UWEB REU/RECCS Research Symposium
August 16, 2006 (Wednesday)
UW Foege Bldg., Room N130, 9:00 am – 12:30 pm
Time
9:00 – 9:05
9:05 – 9:20
9:20 – 9:35
9:35 – 9:50
9:50 - 10:05
Introduction/Welcome: Eric H. Chudler, Ph.D., UWEB Director of Education and Outreach
White, A., Shastry, A., Simonovsky, F., Ratner, B.D., Bohringer, K. Derivatization of pillared
silicon substrates using poly (ethylene glycol) and 1-dodecanethiol. Surfaces that exhibit
hydrophobic and hydrophilic characteristics are essential when developing Lab-on-chip
technologies. The availability of a droplet to be maneuvered on a surface develops from
hydrophobic surface characteristics, while non-protein fouling characteristics are found in
hydrophilic surfaces. By combining these traits an ideal
surface can be produced for biomedical
applications. To satisfy these surface requirements we have attached 8-arm hydroxyl-terminated
poly (ethylene glycol) to silicon oxide and methyl-terminated 1-dodecanethiol to gold. By applying
these techniques to a pillared substrate the result is hydrophilic poly(ethylene glycol) covalently
bonded to pillar tops and hydrophobic 1-dodecanethiol bound to pillar sides and troughs. This
creates a surface where the hydrophobic pillar sides and troughs suspend a droplet for movement
while the hydrophilic pillar tops prevent loss of essential proteins.
LeGare, S.M, Saul, J.M., Linnes, M.P, Giachelli, C.M., and Pun, S.H. Sustained delivery and
expression of plasmid human vascular endothelial growth factor from fibrin scaffolds. The
growth of new tissue is often dependant on the presence of key growth factors that initiate and
regulate cellular processes. Tissue engineering scaffolds provide a matrix for cell attachment and
growth but lack in the presence of growth factors that initiate processes such as angiogenesis.
Our approach is to utilize sphere-templated biodegradable fibrin scaffolds coated with DNA
polymer complexes (polyplexes) for transgene expression of growth factors. The plasmid utilized
in these studies encodes for human vascular endothelial growth factor (hVEGF), which is expressed
as a secreted protein suitable for detection by an enzyme linked immunosorbent assay (ELISA)
and is also a key precursor to angiogenesis. Cells seeded onto fibrin scaffolds coated with
polyethylenimine (PEI)/plasmid encoding hVEGF polyplexes showed VEGF expression after two
days. Sustainability of expression from the fibrin scaffolds was determined by quantifying VEGF
expression with ELISA for 14 days. The amount of DNA used on each scaffold was varied to
control microenvironmental concentrations of VEGF. Successful delivery of growth factors to cells
from biodegradable scaffolds, similar to the VEGF plasmid on fibrin scaffolds, is a promising
starting point for the initiation of new tissue growth, such as angiogenesis.
Perry, L.E., Karp, F.B., Hauch, K.D., Ratner, B.D. Explanted pacemakers: observations of the
long-term foreign body response. Implanted cardiac pacing systems are widely used medical
devices for the treatment of electrophysiological disorders. This study examines morphological and
histological characteristics of the foreign body response observed in postmortem human subjects
with long term implanted cardiac pacing devices. Four implanted pacing systems were retrieved
from cadavers originating from the Willed Body Program at the University of Washington Medical
Center. Tissues were fixed in Formalin, sectioned at 5 m intervals, and stained with Haematoxylin
and Eosin, Wright-Giemsa, and Masson’s Trichrome. Fibrous capsules and inflammatory cells
were identified and characterized; each fibrous capsule was measured for thickness and classified
according to the identity of the material in the tissue-material interface as well as anatomical
location. The surrounding tissues exhibited defined collagen capsules as well as other evidence of
inflammatory cells adjacent to certain materials. Capsule thickness measurements and amount of
cellular activity were found to correlate to position and material in contact with the tissue as well as
duration of implant.
Fink, H., Speer, M., Giachelli, C. Generation of stable myocardin knockdown smooth muscle
cell lines using RNA interference. Calcification of blood vessels occurs in cardiovascular disease
and chronic kidney disease, resulting in hardening of arteries. Smooth muscle is the major cell
population in the blood vessel wall and has been shown to calcify the matrix when cultured in the
presence of elevated inorganic phosphate. This calcified smooth muscle shows loss of smooth
muscle marker genes, such as SM α-actin and SM 22α, which are downstream of the transcriptional
coactivator myocardin. In this study, we use siRNA to create several smooth muscle cell lines with
lowered expression of myocardin. These cells will be interrogated for their expression of SM
10:05 - 10:20
10:20 - 10:35
10:35 - 10:50
10:50 - 11:00
11:00 - 11:15
11:15 - 11:30
marker genes and ease of calcification to determine the effect myocardin may have in the
calcification process of smooth muscle.
Dinan, B., Bhattarai, N., Li, Z., Zhang, M. Characterization of chitosan/PCL based
nanofibrous scaffolds for tissue engineering. Polymeric nanofibers that mimic the structure and
function of the natural extracellular matrix (ECM) are of great interest in tissue engineering as
scaffolding materials to restore, maintain or improve the function of human tissues. Electrospinning
has been recently developed as an effective technique for nanofiber fabrication. In our study, the
electropsinning method is used to create composite nanofibrous scaffolds made of widely accepted
natural biopolymer i.e. chitosan, and
polycaprolactone (PCL), a well-known synthetic
biocompatible polymer. Solutions of chitosan in trifluroacetic acid and PCL in trifluroethanol were
first prepared separately and mixed together to get the final electrospinning solution. To achieve the
fine electrospun nanofibers of chitosan/PCL composite we varied the solution concentration and
adjusted electrospinning variables. The morphology of the scaffold was observed using scanning
electron microscopy (SEM) and the mechanical strength and elasticity of the material in addition to
solution viscosities were measured. Potential use of this nanofibrous matrix for tissue engineering
was studied by examining the integrity of genipin crosslinked nanofibrous mat and noncrosslinked
mat in water, PBS, and cell culture medium and the cellular compatibility.
Espinoza, L., Hower, J.C., Jiang, S. Influence of salt and pH on the adsorption of fibrinogen
and lysozyme to self-assembled monolayers using a surface plasmon resonance sensor. This
work examines protein adsorption on surfaces to provide a fundamental understanding of the
molecular-level mechanism for protein interactions with biomaterials. Carboxylic acid, amine, and
hydroxyl terminated self-assembled monolayers (SAMs) are used to model negatively charged,
positively charged, and hydrophilic/neutral surfaces, respectively. The adsorption of fibrinogen and
lysozyme on these SAM surfaces under different ionic strengths and pH values is studied using a
surface plasmon resonance (SPR) sensor. It is shown that variations in ionic strength have little
effect on the adsorbed amount of fibrinogen and lysozyme on these SAMs at a fixed pH value.
Analysis of the effect of pH on protein adsorption at a fixed ionic strength is ongoing. These studies
will provide insights into the role of hydrogen bonding and electrostatic interactions in protein
adsorption.
Piñón, T., Katzenmeyer, K., Callihan, J., Bryers, J.D. Isolation and characterization of a
fibronectin-binding receptor from Staphylococcus epidermidis. Staphylococcus epidermidis is the
leading cause of nosocomial or implant-related infections, and is responsible for serious
complications including sepsis, bacterial emboli, and even death. Bacterial adhesion is mediated by
specific interactions between the cell surface receptors and proteins, such as fibronectin (Fn), that
initially adhere onto an implanted medical
device. Here we show the isolation and partial
characterization of a recombinant S. epidermidis fibronectin-binding protein, FnR, previously
cloned in Escherichia coli M607. The His-tagged fibronectin receptor was extracted and purified
via nickel chelate affinity chromatography. The molecular weight of the isolated FnR was
determined to be 32.5 kDa via SDS-PAGE and from the known amino acid sequence. Inhibition
assays verified the biological activity of the FnR and also showed the protein can successfully
block binding of S. epidermidis to Fn. Results provide a basis for future studies of bacterial
adhesion that will aid in the development of anti-infective medical implants and improve
therapeutic treatments for device-related infections.
BREAK
Deryckx, K.S., Riegel, A.E., Naeemi, E.D. Solid phase synthesis of 11-mercapto-1-undecanoic
acid and 1-dodecane thiol for in situ assembly of monolayers on gold. Synthesis and purification
of thiols can be a time consuming and complicated process. Utilizing solid phase synthesis
techniques, commonly used for protein synthesis, could increase the purity of thiols in a more
efficient way. In situ assembly of monolayers from resin-bound thiols also prevents the unpleasant
odor associated with thiols and eases the self assembly process. In this experiment, 11-mercapto-1undecanoic acid and 1-dodecane thiol are synthesized from thiol trityl resin, and their identities are
verified using Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR), and Gas
Chromotography/Mass Spectrometry (GC/MS). Thin Layer Chromatography (TLC) and Ellman’s
Reagent are used as secondary thiol detectors. In situ assembly of monolayers from these thiols is
explored using contact angle comparisons to positive and negative controls.
Parker, S., Boeckl, M., Solomon, R., Naeemi, E. Spectrophotometric analysis of alkanethiols
11:30 - 11:45
11:45 - 12:00
12:00 - 12:15
12:15 - 12:30
using DTNB for HPLC. This analytical assay was done in two parts. The first was a preparation of
concentration curves of various alkenethiols using the colorimetric reagent DTNB (Ellman's
reagent) at 412 nm in a UV/VIS spectrophotometer. The second half was adapting those curves to
approximate concentrations in a UV/VIS detector for HPLC, and developing a highly accurate
detection for the concentration of thiols.
Kirkman, J., Rockhill, W., Bosma, M.M. Propagation patterns of spontaneous synchronous
activity and the respective driving neuronal populations in embryonic mouse midbrain.
Spontaneous synchronous activity has been recorded during development in many parts of the
central nervous system, including the hindbrain, cortex and visual system. This activity influences
neural pathfinding, synapse stabilization and correct circuit formation; and has been shown to occur
in embryonic mouse midbrain. The majority of the midbrain spontaneous activity occurs on
embryonic day (E) 12.5. Calcium imaging was used to examine the propagation patterns of
spontaneous synchronous activity in embryonic mouse midbrain from E11.5 to E14.5. The activity
patterns were compared with neuronal populations identified through immunocytochemistry to
propose specific populations responsible for driving the spontaneous activity.
Le, H., Roberts, B., Boeckl, M.S., Naeemi, E. Optimizing conditions of protecting and
deprotecting alkanethiols for use in self-assembled monolayers. Self-assembled monolayers
(SAMs) have been applied in various fields ranging from nanofabrication to biomaterials as a
convenient method to create surfaces with specific physical and chemical properties. No matter
what type of SAM is desired, purity of the alkanethiol is required for precise, high performance
surface properties. This research focuses on optimizing the conditions of protecting alkanethiols
with different protective groups and conditions of deprotecting by varying concentrations of
acid/base and time of refluxing. Purity was determined by NMR and the quality of the resulting
monolayer was determined contact angle analysis.
Inouye, B., Navratil, A., Usui, M., Underwood, R., Kulikauskas, R., Fleckman, P., Allan, C.
Ki67 and Msx-1 expression in human digit development and in an organ culture model of fetal
digit tip regeneration. Current surgical management of severed limb injuries often result in
compromised limb reconstruction and function. With successful regeneration, post-injury quality of
life for patients could dramatically improve. An upregulated expression of Msx-1, a homeobox
gene coding for a transcriptional repressor, has been seen in cells of regenerating tissues of
amputated digits in mice, and in response to tip amputation in cultured human fetal digits. Our
study explores the localization of Msx-1 and Ki67 (a proliferation marker) in human digits during
development compared to an in vitro organ culture model of amputated fetal digits. Standard
immunohistochemistry techniques were used to stain for Msx-1 and Ki67 in frozen and embedded
fetal digits. Staining was done on a developmental series ranging from estimated gestational ages
(EGA) of 53 to 110 days, as well as in cultured, amputated digits of EGA 53 and 87 days to
determine whether Msx-1 expressing cells, associated with injury response and localized at the
amputation site, arise through migration or on site proliferation. Our results show mutually
exclusive expression, indicating that Msx-1 stained cells present at the amputation site are not
proliferating. Instead this suggests a migration or a localized upregulation of Msx-1 expressive
activity of those cells.
Cordova, L.H., McGonigle, J.S., Giachelli, C.M., Scatena, M. Vascular endothelial cell
signaling responses to OPG and RANKL. Angiogenesis is a vital mechanism in the body’s ability
to respond to circulatory dysfunction. The body’s own regulation of angiogenesis is controlled by
many cellular and extracellular signals. The pair of molecules osteoprotegerin (OPG) and receptor
activator of NF-κB ligand (RANKL) is well established as a osteoclast regulatory mechanism, and
they have recently showed promise in the induction of angiogenesis. In this study we evaluate the
potency of these molecules to induce two pathways associated with angiogenesis: p38 MAPk and
MEK/Erk. Cultured vascular endothelial cells were treated with RANKL or OPG before lysing the
cells for protein collection. Western blots were performed on the lysates and probed for the
phosphorylated forms of p38 MAPk and Erk. OPG was shown to activate the MEK/Erk pathway,
a known essential signal for angiogenesis, while it did not have an effect on the pro-apoptotic
pathway through p38 MAPk. RANKL conversely stimulated p38 MAPk without activating
MEK/Erk. These results indicate possible mechanisms through which OPG could act to promote
angiogenesis.