Electrospinning aligned fibers with non-ionic surfactant for additional control of scaffold physical properties, protein adsorption, and cell behavior.

Introduction:: Electrospinning produces extracellular matrix-like scaffolds with tunable properties including degree of alignment for connective fibrous tissue in vitro injury models. Alignment is typically obtained by modulating rotating collector speed; however, other factors like addition of non-ionic surfactant also contribute to fiber alignment and mesh morphology. We have previously shown that non-ionic surfactants in electrospun systems promote fine-tuned control of properties including fiber diameter, morphology, surface roughness and drug encapsulation.^1,2 Further, surfactant in these fibers alters protein adsorption and impacts resulting cell adhesion.¹

Many electrospinning systems require surfactant for modulation of solution properties for specific electrospun fiber morphology and scaffold characteristics. By modulating processing parameters to obtain varying alignment with non-ionic surfactant and identifying impact on protein adsorption and cell morphology, we offer further insight to optimization of these systems to obtained desired surface and bulk properties. Prior results in a control system without surfactant have shown that increased mandrel diameter modifies the impact of processing parameters like collection voltage, collection distance, and rotational speed on fiber diameter and level of alignment. The objective of this study is to elucidate the effects of non-ionic surfactant on fiber alignment, mesh morphology, tensile properties, protein adsorption, and cell interactions. It is hypothesized that the addition of non-ionic surfactant reduces the retained charge in the fibers and in a rotating collector system modifies retained charge polarity significantly to impact protein adsorption and resulting cell morphology.

Materials and Methods:: Sample Collection: Solutions of 20% w/v polycaprolactone (PCL) in chloroform (CHCl₃) for the PCL only and with 30% w/w Span 80 (S80) with respect to PCL for PCL + S80 were prepared. Preliminary samples were spun at 65% RH (±10%) for PCL only and 35% RH (±10%) for PCL+S80, 20℃, for 15 minutes at 30cm collection distance, 15 kVs for PCL only and 12 and 15kVs for PCL+S80, and at 780, and ~1000 RPMs on a ¾”, 1” and 2” diameter spinning mandrel. For increased thickness spinning time was extended to 30 min for all samples mechanically tested. Fiber Diameter: Meshes were imaged with Phenom Pro Desktop scanning electron microscope: 1000x, 10kV accelerating voltage, backscatter detector, and micrographs analyzed with ImageJ and DiameterJ. Tensile test: Strain to failure was conducted on a RSA3 Dynamic Mechanical Analyzer at a rate of 0.5 mm/s and initial pre-strain of 10%. Contact Angle Measurements: 3 specimens tested per mesh sample. Specimens mounted on glass microscope slides and tensiometer used to measure contact angle of ultra-pure water droplet placed on each sample. Cell Morphology and Focal Adhesions: Meniscal fibrochondrocytes (male, 17 years old, P4) were used as an initial model tissue as the meniscus is composed of extracellular macromolecules with ranging degrees of alignment. PCL only samples from prior experiments were cultured with MFCs at 25,000 cells/mesh for 3 days before fixing and staining with DAPI, Paxillin, Vinculin, and Phalloidin (actin).

Results, Conclusions, and Discussions:: In PCL only samples, increasing mandrel diameter resulted in increased alignment with stabilizing impact on other parameters including speed and collection voltage. In lower diameter mandrels, increased rotational speed increased alignment, however, this dependence of alignment on speed can be disrupted by varying collection voltage as shown in Figure 1A. In surfactant samples, alignment was only moderately achievable with decreased voltage, increased rotational speed, and decreased flow rate. Samples at comparable conditions to the PCL only control are shown in Figure 1A. Preliminary protein adsorption studies show increased protein adsorption on aligned PCL samples compared to unaligned and a similar trend in PCL+S80 samples. This indicates the ability to control the effect of S80 on protein adsorption and resulting cell adhesion via integrin binding. Increased adsorption was seen overall in PCL only samples and this is attributed to increased roughness and hydrophobicity compared to surfactant samples. Electrospun scaffolds can retain charge for long periods of time which may influence cell adhesion and morphology and aligned fibers may have aligned polarity of charges from the rotational collection.³ It is thought that aligned fibers containing non-ionic surfactant may have a more variable response in charge retention which may alleviate the commonly noted decrease in protein and cell adhesion with surfactant fibers. Increased MFC alignment and reduced paxillin was observed with increasing fiber alignment (Figure 1B&C) likely indicating altered strain sensing through the focal adhesion-actin complex. Future work will determine the impact of protein adsorption on cell adhesion and focal adhesion complexes in aligned fibers with S80 in both static and dynamic strain environments using a Mechanoculture J1 culture device. It is expected that not only will surfactant have micro level effects but that macroscopically the strain stiffening behavior observed in the PCL+S80 scaffolds will also impact cell behavior.
Addition of non-ionic surfactant modifies scaffold morphology, mechanical properties, and fiber surface charge. In an aligned system, fiber alignment and charge polarity create increased protein adsorption for hydrophilic scaffolds containing surfactant. This adsorption behavior in conjunction with differing macroscopic tensile properties is expected to modify MFC adhesion and regulation of mechanosensitive signaling.


Acknowledgements (Optional): :

References (Optional): : 1. Johnson, P.M.; et al. Biomater. Sci. , 2021, 9(4), 13

2. Johnson, P.M.; et al. Macromolecules, 2022

3. Chaurey, V.; et al. Acta Biomater. 2012