Easy fabrication of aligned PLLA nanofibers-based

Materials Science and Engineering C 62 (2016) 301–306

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Easy fabrication of aligned PLLA nanofibers-based 2D scaffolds suitable for cell contact guidance studies John Mohanraj a, Luca Puzzi a, Ennio Capria b, Stefania Corvaglia b, Loredana Casalis b, Luisa Mestroni c, Orfeo Sbaizero a, Alessandro Fraleoni-Morgera a,b,d,⁎ a

Department of Engineering and Architecture, Univ. of Trieste — V. Valerio 10, 34100 Trieste, Italy Sincrotrone Trieste SCpA, s.s. 14 km 163,5, 34149 Basovizza, TS, Italy University of Colorado Denver AMC, 13001 E17th, Aurora, CO 80045, USA d CNR Nano S3, Via Campi 213/A, 41125 Modena, Italy b c

a r t i c l e

i n f o

Article history: Received 16 July 2015 Received in revised form 3 November 2015 Accepted 18 December 2015 Available online 23 December 2015 Keywords: Scaffold PLLA ASB-SANS HeLa NIH-3T3 Contact guidance

a b s t r a c t An easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(L-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns, with fiber widths between 40 and 500 nm and lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and, more in general, the behavior of cells onto nanofibrous scaffolds), at very low costs and in an extremely easy way, accessible to virtually any laboratory. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tissue engineering has been a very active field of investigation in the last twenty years. This is due to factors like the increase of chronic diseases related to tissue degeneration [1–3], a growing number of findings over development, differentiation, communication and functions of cultured cells, which are opening multiple exciting research paths [4–6], and the progressive reduction in the use of experimentation animals that calls for alternative means for drugs testing [7,8]. Besides advances in molecular biology and physiology, a prominent position in tissue engineering is held by scaffolds, i.e. artificial structures providing cells a suitable environment for structural support, proliferation, growth, communication and migration. In nature, these functions are performed by the extracellular matrix (ECM), a complex construct constituted mainly by polysaccharides and fibrous proteins, organized at several dimensional scales, from micro to nano, most often as fibrous structures. The larger features of the ECM (down to a few hundreds of nm in width, up to several tens of microns long) offer structural support for cells and allow their migration and development, while the smaller ones (ranging from a few hundreds to a few units of nm) are the basis for ⁎ Corresponding author at: Department of Engineering and Architecture, Univ. of Trieste — V, Valerio 10, 34100 Trieste, Italy. E-mail address: [email protected] (A. Fraleoni-Morgera).

http://dx.doi.org/10.1016/j.msec.2015.12.042 0928-4931/© 2015 Elsevier B.V. All rights reserved.

diffusing molecular signals, nutrients and metabolites to and from the cells. In addition, the physical and chemical properties of the ECMcomposing materials contribute to control cell activities and metabolism [9–11]. Recently, several indications about the influence of nanoscale-patterns in promoting cell differentiation, development and behavior have been reported. For example, cardiomyocytes grown on non-structured scaffolds present poorer contractile activity than those of their analogues grown onto natural ECMs, apparently due to the lack of both dynamic-mechanical environment and topological cell alignment/guidance features [12]. Similarly, neurons grown on artificial, non-structured culturing media present marked morphological differences with respect to those grown in vivo, resulting in a lesser participation to the synaptic transmission process [9]. Moreover, in some instances functional nanostructures showed intriguing features in terms of potentiating cell physiological activity, as is the case for neural cells grown onto conducting carbon nanotubes, which exhibited notable increase of firing frequencies with respect to those grown onto standard culturing media [13]. In this view, the fabrication of scaffolds with controllable fibrillar nanostructures is assuming a growing importance. This type of fabrication can be carried out in a variety of ways, ranging from well-established lithographic techniques [14] to more recently developed methods, like nano-imprint-based soft lithography [15], electro-spinning [16], phase separation-induced nanostructuring [17]. However, these methods often show problems related to high

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processing costs, low through-put, long scaffold development times, need for complex instruments (as is the case for lithographic-based processes), limited scaffold surface area (i.e., a few square mm at the maximum), or different combinations of these limitations. In particular, a currently very difficult task is to produce combinations of micro- and nano-sized features, typical of native ECM, in an easy, low cost and straightforward way. While interesting approaches towards this goal have been presented recently [18,19], the growing need for viable and economical methods to reproduce the peculiar multiscale fibrillar morphological structure of ECM encourages to look for novel scaffold fabrication strategies. In this view, a method named ASB-SANS (Auxiliary Solvent-Based Sublimation Aided Nano-Structuring), based exclusively on wet processing a solution, was shown to be able to deliver nanofibrous structures with feature sizes down to the nanoscale, over large areas (tens of cm2), in minutes and at a very low cost [20,21]. The method allows to pattern several different materials, including biodegradable/bioresorbable ones, like polyglycolic acid (PGA) or poly-Llactic acid (PLLA), known to favor cell growth, attachment and migration via progressive resorption of the artificial scaffold [22,23]. A notable feature of ASB-SANS is that it is able to produce in a single fabrication step hierarchically organized micro- and nano-sized fibers (from a few microns down to less than 30 nm in diameter), with a topology controllable via simple variation of the solution component concentrations. [20,21]. Herein, we describe the fabrication of PLLA fibrillar patterns via ASBSANS with hierarchical, multiscale organization, characterized by arrays of parallel primary, relatively large fibers (width of about 300–500 nm) from which secondary smaller fibers (down to a few tens of nm width) develop in an approximately orthogonal fashion. The biocompatibility of the so-obtained patterns has been demonstrated using them as scaffolds for the growth of two immortalized cell lines, deriving from human (HeLa ATCC CCL-2 epithelial cells, briefly HeLa) and from mouse (NIH-3T3 ATCC CRL-1658 embryonic fibroblasts, briefly 3T3). Clear effects of contact guidance attributable to these ordered fibrous patterns, as well as effects of the hierarchical fibrillar patterns on both the cells growth features and their behavior, have also been observed and discussed. 2. Materials and methods 2.1. Reagents and equipment PLLA (MW = 258,700), para-dichlorobenzene (PDCB) and chloroform (CHCl3, 99.8%) were purchased from Sigma-Aldrich and used without further purification. Either microscopic glass slips (for HeLa cells) or Si/SiOx wafers (for NIH-3T3) were used as substrates for the scaffold preparation. The Si/SiOx wafers were purchased from ITME (Poland). The glass slides were washed with soap solution followed by copious amounts of deionized water, while the Si/SiOx slides were dipped in sulfuric acid in order to remove possible traces of organic residues, and then rinsed thoroughly with de-ionized water. The SEM images were taken with a Carl Zeiss Supra 40 Scanning Electron Microscope. The optical microscopic images were captured with an Olympus BH-2 microscope equipped with an Olympus camera. 2.2. Preparation of PLLA scaffolds via ASB-SANS method To prepare micro and nanostructured patterns via the ASB SANS technique, a ternary solution containing PLLA, CHCl3 and PDCB was used. A detailed description of the method has been given previously [20,21]. A stock solution was prepared by adding 1 mg of PLLA to 1 mL of CHCl3 and stirring overnight to allow complete polymer dissolution. Precise quantities of PDCB were weighed and added to the aliquot amounts of PLLA stock solution to prepare ternary systems with PDCB/ PLLA weight ratio of 50:1 (PLLA-1 solution) and 100:1 (PLLA-2 solution), respectively. Approximately 5 μL of PLLA-1 (PLLA-2) solution

were drop casted onto clean standard microscope glass slides (clean Si/SiOx chips). Then, the prepared substrates were left at ambient conditions under the fume hood for 6 h. During this period, at first the CHCl3 molecules slowly evaporate, leading to the formation of a film of solid PDCB crystals incorporating PLLA on the substrates. The formed crystalline structures act hence as a temporary template for the PLLA chains dissolved in the solid PDCB. The subsequent sublimation of the PDCB crystals leave micro and nanostructured patterns of PLLA on the substrates, allowing at the same time a clean and quantitative elimination of the templating material (i.e., PDCB). The complete removal of PDCB from the samples was verified by scratching the developed fibers from sacrificial samples and analyzing them with Fourier-Transform Infrared (FTIR) spectroscopy, which did not reveal any appreciable presence of the molecule [21]. 2.3. Cell cultures HeLa (ATCC CCL-2) and NIH-3T3 (ATCC CRL-1658) were cultured according to the standard procedure at 37 °C and 5% CO2 in Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mix. Both HeLa and NIH3T3 (3 × 104 cells/mL) were plated onto the UV sterilized (1 h) ASBSANS prepared scaffolds, then grown in an incubator for 48 h, fixed with 4% PFA and stained with Coomassie brilliant blue G250 (SigmaAldrich). 3. Results PLLA was chosen as the scaffold material due to its bio-resorbability and its favorable properties in terms of cells adhesion, viability and proliferation. In fact, it has been demonstrated that this polymer is suitable for prolonged cell culture, even for lineages very sensitive to toxins [24, 25]. PLLA nanopatterns were hence fabricated onto glass slides using the PLLA-1 solution, following the general ASB-SANS procedure, described in previous work [20,21]. ASB-SANS is characterized by a wetprocessing approach, which allows to cover large substrate areas (up to several cm2), a good versatility (the method can address several different polymers, as well as carbon nanotubes), and an easy removal of the templating material (in this case PDCB). [20,21] All these features led to rapidly obtain micro and nanofibrous PLLA patterns on the glass slides (typically, patterns are ready after about 5 min from the ternary solution deposition; more time for using the resulting scaffold is waited only as a prudential measure for ensuring that the sublimating substance has completely left the scaffold), covering a total area of several thousands of μm2, with the fibers occupying approximately 20% of the substrate (calculated using an image processing software, see Supplementary Material, Fig. S1). As evidenced by an optical microscope analysis (Fig. 1a), the patterns developed on glass consist in a series of parallel primary fibrous structures, having lengths of several tens of microns and widths well below 1 μm. From these primary fibers, secondary ones stem in an almost orthogonal fashion, with lengths up to a few tens of microns and widths visibly smaller than those of the primary ones. In order to get a clear view of these morphological details, the same PLLA-1 solution used for the glass substrates was deposited onto polished Si/SiOx chips, but due to the difference in intrinsic surface properties between bare glass and SiOx, and likely to a dedicated washing procedure for the latter (see Materials and Methods), the patterns developed on the chips with this solution were irregular and poorly defined. Therefore, a modification of the PLLA-1 solution was made, realizing a PLLA-2 solution characterized by a higher PDCB/PMMA weight ratio (see Materials and Methods). Once deposited onto the Si/SiOx chips, the PLLA-2 solution originated well defined patterns with a morphology (fibers size and branching type and extent) closely resembling that achieved on glass using the PLLA-1 solution, according to optical microscope characterization (Fig. 1b; compare also Fig. 1 and the further


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Fig. 1. PLLA patterns at different magnifications. a: PLLA-1 solution-originated patterns developed onto glass slides (optical microscope view); b,c: PLLA-2 solution-originated patterns as seen by SEM imaging; c: close-up of the patterns visible within the red rectangle shown in Fig. 1b, where a main fiber is visible as the zigzagging line at the bottom of the image, and both secondary patterns (vertical fibers stemming almost perpendicularly from the main one) and tertiary ones (tiny and short fiber-like features stemming from the secondary ones, again perpendicular to the latter) are also recognizable.

Fig. 3a-h). The subsequent SEM imaging of the PLLA-2 solutionoriginated patterns, even though complicated by non-negligible polymer charging problems caused by the electronic beam, allowed to define a width range of about 300–500 nm for primary fibers, and of about 100–400 nm for secondary ones. Moreover, a further level of finer tertiary structures, again stemming almost orthogonally from the secondary ones, was observed (Fig. 1c), with widths below 100 nm and lengths in the range of several microns. Overall, the morphological features of these fibrous patterns, with hierarchical organization, lengths exceeding tens/hundreds of microns and sizes ranging from a few hundreds to several tens of nanometers, appear resembling those found in natural ECM, except for the fact that the here presented ASBSANS-generated patterns are almost 2-dimensional, while natural ECM is developed in a full 3D fashion. Due to the peculiarities of the ASB-SANS-fabricated nanofibers (which are grown by self-assembly directly onto the substrate, are inherently branched and leave large areas of the bare substrate exposed to the cells and to the culture medium), it was not possible to estimate their hydrophilicity, nor to measure their mechanical properties. Nonetheless, it has been reported that, in addition to the known viability of standard PLLA layers [24,25], also electrospun PLLA nanofibers of sizes analogous to those of the ASBSANS-obtained ones are fully compatible with cells growth, despite their relatively low hydrophilicity (water contact angles N 110°) and the fact that chemical or physical treatments aimed at increasing the hydrophilicity of the fibers can further promote the scaffold biocompatibility [26–28]. Also the tensile stress of PLLA nanofibers with diameters in

the 300–500 nm range has been assessed, showing values in the range of a few hundreds of MPa for single nanofibers [29–31] and much lower values (a few MPa) for electrospun fibrous mats, where again the biocompatibility of the so-obtained scaffold was demonstrated [27]. The PLLA patterns obtained on glass have been cultured with HeLa cells. After two days in culture, cells have been fixed and stained with Coomassie Blue to improve the cell contrast for optical microscopic studies. As is visible from Fig. 2, the ASB-SANS-generated PLLA patterns delivered both sparse and denser cell aggregates, evidencing a good viability of the scaffold. Interestingly, the cells showed a marked tendency to grow aligned to the PLLA patterns, independently from the crowding level, forming pretty peculiar head-to-tail queues in the grooves found between couples of primary fibers, and sometimes also on the secondary smaller fibers, perpendicularly to the primary ones (see Fig. 2a, where white dashed lines have been drawn as a guide to the eye). This behavior testifies for contact guidance of the PLLA nanostructures over the cell growth patterns of HeLa, in line with several reports stating the influence of topographic features over cell alignment [32–34]. In order to verify that the observed alignment was actually due to the PLLA patterns, two control samples, one based on a bare glass slide and one consisting of a plain (i.e., not patterned) layer of PLLA deposited onto the same type of glass, were prepared. As is visible in Fig. 2c and d, both control samples showed satisfactory but completely random cell growth, clearly indicating that PLLA is neither detrimental to HeLa nor intrinsically delivering any kind of preferential cell alignment when not presenting defined patterns.

Fig. 2. a, b: Optical microscopic images of HeLa cells grown onto PLLA patterns on glass substrates. The white dashed lines highlight the zones in which cell alignment is clearly recognizable. c: HeLa cells grown onto bare glass. d: HeLa cells grown onto plain (i.e., not patterned) PLLA.

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HeLa cells are very robust and thrive easily on several different substrates [35], therefore their growth onto the ASB-SANS-generated patterns does not necessarily constitute a proof of general cell viability of the scaffolds fabricated with this technique. In this view, also NIH-3T3 cells, a type of mouse embryonic fibroblasts, have been cultured onto PLLA-based patterns prepared by ASB-SANS. These cells represent a model for large cell populations, and they are known to be sensitive to the topography of the underlying substrate [36–40], hence they are a perfect testbed for validating ASB-SANS generated patterns as scaffolds for testing contact guidance effects on cells. Moreover, they are apt for in-situ biochemical signaling and mechanical properties investigations [41,42]. In order to later collect SEM images of the grown cells, the cultures have been carried out directly using conductive Si/SiOx chips as substrates, using PLLA-2 type solutions in order to have patterns similar to those tested for HeLa on glass. The 3T3 cells grew well on the patterns, evidencing their good viability, and several fibroblasts showed a pronounced elongated structure. In most cases the elongation axis was parallel either to the main or to the secondary fibers of the PLLA pattern, signaling that for 3T3 the ASBSANS-generated micro/nanostructure has an actual contact guidance effect on the cell activity (Fig. 3). However, it was not possible to identify whether the major topographic guidance of the patterned substrate was influenced by the main or secondary fibers (previous studies evidenced that when patterns at different length scales are present, the found cellular responses to topographic cues are well detectable but not easily controllable [32]). When cultured in the same conditions on plain PLLA or on bare Si/SiOx chips, the NIH-3T3 cells showed neither any pronounced elongation, nor any preferential orientation (Fig. 3i,l), confirming that the elongation and alignment effects are actually due to the ASB-SANS-generated patterns. Interestingly, in several instances the 3T3 pseudopodia followed rather closely the PLLA patterns, as seen in Fig. 3c,g,h. On the contrary, the control samples with plain PLLA and bare Si/SiOx chips did not show any preferential direction in the pseudopodia development (Fig. 3i,l).

4. Discussion The aforementioned results show that ASB-SANS-generated patterns allow a clear cell viability, and that the obtained aligned PLLA fibers have a sizeable effect of contact guidance towards the cells development, an effect attributed to the geometrical features of the patterns. In fact, it is observable (Fig. 2a,b) that for HeLa cells the populated grooves have width of about 20–30 μm, pretty well coincident with the cells size, confirming known effects of physical confinement of cells along grooved structures. Also rarely formed curved grooves have been colonized by HeLa queues (Fig. 2b, left), underlining the effect of the ASB-SANS-generated motifs in determining the cell growth pattern. The 3T3 cultures confirm the topographic guidance induced by the patterned substrate, and they suggest further interesting considerations. In particular, in this case the elongation induced by PLLA nanofibrous patterns is coupled to an evident contact guidance effect towards the pseudopodia development. This is in line with reports showing that topography can provide guidance to pseudopodia development [43,44]. SEM analysis of a few cells confirmed this point, as is visible in Fig. 4, where pseudopodia developed by the cells on PLLA-2 solutionoriginated scaffolds follow the patterns keeping the alignment for remarkably long distances (up to hundreds of microns). More interestingly, a closer look at the pseudopodia showed filopodia stemming perpendicularly from both sides of the pseudopodia at rather regular distances of a few microns (Fig. 4b,d), well aligned with the secondary nanofibrous structures of the PLLA patterns, which have widths ranging between 100 and 400 nm (Fig. 4c,d), well matching those of filopodia [45]. It is known that filopodia have an important role in sensing the external environment and it is hypothesized that they actively contribute to the contact guidance behavior of cells, although this point is under debate and the basic mechanisms involved are not yet clear [44,46–50]. Standard investigations over contact guidance phenomena require large area nanostructures with appropriate sizes and topological features [51], and the currently available

Fig. 3. a-h: NIH-3T3 fibroblasts cultured on PLLA patterns developed on Si/SiOx chips via ASB-SANS, showing enhanced elongation and variable degrees of alignment along the nanofibers. Each panel shows cells grown either in a different part of a substrate, or on a different chip. In some instance a recognizable alignment of the cells pseudopodia along the patterns is also visible (c, g, h). i: NIH-3T3 cells grown onto bare Si/SiOx slides. l: NIH-3T3 grown onto plain (i.e., non-patterned) PLLA. Both the latter cultures have been carried out in the same conditions of those shown in (a–h).


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Fig. 4. SEM images of 3T3 cells fixed onto ASB-SANS-generated PLLA patterns. a,c: wide view of the cells evidencing their pseudopodia; b,d: close up of selected zones of the respective pseudopodia, where it is possible to recognize several filopodia stemming perpendicularly from the pseudopodia, at both the start of the pseudopod and at its terminal end (in d red circles have been added to highlight the filopodia position).

patterning techniques are not optimized for delivering at the same time aligned nanostructures on large substrate areas at low costs; in other words, these studies require to sustain non-negligible costs associated with the production of large area aligned nanostructured scaffolds. On the other hand, as above described, the ASB-SANS technique easily delivers ready-made 2D scaffolds fully matching these requirements, allowing to observe contact guidance effects on a scale of several mm2, i.e., for significant numbers of cells. Notably, this result is achieved with very accessible equipment (common glassware and chemical reagents, plus the obviously needed biocompatible polymer to be nanostructured), and in a very short time, allowing a vast number of laboratories to carry out extensive testing related to contact guidance phenomena. In the here discussed case, we speculate that the nanosized secondary PLLA fibers, having widths commensurate with those of the protruding filopodia, provide an actual source of topographic guidance for the latter ones. However, the presented data are not conclusive yet, and more work on this point is ongoing. 5. Conclusions Highly ordered long range patterned fibrillar scaffolds, with a hierarchical organization resembling that of the natural ECM, though in 2D instead of 3D, were fabricated from PLLA by means of a facile, low-cost and fast wet processing-based method named ASB-SANS. The prepared patterns were used as scaffolds for the growth of both HeLa cells and NIH-3T3 mouse embryonic fibroblasts, which were successfully cultured, evidencing a good biocomptibility of the ASB-SANS-generated patterns. Interestingly, the fibrillar patterns evidenced for both cell lines a clear contact guidance effect. In particular, HeLa cells lined up in peculiar queues within the pattern grooves, while 3T3 assumed pronounced elongated shapes and showed protruded pseudopodia aligned with the fibrillar patterns. Moreover, SEM images revealed that nanometric filopodia stem from both sides of the pseudopodia at regular intervals, following the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. Likely, these nanopatterns provide guidance to the filopodia development. Overall, ASB-SANS carried out on PLLA proved to be a fast and affordable technique to realize ordered and biologically viable 2D scaffolds. In particular, it has been shown that

ASB-SANS is able to produce patterns based on biocompatible materials at multiple size scales, from several hundreds of nanometers wide, several tens of microns long (primary fibers), to 100 (and even less) nm wide, several units of microns long (secondary fibers), in a single process step. Remarkably, this ability is coupled to rapidity of development (the patterns are physically ready in minutes from the solution deposition) and to extremely low costs (the whole process is carried out using common glassware and inexpensive reagents). Therefore, ASB-SANS is proposed as a viable technique to easily and quickly provide oriented scaffolds suitable for studies over phenomena like the influence of topographic guidance on pseudopodia and filopodia development, and possibly also more complex ones like stem cell development and differentiation. Acknowledgments JM gratefully acknowledges the University of Trieste for financial support. OS, LM and LP gratefully acknowledge financial support from the Foundation Leducq, Transatlantic Network of Excellence (14-CVD 03). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.12.042. References [1] D.R. Garris, B.L. Garris, Diabetes-induced, progressive endometrial involution characterization of periluminal epithelial lipoatrophy, Diabetes 52 (2003) 51–58. [2] G. Riley, Chronic tendon pathology: molecular basis and therapeutic implications, Expert Rev. Mol. Med. 7 (2005) 1–25. [3] O. Golubnitschaja, Cell cycle checkpoints: the role and evaluation for early diagnosis of senescence, cardiovascular, cancer, and neurodegenerative diseases, Amino Acids 32 (2007) 359–371. [4] A. Kotwal, C.E. Schmidt, Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials, Biomaterials 22 (2001) 1055–1064. [5] M.P. Lutolf, J.A. Hubbell, Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering, Nat. Biotechnol. 23 (2005) 47–55.

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