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Biomacromolecules 2008, 9, 1044–1049
Electrospinning of Highly Porous Scaffolds for Cartilage Regeneration Anna Thorvaldsson,*,† Hanna Stenhamre,‡,§ Paul Gatenholm,‡ and Pernilla Walkenström† Swerea IVF, Box 104, SE-431 22 Mölndal, Sweden, Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden, and Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden Received November 7, 2007; Revised Manuscript Received December 21, 2007
This study presents a new innovative method where electrospinning is used to coat single microfibers with nanofibers. The nanofiber-coated microfibers can be formed into scaffolds with the combined benefits of tailored porosity for cellular infiltration and nanostructured surface morphology for cell growth. The nanofiber coating is obtained by using a grounded collector rotating around the microfiber, to establish an electrical field yet allow collection of nanofibers on the microfiber. A Teflon tube surrounding the fibers and collector is used to force the nanofibers to the microfiber. Polycaprolactone nanofibers were electrospun onto polylactic acid microfibers and scaffolds of 95 and 97% porosities were made. Human chondrocytes were seeded on these scaffolds and on reference scaffolds of purely nanofibers and microfibers. Thereafter, cellular infiltration was investigated. The results indicated that scaffold porosity had great effects on cellular infiltration, with higher porosity resulting in increased infiltration, thereby confirming the advantage of the presented method.
Introduction Scaffolds to be used in tissue engineering are threedimensional structures that can be produced by a number of different methods. Electrospinning is one method that in recent years has gained increased attention. It is an efficient method used to create fibers on micro- and nanoscales, the methodology and theory behind it being extensively described by, for example, Reneker et al.1,2 In electrospinning a polymer solution or melt is fed through a syringe and charged by a high voltage. The charged solution is directed toward a grounded collector and, as solvent evaporates on the way to the collector, dry fibers are collected. The flexibility of the electrospinning process is one of its advantages. A wide variety of materials can be electrospun and the fiber morphology can be easily controlled by parameters such as polymer molecular weight, polymer concentration, solvent properties, electrical field applied, and solution feed rate.3–6 Incorporation of particles in the fibers is also possible, useful in tissue engineering applications when it comes to, for example, a controlled release of growth factors or other substances.7–10 More importantly though, when applied in the field of tissue engineering, electrospinning has the great advantage of producing nanofibrous scaffolds resembling a natural extracellular matrix (ECM). The size range of electrospun fibers and the great surface area of the constructs they form are two traits shared with natural ECM. There are studies indicating that cell adhesion and proliferation are enhanced on nanofibers compared to microfibers.11–17 Furthermore, the threedimensional (3D) structure of the electrospun scaffolds allows the cells to fully differentiate, in turn calling for a maintenance * Corresponding author. E-mail:
[email protected]. † Swerea IVF, Box 104, SE-431 22 Mölndal, Sweden. ‡ Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden. § Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden.
of normal biological activity of the cells that is not always possible in a 2D environment.18,19 In order to form a desired 3D structure of growing cells, the infiltration of cells into the scaffold must be efficient. Therefore, the scaffold porosity is a critical feature and has to be high enough as to allow cells to freely migrate. The pore sizes required by different cells vary but must generally be at least the size of a cell, i.e., about 10 µm.3,20 However, in many cases pore sizes of several 100 µm are necessary for optimal migration.21,13 The pore sizes have also been seen to affect the cell development when it comes to differentiation and matrix production.22–24 High porosity and large pore sizes are beneficial in many aspects, although one must be careful not to increase them too much. If the distances between the fibers get too big, they might not be possible for the cells to bridge over, hence affecting cell growth negatively.13 Due to different cells having different needs, being able to tailor the porosity would be a great advantage and could in many cases even determine failure from success. The size range of nanofibers is, as mentioned, an attractive trait in many aspects. Small fiber diameters do however result in low porosities and small pore sizes of the matrices, one of the limitations in using nanofibrous scaffolds. Methods previously used to increase porosity of electrospun nanofibrous scaffolds have included multilayering and mixing electrospinning, both creating bicomponent scaffolds in which one component can be leached out to generate a porous construct.25 Also, electrospinning combined with salt leaching has been a well used technique.26 Leaching out a component might however affect the structural morphology of both fibers and scaffolds.25,27,28 Another approach is to increase the diameter of the fibers. It is easily achieved with electrospinning, as for example higher polymer concentrations result in larger fiber diameters.20,29,30 Larger fiber diameters in turn increase the porosity of the structure but might have a negative effect on cell adherence and proliferation.11,12,15 Recently published studies show that combining nano- and microfibers in a scaffold
10.1021/bm701225a CCC: $40.75 2008 American Chemical Society Published on Web 02/09/2008
Highly Porous Scaffolds for Cartilage Regeneration
Biomacromolecules, Vol. 9, No. 3, 2008 1045
Figure 1. Setup for electrospinning PCL nanofibers onto a single PLA microfiber.
may be a promising alternative.16,31 The nanofibers then provide surfaces for cell attachment and proliferation, while the microfibers provide the structural environment. This is beneficial in that the porosity of the structure can be easily varied by change of nanofiber/microfiber ratio; thus it provides desired control over a very important parameter. However, there are problems concerning the methods by which nanofibers and microfiber are mixed. Previous attempts have been based on multilayering or mixing electrospinning of fibers in different size ranges. Upon sequential electrospinning the nanofibers form sheetlike structures over the microfibers, thus no mixing occurs between the two fiber types and the nanofiber layers prevent adequate infiltration.25,31 Using two spinnerets to simultanously electrospin both nano- and microfibers is also difficult due to the electrostatic repulsion between the two fiber jets.25 This study describes a new and innovative tecnique for creating highly porous scaffolds with a suitable combination of nano- and microfibers. It makes use of the possibility of electrospinning nanofibers directly onto single microfibers, thus eliminating problems with layering and providing a very flexible tecnique for creating structures of variable morphologies. By electrospinning nanofibers onto single microfibers, one ends up with long fibers containg the best of two worlds. The nanofibers are present to enhance cell adhesion and spreading although by collecting them on a microfiber they can easily be formed into any shape and size and, most importantly, into scaffolds of any porosity. Polycaprolactone (PCL) nanofibers were in the study electrospun onto polylactic acid (PLA) microfibers. PCL and PLA are both biopolymers that are biocompatible and biodegradable. Furthermore, they have been previously electrospun and proven to support growth of a wide variety of cells when used in nanofibrous scaffolds for tissue engineering purposes.3,8,25,28–31 The nanofiber-coated microfibers were formed into scaffolds of two different porosities and characterized using electron microscopy. The effects on cellular infiltration were evaluated as a preliminary chondrocyte cell infiltration study was performed, including scaffolds of pure microfibers and nanofibers for comparison.
Materials PLA (100%) (Terramac, 70000–100000 g/mol, % D
