1. Effects of BMP-2 and Estradiol on 3T3 Fibroblast Cell Line
Joe Ebbert
Central Catholic HS
5th Year in PJAS

2. Fibroblasts
A type of cell that synthesizes the extracellular matrix and collagen, the structural framework for animal tissues
Plays a critical role in wound healing
Most common cells of connective tissue in animals
Tissue damage stimulates fibrocytes and induces the mitosis of fibroblasts
The suffix "-blast" is used in cellular biology to denote a stem cell or a cell in an activated state of metabolism

3. 3T3 Cells Cell line established from Swiss mouse embryo tissue
Standard fibroblast cell line
Do not differentiate, produce ECM parts and structural proteins
Often used in the cultivation of keratinocytes, with the 3T3 cells secreting growth factors favorable to these kinds of cells.

4. Bone Morphogenetic Protein 2
Plays an important role in the development of bone and cartilage
Involved in the hedgehog pathway, TGF beta signaling pathway, and in cytokine- cytokine receptor interaction
Used clinically to stimulate the production of bone
Negative regulation of cell proliferation

5. Estradiol A steroid, an estrogen, and the primary female sex hormone
Important in the regulation of the menstrual female reproductive cycle
Essential for the development and maintenance of female reproductive organs
Used as a medication in hormone replacement therapy
Induce proliferation of cells

6. Research Problems
How does the interaction of BMP-2 and estradiol affect the proliferation of the cells?
What does this tell us about BMP-2 levels and how they may affect cell proliferation during clinical use of estradiol?
Does one variable offset the other in regards to negative or positive regulation of cell proliferation?

7. Purpose To determine the effects of BMP-2 on 3T3 cell proliferation
To determine the effects of estradiol on 3T3 cell proliferation
To determine if synergy exists between BMP-2 and estradiol in their effects on 3T3 cell proliferation

8. Hypotheses Null hypotheses: Alternative hypotheses:
BMP-2 will not have a significant effect on the proliferation of 3T3 cells
Estradiol will not have a significant effect on the proliferation of 3T3 cells
Synergistic effects on 3T3 cells will not exist between BMP-2 and estradiol
Alternative hypotheses:
BMP-2 will have a significant effect on the proliferation of 3T3 cells
Estradiol will have a significant effect on the proliferation of 3T3 cells
Synergistic effects on 3T3 cells will exist between BMP-2 and estradiol

9. Materials Cryotank 75mm2 tissue culture treated flasks
Fetal bovine serum (FBS)
3T3 Fibroblastic Cell Line
Trypsin
Macropipettes + sterile macropipette tips (2 mL, 5 mL, 10 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])
75 mL culture flask
BMP-2
Estradiol
Incubator
Nikon Inverted Microscope with imaging technology
Laminar Flow Hood
Laminar Flow Hood UV Sterilizing Lamp
Sharpie pen
Hemocytometers
Sterile PBS
Ethanol (70%)
Sterile Water
Purple Nitrile gloves

10. Procedure (cell culture)
A 1 mL aliquot of 3T3 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm2 culture flask yielding a cell density of approximately 106 to 2x106 cells
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 4x106 to 5x106 cells/mL was reached
The culture was passed into flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO2

11. Procedure (day 0: variable addition)
Two T75 flasks of cell suspension were pooled into one flask to be used to distribute to T25 flasks
4 mL of DMEM media were added to each of 36 T25 flasks (18 for day 1 counts, 18 for day 2), and 1 mL of cell suspension was added to each of 36 T25 flasks
Flasks were placed back into incubator and cells were allowed to attach for several hours
Variables (BMP-2 and estradiol) were added to flasks at varying concentrations in 50 µL with a micropipettes, demonstrated by the following chart

12. Concentrations Chart Zero (0 M) Low (2×10-6 M) High (2×10-5 M)
Estradiol⇒
BMP-2⇓
Zero (0 M)
Low (2×10-6 M)
High (2×10-5 M)
Zero BMP-2, Zero E2
Low BMP-2, Zero E2
High BMP-2, Zero E2
Zero BMP-2, Low E2
Low BMP-2, Low E2
High BMP-2, Low E2
Zero BMP-2, High E2
Low BMP-2, High E2
High BMP-2, High E2

13. Procedure (cell counts)
Days 1 and 2
Cell densities were determined as follows:
The cells were trypsinized and collected into cell suspension.
10 µl aliquots were transferred to a Hemocytometer for quantification (eight total counts).
Total count multiplied by 5,000 to determine the total number of cells per flask

14. Effects of BMP-2 on 3T3 Day 1 p-value: 2.566×10-8
Focus on day 2 results

15. Effects of Estradiol on 3T3
Day 1 p-value: 1.633×10-5
Focus on day 2 results

16. Effects of BMP-2 and Estradiol on 3T3
Day 1 p-value: 2.515×10-16
Day 2 p-value: 8.274×10-9

17. Dunnett’s Test Results
t-crit=2.92
BMP
T-value
Significance
Day 1
Low
9.483
Significant
High
4.881
Day 2
8.788
17.638
Estradiol
T-value
Significance
Day 1
Low
4.985
Significant
High
5.75
Day 2 --
Not Significant

18. P-Values Overview
BMP-2 (Day 1)
Single Factor
BMP-2 (Day 2)
Estradiol (Day 1)
Estradiol (Day 2)
Total (Day 1)
Two Factor
Total (Day 2)
2.566×10-8
2.572×10-13
1.633×10-5
0.780
2.515×10-16
8.274×10-9
Significant
Not Significant
Hypotheses regarding BMP-2 and synergy should be rejected, hypothesis regarding estradiol should be accepted

19. Conclusions BMP-2 significantly affected the proliferation of 3T3
Estradiol significantly affected the proliferation of 3T3 on day 1, but did not significantly affect the proliferation of 3T3 after day 2
Total p-values taken from two-factor ANOVA, which tests for synergy between the two variables used
P-values extremely low, suggesting negative synergy between the variables
High level of BMP-2, no E2 varied most from the control
Low level of E2, no BMP-2 varied least from the control

20. Conclusions on Synergy
When any level of BMP-2 was added, raising the level of estradiol decreased the variability from the control.
Therefore, estradiol relieves the negative regulation of proliferation caused by the bone morphogenetic protein 2.
There was a negative level of synergy produced because the cells proliferated less when the variables were at the same level together than they did when the variables were alone.

21. Applications
In clinical use of the bone morphogenetic protein 2, production of fibroblast cells would increase if higher levels of the estradiol hormone exist.
In clinical use of estradiol, lower levels of BMP-2 would induce greater proliferation of fibroblasts.
In reparation of injuries, fibroblast cells are vital. If high levels of BMP- 2 exist in that scenario, the fibroblasts would not grow adequately and would consequently stunt the procedure of healing.
Conversely, estradiol levels would not significantly alter the rate of fibroblast proliferation and would therefore not stunt the procedure of healing.

22. Limitations Hemocytometer counts may vary Clumping of cells
Number of replicates
Lag time in cell transferring
Discrepancies in cell counting
Length of experimentation time

23. Extensions Greater number of replicates
Greater number of variable concentrations
Longer experimentation time
Use different cell lines (C2C12 stem cells, MG63 cancer cells)
Test synergistic effects of different growth factors, hormones
Extracellular Matrix Assay

24. Acknowledgements and Sources
Dr. Phil Campbell, Ph.D., Carnegie Mellon University
Mr. Mark Krotec, Sponsor
(BMP-2)

25. Anova: Two-Factor With Replication
SUMMARY
Low
High
Total
Count 8 24
Sum
276
504
539
1319
Average
34.5 63
67.375
Variance
130
684
405
250
1339
85.5
50.625
31.25
47.125
2.679
0.54
486
499
752
1737
60.75
62.375 94
72.375
4.632
9.887
1446
1408
1541
60.25
ANOVA
Source of Variation SS df MS F
P-value
F crit
Sample 2
E-06
Columns
Interaction 4
E-16
Within 71

26. Anova: Two-Factor With Replication
SUMMARY
Low
High
Total
Count 8 24
Sum
1592
1535
1580
4707
Average
199
197.5
Variance
722
230
879
1056
1140
3075
132
142.5
161
416
957
1534
20.125 52
2632
3007
3677
ANOVA
Source of Variation SS df MS F
P-value
F crit
Sample 2
E-32
Columns
3.0186E-10
Interaction 4
E-09
Within 63 71

27. Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 8
276
34.5
Low (1%)
684
85.5
9.483
High (10%)
486
60.75
4.881
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
10407 2
5203.5
E-08
Within Groups
2429.5 21
Total 23

28. Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1 8
1592
199
722
Column 2
879
8.788
Column 3
161
20.125
17.638
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 2
E-13
Within Groups 21
Total 23

29. Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 8
276
34.5
Low (1%)
504 63
130
4.985
High (10%)
539
67.375
5.75
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 2
E-05
Within Groups 21
Total 23

30. Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1 8
1592
199
722
Column 2
1535
Column 3
1580
197.5
230
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
225.75 2
Within Groups 21
Total 23