1. Frank Wolf Pittsburgh Central Catholic High School Grade 11 PJAS 2010

2.  What is TE? ◦ The development and manipulation of artificial implants, laboratory-grown tissues, and genetically engineered cells and/or molecules to replace or support the function of defective or injured body parts  Why is TE important? ◦ It has the potential to replace or supplement the function of tissues destroyed or compromised in any variety of ways, including:  Inherent design flaws  Hereditary/congenital defects or conditions  Disease  Trauma  Damage from an individual’s environment  Aging  TE has great potential for supplementing muscle tissue.

3. Cells ECM Hormones Blood Supply Defect Regeneration Phil Campbell, Carnegie Mellon

5.  Direct Physical Trauma ◦ Laceration – cutting/tearing of tissue ◦ Contusion - bruise ◦ Strain – “pulling a muscle”  Biological (Inherited Abnormalities) ◦ Muscular Dystrophies (DMD, BMD) ◦ Neurological Disorders (multiple sclerosis, Cerebral palsy) ◦ Storage Diseases (mucopolysaccharidoses, lipidoses)

7.  Male sex hormone produced in the sex organs and adrenal cortex of the adrenal gland  Androgen ◦ Any hormone that stimulates/maintains male characteristics in vertebrates  Steroid hormone ◦ Passes through cellular and nuclear membranes and affects transcription  Acts by binding to androgen receptor ◦ Androgen receptor – transcription factor  Stimulates myoblast formation (cell differentiation)  Particular testosterone molecule used: testosterone C-III ◦ Anabolic steroid  Drug meant to mimic the effects of bodily-produced testosterone  Increases skeletal muscle mass

8.  Subclone of the mus musculus (mouse) myoblast cell line.  Mouse stem cell line is frequently used as a model in tissue engineering experiments. 1.Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins. 2.Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells. 3.Expresses the androgen receptor (AR).  AR- DNA binding transcription factor which regulates gene expression.

9.  The purpose of this study is to observe the effects of the anabolic steroid testosterone C-III on the proliferation, differentiation, and survivorship of C2C12 stem cells.

10.  Alternative Hypothesis: The addition of testosterone C-III WILL affect the proliferation, differentiation, and survivorship of C2C12 stem cells.  Null Hypothesis: The addition of testosterone C-III WILL NOT affect the proliferation, differentiation, and survivorship of C2C12 stem cells.

11.  Cryotank  75mm 2 tissue culture treated flasks  Twenty 25 mm 2 tissue culture treated flasks  Fetal bovine serum (FBS)  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 Serum - 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])  Testosterone C-III (powder)  75 mL culture flask  Incubator  Nikon Inverted Microscope  Aspirating Vacuum Line  Laminar Flow Hood  Laminar Flow Hood UV Sterilizing Lamp  Labeling Tape  Hemocytometer  Sterile PBS  Ethanol (70% and 100%)  Distilled water

12.  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.  After trypsinization, cells from all of the flasks were pooled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL).  0.1 mL of the cell suspension was added to 20 25 mm 2 tissue culture treated flasks containing 5 mL of DMEM (com) media, creating a cell density of approximately 10 5 cells per flask.  The 1 M stock solution of testosterone C-III was created using 1 mL of ethanol and 0.28842 grams of testosterone C-III.  The 10 -6 M, 10 -7 M, and 10 -8 M concentrations were created from the stock, where 10 -6 M is the suggested working concentration of the steroid.  The 3 experimental groups and the control group were created by adding:  20 µl of the 10 -6 M solution to 5 flasks  20 µl of the 10 -7 M solution to 5 flasks  20 µl of the 10 -8 M solution to 5 flasks  20 µl of ethanol to 5 flasks (Control)  The cells were incubated at 37°C, 5% CO 2 for the remainder of the study.  Three flasks from each group were used in the Proliferation Experiment and two flasks from each group were used in the Differentiation Experiment.

14.  Day 1 ◦ Using one flask from each group, cell densities were determined as follows:  The cells were trypsinized and collected into cell suspension.  25 µl aliquots were transferred to a Hemocytometer for quantification (six counts per flask).  Day 1 and Day 6 ◦ Using the Nikon Inverted Microscope, images of eight representative areas of each flask were taken.  Day 1 and Day 6  Using the Nikon Inverted Microscope, images of eight representative areas of each of the flasks were taken.  Day 2  The original media was removed and replaced with 1% DMEM media (serum starvation) to induce myotube differentiation.

15. p-value: 1.78E-08

16.  ANOVA ◦ Compares variation within groups to variation between groups ◦ p-value: 1.78E-08 ◦ REJECT NULL ◦ Significant variation  Dunnett’s Test ◦ Compares each experimental group to control individually ◦ 0.05 t-critical value: 2.71 Groupt-valueVariation 10 -6 M9.82Significant 10 -7 M3.11Significant 10 -8 M1.19Insignificant

17. Day 1Day 6

18. Day 1Day 6

19. Day 1Day 6

20. Day 1Day 6

21. Control10 -6 M 10 -8 M10 -7 M Most significant differentiation (qualitative – most dramatic myotubule formation) Relatively comparable degrees of differentiation (qualitative)

22. Results Consistent with Prior Study  Proliferation Experiment ◦ From the ANOVA and the subsequent Dunnett’s tests, the addition of testosterone C-III induced a statistically significant increase in proliferation in the C2C12 cells when it is added at its suggested working concentration, 10 -6 M, and one tenth this concentration, 10 -7 M.  Differentiation Experiment ◦ From the qualitative analysis of the images gathered from the flasks, it appears that the addition of testosterone C-III induced myotubule formation. This was especially apparent in the 10 -6 M, while the 10 -7 M and 10 -8 M concentrations showed less dramatic differentiation in comparison.

23.  Differentiation Experiment ◦ Evaluation of images – qualitative and imprecise ◦ Solution – quantitative differentiation assay, e.g. MyoD tagging ◦ CyQUANT™ Cell Proliferation Assay  More quantitative than counting cells on a Hemocytometer  Fluorescent dye binds to nucleic acid in the cell  Evaluate concentrations of testosterone C-III greater than suggested dosage to determine if the steroid is hazardous in excess

24.  Dr. Phil Campbell  Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University  Mark Krotec, PTEI  C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression – German Sport University, Cologne, Germany  Chen Y, Zajac JD Maclean HE 2005 Androgen regulation of satellite cell function. Journal of Endocrinology 186 21-31.  Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng W and Bhasin S 2004 Androgen receptor in human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. Journal of Clinical Endocrinology and Metabolism 89 5245-5255.