Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs
Jacqueline Mimnaugh, RETNeuqua Valley High School
Dr. Richard GemeinhartMelanie KollmerUIC Department of Biopharmaceutical Sciences
Tracy Chuong, REUUniversity of California Berkley
hMSCs and Tissue EngineeringHuman Mesenchymal Stem Cells (hMSCs)
Can differentiate into bone, cartilage, fat, and other cells
Tissue Engineering↓
Bone diseases/defects, trauma, cancer, mal-union/non-union fractures
Osteogenesis → Induce hMSCs to develop into bone cells
Cells in the lab are typically cultured on plates
2 D
The Problem
Cells in vivo (in an organism)
3 DIt is possible that cells grown in a 3D scaffold would:
1. Be more like cells in vivo2. Would not reach confluence as quickly3. Could be implanted directly
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?
Research Project
Superporous Hydrogels
• Poly (ethylene glycol) diacrylate or PEGDA
• Polymer network, hydrophilic
• Pores from 100 µm to 600 µm created by gel-foaming
Creating a 3d Scaffold
1. Seed hMSCs onto 2D plates and 3D hydrogels
Project Overview
2. Add osteogenic differentiation medium
3. Compare cells at day 2, 7, 14, 21 and 28
After 24 hours
After 24 hours
1. MTS – Cell Viability
2. BCA – Protein Levels
3. Calcium
4. Alkaline Phosphatase – Early Marker
5. ELISA: Osteopontin – Early and Late MarkerOsteocalcin – Late Marker
Comparing the cells
6. qPCR – Determine Gene Expression- ALPL, RUNX2, OC, OP, BMP2
7. Von Kossa Staining - Mineralization
Cell Viability/Proliferation – Cells breakdown MTS into a product that can be detected by a spectrophotometer
higher absorbance = more viable cells
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
MTS Results
Alkaline Phosphatase – ALP is a byproduct of osteoblast activity. ALP cleaves a substrate to product a fluorescent product
higher fluorescence = more ALP activity
ALP Results
Von Kossa stain binds to phosphate and calcium in the sample More dark spots = more mineralization
Von Kossa Results
2D Plate, 40 X 3D Plate, 40XDay 7
Von Kossa stain binds to phosphate and calcium in the sample More dark spots = more mineralization
Von Kossa Results
2D Plate, 40 X 3D Plate, 40XDay 14
Von Kossa Results
2D Plate, 40 X 3D Plate, 40 X
Day 7
Day 14
1. Standard deviation for SPH samples – H 39 vs. Smoker
2. Create a standard curve for MTS to determine actual number of cells?
Considerations