Hierarchical Micro- and Nano-Patterning of Metallic Glass to Engineer

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Hierarchical Micro- and Nano-Patterning of Metallic Glass to Engineer Cellular Responses Jennie Wang, Ayomiposi M Loye, Jittisa Ketkaew, Jan Schroers, and Themis R. Kyriakides ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00007 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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ACS Applied Bio Materials

Hierarchical Micro- and Nano-Patterning of Metallic Glass to Engineer Cellular Responses Jennie Wanga,b#, Ayomiposi M. Loye a,c#, Jittisa Ketkaewa,b, Jan Schroersa,b Themis R. Kyriakides a,b,d* a. Center for Research on Interface Structures and Phenomena, Yale University, New Haven, CT, 06520, United States b. Department of Mechanical Engineering & Materials Science, Yale University, New Haven, CT, 06511, United States c. Department of Biomedical Engineering, Yale University, New Haven, CT, 06511, United States d. Department of Pathology, Yale University, New Haven, CT, 06511, United States #

These authors contributed equally to this work Address all correspondence to [email protected]

*

Keywords: Micro-topography; nano-topography; bulk metallic glass; macrophage, foreign body response; cell-cell fusion.

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Abstract Nano- and micro-patterning of biomaterials is a rapidly evolving technology used in the engineering sciences to control cell behavior. Specifically, altering the topographies and hence, surface mechanical properties has been shown to induce changes in cell morphology and function. Here, we show a method for fabricating hierarchical microand nano-patterns of Pt57.5Cu14.7Ni5.3P22.5 (Pt-BMG) on the relevant length scales comparable to that of the proteins and cells. Leveraging, the amorphous nature of PtBMGs, we have a versatile toolbox to manipulate patterns on the nano, micro level and combine multiple length scales to examine specific cell responses.

We assay

morphology of macrophages and fibroblasts, two cell types critical to the foreign body response. Furthermore, we show that nano-topography is critical to reducing macrophage fusion and that high levels of fusion on both un-patterned and micropatterned substrates can be mitigated with the addition of nano-topographical features. Interestingly, we show that wetting ability of the substrates does not correlate with cellular responses on theses substrates. Our results suggest that the different topographical length scales can be used to systematically affect corresponding celltype-specific responses.

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1. Introduction

The foreign body response (FBR) is a precursor of implant failure characterized by the fusion of macrophages into foreign body giant cells and the formation of a fibrotic capsule deposited by fibroblasts.1 Macrophages fuse and secrete damaging cell products that harm surfaces and impair the function and lifespan of biomaterials.2 Interactions that take place during the FBR involve multiple characteristic length scales. For example, while cell sizes are on the order of microns, proteins and fibrils that reside in the extracellular matrix (ECM) have lengths that are on the order of nanometers.3 Numerous studies have identified the unique roles of different surface topographies, shapes, and length scales as well as contrasts in ECM rigidity and extracellular forces in influencing cell morphology, proliferation, and differentiation.4-9 Therefore, having a toolbox that would allow for the manipulation of surface topography in a controlled manner on these two length scales is critical for engineering high moduli implantable devices that can be tuned for specific cellular functions. On the nano-scale, several studies have shown that nano-structures such as grooves, nodes, pores, and pillars produced using lithographic techniques can mimic the topography of fibers, particulates, and features that cells interact with in vivo.10-13 These structures can, in turn, influence the cells’ resulting morphology, spreading, and orientation. We showed previously that fibroblasts, macrophages, and endothelial cells were able to detect amorphous Pt57.5Cu14.7Ni5.3P22.5 BMG substrates patterned with nanorods ranging from 55 nm to 200 nm in diameter.14 Specifically, fibroblasts responded by decreasing in cell size, becoming more circular in morphology, and

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secreting less collagen, while macrophages displayed more elongated morphologies and endothelial cells significantly decreased their cell size. Towards the goal of engineering the cell microenvironment, it is important to understand the effect of compositional, biochemical, and biophysical cues on cell behavior. Biophysical design considerations include structural features, electrical conductivity, degradability, and mechanical properties.15 Mechanical properties of substrates have been shown to affect specific cell fate and signaling. For example, PDMS elastomeric micro-post rigidity affects mesenchymal stem cell (MSC) traction force, focal adhesion structures and differentiation.16 Magnetic hydrogels have been shown to affect mechanical stimuli through interaction with magnetic fields to direct adipogenesis, cell growth and metabolic activity.17 Furthermore, mechanical cues transduced to the nucleus through nano-pillar mediated nuclear deformation have a significant role in controlling cell behavior.18 The effects of topographical micro-scale patterns on cell behavior have also been explored.19-21 Several fabrication techniques have been employed to this end, including photolithography and dry and wet etching of semiconductor materials like silicon as well as soft lithography and stamping for polymers such as poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) PEG diacrylate hydrogel.22-26 For example, it has been shown that the height and spacing of micro-grooves of PDMS can significantly decrease human osteosarcoma (HOS) cell proliferation as compared to a flat surface, and the micro-patterned substrate also influenced HOS cell adhesion and spreading.12,27,28 While various methods of hierarchically patterning nano-scale features on top of micro-scale features have been explored (e.g. molding and imprinting, chemical etching,

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laser-assisted etching, photolithography, and self-assembly, among other techniques), the achieved nano-scale secondary features are often randomly dispersed and oriented and also differ in size and shape.29-37 For example, Kyle et al used biomimetic silicone surfaces to evaluate in vitro FBR responses using soft lithography that created randomly dispersed surfaces.38 Furthermore, the dimensions of the micro-features in many cases are larger than those of individual cells, making it difficult to understand how cells behave in response to hierarchical patterns that are comparable in size. While electron beam lithography and soft imprinting have been demonstrated to produce uniform and regular secondary features, the resulting primary features are still large in comparison to the size of interest for studying cellular responses.36,39-43 The bulk of previous studies focusing on micro- and nano-patterning for cellular applications were employed with polymer or semiconductor materials. However, it would be ideal to form micro- and nano-patterns from a material with metal-like mechanical properties because polymers display comparably low yield strengths and elastic moduli, and silicon lacks both sufficient levels of strength and biocompatibility necessary for structurally sound medical implants.44-47 Although conventional metals like steel and titanium display high levels of strength and toughness, techniques to shape them on the micro and nano length scale are from a performance point of view, intrinsically limited by grain boundaries. 48,49 Efforts to create micro-nanoscale patterns on porous and solid nanostructured titanium implants require difficult processing using laser irradiation and hydrothermal treatment and lead to the creation of structures that are randomly dispersed. 50, 51

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A material class that exhibits the desirable properties of metals paired with processing methods suitable on the micro- and nano-scale and even high levels of biocompatibility in some cases is bulk metallic glasses (BMGs).52-56 In addition to possessing superb mechanical properties such as strength, elasticity, and in several cases toughness and ductility, the high stability against crystallization of BMGs enables the material to be thermoplastically formed (TPF) in its super-cooled liquid region, at temperatures significantly below the melting temperature.54,57-59 This allows for easy creation of highly versatile micro- and nano-scale molding in a nonrestrictive setting with accurate and controllable topography. A method to hierarchically pattern BMGs has been demonstrated by sequentially embossing nano-pillars on top of micro-scale features via a TPF process.53 Despite proof of the hierarchical patterning ability for BMGs, patterning through this method has been limited to 10 microns or above for the microscale features. However, in order to stimulate cells, one would need a microstructure pattern that is a fraction of the length of a cell of ~10 microns. Moreover, because proteins exist on the nano-scale, addition of a nano-topography could modulate protein responses to affect cell behavior. To understand the effect of biomedical implants that mimic in vivo topography alone, we show here that hierarchically patterned BMGs comprising tunable nanopatterns and micro-patterns can be fabricated in a highly versatile manner with micro dimensions down to 1 micron and nano-dimensions of 40 nm comparable to the length scale of relevant proteins. Specifically, for the study of the cellular response, we utilize 40 nm-diameter nanorods sequentially patterned onto 1 µm-diameter micro-pillars to investigate preferential pattern detection for two different cell types on length scales

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similar to those of cells and proteins. Fibroblasts and macrophages were plated on four different substrates – flat BMGs, nano-patterned BMGs, micro-patterned BMGs, and hierarchical micro- and nano-patterned BMGs. We found that significant differences in cell morphology and function among the topographies and for each cell type were observed. Additionally, we see that nanotopography has a greater effect on reducing macrophage fusion in comparison to microtopographical and smooth surfaces.

2. Materials and Methods 2.1 Materials The amorphous Pt57.5Cu14.7Ni5.3P22.5 metallic glass (Pt-BMG) alloy was prepared by induction melting combining Platinum: 99.95, Copper: 99.999, Nickel: 99.5, Phosphorus: 99.999 purity in a 2 mm-diameter quartz tube under an argon atmosphere. All composition values are given in atomic percent. The alloy was subsequently fluxed with B2O3 for 20 minutes at 930°C. Water quenching resulted in an amorphous sample and its amorphous nature was confirmed by x-ray diffraction and thermal analysis. Anodic aluminum oxide (AAO), or alumina, templates with a 10 mm-diameter disc, 50 µm wafer thickness, and 40 nm-diameter pores were purchased from InRedox. Silicon wafers containing 1 µm-diameter pores were fabricated by photolithography and deep reaction etching technique. Cell culture media, ACK lysing buffer, rhodaminephallodin, and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Fischer Scientific. Recombinant human macrophage colony stimulating factor (M-CSF) and recombinant mouse interleukin 4 (IL-4) were purchased from R&D systems. 2.2 Fabrication of hierarchical micro- and nano-patterned BMG

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Pt-BMG was used to fabricate the various patterned surfaces because the material offers several advantages of strength, elasticity, biocompatibility, corrosion resistance as well as resistance to surface oxidation during thermoplastic forming.60,61 As-cast Pt-BMG rods were cut into cylindrical samples 2 mm in diameter and 2.5 mm tall, after which they were cleaned in acetone and methanol. Thermoplastic forming of Pt-BMG into silicon and alumina molds was carried out using a custom-made forming system which is integrated with a Instron Model 5569. PID controlled heaters enable precise temperature in the temperature region between the glass transition temperature, Tg = 215°C, and crystallization temperature, Tx = 295°C, of the Pt-BMG and pressures that were both calculated and experimentally optimized. The hierarchical micro- and nano-patterns of Pt-BMG were fabricated by first forming an array of nanorods using alumina templates with 40 nm nominal pore sizes (Figure 1a) at 270°C for 120 seconds and at an applied pressure of 100 MPa. The alumina mold was subsequently dissolved in a potassium hydroxide solution. Afterwards, the micro- and nano-pattern combinations were created by replacing the Pt-BMG sample with the nanorod array created in the first part and forming this over a silicon wafer containing negative micro-scale features at 235°C (Figure 1b). We utilized the trapped air between the nano- and micro-scale features as a cushion to prevent direct contact between the two surfaces (micromold and nanopattern) which would damage the nanopattern. The micro-features contained in the silicon wafer were designed using LayoutEditor, and photolithography was employed to create the wafer (Figure 1c). Resulting scanning electron micrographs of each step are shown in the right column of Figure 1. Nanopatterned surfaces were created using the procedure in Fig. 1a and micro-patterns

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using the procedure in Fig. 1b, except that a flat BMG disc was used on top instead of a nanorod array. After each batch of samples was fabricated, they were characterized using scanning electron microscopy (SEM) to ensure that the structures were uniform and consistent among sets. 2.3 Measurement of contact angle Water contact angles of each surface pattern were measured using a VCA 2500 mp Video Contact Angle System connected to a Go-5 QImaging camera and an optical light distributed by Nikon, Inc using a well-established procedure that has been described earlier.62 A 3 µl drop of water was added on samples using a pipette, and the contact angle measurements were made using ImageJ.

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Figure 1. Fabrication of hierarchical micro- nano-patterns of Pt-BMG. a) Silicon mold fabrication with micro-features using photolithography. b) Bulk Pt-BMG is placed on porous alumina mold with 40 nm-diameter pores between planarized metal plates heated to 270°C (above Tg). T represents temperature. System is subject to 7.5 kN of compressive force for 2.5 minutes, allowing the Pt-BMG to flow into the pores of alumina mold. P represents pressure. The alumina mold is dissolved in a potassium hydroxide solution, yielding Pt-BMG nanorods. c) Pt-BMG nanorods fabricated in b) are placed on silicon mold with 1 µm-diameter micro-pillars and system is subject to 4.5 kN of compressive force for 2.5 minutes at 235°C. Nanorods are much shorter than microfeatures, so nanorod regions cushioned by air are preserved while the remaining regions are flattened. Silicon wafer is dissolved in a potassium hydroxide solution. Scanning electron micrographs of each step are shown on the right.

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2.4 In vitro cell culture For analysis of cell morphology, NIH 3T3 fibroblasts were plated at a density of 20,000 cells per well in a 24 well plate for 24 hours. Fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Animal protocols were approved by Institutional Care and Use Committee at Yale University. Primary mouse macrophages were obtained from the femurs of C57BL/6 mice. Femurs were removed and bone marrow was flushed using a 25G needle. Red blood cells were lysed using ACK lysing buffer. The bone marrow cell suspension was plated on petri plastic overnight and the MCSF dependent non-adherent fraction was expanded in Iscove's Modified Dulbecco's Media (IMDM) with 10% FBS, 1% penicillin-streptomycin, and 1.5ng/ml M-CSF, fed on day 5 and collected at day 10. Then, macrophages were plated in expansion media at a density of 20,000 cells per well in a 24 well plate for 24 hours. For analysis of macrophage fusion, primary expanded macrophages were cultured in IMDM with 10% FBS, 1% penicillinstreptomycin, 10 ng/ml M-CSF, and 10ng/ml IL4 for 10 days. Media was replaced on day 3, 5, and 7. 2.5 Immunofluorescence Analysis At the appropriate endpoint, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 1% Triton X-100, and stained with rhodamine-phalloidin and DAPI that stain the actin cytoskeleton and nucleus, respectively. Substrates were mounted on a glass slide and imaged using a Zeiss fluorescent microscope. 2.6 Quantification of cell behavior

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Fibroblast

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quantified

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ImageJ.

Approximately 50 cells per substrate were manually outlined and area, perimeter, circularity, and elongation factor were quantified. Circularity and elongation factor were determined as previously described.12 Briefly, circularity is quantified as [4π (cell area)/cell perimeter)2] and elongation factor is described as the length of the longest axis divided by the longest axis perpendicular to it. FBGCs were quantified using ImageJ and fusion index, average area per FBGC, nuclei per FBGC and the number of fused cells per image was determined. Fusion index is defined as the ratio of fused nuclei to total nuclei. 2.7 Statistical Analysis Results are presented as mean ± standard error of mean (S.E.M). Two-way analysis of variance (ANOVA) with Turkey’s posthoc analysis was used to compare between experimental groups. P values less than 0.05 were considered significant.

3. Results and discussion 3.1 Fabricated Pt-BMG surface topographies and impact of length scales on wettability The four substrates that were plated with fibroblasts and macrophages to examine the effects of hierarchical micro- and nano-scale surface topographies on cell behavior are shown in Figure 1. These include (a) a flat Pt-BMG sample, (b) a nanopatterned Pt-BMG sample with 40 nm-diameter rods, (c) a micro-patterned Pt-BMG sample with 1 µm-diameter pillars, and (d) a hierarchical micro- and nano-patterned PtBMG sample with 40 nm rods superimposed on top of 1 µm pillars. Nanorods are about 250 nm tall and the micro-pillars are approximately 1 µm tall (heights measured using

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ImageJ). The relative hydrophobicity of biomaterial surfaces is an important consideration for engineering controlled cellular responses. Many studies have investigated the effects of wettability on cell adhesion, proliferation, and spreading, and these studies have reported significant differences in cell behavior depending on the observed water contact angle.61-63 To further characterize the fabricated structures and correlate their wettability to cell responses, measurements of water contact angle were conducted (Figure 2). Flat and micro-patterned Pt-BMG surfaces are relatively hydrophilic (contact angles of 65° and 63°, respectively). Nanopatterning results in extremely hydrophilic (contact angle of 23°), whereas addition of the nanorods on the micro-pillars caused the surface to become more hydrophobic (contact angle of 79°). These measurements are consistent with wettability trends in patterned BMGs observed previously.35,43,64-66

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Figure 2. Effect of surface patterning on water contact angles for Pt-BMG. a) Flat surface is hydrophilic (contact angle = 65 ± 2°). b) Nano-patterned surface is extremely hydrophilic (contact angle = 23 ± 3°). c) Micro-patterned surface remains hydrophilic (contact angle = 63 ± 2°). d) Sequentially patterning micro-pillars with nanorods turns the surface more hydrophobic (contact angle = 79 ± 2°).

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3.2 Morphological responses to patterned Pt-BMG surface topographies The influence of various surface topographies on cellular morphology was determined by seeding fibroblasts and macrophages on the substrates and monitoring subsequent cytoskeletal remodeling. Significant and distinct changes in morphology were observed on the surfaces for each cell type. Fibroblasts exhibited more circular and less activated morphologies with decreased area and perimeter values on the hierarchical micro- and nano-pattern as compared to the micro-pattern alone, and they also displayed elongated morphologies on the flat Pt-BMG substrate (Figure 3, S1). Despite the large differences in wetting between patterns the fibroblast response is similar for micro, nano and Micro-nano pattern. Fibroblasts exhibited more circular and less activated morphologies with decreased area and perimeter values on the hierarchical micro- and nano-pattern as compared to the micro-pattern alone, and they also displayed elongated morphologies on the flat Pt-BMG substrate (Figure 3). Interestingly, macrophages demonstrated much more dramatic increases in circularity and corresponding decreases in area and perimeter going from the hierarchical micro- and nano-pattern to the micro-pattern alone as compared to the fibroblasts, and the macrophage morphologies on the flat Pt-BMG surface were relatively circular and unexpressed (Figure 4).

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Figure 3. Characterization of fibroblast morphology on micro-patterned, micro- and nano-patterned, nano-patterned, and flat BMG. Representative immunofluorescence images of rhodamine-phalloidin (cytoskeleton) and DAPI (nuclei) of (a) fibroblasts. Quantification of (b) area, (c) perimeter, (d) circularity, and (e) elongation factor. n=50 cells, ANOVA with Turkey’s posthoc analysis, #, p

Hierarchical Micro- and Nano-Patterning of Metallic Glass to Engineer

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