Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Summary

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO₂ fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Abstract

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO₂ fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO₂ expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

Introduction

The concept of developing a synthetic scaffold that can be implanted into patients to aid in tissue repair and regeneration is one that has permeated the regenerative medicine field for decades. The ideal synthetic scaffold serves to induce cell migration from surrounding healthy tissue, provides an architecture for cell seeding, adhesion, signaling, proliferation, and differentiation, supports vascularization, allows for adequate oxygenation and nutrition delivery, and promotes host immune activity to ensure success after implantation¹. Additionally, it can be used as a carrier for embedding antimicrobial molecules to assist in wound healing

Protocol

All in vivo procedures outlined below were approved by the IACUC Committee at the University of Nebraska Medical Center.

1. Prepare the Solutions for Standard Electrospinning

1. In a 20 mL glass tube, dissolve 2 g of poly(ε-caprolactone) (PCL, Mw= 80 kDa) in a solvent mixture of dichloromethane (DCM) and N,N-dimethylformamide (DMF) with a 4:1 ration (v/v) at a concentration of 10% (w/v).
   CAUTION: Handle DCM and DMF in a well-ventilated hood to avoid exposure to fumes. Do not expose DCM to plastic materials.
2. Place the glass tube into a lab rotator until the solution becomes clear. The solution may mix ....

Results

The efficacy of expanding traditional 2D electrospun nanofiber mats into 3D scaffolds via depressurization of subcritical CO₂ fluid was demonstrated in different capacities: the thickness of the scaffolds increased from 1 mm when untreated to 2.5 mm and 19.2 mm with one and two CO₂ treatments, respectively (Figure 3A-C). The porosity-a characteristic of the architecture critical for cell seeding-also increased in a manne.......

Discussion

Transforming traditional 2D electrospun nanofiber mats into expanded 3D scaffolds via CO₂ depressurization was investigated. Traditional 2D nanofiber mats are successfully expanded via subcritical CO₂ fluid. The critical steps are to fabricate 2D nanofiber mats under an optimized condition and cut the mats without deforming the edges (e.g., using sharp surgical scissors). This CO₂-expanded nanofiber scaffolds have many benefits over traditional 2D mats including.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polycaprolactone	Sigma-Aldrich	440744
N,N-Dimethlyformamide	Fisher Chemical	D-199-1
Dichloromethane	Fisher Chemical	AC61093-1000
Coumarin 6	Sigma-Aldrich	546283
Rotating Steel Drum	customized		This serves as a collector during electrospinning.
Syringe Pump	Fisher Scientific	14-831-200	Coaxial spinning requires two single syringe pumps.
Revolver	Lab Net International	H5600	Adjustable lab rotator for mixing solutions
Hypodermic Needle (27G x 1 1/2")	EXCELINT International Co	26426	This is part of the example customized coaxial nozzel shown.
Hypodermic Needle (21G x 1 1/2")	EXCELINT International Co	26416	This is part of the example customized coaxial nozzel shown.
High Voltage DC Power Supply	Gamma High Voltage Research	ES30
Scanning Electron Microscope	FEI	Nova 2300
Fluorescence Microscope	Zeiss	Axio Imager 2
LL 37 ELISA Kit	Hycult Biotech	HK321-02