Influence of 2D and 3D Environments on Osteogeneic Differentiation in hMSCs

Jacqueline Mimnaugh, RETNeuqua Valley High School

Dr. Richard GemeinhartMelanie KöllmerUIC 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

Objective: Compare the osteogenic differentiation of cells

in 2D and 3D culture systems.

2D Culture Plate

3D Superporus Hydrogel

Viability?Compare Cells: Proteins? Mineralization?

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 – Mid-phase 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 plate reader

higher absorbance = more viable cells

MTS Results

Considerations:1. Cell seeding

number

2. Cells from SPH lost over time

3. Deviation – two donors

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

Considerations:

1. Normalized with MTS

2. Early marker of differentiation

3. Day 21 – Time

Day 7 Day 21 Day 28

2D

SPH

Von Kossa stain binds to phosphate and calcium in the sample More dark spots = more mineralization

Von Kossa Results