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

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

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.

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

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

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?

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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

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

Acknowledgements and Sources Dr. Phil Campbell, Ph.D., Carnegie Mellon University Mr. Mark Krotec, Sponsor https://www.ncbi.nlm.nih.gov/gene/650 (BMP-2) https://www.drugs.com/estradiol.html http://www.nih3t3.com/

Anova: Two-Factor With Replication SUMMARY Low High Total   Count 8 24 Sum 276 504 539 1319 Average 34.5 63 67.375 54.95833333 Variance 98.85714286 130 163.4107143 341.0851449 684 405 250 1339 85.5 50.625 31.25 55.79166667 158.5714286 47.125 83.92857143 613.9112319 2.679 0.54 486 499 752 1737 60.75 62.375 94 72.375 89.64285714 465.6964286 66.57142857 433.7228261 4.632 9.887 1446 1408 1541 60.25 58.66666667 64.20833333 558.1086957 229.4492754 785.5634058 ANOVA Source of Variation SS df MS F P-value F crit Sample 4632.333333 2 2316.166667 15.98822128 2.42285E-06 3.142808517 Columns 391.0833333 195.5416667 1.34980072 0.266686576 Interaction 22422.83333 4 5605.708333 38.69553367 2.51486E-16 2.517670458 Within 9126.625 144.8670635 36572.875 71

Anova: Two-Factor With Replication SUMMARY Low High Total   Count 8 24 Sum 1592 1535 1580 4707 Average 199 191.875 197.5 196.125 Variance 722 396.6964286 230 420.2880435 879 1056 1140 3075 109.875 132 142.5 128.125 479.2678571 157.7142857 757.1428571 617.2445652 161 416 957 1534 20.125 52 119.625 63.91666667 32.98214286 85.42857143 456.8392857 1970.949275 2632 3007 3677 109.6666667 125.2916667 153.2083333 5940.231884 3620.824275 1553.998188 ANOVA Source of Variation SS df MS F P-value F crit Sample 209806.0278 2 104903.0139 284.5409285 2.85015E-32 3.142808517 Columns 23354.86111 11677.43056 31.67408456 3.0186E-10 Interaction 22613.72222 4 5653.430556 15.33447248 8.27444E-09 2.517670458 Within 23226.5 63 368.6746032 279001.1111 71

Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 276 34.5 98.85714286 Low (1%) 684 85.5 158.5714286 9.483 High (10%) 486 60.75 89.64285714 4.881 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 10407 2 5203.5 44.9777732 2.56589E-08 3.466800112 Within Groups 2429.5 21 115.6904762 Total 12836.5 23  

Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 8 1592 199 722 Column 2 879 109.875 479.2678571 8.788 Column 3 161 20.125 32.98214286 17.638 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 127985.5833 2 63992.79167 155.542536 2.57149E-13 3.466800112 Within Groups 8639.75 21 411.4166667 Total 136625.3333 23  

Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 276 34.5 98.85714286 Low (1%) 504 63 130 4.985 High (10%) 539 67.375 163.4107143 5.75 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 5099.083333 2 2549.541667 19.49847499 1.63285E-05 3.466800112 Within Groups 2745.875 21 130.7559524 Total 7844.958333 23  

Anova: Single Factor SUMMARY Groups Count Sum Average Variance Column 1 8 1592 199 722 Column 2 1535 191.875 396.6964286 Column 3 1580 197.5 230 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 225.75 2 112.875 0.251075774 0.780265583 3.466800112 Within Groups 9440.875 21 449.5654762 Total 9666.625 23