Received: 4 March 2019 Revised: 8 May 2019 Accepted: 10 May 2019 DOI: 10.1002/term.2911 RESEARCH ARTICLE Unidirectional neuronal cell growth and differentiation on aligned polyhydroxyalkanoate blend microfibres with varying diameters Lorena R. Lizarraga‐Valderrama1 John W. Haycock2 | | Caroline S. Taylor2 Jonathan C. Knowles3,4,5,6 | | Frederik Claeyssens2 | Ipsita Roy1 1 Applied Biotechnology Research Group, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, London, UK 2 Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK Abstract Polyhydroxyalkanoates (PHAs) are a family of prokaryotic‐derived biodegradable and biocompatible natural polymers known to exhibit neuroregenerative properties. In this work, poly(3‐hydroxybutyrate), P(3HB), and poly(3‐hydroxyoctanoate), P(3HO), have been combined to form blend fibres for directional guidance of neuronal cell 3 Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, London, UK 4 Department of Nanobiomedical Science and BK21 Plus NBM, Global Research Center for Regenerative Medicine, Dankook University, Cheonan, South Korea growth and differentiation. A 25:75 P(3HO)/P(3HB) blend (PHA blend) was used for the manufacturing of electrospun fibres as resorbable scaffolds to be used as internal guidance lumen structures in nerve conduits. The biocompatibility of these fibres was studied using neuronal and Schwann cells. Highly aligned and uniform fibres with varying diameters were fabricated by controlling electrospinning parame- 5 The Discoveries Centre for Regenerative and Precision Medicine, UCL Campus, London, UK 6 UCL Eastman‐Korea Dental Medicine Innovation Centre, Dankook University, Cheonan, South Korea Correspondence Ipsita Roy, Applied Biotechnology Research Group, School of Life Sciences, College of Liberal Arts and Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK. Email: i.roy01@westminster.ac.uk Funding information Neurimp, Grant/Award Number: No. 604450 604450 ters. The resulting fibre diameters were 2.4 ± 0.3, 3.7 ± 0.3, and 13.5 ± 2.3 μm for small, medium, and large diameter fibres, respectively. The cell response to these electrospun fibres was investigated with respect to growth and differentiation. Cell migration observed on the electrospun fibres showed topographical guidance in accordance with the direction of the fibres. The correlation between fibre diameter and neuronal growth under two conditions, individually and in coculture with Schwann cells, was evaluated. Results obtained from both assays revealed that all PHA blend fibre groups were able to support growth and guide aligned distribution of neuronal cells, and there was a direct correlation between the fibre diameter and neuronal growth and differentiation. This work has led to the development of a family of unique biodegradable and highly biocompatible 3D substrates capable of guiding and facilitating the growth, proliferation, and differentiation of neuronal cells as internal structures within nerve conduits. K E Y W OR D S electrospun fibres, nerve regeneration, peripheral nerves, polyhydroxyalkanoates, topographical guidance -------------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd J Tissue Eng Regen Med. 2019;13:1581–1594. wileyonlinelibrary.com/journal/term 1581 LIZARRAGA‐VALDERRAMA 1582 1 | I N T RO D U CT I O N ET AL. adhesion, proliferation, and differentiation, so the scaffold can become a mature and functioning construct. In the body, axons are surrounded Engineered scaffolds are designed to closely mimic the topography, by uniaxial aligned lipoprotein sheaths composed of myelin (Haycock, spatial distribution, and chemical cues corresponding to the native 2011). For this reason, research in scaffolds for nerve tissue extracellular matrix (ECM) of the intended tissue in order to support engineering consists predominantly of 3D structures based on aligned cell growth and differentiation. In tissue engineering, both three‐ fibres. Electrospinning is a versatile manufacturing method used to dimensional (3D) and two‐dimensional (2D) cell cultures are used. produce random or aligned fibres with either nanoscale or microscale Porous scaffolds facilitate mass transfer and exchange of nutrients, diameters metabolites, and gases. Additionally, their high surface area enhances electrospinning is an ideal technique to reproduce aligned fibres to cell adhesion and their interconnected porosity enables 3D cell mimic the extracellular matrix environment of neuronal cells and serve ingrowth, which can be spatially controlled. Although the use of scaf- as a nucleating environment. Additionally, this technique is also used folds with cocultures in 3D has been widely applied to regenerate a to produce fibrous structures with random distribution, characteristic broad variety of tissues, these techniques have been scarcely used of the native ECM fibres found in majority of tissues such as the for nerve tissue regeneration. Three‐dimensional culture techniques breast, liver, bladder, and lung (Cai, Yan, Liu, Yuan, & Xiao, 2013). using a vast diversity of materials. Therefore, would not only allow a better understanding of neuron–glial cell com- It has been shown that hollow NGCs are able to bridge nerve stumps munication but could also contribute towards the development of resulting from severed nerves when the gaps are less than 10 mm by scaffolds for peripheral nerve regeneration (Daud, Pawar, Claeyssens, facilitating the formation of a fibrin cable. This fibrin cable supports Ryan, & Haycock, 2012). the migration of Schwann cells permitting the recreation of longitudi- The use of nerve guidance conduits (NGCs) to reconnect peripheral nally oriented bands of Büngner, which are aligned columns of Schwann nerve gaps has been extensively investigated in the last 20 years. Nota- cells and laminin. In this way, bands of Büngner serve not only as a ble efforts have been made to overcome the limitations of using the source of neurotrophic factors but also as a guiding substrate that pro- standard treatment, autografting, including donor site morbidity, scar tis- motes axonal regrowth (Kim, Haftel, Kumar, & Bellamkonda, 2008). sue formation, scarcity of donor nerves, inadequate return of function, In vitro cells have been shown to respond differently to diverse and aberrant regeneration. Although some NGCs made from natural topographic scales, for example, nanoscale, microscale, and macroscales. and synthetic materials have been clinically approved, the regeneration Changes in the topography of a substrate can alter the biological behav- obtained with them is only comparable with that using autologous grafts iour due to different sensitivity scales of cells as a consequence of the when the gaps are short (less than 5 mm). Commercial NGCs are all hol- variability in cell sizes, cell matrix, and filopodia (Sun et al., 2006). Several low tubes and can induce scar tissue and release compounds detrimen- studies have demonstrated that aligned electrospun fibres can provide tal for the nerve regeneration process. Several research groups have contact guidance to cultured cells, particularly leading to the elongation investigated the introduction of structures within the lumen to improve and alignment of cells along the axes of fibres. The resulting elongation neuronal regeneration such as luminal filaments, fibres, and multichannel along the fibres emulates the structure of bands of Büngner (Chew, Mi, structures (de Ruiter, Malessy, Yaszemski, Windebank, & Spinner, 2009; Hoke, & Leong, 2007). Furthermore, it has been shown that aligned Jiang, Lim, Mao, & Chew, 2010). fibres are able to induce the orientation of focal adhesion contacts Schwann cells are the glial cells of the peripheral nervous system. They insulate axons through wrapped layers of the myelin membrane, and the cell actin cytoskeleton through contact guidance (Badami, Kreke, Thompson, Riffle, & Goldstein, 2006). permitting and accelerating impulse conduction, compared with unmy- A wide diversity of biodegradable materials has been used for the elinated axons. It is well known that the two‐way communication manufacturing of scaffolds for nerve tissue engineering applications. between neurons and glial cells is crucial for normal functioning of Fibres of both synthetic polymers (aliphatic polyesters, polylactic the nervous system. Axonal conduction, synaptic transmission, and acids, and polycaprolactones [PCLs]) and natural polymers (gelatin information processing are controlled by neuron–glial interaction. and silk) have been already used as lumen modifications of NGCs. Neurons and glia communicate through cell adhesion molecules, The use of biodegradable polymers for the manufacture of implants neurotransmitters, ion fluxes, and specialized signalling molecules, avoids a second surgery after implantation. After the implant is whereas glial–glial cell communication relies on intracellular waves of sutured at the nerve stumps, the scaffold is populated and remodelled calcium and intracellular diffusion of chemical messengers (Fields & by neuronal cells and eventually replaced by native tissue; hence, the Stevens‐Graham, 2002). original function can be restored. Although polyhydroxyalkanoates As the design of scaffolds should reproduce the tissue of interest, (PHAs), in particular poly(3‐hydroxybutyrate) (P(3HB)), have also been the native environment of neurons must be taken into consideration investigated for peripheral nerve regeneration applications (Hart, (Haycock, 2011). Although tissue‐engineered scaffolds may not Wiberg, & Terenghi, 2003; Hazari, Wiberg, Johansson‐Rudén, Green, exactly reproduce the target tissue, they have shown to provide a & Terenghi, 1999; Mohanna, Terenghi, & Wiberg, 2005; Mosahebi, “nucleation structure” that trigger cellular self‐organization (Sun, Wiberg, & Terenghi, 2003; Mosahebi, Fuller, Wiberg, & Terenghi, Jackson, Haycock, & MacNeil, 2006). In tissue engineering, the main 2002; Mosahebi, Woodward, Wiberg, Martin, & Terenghi, 2001; Ren approach is to generate this “nucleating environment” in which 3D et al., 2013), studies have focused on the fabrication of hollow NGCs structures contain enough information for permitting cellular without lumen modifications. LIZARRAGA‐VALDERRAMA ET AL. 1583 The aim of this work was to manufacture 25:75 poly(3‐ hydroxyoctanoate) (P(3HO))/P(3HB) blend (PHA blend) electrospun 2.4 | Manufacturing of aligned P(3HO)/P(3HB) blend fibres by electrospinning fibres as resorbable scaffolds for their use in the manufacture of inner guidance fibres to be inserted in the lumen of NGCs. An in‐depth Electrospinning was performed using a high voltage power supply study on the effect of the PHA blend fibre diameter on cell growth (Genvolt, UK) with a syringe pump (WPI, USA) and a rotating and differentiation of NG108‐15 neuronal and RN22 Schwann cells cylindrical collector (IKA, UK). A 1‐ml plastic syringe (Intertronics, was carried out. The choice of this particular PHA blend was driven UK) with a blunt needle (20 G) connected to the power supply by our work that we have published earlier (Lizarraga‐Valderrama was used to fabricate the fibres. All the fibres were collected on a et al., 2015), in which this blend was shown to be the most biocompat- sheet of aluminium foil, which was used to wrap the electrically ible with respect to neuronal cells when compared with the widely grounded collector. Aligned fibres of PHA blend were produced commercialized PCL and other P(3HO)/P(3HB) blend compositions. using varying polymer concentrations (15%, 25%, and 30% and The biodegradation product resulting from the breakdown of 35% w/v) dissolved in chloroform under different voltage conditions P(3HB), 3‐hydroxybutyric acid, is a known natural metabolite found (12 and 18 kV) and collector speed (1,500 and 2,000 rpm). All PHA in the human body, hence is expected to have minimal toxicity and solutions were electrospun at a distance of 10 cm from the collec- immunogenic response (Newman & Verdin, 2014). Also, the hydrolytic tor for 1 min with a syringe pump flow rate of 1 ml/hr. The degradation of P(3HO) leads to the formation of 3‐hydroxyoctanoyl‐ electrospun sheet dimensions after electrospinning were 5 cm × 20 cm, CoA, which is a natural metabolite found in the fatty acid beta from which squares of 1.5 cm × 1.5 cm were removed for SEM oxidation pathway. Here, the enzyme 3‐hydroxyacyl‐CoA dehydroge- analysis and cell culture experiments. Table 1 summarizes the pro- nase can convert it to the corresponding enoyl‐CoA derivative cessing conditions used to obtain PHA blend fibres with different (Houten & Wanders, 2010). diameters using varying polymer concentrations (15, 25, 30, and 35 wt%). Three representative diameters were chosen for cell cul- 2 ture studies on the basis of the uniformity in fibre size distribution: MATERIALS AND METHODS | large (13.5 ± 2.3 μm), medium (3.7 ± 0.3 μm), and small (2.4 ± 0.3 μm). 2.1 | Production and extraction of P(3HO) and P(3HB) Production, extraction, and purification of P(3HO) and P(3HB) were performed as described previously (Rai, Keshavarz, Roether, Boccaccini, & Roy, 2011). The determination of lipopolysaccharides was carried out as described by Rai et al. (2011). 2.5 | Characterization of aligned P(3HO)/P(3HB) blend fibres by SEM Three separated batches of samples belonging to the three fibre groups with different diameters were characterized using a Philips 2.2 | Film preparation FEI XL30 Field Emission Gun Scanning Electron Microscope (Philips, Netherlands). Three parameters were analysed: (a) fibre diameter, Films of PHA blends and PCL were prepared using the solvent‐casting (b) fibre density, and (c) fibre alignment. An average of 27 fibres method reported previously (Lizarraga‐Valderrama et al., 2015). per fibre group were studied for the determination of fibre diameter and fibre alignment. The same images used to determine parameters 2.3 Scanning electron microscopy of films and scaffolds | (a) and (c) were analysed to measure the density, which was defined as the number of fibres per unit area of the image. Fibre alignment was determined by measuring the angular variance between the Surface topography of the films and electrospun fibres was analysed fibres. Therefore, a reference line was drawn parallel to a central using scanning electron microscopy (SEM) as described previously fibre, from which the angular difference was measured with respect (Lizarraga‐Valderrama et al., 2015). to each fibre across the sample. The resulting data were collected TABLE 1 Summary of electrospinning conditions used to manufacture aligned P(3HO)/P(3HB) blend fibres of varying diameters using different polymer concentrations Fibre diameter Voltage (kV) Collector speed (RPM) 12 18 Polymer concentration 15 wt% 25 wt% 30 wt% 35 wt% 2,000 2.4 ± 0.3 3.5 ± 0.3 4.3 ± 0.2 12.1 ± 3.6 1,500 3.5 ± 0.4 4.1 ± 0.3 4.4 ± 0.3 13.5 ± 2.3 2,000 3.1 ± 0.3 3.4 ± 0.3 4.4 ± 0.8 14.0 ± 3.5 1,500 3.4 ± 0.2 3.7 ± 0.3 5.1 ± 0.9 15.9 ± 8.0 LIZARRAGA‐VALDERRAMA 1584 ET AL. in four groups classified by their angular difference (±0°, 1°, 2°, Sigma‐Aldrich, Gillingham, UK). Experimentally differentiated neuronal and 3°). cells were then counted using ImageJ and identified as neuronal cells expressing neurites. 2.6 | NG108‐15 neuronal cell culture 2.9 | Statistical analysis NG108‐15 neuronal cells were prepared as described previously Statistical analysis was conducted using GraphPad Prism 6 software. (Daud et al., 2012). A Shapiro–Wilk and Bartlett's test was previously performed to verify 2.7 | Live/dead measurement of NG108‐15 neuronal cells Live and dead cell measurements were carried out as previously the normality and homogeneity of the data. To analyse the difference between data, a one‐way analysis of variance test (p < .05) was conducted followed by Tukey's posttest (p < .05). Data were reported as mean ± SEM. reported (Daud et al., 2012). 3 2.8 | Immunolabelling of NG108‐15 neuronal cells and RN22 Schwann cells | RESULTS 3.1 | Physical characterization of aligned PHA blend microfibres by SEM To assess the differentiation of neuronal cells, samples were immunolabelled using β‐III‐tubulin as the primary antibody and with Three scaffolds consisting of aligned PHA blend microfibres with Alexa Fluor® 488 goat antimouse IgG as the secondary antibody varying diameters were manufactured by electrospinning using three (Sigma‐Aldrich, Gillingham, UK). The samples containing cultures of different processing conditions summarized in Table 1. The fibre NG108‐15 neuronal cells were washed with phosphate buffered saline architecture was studied using a field emission scanning electron (PBS; ×3; Sigma‐Aldrich, Gillingham, UK) and fixed with 4% (v/v) microscope to analyse three physical features using the NIH ImageJ paraformaldehyde for 20 min. Then they were permeabilized with software: (a) fibre diameter, (b) fibre density, and (c) fibre alignment. 0.1% (v/v) Triton X‐100 (Sigma‐Aldrich, Gillingham, UK) for 20 min, SEM micrographs of small, medium, and large fibres are shown in before being washed with PBS (×3). Unreactive binding sites Figure 1a,b; Figure 1c,d; and Figure 1e,f, respectively. After analysis were blocked with 3% (w/v) bovine serum albumin (BSA; Sigma‐Aldrich, of the scaffolds, the resulting average fibre diameters for small, Gillingham, UK) with the cells being incubated overnight with mouse medium, and large fibre groups were measured to be 2.4 ± 0.3, anti‐β‐III‐tubulin antibody (1:1000; Promega, Madison, USA) diluted in 3.7 ± 0.3, and 13.5 ± 2.3 μm, respectively (Figure 1g). Statistical anal- 1% BSA at 4°C. Cells were then washed three times with PBS before ysis showed that the difference in fibre diameter between the small ® being incubated either with Alexa Fluor 488 goat antimouse IgG anti- and medium fibre group compared with the large fibre group was bodies (1:200 in 1% BSA; Sigma‐Aldrich, Gillingham, UK) or Texas Red‐ significant (2.4 ± 0.3 vs. 13.5 ± 2.3 μm, *p < .05, and 3.7 ± 0.3 vs. conjugated antimouse IgG antibody (1:100 dilution in 1% BSA) for 13.5 ± 2.3 μm, **p < .05). 90 min (Sigma‐Aldrich, Gillingham, UK). After washing the cells once Fibre density determination was also carried out on the basis of with PBS, 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI; the SEM micrograph images. It can be seen in Figure 1h that the 1:500 dilution in PBS; Sigma‐Aldrich, Gillingham, UK) was added to label medium‐sized fibre group (0.30 ± 0.03 fibres per micrometre) nuclei along with phallodin‐FITC (1:1000 dilution in PBS; Sigma‐Aldrich, exhibited the highest fibre density compared with the small Gillingham, UK) to label RN22 Schwann cells when required. Cells (0.22 ± 0.04 fibres per micrometre, *p < .05) and the large fibre groups were then incubated for 30 min at room temperature before being (0.21 ± 0.02 fibres per micrometre, **p < .05). However, no significant washed again with PBS (×3). Cells were then imaged using an difference was found between the small and large fibre groups. upright Zeiss LSM 510 confocal microscope (Zeiss, Oberkochen, Fibre alignment estimation was assessed by measuring the Germany) in the Kroto Research Institute imaging facility at University angular difference between the fibres and an assigned central refer- of Sheffield. Nuclei were visualized by two‐photon excitation using a ence line. For each size fibre group, an average of 27 fibres was Ti:sapphire laser (716 nm) for DAPI (λex = 358 nm/λem = 461 nm; analysed to determine the mean of each angular difference group Sigma‐Aldrich, Gillingham, UK). For imaging the neuronal cell body and and is presented in a histogram (Figure 1i). neurites, a helium–neon laser (543 nm) was used to detect Texas Red‐ A major proportion of fibres for the three size groups presented an conjugated antimouse IgG antibody (1:100 dilution in 1% BSA; angular difference of 0°. Medium and large fibres showed the highest λex = 589 nm/λem = 615 nm; Sigma‐Aldrich, Gillingham, UK) and an number of fibres with an angular difference of 0° when compared with argon ion laser (488 nm) to detect Alexa Fluor® 488 goat antimouse small fibres (*p < .05 in comparison with medium fibres and large IgG (λex = 495 nm/λem = 519 nm; Sigma‐Aldrich, Gillingham, UK). For fibres). The proportion of fibres with increasing angle of variance imaging RN22 Schwann cell cytoskeleton, argon ion laser (488 nm) dropped markedly, showing that the majority of the fibres were was used to detect phallodin‐FITC (λex = 495 nm/λem = 521 nm; arranged in a straight line and parallel to each other (Figure 1a,c,e,h). LIZARRAGA‐VALDERRAMA ET AL. 1585 FIGURE 1 Characterization of electrospun PHA blend fibres by scanning electron microscopy. Diameter, density, and alignment of fibres were determined by image analysis. (a,b), (c,d), and (e,f) show the micrographs of the electrospun fibre mats corresponding to the small, medium, and large PHA blend fibre diameters, respectively. (g) Graph characterizing the fibre diameter for the different electrospinning conditions. The average diameters for the small, medium, and large fibres were 2.42 ± 0.34, 3.68 ± 0.26, and 13.50 ± 2.33 μm, respectively. A total of approximately 27 fibres were analysed for each fibre diameter group to determine the mean of the diameters (mean ± SD, n = 3 samples independently fabricated, * p < .05 compared with small fibres, **p < .05 in relation to medium fibres). (h) Mean fibre density measurement for each fibre group. The number of fibres contained in an area of 152 μm2 was measured (mean ± SEM, n = 3 samples independently fabricated). (i) Fibre alignment estimation. Angular difference between fibres and an assigned central reference fibre was measured. An average of 27 fibres was analysed for each fibre size group to determine the mean of each angular difference range (mean ± SEM, n = 3 samples independently fabricated, *p < .05 in comparison with medium fibres and large fibres). Scale bar = 50 μm 3.2 | Live/dead measurement of NG108‐15 neuronal cells on PHA blend electrospun fibres The percentage of live cells on small fibres (97.76 ± 0.58%), medium fibres (99.77 ± 0.22%), large fibres (98.87 ± 0.60%), flat PHA blend film (95.89 ± 1.43%), and flat PCL film (94.72 ± 1.68%) Live/dead cell assays were performed in order to compare the attach- was higher when compared with glass (62.28 ± 13.16%, ♦p < .05; ment and survival of NG108‐15 neuronal cells on the electrospun Figure 2g). However, no significant differences in percentage of live PHA blend sheets with varying fibre diameters. In Figure 2, represen- cells were found between the fibre groups. The number of neuronal tative confocal images of neuronal cells grown on different substrates cells that grew on the small (142.20 ± 11.42; *p < .05), medium are shown. (142.67 ± 6.36, **p < .05), and large (188.58 ± 13.00, Figure 2a–c corresponds to cells grown on the small, medium, and large PHA blend fibres, respectively. It can be seen that neuronal cell *** p < .05) fibre groups was higher when compared with that obtained on the glass control (27.20 ± 8.40; Figure 2h). growth had an aligned distribution on all three fibre groups (Figure 2 As seen in Figure 2h, the number of neuronal cells that adhered to a–c). On the other hand, random growth of cells was observed on and grew on the small (142.20 ± 11.42, *p < .05) and large the flat substrates of PHA blend (Figure 2d), PCL (Figure 2e), and glass (188.58 ± 13.00, ***p < .05) fibre groups was higher when compared (Figure 2f), in which several clusters of cells were found. with that obtained on the PHA blend (76.89 ± 5.60) and PCL 1586 LIZARRAGA‐VALDERRAMA ET AL. FIGURE 2 Confocal micrographs of NG108‐15 neuronal cells labelled with propidium iodide (red; dead cells) and Syto‐9 (green; live cells) after 4 days in culture on aligned PHA blend fibres and live/dead cell analysis. (a) Small fibres (2.42 ± 0.34 μm). (b) Medium fibres (3.68 ± 0.26 μm). (c) Large fibres (13.50 ± 2.33 μm). (d) PHA blend film. (e) PCL film. (f) Glass. Scale bar = 50 μm. (g) Live/dead analysis of neuronal cells on fibres with varying diameters and controls. Percentage of live neuronal cells on the fibres, PHA blend film, and PCL film was higher in comparison with glass (control; mean ± SEM, n = 9 independent experiments, ♦p < .05). (h) Histograms showing numbers of neuronal cells on fibres with varying diameters and controls [Colour figure can be viewed at wileyonlinelibrary.com] (78.00 ± 12.04) flat substrates (Figure 2h). No significant difference was found between the number of cells grown on medium fibres (140.30 ± 10.22) when compared with those grown on either small 3.3 | Neurite outgrowth assessment on NG108‐15 neuronal cell culture grown on PHA blend electrospun fibres or large fibres. Large fibres supported the highest number of neuronal cells (188.58 ± 13.00) when compared with the controls (PHA blend, NG108‐15 neuronal cells were grown on the scaffolds and PCL flat substrates, and glass, ***p < .05) and small fibres (*p < .05). immunolabelled with the anti‐β III‐tubulin antibody to assess neurite The number of cells grown on medium fibres was only significantly outgrowth and differentiation. Figure 3a–f shows the confocal images different to the number of cells grown on glass. No significant of immunolabelled neuronal cells grown on all the substrates. difference was found between the number of neuronal cells grown on the controls (PHA blend, PCL, and flat substrates and glass). Differentiation was observed in all the neuronal cells that were positive for anti‐β‐III‐tubulin antibody in all the substrates including LIZARRAGA‐VALDERRAMA ET AL. 1587 FIGURE 3 Confocal micrographs of NG108‐15 neuronal cells inmunolabelled for β‐III‐tubulin after 4 days in culture on aligned PHA blend fibres, PHA, PCL flat substrate, and glass. Nuclei are counterlabelled with DAPI. (a) Small fibres (2.42 ± 0.34 μm). (b) Medium fibres (3.68 ± 0.26 μm). (c) Large fibres (13.50 ± 2.33 μm). (d) PHA blend film (flat substrate). (e) PCL film (flat substrate). (f) Glass. Scale bar = 50 μm. (g) Number of neuronal cells with neurites on P(3HO)/P(3HB) blend fibres, PCL, and glass (control; mean ± SEM, n = 9 independent experiments, p < .05). The number of differentiated cells grown on the large fibre substrate was significantly higher when compared with the small fibre substrate and controls (*p < .05). The number of neuronal cells on medium fibres was significantly different only when compared with that on large fibres and controls (**p < .05). The number of differentiated neuronal cells that grew on large fibres was significantly different when compared with the rest of electrospun fibrous substrates (small and medium) and flat substrates (PHA blend, PCL, and glass; *p < .05, **p < .05, ***p < .05) [Colour figure can be viewed at wileyonlinelibrary.com] the controls: PHA blend (Figure 3d), PCL (Figure 3e), and flat sub- Figure 3g shows the number of differentiated neuronal cells on strates and glass (Figure 3f). Alignment of cell growth was clearly each substrate. The large fibre group displayed the highest number seen on the three fibre groups—small fibres (Figure 3a), medium of differentiated neuronal cells (225.67 ± 24.85) compared with the fibres (Figure 3b), and large fibres (Figure 3c)—whereas a random small (129.13 ± 16.58, *p < .05) and medium (125.33 ± 2.40, distribution of cells was observed in the controls: PHA blend **p < .05) fibre groups. Statistical analysis showed no significant differ- (Figure 3d), PCL (Figure 3e), and flat substrates and glass (Figure 3f). ence between the number of neuronal cells that grew on the small 1588 (129.13 ± 16.58) and medium (125.33 ± 2.40) fibre groups. The number of differentiated neuronal cells on the small (129.13 ± 16.58, *p < .05) and large (225.67 ± 24.85, ***p < .05) fibre groups was found LIZARRAGA‐VALDERRAMA ET AL. 3.4 | Neurite outgrowth assessment on NG108‐15 neuronal cell/Schwann cell cocultures grown on PHA blend electrospun fibres significantly different to those measured on the flat substrates, the PHA blend (79.88 ± 24.49), PCL (51.25 ± 7.87), and glass Confocal micrograph images of NG108‐15 neuronal cells (red) grown (10.13 ± 2.96). However, no significant difference was found between in coculture with RN22 Schwann cells (green) on small fibres the number of differentiated neuronal cells found on the medium fibre (Figure 4a,d,g), medium fibres (Figure 4b,e,h), and large fibres group with respect to PHA blend and PCL flat substrates (Figure 3). No (Figure 4c,f,i) are shown in Figure 4. Only a few Schwann cells were significant difference was found between any of the flat substrates, detected after 4 days in coculture and were observed to attach to all PHA blend, PCL, and glass. In Figure S1, differentiated neuronal cells three fibre groups: small fibres (Figure 4d), medium fibres (Figure 4e), grown on the three fibre groups are shown with a higher magnification and large fibres (Figure 4f). Although only a few Schwann cells were (40×). Neurite‐bearing cells can be observed forming parallel aligned detected, the growth of neuronal cells confirmed that these two cell groups of cells in the three fibre groups: small fibre (Figure S1A–J), lines were able to coexist on all the fibre groups. Alignment of neuro- medium fibre (Figure S1B–K), and large fibre (Figure S1C–L). On the nal cell growth was observed in all fibre groups: small fibres (Figure 4 other hand, clusters of cells were observed on the controls, PHA blend a), medium fibres (Figure 4b), and large fibres (Figure 4c). However, (Figure S2A,D), PCL (Figure S2B,E), and flat substrates and glass random growth was observed on the control substrates: PHA blend (Figure S2C,F) (Figure 5a), PCL (Figure 5b), and flat substrates and glass (Figure 5c). FIGURE 4 Confocal micrographs of NG108‐15 neuronal cells (red) grown with RN22 Schwann cells (green) inmunolabelled for β‐III tubulin and stained with phalloidin and DAPI after 4 days in culture on aligned PHA blend fibres. (a,d,g) Small fibres (2.42 ± 0.34 μm), (b,e,h) medium fibres (3.68 ± 0.26 μm), and (c,f,i) large fibres (13.50 ± 2.33 μm). Alignment of neuronal cells was observed in all three fibre groups. Scale bar = 50 μm [Colour figure can be viewed at wileyonlinelibrary.com] LIZARRAGA‐VALDERRAMA ET AL. 1589 FIGURE 5 Confocal micrographs of NG108‐15 neuronal cells (red) grown with RN22 Schwann cells (green) inmunolabelled for β‐III tubulin and stained with phalloidin (F‐actin) and DAPI (nuclei), after 4 days in culture on flat substrates (controls) consisting of (a,d,g) PHA blend film, (b,i,h) PCL film, and (c,f,i) glass. Cell growth was randomly oriented on the flat substrates, the PHA blend film, PCL film flat substrates, and glass. Scale bar = 50 μm [Colour figure can be viewed at wileyonlinelibrary.com] After analysis of confocal images using ImageJ, all neuronal cells were (274.43 ± 25.36) compared with the number of NG108‐15 grown found to be differentiated on all the substrates: small fibres (Figure 4 on their own (125.33 ± 2.40, p < .05; Figure 6b). Similarly, the number g), medium fibres (Figure 4h), large fibres (Figure 5i), PHA blend of neuronal cells grown on large fibres (225.6 ± 24.85) increased (Figure 5g), PCL (Figure 5h), and flat substrates and glass (Figure 5i). when they were grown with Schwann cells (423.25 ± 16.90, p < .05; Similarly, the number of NG108‐15 neuronal cells that adhered Figure 6b). and grew on the medium fibres (274.43 ± 25.36) was higher than that The number of neuronal cells measured on the P(3HO)/P(3HB) measured on small fibres (191.00 ± 9.50, *p < .05; Figure 6a). The blend control (79.88 ± 24.49) was also significantly different when resulting number of neuronal cells found on the medium (**p < .05) grown along with RN22 (102.00 ± 27.13, p < .05; Figure 6b). No sig- and large fibre groups (***p < .05) was significantly higher than those nificant increment in the number of neuronal cells was found when measured on the control substrates: PHA blend, PCL flat substrates, NG108‐15 were cocultured with RN22 cells on small fibres and glass. Significant differences were also found in the number of (129.13 ± 16.58 vs. 191.00 ± 9.50), PCL (51.25 ± 7.87 vs. 102.00 neuronal cells grown on the P(3HO)/P(3HB) blend (171.00 ± 2.65, 27.13), and glass (10.13 ± 2.96 vs. 16.67 ± 7.04; Figure 6b). • p < .05) and PCL (102.00 ± 27.13 •• p < .05) flat substrates with respect to glass (16.67 ± 7.04). Figure 6b shows the number of neuronal cells grown on all substrates in coculture with Schwann cells 4 | DISCUSSION versus the number of neuronal cells grown on all the substrates without Schwann cells. Interestingly, statistical analysis showed that This investigation shows that the development of a 3D scaffold the number of neuronal cells grown on medium fibres was significantly consisting of aligned microfibres results in the successful alignment higher when these cells were grown in coculture with Schwann cells of neuronal cell growth either individually or in coculture with RN22 LIZARRAGA‐VALDERRAMA 1590 ET AL. FIGURE 6 Number of neuronal cells grown with Schwann cells on all the substrates. Statistical analysis showed that the numbers of neuronal cells found on the three fibre groups was significantly different. Also, the number of neuronal cells grown on the fibre groups increased as the fibre diameter increased (Figure 6a). The number of neuronal cells found on large fibres (423.25 ± 16.90) was significantly higher than those found on small (191.00 ± 9.50, *p < .05) and medium fibres (274.43 ± 25.36, ***p < .05; Figure 6a). (b) Number of neuronal cells grown on their own compared with number of neuronal cells grown in coculture with Schwann cells Schwann cells. Because PHA blends had previously been shown to grown in a 2D culture environment (Edmondson et al., 2014). To display superior biocompatibility with neuronal NG108‐15 cells date, 3D cultures have been used to study more than 380 cell lines compared with the commercial PCL, other PHA blends, and neat (Ravi, Paramesh, Kaviya, Anuradha, & Paul Solomon, 2015). However, PHAs, this blend was chosen for the fabrication of microfibres to be most of cell studies are still based on the use of 2D cultures using flat used for lumen modification of NGCs. Several fibre diameters were and rigid substrates, which considerably differ from the native produced using PHA blends by electrospinning in order to assess the environment of the cells. As a result, it has been shown that 2D cell effect of fibre diameter on the response of neuronal cells. culture assays can provide misleading and nonpredictive data for Three‐dimensional cultures provide an effective approach to in vivo cell behaviour. develop a functional construct that mimics the natural architecture Three‐dimensional cultures influence signal transduction by of a multicellular/tissue environment. In their native environment, affecting the spatial organization of the cell surface receptors involved the majority of cells are surrounded by an extracellular matrix in cell interaction. This will ultimately induce a specific gene expres- together with phenotypically dissimilar stromal cells in a 3D fashion. sion profile, resulting in a particular cell behaviour (Edmondson et al., Three‐dimensional scaffolds should be able to provide physical cues 2014). In spite of the fact that limitations of 2D cultures are well that promote topographical stimuli to trigger cell adhesion, growth, known, only few 3D models have been developed for nerve regener- and differentiation. Also, the scaffold should be designed to permit ation research. A further reduced number of studies are based on the exchange of vital nutrients and gases with the external the use of coculture models using natural polymers to investigate environment. Development of 3D cultures is rapidly gaining relevance peripheral nerve repair with the ultimate aim of manufacturing NGCs. as they mimic more accurately the native environment of cells Several scaffolds have been developed for peripheral nerve regenera- compared with 2D cultures. tion research including porous matrices, random fibres, and hydrogels. Drug discovery research has driven a considerable amount of However, only few studies have described the use of aligned fibres innovations in 3D culture systems over the past 5 years (Edmondson, using more than one cell type (Daud et al., 2012; Wang, Mullins, Broglie, Adcock, & Yang, 2014). Also, tissue engineering, cancer, and Cregg, McCarthy, & Gilbert, 2010). In the majority of the studies, stem cell research have adopted and adapted 3D culture methods to aligned fibres are used and have focused on the use of a single cell obtain more predictable in vivo data. It has been shown that cell type such as primary neurons extracted from dorsal root ganglia responses to 2D and 3D environments are different. Focal adhesion, (Corey et al., 2007), PC12 cells (Mohanna et al., 2005), and primary proliferation, population, differentiation, and gene and protein expres- human Schwann cells isolated from sciatic nerves of human foetuses sion have been shown to be different for the same cell type when (Behbehani et al., 2018; Chew et al., 2007). grown on a 3D scaffold compared with that on a 2D construct Previous studies have shown that the diameter of fibres can affect (Sun et al., 2006). In fact, it has been shown that cells in the 3D culture cell growth and function not only in neuronal cells (Gnavi et al., 2015) environment differ morphologically and physiologically from cells but also in different cell types such as human dermal fibroblasts (Liu LIZARRAGA‐VALDERRAMA ET AL. 1591 et al., 2009) and osteoblasts (Badami et al., 2006). In the case of on all the electrospun fibre sizes had a more uniform arrangement peripheral nerve repair, it is still not clear whether nanofibres or when compared with that on the surface of the PHA blend film. microfibres support better nerve regeneration. Some researchers This finding agreed with previous studies in which electrospun fibres argue that nanofibres should be better scaffolds because their dimen- have been shown to affect cell proliferation, differentiation, and sions and architecture are closer to the native structure of the migration (Daud et al., 2012). No cytotoxic effect was observed extracellular matrix of neurons (Jiang, Mi, Hoke, & Chew, 2014). when neuronal cells were grown on any of the studied substrates. Nevertheless, comparative studies are not conclusive and have shown Thereafter, the correlation between PHA blend microfibre diameter that despite the resemblance of nanofibres to the ECM of neurons, and neuronal growth under two conditions, individually and in cocul- they have not displayed a satisfactory outcome. Optimal results in ture with RN22 Schwann cells, was evaluated. This was investigated nerve regeneration have been reported by using both nanofibres using two types of cell staining, live/dead cell test and anti‐β tubulin (Wang et al., 2010; Wang et al., 2011) and microfibres (Hurtado immunolabelling, to identify neuronal neurite formation. Results from et al., 2011); however, studies of fibre size effect in nerve regenera- both studies revealed that all PHA blend fibre groups were able to tion remain limited. In fact, Yao et al. (2009) fabricated PLLA fibres support growth well and to guide aligned distribution of neuronal cells of varying diameters and found that neurite outgrowth and cell when grown individually and in the presence of RN22 Schwann cells. migration were inhibited when fibres of 200‐nm diameter were used. All the substrates, including the controls, were found to support Conversely, Wen and Tresco found good alignment and outgrowth neurite outgrowth but with different levels of efficiency. Electrospun of neurites on microfibres with diameters ranging from 30 μm fibre substrates were shown to support better both cell growth and (comparable with cellular size) to 5 μm. However, Schwann cell migra- differentiation. Although the counting of the neurite bearing cells tion and neurite outgrowth were inhibited when the diameter of the was not possible, higher abundance of neurite bearing cells was microfibres was greater than 30 μm (Wen & Tresco, 2006). In an observed in the fibrous scaffolds (Figure S1). Although the in vitro study, small‐diameter fibres resulted in a decreased neurite electrospinning technique allows optimal fabrication of aligned fibres length of 42% and 36% compared with the large (1,325 + 383 nm) and there are significant benefits to having tissue engineering and intermediate (759 + 179 nm) diameter fibres, after 5 days of cul- constructs at the length scales, its commercial use is limited due to turing chick dorsal root ganglion (DRG; Wang et al., 2010). However, poor replicability and constraints for scaling‐up. Furthermore, the in an in vivo study, nanofibres exhibited better results compared with need of high voltages and formation of random alignment during microfibres when used as lumen scaffolds in NGCs for repairing 15‐ the process confine this technique to the academic niche. An innova- mm critical defect gaps. Nanofibre conduits (251 ± 32 nm) promoted tive technique, pressurized gyration, established in 2013, could be a significantly higher number of myelinated axons and thicker myelin used to produce more replicable aligned fibres and at much higher sheaths compared with microfibre conduits (981 ± 83 nm). Addition- throughput, for applications in the field of nerve tissue engineering. ally, nanofibre conduits produced an increased number of regenerated Pressurized gyration benefits from an increased number of parameters DRG sensory neurons (1.93 ± 0.71 × 103) compared with microfibre that can be modified for manufacturing large quantities of homoge- 3 conduits (0.98 ± 0.30 × 10 ; Jiang et al., 2014). Conversely, in a 3D nous fibers, resulting in a greater control over the final morphology in vitro model, microfibres with higher diameter (8 μm) supported a (Heseltine, Ahmed, & Edirisinghe, 2018). better outcome of neurites outgrowth than fibres with 5‐ and 1‐μm Results revealed a direct correlation between fibre diameter and diameter. However, when neurons were grown in coculture along with neuronal growth and differentiation. Although neuronal cell viability Schwann cells or as DRG explants, the smallest fibres (1 mm) displayed was similar for all the substrates (approximately 99%) except on superior performance in neurite outgrowth and Schwann cell glass, large fibres supported the highest number of live neuronal cells migration (Daud et al., 2012). In the present study, highly aligned large grown individually compared with the rest of substrates. However, no fibres (13.50 ± 2.33 μm), which displayed the best performance of significant difference was found between the number of live neuronal neurite outgrowth, resemble α‐fibres in diameter (12–22 μm; cells grown on small fibres and medium fibres. A similar outcome was Woessner, 2006), replicating to some extent the microtopography found when neuronal cell differentiation was assessed in the single that surrounds axon fibres inside the fascicles. Despite the cell type culture. The greatest extent of neuronal cell differentiation extensive development of manufacturing techniques in the field of was displayed on large fibres, whereas no significant difference was tissue engineering, the complex mixture of nanotopography and found between small and medium fibres (Figure 6a). Interestingly, microtopography characterizing the neuronal cellular environment when neuronal cells were grown in coculture with RN22 Schwann has not yet been replicated. cells, the number of NG108‐15 cells increased as the fibre diameter In this investigation, highly aligned and uniform PHA blend fibres increased (Figure 6b). These findings correlated with previous studies with varying diameters were successfully fabricated by controlling in which variation on the diameter of electrospun fibres made affected electrospinning parameters summarized in Table 1. In a preliminary the neurite outgrowth (Wang et al., 2010). Ren et al. (2013) found an study, the effect of fibres on the growth of NG108‐15 neuronal cells enhanced differentiation of human neural crest stem cells towards the was investigated by a live/dead test. Cell growth and migration Schwann cell lineage when an aligned electrospun fibre matrix was observed on the electrospun fibres showed directional alignment in used as the substrate. In a similar study, Du et al. (2017) found rapid accordance with the direction of the fibres. The distribution of cells directional cell adhesion and migration of both Schwann cells and LIZARRAGA‐VALDERRAMA 1592 ET AL. DRGs grown on aligned electrospun nanofibre hydrogel matrix. The revealed a direct relationship between fibre diameter and neuronal aligned topography of the matrix accelerated axonal cell outgrowth growth and differentiation. The greatest number of neuronal cells when compared with a random fibre matrix (Du et al., 2017). Wang was displayed on large fibres (13.50 ± 2.33 μm) when grown individu- et al. (2011) grew human embryonic stem cells on Tussah silk fibroin ally and in coculture. The number of NG108‐15 cells increased on all scaffold using both random and aligned orientation with diameters the substrates when cocultured with RN22 cells. Thus, aligned large of 400 and 800 nm, to study the effect of fibre alignment and diame- fibre‐based constructs are a potential alternative for the efficient ter on cell viability and neuronal differentiation. They found that the growth and differentiation of neuronal cells, especially in the context aligned Tussah silk fibroin scaffold with 400‐nm fibres displayed the of inner structures of NGCs, which can act as highly efficient alterna- best neuronal differentiation (Wang et al., 2010). In a similar study, tives to the standard autografting procedures used to repair nerve Panahi‐Joo et al. (2016) fabricated random, semialigned, and highly gaps. In future, because the PHA blend fibres fabricated in this study aligned PCL fibres using two‐pole electrospinning to study attach- have proven to be optimal guidance cues, these can be used as the ment, proliferation, and migration of PC12 neuronal like cells. lumen structures of NGCs. Varying microfibre scaffolds (2.0– They observed both directional growth and elongation in PC12 13.0 μm) will be produced as internal structures within NGCs and neuronal cells when grown on PCL aligned fibres (Panahi‐Joo et al., implanted in 10‐mm defect gaps in median nerves, using rats as 2016). In another study, neural stem cells displayed increased the animal model. Peripheral nerve regeneration will be assessed oligodendrocyte differentiation on 283‐nm fibres, whereas increased after 1, 3, and 6 months of implantation using morphological, neuronal differentiation was observed when grown on 749‐nm fibres morphoquantitative, and stereological analysis. Subject to positive (Christopherson, Song, & Mao, 2009). results, clinical trials will be the final outcome. Although RN22 Schwann cells were scarcely detected, statistical analysis showed a significant increase in the number of NG108‐15 neuronal cells on the three substrates when grown in coculture with Schwann cells (Figure 6b). The number of neuronal cells increased significantly on medium fibres, large fibres, and PHA blend films when cocultured with Schwann cells. These findings suggest that Schwann cells are able to enhance neuronal cell growth significantly. This could be due to the secretion of a combination of neurotrophic factors (e.g., nerve growth factor and/or brain‐derived neurotrophic factor) from Schwann cells into the culture medium with paracrine signalling ACKNOWLEDGEMENTS The authors would like to thank the Department of Materials Science and Engineering (Kroto Research Institute, University of Sheffield, UK); NEURIMP, grant agreement no. 604450, a Framework 7 project funded by the EC; and University of Westminster for providing the facilities, materials, and funding for this research work. The authors would also like to acknowledge Rosa Angelica L. Valderrama and Raul Lizarraga for providing funding for this investigation. to the neuronal NG108‐15 cells. CONFLIC T OF INT E RE ST The authors declare no competing financial interest. 5 | C O N CL U S I O N S ORCID Highly aligned and uniform fibres with varying diameters were successfully fabricated by controlling electrospinning parameters. Preliminary studies were carried out to evaluate the effect of fibres Lorena R. Lizarraga‐Valderrama https://orcid.org/0000-0002-3200- 5733 Ipsita Roy https://orcid.org/0000-0001-5602-1714 on the growth of NG108‐15 neuronal cells by live/dead tests. Cell migration observed on the electrospun fibres showed directional alignment in accordance with the direction of the fibres. The distribution of cells on the electrospun fibres had a more uniform aligned arrangement when compared with that on the surface of the PHA blend films. This finding agreed with previous studies in which electrospun fibres have been shown to affect cell proliferation, differentiation, and migration. Additionally, no cytotoxic effect was found when neuronal cells were grown on any of the studied substrates. Thereafter, the relationship between PHA blend microfibre diameter and neuronal growth under two conditions, individually and in coculture with RN22 Schwann cells, was evaluated. 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A) Neuronal cells inmunolabelled for beta‐III tubulin grown on P(3HB)/ P(3HO) blend flat film. B) Neuronal cells inmunolabelled for beta‐III tubulin grown on PCL. C) Neuronal cells inmunolabelled for beta‐III tubulin grown on glass. D) Neuronal cells inmunolabelled for beta‐III tubulin + DAPI grown on P(3HB) blend flat film. E) Neuronal cells inmunolabelled for beta‐III tubulin + DAPI grown on PCL. F) Neuronal cells inmunolabelled for beta‐III tubulin + DAPI grown on glass. Cell growth was randomly ori- SUPPORTING INFORMATION Additional supporting information may be found online in the ented on each of the flat surfaces and clusters of neuronal cells connected through neurites were observed. Scale bar = 12.5 μm. Supporting Information section at the end of the article. Figure S1. Confocal micrographs of NG108‐15 neuronal cells ummunolabelled for beta‐III tubulin after four days in culture on aligned PHA blend fibres. (A, G), Neuronal cells inmunolabelled for beta‐III tubulin grown on small fibres. (B, H), Neuronal cells inmunolabelled for beta‐III tubulin grown on medium fibres. (C, I) Neuronal cells inmunolabelled for beta‐III tubulin on large fibres 2. (D, J), Neuronal cells ummunolabelled for beta‐III tubulin + DAPI grown on small fibres. (E, K), Neuronal cells inmunolabelled for beta‐III tubulin How to cite this article: Lizarraga‐Valderrama LR, Taylor CS, Claeyssens F, Haycock JW, Knowles JC, Roy I. Unidirectional neuronal cell growth and differentiation on aligned polyhydroxyalkanoate blend microfibres with varying diameters. J Tissue Eng Regen Med. 2019;13:1581–1594. https:// doi.org/10.1002/term.2911