Synchrotron Microbeam Diffraction Studies on the Alignment within 3D-Printed Smectic-A Liquid Crystal Elastomer Filaments during Extrusion

Abstract

3D printing of novel and smart materials has received considerable attention due to its applications within biological and medical fields, mostly as they can be used to print complex architectures and particular designs. However, the internal structure during 3D printing can be problematic to resolve. We present here how time-resolved synchrotron microbeam Small-Angle X-ray Diffraction (μ-SAXD) allows us to elucidate the local orientational structure of a liquid crystal elastomer-based printed scaffold. Most reported 3D-printed liquid crystal elastomers are mainly nematic; here, we present a Smectic-A 3D-printed liquid crystal elastomer that has previously been reported to promote cell proliferation and alignment. The data obtained on the 3D-printed filaments will provide insights into the internal structure of the liquid crystal elastomer for the future fabrication of liquid crystal elastomers as responsive and anisotropic 3D cell scaffolds.

1. Introduction

Synchrotron X-ray microbeam diffraction has been widely used to correlate optical microscopy images with local X-ray structure. One of the earliest studies was done on beamline 4A at the Photon Factory to characterize the local layer structure across a broad wall in a surface-stabilized ferroelectric liquid crystal (FLC) [1]. Other X-ray microbeam liquid crystal (LC) studies include the time-resolved study of an FLC [2], elucidation of the structure of LC phases derived from bent-core molecules [3], a combined X-ray microbeam, polarized optical microscopy, a scanning electron microscopy study of the internal structure of periodic smectic-A (Sm-A) zig-zag defects from mesoscopic scale to molecular level [4], and the determination of orientational periodicities in the LC smectic-C* (Sm-C*) subphases by using an X-ray energy at the resonant edge of selenium-containing compounds [5]. One of the authors (RP) performed his first X-ray microbeam experiment with Professor Clark’s group to correlate polarized optical microscopy images with local X-ray microbeam diffraction from short DNA oligomers [6]. This was a highly memorable project that was later highlighted in an engaging book [7]. In the current work, X-ray microbeam diffraction is used to elucidate the evolving local structure as a liquid crystal elastomer (LCE) composite ink is three-dimensional (3D) printed.

Recently, new software and hardware advances in additive manufacturing (AM) or 3D printing, have been explored to pattern novel and smart materials with complex [8] and specific requirements increasing 3D printing applications into biological and medical fields [9,10]. Extrusion-based printing of semi-crystalline polymers is usually performed by the fused filament fabrication (FFF) technique, while the printing of pastes and structured fluids is done through continuous direct ink writing (DIW) [11,12]. Information about the structure formation during 3D printing is difficult to resolve, especially at the nanoscale. Thermal imaging has been combined with in situ small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) to observe crystallization in thermoplastic polymers printed by FFF, where the local temperature was correlated to the degree of crystallinity [13,14]. Recent examples of coherent X-ray techniques have revealed unprecedented access to help in the understanding of real-time structural transitions that occur during the 3D printing of amorphous polymer composites and ceramics [15,16,17].

3D biological constructs require functional and structural architectures for their very particular applications, and in terms of their utility require specific (matching) mechanical properties, biocompatibility and, in most cases, biodegradability [18,19]. As part of the exclusive group of smart materials, liquid crystal elastomers (LCE) have been identified as materials capable of reversible actuation and have recently been studied as viscous inks formed from un-crosslinked liquid crystal (LC) oligomers for soft robotics [20,21]. The LCE actuation comes from the alignment of the liquid crystal mesogens, that have long been used as agents for thermal stability [22], sensors [23], or actuators [24,25] during phase transition or extrusion during the 3D printing process. However, LCEs have been studied in more detail for applications in the biological field as cell scaffolds. LCEs’ orientational order, optical, and anisotropic elastic properties [26,27] make them suitable for biological application as they promote the development of the extracellular matrix (ECM) [13,26,28], cell attachment and proliferation [29,30], as well as promoters of cell orientation and maturation [31,32]. Very recently, LCEs have been shown to control the dynamics of bacteria [33,34]. Most likely, the growing interest in LCEs is because they can be used to create complex 3D architectures representative of organs or animal models [35,36], or function as long-term multi-responsive cell scaffolds [13,28,37]. Their applications can be expanded to anticancer drug screening, drug discovery, and other pharmaceutical applications [38,39] as new tools for preventative medicine.

There are two main strategies to synthesize LCEs: when mesogenic moieties are attached as part of the polymer backbone, they are termed main-chain LC elastomers [36,40], while if they are attached as a side-chain (pendant) to the backbone, they are termed side-chain LC elastomers [40]. Depending on the organization of the mesogenic moieties within the bulk, a formation of nematic, smectic, or cholesteric forms, among others, will be observed [41]. When the mesogenic pendant moieties are end-on side-chain elastomers, an LCE smectic phase could be favored, while side-on mesogenic pendant moieties or main-chain mesogenic moieties would more preferably form a nematic phase because they prefer the long-range positional order [42]. In any case, the orientation of the mesogenic moieties is transferred to the bulk of the LCE. The alignment of LCEs has been mainly studied in films as the result of an external stimuli [43,44] and very recently on filaments from nematic LCEs’ inks [31,33]. We have previously reported a biocompatible and biodegradable side-chain Sm-A LCE-based ink for 3D printing as a 3D cell scaffold. The photocrosslinkable 3D polycaprolactone (PCL)-based LCEs have been shown to induce anisotropic cell behavior without any external stimulus applied [45,46]. In this study, we used a photosensitive Sm-A LCE as ink for 3D printing and studied its Sm-A organization (anisotropy) as it extruded at room temperature forming filaments in situ inside the beamline hutch. We also report here the synchrotron microbeam Small-Angle X-ray Diffraction (μ-SAXD) data revealing the alignment of the Sm-A LC within the printed filament during extrusion. This work provides an entirely new platform to study the 3D printing of Sm-A LCE elastomers and evaluates the effect of extrusion using synchrotron x-ray microbeam diffraction.

2. Materials and Methods

All air-sensitive manipulations and synthetic steps were performed under nitrogen gas. ε-Caprolactone (ε-CL, purchased from Alfa Aesar) was dried over calcium hydride (from Sigma-Aldrich) and distilled under reduced pressure. D, L-Lactide was used as received (from Alfa Aesar). Dipentaerythritol, triethylamine, methacryloyl chloride, and Irgacure 819 (Bis (2,4,6-trimethylbenzoyl)-phenylphosphine oxide) were used as received (Sigma-Aldrich). Cholesterol (purchased from Sigma-Aldrich) was modified to cholesteryl 5-hexynoate by following previous work [47,48]. All solvents used for the synthesis and purification were EMD Millipore grade purified by a Pure-Solv solvent purification system (Innovative Technology Inc., Oldham, UK).

Amber syringe barrel, white polyethylene piston, end cap and blue dispense tip with an internal diameter of 0.41 mm (Nordson PN 7018272) were purchased from Nordson.

The print was obtained with our in-house 405 nm photosensitive LCE-based ink containing Irgacure 819 photoinitiator (Bis (2,4,6-trimethylbenzoyl)-phenylphosphine oxide). A curing of 75 seconds was applied in situ followed by 15 minutes ex situ post-curing. The UV system consisted of an Omnicure S1500 UV lamp (200 W Mercury lamp with a 320–500 nm filter) at 75% intensity and used a light guide placed at 13 cm from the sample in situ and 20 cm during ex situ post-curing. The print was made onto a 25 μm thick polyimide substrate (CAPLINQ PN PIT2A).

Scanning electron microscopy (SEM) was performed on samples which were gold-coated (700 Å) using a sputter coater (Hummer VI-A, Anatech Ltd., Springfield, VA, USA) at 10 mA DC for 3 min. Images were obtained using Quanta 450 FEG SEM under high vacuum. ¹H and ¹³C NMR of copolymers and elastomers were recorded in CDCl₃ at room temperature on a Bruker DMX 400 MHz instrument and referenced internally to residual peaks at 7.26 (¹H).

Synchrotron µ-SAXD

Measurements were performed in transmission mode of the Soft Matter Interfaces beamline (12-ID SMI) at the National Synchrotron Light Source II (NSLS-II) [49]. Scattering patterns were measured at 16.1 keV with a beam size at the sample position of 2.5 µm × 25 µm (V × H) and recorded on a Pilatus 300 K−W detector, consisting of 0.172 mm square pixels in a 1475 × 195 array, mounted at a fixed distance of 0.275 m from the sample position. The 2D raw SAXS images were acquired in-air and converted into q-space, followed by a fixed background subtraction. Local variations in thickness, liquid crystal concentration, and morphology may have impacted the baseline after subtraction of a fixed background. Reduced images were visualized in ** of the Sm-A orientation with layer normal approximately perpendicular to the incident X-ray beam. We plan to conduct further ex situ studies rotating the sample with respect to the incident X-ray beam for a full 3D orientation map. In future work, we will explore the Sm-A layering alignment in rows of 3D-printed filaments intending to fabricate a fully active and responsive cell support structure into architectures to control cell directionality and anisotropy. This study will involve the integration of a multitude of cell types into this elastomer-ink, to help it become a bio-ink, and not only provide but also stimulate cell alignment. Our goal is to fabricate, among other tissues, complex vascular networks from the LCEs using 3D-printing containing cells.