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

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

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.

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.

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.

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

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.

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.

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.

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.

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

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 (0.5-0.7 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.

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.