BySonali Dadoo

SchoolPine-Richland High School

Grade11

CategoryMicrobiology

ADVIL EFFECTS ON 3T3 CELL

PROLIFERATION, SURVIVORSHIP,

AND REGENERATION

WOUND HEALING:“PATHOPHYSIOLOGY OF TISSUE REPAIR AND TRANSFER” BY

EDOARDO AUSTONI, V INCENZO GIANNOCCARO

Photo Credit to: Clini Medhttp://www.clinimed.co.uk/

Platelets, PMN

Macrophages, Fibroblasts, Capillaries

What is a scar?Body’s natural healing mechanism in

response to injury, trauma, etc.Tissue formed during healing process

Scar Formation ComplicationsCollagen fibers arranged randomly as opposed

to linear, parallel formation

WOUND HEALING OVERPRODUCTION OF SCAR TISSUE

Photo Credit: wiseGEEK.com

Photo Credit: Schweiger Dermatology

Myofibroblast TheoryFibroblast differentiation into myofibroblastsMyofibroblasts contract with alpha-smooth

muscle actinContracts edges of wound to repairLack of apoptosis leads to keloid and

hypertrophic scar

Fibronectin TheoryFibronectin structure precedes wound

contractionProvides structure for fibroblasts

CAUSES OF SCAR FORMATION COMPLICATIONS

Corticosteroid Injections Reduce collagen synthesis,

infl ammatory mediators, and fi broblast proliferation

5-Fluorouracil Pyrimidine analogue with

antimetabolite activity Reduces fi broblastic proliferation in

tissue culture and postoperative scarring

Tamoxifen Nonsteroidal antiestrogen used to treat

breast cancer Inhibit proliferation of keloid

fi broblasts and their collagen synthesis

Manipulation Physically bending joint to break scar

tissue

SOME CURRENT TREATMENTS

Photo credit: Musculoskeletal Network

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

VARIABLEADVIL

Photo credit: Thisthatbynat

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

3T3 CELLS

Photo credit: National Institute of Health

To examine the effects of Advil on 3T3 cell proliferation, survivorship, and regeneration

PURPOSE

Photo credit: Advil.com

Null HypothesisThe addition of Advil (Ibuprofen) will not affect the proliferation, survivorship, and regeneration of 3T3 cells

Alternate HypothesisThe addition of Advil (Ibuprofen) will significantly reduce the proliferation, survivorship, and regeneration of 3T3 cells

HYPOTHESES

Cryotank One 75mm2 tissue

culture treated fl asks

Six 25 mm2 tissue culture treated fl asks

10% fetal bovine serum

3T3 Cell Line Trypsin-EDTA Pen/strep Macropipette +

sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)

Micropipettes + sterile tips

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])

Incubator Aspirating Vacuum

Line Nikon Inverted

Compound Optical Scope

Laminar Flow Hood Laminar Flow Hood

UV Sterilizing Lamp Labeling Tape Sterile PBS Ethanol (70% and

100%)

Distilled water Nikon Inverted

Microscope Aspirating Vacuum

Line Hemocytometer Permanent marker 24 well plate Test tube rack Sterile Filter Lint wipes Advil Capsules

MATERIALS

Photo credit: New York Microscope Company

Liquified five 200mg capsules of Advil – about 1 gram total

X represents the average dose of Advil taken in a given day

= X100X stock solution of Advil was made and

purified

PROCEDURE: PREPARING VARIABLE STOCK

Photo credit: Advil.net

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 fl asks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2.

PROCEDURE: PREPARING THE CELLS

Photo credit: BioResource

Seeded 15 fl asks 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 fl asks to proliferate in incubator overnight

PROCEDURE: PROLIFERATION

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

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 pipetteLoaded hemocytometer with 25uL from fl askTook 8 counts per fl askCounted cells in fi eld of view of hemocytometer under

Nikon inverted microscope and multiplied count by 10 3 for total cells/mL

Repeated on Day 3

PROCEDURE: PROLIFERATION (CONTINUED)

Seeded 15 wells: 1mL of 3T3 cell suspension in 10% DMEM media Allowed cells to proliferate until reached 80%

confluenceMade scratch in all plates

WellScratch

Kept 5 control wellsTook images of plates with Nikon Inverted

Microscope Day 1, Day 3 and Day 5 of scratchCompared regeneration over scratch on each well

PROCEDURE: REGENERATION

Fixed and stained each plate after imagingRemoved media1mL PBS Wash1mL Ethanol Wash for 2 minutesAdded 0.5mL of Toluidine Blue StainRemoved Stain1mL PBS Wash

Compared regeneration over scratch on each well and re-imaged on Day 1, Day 3, and Day 5

PROCEDURE: REGENERATION (CONTINUED)

Photo credit: Shutterstock.com

PROLIFERATION RESULTS

Control Low High0

5

10

15

20

25

30

Proliferation Cell Counts

Day 1Day 3

Concentration of VariableCell C

ou

nts

(in

th

ou

san

ds)

Day 1 p-value: 2.16E-07

Day 3 p-value: 0.000259

PROLIFERATION RESULTS (CONTINUED)

ANOVA

Con-trol

Low High05

1015202530

Proliferation Cell Counts

Day 1Day 3

Concentration of VariableC

ell C

ou

nts

(in

th

ou

san

ds)

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

Concentration

T-Value T-Critical Significance

Low – 0.1X 6.63 2.42 Yes

High - X 1.64 2.42 NoConcentration

T-Value T-Critical Significance

Low – 0.1X 2.12 2.42 No

High - X 2.54 2.42 Yes

Day 1

Day 3

REGENERATION RESULTSDAY 1

Control Group

Low Concentration 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

ProliferationAdvil 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.

RegenerationAdvil appeared to have little eff ect on 3T3

cell regeneration based on qualitative analysis of images

CONCLUSION

LimitationsTwo concentrations of variable usedTwo days of cell counting Cell counts have small, inherent errorsRegeneration analysis was qualitative

ExtensionsWider range of variable concentrationsEarlier exposure to test eff ects on cell attachmentCount cells using time lapse imagingExplore synergistic eff ects of Advil with other

drugs

LIMITATIONS AND EXTENSIONS

Mr. Mark KrotecPittsburgh Tissue Engineering InitiativeConrad 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. 25Jan. 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, 17Sept. 2012. Web. 25 Jan. 2013.

http://www.breastcancer.org/>.

ACKNOWLEDGMENTS AND REFERENCES