Increasing the Pore Size of Electrospun Scaffolds

Abstract

Electrospinning has gained much attention in the past decade as an effective means of generating nano- to micro-scale polymer fibers that resemble native extracellular matrix. High porosity, pore interconnectivity, and large surface area to volume ratio of electrospun scaffolds make them highly conducive to cellular adhesion and growth. However, inherently small pores of electrospun scaffolds do not promote adequate cellular infiltration and tissue ingrowth. Cellular infiltration into the scaffold is essential for a range of tissue engineering applications and is particularly important in skin and musculoskeletal engineering. Pore size, porosity, and pore interconnectivity dictate the extent of cellular infiltration and tissue ingrowth into the scaffold; influence a range of cellular processes; and are crucial for diffusion of nutrients, metabolites, and waste products. A number of electrospinning techniques and postelectrospinning modifications have, therefore, been developed in order to increase the pore size of electrospun scaffolds. Diverse techniques ranging from simple variations in the electrospinning parameters to complex methodologies requiring highly specialized equipment have been explored and are described in this article.

Importance of Porosity, Pore Size, and Pore Interconnectivity for Tissue-Engineered Scaffolds

Pore size, porosity, and pore interconnectivity are important features of tissue-engineered scaffolds that strongly influence cellular activity. In particular, they dictate the extent of cellular infiltration and tissue ingrowth into the scaffold. This has been demonstrated in constructs made from a range of synthetic polymers, including polycaprolactone (PCL),^1–4 polyurethane,^5,6 poly(lactic-co-glycolic acid) (PLGA),⁷ and poly(lactic acid),⁵ and natural proteins, including gelatin^8,9 and tropoelastin^10,11, and employing a number of cell types, including fibroblasts,^{4,7,8,10–12} mesenchymal stem cells (MSCs),^1,3 CFK2 chondrocytic cells², and MG63 human osteosarcoma cells.⁹ These parameters also influence a range of cellular processes, including cell adhesion,¹³ migration,^5,9–11 proliferation,^1,7,13 morphology, phenotypic expression,^9,13–16 DNA synthesis,¹⁶ and extracellular matrix (ECM) deposition.^1,5,13,15

Cellular infiltration into the scaffold is essential for a range of tissue-engineering applications and is particularly important in skin and musculoskeletal engineering. For example, dermal substitute scaffolds are expected to promote dermal fibroblast adhesion, growth, and infiltration, as the presence of fibroblasts in dermal substitutes accelerates and enhances dermal and epidermal regeneration.^17–24 In addition to their importance in promoting cellular infiltration, pore size and pore interconnectivity are crucial for diffusion of nutrients, metabolites, and waste products.^8,25 Nutrient, oxygen, and waste transport is particularly important for in vitro cell culture and immediately post implantation in vivo before the blood supply is re-established.²⁶

Electrospinning As a Novel Technique for Generating Highly Porous Scaffolds

Electrospinning is an efficient way of producing nano- to micro-scale fibers from a variety of natural and synthetic polymers.²⁷ It has generated much interest in the tissue-engineering field as a simple, inexpensive means of mimicking native ECM fibers.²⁸ Electrospun constructs are currently being investigated as means of replacing and regenerating a number of tissues including skin, tendon, bone, cartilage, and blood vessels (for reviews see refs.^29–32).

Electrospinning utilizes a strong electric field to process a polymer solution into a fibrous construct.²⁸ In a typical electrospinning setup, a polymer solution is ejected through a blunt-ended needle using a syringe pump. The needle is connected to a high positive voltage power supply typically capable of producing voltages between 1 and 30 kV. When the electrostatic charge becomes higher than the surface tension of the polymer droplet at the needle tip, the droplet is deformed into a conical shape called a Taylor cone. Beyond a critical charge density, a Taylor cone becomes unstable, and a polymer jet is emitted from the tip of the cone. This jet is accelerated from the needle tip to a grounded (or negatively charged) collector, generating a nonwoven construct composed of continuous nano- to micro-scale polymer fibers. Typically, the polymer is dissolved in an organic solvent such as 1,1,1,3,3,3 hexafluoro-2-propanol, which evaporates as the material is driven across the air gap between the needle tip and the collector. Continuous fiber accumulation at the collector generates a three-dimensional (3D) scaffold.^27,29,33,34

The resulting electrospun scaffold is composed of thin cylindrical (circular cross-section) or ribbon-like (rectangular cross-section) fibers with diameters ranging from 0.01 to 10 μm and highly interconnected pores.^27,35 Physical properties of electrospun scaffolds, including fiber morphology and diameter, porosity, pore size, and mechanical properties, depend on the electrospinning parameters and postelectrospinning modifications. Electrospinning parameters can be classified as system parameters and process parameters (listed in Table 1).³⁰ Variations in one or more of these parameters translate into variations in fiber and scaffold properties. For example, an increase in polymer concentration is known to result in greater fiber diameter,³⁶ whereas the shape of the collector has a significant effect on the final fiber alignment.³³ In a basic electro-spinning setup, a flat collector accumulates randomly positioned fibers, whereas a rotating mandrel collector can result in aligned fibers.^37,38

Table 1.

Electrospinning Parameters (Adapted from Ref.³⁰)

System parameters	Process parameters
Molecular weight of the polymer	Electric potential
Molecular-weight distribution and architecture of the polymer in the solution	Flow rate of the polymer through the needle
Polymer solution properties including viscosity, conductivity, and surface tension	Distance between the needle tip and the collector
	Collector shape
	Ambient parameters such as temperature, humidity, and air velocity

High porosity, pore interconnectivity, and large surface area to volume ratio of electrospun scaffolds make them highly conducive to cellular adhesion and growth.^29,34 However, a common problem encountered with many electrospun materials is that inherently small pores of electrospun scaffolds do not promote adequate cellular infiltration and tissue ingrowth. The ability to increase the pore size of electrospun scaffolds while maintaining fiber morphology and pore interconnectivity is an important area of research that has gained attention over the past 5 years. A number of electrospinning techniques and postelectrospinning modifications have, therefore, been developed in order to increase the pore size of electrospun scaffolds. Diverse techniques ranging from simple variations in the electrospinning parameters to complex methodologies requiring highly specialized equipment have been explored and are described here (Table 2).

Table 2.

Summary of Techniques Used to Increase the Pore Size of Electrospun Scaffolds Described in This Review

Technique	Material	Pore size	Tissue-engineering application and/or cell type	Reference
Increased fiber diameter	PCL	10–45 μm	Not specified/MSCs	3
	Gelatin	1–10.7 μm	Bone/MG63 cells	9
		11.8 μm	Skin/fibroblasts, keratinocytes	8
	Tropoelastin	1.6–27.9 μm	Dermis/fibroblasts	10,11
Rotating mandrel collector	PLGA	132 μm	Skin/fibroblasts	7
Wet electrospinning	PGA	Not specified	n/a	52
	Silk fibroin	586–931 μm	Bone/MC3T3-E1 preosteoblasts	53
FLUF	PCL	2–5 μm	Not specified INS-1(832/13) cells	51
Sacrificial material electrospinning	PCL/PEO	Not specified	Not specified/MSCs	55
	PCL/gelatin	Not specified	Not specified/ bone marrow stromal cells	56
Salt leaching	PCL	100–200 μm^a	Cartilage/ CFK2 cell line	2
Gas foaming	PCL	50–70 μm^b	Not specified/ fibroblasts	58
Cryogenic electrospinning	PLA	10–500 μm	Not specified/ 3T3/NIH fibroblasts	12
	PLGA	∼37 μm	n/a	59
	PEU	∼60 μm	n/a
DPMD and electrospinning	PCL/collagen	Not specified	Not specified chondrocytes	60
Electrospinning and cell electrospraying	PEUU/cells	Not specified	Blood vessels and other soft tissues/SMCs	61

Gap sizes where the scaffolds have delaminated.

Sizes of holes made in the scaffolds by the blowing agent.

FLUF, focused, low density, uncompressed nanofiber; DPMD, direct polymer melt deposition; PCL, polycaprolactone; PLGA, poly(lactic-co-glycolic acid); PGA, poly(glycolic acid); PEO, poly(ethylene oxide); PEUU, poly(ester urethane) urea; MSCs, mesenchymal stem cells; SMCs, smooth muscle cells; PLA, poly(lactic acid); n/a, not applicable.

Increasing the Pore Size of Electrospun Scaffolds

Modified electrospinning parameters

The most straightforward means of increasing the pore size of electrospun scaffolds involves careful modification of system or process parameters during the electrospinning process. Larger pores can be achieved by increasing the diameter of electrospun fibers, a technique that is described by a number of groups as means of increasing the pore size of scaffolds electrospun from both synthetic and natural polymers.^9–11,39,40 Statistical modeling predicts a relationship between the fiber diameter and the pore size of electrospun scaffolds, where a larger fiber diameter correlates with an increase in the pore size.⁴¹ This is in agreement with experimental data obtained for a range of polymers including PCL,^1,3,4,39 gelatin,^8,9 and tropoelastin.¹⁰ For example, a 2.5-fold increase in the diameter of electrospun PCL fibers results in a 2.25-fold increase in the average pore size, resulting in an average pore diameter of 45 μm.³ Similarly, a 1.4-fold increase in the tropoelastin fiber diameter corresponds to a 1.5-fold increase in the average pore size and a 2.3-fold increase in the overall scaffold porosity.¹¹ Thicker electrospun fibers are most commonly obtained by increasing polymer concentration or flow rate during the electrospinning process.^{3,4,11,36,39,42,43} This technique has been utilized to enhance the infiltration of MSCs,^3,4 myofibroblasts,³⁹ osteoblastic MG63 cells,⁹ and dermal fibroblasts^10,11 into electrospun scaffolds.

However, it has historically been accepted that nanofibers accelerate and increase cell adhesion and proliferation compared with their microscale counterparts,^44–47 which raises concern about utilizing increased fiber diameter as a means of increasing the pore size of electrospun scaffolds. This has led to predominant electrospinning of nanofibers rather than wider diameter microfibers. However, recent studies have shown that the relationship between fiber diameter and cellular interactions is much more complex, especially at a microfiber scale. A number of groups have found superior cell proliferation on microfibers compared with nanofibers.^3,4,44,45 Cell growth kinetics decrease with increasing fiber diameter on nanofibers, but not on microfibers⁴⁵; whereas human cells can attach and organize well around fibers with diameters smaller than those of the cell.^48,49 Further, most studies investigating the relationship between cell interactive properties and fiber diameter were performed on synthetic polymers that have no inherent cell interactive groups. Cell attachment on such scaffolds is likely to depend on adsorption of cell interactive proteins, such as vitronectin and fibronectin, from fetal calf serum.⁴⁶ Nanofibers have larger surface area to volume ratios that promote higher protein adsorption and, therefore, accelerate cell attachment.⁴⁵ Additionally, one study has found that MSCs attach best to PCL fibers with diameters of 2.6 μm, compared with 0.3 and 5.2 μm. Such findings suggest that the relationship between cell interactive properties and fiber diameter of electrospun scaffolds is much more complex than was originally thought and should be explored for each scaffold and cell type individually, taking into account the trade-off between pore size and fiber diameter.⁴

A number of groups have combined micro- and nano-scale fibers within the same scaffold to utilize the inherent advantages of both fiber types. Such scaffolds can possess a range of pore distributions, thus allowing culture of different cell types or creation of multiple cell interfaces on a single scaffold to direct different cellular functions. For example, in skin tissue engineering, a porous scaffold would allow fibroblast and vascular cell infiltration; whereas a less porous surface would promote migration and differentiation of multiple keratinocyte layers. Similarly, in bone tissue engineering, such scaffolds could promote osteogenesis and vascularization on one side of the scaffold and osteochondral ossification on the other.^3,50

A different approach for increasing the pore size of electrospun scaffolds by changing the electrospinning parameters involves changes to the collector shape, including the utilization of a rotating mandrel collector or a spherical dish collector, as well as use of solvent baths as fiber collectors in wet electrospinning. A novel rotating frame collector consisting of metal struts (rotating mandrel collector) was developed in an attempt to increase the pore size of electrospun PLGA scaffolds.⁷ This setup results in electric field changes that favor lower fiber density deposition between metal struts resulting in scaffolds with pore sizes up to 132 μm. The resultant scaffolds allow for fibroblast infiltration to more than 100 μm into the scaffold by day 5 postseeding. However, fiber deposition in this setup is not uniform across the whole collector, thus resulting in areas of high- and low-fiber densities and, therefore, heterogeneity in fiber and pore size distribution.

Focused, low density, uncompressed nanofiber (FLUF) electrospinning, on the other hand, utilizes a collection system consisting of an array of metal probes embedded in a nonconductive spherical dish (Fig. 1). This setup, therefore, overcomes the limitations of flat collectors by allowing the creation of 3D cotton ball-like scaffolds consisting of loosely packed nanofibers and deep interconnected pores. FLUF electrospun PCL scaffolds promote the growth and infiltration of rat insulinoma INS-1 (823/13) cells up to 300 μm into the scaffold and show promise for the study of pancreatic tissue engineering.⁵¹ FLUF electrospinning proves to be a novel and relatively simple means of increasing the porosity and pore size of electrospun scaffolds, but it remains to be seen whether the technique can be applied to natural polymers.

FIG. 1.

(A) Traditional electrospinning setup showing the syringe nozzle (i) Taylor cone formation, (ii) the voltage source, (iii) a flat collector, (iv) (B) a flat PCL scaffold generated by traditional electrospinning, (C) FLUF electrospinning setup showing the spherical dish collector, and (v) (D) a cotton-ball like PCL scaffold generated by FLUF electrospinning. Images modified from ref.⁵¹. PCL, polycaprolactone; FLUF, focused, low density, uncompressed nanofiber. Color images available online at www.liebertonline.com/teb

Wet electrospinning has gained considerable attention in the tissue-engineering field as a means of increasing the porosity and pore size, as well as of reducing the bulk density of electrospun scaffolds. Unlike conventional electrospinning, where the collector is a conductive plate, wet electrospinning utilizes a wet bath containing a range of solvents, such as water, tertiary-butyl⁵² alcohol, or methanol⁵³ as a grounded fiber collector.⁵⁴ The fiber dispersion is solidified by lyophilization after electrospinning. This methodology is an effective means of increasing the porosity and pore size of electrospun poly(glycolic acid) and silk fibroin scaffolds, both of which are promising bone-tissue-engineering constructs. Pore size can be further increased by using a porogen, such as sodium chloride, in the collector bath. For example, the pore size of electrospun silk fibroin scaffolds was increased from 1.3–2.4 μm in conventional electrospinning to 586–931 μm in wet electrospinning combined with the use of a porogen.⁵³ Widespread use of this technique is likely to depend on identifying appropriate solvents to use as collectors that do not dissolve natural polymers before cross-linking.

Postelectrospinning modifications

A number of attempts at increasing the pore size of electrospun scaffolds involve changes to the scaffold structure after electrospinning. Many of these postelectrospinning modifications, such as salt leaching and lyophilization, involve adaptation of the well-established hydrogel modification techniques to electrospun scaffolds. The simplest of these techniques is electrospinning of sacrificial materials, where the polymer of interest is co-electrospun with another sacrificial polymer. The sacrificial polymer is then dissolved away in a solvent that has no effect on the structure of the polymer of interest. For example, PCL, a slow degrading polyester, can be co-electrospun with poly(ethylene oxide) (PEO), a high-molecular-weight water-soluble polymer. When PEO is dissolved in water, a PCL scaffold with large pores that promotes cellular infiltration remains.^1,55 PEO is a common sacrificial polymer due to its aqueous solubility, but other materials, such as gelatin, can also be employed.⁵⁶ The use of sacrificial fibers is a simple means of increasing the porosity and pore size of electrospun scaffolds if appropriate sacrificial materials can be identified. The technique, however, may not be easily applied to increasing the pore size of natural polymer scaffolds, as natural polymers tend to be soluble in water before chemical cross-linking.²⁸

Salt leaching is a common means of increasing the porosity and pore size of hydrogels. Salt particles are dispersed throughout the polymer solution and leached out or dissolved away in water once the polymer network has solidified.⁵⁷ This methodology has been tailored to introduce large pores into electrospun PCL scaffolds. Salt particles are introduced to the Taylor cone during the electrospinning process and leached out in water once the scaffold is electrospun.² The resultant scaffolds consist of partially delaminated fiber layers with alternating high- and low-fiber density distribution (Fig. 2). They allow for cell infiltration up to 4 mm into the scaffold, but cell distribution is heterogeneous. This technique would benefit from improvements in the control of salt particle delivery, as it results in non-uniform pore distribution. This is likely to be feasible, as salt particle delivery has been studied and optimized for many years in hydrogel systems.

FIG. 2.

Electrospun PCL scaffold produced using electrospinning and salt leaching. (A) An electrospun PCL scaffold containing salt particles and (B) after salt particle removal. Image adapted from ref.²

Gas foaming is another technique commonly applied to hydrogel materials to increase their porosity and pore size. A chemical blowing or foaming agent is introduced into the polymer solution and the nucleation and growth of gas bubbles that result from the decomposition of the blowing agent creates pores in the polymer network.⁵⁷ A chemical blowing agent in a PCL solution can increase the pore size of electrospun PCL scaffolds.⁵⁸ The final scaffold is fibrous with regions of 50–70 μm holes resulting from fibers dispersed by the blowing agent. Fibroblasts interact with these modified scaffolds, but with no apparent cell infiltration into the scaffold. The blowing agent can have a negative impact on the electrospinning process and requires modifications to the electrospinning setup. Further, it results in fiber morphology changes and creates heterogeneous porosity that does not necessarily improve cell infiltration. Similar to salt leaching, this technique could benefit from optimization by using the information from the well-established hydrogel field.

Cryogenic electrospinning involves simultaneous deposition of polymer fibers and ice crystals onto a chilled collector during the electrospinning process.^12,59 After electrospinning, ice crystals are removed by lyophilization, thus leaving behind pores ranging between 10 and 500 μm. The principles behind this methodology are quite simple, but difficulties arise in achieving the correct balance between fiber deposition and ice crystal formation, and it is reliant on tight regulation of the chamber temperature and humidity during the electrospinning process.¹² When such balance is not achieved, the scaffold separates into layers of high- and low-fiber density, resulting in heterogeneous scaffold morphology.⁵⁹

Miscellaneous techniques

A hybrid process utilizing direct polymer melt deposition (DPMD) and electrospinning has been investigated by one group to improve the scaffold pore size, while retaining the cell adhesive and ECM mimicking properties of electrospun scaffolds.⁶⁰ DPMD involves extrusion of thick, microscale polymer fibers (400 μm diameter in this case) through a nozzle using compressed air. The system fabricates 3D log-like PCL fibers that determine the overall 3D architecture of the scaffold. Layers of DPMD-generated fibers are interdispersed with nanoscale electrospun collagen and PCL fibers (Fig. 3). This novel combination of different scaffold generation techniques brings together beneficial features from both methodologies, but the fiber morphology differs considerably from native ECM fibers. Further, PCL has to be heated to 150°C to obtain a working solution for DPMD. Such temperatures could not be feasibly applied to proteins without causing protein denaturation and loss of function. However, this technique is a good way of generating hybrid synthetic and natural polymer scaffolds, where synthetic polymers are processed through DPMD and natural polymers are electrospun.

FIG. 3.

DPMD and electrospinning as a means of increasing the scaffold pore size. (A) Schematic representation of the fiber deposition technology, (B) schematic representation of the scaffold consisting of microscale fibers (left) produced by DPMD and nanoscale fiber (middle) produced by electrospinning, and (C) final scaffold produced by this technology. Image adapted from ref.⁶⁰ DPMD, direct polymer melt deposition. Color images available online at www.liebertonline.com/teb

A novel approach toward increasing cell density inside electrospun scaffolds without changing scaffold pore size involves concurrent polymer electrospinning and cell electrospraying. A proof of principle study was performed by electrospinning poly(ester urethane) urea (PEUU) and electrospraying of vascular smooth muscle cells to obtain cell-infiltrated scaffolds. The final scaffold is impregnated with viable cells that are maintained up to 7 days in cell culture.⁶¹ Although successful cell-impregnated scaffolds were demonstrated, the setup described is complex and maintenance of cell viability and sterility under such circumstances is an issue. Further, PEUU does not require postelectrospinning modifications, such as cross-linking, in contrast to most natural polymers.²⁸ It is likely that uncross-linked electrospun proteins would not maintain their fiber morphology when exposed to aqueous cell culture media in a similar electrospinning setup.

Quantifying the Porosity and Pore Size of Electrospun Scaffolds

Research into techniques aimed at increasing the porosity and pore size of electrospun scaffolds has seen major progress in the past 5 years, and new techniques are constantly emerging. However, an area of research that would benefit from major advancements involves techniques to quantify the porosity and pore size of electrospun scaffolds. Currently, well-established techniques used to characterize other scaffold types, such as hydrogels and ceramics, are typically adapted to electrospun scaffolds. These include mercury porosimetry, liquid intrusion, and gravimetry. However, the applicability of these techniques to electrospun scaffolds is questionable.

Mercury porosimetry and liquid intrusion rely on intrusion of mercury or ethanol into the scaffold void volumes (pores), whereas gravimetry calculates porosity as a difference between predicted scaffold density and the polymer density. These techniques are well suited for scaffolds with large, distinct, solid pores, but not for electrospun scaffolds. Since the pores of electrospun scaffolds are not solid similar to those of hydrogels or ceramics, they expand due to fiber flexibility on mercury intrusion.⁴⁵ Further, mercury often cannot enter pores smaller than 4 μm without pressures that cause scaffold collapse, thereby underestimating scaffold porosity.³ In contrast, liquid intrusion overestimates scaffold porosity as a result of ethanol diffusion into the fibers.³

Microscopy techniques such as confocal lazer scanning microscopy (CLSM) and computed microtomography (CMT) are increasingly utilized to quantify scaffold porosity and pore size, but CLSM is limited by the low scanning depth and CMT by low resolution.²⁶ Improvements in technological ability, such as the introduction of nano X-ray tomography, may overcome resolution problems encountered with the currently available techniques. In the meantime, a number of groups are using combinations of multiple techniques to quantify scaffold porosity, as well as introducing a range of inventive techniques. For example, Soliman and colleagues⁴ proposed an additional method of determining scaffold porosity, which involved measuring the percent void fraction in top-view scanning electron micrographs of electrospun scaffolds. Scanning electron microscope (SEM) images were converted to black and white binary images, and the percentage of white pixels in each image was determined. We quantified the porosity of electrospun elastin scaffolds by using a similar technique but with the help of scaffold cross-sections rather than SEM images of scaffold surfaces.¹¹

Conclusions

Electrospinning has emerged as one of the leading scaffold fabrication techniques in the tissue-engineering field. It is a simple, convenient, and inexpensive means of fabricating scaffolds consisting of nano- to micro-scale polymer fibers resembling the fibers of native ECM. However, an inherent limitation of electrospun scaffolds is the relatively small pore size that does not promote cellular infiltration and tissue ingrowth into the scaffold and, therefore, limits the utility of electrospun constructs for widespread tissue-engineering applications. Innovative techniques were consequentially developed in the past decade to increase the pore size of electrospun scaffolds. These techniques range from simple parameter alterations in the electrospinning setup, through postelectrospinning modifications that largely stem from hydrogel and other scaffold fabrication technologies, to a number of highly innovative techniques that successfully bridge electrospinning with other novel technologies in the tissue-engineering field.

This area of research is relatively new, and most techniques have so far only been tested on a limited number of synthetic polymers, with very little information on how they apply to proteins and other natural polymers. However, considering the potential of electrospinning, its relative merits compared with other scaffold fabrication techniques, and the progress made thus far, the development of a comprehensive array of methodologies for increasing the pore size of electrospun scaffolds is only a matter of time.

Footnotes

Acknowledgments

This work was supported by grants from the Australian Research Council and the National Health and Medical Research Council. The authors acknowledge the facilities as well as scientific and technical assistance from staff in the AMMRF (Australian Microscopy and Microanalysis Research Facility) at the Australian Center for Microscopy and Microanalysis, The University of Sydney. They are grateful to Ms. Yannie Poon for technical assistance.

Disclosure Statement

No competing financial interests exist.

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