1. Team ELECTRODE Mentor: 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

2. Diabetes  Prevalence of Diabetes  In 2010, 1.9 million Americans over the age of 20 were diagnosed with diabetes  Formation of Diabetic Ulcers  Nerve damage, poor circulation, lack of tissue repair  Issues with Current Treatments  Requires constant application by the patient, doesn’t directly target the wound, can be very expensive http://www.executivehm.com/news/diabetes-in-the-us/

3. EFFECTS of VEGF & bFGF in wound healing  Body’s natural bioelectric healing system  Angiogenesis and Inflammation  Wound healing and electrical stimulation Epithelial Cells

4. 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.

5. We are examining the effects of… Cell proliferation, Cell migration, VEGF expression, bFGF expression 1) An optimized linearly applied electric field (in vitro) on… 2) An optimized radially applied electric field (in vitro) on… 3) an optimized (either radially or linearly) applied electric field (in vivo, using diabetic rat models) on…

6. In Vitro Experiments Cell 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

7. Device Design  Use best parameters determined from experimental trials  Compare effectiveness of a linear setup vs. a radial setup  Cheap and small prototype for a more marketable device Electrical Circuit Battery Flow +

8. 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.

9. In Vitro Experiments: Parameters  Test voltages:  0.01 V  0.1 V  1.0 V  50 Hz  Applied for one 30-minute period  6 wells: 3 control, 3 experimental Pulsed Monophasic Current Time Voltage

10. 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

11. 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

12. Cellular Proliferation Control Experimental

13. 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 Analysis 1. Live/Dead 2. mRNA Extraction 3. Gene Expression 4. ELISA (Protein) 5. VEGF (vascular endothelial growth factor) 6. BFGF (basic fibroblast growth factor)

14. 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

15. 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

16. Migration data, 10/9/12 Left side view, scraped line of cells at edge

17. 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/

18. 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!

19. 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

20. QUESTIONS?  Please visit our website at: http://teams.gemstone.um d.edu/classof2014/electrod e/index.html http://teams.gemstone.um d.edu/classof2014/electrod e/index.html