Interdisciplinary Seminar of the IME
In academic year 98/99, the IME hosted 26 seminars by leading international and national investigators and 20 seminars by Penn faculty. The seminars, of which half were co-sponsored with departments, have been consistently well attended by Penn faculty, fellows, students and research staff with interdisciplinary interests. The Seminars are supported in part by a grant from the Interdisciplinary Seminar Fund of the Vice Provost for Research.
"Policy" lectures in Biomedical Engineering by University of Washington Provost (and Bioengineering) Lee Huntsman, and Bioethics by Penn's Art Caplan drew audiences of over 100. Also attracting large audiences were the IME/LRSM Joint Lecture(November 1998) delivered by David A. Tirrell (CalTech), who spoke on "Artificial Proteins: Bridging the Gap between Natural and Synthetic Macromolecular Materials", and the 4th Britton Chance Lecture in Engineering and Medicine, (February 1999), co-sponsored with the Department of Chemical Engineering, where Jeffrey A. Hubbell (Swiss Federal Institute of Technology) spoke on "Biomaterials in Engineering Tissue Responses: Blocking, Presenting and Receiving Biological Information".
The informal IME Chalk Talk Series continues to feature presentations by IME Members and postdocs (and occasionally graduate students). Functioning as a discussion group of current research, 30-45 attendees come to these Friday meetings that promote interdisciplinary research in biomedical engineering in a less formal setting.
Symposiumon Nanotechnology in Biomedicine
A research retreat on Applications of Nanotechnology to the Biosciences, organized by the IME and the PMI and supported by the Retreat Fund of the Medical School, was held in May 1999. The objective was to stimulate discussion concerning the detection and manipulation of single macromolecules, particularly in their native state, and consideration of the application of nanotube technology in the biomedical sciences. Both areas are rich in opportunities to design and harness nanoscale systems for biomedical application.
Dr. Richard Tannen, Senior Vice Dean of the Medical School, opened the day. Experts from out-side Penn presented state-of-the-art lectures.
Toshio Yanagida (Osaka, Japan) talked on manipulation of single protein molecules by optical and scanning probe microscopes. Steven Wilson (NHU) reviewed nanostructures in biomedicine; Jan Hoh (Hopkins) discussed spatially resolved force spectroscopy using AFM, and Steve Block (Princeton) talked on optical trap studies of molecular motors. Penn speakers were Drs. Alan T. (Charlie) Johnson (Physics), Joseph Forkey (PMI) and Henry Shuman (Physiology). The day concluded with a panal discussion led by Peter F. Davies and Yale Goldman (IME and PMI Directors).
First IME Retreat
The first IME Retreat took place on February 2, 1999, in the Sugarloaf Conference Center in Chestnut Hill. The central theme was tissue engineering. The keynote speakers were Robert Reneman, M.D., Ph.D. (Masstricht, The Netherlands), and Martin Yarmush, M.D., Ph.D., (Mass. General, Boston). The retreat also featured talks by five IME members. Graduate student, postdoctoral and junior faculty research was presented in a poster sission (31 posters submitted). Hyun-Jeong Ra (Discher Lab, MS in Biotechnology student) and Brian Helmke, Ph.D. (Davies Lab, BE post doc) received the "Best Poster" awards in the pre- and postdoctoral categories, respectively. Eighty fivfe researchers attended the retreat.
Stay tuned for more details on the 2nd IME retreat!
Spatial and Temporal Regulation of Gap Junction Connexin43 in Vascular Endothelial Cells Exposed to Controlled Disturbed Flows In Vitro
Natacha DePaola, Peter F. Davies, William F. Pritchard, Lucio Florez, Nadeene Harbeck, Denise C. Polacek
Endothelial cell communication plays an important role in vascular physiology and atherosclerosis. It occurs by a combination of humoral exchange and by direct transfer of signals between neighboring cells through transmembrane gap junctional assemblies 
composed of connexin (Cx) proteins. The lab of Drs. Peter F. Davies and Denise Polacek in collaboration with the lab of Dr. Natacha DePaola at the Rensselaer Polytechnic Institute studied the molecular regulation of endothelial communication in relation to hemodynamic forces in a unique culture system that precisely models complex flows near arterial branches. RNA transcription and protein assembly of the principal endothelial connexin, Cx43, was measured in regions of flow disturbance compared with undisturbed flow, the former simulating flow separations and gradients of hemodynamic shear stresses observed in arteries. Significant spatial and temporal differences in molecular expression were noted between regions, particularly a sustained disassembly of gap junction Cx43 in disturbed flow. The differences were extended to functional communication by measuring the passage of fluorescent dyes between cells; communication was impaired in disturbed flow but not in unidirectional undisturbed shear stress. The results, published in the Proceedings of the National Academy of Sciences, USA (96:3154-3159, 1999), suggest a prominent role for hemodynamics in determining regional differences in cell-cell communication. This may contribute to vascular pathophysiological changes in regions of flow disturbance which are classic sites of atherosclerotic lesion development.
Inhibition of Nitric Oxide But Not Prostacyclin Prevents Poststenotic Dilatation in Rabbit Femoral Artery
William J. Calvo, George Hajduczok, James A. Russell, Scott L. Diamond
In 1842, physicians at a Paris hospital observed that if they put a band around a blood vessel to mimic a constricted area, the vessel unexpectedly hyperdilated downstream of the narrowing. Although the hemodynamic origin of poststenotic dilatation (PSD) is generally undisputed, the precise mechanisms have been controversial and the mediators unknown. The downstream region has low pressure where recirculation eddies oscillate in size during the cardiac cycle. Dr. Scott Diamond together with collaborators at SUNY Buffalo proposed that nitric oxide elicited by fluid shear stress acting on the endothelium in the throat of the stenosis and/or at focal sites of flow reattachment may be an important mediator of PSD.
Rabbit paired femoral arteries were banded to reduce diameter by 71%. A gel was painted onto the outside of the arteries that contained either nitric oxide inhibitor or a placebo. PSD, measured after 3 days, was reduced to 23% of the control levels by the nitric oxide inhibitor whereas an inhibitor of prostaglandin synthesis caused no significant reduction of PSD. The findings identify nitric oxide as a key molecular mediator of PSD. The work was published in the journal Circulation (99: 1069-1076, 1999).
The Effects of Overuse Combined with Intrinsic or Extrinsic Alterations in an Animal Model of Rotator Cuff Tendinosis
James E. Carpenter, Colleen L. Flanagan, Stavros Thomopoulos. Edward H. Yian, and Louis J. Soslowsky
This publication received the Jack Hughston Award from the American Orthopaedic Society for Sports Medicine, for the "Best Paper Published in the American Journal of Sports Medicine in 1998".
Dr. Louis Soslowsky together with collaborators at the University of Michigan used an in vivo rat model to evaluate overuse and overuse plus intrinsic tendon injury or extrinsic tendon compression in the development of rotator cuff injury. One shoulder of each rat received an intrinsic or extrinsic injury plus overuse (treadmill running), while the other received only overuse. Supraspinatus tendons were evaluated histologically or geometrically and biomechanically, and compared to those of rats from a cage-activity control group. Supraspinatus tendons of rats in both experimental groups had increases in cellularity and collagen disorganization and changes in cell shape compared with control tendons. Tendons with injury plus overuse exhibited a worse histologic grade than those with overuse alone. The cross-sectional area of the tendons in the experimental rats was significantly more than in control tendons, with increased area in the injured vs. overuse-alone tendons. Biomechanically, the tissue moduli of the overuse/injury tendons were significantly lower than in the overuse-alone tendons. This study demonstrated that damage to the supraspinatus tendon can be caused by overuse and intrinsic injury, overuse and extrinsic compression, and overuse alone. The work appeared in the American Journal of Sports Medicine (26: 801-7, 1998).
Atomic Force Microscope for Studies of Biological Systems
The NIH/NCRR awarded the IME (Principal Investigator: Peter F. Davies) $246,000 For the purchase of an atomic force microscope (AFM) dedicated to soft materials studies. The Digital Instruments (Santa Barbara, CA) Nanoscope IIIa Atomic Force Microscopy system will consist of a Bioscope AFM unit/inverted optical microscope principally for intact cell work, and a MultiMode AFM unit for highest resolution molecular and biomembrane investigations. The system will be located in the IME, on the first floor of the Vagelos Research Labs. The facility will provide Precise measurements of surface topography and force-related events in biological systems ranging from single molecules to intact cells, simultaneous with direct observation and quantitation of molecular dynamics through fluorophore reporter molecules. In addition, it will also provide interdisciplinary research training opportunities for graduate students and postdoctoral fellows.
A Spatial Approach to Transcriptional Profiling: Mechanotransduction and the Focal Origin of Atherosclerosis
Peter F. Davies, Denise C. Polacek, Jeffrey S. Handen, Brian P. Helmke, and Natacha DePaola
The initiation and progression of focal atherosclerotic lesions has long been known to be associated with regions of disturbed blood flow. The lab of IME Director Peter Davies and Senior Research Investigator Denise Polacek has recently combined precision in experimental models of spatially defined flow with regional and single-cell gene-expression ("transcriptional profiling") to investigate the relationships linking hemodynamics to blood vessel pathobiology. Single cells can be isolated 
from regions of well-defined flow conditions in vivo or in vitro, and analyzed to reveal changes in gene expression under different hemodynamic and pathologic conditions, achieved by the application of the amplified antisense mRNA (aRNA) technique developed by IME member Jim Eberwine and colleagues. Quantitative profiles of gene expression, including the use of high-throughput hybridization to screen many genes simultaneously, allow endothelial heterogeneity to be addressed in a detailed (single cell) yet comprehensive (multiple genes, high-throughput) approach that increases the probability of finding new therapeutic targets. The aRNA technique provides near-linear amplification of the original mRNA population of the cell, in contrast to RT-PCR which is unable to amplify all messages accurately because of misincorporations during the exponential amplification process and a bias towards smaller cDNA sizes. The accompanying figure illustrates the use of the technique to show transcriptional profiles for 40-genes in six morphologically identical endothelial cells individually dissected from a confluent monolayer following 16 hours of unidirectional shear stress. The precision that this approach brings to spatial localization of vascular cell gene expression in the context of hemodynamics is powerful, particularly when extended to thousands of genes through microarray technology. This work will be published in the September 1999 issue of Trends in Biotechnology (17:347-351).
Reciprocal Interactions Between b1-Integrin and Epidermal Growth Factor Receptor in Three-Dimensional Basement Membrane Breast Cultures: A Different Perspective in Epithelial Biology
Fei Wang*, Valerie M. Weaver*, Ole W. Petersen, Carolyn A. Larabell, Shoukat Dedhar, Per Briand, Ruth Lupu, and Mina J. Bissell
*Both authors contributed equally to the work
Cell adhesion and growth factor independence allow breast tumors to metastasize and grow. Dr. Valerie Weaver with colleagues and collaborators at Lawrence Berkeley National Laboratory in California, The Panum Institute and Danish Cancer Society in Denmark, and The Jack Bell Research Center in Canada have shown that when breast epithelial cells are organized into three-dimensional tissue-like structures by a basement membrane extracellular matrix in culture, growth and adhesion pathways are coupled dynamically and reciprocally. The work, published in the Proceedings of the National Academy of Sciences (95: 14821-14826, 1998), demonstrates a bidirectional cross-modulation of b1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway, when nonmalignant or tumorigenic mammary cells are grown to form three-dimensional, tissue-like structures, but not in undifferentiated monolayers in tissue culture. Inhibition of the signaling activity associated with either of these receptors, or inhibition of MAPK kinase activity, induced a concomitant down-regulation of normal breast tissue structure and behavior. The findings explain why breast cancer cells fail to differentiate when grown as monolayers. They indicate that both 'inside out' and 'outside in' signaling can correct aberrant behavior and restore normal function to tumor cells in a three-dimensional basement membrane. These results may prove useful in devising alternative therapeutic strategies for early stages of breast cancer progression.
Center for Bioactive Materials and Tissue Engineering
Of the various musculoskeletal conditions requiring treatment, spinal problems are among the most prevalent and difficult to manage. Surgical treatments for herniated discs include laminectomy, spinal fusion, or disc replacement with prostheses, none of which are entirely satisfactory. The Center has developed resorbable scaffold materials that stimulate bone cell function and lead to mechanically functional repair in a rodent model. We are now pursuing a preclinical tissue-engineering project to evaluate the behavior of this scaffold seeded with osteoprogenitor cells.
Events at the surface between materials and osteoblasts are modeled using self-assembled monolayer surfaces, which allow a high degree of control over surface composition. Although materials currently used for musculoskeletal repair are very diverse, the incorporation or introduction of hydroxyl groups is easily accomplished in most materials. By investigating hydroxyl group regulation of biological material absorption at surfaces, we create tools to design the surface of resorbable materials for enhanced tissue repair.
The long-range goal of the Center to address degenerative disc disease includes a novel treatment in which the nucleus pulposus tissue of the intervertebral disc is regenerated. These cells can proliferate in vitro with retention of the phenotype. We implant hybrid cell - resorbable biomaterial constructs and study the cell and molecular biological mechanisms associated with the physical restoration of the intervertebral disc.
For more information, contact Dr. Paul Ducheyne.
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