1. Adult Stem Cell Interaction With Engineered Scaffolds
By: Kellen Carleton
Pittsburgh Central Catholic
10th Grade

2. Introduction: Scaffolds
Provide temporary framework for cell proliferation, migration, and differentiation
Type/composition varies with purpose, seeded cells, and host tissue compartment
Many different materials (natural and synthetic) have been investigated for use in tissue regeneration
The scaffold provides a framework for growing cells in tissue regeneration. The type of scaffold varies with the purpose, tissue type, and implant location. Many different materials, both synthetic and natural, have been investigated. One such material, collagen, has been used for biocompatible medical sutures before the advent of tissue engineering.
Scaffold for Tissue Engineering

3. Scaffold Introduction (cont.)
PLA Scaffolds– popularly used in tissue engineering because of its biodegradability
PCL Scaffolds– biodegradable polymer which has been used in the fabrication of surgical implants. It has also been extensively studied as a biodegradable scaffold material for tissue engineering applications.

4. Scaffold Introduction (cont.)
UBM Scaffolds—Urinary Bladder Matrix is an extracellular matrix (ECM) scaffold. It is now used in wound care management of partial and full-thickness wounds where conventional methods for wound care usually fail to give satisfactory results.
PMMA Material– like “bone cement,” used in full reconstruction of bones in the body. Not a true scaffold for regeneration.

5. What is Tissue Engineering/ Regenerative Medicine?
Replacing diseased or injured tissues with tissue constructs designed and fabricated for the specific needs of each individual patient.

6. Principles of Tissue Engineering
Cells
ECM
Defect
Regeneration
Blood
Supply
Hormones
Phil Campbell, Carnegie Mellon

7. Stem Cells
Cell that can produce lineages more specialized than themselves and can renew itself
Spectrum of Stem Cell Behavior:
totipotent embryonic cells
pluripotent cells
multipotent cells
unipotent cells.

8. C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line.
Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.
Used as a model in many tissue engineering experiments.
Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.
Expresses muscle proteins and the androgen receptor (AR).
AR- DNA binding transcription factor which regulates gene expression.

9. Objective
To investigate which material of scaffold (PLA, PCL, PMMA, UBM) is best for stem cell growth.
Specifically, to investigate which scaffold attracts the most cells and is ultimately best for stem cell attachment and growth.

10. Hypothesis
Null Hypothesis– None of the scaffolds will permit Stem Cell attachment and subsequent growth
Alternate Hypothesis– Scaffolds will vary in promoting cell attachment and growth, and UBM will yield the best result.

11. Materials PMMA Scaffold Material Well Plates PCL Scaffold Material
C2C12 Myoblastic Stem Cell Line
PLA Scaffold Material
DMEM Media (10% Calf Serum)
UBM Scaffold Material
DMEM Media (1% Calf Serum)
Hot Plate
Ethanol
Beaker
Rubber Gloves
Tongs
Microscope
Flasks
Trypsin
Pipets
Aspirator
Stain
Water
Test Tube Rack
Sterile Pipet Tips
100 mL Graduated Cylinder

12. Procedure C2C12 Culture Preparation
Adult Stem Cells were grown in Four T-75 Culture Flasks, each containing about 4-6 Million Cells.
At culture confluency of approximately 50%, cells were trypsinized and resuspended in 5mL of 10%DMEM media.
1 mL of cell suspension ( million cells) was loaded into each well/scaffold.
Images were captured on days 1, 5, and 10 using a Nikon inverted scope/computer interface.
Cells/scaffold were stained with toluidine blue on day 10 to enhance visualization. Images were captured using Nikon system.

13. Procedure Scaffold Preparation
As cells were being grown in flasks, PLA Scaffolds were created using PLA crystals. The PLA crystals were melted down using a hot water/watch glass system. Crystals were formed and dried, then sterilized with 95% ethanol and uv irradiation.
The PCL, PMMA, and UBM scaffolds were cut from larger preformed samples into four separate smaller scaffolds. Each was sterilized with ethanol and uv irradiation.
Scaffolds were transferred to the well plates, stored in 1mL sterile PBS until cell seeding.