Postproduction Processing of Electrospun Fibres for Tissue Engineering

Summary

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Abstract

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.^{1, 2, 3} Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.

Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).

Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.⁴ An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Protocol

1. Electrospinning of Random and Aligned Fibres

Electrospinning creates fine fibrous networks by using electric potential to draw a polymer solution towards an earthed collector. Collectors can be in very many shapes and can be static or, more commonly, rotating. The solvent evaporates before the solution arrives at the collector and the jet solidifies into a fibre.

Each polymer requires its own set of conditions to produce a given type of fibre. The concentration of the polymer, the solvent, the distance between the pumped solution and the earthed collector, the potential difference between the two, the velocity....

Discussion

Electrospinning is a very popular technique for producing scaffolds for tissue engineering.^{14, 15, 16} While it is relatively simple to produce basic electrospun scaffolds for experimental use the technique is also complex and multifaceted with many variables.⁶ There are many studies describing how the electrospinning parameters determine the scaffold produced. In this study the focus is on the considerable challenges post production to make scaffolds of appropriate architectures and mechanical pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly lactic-co-glycolic acid	Sigma Aldrich
Poly lactic acid	Sigma Aldrich	81273	Inherent viscosity ~2.0dl/g
Poly ε-caprolactone	Sigma Aldrich
Poly hydroxybutyrate-co-hydroxyvalerate 12:1	Goodfellow	578-446-59	PHB88/PHV12
Dichloromethane	Sigma Aldrich or Fisher	270997 or D/1850/17	>99.8% contains 50-150ppm amylene stabiliser
50 multi coloured balloons	Wilkinson’s Hardware Stores Ltd.	0105790
Goat anti-rabbit IgG (FC):FITC	AbDserotec	STAR121F
Rabbit anti-human alpha elastin	AbDserotec	4060-1060
Screw Cap GL45 PP 2 Port, pk/2	SLS	1129750
4′,6-Diamidino-2-phenylindole dihydrochloride	Sigma Aldrich	32670
CellTracker green CMFDA	Invitrogen	C7025
CellTracker red CMTX	Invitrogen	C34552