Strategies to Tune Electrospun Scaffold Porosity for Effective Cell Response in Tissue Engineering

Abstract

Tissue engineering aims to develop artificial human tissues by culturing cells on a scaffold in the presence of biochemical cues. Properties of scaffold such as architecture and composition highly influence the overall cell response. Electrospinning has emerged as one of the most affordable, versatile, and successful approaches to develop nonwoven nano/microscale fibrous scaffolds whose structural features resemble that of the native extracellular matrix. However, dense packing of the fibers leads to small-sized pores which obstruct cell infiltration and therefore is a major limitation for their use in tissue engineering applications. To this end, a variety of approaches have been investigated to enhance the pore properties of the electrospun scaffolds. In this review, we collect state-of-the-art modification methods and summarize them into six classes as follows: approaches focused on optimization of packing density by (a) conventional setup, (b) sequential or co-electrospinning setups, (c) involving sacrificial elements, (d) using special collectors, (e) post-production processing, and (f) other specialized methods. Overall, this review covers historical as well as latest methodologies in the field and therefore acts as a quick reference for those interested in electrospinning matrices for tissue engineering and beyond.

Keywords: 3D printing; air impedance; anisotropic pores; electrospray; gas foaming; laser ablation; liquid bath collector; sacrificial fibers; salt leaching; ultrasonication.

Figure 1

Trends in electrospinning: Number of publications on electrospinning has seen an exponential increase in recent years (a), and the areas of applications have been diverse across various disciplines (b) (publication trend was obtained from WoS Core Collection, search string “electrospinning” in “Title”, time span “1984–2019”).

Figure 2

Electrospinning setup and working principle: A typical setup includes a syringe pump, a syringe with blunt end needle, a high voltage power supply, and a grounded collector (a). In principle, upon applying high voltage, the electrostatic repulsions dominate the surface tension and lead to the formation of a smooth continuous jet (b). Before the jet reaches the grounded collector, the solvent evaporates and thus results in the formation of fibrous network.

Figure 3

Electrospun matrices in tissue engineering: (a) Tissue engineering deals with a combination of cells, natural or synthetic scaffolds, and physiological factors to build a three-dimensional living tissue construct that mimics native tissue. (b) By electrospinning technique, different forms of scaffolds can be prepared with natural/synthetic materials that can be used in tissue engineering applications.

Figure 4

Porosity challenge in electrospun scaffolds: (a) Dense packing of electrospun fibers reduces cell infiltration and therefore lead to suboptimal cell response in the conventional setup. (b) By any modification in the process setup, any enhancement in the scaffold porosity would enrich cell infiltration and subsequent cell–cell and cell–matrix interactions and therefore would lead to optimal cell response.

Figure 5

Conventional electrospinning parameters affecting scaffold porosity: solvent properties, polymer properties, processing additives, flow rate, voltage applied, needle gauge, distance from jet evolution point to deposition point, and other parameters such as ambient temperature and humidity are conventionally known to influence the fiber diameter, density and therefore the porosity.

Figure 6

Flow rate induced changes in porosity of electrospun synthetic human elastin scaffold: Increase in flow rate led to an increase in pore diameter and porosity as evident from Image J analysis of SEM pictures (a: processed data, b: cross-section images). Dermal fibroblast cells largely remained on the surface in case of low porous scaffold, whereas the cells infiltrated deep in case of high porous scaffold (c: fluorescence images, blue: DAPI staining, red: scaffold autofluorescence). Adapted with permission from Rnjak-Kovacina et al., 2011 [50] Copyright ©Elsevier 2011.

Figure 7

Schematic of sequential and concurrent electrospinning approaches: Two-syringe system can be sequentially used to fabricate layer-by-layer structure of micro- and nanofibers (a) or can be concurrently used to fabricate mixed fibrous structure (c). Alternatively, single syringe system loaded with two or more portions of variably concentrated polymer solutions can be explored to fabricate a structure with gradient structure (b).

Figure 8

Cell response on a micro/nanofibrous mat prepared by dual electrospinning approach: Actin cytoskeleton staining indicated that MC3T3-E1 cell response was higher on micro/nanofibrous poly lactic-co-glycolic acid (PLGA)-Col (b,d) and PLGA-Col-HA (c,e) scaffolds than on microfibrous PLGA scaffold (a). The same observation was confirmed quantitatively by cell viability assay (f). Adapted from Kwak et al., 2016 [57] © The Authors 2016.

Figure 9

Schematic of approaches involving sacrificial elements: Fibrous sacrificial component could be incorporated by sequential or concurrent electrospinning along with the fibrous component of interest (a). Alternatively, particulate sacrificial component could be incorporated either by direct deposition (b) or by sequential or concurrent electrospraying (c).

Figure 10

Porosity and cellular infiltration in scaffolds prepared with sacrificial elements: SEM images of PEO/PLLA (75% PEO) before fiber removal (a) and after fiber removal (b), particulate matter was indicative of biomineralization). Cellular infiltration was complete throughout the scaffold after removal of sacrificial fibers (c). Adapted with permission from Whited et al 2011 [63] Copyright ©Elsevier 2011.

Figure 11

Schematic of approaches involving special collectors: a variety of collectors such as rotating frame cylinder (a), patterned grid (b), needle-like array dish (c), cryogenic plate/mandrel (d), and liquid bath collector (e), have been used in order to produce electrospun scaffolds with enhanced porosity.

Figure 12

Cell response on electrospun scaffolds prepared by patterned collectors: Compared to the conventional electrospun scaffold (a), the scaffolds prepared by patterned collectors were having zones with a lower fiber density of 10-fold increased porosity (d,g), and as a result, fibroblasts infiltration was relatively enhanced (b,c: normal, e,f: and h,i: patterned). Adapted with permission from Vaquette and Cooper-White, 2011 [76]. Copyright ©Elsevier 2011.

Figure 13

Schematic of approaches involving post-production processing: Controlled exposure of the electrospun scaffold to ultrasonic treatment (a), incorporation of sodium borohydride or similar salt in the scaffold and subsequent incubation in appropriate bath to allow gas foaming (b), and controlled laser ablation treatment (c) are found to be effective in manipulating the pore size after the production.

Figure 14

Laser ablation approach to enhance the porosity in electrospun scaffolds: Femtosecond laser ablation approach created pores of defined size without any fiber melting and blockage of porous structure. Adhesion, morphology, and viability of human mesenchymal stem cells (hMSCs) were influenced by pore sizes created by laser ablation (a–f: SEM images, g–m: fluorescence images, n: cell proliferation data). Adapted with permission from Lee et al., 2012 [92] Copyright ©Elsevier 2012.

Figure 15

Schematic of electrospinning approaches involving unique concepts: Alternative electrospinning and electrospraying process (a), emulsion electrospinning method (b), and alternative 3D printing and electrospinning approach (c) are reported to yield scaffolds with relatively superior pore properties.

Figure 16

Macro and microporous scaffolds prepared by a combination of electrospinning and 3D printing: Macroscopic images of 3D printed grid without and with patterned and classical electrospun mat (a–c respectively). SEM images of 3D printed grid with patterned electrospun mat (d,e—low magnification, g—high magnification), and classical electrospun mat (f—low magnification, h—high magnification). Adapted from Rampichova et al. 2018 [98].