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