1. Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs
Jacqueline Mimnaugh, RET
Neuqua Valley High School
Dr. Richard Gemeinhart
Melanie Kollmer
UIC Department of Biopharmaceutical Sciences
Tracy Chuong, REU
University of California Berkley

2. hMSCs and Tissue Engineering
Human Mesenchymal Stem Cells (hMSCs)
Can differentiate into bone, cartilage, fat, and other cells
Osteogenesis → Induce hMSCs to develop into bone cells
Tissue Engineering ↓
Bone diseases/defects, trauma, cancer, mal-union/non-union fractures

3. The Problem 2 D 3 D Cells in vivo (in an organism)
Cells in the lab are typically cultured on plates
2 D
Cells in vivo (in an organism)
3 D
It is possible that cells grown in a 3D scaffold would:
Be more like cells in vivo
Would not reach confluence as quickly
Could be implanted directly

4. Research Project
How will a 2D and 3D environment effect the osteogenic differentiation of hMSCs?
Compare cells grown in a 2D and a 3D environment:
Viability?
Proteins?
Genes expressed?
Mineralization?

5. Creating a 3d Scaffold Poly (ethylene glycol) diacrylate or PEGDA
Superporous Hydrogels
Poly (ethylene glycol) diacrylate or PEGDA
Polymer network, hydrophilic
Pores from 100 µm to 600 µm created by gel-foaming

6. Project Overview 2. Add osteogenic differentiation medium
1. Seed hMSCs onto 2D plates and 3D hydrogels
After 24 hours
2. Add osteogenic differentiation medium
After 24 hours
3. Compare cells at day 2, 7, 14, 21 and 28

7. Comparing the cells
1. MTS – Cell Viability 2. BCA – Protein Levels 3. Calcium 4. Alkaline Phosphatase – Early Marker 5. ELISA: Osteopontin – Early and Late Marker Osteocalcin – Late Marker
6. qPCR – Determine Gene Expression
- ALPL, RUNX2, OC, OP, BMP2
7. Von Kossa Staining - Mineralization

8. higher absorbance = more viable cells
MTS Results
Cell Viability/Proliferation – Cells breakdown MTS into a product that can be detected by a spectrophotometer
higher absorbance = more viable cells

9. MTS Results But wait a minute! These results are misleading…
Literature: 3000 cells/cm2
2D wells: 0.75 cm2 → 2250 cells
3D gels: 150 cm2 → 450,000 cells

10. higher fluorescence = more ALP activity
ALP Results
Alkaline Phosphatase – ALP is a byproduct of osteoblast activity.
ALP cleaves a substrate to product a fluorescent product
higher fluorescence = more ALP activity

11. More dark spots = more mineralization
Von Kossa Results
Von Kossa stain binds to phosphate and calcium in the sample
More dark spots = more mineralization
2D Plate, 40 X D Plate, 40X
Day 7

12. More dark spots = more mineralization
Von Kossa Results
Von Kossa stain binds to phosphate and calcium in the sample
More dark spots = more mineralization
2D Plate, 40 X D Plate, 40X
Day 14

13. Von Kossa Results
Day 7
Day 14
2D Plate, 40 X 3D Plate, 40 X

14. Considerations
Standard deviation for SPH samples – H 39 vs. Smoker
2. Create a standard curve for MTS to determine actual number of cells?