Advil Effects on 3T3 Cell Proliferation, Survivorship, and Regeneration By Sonali Dadoo School Pine-Richland High School Grade 11 Category Microbiology

Macrophages, Fibroblasts, Capillaries Wound healing: “Pathophysiology of Tissue Repair and Transfer” by Edoardo Austoni, Vincenzo Giannoccaro Platelets, PMN Proposed 5 Steps of Wound Healing 1) Platelets: accumulate at wound sight, activate thrombin 2) PMN: Wound healing, killing bacteria 3) Macrophages: Phagoctyize bacteria, promote angiogenesis 4) Fibroblasts: formation of collagen scaffold and bundles 5) Capillaries: angiogenesis, proper supply of blood to granulation tissue PMN: Polymorphonuclear Neutrophils Thrombin: clots blood by converting fibrinogen to fibrin Granulation tissue: new connective tissue and tiny blood vessels that form on the surfaces of a wound during the healing process Angiogenesis: formation of new blood vessels from old ones Lymphoctyes: not necessary – but would come after macrophages, function in immune response thus increasing host’s susceptibility to disease Fibroblasts: Myofibroblasts – another theory for fibroblast function in which fibroblasts differentiate into myofibroblasts Macrophages, Fibroblasts, Capillaries Photo Credit to: Clini Med http://www.clinimed.co.uk/

Wound healing overproduction of scar tissue What is a scar? Body’s natural healing mechanism in response to injury, trauma, etc. Tissue formed during healing process Scar Formation Complications Collagen fibers arranged randomly as opposed to linear, parallel formation Both of these pictures are a result of an overproduction of scar tissue. This means that during the collagen accumulation phase of Maturation during wound healing, too much collagen was produced because the collagen got stuck in collagen bundles Picture Left: Hypertrophic Scar Picture Right: Keloid Scar 2 Main differences: Hypertrophic Scars do not extend past the original wound, keloid scars extend past the original wound, Hypertrophic Scars tend to randomly flatten in between but keloids do not Photo Credit: wiseGEEK.com Photo Credit: Schweiger Dermatology

CAUSES Of scar formation complications Myofibroblast Theory Fibroblast differentiation into myofibroblasts Myofibroblasts contract with alpha-smooth muscle actin Contracts edges of wound to repair Lack of apoptosis leads to keloid and hypertrophic scar Fibronectin Theory Fibronectin structure precedes wound contraction Provides structure for fibroblasts Myofibroblasts can contract by using smooth muscle type actin-myosin complex, rich in a form of actin called alpha-smooth muscle actin. These cells are then capable of speeding wound repair by contracting the edges of the wound. Fibronectin degrades after Collagen III comes in and replaces Collagen I

Some current treatments Corticosteroid Injections Reduce collagen synthesis, inflammatory mediators, and fibroblast proliferation 5-Fluorouracil Pyrimidine analogue with antimetabolite activity Reduces fibroblastic proliferation in tissue culture and postoperative scarring Tamoxifen Nonsteroidal antiestrogen used to treat breast cancer Inhibit proliferation of keloid fibroblasts and their collagen synthesis Manipulation Physically bending joint to break scar tissue Not by any means a conclusive list Antimetabolite – reduces cell proliferation generally; generally used in chemotherapy for cancer Photo credit: Musculoskeletal Network

Variable Advil Reduces inflammatory hormones in body Generally given to patients in strong doses after surgery to reduce inflammation/trauma and restore joint mobility Average dose 200mg – 1200mg Photo credit: Thisthatbynat

3t3 cells Mouse derived fibroblastic cell line Standard line used to simulate human fibroblasts Cells proliferate extremely rapidly, but growth stops when cell-to-cell contacts are formed Widely used cell line in research Biologically immortalized cell line A biologically immortal living thing can still die from means other than senescence (old age) such as through injury or disease. Photo credit: National Institute of Health

purpose To examine the effects of Advil on 3T3 cell proliferation, survivorship, and regeneration Photo credit: Advil.com

Null Hypothesis Alternate Hypothesis hypotheses The addition of Advil (Ibuprofen) will not affect the proliferation, survivorship, and regeneration of 3T3 cells Alternate Hypothesis The addition of Advil (Ibuprofen) will significantly reduce the proliferation, survivorship, and regeneration of 3T3 cells

materials DMEM 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]) Cryotank One 75mm2 tissue culture treated flasks Hemocytometer Permanent marker Six 25 mm2 tissue culture treated flasks 24 well plate Incubator Test tube rack Aspirating Vacuum Line Sterile Filter 10% fetal bovine serum Nikon Inverted Compound Optical Scope Lint wipes 3T3 Cell Line Advil Capsules Trypsin-EDTA Laminar Flow Hood Pen/strep Laminar Flow Hood UV Sterilizing Lamp Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL) Labeling Tape Sterile PBS Micropipettes + sterile tips Ethanol (70% and 100%) Distilled water Nikon Inverted Microscope Photo credit: New York Microscope Company

Procedure: Preparing variable stock Liquified five 200mg capsules of Advil – about 1 gram total X represents the average dose of Advil taken in a given day 1 𝑔𝑟𝑎𝑚 10 𝐿 𝑜𝑓 𝑏𝑜𝑑𝑦 𝑓𝑙𝑢𝑖𝑑 = X 100X stock solution of Advil was made and purified Photo credit: Advil.net

Procedure: preparing the cells A 1 mL sample of 3T3 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask. The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO2) for 2 days until a cell density of approximately 106 to 2x106 cells/mL was reached. The culture was passed into 6 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2. The cells were thawed and grown in 10% DMEM Serum. The media was then replaced to remove the cryo-freezing fluid and the culture was then passed into six flasks in preparation for experimentation. Questions: What is cryo-freezing fluid? Liquid nitrogen that freezes and preserves cells Why 37 degrees C? and Why 5% CO2? Standard operating procedure, used to high concentrations of CO2 cause of metabolism, adapted to high levels How did you know when density was reached? Why that density? Not too little and not too much C2C12s are from C3H mice DMEM Stands for Dulbecco's Modified Eagle's Medium (modified version of EMEM Eagle’s Minimal Essential Medium (Eagle as in Henry Eagle, the discoverer) Photo credit: BioResource

Procedure: proliferation Seeded 15 flasks with 3T3 cells from the original flasks in 5mL of 10% DMEM media each Allowed cells to incubate in CO2 incubator overnight and adhere to bottom of flask Allowed flasks to proliferate in incubator overnight Control group: 5 flasks Low concentration of variable group: 5 flasks Removed 5μL media Added 5μL variable High concentration of variable group: 5 flasks Removed 50μL media Added 50μL variable

Procedure: proliferation (continued) Next day, trypsinized cells and performed cell counts on the cell suspension Rinsed with 1mL of trypsin, pipetted out Added 1mL trypsin, incubate for 5 minutes Slap flask and confirm with microscope that cells are no longer adhered to bottom of flask Quenched reaction with 5mL media. Re-added variable. Re-suspended cells with 1mL trypsin wash before taking counts using pipette Loaded hemocytometer with 25uL from flask Took 8 counts per flask Counted cells in field of view of hemocytometer under Nikon inverted microscope and multiplied count by 103 for total cells/mL Repeated on Day 3

Procedure: regeneration Seeded 15 wells: 1mL of 3T3 cell suspension in 10% DMEM media Allowed cells to proliferate until reached 80% confluence Made scratch in all plates Well Scratch Kept 5 control wells Took images of plates with Nikon Inverted Microscope Day 1, Day 3 and Day 5 of scratch Compared regeneration over scratch on each well Why 80% confluence? Standard measurement, 80% well covered, but still room for growth Why do we use Pen/Strep? To keep bacteria from forming 10% - 10% FBS, 89%DMEM, and 1% Pen/Strep

Procedure: regeneration (continued) Fixed and stained each plate after imaging Removed media 1mL PBS Wash 1mL Ethanol Wash for 2 minutes Added 0.5mL of Toluidine Blue Stain Removed Stain Compared regeneration over scratch on each well and re-imaged on Day 1, Day 3, and Day 5 Toluidine Blue Photo credit: Shutterstock.com

Proliferation results This graph shows the results from the cell counts from the proliferation assay. The title of the graph is….The y axis shows….The x axis shows…The dark bars represent….and the light bars represent….Each bar in the graph represents an average of 8 replicates. From the graph it appears that proliferation increased from Day 1 to Day 3 for the control and low groups, for the high, however, it seemed to decrease. For Day 1, it appears that the low concentration increased proliferation when compared to the control and high concentration decreased proliferation greatly when compared to the control. Similarly for Day 3, it appears that there was a slight increase in the low concentration and a slight decrease in the high concentration when compared to the control.

Proliferation results (continued) ANOVA Day 1 p-value: 2.16E-07 Day 3 p-value: 0.000259 MOVE P VALUES Statistical analyses were performed to see if these effects were significant. An analysis of variance, known as an ANOVA test, was performed. The resulting P-Values were taken from days 1 and 3. Day 1 compares the control low and high concentrations for Day 1. The Day 3 p-value compares the control, low, and high concentrations for Day 3. All of the p-values were lower than 0.05, so this means that at least two of the means varied significantly when compared to the control.

Proliferation results (continued) dunnett’s test T-Value Difference of Average of Experimental Group and Average of Control Group Square Root of two times the MS Value divide by the number of replicates So, a further pairwise test, called the Dunnetts Test, was performed to see what concentration might have produced significant variation when compared to the control. This formula is used to determine the T-Value. If the T-value is greater than the T-Critical value, that means the means varied significantly. For day 1, the low concentration varied significantly when compared to the control, but the high concentration did not vary significantly when compared to the control. For day 3, the low concentration did not vary significantly with the control either, however, the high concentration varied significantly when compared to the control. These results suggest that a low dose of Advil may significantly increase proliferation, while a high dose of Advil may significantly reduce proliferation. T-critical value based on number of groups and replicates, so I had 3 groups for proliferation and 5 replicates of each Significant effects include ones that could not have occurred by chance Concentration T-Value T-Critical Significance Low – 0.1X 6.63 2.42 Yes High - X 1.64 No Day 1 Concentration T-Value T-Critical Significance Low – 0.1X 2.12 2.42 No High - X 2.54 Yes Day 3

Regeneration results Day 1 Low Concentration Group Control Group High Concentration Group

Regeneration results (continued) day 3 Control Group

Regeneration results (continued) day 3 Low concentration of variable

Regeneration results (continued) day 3 high concentration of variable

Regeneration results (continued) day 5 control group

Regeneration results (continued) day 5 low concentration of variable

Regeneration results (continued) day 5 High concentration of variable

conclusion Proliferation Regeneration Advil significantly reduced 3T3 cell growth by Day 3 of the High Concentration and significantly increased proliferation for the Low Concentration on Day 1. The High Concentration on Day 1 and the Low Concentration on Day 3 both appeared to have no significant effect when compared to the control. Regeneration Advil appeared to have little effect on 3T3 cell regeneration based on qualitative analysis of images

Limitations and extensions Two concentrations of variable used Two days of cell counting Cell counts have small, inherent errors Regeneration analysis was qualitative Extensions Wider range of variable concentrations Earlier exposure to test effects on cell attachment Count cells using time lapse imaging Explore synergistic effects of Advil with other drugs Synergistic: used especially of drugs or muscles that work together so the total effect is greater than the sum of the two (or more) TIME LAPSE IMAGING – COUNT CELLS

Acknowledgments and references Mr. Mark Krotec Pittsburgh Tissue Engineering Initiative Conrad M. Zapanta, Ph.D. Biomedical Engineering Laboratory, Carnegie Mellon University "Hypertrophic Scarring and Keloids." Medscape. N.p., 2013. Web. 25 Jan. 2013. <http://emedicine.medscape.com/>. "Keloids & Hypertrophic Scars." DermNet NZ. N.p., n.d. Web. 25 Jan. 2013. <http://www.dermnetnz.org/>. "Keloid Scars." Schweiger Dermatology. N.p., n.d. Web. 25 Jan. 2013. <http://www.nyccosmeticdermatology.com/>. Knapp, Daniels, and Kaplan. "Pathologic Scar Formation." National Center for Biotechnology Information. U.S. National Library of Medicine, Jan. 1977. Web. 25 Jan. 2013. <http://www.ncbi.nlm.nih.gov/>. "Scar Tissue Formation." Breastcancer.org. Breastcancer.org, 17 Sept. 2012. Web. 25 Jan. 2013. http://www.breastcancer.org/>.