Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Summary

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Abstract

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task.  Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named poly(glycerol-dodecanoate) (PGD)¹ by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Introduction

Electrospinning is one of the effective processing methods to produce micro-to-nanometer size fiber scaffolds. The basic principle of electrospinning involves a Taylor cone of solution that is held at the orifice of a needle by applying high voltage between the tip of the needle and a grounded collector. When the electrostatic repulsion in the solution overcomes the surface tension, a charged fluid jet is ejected out of the needle tip, travels through the air with solvent evaporation, and is finally deposited on the grounded collector. The syringe pump provides a continuous flow of solution emerging from the spinneret and thus multiple copies of the electrospun fibers....

Protocol

1. Electrospinning Collector Setup

1. Cut the aluminum foil into a rectangular piece.
2. Fold the rectangular piece into a rectangular strip, and attach it perpendicular to a flat metal plate with tape (Figure 1). Note: The size of the fiber mat depends on the length and width of the strip. Thus, the strip dimensions can be adjusted as needed.

2. Polymeric Solution Preparation

1. Mix glycerol and dodecanedioic acid (DDA) in 1:1 molar ratio in a beaker at 120 °C for 100 hr to obtain PGD polymer.
2. Dissolve poly(ethylene oxide) (PEO) and gelatin in 65% ethanol with a weight ratio o....

Results

The major components of the electrospinning are shown in Figure 1. A large size fiber mat was typically obtained through the perpendicularly attached aluminum foil strip and a flat metal plate. Figure 2 shows the collector design and the electrospinning fiber mat. The width and length can be adjusted for different applications. The length of the fiber made with PGD polymer and basal solution mixture is up to 10 cm. The morphology of electrospun fibers is shown in Figure 3

Discussion

The limitations of simple collectors or the complexities of rotating collectors that are currently used for electrospinning increase the restriction of obtaining the desired length and size of fiber mat for some applications. Additionally, transferring fibers from the ground collector to the culture dish or other substrates is a challenge⁵. In this report, a newly designed collector, made simply by attaching an aluminum foil strip to the grounded collector, was able to obtain large size fiber mats up to 10 cm .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glycerol	Sigma-Aldrich	G7757
Dodecanedioic acid	Sigma-Aldrich	D1009
Gelatin	Sigma-Aldrich	D1890
Poly(ethylene oxide) (PEO)	Sigma-Aldrich	182028
Riboflavin	Sigma-Aldrich	132350250	0.10%
Mouse embryonic stem cells	GlobalStem	GSC-5002
Matrigel	Becton Dickinson	356234
DMEM/F12	Thermo Scientific	SH30272.02
N2 supplement	Invitrogen	17502048	1%
FGF2	Stemgent	03-0002	10 ng/ml
Accutase	Invitrogen	A11105-01
Phosphate buffered saline (PBS)	Invitrogen	10010-031
Resazurin fluorescence dye	Sigma-Aldrich	62758-13-8
SV Total RNA Isolation System	Promega	Z3100
GoScript Reverse Transcription System	Promega	A5000
GoTaq qPCR Master Mix	Promega	A6001
Syringe pump	Fisher scientific	14-831-200
High voltage power source	Spellman High Voltage Electronics Corporation		SL30
UV light	Philips	308643	15W/G15T8
Synergy HT Multi-Mode Microplate Reader	BioTek
Perkin Elmer GeneAmp PCR System 9600	Perkin Elmer	8488
StepOne Real-time PCR System	Applied Biosystems	4376357