEAR FLOW M. Sato, N. Sakamoto, K. Takeda, and N. Kataoka Grad. School of Mech. Eng., Tohoku University, Sendai 980-77, Japan

The objective of this study was to design a new apparatus to allow the control of the magnitude and frequency of dynamic stretch applied uniformly to cells cultured on a silicon elastic membrane. The apparatus was designed to produce equi-biaxial dynamic stretches with area changes ranging from 0 to 55% and frequencies ranging from 0 to 2 Hz. Homogeneous finite strain analysis using triangles of markers was performed in both dynamic and static conditions to compute the symmetric two-dimensional Lagrangian strain tensor on the membrane. Strain measurements showed that the shear component of the strain tensor (E~) was near zero, and that there was no significant difference between radial (E~,) and circumferential (E~c) components, indicating the attainment of equi-biaxial strain. Bovine aortic endothelial cells were transiently transfected with a chimeric construct in which the luciferase reporter is driven by TPA-responsive elements. The application of cyclic stretch at 1 Hz to the transfected cells cultured on the membrane led to a significant increase in luciferase activity only when the cell area was stretched by 15% or more. Cells in different locations of the membrane showed similar induction of luciferase activities, confirming that the strain is uniform and equi-biaxial across the membrane.

We will report preliminary results on the morphological response of endothelial cells to fluid shear flow using excised rabbit aortic segments. The specimen with a new longitudinal axis 90 ~ from the original vessel axis was carefully fixed on a silicone rubber with holder, which will be set to a flow chamber. In this study shear stress of 1 Pa was applied for 24, 48 and 72 hours. After flow exposure endothelial cell junctions were stained with silver nitrate and the cell shape and orientation were measured. As a control, aortic segments were kept in an incubator under no flow condition. Just after excision of aortic walls shape index of endothelial ceils was 0.36~0.07 (n=104, mean+SD), and angle of cell orientation was 4.9*• ~. In the segments exposed to flow the shape indices were 0.48+0.10 (n=108) for 24 hours, 0.54• (n=61) for 48 hours and 0.~.10 (n=99) for 72 hours. This time course change in shape index was similar to the case of control experiments. However, the shape index values at 48 and 72 hours in flow exposed cells were significantly larger than those in control conditions. Although cell alignment in flow exposure took almost the similar time course changes to the control until 48 hours, around 40% cells strongly aligned to the flow direction (>_80") at 72 hours.

284 SKIN ADAPTATION TO MECHANICAL STRESS: LIGHT AND ELECTRON MICROSCOPY RESULTS J.E Sanders and B.S. Goldstein Departments of Bioengineering and Rehabilitation Medicine, University of Washington, Seattle, W A

287 BLADDER SMOOTH MUSCLE CELL RESPONSES TO SUSTAINED HYDROSTATIC PRESSURE Karen M. Haberstroh, Martin Kaefer', Alan B. Retik', Michael R. Freeman', and Rena Bizios Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, "Department of Urology, Boston Children's Hospital and Harvard Medical School, Boston, MA

The skin of people who use prosthetic limbs, wheelchairs, or orthoses is at risk of breakdown because of the high-magnitude or long-duration repetitive mechanical stresses that are applied However, under the proper conditions skin can adapt to become more load-tolerant. The purpose of this research is to investigate skin adaptation at a structural level. Skin on the lower lateral hind limb of 15 farm pigs (initial mass --7 kg) was loaded ' at 1 Hz with normal and shear force waveforms similar to those that occur at the stump-socket interface of lower-limb amputees ambulating with prostheses. Loads (maximum normal force 13 N, maximum shear force 3 N) were applied to a 7 mm x 8 mm pad for 1 hour/day 5 days/week for 4 weeks. Quantitative morphological analysis comparing adapted and control skin was conducted to evaluate: skin thickness, elastin density, epidermal cellular layer thickness, basement membrane surface area, collagen density, cell density, collagen fibril thickness, and collagen fibril orientation. Results from this analysis will be discussed.

285 MORPHOLOGICAL RESPONSES OF CULTURED AORTIC ENDOTHELIAL CELLS TO THE CHANGE OF FLOW DIRECTION N. Kataoka, H. Miyano, S. Ujita and M. Sato Grad. School of Meek Eng., Tohoku University, Sendai 980-77, Japan The effects of the change of flow direction on the morphology of cultured bovine aortic endothelial cells were studied. Fully confluent endothelial cells cultured on glass plates were subjected to fluid-imposed shear stress of 2 Pa for 24 hours. Experiments on shear flow exposure were performed for alternately orthogonal flow with a 3, 30 rain and 1 hour interval. After flow exposure, endothelial cells were fixed and F-actin filaments were stained with rhodamine phalloidin and shape index (SI) and angle of cell orientation were measured. After application of 2 Pa shear stress for 24 hours in the alternately orthogonal flow with 30 rain interval the endothelial cells did not elongate so strongly (SI = 0.63 • 0.14) and slightly aligned with the center of two flow directions. With 1 hour interval, cells were more elongated (0.55 + 0.16) than the cells with 30 min and aligned with the center of two flow directions. In the both flow conditions, thick and clear stress fibers which aligned with cell axes were observed in central portion of the cells. In the flow experiments with 3 min interval, many cells were removed from the glass plates. Moreover, in the remained cells on the glass plates after 24 hours, stress fiber was not observed. The results of this study provide evidences that endothelial cells reflect the local blood flow pattern.

An in vitro cellular model was designed to simulate conditions pertinent to bladder physiology and used to investigate the responses of bladder smooth muscle cells to pressure. Ovine bladder smooth muscle cells (passage number four) were either maintained under 0.5 cm H20 (control) or exposed to sustained hydrostatic pressure of 2, 4, 6, and 8.5 cm H20 under standard cell culture conditions for time periods of 1, 3, 5, and 7 days. Under these conditions, bladder smooth muscle ceils proliferate as a function of both pressure level and duration of exposure to the mechanical stimulus. Transferable, bioactive, soluble, and mitogenic compound(s) is(are) involved in the mechanism(s) of this response. These findings provide cellular/molecular level information regarding bladder tissue physiology and could have significant implications in the field of clinical urology.

288 MEASUREMENT AND MODELING OF COUPLED OSMOTIC AND MECHANICAL BEHAVIOR OF PLANT CELLS DURING CRYOPRESERVATION J.R. Walsh, K.R. Diller and J.J. Brand Univ. Texas, Biomedical Engineering Prog. & Dept. Botany, Austin, TX. In conjunction with the development of cryopreservation protocols for more than 2000 strains for algae in the UTEX culture collection, we have analyzed the governing biophysical behavior of cells with a semipermeable membrane and an outer elastic wall during the osmotic stresses imposed during the addition and removal of cryoprotective agents and during freezing and thawing. Osmotic challenge tests were conducted on the algae Chlorococcum texanum to subzero temperatures on a perfusion cryomicroscope system. Transient volumetric changes were quantified via computer analysis of video micrographs. A network thermodynamic model of the process coupled between the osmotic and mechanical energy domains was inverse fit to the data to determine the constitutive properties for the cells and the temperature coefficients. The cells demonstrated a clear biphasic behavior. At relatively large diameters for which the cell wall and membrane connection was intact, the elastic properties of the wall dominated volume changes in response to osmotic stress. At smaller cell diameters below the state at which plasmolysis occurs, the transient volume was dictated by the osmotic properties of the membrane. This research was sponsored by NSF grant no. DBI-9512746 and CTS9632378 and Texas Advanced Research Program no. 003658-158.

Cell and T i s s u e E n g i n e e r i n g - Posters

289

HYDRODYNAMICS OF M I C R O P I P E T ASPIRATION Jeanie Drury and Micah Dembo Boston University, Dept. of BME, Boston, MA Experiments on the micropipet aspiration of human leukocytes have led to a class of simple mechanical models in which the cell is approximated as a "slippery droplet" of viscous fluid. Here. we present computations that reveal the detailed predictions of the simplest and most idealized case of such a scheme; namely, the case where the fluid of the droplet is homogenous and Newtonian. and the surface tension of the droplet is constant. We have investigated the behavior of the model as a function of surface tension, droplet radius, viscosity, aspiration pressure, and pipet radius. Computations were carried out using a low Reynold number hydrodynamics transport code based on finite element method. Although idealized and simplistic, we find that the slippery droplet model predicts many observed features of leukocyte aspiration. However, there are certain features that are not observed in leukocytes. In particular, at high aspiration pressures, the model predicts dilation of the membrane past the point of being continuous, as well as a reentrant jet.

290 ALTERATION OF GENE EXPRESSION IN ORGANOTYPIC BRAIN CULTURES IN RESPONSE TO MECHANICAL INJURY -B. Morrison III, *J. H. Eberwine, -D. F. Meaney, +T. K. Mclntosh Departments of -+Bioengineering, +Neurosurgery, and *Pharmacology University of Pennsylvania, Phila., PA 19104 Mechanical brain injuries typically occur in less than 50ms as a result of tissue strain between 10% to 50% and strain rate between 10 and 50Hz. Many changes after brain injury have been documented, and it is hypothesized that these changes, occurring as a result of the initial mechanical trauma, ultimately lead to altered gene expression. These changes in gene expression in brain tissue have been documented in an in vitro model of a purely mechanical injury. Organotypic brain slice cultures, 350urn thick, were allowed to attach to a deformable substrate mounted in a custom built culture well. Cultures were maintained in vitro for 18 days before experimental injury. Cultures were aseptically injured by a single mechanical event at a strain of 28% and a maximum strain rate of 17Hz. Cultures were then returned to the incubator for 2 days before being harvested. Isolated RNA was amplified, labeled, and probed by means of Reverse Northern Hybridization. In preliminary experiments, the expression of CKII, PKClb, and Ubiquitin was decreased whereas the expression of NGF was increased after injury as compared to uninjured control cultures. Supported by NINDS grants P01-NS08803 and R01-NS26818.

291 RESPONSE OF NEURAL CELLS TO AN ACUTE MECHANICAL DEFORMATION Yougandh Chitre, Harry Keo Springer and Robert S Cargill 1I, George W. Woodruff School o f Mechanical Engineering, Georgia Institute of Technology, Atlanta, G A 30332-0405 Traumatic brain injury (TBI) accounts for as many as two million deaths annually in the Umted States. The dearth of quantifiable data on the response of neural cells to an acute mechanical deformation produced in TBt has prompted our investigation. We present here a device that induces a dynamic deformation to a culture of differentiated NG 108-15, neuroblastoma cross-glial hybrid cells. Our device produces a deformation to a 0 2 0 mm thick Sylgard elastomeric membrane by vertically deflecting it over a fixed, cylindrical, hollow form, resulting in an equibiaxial strain field on the membrane surface. This allows cellular strain to be independent of cell orientation and location. While strained, the cells remain in a single plane-of-focus permitting real-time measurements with an epifluorescence microscopy system Thus, increases n intracellular free calcium concentrations and changes in membrane potential, indicators of response and functional impairment, can be measured. Qualitative static and dynamic tests have demonstrated the attachment of a NGI08-15 culture to the membrane surface treated with laminin, an adhesion promoter. We are currently quantifying the (esponse~ of these cells for stratus up to 50% with strain rates of either 0. Is" or 10 s . The insight gained from our cellular level research will increase our understanding of the mechanisms involved in the loss of neuronal function resulting from TBI.

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292 MYOTUBE STRETCH INDUCES A STRAIN RATE DEPENDENT INCREASE IN PROTEIN SYNTHESIS T.J. Burkholder, C. B. Clark and J. A. Frangos UCSD, Dept. of Bioengineering, San Diego, CA. Mechanical signals are essential to skeletal muscle morphogenesis and maintenance. In vitro systems have demonstrated that strain patterns influence the alignment and fusion of myoblasts into myotubes and the long term condition of those myotubes. Similarly, cyclical passive stretch stimulates whole muscle growth in vivo. We investigated whether the stretch induced increase in protein synthesis is strain rate dependent. C2C12 skeletal myoblasts were seeded onto collagen coated silastic membranes and induced to fuse. Two days after fusion, cells were exposed to periodic cyclic stretch in a uniform linear cell stretcher. Cultures were switched to serum free media six days after fusion and amino acid incorporation was evaluated 24 hours later by incorporation of tritiated amino acids. Radioactivity in purified protein was enhanced in myotubes cycled at strain rates less than 25% s -1 (p < 0.001), by an average of 32% over static controls. At strain rates greater than 30% s "l, labeled amino acid incorporation was slightly but nonsignificanfly reduced, averaging 90% of controls. The biphasic nature of this stretch response has implications for the investigation of strain induced effects. It highlights the importance of maintaining a defined strain rate when altering strain magnitude.

293

EXPOSURE OF HUMAN ENDOTHELIAL CELLS TO SUSTAINED PRESSURE AFFECTED INTEGRIN-MEDIATED CELL FUNCTIONS E.A. Schwartz*, R. Bizios*, and M.E. Gerritsen:l: *Dept. of Biomed. Eng., Rensselaer Polytechnic Institute, Troy, NY. ~IBJD&C, Bayer Corporation, West Haven, CT. Endothelial cells in vivo are exposed to mechanical forces (i.e., fluid shear, tensile stress, and hydrostatic pressure) which affect cell function; however, the molecular mechanism(s) by which cells respond to these forces are poorly understood. The current research aims to elucidate these mechanisms by exposing cultured human umbilical vein endothelial cells to 1 - 4 cm H20 sustained hydrostatic pressure for time periods of 0.2 - 4 days, and, subsequently, by examining protein expression by these cells. Western blot and immunoprecipitation techniques are used to quantitate the total amount and phosphorylation states of both integrinassociated focal adhesion plaque molecules and immediate-early response transcription factors associated with intracellular transduction of mechanical forces. These results contribute to the current knowledge of endothelial cell function and have important implications in vascular pathophysiology.

294 EFFECTS OF TISSUE CONSTRAINING ON L I T H O T R I P S Y INDUCED BUBBLE OSCILLATION I N VIVO P. Zhong , , I. Cioanta #, F.H. Cocks# and G.M. Premlnger Depts. of #Mech. Engr. & Mat. Sci. and *Urology, Duke University Using a focused hydrophone (fr = I MHz), we have measured the acoustic emission (AE) associated with the rapid oscillation of cavitation bubbles induced by lithotripter shock waves in renal parenchyma and within the kidney pelvis of a swine model, and compared this with the AE produced in water under the same lithotripsy conditions. Although in general, a similar temporal pattern of AE signals were detected in vivo, as compared to that of in vitro, substantial differences in the duration of bubble expansion and subsequent ringing after its primary collapse were observed. At 20 kV output voltage (40 MPa peak pressure), bubble expansion duration was found to decrease from 301+_17 Its in water to 114+-3 and 67+4 Its, in pelvis and renal parenchyma, respectively. While the ratio of subsequent ringing to bubble expansion duration was found to increase from 0.7 to I~0 and 15, in the corresponding sequence. This finding indicates a clear tissue-constraining effect on lithotripsy-induced bubble oscillations in vivo. The results also suggest that cavitation nuclei (1-3 Itm) in capillary vessels (8-30 Itm) may expand larger than 500 Itm. Such a large expansion of the bubble inside the vessel may constitute the mechanism for capillary rupture. (Supported by the Whitaker Foundation and NIH grant PO1-DK 20543)

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Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

295 TROPOMODULIN-TROPOMYOSIN COMPLEX AS A MOLECULAR RULER FOR THE PROTOFILAMENTS IN THE ERYTHROCYTE MEMBRANE SKELETON L. A. Sung, J. J.-C. Lin, L. J. Yee, and M. Mehrpouryan University of California, San Diego, Dept. of Bioengineering, La Jolla, CA and University of Iowa, Dept. of Biological Sciences, Iowa City, IA The erythrocyte membrane skeleton is composed of hexagonal protein lattices with F-actin protofilaments positioned in the junctional complexes. The protofilaments consist of mainly actin, tropomyosin (TM) and tropomodulin and have a uniform length of 33-35 nm. We have previously cloned the cDNA for human erythrocyte tropomodulin. We discovered that recombinant tropomodulin binds within the N-terminal 18 residues of TM isoform 5 (TM5), preventing the head-to-tail association of TM. We further identified TM5 and TM5b, a product of the ~' and c~ gene, respectively, as the major isoforms in human erythrocytes (Sung et at., in preparation). Our findings that ( 1) both TM isoforms in erythrocytes are of low MW TM with 33-35 mn in length; (2) tropomodulin is capable of binding both TM5 and TM5b, which form homodimers and heterodimers; and (3) erythroleukemia cells increase tropomodulin expression upon induction of differentiation, support our hypothesis that the complex formed by tropomodulin, a capping protein, and TM5 or TM5b, a 33-nm long rod-like molecule, may function as a molecular ruler. Such a ruler may meter-off long actin filaments to short protofilaments of 33-35 nm during terminal differentiation and thus, alter the mechanical properties of maturing erythrocytes.

296 CHLORPROMAZIN MODULATES THE MORPHOLOGICAL MACRO- AND MICROSTRUCTURE OF ENDOTHELIAL CELLS I.S.Hueck, H.G.Hollweg*, G.W.Schmid-Schtnbein**, U.Sahm And G.M.Artmann Cell Biophysics, Aachen University of Appl. Sciences, *Institute for Pathology, RWTH Aachen, GER; Dept.of Bioeng.**,UCSD The amphipathic cationic drug Chlorpromazin (CP) causes red cell membrane bending with formation of stomatocytic shapes. In this study we in-vestigated the effects of CP on the shape of bovine aortic endothelial cells (BAEC) in confluent monolayers and their submicroscopic plasma mere-brahe curvature with image analysis of phase contrast- and TEM. Above 10 gM CP, a steady decrease of the relative cell area (area before/after CP-treatment) occured. The cell area circularity index (ACI, perimeter2/cell area) increased at 10 gM CP. Between 20 I.tM and 150 p.M CP, the ACI decreased continuously leading to a perfect spherical cell shape. Distinct submicroscopic curvature changes were observed with nanomolar CP con-centrations: The membrane assumed an irregular shape developing more intracellular vesicles and pseudopodia. At higher concentrations, the macroscopic changes in membrane curvature may be due to loss of cell junctions, while at lower concentrations bilayer bending may play a more significant role with a possible influence on transendothelium transport.

298 PARTITION COEFFICIENT OF MACROMOLECULES IN POLYMER GELS AND TUMOR TISSUES. Fan Yuan and Ava Krol. Department of Biomedical Engineering, Duke University, Durham, NC 27708 Despite its clinical importance, them is a paucity of data available in the literature for the partition coefficient of macromolecules in tumor tissues. In this study, we quantified the partition coefficient of bovine serum albumin (BSA) and dextran molecules with different molecular weights, ranging from 10,000 to 2,000,000 in agarose and gelatin gels as well as tumor tissues. The measurement involved (a) sectioning of gels or tumor tissues into thin slices (- 500 p.m) using the Vibratome | (b) ex vivo incubation of the slices in solutions containing rhodamine labeled tracer molecules, and (c) quantification of the equilibrium tracer concentrations in both slices and solutions. The concentrations of tracers were determined via fluorescence intensity measurement using a fluorescence microscope equipped with an intensified CCD camera and an image processing system. Our preliminary data indicated that the partition coefficient decreases in general when the molecular size was increased. However, the partition coefficient in tumor tissues was insensitive to the molecular weight of dextran in the range between 10,000 and 40,000. In addition, the partition coefficient of BSA (MW = 67,000) is larger than that of dextran 70,000 in different gel preparations and in a rat mammary carcinoma R3230. But this difference is reversed in a rat fibrosarcoma. Our hypothesis is that the discrepancy in the data between different tissues and/or gels can be explained by the molecular charge effect.

299 DIFFUSION OF PARTICLES THORUGH DILUTE AGAROSE GELS Nilesh Ron and Curt Thies Biological Transport Laboratory, Washington University, St. Louis, MO This study investigated the diffusion behavior of particles through dilute agarose gels and determined that such gels have a fiber diameter similar to that of mucus. Average pore sizes of the gels were estimated from rate of particle diffusion measurements. Agarose gels of varying pore size can be made by altering gel concentration and thermal history. Experimental data for particle diffusion through many dilute agarose gels fit the Renkin model for hindered diffusion through a cylindrical pore. Based on this model, mucos composed of = 5% mucin with an average fiber diameter of 8 nm should be impermeable to _> 20 nm particles. Smaller particles should exhibit reduced diffusivity in mucus as governed by the Renkin model.

297 ESTIMATING INTER-CELLULAR COMMUNICATION DISTANCES; THE IMPORTANCE OF CYTOKINE SECRETION AND DIFFUSION TIME CONSTANTS. K. Francis and B. O. Palsson UCSD, Department of Bioengineering, La Jolla, CA

300 MICROVASCULAR NETWORK ANATOMY AND HEMODYNAMIC SIMULATION OF A RAT PEDICLE SKIN FLAP. Patrick S. Cottler, Richard J. Price, and Thomas C. Skalak. University of Virginia, Department of BME, Charlottesville, VA.

An important mode of intercellular communication is the release of soluble cyto- and chemokines. Once secreted, these signaling molecules diffuse through the surrounding medium and eventually bind to neighboring cell's receptors whereby the signal is received. This mode of communication is regulated both by physcio-chemical transport processes, and cellular secretion rates, which in turn are determined by genetic and biologic processes. The present study uses a "solitary cell" model to estimate the effective communication distance over which a single cell can meaningfully propagate a soluble signal. The analysis reveals; i) that this process is governed by a single key dimensionless group which is a ratio of biological parameters and physico-chemical determinants; 2) that this ratio has a maximal value; 3) that for realistic values of the parameters contained in this dimensionless group, it is estimated that the domain that a single cell can effectively communicate in is about 250 micron in size; and 4) that the communication within this domain takes place in 10 to 30 minutes. These results have fundamental implications for interpretation of organ physiology and for engineering tissue function ex vivo.

Pedicle skin flaps are often utilized in plastic surgery to repair deep defects. To better understand the hemodynamics underlying successful flap design, a network model of a viable rat pedicle skin flap has been developed. A map was constructed from an arterial vascular cast to determine the connectivity, vessel dimensions (length and diameter), and transverse arterial (TA) and arcade arterial (AA) spacing. The model mirrors the arterial vessels with a venous network of larger diameter, with the arteriovenous capillary units included as equivalent resistors. A networksolving program was used to compute the pressure and flow distribution. The computed total flow agreed well with experimental measurements using ultrasonic flowmetry (0.31 vs. 0.26ml/min/g). Regional micmvascular flows were relatively homogeneous, averaging 0.10+/.0151al/s in widely separated regions. Anastomotic arteriolar connections are responsible for producing adequate regional flows in this viable flap, suggesting that microvascular architecture is important in flap design. Supported by The Whitaker Foundation and NIH HL52309.

Cell and Tissue E n g i n e e r i n g - Posters

301 TISSUE-ENGINEERED MICROVASCULAR NETWORKS FOR GENE THERAPY K. J. Goochl, R. F. Paderal, S. Feng2, R. C. Mulligan2, and R. Langerl 1Department of Chemical Engineering, MIT, Cambridge, MA 02139 2Harvard, Cambridge, MA 02139 Towards the aim of developing a method for systemic delivery of proteins for gene-therapy applications, we have explored the potential of tissue-engineered microvascular networks. Endothelial cells were seeded onto nonwoven meshes of polyglycolic acid (PGA) fibers and cultured in vitro for 1 wk. When placed subcutaneously in athymic rats the endothelial celI/PGA constructs were invaded by host tissue and vasculature. Antibody staining of histological sections revealed the presence of endothelial cells in the extravascular space. In subsequent experiments using endothelial cells expressing the lacZ-reporter gene, a fraction of the lacZ positive cells incorporated into the host vasculature as evidenced by blue-stained capillaries containing red blood cells. These results indicate tissue-engineered microvascular networks incorporate into the host vasculature and may provide an ideal system for systemic delivery of proteins in gene-therapy applications.

302 ANALYSIS OF GPIIb/IIIa-FIBRINOGEN CROSSBRIDGING RATE OF PLATELETS AT CONSTANT SHEAR RATE Pushkar Tandon and Scott L. Diamond Institute for Medicine & Engineering, University of Pennsylvania We have modeled platelet aggregation in flow by accounting for shear flow hydrodynamics and platelet receptor biology. Considering platelets and their aggregates as unequal sized spheres with DLVO interactions (platelet surface potential = -15 mV, Hamaker constant = 10 -19 J), trajectory calculations provided the upstream collision cross-sectional area and collision frequency. GP IIb/IIIa mediated aggregation has been modeled as a kinetic process where the forward rate of bond formation was estimated from the diffusional limited rate multiplied by an effectiveness factor, 11. For a value of ~=0.007, we calculate the overall efficiency (including both receptor binding and hydrodynamics effects) for equal sized particles to be 0.357 for G=41.9 s -t, 0.0196 for G=335 s -1 and, 0.0081 for G=1920 s -1, values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. The calculated collision frequencies have been included in a population balance framework and our predictions give agreement with observed evolution of size distributions of platelets aggregating in a shear field. The overall method provides a hydrodynamic and receptor correction of the Smoluckowski collision kernel and gives, to our knowledge, the first estimate of 11 for the GP IIb/Illa cross-bridging of platelets based on quantitative hydrodynamic calculations.

303 EFFECTS OF FIBRIN NETWORK MORPHOLOGY AND CROSSLINKING ON CLOT RHEOLOGY E.K. Ang i, J.W. Weisel 3, L. Lorand 2, and L.F. Mockros 1 IDepartment of Biomedical Engineering and 2Department of Cell and Molecular Biology, Northwestern University; 3Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine The structural origins of fibrin clot viscoelasticity were examined. Fibrin network morphology was manipulated by varying fibrinogen, thrombin, and calcium ion concentrations, and features of the network architecture (fiber lengths, fiber diameters, and branching densities) were analyzed and quantified using computerized three-dimensional reconstructions from stereo scanning electron micrographs. Degrees of crosslinking and crosslinking types (y-, a-, and c~y-hybrid crosslinks) in the clots were controlled using an active-center inhibitor of factor XIlla and analyzed by SDS-PAGE. The viscoelastic behavior of clots resulting from these alterations of architecture and crosslinking were examined with a cone/plate rheometer. By correlating the shear modulus of the clots to the distributions of crosslink types and structural features found in them, the individual contributions made by the various crosslinks, fiber sizes, and degrees of branching to clot elasticity were identified.

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304 CALCIUM TRANSIENTS DURING NEUTROPHIL MOTILITY RESPONSES A. M. Alteraifi and D. V. Zhelev Dept. Mech. Eng. Mat Sci., Duke University, Durham, North Carolina The release of free cytosolic calcium is a secondary messenger for many cell functions. Here we study the coupling between the release of intracellular calcium and motility responses of the human neutrophil. Two groups of motility responses are studied -- motility responses in presence of adhesion, such as crawling and phagocytosis, and motility responses "in suspension", such as pseudopod formation. The motility responses are stimulated by N-formyl-methionyl-leucyl-phenylalanine (tMLP) and the release of calcium is monitored by measuring the fluorescence from fluo-3. FMLP induces a single release of free cytosolic calcium both in suspended ceils and crawling cells. This is a threshold process which occurs at 100 nM tMLP for suspended cells and 5 nM fiklLP for crawling cells. The threshold for calcium release in suspended cells is larger than the minimum fMLP concentration (on the order of 0.1 nM) for initiating pseudopod formation. So, there is a range of tMLP concentrations where pseudopod formation occurs without calcium release. To explore this relationship further we studied neutrophil desensitization. Our results show that the release of calcium is desensitized bytMLP while pseudopod extension is not. All the results taken together suggest that the release of free cytosolic calcium and the motility responses of the neutrophil follow different signaling pathways.

305 MODELING OF PLATELET-MEDIATED THROMBOGENESIS Erik Sorensen, Greg Burgreen and James Antaki Program in Bioengineering, Univ. Of Pittsburgh, Pittsburgh, PA A comprehensive model of platelet-mediated thrombogenesis requires parameters to describe platelet activation (via shear and agonists), platelet transport in flowing blood, effects of artificial surfaces (if present), kinetics and mechanics of platelet-platelet and platelet-surface adhesion, alterations in local fluid dynamics due to the presence of a thrombus, and interplay between platelet- and cascade-mediated hemostatic mechanisms. To date, most models in the literature have focused on these components individually without attempting ot integrate them into a single, comprehensive approach. Furthermore, no attempt to model the distinct interactions between platelets, adhesive proteins, and biomaterials of differing chemical composition has been attempted. To date, we have developed a finite element-based simulation of agonist transport near growing thrombi which is capable of accurately reproducing agonist concentration profiles reported by other investigators. Current efforts are focused on extending this model to account for platelet transport, including near-wall excess platelet concentrations, shear-induced platelet activation, and multicomponent agonist interactions and their effect on platelet aggregation and surface adhesion by incorporating components of previously reported models into a single, multifactorial, computational model of thrombogenesis. Future efforts will focus on developing a coherent predictor of biomaterial-specific platelet adhesion events.

306 EFFECTS OF RADIOTHERAPY ON M1CROVASCULAR NETWORK BLOOD FLOW IN NORMAL TISSUE Vinh Nguyen, Noah M. Roth, and Mohammad F. Kiani School of Biomedical Engineering and Department of Radiation Oncology, University of Tennessee, Memphis, TN 38163 Ionizing radiation damages microvascular networks in normal tissue and alters their structure and function. Cremaster muscles of hamsters were locally irradiated with a 10Gy single dose. Intravital microscopy was used to measure structural and functional parameters in all vessels and to establish vascular interconnection in selected networks. Our in-vivo results indicate a decrease in RBC velocity (138.0• Mean+SEM) and vessel diameter (10.4+0.6p.m) at 3 days post-irradiation compared to age-matched controls (337.9+19.9p_m/s, 12.5~:0.51am). At 7 days post-irradiation RBC velocity (181.7+22.9gm/s) and diameter (8.3• of capillaries were significantly lower compared to age-matched controls (236.2• 10.2• Similar changes were observed 30 days post-irradiation. Capillaries were effected the most by irradiation. A mathematical model which accounts for the non-linear characteristics of blood was developed to simulate blood flow in irradiated and control microvascular networks. This model estimates effects of irradiation on parameters, such as vessel wall stress, that can not be measured in vivo. Supported by ACS and NIH.

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Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

307 A NEW UNDERSTANDING OF STARLING'S PRINCIPLE AT THE MICROSTRUCTURAL LEVEL X. Hu and S. Weinbaum CUNY Graduate School and Department of Mech~icaI Engineering, The City College of New York, New York, NY 10031 In this paper a new microstructural model is proposed to explain a fundamental paradox in Starling's law. Contrary to century old beliefs, recent measurements of tissue osmotic pressure reveal, surprisingly, that there is no osmotic reabsorption of water in the venous capillaries except for the peritubular capillaries in the kidney. Our new model describes for the fh'st time the detailed structure of osmotic gradients and flows across the endothelial surface glycocaiyx, the interendothelial cleft with its junction strand and the cleft exit. It is shown that contrary to widely held views, the osmotic force is not felt across the entire endothelial cell layer, but across highly localized shielded regions behind the matrix where there is a high water flux due to the infrequent breaks in the junction strand. On the venular side the osmotic force is decreased because back diffusion through the breaks leads to an elevated protein concentration behind the matrix layer, so the smaller filtration flow leads to a small net water flow. Thus except for rapid transient phenomena, the classic Landis-Starling diagram does not apply and there is no venous reabsorption.

308 A NEW ANIMAL MODEL OF ERYTHROCYTE SENESCENCE : STUDIES ON ALTERATIONS OF OSMOTIC FRAGILITY AND RHEOLOGICAL PROPERTIES OF ERYTHROCYTE DURING ITS LIFE SPAN Lu Zhihong, Wen Zong-Yao, Song Lichan, Sun Dagong Department of Medical Physics, Beijing Medical University, Beijing 100083, P.R. China Tel: (8610)62092419 Fax: (8610)620115582 E-mail: [email protected] mu.edu.cn Adopting Mohandas method of inducing spherocytic anemia of animal by using antibody, a new model of red blood cells (RBCs) synchronous senescence under nearly normal physiological conditions was proposed. Using this model, we studied the changes of RBCs osmotic fragility with their aging. The results indicate that the osmotic fragility of RBCs increased with RBCs aging. These changes were not linear during RBC aging. The indexes of DI, IDI, which reflect RBCs rheological behaviors during their aging process, were measured with an Ektacytometry. Both indexes decreased with RBC aging, but the relationship was not linear.

309 RED CELL AGGREGATION AND VENOUS VASCULAR RESISTANCE B. Liu, C Flarity, A. Popel, M Intaglietta and P C Johnson. Dept of Bioengineering, UC San Diego, La Jolla, CA 92093-0412 Recent studies in our laboratory have shown that venous vascular resistance is significantly increased by red cell aggregation (Cabel et al, AmJ. Physiol. HI020-HI032, 1997). To examine this phenomenon further we have developed a method to measure size of red cell aggregates in venules in vivo by a correlation method. The estimates of aggregate size obtained agree with the values obtained by direct measurement from stopframe analysis of video images. Analysis of video data from venules in cat sartorius muscle shows a significant increase in aggregate size as flow rate is reduced, which may explain, at least in part, the rise in venous resistance at low flows. Studies in rat spinotrapezius muscle venules using polyvinylpyrrolidone (row. 360,000) to induce aggregation reveal that aggregates entering a venule from a side branch tend to remain in the peripheral flow layer, which may increase energy loss in the venular network when aggregates are present. (Supported by HL52684)

310 A PROBABILISTIC MODEL OF ROSETTE FORMATION M, Long 1,2, E. Moses 1 and C. Zhu 1 IGeorgia Institute of Technology, ME, Atlanta, GA 30332-0363, USA: 2Chongqing University, Bioengi. Center, Chongqing 630044, PRC. A probabilistic model has been developed for rosette formation, which is an assay for specific cell-cell adhesion commonly used in immunology. The basic idea is the probability of adhesion, which is a measure of the ability for a receptor expressing cell (target cell) to adhere to a ligand coating red blood cell (RBC) when the two are put into contact. Assuming uniform distributions of receptors and ligands on the surfaces of target and red cells and equal opportunity for a target cell to make contact with as many RBCs as allowable by the geometric constraint, the probability of having n RBCs to simultaneously adhere a target cell is binomially distributed. Two cellular and molecular systems were used, one consisting of Chinese hamster ovary cells transfected to express CD16A rosetting with human RBCs coated with human IgG, another human colon carcinoma cells expressing sLe x rosetting with RBCs coated with E-selectin. Excellent agreement was found between theory and experiment for the rosette size distribution. The binding affinity was determined by quantitatively relating the rosette size distribution to the densities of receptors and ligands and their binding affinity. (Supported by NSF 9350370, NIH AI38282, and Scholarship of SEdC of PRC).

311 RBC PACKING STUDIED BY ULTRASONIC INTERFEROMETRY S.M. Razavian, R.B. Wenby and H.J. Meiselman Department of Physiology and Biophysics, USC School of Medicine, Los Angeles, CA 90033, USA The physical properties of sediments resulting from red blood cell (RBC) sedimentation can provide important information on cell-cell interactions (e.g., aggregation). We have thus employed the Echo-Cell system (A-mode echography, 8 MHz transducer, 2 kHz rate) to examine the degree of packing of RBC sediments using normal and fixed human RBC suspended in isotonic buffer (PBS), autologous plasma, dextran solutions or bloodgroup mismatched media. Our results indicate that RBC packing increases with increasing aggregation tendency (22% higher for cells in plasma vs. PBS, p<0.01). Dextran fractions known to be non-aggregating (i.e., 10 or 20 kD MW) had no effect on packing, whereas dextran of 70,000 MW had a bi-phasic effect with a maximum increase at a dextran concentration of 2%. RBC made less deformable via low levels of glutaraldehyde (GA) exhibited a dose-related decrease (0.005%=-6%; 0.02%=-14%) when suspended in PBS. ABO blood-group mismatching resulted in RBC agglutination and a titer-related increase of RBC packing. Our results thus indicate the merit of this new method for studies of RBC-RBC interactions.

312 POLYETHYLENE GLYCOL COATING OF HUMAN RED BLOOD CELLS ABOLISHES AGGREGATION AND REDUCES LOWSHEAR BLOOD VISCOSITY. J.K.Armstrong, T.C.Fisher and H.JMeisehnan. Dept. of Physiology and Biophysics, USC School of Medicine, Los Angeles, California. The covalent attachinent of polyethylene glycol (PEG) to red blood cells (RBC) using cyanuric chloride derivatives (PEG-CN) was investigated over a range of PEG molecular weights (5k-35kD) and concentrations (0.25-20 mg of polymer per mL of RBC). The PEG-coating was achieved under mild conditions (washed RBC at 50% hot in buffer at pH 8.3, 25~ The PEG-coated RBC were resuspended in autologous plasma at 40% het. RBC aggregation and blood viscosity were measured using a Myrenne aggre~ometer and Contraves low shear viscometer (shear rates 0.08-98.5s- ). The PEG-coating reduced both aggregation and low shear viscosity in a dose-dependent manner. RBC aggregation was abolished between 1-2.5 mg/mL, dependent on the PEG MW. On an equimolar basis, higher MW PEGs were more effective inhibitors of aggregation. At concentrations of 5-20mg/mL low shear viscosity was reduced from 70-90mPa.s (control) to 10-15mPa.s (PEGcoated RBC). RBC deformability, morphology, 02 transfer and high shear viscosity were unaffected by the PEG coating. This simple technique effectively reduces viscosity, and may prove to be a clinically useful treatment to improve blood flow after acute ischemia.

Cell a n d T i s s u e E n g i n e e r i n g - P o s t e r s

313 THE EFFECTS OF POLYETHYLENE GLYCOL AND POLOXAMERS ON RED BLOOD CELL AGGREGATION J.K.Armstrong, T.C.Fisher and H.J.Meiselman. Dept. of Physiology and Biophysics, USC School of Medicine, Los Angeles, California. The effects on RBC aggregation of polyethylene glycol (PEG) over a range of molecular weights (MW 1k-1000kD) and a series of poloxamers (P108, P188, P238, P288 and P338) of increasing MW (4.6-15kD) were studied as a function of concentration and temperature. Poloxamers are triblock copolymers, composed of a central block of polypropylene glycol (PPG) flanked by two PEG blocks. PEG homopolymers remain as single chains at all temperatures studied, while this series of poloxamers form micelles at a critical miceIlization temperature (CMT) which decreases with increasing poloxamer MW. Aggregation and low shear viscosity of human RBC suspended in autologous plasma at 40% hematocrit was investigated at polymer concentrations up to 10 mg/mL of plasma. PEGs <18.5kD were anti-aggregant while higher MW PEGs promoted RBC aggregation. All poloxamers were anti-aggregant below the CMT and became pro-aggregant above the CMT. The anti-aggregant effects of poloxamers were directly related to total PEG chain length, while the pro-aggregant effects could be attributed to the formation of micelles and were comparable to high molecular weight PEGs (>35kD). The anti-aggregant effect of PEG 8k and 10kD are identical to the effect of P188 (8.4kD) and indicate that the effects of poloxamers on RBC aggregation are solely due to the PEG portion of the copolymer.

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316 EDDY FLOW-INDUCED ACTIVATION OF THE MAPK AND THE c-jun GENE IN RAT AORTAS IN VIVO M. Katada~ and S. Q. Liu Dept. of Blomed. Eng., Northwestern University, Evanston, IL Fluid shear stress in vitro has been shown to influence the activities of the mitogen signal transduction pathways, that play an important role in the regulation of cell proliferation. This study focused on the influence of eddy blood flow, that is associated with reversed and lowered fluid shear stress, on phosphorylation of the mitogen activated protein kinase (MAPK) and expression of the c-jun gene, key elements of the Ras-MAPK-immediate earlygene signaling pathway, in the aortic endothelial cells in vivo. The rat abdominalaorta was narrowed by using an aneurysm clip, leading to the formation of eddy flow at the region distal to the clip. Phosphorylated and nonphosphorylated MAPKs were labeled by using specific monoclonal antibodies and the cjun mRNA was labeled in site by using a digoxigenin-conjugated c-jun cDNA probe. In an en face examination, the nonphosphorylated MAPK was detected in the endothelial cells at the proximal and distal as well as the narrowed regions, whereas the phosphorylated MAPK was mainly detected at the distal region, where eddy blood flow was found, at 30 and 60 min after surgery. The c-jun mRNA was up-regulated in the endothelial cells at the distal region, but not at the proximal and the narrowed regions, from 3 to 12hours. These results indicate that eddy blood flow plays a role in the activation of the Ras-MAPK-immediate early gene signaling pathway in the endothelial cells in vivo.

314 EFFECT OF SALICYLIC ACID DERIVATIVES ON RED BLOOD CELL SHAPE, STIFFNESS AND RELAXATION TIME Al. Li, H. Seipelt * U. Sahm and G.M. Artmann Cell Biophysics, Aachen University of Applied Sciences, Germany; 9 Pediatrics Hospital, Univ. Greifswald, Germany

317 DOES ELEVATED NO ENHANCE PGI2 PRODUCTION BY SHEAR STRESSED AORTIC ENDOTHELIAL CELLS? Weiguo Wang and Scott L. Diamond Bioengineering Laboratory, ChE Dept.,SUNY, Buffalo, NY 14260 Inst. for Medicine & Engineering, Univ. Pennsylvania, Phila., PA 19104.

Salicylamide (SAM), Sodium salicylate (SA) and Acetylsalicylic acid (ASA) have different suhstituents on the benzene ring. Using automated RBC shape analyzing systems, we investigated how these compounds(conc.:0.5-20 raM) affected red blood cell (RBC) shape, stiffness (S) and relaxation-time (RT). SAM showed a maximum echinocytic shape at 5 mM and reduced shape effects with both higher and lower concentrations. SA progressively induced echinocytes with increasing concentrations. All shape changes occurred within 2 rain. of incubation and were reversible after reincubating in buffer. In contrast, the shape was not affected by ASA. These tendencies were in parallel to a RBC stiffening. RT, however, continuously increased in both SAM and SA, with SAM always being higher. ASA showed no effect. A postulated net excess of SAM or SA molecules within the outer leaflet may explain the shape changes as well as the loss of fluidity (S and RT increase). In contrast to ASA, SA and SAM are phenols and furthermore able to form intramolecular hydrogen bonds which increase molecular hydrophobicity and thus the uptake into the bilayer.

In light of evidence that nitric oxide (NO) can directly enhance cyelooxygenase activity, we tested the role of shear stress induced production of NO by endothelium as an autocrine mediator of prostacyclin (PGI2) production. Early passage bovine aortic endothelial cells were exposed to shear stress (25 dynes per square cm, dpc) in arginine-free physiological saline buffer in the presence or absence of L-arginine inhibitors that block NO production. As expected, shear stress elicited a marked burst of NO (measured by DAN reaction for nitrite) over the first 5 min of flow with sustained elevated NO production for the following 60 min. Cumulative NO production after 60 min with shear was 0.1920 n m o l e / c m 2 - B A E C versus 0.0257 nmole/cm 2 for controls (7.5-fold elevation, n = 4, p < 0.001). NO production was reduced by 90 % (n = 3) with either L-nitro arginine (LNA, 10 I~M) or L-nitro arginine methyl ester (LNAME, 100 gM). Shear stress elevated cumulative PGI2 production at 60 min from 0.0307 to 0.281 ng/cm 2 (9.1-fold elevation, n = 3, p < 0.001). This cumulative PGI2 production at 60 min was reduced to only a 4.1-fold elevation (n =3, p < 0.001 ) with LNA and 5.0-fold elevation (n = 3, p < 0.001) with LNAME. Thus, NO induced by shear stress is responsible for at least half of the PGI2 elicited by shear stress.

315 NO REGULATES PDGF-A AND MCP-I EXPRESSION INDUCED BY GRADIENTS IN SHEAR STRESS IN ENDOTHELIAL CELLS X.P. Bao and J.A. Frangos Dept. Of Bioeng., Univ. Of California/San Diego, La Jolla, CA 92093

318 SHEAR-INDUCED INCREASE IN ENDOTHELIAL DIFFUSIVE ALBUMIN PERMEABILITY IS INDEPENDENT OF NITRIC OXIDE. Yong S. Chang and John M. Tarbell Dept. of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802

A novel approach using three well-defined laminar flow profiles has been used to distinguish the influences of steady and gradients in shear stress with respect to expression of c-fos, PDGF-A and MCP-I in primary cultured human umbilical vein endothelial cells. The three flow profiles were ramp flow (shear stress of 16 dyn/cm2 smoothly transitioned at flow onset), step flow (shear stress of 16 dyn/cm2 abruptly applied at flow onset), and impulse flow (shear stress of 16 dyn/cm2 abruptly applied for 3 seconds only). Relative to the levels of gene expression induced by ramp flow, impulse flow increased c-fos, FDGF-A, and MCP-1 expression levels 6, 3.5, and 3.5-fold, respectively (p<0.5), relative to ramp flow. To explore the apparent inhibitory effect of steady shear stress on the expression of these three genes, the possible regulatory role of nitric oxide was investigated. The NO donor spermine-NO (100 I.tM) dramatically reduced MCP-I and PDGF-A expression levels induced by impulse flow to levels similar to those produced by step flow. Conversely, treatment with NO synthase inhibitor IqC-Amino-L-arginine.HCl (300 gM) markedly enhanced MCP-1 and PDGF-A expression levels induced by step and impulse flow. The observations indicate that temporal shear stress gradients stimulate expression of endothelial genes, while steady shear stress may regulate endothelial gene expression induced by temporal gradients in shear stress through a feedback mechanism involving NO production.

Recent studies provide evidence that endothelial monolayer transport properties are sensitive to alterations of fluid shear stress on the surface of the cells. Previously, using bovine aortic endothelial cells (BAECs) grown on porous polycarbonate filters, we demonstrated that shear-induced increase in hydraulic conductivity (Lp) can be attenuated with addition of nitric oxide synthase inhibitors such as L-NMMA. This study addresses the role of nitric oxide in mediating the shear-induced increase in endothelial albumin diffusive permeability (P0. Pe was determined by measuring the flux of fluorescein isothiocyanate conjugated to bovine serum albumin across endothelial monolayers with zero pressure gradient. Application o f ] 0 and 20 dyn/em2 shear stress elicited a t0-fold increase in Pe at the end of three hours. Surprisingly, however, addition of L-NMMA (100~,1) had no significant effect in altering shear-induced Po. These data coupled with our previous findings, indicate that in our model system, endothelial monolayers regulate shear-induced increases in Po and Lp by different mechanisms. (Supported by NIH Grant HL35549 and NASA Grant NAG3-1871 )

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319 INTEGRINS ARE INVOLVED IN THE SHEAR STRESS ACTIVATION OF MITOGEN ACTIVATED PROTEIN K1NASES S. Jalali; S. Li; Y-J. Shyy and Shu Chien University of California, San Diego, Department of Bioengineering and Institute for Biomedical Engineering, La Jolla, CA

322 DISTURBED FLOW ENHANCES ENDOTHELIAL CELL PROLIFERATION P. Hsu, J. Shyy, S. Usami and S. Chien UCSD, Institute of Biomedical Engineering and Dept. of Bioengineering, La Jolla, CA.

Hemodynamic forces constitute a risk factor for cardiovascular diseases. We have previously shown that fluid shear stress induces p60src-MAPKs signaling pathways and increases the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK). The objective of this study is to test the involvement of integrins in shear stress-induced signal transduction. Extracellular matrix proteins were adsorbed onto sterile glass slides: vitronectin (0.5 gg/cm~L fibronectin (5 gg/cmb, collagen (6 I.tg/cm~), and gelatin (0.6 gg/cmb. Bovine aortic endothelial cells were then seeded on the slides in the absence of serum. After 8-hr incubation, cells were either sheared in a flow channel for 30 min using a circulating flow system or kept as static controls. Shear stress caused a significant increase in the induction of JNK on glass slides coated with vitronectin, fibronectin, and gelatin, but not on the ones with collagen. These results suggest that integrins on the abluminal cell surface may be involved in the shear induction of JNK. To test the roles of fibronectin and vitronectin receptors specifically, blocking antibodies against O~v~3and [31 were found to have an inhibitory effect in the shear activation of ERK and JNK. Our findings suggest that integrins are involved in the signaling in response to shear stress.

Atherosclerotic lesions occur in branching areas of blood vessels where the blood flow is disturbed with secondary flow and flow reattachment. At these branching areas, endothelial cell turnover rate is greater than the straight part of the vessel. In this study we investigated the mechanism by which disturbed flow enhances endothelial proliferation in vitro. We used a vertical step-flow chamber to create disturbed flow. Confluent bovine aortic endothelial cells (BAEC) were subjected to the flow for 24 hours. In the flow reattachrnent area, there was an increase in 5-bromo-2'deoxyuridine (BrdU) incorporation, indicating enhanced cell proliferation. To study the signal transduction event, we labeled the cells with an antibody against phosphorylated extracellular signal related kinase (ERK). There was an increase in ERK phosphorylation in flow reattachment area, suggesting the activation of the ERK signal transduction pathway. Our results showed that the disturbed flow enhances endothelial cell proliferation by activating the ERK.

320 FLUID FLOW STIMULATES PROSTAGLANDIN E 2 AND NITRIC OXIDE RELEASE IN PRE-OSTEOCLASTS T. B. Du, T.N. McAllister and J.A. Frangos Dept. of Bioengineering, UC San Diego, La Jolla, CA 92093

323 TIlE INFLUENCE OF FLUID STRESSES ON CELLS ATTACHED TO A MICROCIIANNEL WALL Donald P. Gaver and Stephanie M. Kute Dept. of BiomedicalEng., Tulane University,New Orleans, LA 70118 Adhesion of a cell or group of cells to a channel wall is an important event relevant to a variety of situations from leukocyte adhesion in capillaries to biofilm formation in porous media. Cells remain attached if their adhesion strength exceeds the effect of hydrodynamic stresses. Also, mechanotransduction may depend upon the magnitude and distribution of stresses on the cell wall. We aim to quantify the influence of hydrodynamic stresses on individual cells, and determine the fluid-mediated interaction between pairs of cells in a microchannel. We model this system as a single microchannel of length L and height H. Flow of a viscous incompressible fluid is driven within the channel with an applied pressure difference AP. Discrete cells of height R attach to the channel wall, modifying the "effective" wall shape and influencing the flow field. By using a pressure-based velocity scale, U = APH2/(8,uL), only two dimensionless parameters exist: fl = H/L, the channel aspect ratio, and 7 = R//-/, the cell to channel-height aspect ratio. For small t , flow disruption leads to large amplification of the shear stress, force, and torque exerted onto individual cells: Interactions between cells adhering to opposite walls extend only a distance 2R, but greatly increases the magnitudes of the fluid stresses on the cells. Neighboring cells on the same wall have larger interaction distances (-1 OR), and results in significant stress-shielding. These models will be used to predict aggregation behavior in microchannels.

Mechanical strain-induced or fluid flow-induced bone remodeling studies have focused on the role of osteoblasts in mechanochemical signal transduction. Conversely, investigation of mechanically regulated humoral agents has concentrated on osteoclasts, since osteoclast recruitment and migration is much more rapid than osteoblast deposition. PGE 2 and NO in particular, have been identified as autocrine/paracrine agents which inhibit osteoclast activity. We hypothesized that fluid flow may direcdy inhibit resorption by stimulating PGE 2 and NO release in osteoclasts. We have developed novel methods to isolate and culture osteoclast precursors and to induce fusion into multinucleated osteoclasts. Bone marrow from prenatal Sprague Dawley rats was plated onto untreated petri dishes. Preosteoclasts, as verified by morphology and TRACP positive staining, preferentially adhere to the petri dish. Fusion was induced by co-culture with osteosarcoma cells (MG63) grown on a glass slide and separated from the osteoclast precursors by a silicon spacer. In this study, we report that fluid flow-induced shear stress (12 dynes/cm2) stimulates a 21 fold increase in PGE 2 release (35 ng/mg/hr) and a 2.25 fold increase in NO release (4.7 nmol/mg/hr). NO production is completely abolished with the L-arginine analog L-NAA (100~tM). This suggests that fluid flow directly influences osteoclast function through autocrine mechanisms.

321 RAS IS NOT NECESSARY IN SHEAR STRESS-INDUCED CELL ALIGNMENT IN ENDOTHELIALCELLS Song Li, StephenZ.-H. Wu, Suli Yuan, Pin-pin Hsu, John Y.-J. Shyy, and Shu Chien Department of Bioengineering and Institute for Biomedical Engineenng, University of California, San Diego, La Jolla, CA 92093 Ras plays a critical role in shear stress-induced signal transduction in vascular endothelial cells (EC), e.g., activation of mitogen-activatcd protein kinases. The aim of this study is to test the role of Ras in shear stress-induced EC structure change, especially coil alignment with flow direction. The expression plasmids encoding Ras(N17), a dominant negative mutant of Ras, were co-transfected with plasmids encoding [5galactosidase ([5-gal) into cultured bovine aortic EC (BAEC). Confluent transfected BAEC were subjected to shear stress at 12 dyrgcm2 for 16 hr. The BAEC were then fixed and the transfected ceils were identified by [5gal staining. Under static condition, cell shape indexes are 0.79_+0.10 (mean+S.D.), and cell orientations are randomly distributed. After 16 hr sheanng, cell shape indexes shifted to 0.62_+0.09 and 0.63_+0.10 for nontransfected and transfected cells, respectively, indicating that the cells were more elongated. More than 80% of both non-transfected and transfected cells had an orientation angle less than 15~ Both liposomc and adenovirus tmnsfection have the similar results. These data suggest that, while Ras is critical in the shear stress-induced signaling, it is not necessary for shear stress-induced cytoskeleton remodeling.

This research was funded by NSF BES-9358207 and DoE-FGO1-93-EW53023

324 VELOCITY PROFILE IN D-SHAPED MICROCHANNELS. Mary D.S. Frame, Gary B. Chapman, Yoshia Makino. Dept. of Anesthesiology, Univ. of Rochester, Rochester, NY, 14642 The fluid velocity profile in a D-shaped microchannel was measured, and compared to the predicted profile (finite element package, CFD-ACE, CFD Research Corp., Huntsville, AL). D-channels (21+lp.m rad, scanning electron microscopy) were perfnsed (5-100nl/min) with fluorescently labeled microspheres (0.5p.m dia., <1% vol/vol) (n---9), modeling the flow of the temunal arterioles of the microcirculation. Steady state microsphere flow through the channel was videotaped. Microsphere velocity (>250 per channel) was measured off-line as a function of 2-dimensional radial position across the channel. Velocity profiles were detemained by dividing individual velocities by the maximum (Vmax), or by the harmonic z01 . v/Vave mean velocity (Vave), for each 5% across the channel radially. Figure: predicted values are ,~ i ~, the lines, data are mean:i:SEM (n-~)). Using ~2 ~ . v/Vmax Vmax, predictions were higher than data, o, ~ predictedexcept at the channel wall (dotted line, open o, . .... t. circles). Using Vave, predictions were higher ..... "--. 9 than data in the centerline and lower at the 0. . . . ~":~ wall (solid line, squares). Vave data were not o~ 0~ o, 0~ 0~ , 0 different from the reported profile for dual slit data (Baker&Wayland, MVR 7,131,1974). This study was supported by The Whitaker Foundation.

ll~1

__ predicted

i

02

~''r radialposition/radius

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325 FLUID SHEAR EFFECTS ON TISSUE F A C T O R ACTIVITY IN H U M A N MONOCYTES. S. Peckham 1, Y. Nemerson2 and V. T u n- t t o1. Department of Biomedical Engineering, The University of Memphis, Memphis, TN and Dlvlsmn of Thrombosis Research, Mt. Sinai Medical Center, New York, NY 2. 9

1

9

.

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328 ENDOTHELIAL POTASSIUM CHANNEL mRNA EXPRESSION LEVEL IS REGULATED BY SHEAR STRESS W. Lindsley, S. Forsyth, J. Hoger*, and A. Hoger Univ. Of Calif., San Diego, La Jolla, CA 92093, and *Hitachi Chemical Research Center, Irvine, CA 92612

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The goal of this research is to determine the extent to which tissue factor (TF) activty in human monocytes may be modulated by the mechanical stresses of fluid flow. Tissue factor is a transmembrane glycoprotein responsible for the initiation of the extrinsic pathway of blood coagulation. Human peripheral blood monocytes were isolated for use in these experiments. Stimulated and unstimulated cells were sheared in a Hercules viscometer at a shear stress of 100 dyne/cm2 for 20 minutes. Conversion of FX to FXa by sheared and control cells was measured upon addition of buffer containing FX, FVIIa and calcium. Both stimulated and unstimulated monocytes showed significant increases in TF activity after exposure to a shear stress of 100 dyne/cm2 for 20 minutes. The results suggest the possibility that this level of shear may increase the activity of TF already present in stimulated monocytes as well as increase the production of TF in unstimulated cells.

326 ENDOTHELIAL WOUND HEALING UNDER SHEAR STRESS Annette B. Branger and Christopher M. Waters Northwestern University, Depts. of Anesthesiolgy and Biomedical Engineering, Chicago, IL

Endothelial cells sense and react to the shear stress generated by the flow of blood in the vasculature. The responses of endothelial cells to shear include changes in membrane ion currents, regulation of various genes, changes in the release of vasoactive substances and intracellular signaling molecules, and changes in celt morphology. One of the most rapid responses to shear in endothelial cells is the opening of K + ion channels and the subsequent hyperpolarization of the cell. Previously, we have reported the first cloning and sequencing of an endothelial cell K+ channel (BIK). Subsequent experiments have shown that BIK channel activity is responsive to shear stress when the channel is expressed in Xenopus laevis oocytes. We now report that the level of the mRNA transcript coding for this channel is sensitive to shear stress. Confluent monolayers of bovine aortic endothelial cells were grown on gelatin-coated slides and subjected to 30 dyn/cm2 shear stress for 2 and 4 hours. After 2 hours, the ratio of BIK mRNA levels in sheared cells to those in static controls was 0.96 (+0.14, n=3). However, by 4 hours, this ratio had dropped to 0.38 (+_0.01, n=3), indicating that expression levels of BIK were significantly reduced in the cells exposed to flow.

329 THE LEUKOCYTE RESPONSE TO SHEAR STRESS AFTER APPLICATION OF INFLAMMATORY STIMULATORS Shunichi Fukuda, Benjamin W. Zweifach and Geert W. Schmid-SchOnbein Department of Bioengineering and Institute for Biomedical Engineering, University of Calitomia San Diego, La Jolla, California, USA 92093-0412

Endothelial cell damage can occur m vivo during catheterization and other invasive procedures. While endothelial cell wound repair has been investigated m vivo and under static conditions in vitro, less is known about wound closure rates under controlled shear stresses in vitro. We now report a parallel plate flow chamber that allows real-time observation of the migration and cell spreading of endothelial cells into a wound under a uniform shear stress. In addition, dynamic changes in the focal adhesion sites of the cells in the flow chamber can be visualized using interference reflection microscopy. Preliminary studies indicate that the rate of wound healing is accelerated under shear stress. There does not appear to be a significant difference in the dynamic remodeling of focal adhesion sites between cells under shear and static conditions at the leading edge of the wound. This flow chamber is an effective design for studying endothelial cell wound repair in vitro. Supported by the Feinberg Cardiovascular Research Institute and Whitaker Foundation

Our recent evidence suggests that fluid shear stress in the circulation acts to induce pseudopod retraction of human neutrophils. The present study was carried out in order to clarify the neutrophil response to shear stress after application of cell activators in vitro. When fluid stress was applied to adherent neutrophils which had spread on the glass surface, all the cells retracted their pseudopods. Platelet activating factor, formyl-methionlleucyl-phenylalanine and tumor necrosis factor, respectively, suppressed the response to shear stress in a dose-dependent manner. We could observe an association between the size of spreading cells and their response to shear stress. Subpopulation of cells that had fully spread on the substrate ceased to respond to shear stress at high concentrations of activators. These results suggest that in the majority of neutrophils stimulation does not eliminate the response to shear stress unless the cells have fully spread on the substmte. [Supported by HL 43026]

327 INTERCELLULAR CALCIUM CHANGES IN ENDOTHELIAL CELLS EXPOSED TO FLOW AND AGONIST STIMULATION Laura Worthen and Dr. M.U. Nollert University of Oklahoma, School of Chemical Engineering and Materials Science, Norman, OK

330 XANTHINE OXIDASE IS A SOURCE OF HYDROGEN PEROXIDE IN HUMAN BLOOD PLASMA F. Lacy, D. A. Gough, B. W. Zweifach, and G. W. Schmid-Sch6nbein Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, CA

Florescence microscopy was used to study the intracellular calcium changes in primary cultures of human umbilical vein endothelial cells9 The cells were exposed to shear stress with and without the inflammatory mediator histamine. The control experiments showed that flow without an agonist does not affect the intracellular calcium levels. Cells exposed to histamine, an inflammatory mediator, showed a sharp increase of at least 100 nM of free calcium concentration at the initiation of the flow. Histamine concentrations of 10-7M to 10-9M were used for the experiments. This increase was present at shear stress levels of 0.2 dynes/cm2 up to 20 dynes/cm2. Our conclusion is that the intracellular calcium changes are not affected by flow alone, but are affected by flow and the presence of inflammatory mediators.

Recent evidence indicates that oxygen free radicals play a role in hypertension and that xanthine oxidase may be the source of these reactive species. An electrochemical technique using a Clark electrode to measure hydrogen peroxide (H202) was used to determine levels in blood plasma. Baseline levels of hydrogen peroxide were measured in 5 human blood samples (1.70 + 0.11). Superoxide dismutase (SOD) was added to aliquots of these plasma samples and the peroxide levels increased to 2.35 + 0.26 (p=0.03). When allopurinol was added to aliquots of the plasma samples, the measured hydrogen peroxide levels decreased to 0.67 + 0.23 (p=0.03). Since superoxide is produced in xanthine oxidase reactions, and since allopurinol is an inhibitor of xanthine oxidase, these experiments suggested that xanthine oxidase may be the source of the hydrogen peroxide. Therefore, additional experiments were performed in which xanthine oxidase was added to plasma samples while recording hydrogen peroxide production over time. This resulted in 201.tM hydrogen peroxide production. Therefore, xanthine oxidase may catalyze reactions in human plasma which are sources of hydrogen peroxide. Support by U.S.P.H.S. Grant HL-11)881.

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Cell a n d T i s s u e E n g i n e e r i n g - P o s t e r s

331 IN VITRO CHEMILUMINESCENCE MEASUREMENTS OF PLASMA SUPEROXIDE PRODUCTION BY PANCREATIC ACTIVATING FACTOR(S) E. B. Kistler, F. Lacy, R. Suzuki, A. M. Lefer, G. W. SchmidSchrnbein, and B. W. Zweifach Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Julia, CA Activators for blood cells in the circulation are currently not well understood. We recently found that pancreas homogenate and not other organs studied (heart, liver, spleen, intestine, adrenals, kidney) will activate naive donor neutrophils, as measured by pseudopod formation assays. We examined whether neutrophil activation could be detected by lucigenin chemiluminescence (CL) of superoxide production. The pancreases of six rats were homogenized in 1:9 (w/v) Krebs-Henseleit buffer, incubated for 2.5 hours at 38~ and aliquots filtered with a 3 kD cutoff. Samples were measured for superoxide CL in donor plasma. Results indicate a significant increase (p<0.001) in superoxide induced CL by pancreatic homogenate. In other experiments CL of isolated neutrophils was measured in varying concentrations of autologous plasma, both with and without pancreatic homogenate. Results indicate temporal and spatial differences in superoxide production due to buffering effect of plasma. Measurement of superoxide induced CL in plasma is a straightforward method of quantifying superoxide production in vitro. Support by U.S.P.H.S. Grant HL-43024.

332 IN VITRO NEUTROPHIL ACTIVATING FACTORS PRESENT IN PORCINE PANCREAS E. B. Kistler, A. Brondeau, P. Pfeiffer, A. M. Lefer, B. W. Zweifach, and G. W. Schmid-Schrnbein Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, CA Hemorrhagic and endotoxic shock result in upregulated levels of leukocyte activation, as measured by pseudopod formation or nitroblue tetrazolium (NBT) tests in donor neutrophils exposed to shock plasma. We have recently found that homogenates of rat pancreas and not of other organs activate naive neutrophils. It was of interest to determine whether such activation occurs in pancreatic homogenate of other species. In five male pigs randomly selected the pancreas was removed and put into a .25 M sucrose-saline solution pending homogenization. The organs were homogenized in 1:4 (w/v) saline solution. Samples were incubated for 2.5 hours at 38~ and aliquots were filtered with a 3.000 MW cutoff and tested for neutrophil pseudopod formation. Results indicate a significant increase (p< 0.001) in leukocyte activation by incubated pancreatic homogenate, both non-filtered and the low-molecular weight fraction. These results indicate that porcine pencreas, like rat pancreas contains factors that activate neutrophils in vi'ro, including a low molecular weight activator. The pancreas may serve as an endogenous source for neutrophil activator(s) in shock and in inflammatory conditions. Support by U.S.P.H.S. Grant HL-43024.

333 MECHANISMS FOR HUMAN VENOUS VALVE DESTRUCTION: EXPRESSION OF ADHESION MOLECULES AND CYTOKINES Shinya Takase, John J Bergan, B. W. Zweifach and G. W. SchmidSchrnbein Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla, CA In venous disease valves are destroyed so that reflux occurs and venous pressures become elevated. We recently obtained evidence to suggest that monocytes/macrophages migrate into the venous wails of patients with chronic venous insufficiency. However, it remains uncertain whether these ceils contribute to the actual destruction of the venous valves. To examine indices for monocyte/macrophage and endothe|ium activation, immunohistochemistry for ICAM-1 and E-selectin and cytokines was carried out. Both adhesion molecules were expressed on the luminal endothelium and in adventitial capillaries. The expression of TNF-alpha and IL-la were weak excepts on macrophages in the venous wall. These results suggest that activated endothelium and monocyte/macrophage may contribute to the destruction of the venous valves and wall in man. Supported by HL 43026.

334 PAF-INDUCED CAPILLARY FILTRATION: SITE OF REQU/SITE

NEUTROPHIL A D H E S I O N Norman R. Harrisand RobertD. Spccian LSUMC, Departmentof Molecular& CellularPhysiology,Shreveport,L A Fluid filtration rate (JJS) from capillaries in the rat mesentery increases significantly in response to platelet-activating factor (PAF) through a mechanism that includes neutrophil-endothelial cell adhesion (Am J Physiol 265, 1993). However, it is not clear whether the required neutrophil adhesion occurs within the affected capillaries, or whether postcapillary neutrophil adhesion initiates an upstream communicated response. In the present study, we demonstrate (using immunohistochemistry and confocal microscopy) that the endothelial adhesion molecules P-selectin and ICAM1 are expressed in mesenteric capillaries (N=6 rats). Even so, neutrophil adhesion induced by 100 nM PAF was not observed in the capillaries, but only in postcapillary venules. Furthermore, measurements were made of PAF-induced J,,/S from 8 capillaries in which the flux (transit; #/rain) of fluorescently-labeled leukocytes ranged from 4 to 18/rain. In these capillaries, there was no tendency toward a positive correlation between flux and normalized J,JS (slope = -.14 rain, r2=.27): a positive slop.e would be expected if leukocytes mediate the increased J,/S during transm Therefore, even though adhesion molecules are expressed on capillary endothelial cells, PAF-induced increases in capillary ftlwation may be initiated by postcapillary, rather than capillary, neutrophil adhesion. Supported by NIH HL55255 and DK43785.

335 LEUKOCYTE-LEUKOCYTE INTERACTIONS CAN ENHANCE CELL ATTACHMENT UNDER FLOW Brad Fodow and Matthias Nollert School of Chemical Engineering and Materials Science, University of Oklahoma, Norman, OK The recruitment of leukocytes to sites of injury or tissue damage is an important role of the immune response system. Circulating leukocytes initially attach and roll on the blood vessel wall by means of the selectin family of adhesion molecules. However, once the selectin sites on the vessel wall are covered by leukocytes, other receptors on the adherent leukocytes may mediate further recruitment of circulating cells. We have reproduced the rolling event in an in vitro parallel plate flow chamber using HL-60 ceils flowing over surfaces containing either P-selectin or its ligand, PSGL-1. In other experiments, HL-60 cells were immobilized on the surface with an antibody to CD43. The results indicate that cell-cell interactions depend on PSGL-1 interacting with an undetermined ligand on HL-60 cells. The undetermined ligand is not L-selectin, which is important in neutrophil-neutrophil interactions.

336 IN VITRO MOTILE ACTIVITIES OF HUMAN TUMOR CELLS IN RESPONSE TO CONTACT WITH HUMAN ENDOTHELIAL CELLS A. Wright, Y-H. Li, and C. Zhu Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA The motile response of Calu-1 epithelial lung carcinoma cells when placed in co-culture with human umbilical endothelial cells (ECs) was observed using time lapse video microscopy. Motility was quantified by fitting cell track data to a persistent random walk model of motility, from which a motility coefficient and a persistence time index were derived. The goal of these experiments was to identify the major factor(s) causing an upregulation in Calu-1 motility. Candidates include: soluble factors secreted by the ECs - both expressed constitutively and induced upon contact with the tumor cells - and factors secreted by the ECs to the extracellular matrix (ECM). Determination of the relative importance of these factors was made by assessing and comparing tumor cell motility under multiple conditions. Tumor cells were tested on plastic, on EC monolayers, and on ECM proteins. In addition, plain media, EC conditioned media, and media in contact with ECs was used.

Cell and T i s s u e E n g i n e e r i n g - Posters

337 A System to Measure Neutrophii Adhesion and Migration Under Well-defined Chemotaetlc Gradients Louis H. Strong, Martin L. Yarmush, and Francois Berthiaume Massachusetts General Hospital and Shriners Burns Institute, Boston

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340 NONLINEAR SYSTEM IDENTIFICATION OF NEURONAL NETWORKS IN THE HIPPOCAMUS M.T. Chian*, V.Z. Marmarelis, T.W. Berger Dept. of Biomed. Eng. & Prog. in Neurosci., Univ. Of Southern California, Los Angeles, CA 90089

Leukocytes sequestered at sites of injury are exposed to chemotactic agents which govern their migration into the tissue space. We have developed an in vitro system to examine the process of leukocyte migration across an endothelial monolayer under well-defined chemotactic gradients. This system consists of a confluent endothelial monolayer cultured on a porous polycarbonate membrane which rests on a collagen/agarose composite gel wherein a gradient of chemoattractant is maintained to mimic the release of chemotactic factors from injured tissue. The chemoattractant concentration profile is monitored using a fluorescently labeled chemoattractant in conjunction with a laser confocal detection system attached to an inverted microscope. The raw fluorescence data is "deconvolved" using a point spread function and exponential attenuation functions of the excitation and emission to give an accurate mapping of the fluorophore concentration distribution throughout the gel. Preliminary data using neutrophils indicate that migration into the gel is observed only in the presence of a chemotactic gradient. This system will be useful to investigate the combined effects of inflammatory mediators and chemoattractants on neutrophil adhesion and migration.

The nonlinear characteristics of subsystems in the hippocampal formation can be defined by expressing the input/output relation as a generalized functional power series. The kernels of these functional series represent the n 'h order impulse responses which are computed from the recording of electrophysiological responses to random impulse train stimulations. Characterizing unobservable systems is accomplished through nonlinear systems analysis techniques which utilize n 'h order discreet Fourier transforms in the frequency domain. Identification of unobservable elements for the three fundamental nonlinear system configurations (addition, cascade, feedback) has been tested using simulated data. For example, a simulated cascade system was created where the output of subsystem (A) acted as the input to subsystem (B) to produce an overall system. If the estimated properties of subsystem A and the overall system are known, then the dynamic properites of subsystem B may be mathematically deduced without direct observation. Successful identification of unobservable elements depends on the accuracy of the initial estimates of observable systems which were observed to be most accurate for the addition model. Since the mossy fiber output of the dentate gyrus provide the primary input to CA3, the dynamic properites of the CA3 subsystem can be mathematically deduced without mossy fiber stimulation.

338 A FLOW SYSTEM TO STUDY THE DYNAMIC INTERACTION BETWEEN GP-Ibe AND SURFACE BOUND VWF E. Saldlvar, P. Marchese and Z.M. Ruggeri The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA.

341 D E L I N E A T I O N OF A POSITIVE F E E D B A C K LOOP THAT R E G U L A T E S AMPA A N D NMDA R E C E P T O R DYNAMICS. S.S. Dalal, V.Z. Marmarelis, and T.W. Berger. University of Southern Califomia, Dept. of Biomedical Engineering and Program in Neuroscience, Los Angeles, CA 90089-1451.

A novel system has been implemented to study the dynamic properties of mechanoreceptor molecular pairs (receptor-ligand) under flow, allowing the study of the interaction between platelet glycoproteins and surface bound ligands. The method is based on the adsorption of a recombinant fragment of the glycoprotein Ibr162 (GP-Ibe) onto the surface of fluorescent latex beads, and the suspension of these beads in human blood previously treated to remove virtually all platelets; this suspension is perfused onto a von Willebrand Factor (vWF) coated surface. With fluorescence microscopy it is possible to visualize the transient interaction (translocation) of the latex particles. The interaction between GP-Ib~ and vWF can not be detected by traditional methods (i.e. ELISA) in the absence of external modulators (botrocetin or ristocetin), presumably due to the high dissociation rate of the reaction. This method allows the analysis of the dynamic interactions of mechanoreceptors native to platelets in spherical rigid particles of known size, avoiding the theoretical complication of including platelet viscosity and platelet geometry.

The perforant pathway provides glutamatergic input to the granule cells of the dentate gyms. Electrical stimulation of this pathway yields a granule cell EPSP comprised of AMPA and NMDA receptor-mediated components. Both components of the system are nonlinear with respect to interstimulus interval and have a functional interaction that is due to the voltage-dependent Mg 2. blockade inherent to the NMDA channel. The nonlinearity of each subsystem and their coupling creates the problem of quantifying the properties of the functional interaction between the two receptor subtypes. We pharmacologically isolated the AMPA and NMDA components, and experimentally characterized the dynamics of each subclass of receptor using random impulse train stimulation. Nonlinearities in the input/output relation were represented as the Laguerre expansions of the kernels of a Volterra functional power series. Results showed that a model that included positive feedback of the intracellular membrane potential onto the NMDA subsystem accurately represented the system, whereas simple summation of the AMPA and NMDA subsystems proved inadequate. Supported by ONR, NCRR, and NIMH.

339 BIOLOGICALY INSPIRED ALGORITHM FOR SOLVING MULTIPLE PROBLEMS USING A LIMITED POOL OF NEURONS Wojciech M. Grohman and Krzysztof J. Cios The University of Toledo, Department of Bioengineering, Toledo, OH

342 WIDEBAND INHIBITION OF DORSAL COCHLEAR NUCLEUS TYPE IV UNITS: A COMPUTATIONAL MODEL. K.E. Hancock and H.F. Voigt Department of Biomedical Engineering, Boston University, Boston MA

Humans can quickly recognize a variety of object classes like cars, people, etc., without conscious thinking. Although there are many specialized networks of neurons in the brain, no specific network is responsible for dealing with only one category of objects. On the other hand, the artificial counterpart of the brain, artificial neural networks (ANNs) are developed for performing only one specific function, i.e., there are networks designed for classifying trees, faces, etc. In this paper we show a way of packing many ANNs into a limited pool of neurons. The key idea behind the developed algorithm is to re-use as many neurons as possible in order to solve several different problems using fixed number of neurons. The algorithm yields encouraging results that show a significant decrease in the total required number of neurons needed to perform several tasks. For instance, we solved simple 2-D and 3-D classification problems that resulted in over 25% reduction of neurons used.

A computational model of the dorsal cochlear nucleus (DCN) was implemented to investigate the role of wideband inhibitory inputs in shaping type IV unit activity. Type IV units are mostly inhibited by tones and excited by noise. This can be explained by inhibition from type II units, which are excited by tones but respond weakly to noise. Notchednoise responses suggest that type IV units also receive wideband inhibitory input in some form. The wideband inhibition (WBI) model consists of a population of cells that are each broadly tuned. The lateral inhibition (LI) model has narrowly tuned cells from a broad frequency range; the population as a whole has wideband properties. The responses of type IV units to BF-tones and broadband noise can be simulated accurately using inhibition from type II units alone. Simulation of type IV unit notched noise responses, however, requires input from either of the wideband inhibitory sources. An experiment is demonstrated that takes advantage of facilitation to distinguish between these two wideband inhibitory mechanisms.

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Cell and T i s s u e E n g i n e e r i n g - P o s t e r s

343 A BIPHASIC MODEL OF THE CUPULA AND THE RESPONSE DYNAMICS OF THE VESTIBULAR SEMICIRCULAR CANAL E. R. Damiano Department of BME, University of Virginia, Charlottesville, VA Angular acceleration of the fluid-filled vestibular semicircular canals invokes inertial pressure that acts directly across the cupula partition which traverses a cross-section of the canal. Mechanotransduction arises from the micromechanical deformation of strain-sensitive sensory hair-cell stereocilia embedded in the cupula. An analysis is presented in which the cupula is modeled as a biphasic mixture of interpenetrating solid and fluid constituents. A regular perturbation analysis of the fluid dynamics in the canal provides a dynamic boundary condition which acts across the cupula partition. Results indicate that at frequencies above 1 Hz, the fluid phase of the cnpula is dynamically entrained by the solid phase resulting in a high-frequency response that is consistent with the response predicted by single-phase viscoelastic models of the cupula. Below 1 Hz, the dynamic coupling between the two phases of the mixture weakens and t.he tr~,nscuputar differential pressure is sufficient to force the fluid phase through the mixture with little deformation of the solid phase. This permeation dynamics gives rise to the same low-frequency response as would occur with an impermeable viscoelastic structure having a much greater stiffness. Current estimates of the inechanical stiffness of the cupula, based on afferent nerve data, may therefore overestimate the true value intrinsic to the solid phase by several orders of magnitucte.

344 NITRIC OXIDE PRODUCTION AND FLUCTUATIONS IN RAT BRAIN ARE TRANSIENTLY AUGMENTED AFTER HYPOXIA D.G. Buerk t, R. Ghodadra 2, L. Hao z, Y. Mohadjer 2, and N. Thakor z tDepts. Physiol., Bioeng., and Inst. for Environ. Med., Univ. of Pennsylvania School of Med., Philadelphia, PA and ZDept. Biomed. Eng., Johns Hopkins Univ. School of Med., Baltimore, MD. Nitric oxide (NO) was measured in rat parietal cortex (n = 6) with recessed electrochemical microsensors before and after global hypoxia produced by asphyxia for 3 min. Relative blood flow changes were measured by laser Doppler flowmetry, simultaneously with EEGs. Prior to hypoxia, N O levels were 1.16 _ 0.15 (SE) /zM, increasing to a peak of 2.37 _ 0.71 p M during blood flow hyperemia after restoring respiration. N O returned to baseline within 30-45 min with good E E G recovery in 4 rats, but remained elevated ( > 3 txM) in 2 rats with poor E E G recovery. Fourier analysis of low frequency NO fluctuations during early recovery had power spectral densities with higher magnitudes and a greater number of frequencies compared to spectra prior to hypoxia and spectra after recovery, possibly due to increased neuronal NO production. Experimental results provide insight into the role of NO in brain injury.

345 FLEXIBLE MICROELECTRODE ARRAYS WITH INTEGRATED INSERTION DEVICES David O'Brien, Mark Allen and Richard Nichols A novel, inexpensive process for fabricating flexible metal microelectrode arrays with integrated, rigid insertion devices was successfully developed using photolithography, sputtered Au, electrodeposition, and wet and plasma etching. Flexible arrays can deform with neural tissue to minimize chronic irritation. Previously reported flexible arrays must go through a post-processing step where a rigid structure is attached, by hand, to the array. This limits minimization of device dimensions, reproducibility, and yield. The innovation of this work is the inclusion of photolithographically defined, electrodeposited rigid insertion devices that are attached to an underlying flexible array during an integrated sequence of fabrication steps and allows for batch fabrication. Using photolithography, one can create a variety of insertion device and flexible array geometries and can precisely control where the flexible array is attached to the insertion device. These arrays can be inserted into neural structures having different surgical presentations (e.g. the cortical surface or individual nerve fascicles). After insertion, the rigid device is detached and removed from the site. We have demonstrated that arrays of 3.31.tm thick, 20gm wide flexible conducting lines can be individually attached to 25gm thick, 30gin wide insertion needles.

346 DEVELOPMENT OF A TISSUE ENGINEERED CORNEAL REPLACEMENT Elizabeth Cornelius and Allison Hubel, Ph.D. Biomedical Engineering Center, University of Minnesota, Minneapolis, MN

Present treatments for corneal opacification have typically involved corneal transplants. Concerns about tissue availability and disease transmission limits the applicability of keratoplasty. As a result, we are investigating the potential for the reconstitution of a tissue engineered keratoprosthesis which would provide a more compatible matrix as well as an active cellular component. The objective of this project is to determine corneal cell behavior in an engineered collagen matrix as a first step toward the development of an artificial cornea. The three types of corneal cells were seeded separately on collagen sponges with a pore size of approximately 0.I mm at a seeding density of 115,000 cells/cm2. Fibroblast cultures exhibited appropriate behavior by illustrating increased matrix synthesis over time as well as proliferation and repopulation of the collagen sponge. Epithelial cultures formed 2-3 continuous cell layers on top of the collagen sponge and both epithelial and endothelial cultures began to take on the morphology of the actual corneal surfaces. These studies provide evidence of normal cell function when cultured in a collagen matrix and support the idea that we can create an engineered corneal construct.

347 AN IDEALIZED MODEL OF THE POSTERIOR EYE AND SCLERAL CANAL A.J. Bellezza, R.T. Hart, M.L. Martinez, and C.F. Burgoyne Tulane University, Department of Biomedical Engineering, New Orleans, LA, and LSU Eye Center, New Orleans, LA The scleral canal, where retinal axons converge to form the optic nerve, may be the site of intraocular pressure (IOP)-related local stress concentrations, possibly leading to preferential axonal death in these areas. In this study, our goal was to create an idealized finite element model of the posterior eye, using an elliptical plug to model the effects of the scleral canal geometry. Isotropic material properties for both the surrounding sclera and scleral canal were used, with a modulus of elasticity of 5,5E4 N/m^2 for the scleral canal and 5.5E6 N/m"2 for the surrounding sclera, Simulations were run varying the major to minor axes ratios of the canal opening to reflect human canal anatomy (ranging from 1.75mm/1.75mm to 2,0 mm/1.5mm) and IOP levels to reflect normal (1999.3 Pa) and g[aucomatous (3998.7 Pa) eyes. As anticipated, maximum hoop stresses in the tissue adjacent to the canal were 62.9% greater than stresses in the sclera away from the canal, with a ratio of the major to minor axes of the scleral canal opening of 4:3. Interestingly, within this initial model, the regions of greatest stress concentrations correspond to the regions of early axonal death in glaucoma.

348 INTERACTION BETWEEN AQUEOUS HUMOR FLOW AND IRIS DEFORMATION Victor H. Barocas*, Jeffrey J. Heys* and Michael J. Taravella+ * Department of Chemical Engineering, University of Colorado, Boulder, CO; + Department of Ophthalmology, University of Colorado Health Sciences Center, Denver, CO Pigmentary dispersion syndrome and pigmentary glaucoma are caused by severe posterior displacement of the iris leading to erosion of pigment from the iris. Pupillary block angle closure glaucoma, in contrast, is caused by anterior displacement of the iris, leading to a high pressure drop for aqueous humor exiting the anterior chamber. In both cases, interaction with the flowing aqueous humor is believed to contribute to the displacement of the iris. We have used finite elements to simulate the interaction between the (viscous) aqueous humor and the (elastic) iris. The combined problem was solved using a mixed formulation in each domain, and remeshing during the solution of the model equations was accomplished by the introduction of a pseudo-elastic grid based on that described by Sackinger. We were thus able to solve simultaneously for the steady-state flow field of the aqueous humor and the deformation of the iris. We present results for displacement of the iris in response to variation in the dimensions and properties of eye component tissues.