Team ELECTRODEMentor: Dr. John P. Fisher

Students: Sagah Ahmed, Natalie Anzures, Zach Bosley, Brendan Bui, Ariana Feizi, Sudi Jawahery, Courtney Koenig, Katie Lakomy, Megan Lin, Poorna Natarajan, Eisha Nathan, Hiba Sayed, Eduardo Solano

Evaluating Linear-Radial Electrode Conformations for Tissue Repair and Organizing a Device for Experimentation

Diabetes

Prevalence of Diabetes In 2010, 1.9 million Americans over the

age of 20 were diagnosed with diabetes

Formation of Diabetic UlcersNerve damage, poor circulation, lack of

tissue repair

Issues with Current TreatmentsRequires constant application by the

patient, doesn’t directly target the wound, can be very expensive

http://www.executivehm.com/news/diabetes-in-the-us/

EFFECTS of VEGF & bFGF in wound healing

Body’s natural bioelectric healing system

Angiogenesis and Inflammation

Wound healing and electrical stimulation

Epithelial Cells

Hypothesis

Inflammation of damaged tissue is the major factor suppressing angiogenesis in diabetic ulcers

The application of an electrical stimulus will alleviate the effects of the inflammatory tissue response in wounds

Project Purpose: Create an electrical stimulation device that will improve wound healing in diabetic ulcers.

We are examining the effects of…

In Vitro ExperimentsCell Culture

Freezing/ Thawing Cells are frozen throughout the experiment so a

constant source of cells is available

Nourishment Cells are grown in flasks containing Rat Aortic

Endothelial Cell Medium changed every 48 hours

Passaging Cells are moved to bigger and more flasks as

needed

Plating Before experimentation, cells are plated on 6-well

plate containers

Device Design

ϟUse best parameters determined from experimental trials

ϟCompare effectiveness of a linear setup vs. a radial setup

ϟCheap and small prototype for a more marketable device

Electrical Circuit

Battery

Flow

Flow

+

Device:Linear and Radial Apparatuses

Experimental set-up of a linearly applied electric field. The anode and cathode are 1.5-cm apart.

Experimental set-up of a radially applied electric field. The radius is 1.5 cm.

In Vitro Experiments:Parameters

ϟTest voltages:

ϟ0.01 V

ϟ0.1 V

ϟ1.0 V

ϟ50 Hz

ϟApplied for one 30-minute period

ϟ6 wells: 3 control, 3 experimental

Pulsed Monophasic Current

Time

Volt

ag

e

DEVICE IN ACTION! (electrical stimulation of cells at 0.1 volts, 30 min per well for 3 exp wells)

Negative electrode

Positive electrode

Control(no stimulation)

Experimental

Cell ANALYSIS

Measuring Cell Proliferation

Fluorescently stain live (green) and dead cells (red) in our sample

Live/Dead samples are examined under a fluorescent microscope and cells counted using Computer Software Image J

No significant cell death has been observed after successful cell culture has been achieved

Live/Dead Assay Picture 10x, taken on 10/10/12

100 um

Cellular Proliferation

Control

Experimental

Measuring VEGF/bFGF Expression Levels

Extraction of mRNA and samples are frozen for further testing (ELISA, qRT-PCR)

mRNA levels are tested using the Nanodrop

We are looking for levels of mRNA detection that are high enough to provide us with accurate results in further testing

Cell Migration

Purpose: To observe migration of cell population 3 and 6 hours after electrical stimulation

2 well plates, each with 6 wells.

3 wells control, 3 wells experimental

Cells scraped into 2 cm wide line

Timeline

0 hrs: Stimulation and Plate 1 Imaging

3 hrs: Plate 1 Imaging

6 hrs: Plate 2 Imaging

2 cm cells

No cells

No cells

Migration Imaging/Analysis

Take 3 images along each side of the line of cells (6 pictures per well)

Measure distance from starting position to edge of cell population

Take measurements at several points along the line of cells

Average the distances measured in each image

Image J program

Migration data, 10/9/12

Left side view, scraped line of cells at edge

Current and Future Objectives

FALL 2012:

Experimental testing for Specific Aim 1 (linear device)

Data Analysis for Specific Aim 1

SPRING 2013:

Experimental testing for Specific Aim 2 (radial device)

Protein Cell analysis (ELISA with VEGF and bFGF)

http://www.bmes.org/

Advice for Future Gemstone Teams!

TEAM DYNAMICS

4 Subgroups: Electrical Device, Cell Culture, Cell Migration, Cell Analysis

CURRENT/FUTURE CHALLENGES:

Communication between all teammates/subgroups

Even distribution of workload amongst team members

Coordination having resources prepared and experimental testing times

Keeping up with compiling data and analysis of results

MAKE FRIENDS AND HAVE FUN!!!

GEMSTONE TIPS FOR SUCCESS:

Pick a mentor who is involved in research related to your topic and has RESOURCES

Be flexible with your schedule because plans may change unexpectedly

Apply for grants!

Concluding remarks & THANKS

We would like to thank:

Dr. John P. Fisher, Team ELECTRODE Mentor

Dr. Mario Dagenais, Electrical Device Professor

Mr. Jim Miller, Librarian

Lab Graduate Students:

Kim Ferlin, Bao Nguyen, Martha Wang, and Tiecheng Zhu

Gemstone directors and staff: Dr. Frank Coale, Dr. Kristan Skendall, Mrs. Heather Creek, Dr. James Wallace, Dr. Rebecca Thomas

QUESTIONS?

Please visit our website at: http://teams.gemstone.umd.edu/classof2014/electrode/index.html