Event
Bioengineering Seminar Series: BMES Preview
Friday, October 1, 2010
11:00 a.m.-12:00 p.m.
Room 2108, Chemical and Nuclear Engineering Bldg.
Professor Sameer Shah
sameer@umd.edu
BMES Preview: Oral Presentations
Titles and abstracts to be announced.
Joshua Chetta, Emily Coates, David Hwang and Kimberly Stroka
Graduate Program in Bioengineering
Fischell Department of Bioengineering
University of Maryland
Kimberly Stroka, David Hwang, Joshua Chetta, and Emily Coates, graduate students in Bioengineering at UMCP, will be presenting platform talks at the annual BMES meeting on topics in biophysics, biomechanics, and tissue engineering. These moderated 15 minute talks will follow the conference talk format, which allows 10-12 minutes for presentation followed by questions.
Mechanics of Leukocyte Transmigration through Endothelial Cell Monolayers
Kimberly Stroka and Helim Aranda-Espinoza
Leukocyte transmigration through the vascular endothelium is a crucial step in the normal immune response. However, in cardiovascular disease (CVD), an excess of leukocytes adhere to and transmigrate through the endothelium, take up lipid particles, and deposit them onto the endothelium, resulting in recruitment of more leukocytes and formation of plaques which obstruct blood flow. This process leads to development of the CVD atherosclerosis, an overall stiffening of the arteries, and often heart attack or stroke. In this study we investigate the mechanics of leukocyte transmigration in an in vitro model of the vascular wall. We model healthy versus diseased blood vessels through manipulation of substrate stiffness using polyacrylamide gels, coated with extracellular matrix protein and plated with human umbilical vein endothelial cell (HUVEC) monolayers. The HUVEC monolayers are activated with tumor necrosis factor-alpha to mimic inflammatory conditions. We observe that both leukocyte migration along and transmigration through HUVEC monolayers depend on the stiffness below the endothelium, suggesting that artery stiffening in CVD further perpetuates the cycle of leukocyte transmigration. Using an array of biophysical techniques, we evaluate the morphology, stiffness, cytoskeletal arrangement, adhesion protein expression, migration, cell-substrate adhesion, and permeability of HUVEC monolayers as a function of substrate stiffness. We conclude that altered transmigration occurs due to a combination of both HUVEC and leukocyte mechano-sensing of substrate stiffness.
Changes in Gene Expression of Nucleus Pulposus Cells Subjected to Distinct Load Histories in vivo
David Hwang and Adam Hsieh
Degenerative disc disease is an undiscriminating condition that affects people across all walks of life. The breakdown of the intervertebral disc (IVD) that occurs with aging is characterized by a loss of water content, which reduces the swelling capabilities of the disc and, therefore its ability to respond to loads. At any given time, a discs hydration and biomechanical function is influenced by its prior history of load. Our previous studies have shown that load history changes the ability for the IVD to generate intradiscal pressure (IDP), a stimulus known to regulate cells of the nucleus pulposus (NP). In this study, rat caudal discs were loaded in vivo under different preloading conditions (0 and 0.5 MPa) for one hour, followed by an increase to a final exertion load of 1.0 MPa held for an additional hour. Immediately following the exertion load, RNA was isolated and prepared for real time RT-PCR. Results showed that exertion after preloading upregulates type II collagen and aggrecan expression and downregulates ADAMTS-4, while type I collagen expression remained unchanged This cellular change translates into increased hydration and swelling. Our prior work showed that a higher IDP is generated when discs are loaded without preload, as compared to 0.5 MPa preload. Thus, results were somewhat unexpected, but may be indicative of a response to compensate for lower pressure generation in response to the same exertion load (1 MPa). This may be a cellular adaptation to restore pressure and alleviate shear stress within the NP.
Dynamic Actin Densities in the Axon of Sensory Neurons
Joshua Chetta and Sameer Shah
The axonal cytoskeleton is a well organized heterogeneous scaffold composed of polymer filaments which bear externally and internally applied loads and contribute to force generation as the substrate for the action of motor proteins. Cytoskeletal dynamics have been explored in other cell types, especially during crawling in motile cells and in the growth cone of neurons, but has rarely been imaged in the axon. In order to understand the role of the actin cytoskeleton in the axon during growth, rat sensory neurons were transfected with actin GFP and cultured for 16-20 hours on glass coverslips and axons were imaged during normal unimpeded growth. Novel bursts of actin density were observed. These densification events occurred quickly, on the order of 10 seconds, and frequently migrated a few microns along the axon, in both the anterograde and retrograde directions, before disappearing. While the protrusion of a spine from the side of the axon was always preceded by these types of densifications, the bursts themselves frequently occurred independently of any discernable morphological change. We hypothesize that they may signify regions of nascent or temporary adhesion along the axon. In order to understand the mechanism by which these densities occurred, pharmacological agents were used to modify various cytoskeletal components thought to play a role in their formation.
Zonal Chondrocyte Response to Growth Factor Delivery and Matrix Molecules
Emily Coates and John Fisher
Regenerating articular cartilage with native zonal organization remains a challenge. Retention of stable populations of chondrocytes from each tissue zone can help overcome this challenge. Culture of chondrocytes in monolayer without regard to zonal orientation typically results in a homogenous, fibroblast-like cell population. Three-dimensional culture aids in retention of chondrocyte phenotype, and thus may also help stabilize specific sub-populations. In an effort to understand which microenvironments are ideal for each cell population, insulin-like growth factor-1 delivery and incorporation of matrix molecules within alginate scaffolds were investigated. Populations of superficial, middle, and deep zone chondrocytes were isolated from the femoral condyles of bovine calves. These populations were then separately encapsulated in alginate and IGF-1 was delivered. Results showed approximately 2 times the expression of aggrecan, and 3 times the expression of both type II collagen and endogenous IGF-1 binding protein mRNA in middle and deep zone cells versus superficial cells. Deep zone cells were most responsive to IGF-1. Based on these results, a second experiment was conducted using only two cell populations. Superficial zone cells and middle/deep zone cells were encapsulated in three types of alginate: containing hyaluronic acid, chondroitin sulfate, and type II collagen. Initial results demonstrate high cell viability in all groups, and subsequent investigations will determine the effects of microenvironment on metabolic activity.
