Article pubs.acs.org/Biomac
Engineered Lysozyme Amyloid Fibril Networks Support Cellular Growth and Spreading Nicholas P. Reynolds,*,† Mirren Charnley,‡ Raffaele Mezzenga,§ and Patrick G. Hartley† †
CSIRO, Materials Science and Engineering, Private Bag 10, Bayview Avenue, Clayton, Victoria 3169, Australia Centre for Micro-Photonics and Industrial Research Institute Swinburne, Swinburne University of Technology, Victoria 3122, Australia § ETH, Food & Soft Materials, Department of Health Science and Technology, Schmelzbergstrasse 9, 8092, Zurich, Switzerland ‡
S Supporting Information *
ABSTRACT: Fibrous networks assembled from synthetic peptides are promising candidates for biomimetic cell culture platforms and implantable biomaterials. The ability of the materials to reproduce physiological cell−matrix interactions is essential. However, the synthetic complexity of such systems limits their applications, thus alternative materials are desirable. Here, we design lysozyme derived amyloid fibril networks with controllable topographies, and perform a comprehensive study of the response of cultured fibroblast and epithelial cells. At high surface coverage a favorable increase in spreading and the generation of focal adhesions was observed, due to a combination of biomimetic chemistry and morphology. Their ease of synthesis, makes the nanoscale fibrils presented here ideal materials for future clinical applications whereby large volumes of biomimetic biomaterials are required. Furthermore, the surface chemistry of the fibrils is sufficient for the promotion of focal adhesions with cultured cells, eliminating the need for complex protocols for fibril decoration with bioactive moieties.
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surface.7 However, the design and synthesis of self-assembling short peptide sequences is nontrivial and requires considerable synthetic expertise.15 In nature, nanoscale self-assembled fibrillar structures containing rigid β-sheets motifs (that drive self-assembly) are commonly found in the form of amyloid fibrils.16 However, their connection to neurodegenerative diseases including Parkinson’s17,18 and Alzheimer’s19 has meant they were previously considered unsuitable as ECM mimics. More recent research has provided convincing evidence that the mature fibril is often a nontoxic byproduct of disease.18,20−22 There have also been examples whereby amyloids have been found to possess beneficial functions.23,24 Thus, there has been some investigations into the suitability of using amyloids as self-assembling biomaterials3,4 and biomimetic hybrids.25 Amyloids can be assembled from short synthetic peptides4,26 or from full proteins.3,27 Full protein systems have some advantages over synthetic peptides; first the starting materials are readily available and inexpensive;27 second as many of the proteins used are contained in foodstuffs (β-lactoglobin28 from milk and lysozyme27 from hen eggs), they are generally considered to be nontoxic. Full protein systems, however, offer no control over
INTRODUCTION A major challenge in the field of tissue engineering and regenerative medicine is the development of an artificial extracellular matrix (ECM) mimicking material. If such a material is to have clinical applications (e.g., soft tissue regeneration, or expansion of stem cells), it would need to be able to accurately mimic cell:matrix interactions and be available in large volumes at a reasonable cost.1 These biomaterials should possess a nanoscale topographic morphology reminiscent of the ECM. Moreover the materials should have good cell adhesive properties. Self-assembling peptidic systems offer great potential in the design of nanoscale fibrous biomaterials with both in vitro2−4 and in vivo5,6 applications. These systems are attractive as they can self-assemble into nanoscale fibrillar morphologies that mimic the morphology of the fibrous proteins that make up the ECM.3,7 Self-assembled systems are often preferred over animal derived matrices or coatings such as Matrigel8 or collagen9 due to the highly defined morphology, chemical composition, and purity. There are many examples of self-assembling synthetic peptides2,6,10−14 and one common motif shared by all these systems is the presence of sequences which form β-sheet containing supramolecular structures encouraging the formation of high aspect ratio nanoscale fibrils. Self-assembling peptides are attractive materials as they can be made in high purity and can be designed to display specific functional moieties on the fibril © 2014 American Chemical Society
Received: November 7, 2013 Revised: January 15, 2014 Published: January 16, 2014 599
dx.doi.org/10.1021/bm401646x | Biomacromolecules 2014, 15, 599−608
Biomacromolecules
Article
(MWCO 1000 Da, 24 h, 4 °C) and stored at 4 °C. Amyloid fibril networks (AFNs) were prepared by incubating 50 μL of the fibril solution (pH 7.4) for 10 min onto freshly cleaved mica substrates (1 cm2), followed by rinsing in Milli-Q water (1 mL), and drying under a gentle nitrogen stream. Atomic Force Microscopy. An Asylum Research MFP-3D atomic force microscope (Santa Barbara, CA, USA) was used to measure surface topography. Intermittent contact mode was used for imaging in air with ultrasharp silicon nitride tips (NSC15 noncontact silicon cantilevers, MikroMasch, Spain). The tips had a force constant of 40 N/m and a resonant frequency of 320 kHz. Typically scans were recorded using a set point of 0.7 V at a scan rate of 0.8 Hz. All images were processed (1st order flattening algorithm and roughness parameters) using Igor Pro software, and at least three independent substrates were analyzed when calculating rms (root mean squared) roughness parameters. Fibril thickness measurements were taken from 2 × 2 μm scans, the full width at half-maximum height (fwhm) was recorded for at least 10 fibrils per scan, and average fibril widths were calculated from at least three scans per sample on at least three independent samples. As the same batch of AFM tips was used for all experiments no further corrections for the broadening of the features by AFM (other than the fwhm recordings) for broadening of the features by the AFM tip were considered. Surface coverage measurements were calculated using the ImageJ software package. Briefly, the AFM images were converted to black and white 8-bit images, and the threshold was set to maximize the contrast between the fibrils and the underlying substrate. The percentage coverage of the fibrils was measured using the measure area fraction function in ImageJ. Surface coverage values were calculated from at least 3 images (10 × 10 μm). Cryo-Transmission Electron Microscopy. 200-mesh copper grids coated with a perforated carbon film (Lacey carbon film: ProSciTech, Qld, Australia) were used for all experiments. Grids were cleaned by glow discharge in Nitrogen for 5 s immediately before use. Solutions of fibrils made with varying reaction times (16−30 h) were pipetted (4 μL) onto the copper grids and allowed to adsorb to the grids for 30 s. After adsorption excess fibril solution was removed from the grids by blotting with Whatmann 541 filter paper, for approximately 6−10 s. The adsorbed fibrils were cryo-frozen by plunging the grid into liquid ethane. The frozen grids were stored in liquid nitrogen until required. TEM was performed using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and a Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. An electron dose of 8−10 electrons per Å2 was used for all imaging. Images were recorded using a Megaview III CCD camera and AnalySIS camera control software (Olympus) using magnifications in the range 40 000× to 110 000×. Cell Culture. Samples were placed in the wells of a 24-well plate (Nunc) and then sterilized by immersion in 2X Anti-Anti (Antimycotic-Antibiotic, GIBCO) solution for at least 60 min. An L929 mouse fibroblast monolayer was cultured in media (MEM + GlutaMAX-I, GIBCO), supplemented with 1% v/v NEAA (nonessential amino acids, GIBCO), 1% (v/v) Anti-Anti, and 10% (v/v) FBS (fetal bovine serum, SAFC Biosciences) at 37 °C with 5% CO2/ air atmosphere to 80% confluence. Cells were harvested by trypsinization (2 mL Tryple Express, Invitrogen) and then diluted in 30 mL media followed by centrifugation (300 g for 5 min). Processed cells were counted, resuspended in media (75000 cells/mL), and then added to samples (0.6 mL, 25000 cells/cm2 of well area) and incubated overnight at 37 °C with 5% CO2/air atmosphere. After 24 h incubation, nonadherent cells were removed by rinsing the samples in fresh media and subsequently prepared for either viability assays or immuno-fluorescence assays. Cell Viability Assay. Cell viability was investigated via esterase activity and membrane integrity using the LIVE/DEAD assay. Cells were incubated in LIVE/DEAD reagents (2 μM Calcein AM and 4 μM ethidium homodimer-1, Invitrogen) in Dulbecco’s phosphate buffered saline (DPBS, GIBCO) supplemented with 2% v/v FBS for at least 20 min. Stained adhered cells were imaged on an inverted microscope
the amino acid sequence presented on the fibril surface, and generally the fibrils present no specific sequences that allow adhering cells to generate biomimetic cell:matrix interactions. Biofunctionalization can be achieved by decorating fibrils with short peptide sequences ‘borrowed’ from the proteins within the ECM. The most well-known of these being Arg-Gly-Asp (RGD) from fibronectin (FN), which promotes the formation of integrin mediated focal adhesions (FAs).4 There have been a few examples where self-assembled systems have been successfully fabricated with biofunctional linkers on their surface, and resulted in favorable effects on the physiology of cultured cells.2,4,5 However, progress in this field has been slow as the successful integration and display of functional moieties onto scaffolds poses a number of challenges: first a lack of understanding of the final atomic structures of the supramolecular assemblies makes it difficult to rationally engineer systems that will successfully self-assemble while displaying the desired functionality on the fibril surface and second the fibrillar assemblies are generally held together by noncovalent forces making them fragile and unable to withstand the conditions often required for postassembly chemical/bioconjugation.15 In this study we used an amlyoid fibril system that is formed when hen egg white lysozyme is exposed to high temperature and low pH triggering protein hydrolysis.27,29 The peptide fragments produced have amyloidogenic sequences that promote formation into amyloid fibrils with controllable morphologies.30 Previous studies on a variety of globular proteins has shown that around 80% of the protein monomer is converted into a fibrillar form27,29 and that the amino acid sequence 57−107 from lysozyme is highly amyloidogenic,31 thus it is likely that this sequence is essential to fibril formation. Residues 66−68 within this fragment contain the tripeptide DGR that is analogous to (although in reverse) to the integrin binding RGD sequence from FN. Thus, we proposed that lysozyme fibrils might promote FA formation with cultured cells, without the need for any further decoration. The spacing of adhesive ligands on nanotopographical substrates has also been shown to have profound effects on cellular response.32−34 In order to determine optimum conditions for cellular attachment, here we present a study of a range of different fibrillar morphologies and surface coverages, and an assessment of how these properties impact on cellular response. The different fibrillar surfaces were fully characterized by complementary physical techniques (atomic force microscopy (AFM) and cryogenic transmission electron microscopy (cryo-TEM)), and cellular response to cultured fibroblast or epithelial cell lines was assessed using assays for viability, attachment, morphology of the actin cytoskeleton, and generation of FAs.
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MATERIALS AND METHODS
Amyloid Self-Assembly. In order to obtain protein of sufficient purity to allow a well controlled self-assembly reaction, hen egg white lysozyme (Sigma) was dialyzed according to the protocol used in Jung et al.29 Solutions of 2 wt % lysozyme were prepared in Milli-Q water and adjusted to pH 2, before being filtered through a 0.45 μm membrane in order to remove any pre-existing aggregates before use. Solutions were transferred to sealed glass vials and placed in an oil bath at 90 °C for up to 30 h while undergoing constant stirring (300 rpm, using a 20 × 5 mm Teflon magnetic stirrer bar) as described in Lara et al.27 In order to stop the fibril self-assembly reaction, solutions were quenched in a water-ice mixture. Presence of fibrils was confirmed by a shoulder at around 217 nm in circular dichroism spectra (indicative of extensive β-sheet structures27). The quenched solutions of fibrils were then dialyzed into Milli-Q water at pH 7.4 600
dx.doi.org/10.1021/bm401646x | Biomacromolecules 2014, 15, 599−608
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Figure 1. Fibril width is linearly proportional to reaction time. Measured fibril width is dependent on the hydration state and substrate. (a−c) CryoTEM images of lysozyme fibrils formed at 90 °C for (a) 16 h, (b) 24 h, and (c) 30 h. (d,e) Representative AFM images of dried fibrils on mica, formed at 90 °C for 6 and 30 h respectively. All z-scales in the AFM images =10 nm. (f) Average measured fibril width plotted against fibril reaction time measured by either cryo-TEM (hydrated fibrils, solid line) or AFM (dried fibrils, adsorbed to mica substrates, dashed line). either using a ×10 or ×40 air immersion lens (Nikon Eclipse TE2000U). Cell counts were performed on images recorded with the ×10 lens using the particle analysis function in the software ImageJ. Before the particle analysis, the images were converted to 8-bit black and white images and the threshold set to ensure the software only counted features that were actual cells. The results obtained by the software were compared to images counted by hand and were within 5% accuracy. Statistical analysis of the average number of cells and cell spreading was performed over three separate experiments. The results are reported as attached counts normalized to a mica control surface included in each experimental repeat. Cell area was quantified from images recorded with the ×40 lens using the wand (tracing) tool and measure function in ImageJ. A line bisecting the image was randomly drawn and the first 10 cells along that line were measured in each image. Statistical analysis of both cell numbers and cell spreading area across the three experiments was performed using ANOVA with Tukey’s multiple comparison in Minitab statistical software. Immunofluorescence Assay. Cells were cultured on the different cell culture platforms for 24 h before fixing in paraformaldehdye 4% (w/v) in PBS, for 15 min and permeabilized by the addition of Triton X-100 (0.1% (v/v) in PBS, 10 min, Fluka). Samples were blocked in bovine serum albumin (BSA; 1% (w/v) in PBS, Sigma-Aldrich) for 60 min. Samples were rinsed and incubated with the primary antibody for the focal adhesion protein vinculin (1: 200 dilution in 1% BSA), rinsed and subsequently incubated with Alexa Fluor 568 goat antimouse IgG (1:500 dilution, in 1% BSA Molecular Probes) and Alexa Fluor-488 conjugated Phalloidin (1:20 dilution), for 60 min before a final rinse in PBS and imaged using an Olympus IX71 microscope (Olympus) and 100x objective lens (1.4NA Oil Plan Apochromat).
thickness of the fibrils in a fully hydrated state (cryo-TEM) and also when adsorbed to a solid support (freshly cleaved mica) (AFM). Representative images of the fibrils after various reaction times are shown in Figure 1. Figure 1a−c show cryo-TEM images of the fibrils after 16, 24, and 30 h, respectively. As previously noted,27 the fibrils increase in width with increasing reaction time; this is due to a modular stacking of individual amyloid protofilaments into mature multifilamental structures. As the reaction time increased, a change in fibril morphology can also be seen: In Figure 1a (16 h), almost all the fibrils have a linear fibril structure. As the reaction time increases (Figure 1b), an increasing proportion of the fibrils adopted a multistranded ribbon-like morphology, as previously observed in Lara et al.27 As the reaction time increased further (30 h, Figure 1c), the relative proportion of ribbon-like structures (cf. linear fibrils) increased, as did their width, due to more filaments stacking to form each ribbon. It is this modular addition of filaments to the growing fibrils/ribbons that resulted in the increase of fibril width with increasing reaction time. As the aim of this work was to study how the amyloid fibril networks (AFNs) adsorbed to solid supports affected the culture of eukaryotic cells, we also investigated fibril morphology of the adsorbed fibrils on mica by AFM. Representative AFM images of fibrils formed for either 6 or 30 h before being adsorbed onto mica substrates are shown in Figure 1d, and e, respectively. Once again, these images show that the increasing reaction time results in an increase in fibril width. Figure 1f shows plots of measured fibril width from both the AFM data (dashed line) and the cryoTEM (solid line). Analysis of the fibril widths shows the average measured fibril width increases proportionally with increasing reaction time for both sets of data. The AFM data shows that the fibrils increase from 37.7 ± 0.9 nm at 16 h incubation time to 50.8 ± 1.7 nm at 30 h, while the cryo-TEM
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RESULTS Characterization of Fibril Morphology. The amyloid fibril forming reaction was allowed to progress for 2, 6, 16, 24, or 30 h, and the morphology of the amyloid fibrils was investigated to determine how reaction time affected fibril width. Cryo-TEM and AFM were used to determine the 601
dx.doi.org/10.1021/bm401646x | Biomacromolecules 2014, 15, 599−608
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Figure 2. Surface coverage of amyloid fibril networks generated by incubating solutions of fibrils onto freshly cleaved mica substrates for 10 min, imaged by intermittent contact mode AFM in air. (a) 2 wt % lysozyme solution; amyloid fibril networks formed at 90 °C for (b) 2 h, (c) 6 h, (d) 16 h, (e) 24 h, and (f) 30 h (% coverage also listed on images). All scans have a z-scale = 10 nm.
data shows and increase from 13.9 ± 0.8 nm at 16 h to 26.3 ± 1.2 nm at 30 h. AFM experiments performed on fully hydrated AFN samples (Supporting Information, Figure S1) showed an intermediate thickness of 28.9 ± 2.4 nm (30 h). This discrepancy between the AFM and cryo-TEM data was due to a combination of factors: First, in solution mature fibrils adopt a 3D twisted ribbon structure,27 which eventually close on themselves to form hollow nanotubes.35 High-resolution AFM reveals that this 3D structure is flattened out when the positively charged fibrils are dehydrated and adsorbed to the negatively charged mica substrate27 resulting in an apparent widening of the fibril. The intermediate thickness seen for the AFM performed in a fluid environment arose due to multiple electrostatic interactions between the fibril and the mica causing the fibrils to flatten out on the substrate, but not dehydration effects. Second, the finite diameter of the AFM tip (radius of curvature ∼8 nm) itself resulted in an overestimation of the fibril width. Reaction Time Affects Surface Coverage. The different solutions of fibrils were deposited onto freshly cleaved mica substrates at high concentrations (2 wt %). After 10 min, unattached fibrils were rinsed from the surface, and the dried samples imaged by tapping mode AFM. Representative images of fibrils formed for different reaction times (2−30 h) are shown in Figure 2. In addition to the fibril-coated surfaces, a further surface where monomeric lysozyme was adsorbed to the mica substrates at the same concentration was examined. This substrate was chosen as a control substrate as higher resolution AFM (Supporting Information, Figure S2) revealed that the spaces between adsorbed fibrils in the other substrates were not composed of bare mica but were coated with a film of what are presumably nonaggregated peptides.30,36 Further evidence for the continued presence of nonaggregated peptides after the
self-assembly reaction was seen in the circular dichroism (CD) spectrum of a solution of lysozyme postassembly (Supporting Information, Figure S3). The CD spectrum in Figure S3 displayed a characteristic minimum at around 200 nm typical of unfolded polypeptide chains (due to the presence of nonaggregated short peptides). In addition, the shoulder in the spectrum at around 217 nm is indicative of the presence of βsheet structures from the amyloid fibrils. As the aim of this study was to investigate the effects of changing fibrous topography on a substrate while (to the best of our abilities) keeping the underlying surface chemistry constant, it was decided that the lysozyme control surface would be more appropriate than bare mica. Figure 2a shows a representative AFM image of a mica substrate with adsorbed monomeric lysozyme, at this resolution the surface appears smooth and the measured rms roughness was below 0.5 nm, thus these surfaces were chosen as a ‘flat’ lysozyme coated control surface. Figure 2b−f show representative images at increasing fibril reaction times. What is immediately apparent is that even though the concentration of lysozyme (measured before the self-assembly reaction) was the same in all images the fibril surface coverage were very different. Figure 2b shows the surface coverage after 2 h reaction, at such short reaction times very few fibrils were formed in solution, thus the apparent topography of the substrate was similar to the lysozyme control surface. After 6 h (Figure 2c) considerably more fibrils were present on the mica substrates, and this trend was continued after 16 h (Figure 2d). At reaction times above 24 h (Figure 2e,f) the concentration of fibrils was sufficient to cover the majority of the mica substrate. In order, to gain a more quantitative insight into the surface topography provided by the AFN shown in Figure 2, both the surface coverage and rms roughness (Rrms) of multiple AFM images was calculated for the various different substrates. 602
dx.doi.org/10.1021/bm401646x | Biomacromolecules 2014, 15, 599−608
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For clarity, only the calcein staining is shown (calcein only stains viable cells), and the images of the ethidium homodimer (stains only cells dead or dying cells) stained cells are shown in the Supporting Information (Figure S4). Quantification of the amount of nonviable cells revealed that on all surfaces, less than 1% of the cells remaining attached after rinsing were nonviable, hence the surfaces were deemed noncytotoxic for this cell line at these time scales. The micrographs (Figure 4a−f) showed a moderate increase in the number of attached viable cells as the AFN coverage increased from 0% (monomeric lysozyme coated surfaces) to 86%. The cells cultured on the surfaces with higher percentage AFN coverage (>80%) also appear to adopt more favorable, spread morphologies. In order to quantify these apparent increases in attached cells and spreading, both cell number and the average surface area occupied by each cell was calculated. The results of the cell counting experiments are shown graphically in figure 4g. As seen in the images, increasing the AFN surface coverage resulted in a nonsignificant increase in the number of attached viable cells. This suggests that the presence of the AFN, and hence topography, has no statistically relevant effect on the number of attached cells. In addition to cell number, cell spreading was also investigated, as fibroblast cells are known to adopt more favorable spread morphologies on fibrous nanotopographical surfaces.3 Similar to the cell number results, no significant increases in spreading were seen at AFN coverage’s up to 40% (Figure 4h). However, at the two highest coverages (82 and 86%), the cells adopted a more favorable spread morphology with their average surface area increasing from 289 ± 12.38 μm2 for the 40% AFN to 468 ± 26.23 μm2 for the 82% AFN (p < 0.001). A further increase in cell spreading was seen for the 86% AFN substrates (532 ± 25.27 μm2); however, this increase was not sufficient to be deemed significant compared to the 82% AFN substrate. From these results it appears that at high coverages of AFN, cell spreading is promoted. However, even at the highest coverages, a small fraction of proteins remains in the nonfibrillar morphology, and previous research has shown this to be less than 20%.27,29In order to investigate potential effects this nonfibrillar fraction (or the fibrils themselves) may have on cell attachment and viability, we performed the live/dead assay on fibroblast cells cultured at low seeding density (10 000 cells per mL) for 7 days (Figure S5, Supporting Information). After washing, the vast majority of the remaining attached cells are viable and well spread, indicating that even at longer incubation times there is no unforeseen effect on viability. Amlyoid Fibril Networks Generate Focal Adhesions with Cultured Cells. To determine the origin of the increased cell spreading seen on the AFN coverage’s above 80%, adherent cells were fixed and incubated with phalloidin, to stain the actin cytoskeleton, and antivinculin antibodies, to visualize integrin mediated FAs. Small distinct regions of high antivinculin concentrated around the periphery of the cell (FAs occur predominantly at the cell periphery34) represent FAs between the cell membrane and the underlying substrate. Representative images are shown in Figure 5. Cleaned glass coverslips were chosen as a positive control as previous experiments have shown that glass and other hydrophilic surfaces are capable of generating high levels of FAs when cells are cultured in serum containing media,37 similar those observed when cells are cultured on ECM proteins such as FN.4 As was expected the cells cultured on glass clearly show actin stress fibers running throughout the cell (Figure 5a). Stress fibers are closely connected to the presence
First looking at the surface coverage (Figure 3a), 2 h reaction times provided low coverage with average values of less than
Figure 3. Increasing reaction time causes a higher relative fibril concentration (compared to monomeric or prefibrillar lysozyme) which in turn results in higher fibril coverage. (a) Quantification of surface coverage at different reaction times; at reactions times greater than 24 h the increase in surface coverage becomes statistically insignificant. (b) rms roughness increases with increasing surface coverage at low coverage; however, at surface coverage above 40%, no significant increase in roughness is observed. All values calculated from multiple 10 × 10 μm AFM images. *** p < 0.001; NS = not significant, n = 3.
2%, after 6 h the surface coverage significantly increased to 13%, and after 16 h this further increased to 40%. After 24 h the surface coverage increased to 82%, and at longer reaction times no further statistically significant increases in surface coverage was observable. Henceforth, the various coated surfaces will be referred to based on their average surface coverage (e.g., AFN surfaces formed from fibrils incubated for 30 h, will be referred to as 86% substrates). Initially, a similar trend in rms roughness values was observed. The
