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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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.

18. Proliferation results (continued) ANOVA
Day 1 p-value: 2.16E-07
Day 3 p-value:
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.

19. 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

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

21. Regeneration results (continued) day 3 Control Group

22. Regeneration results (continued) day 3 Low concentration of variable

23. Regeneration results (continued) day 3 high concentration of variable

24. Regeneration results (continued) day 5 control group

25. Regeneration results (continued) day 5 low concentration of variable

26. Regeneration results (continued) day 5 High concentration of variable

27. 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

28. 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

29. 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 <
"Keloids & Hypertrophic Scars." DermNet NZ. N.p., n.d. Web. 25
Jan <
"Keloid Scars." Schweiger Dermatology. N.p., n.d. Web. 25 Jan.
2013. <
Knapp, Daniels, and Kaplan. "Pathologic Scar
Formation." National Center for Biotechnology
Information. U.S. National Library of Medicine, Jan
Web. 25 Jan <
"Scar Tissue Formation." Breastcancer.org. Breastcancer.org, 17
Sept Web. 25 Jan