1. Creatine Effects on Oxidative- Stressed Stem Cells Anthony DeRenzo Pittsburgh Central Catholic HS Grade 11 February 6, 2010

2. What is Tissue Engineering? Broadly Defined: Tissue Engineering is the development and manipulation of artificial implants, laboratory-grown tissues, genetically engineered cells and/or molecules to replace or support the function of defective or injured parts of the body.

3. Cells Principles of Tissue Engineering ECM Hormones Blood Supply DefectRegeneration Phil Campbell, Carnegie Mellon

4. Overview of Skeletal Muscle Structure *Muscle *Muscle Fiber Bundles (fascicles) and the ECM *Muscle Fibers (multinucleated structures) *Basal Lamina Membrane *Satellite Cells (mixed population) *Sarcolemma Membrane *Sarcoglycan Complex *Dystrophin *Actin-Myosin Complex

5. C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line. Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. Mouse stem cell line is used as a model in many tissue engineering experiments. Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells. Expresses muscle proteins and the androgen receptor (AR). AR- DNA binding transcription factor which regulates gene expression.

6. Creatine An organic acid naturally synthesized from amino acids (methionine, glycine, arginine) primarily in the kidney and liver, then transported in the blood for use by muscles. Approximately 95% located in the skeletal muscle. Direct relation to ATP production and storage.

7. Supplemental Creatine Most popular bodybuilding supplement on the market. Has such an impact because it super-hydrates muscle cells with water. Enhances muscle growth, and strengthens fibers. Increased energy levels, strength, and recovery rates. Accelerates weight loss and builds lean body mass.

8. Oxidative Stress and Hydrogen Peroxide Caused by an imbalance between the reproduction of reactive oxygen and the ability to repair toxic damage. All life forms maintain reducing environment within their cells. If disturbed, peroxides and free radicals are produced. These cause harm to cell components (protein, lipids, DNA). Apoptosis Hydrogen peroxide stress Two electron reduction state Formed by dismutation of O 2 - or direct reduction of O 2 Lipid soluble Can be caused through overproduction of oxygen during physical exercise

9. Purpose To examine the effects of Creatine Monohydrate on the proliferation, differentiation, and survivorship of normal and H 2 O 2 stressed C2C12 cells.

10. Hypotheses Nulls: 1. Creatine Monohydrate will not have an effect on the proliferation, differentiation, and survivorship of normal and H 2 O 2 stressed C2C12 cells. 2. There will be no evidence of synergy between Creatine Monohydrate and peroxide stress. Alternates: 1. Creatine Monohydrate will have an effect on the proliferation, differentiation, and survivorship of normal and H 2 O 2 stressed C2C12 cells. 2. There will be significant evidence of synergy between Creatine Monohydrate and peroxide stress.

11. Materials Cryotank Three 75mm 2 tissue culture treated flasks Twenty-four 25 mm 2 tissue culture treated flasks 10% fetal bovine serum C2C12 Myoblastic Stem Cell Line Trypsin-EDTA Pen/strep Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL) Micropipettes + sterile tips DMEM media -1% and Complete Media (4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete]) 75 mL culture flask Incubator Zeis Inverted Compound Optical Scope Aspirating Vacuum Line Laminar Flow Hood Laminar Flow Hood UV Sterilizing Lamp Labeling Tape Creatine Monohydrate Hydrogen Peroxide Hemocytometer Sterile PBS Ethanol (70% and 100%) Distilled water

12. Procedure (Stem Cell Line Preparation) A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached. The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

13. Procedure (Proliferation) After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL). 5 ml of the cell suspension was added to 24 25 mm 2 tissue culture treated flasks, creating a cell density of approximately 10 5 cells per flask. 1 mM stock concentration of hydrogen peroxide created by adding 0.1 ml of peroxide to 8.7 ml of sterile water. The following concentrations of variable (next page) were added to the flasks. 4 flasks for each group (2 for proliferation, 2 for differentiation) The cells were incubated (37°C, 5% CO 2 ) for the remainder of the study.

14. Experimental Groups 0% Peroxide10 uM Peroxide 0% Creatine 0.6 ml sterile water 4.4 ml cell culture and medium 0.05 ml peroxide 0.55 ml sterile water 4.4 ml cell culture and medium.01% Creatine 0.05 ml Creatine 0.55 ml sterile water 4.4 ml cell culture and medium 0.05 ml peroxide 0.05 ml Creatine 0.5 ml sterile water 4.4 ml cell culture and medium 1% Creatine 0.5 ml Creatine 0.1 ml sterile water 4.4 ml cell culture and medium 0.05 ml peroxide 0.5 ml Creatine 0.05 ml sterile water 4.4 ml cell culture and medium

15. Procedure (Differentiation) The differentiation experiment was identical to the proliferation experiment with the following exceptions: On Day 3 of experimentation, the original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation. On Days 1, 3, and 6 pictures of two areas of each flask were taken with a Nikon Inverted Microscope. These pictures are a visual representation of the proliferation and differentiation of the cells. Imaging

16. Proliferation Cell Count (10 4 cells/flask)

17. P Values (Single /Double Factor) Stressed vs. Non Stressed 0.01% Creatine Stressed vs. Non Stressed 0.1% Creatine 2.23 E-07 (SF) Significant 0.013721 (SF) Significant 0.06457 (DF) Insignificant 0.00275 (S) Significant 0.058231 (DF) Insignificant Conclusions: 1.The first null hypothesis can be rejected in every case. 2.The second null hypothesis can be accepted in both cases.

18. Unstressed Stressed Proliferation Unstressed vs. Stressed 0% Creatine Day 1 Day 3

19. Proliferation Unstressed vs. Stressed 0.01% Creatine Unstressed Stressed Day 1 Day 3

20. Proliferation Unstressed vs. Stressed 0.1% Creatine Unstressed Stressed Day 1 Day 3

21. Differentiation Unstressed vs. Stressed 0% Creatine Day 6 Unstressed Stressed

22. Differentiation Unstressed vs. Stressed 0.01% Creatine Day 6 Unstressed Stressed

23. Differentiation Unstressed vs. Stressed 0.1% Creatine Day 6 Unstressed Stressed

24. Qualitative Analysis (Differentiation) Unstressed vs. Stressed 0% Creatine Appearance - Significant Evidence of myotube formation in unstressed test group. Unstressed vs. Stressed 0.01% Creatine Appearance - Significant Evidence of myotube formation in unstressed test group. Unstressed vs. Stressed 0.1% Creatine Appearance - Significant Evidence of myotube formation in unstressed test group. In all examples, creatine was not able to remediate the oxidative stress. Therefore, there were no synergistic effects.

25. Limitations and Extensions Only used qualitative assay of differentiation / Utilize quantitative assay (MyoD expression) Test more variations of concentrations Use other types of stress (UV, heat, infection, various chemicals) CyQUANT™ Cell Proliferation Assay More quantitative than counting cells on a Hemocytometer Fluorescent dye binds to nucleic acid in the cell

26. References John Wilson, Biostatistician for the University of Pittsburgh Conrad M. Zapanta, Ph.D BiomedicalEngineering Laboratory, Carnegie Mellon University Mark Krotec, PTEI http://www.netdoctor.co.uk/focus/nutrition/facts/oxidat ive_stress/oxidativestress.htm http://cropsoil.psu.edu/courses/AGRO518/Oxygen.htm http://www.creatinejournal.com/ http://www.bodybuilding.com/store/creatine.html