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Tissue Engineering and Regenerative Medicine
Introduction of laser interference lithography to make nanopatterned surfaces for fundamental studies on stem cell response Bhavya Kaushik Ekambaram, Marcus S. Niepel, Bodo Fuhrmann, Georg Schmidt, and Thomas Groth ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00060 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018
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ACS Biomaterials Science & Engineering
Introduction of laser interference lithography to make nanopatterned surfaces for fundamental studies on stem cell response Bhavya K. Ekambaram#,†, Marcus S. Niepel#,†,‡, Bodo Fuhrmann‡,§, Georg Schmidt‡,§, and Thomas Groth*,†,‡,+ AUTHOR ADDRESS †
Biomedical Materials Group, Institute of Pharmacy, ‡ Interdisciplinary Centre of Materials
Science, § Institute of Physics, + Interdisciplinary Center of Applied Research, Martin Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany *Email:
[email protected] KEYWORDS Laser interference lithography, nanostructures, self-assembled monolayers, human adipose derived stem cells, cell adhesion, differentiation
ABSTRACT
The extracellular matrix (ECM) is a nanostructured environment that provides chemical, mechanical and topographical stimuli for various cellular functions. Here, we introduce the application of laser interference lithography (LIL) to generate hexagonally-arranged gold nanostructures of three different dimensions on silicon to study the effect of feature 1 ACS Paragon Plus Environment
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dimensions on human adipose-derived stem cells (hADSC) in terms of adhesion, growth, and differentiation. Self-assembled monolayers (SAM) were used to passivate the background silicon surface with a long-chain polyethylene glycol (PEG), while the gold nanostructures were activated with mercaptoundecanoic acid (MUDA) to direct protein adsorption and cell adhesive structures to them, only. It was possible to show that the size and distance of the nanostructures affected the spreading of hADSC with a decrease of cell size with the increase of feature dimensions, which corresponded also to the expression of focal adhesions and presence of the small GTPase RhoA. Effects of these early events, related to outside-in signal transduction, were visible by an enhanced cell growth on smaller feature dimensions and distinct effects on cell differentiation. Owing to the precise control of chemical and topographical cues, the presented system offers great potential to study effects of material topography on stem cell behavior, which may pave the way for applications in tailoring surfaces of implants and tissue engineering scaffolds.
INTRODUCTION Typically, vertebrate cells are able to sense their environment and interplay with the mechanical, chemical, and topographical features of the surrounding extracellular matrix (ECM). The ECM is a complex structure consisting of fibrillar and non-fibrillar proteins and proteoglycans with feature sizes ranging from tens of nanometers to several microns.1 Hence, cells experience a nanostructured environment in vivo, interacting with a tissue-specific presence of collagen, elastin and fibronectin (FN) fibrils of different dimensions.2 Therefore, nanotopographical features on/in biomaterials can be a tool to regulate cell fate through direction of organization of cell adhesive structures and subsequent outside-in signaling.3-6 Methods of nanofabrication of materials are manifold and generally classified into two principle approaches, namely top-down and bottom-up.7-8 The bottom-up methods work by self-assembly or self-organization of smaller entities into higher ordered structures. Methods 2 ACS Paragon Plus Environment
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such as colloidal gold lithography, polymer demixing, phase segregation of silanes, and other self-assembly procedures such as the layer-by-layer technique are common examples for the bottom-up approach to design structures in the sub-micron or even low-nanometer range.8-11 The advantages of these techniques are cost effectiveness and ease of operation, but they may lack precision and generation of randomly arranged structures.8, 12-13 In contrast, top-down methods work by removing material from larger entities. Well-known examples for this approach are photolithography, X-ray lithography, electron beam lithography (EBL) or nanoimprint lithography, which are also capable to create feature dimensions in the extreme low nanometer range ( 80°), which is related to the presence of gold (WCA of plain gold ~60°) and the topographical heterogeneity. The increase in WCA with decreasing feature dimensions from large to small represents probably an effect of the increased quantity of gold. In addition, roughness effects may play a role here according to the Cassie-Baxter model.47 The increased WCA hysteresis during dynamic contact angle measurements on the smallest features can be taken as an indicator for an enhanced topological heterogeneity. It further increases the advancing contact angles representing the hydrophobic gold phase as shown previously.16 The modification of silicon with PEG caused a clear drop in WCA rendering the surfaces more hydrophilic (θ < 50°) due to the hydrophilic nature of PEG.48 Hence, the decreased WCA confirmed also the successful formation of SAM and thus passivation of the surface. It was confirmed also in a previous study on flat silicon surfaces.9 The subsequent modification with MUDA led to a slightly increased WCA on all nanostructured surfaces (θ ~55°), resulting also in hydrophilic surfaces. During both static and dynamic WCA measurements an inverse trend was found for the WCA 28 ACS Paragon Plus Environment
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with change of feature dimensions being lowest on the smallest features, which may be also related to the Wenzel relationship.49 Both silicon and gold were coated by hydrophilic molecules (PEG or MUDA) resembling now a “rough” hydrophilic surfaces. Hence, also the Wenzel relationship may apply here that “rougher” surfaces, which corresponds to the surface with the smallest feature size, have also the lowest advancing contact angles.50 Cell adhesion and proliferation Adhesion and growth of cells is important for the colonization of biomaterial surfaces. It is also well known that cell spreading is a strong regulator of growth and differentiation of cells.51-54 We could show here that size and periodicity of nanostructures affects cell spreading. In contrast, the cytoskeletal arrangement of stem cells was influenced to a lesser extent. In all cases, polymerized actin fibers were oriented longitudinal in hADSC, which went along a strong polarization of cells as indicated by the high aspect ratio. Such strong, parallel organization of the actin cytoskeleton is an indicator for high mechanical tension generated by the cells through the interaction of integrin adhesion receptors clustered in FA with the underlying substratum and actin cytoskeleton.5, 55-57 However, the irregular arranged actin fibers in hADSC on plain silicon are attributed to the missing patterns. Here, the cells have the freedom of choice to move anywhere on the hydrophilic surface.16 Another indicator for mechanical tension is the strong expression of vinculin in FA at the cells edges to which actin stress fibers insert on all structures. However, the number of FA was highest in hADSC on the smallest nanostructures as it was also found in a previous study with fibroblasts seeded on nanostructured surfaces made by NSL.16 This resulted in an enlarged cell area, which declined slightly with increasing feature dimension. Since nanostructures with the smallest feature dimensions are closely arranged to each other, we speculate that it can provide a more homogeneous surface with freedom for the cells to initiate enhanced integrin clustering, leading to larger spreading than on nanostructures with larger dimensions, which affects 29 ACS Paragon Plus Environment
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mechanotransduction processes, too. Here, the visualization of FAK and RhoA after two hours revealed a noticeable difference in dependence on the structure dimensions. FAK is one of the important tyrosine-phosphorylated proteins that is involved in mediating both integrin and receptor tyrosine kinase (RTK) signaling for the regulation of adhesion, cell shape, and cell migration.24, 58 RhoA, as a small G-protein in the Rho family, couples with downstream regulator, ROCK, in response to extracellular signals.59-60 The activation of RhoA/ROCK always results in cytoskeletal tension, which is essential for sensing mechanical stimuli and subsequently affects cell differentiation.60 Here, the expression of FAK was found much pronounced on nanostructures with smallest feature dimensions, which was consistent with previous observations,27 while the presence of RhoA was not dependent on the feature dimensions. Further, FAK was increased on plain silicon due to its topographical homogeneity giving rise to various anchorage points. It should be noted that FAK plays a vital role in regulating cell growth through the activation of mitogen-activated protein kinase pathway (ERK/MAPK).61-62 Hence, the growth of hADSC was enhanced on the smaller features with no significant difference between mean and small feature sizes. However, this represents another important finding of this study that such variation of feature dimensions towards smaller structures may be useful to promote growth of mesenchymal stem cells on implants or tissue engineering scaffolds. The elevated growth on plain silicon surfaces is attributed to the enhanced adsorption of serum proteins, leading to improved adhesion. Due to the blocking of the silicon surface with PEG on the nanostructured surfaces, a reduced protein adsorption can be expected shown by the FITC-FN studies, which results in a lowered adhesion and growth.
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Cell differentiation The effect of RhoA activation in response to the nanotopography on hADSC differentiation towards adipogenic, chondrogenic, and osteogenic lineages was studied in the presence of chemical inducers. In general, adipogenic differentiation is characterized by a decrease in cytoskeletal assembly with a round morphology. Further, it involves activation of PPARγ to regulate adipocyte maturation. The overexpression of PPARγ could induce a rounded morphology, whereas cells elongate when the expression of PPARγ decreases.63 Chondrogenesis is usually associated with the aggregation of cells for pre-cartilage condensation processes by changing the cytoskeletal architecture through up-regulation of Ncadherins and subsequently activating intracellular signaling pathways.64 Morphological changes in the process of osteogenesis play an important role to regulate actin cytoskeleton in early differentiation. Here, cells become more spread and elongated with a high rate of proliferation.65 Further, regulatory transcription factors such as Runx2, β-catenin and Cbfa1 are expressed during osteogenic differentiation,66 which all help to convert progenitor cells to osteoblasts. In a first trial, our study revealed that the differentiation potential of the nanostructures in combination with chemical inducers is limited in case of osteogenesis. However, adipogenic differentiation was induced by the structures, even though with an atypical cell shape. Typically, a round cell shape with disrupted actin cytoskeleton is needed for successful formation of fat vacuoles, where perilipin is located.67 Nevertheless, we also found fat vacuoles in the spread phenotype. Furthermore, the presence of collagen II and aggrecan indicated that the cells started to differentiate into chondrogenic lineage, even though not to a full extent. Typically, a high cell number is needed for chondrogenesis, which is mostly realized by culturing cells in 3D environments.68 Further, soft substrates in combination with mechanical loading support chondrogenesis to a higher extent.68 Nevertheless, the RhoA/ROCK signaling regulates chondrogenic differentiation, too,69 as 31 ACS Paragon Plus Environment
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shown here. Overall, cytoskeletal reorganization indicates that the activation of RhoA/ROCK has a crucial role in the process of differentiation. It acts as a negative regulator for adipogenic and chondrogenic differentiation, while it serves as a mechanotransducer of matrix stiffness and promotes osteogenesis.60, 70 Studies demonstrated that inhibition of RhoA/ROCK with Y-27632 inhibitor and FAK phosphorylation with PF 228 led to a decreased cytoskeletal assembly and impaired subsequent cellular processes.59-60 CONCLUSION The aim of this study was to explore the suitability of laser interference lithography (LIL) to design hexagonally-arranged nanostructures with periodicities similar to the previously used nanosphere lithography (NSL) and to study the effect of nanotopography on mesenchymal stem cell behavior. It was shown that LIL enabled for precise control of size and distance of nanostructures by simply changing the angle of incidence for illumination. A combination of photolithography and LIL could result in lower periodicities (
