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Article
Ice-Templated and Cross-Linked Amyloid Fibril Aerogel Scaffolds for Cell Growth Gustav Nyström, Wye-Khay Fong, and Raffaele Mezzenga Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00792 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017
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Ice-Templated and Cross-Linked Amyloid Fibril
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Aerogel Scaffolds for Cell Growth
3 Gustav Nyström‡, Wye-Khay Fong‡§ and Raffaele Mezzenga*,‡
4 5 6
‡
ETH Zurich, Department of Health Science & Technology, Schmelzbergstrasse 9, 8092 Zurich,
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Switzerland §
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,
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Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.
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Keywords: self-assembly, templated assembly, amyloid fibril, 3D scaffold, cell, microscopy
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*Email:
[email protected] 12 13 14 15 16
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ABSTRACT
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Amyloid fibrils prepared from β-lactoglobulin were used to form freeze-dried and cross-linked
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aerogels. By varying the fibril concentration and freezing gradient, it was possible to control the
4
pore structure and elastic modulus of the aerogels over one order of magnitude from ~2 to ~200
5
kPa. Using butane tetracarboxylic acid as cross-linker these aerogels maintained their monolithic
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shape in aqueous conditions displaying elastic behavior and a modulus in the range of 4–40 kPa.
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When explored as scaffolds for cell growth, the amyloid fibril aerogels demonstrated
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biocompatibility and led to the successful penetration and permeation of two epithelial cell lines
9
(Caco-2 and HT29) throughout the scaffold. These soft, elastic and water-stable biomaterials
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expand the scope of amyloid fibril aerogels, making them suitable for wet state applications such
11
as heterogeneous catalysis, purification membranes and 3D matrices for cell growth.
12 13
1. INTRODUCTION
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Water is the most important liquid available; it is critical to sustain life and is highly useful for
15
dissolving, dispersing, processing and distributing media. Water also has structural properties,
16
showing regular ordering,1 as well as an accessible liquid to solid transition, both of which can
17
be exploited on the nanoscale. By controlling the depth and direction of the freezing gradient,
18
different forms and shapes of solid, amorphous or crystalline2 ice can form. During the growth of
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ice crystals, solutes3 or colloidal particles4 dispersed in the aqueous phase are expelled from the
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ice phase and concentrated in regions surrounding the growing ice front. This phenomenon has
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been used to template and control the pores in composite materials,5 to modify the texture of
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foods,6 as well as a way to control the aggregation7 and to induce phase separation between
2
colloids.8
3
Amyloid fibrils are semi-flexible anisotropic colloids formed through self-assembly from protein
4
aggregates. They were initially discovered in vivo, in relation to neurodegenerative disorders
5
including Alzheimer's and Parkinson's disease, but have recently attracted increasing attention in
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bio-functional materials such as catalytic scaffolds, hormone storage and functional coatings.9–11
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These fibrils are known to self-assemble into nematic liquid crystal phases12,13 and to form
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physical hydrogels at appropriate fibril and salt concentrations.14,15 These gels are typically weak
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with elastic moduli in the order of 10–50 Pa and while they maintain their structural integrity in
10
solution, they are difficult to handle without causing the gel network to break. One option to
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improve the mechanical properties of the gels is to use chemical cross-links. While there are
12
many different alternatives to form the cross-links, a particularly appealing route is to use a
13
solid-state reaction approach,16,17 allowing for the homogenous distribution of cross-linker
14
throughout the gel network, and to go through a freezing and drying step before finally re-
15
dispersing the gel in the medium of interest.
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Cells are known to naturally adhere to amyloid fibrils,18,19 and the existing literature includes the
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use of α-synuclein amyloid fibril hydrogels to promote stem cell differentiation to neurons20 and
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the exploration of heat-induced gelation of lysozyme amyloid fibril hydrogels as cell scaffolds.21
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Lysozyme fibrils have also been used as two-dimensional cell supports for the study of
20
controlled cell attachment,22 spreading and growth of cells,23 as well as substrates for primary
21
human trabecular bone-derived pre-osteoblast cells,24 further demonstrating the viability of these
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fibrils for supported cell growth. Other, more specific peptide-based systems have also been used
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to study the interaction between amyloid fibrils and cells. For instance, thin films of TTR1-
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cycloRGDfK peptide fibrils demonstrated cell adhesion and spreading of cells,25 and hydrogels
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formed using fibrils based on the C-terminal amyloid β-protein (Aβ42) were used for cell culture
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and stem cell differentiation.26
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Here, we use amyloid fibrils prepared from β-lactoglobulin (BLG) as starting colloidal particles
5
and a controlled freezing, drying and cross-linking protocol to prepare porous structural amyloid
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fibril aerogel scaffolds. By changing the particle concentration and morphology of the scaffolds,
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it is possible to control the mechanical stiffness of these materials within one order of magnitude
8
reaching final hydrated stiffness values up to ~40 kPa, three orders of magnitude higher than
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traditional amyloid hydrogels. The chemical cross-linking renders these materials highly stable
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in water, opening up a wide range of post-functionalization treatments and wet state applications
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such as filters, purification membranes27 and catalysis substrates.28 In this paper, we explore
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these porous structural amyloid materials as scaffolds for cell growth using cells from two
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different cell lines. The results, evaluated using laser scanning confocal microscopy and cell
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viability assays, show that these materials are biocompatible, making them good candidates for
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applications as cell growth scaffolds, drug delivery systems and artificial implant materials.
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2. EXPERIMENTAL SECTION
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Materials. Biopure β-lactoglobulin (BLG) monomer (lot JE 003−6−922) was obtained from
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Davisco Foods International, Inc. (Le Sueur, MN). 1,2,3,4 Butanetetracarboxylic acid (BTCA)
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and sodium hypophosphite, NaH2PO2 (SHP) was purchased from Sigma Aldrich. For the cell
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culture and DOX release studies, Dulbecco’s modified Eagle’s media (DMEM), fetal bovine
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serum (FBS), non-essential amino acids (NEAA) and Penicillin-Streptomycin (5,000 U/mL)
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(pen/strep) were acquired from Gibco, Life Technologies, Switzerland. 24 well plates were
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purchased from Bioswisstec AG, Switzerland and the µ-Slide 8 Well, ibiTreat from Vitaris AG,
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Switzerland. The HT29 and Caco2 cell lines were a generous gift from Dr. Tomás de Wouters of
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the Food Biotechnology group at ETH Zürich.
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Preparation of amyloid fibrils. Amyloid fibrils were prepared according to a previous
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protocol.12 In short, 20 g of BLG monomer were dissolved in 180 mL deionized water (Milli-Q
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purification system, Millipore). The pH was set to 4.6 followed by centrifugation (20 000 RCF
7
for 15 min) to remove larger protein aggregates or denatured protein particles. The supernatant
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was collected and the pH was readjusted to 2 followed by filtration through a 0.22 µm cellulose
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filter and finally dialyzed (6000-8000 MWCO, Spectrum Laboratories) against pH 2 Milli-Q
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water and subsequently Milli-Q water for 5 days with daily bath changes. Following the
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purification, the protein monomer solution was incubated at 2 wt% monomer concentration, pH
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2 and 90 °C for 5 hours. During the incubation, the protein monomer unfolds, hydrolyzes and
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self-assembles into amyloid fibrils.
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Preparation of cross-linked amyloid fibril scaffolds. Amyloid fibril dispersions (at
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concentrations of 1–4 wt%) were mixed with dry powder of BTCA and NaH2PO2 at a fibril to
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BTCA weight ratio of 1:0.2 and a NaH2PO2 to BTCA weight ratio of 2:1. For the Amyloid-196
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samples, aliquots of 90 µL were put in 7 mm diameter and 2 mm height stainless steel cylindrical
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molds and frozen using liquid nitrogen. For the Amyloid-20 samples, aliquots of 90 µL were put
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in 7 mm diameter and 2 mm height PDMS molds placed on a stainless-steel bottom plate,
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covered with an isolating PDMS sheet and put to freeze at -20 °C. Following freezing, the
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samples were freeze dried (FreeZone 4.5, Labconco, US) and the dried samples were heat cured
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at 150 °C for 3 min. Prior to use, the scaffolds were thoroughly washed using Milli-Q water to
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remove any remaining chemicals as well as UV sterilized before the cell experiments.
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Characterization. Scanning Electron Microscopy (SEM) was performed using a Leo 1530
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Gemini microscope (Zeiss) operated at 1 kV acceleration voltage and a working distance of 5
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mm. Thin sections of the scaffolds were cut with a razor blade and attached to aluminum stubs
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using adhesive carbon tape and a ~3 nm layer of platinum was deposited on the samples prior to
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imaging. Transmission Electron Microscopy (TEM) was performed using a Morgagni 268 (FEI)
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microscope operated at 100 kV acceleration voltage. The samples were prepared by placing 4 µL
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of 1 g L-1 dispersion on carbon coated copper grids (Quantifoil). After an adsorption time of 60 s
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the grids were blotted, rinsed 2 times with pH 2 Milli-Q water and dried at ambient conditions.
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Fourier Transform Infrared Spectroscopy (FTIR) was performed using a Varian 640
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spectrometer equipped with a Golden Gate diamond ATR stage. Mechanical testing was
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performed using a Z010 (Zwick) operating in compression mode, using a 10 N load cell and a
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compression rate corresponding to 10% of the initial specimen height per min. Compressive
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stress-strain curves were plotted and the Young's modulus was determined from the slope of the
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low strain region. The density and porosity of the amyloid fibril scaffolds were calculated by
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gravimetric determination of the aerogel weight using a balance and careful measurement of the
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aerogel volume using optical microscopy imaging. By taking only the mass given by the balance
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into consideration we obtain the apparent density of the scaffolds as the mass of the solid divided
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by the geometric volume of the scaffolds. The porosity is calculated according to: φ = 1−
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where ρ app is the apparent scaffold density and ρ fib is the density of the amyloid fibrils
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measured by helium pycnometry (Accupyc 1340, Micromeritics) to 1.3 g cm-3.
ρapp ρ fib
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Cell Studies. Prior to seeding, the amyloid fibril scaffolds were sterilized by exposure to UV
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light for 1 h and then hydrated and rinsed with a large volume of autoclaved Milli-Q water to
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ensure clean and sterile samples. Individual hydrated scaffolds were then placed into separate
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wells and rinsed twice with cell culture medium.
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Caco-2 human epithelial colorectal adenocarcinoma and HT29
human colorectal
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adenocarcinoma monolayer cells were cultured in DMEM, supplemented with 1% v/v NEAA
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(nonessential amino acids, Gibco), 1% (v/v) Penicillin Streptomycin, and 10% (v/v) fetal bovine
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serum, (FBS, Gibco) at 37 °C with 10% CO2 to 80% confluence. Cells were harvested by
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trypsinization (2 mL) and then diluted to 10 mL media followed by centrifugation (105 RCF for
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5 min). Processed cells were re-suspended, counted and then seeded into each well at a density of
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10 000 cells/well in 1 mL of media in a 24 well plate or 3000 cells/well in 300 µL media in the
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8-well µ-Slides. For the wells that contained fibril scaffolds, cells were seeded at the same
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density into the fibril network. The plates were then incubated overnight at 37 °C with 10% CO2.
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After 24 h incubation, non-adherent cells were removed by rinsing the samples in fresh media.
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The culture media was changed daily. At 4 days after seeding, the fibril scaffolds were removed
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from the cell culture medium and subsequently then prepared for either viability assays or for
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confocal fluorescence imaging.
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Cell Viability Assay and Imaging. Cell viability was investigated utilizing a lactate
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dehydrogenase (LDH) assay (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega,
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Switzerland). Data were further analyzed using a one-way ANOVA statistical test (Holm-Sidak
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method) in order to determine statistical significance (p < 0.05) between the groups.
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For imaging, cells were labeled using a LIVE/DEAD viability/cytotoxicity staining kit (L-
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3224, Molecular Probes) where the fluorophores, calcein-AM and ethidium homodimer-1, was
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used to stain live and cells respectively. Cells were imaged using an LSM 780 laser scanning
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confocal microscope (Zeiss). A 488 nm argon laser was used as excitation source and the emitted
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photons were collected in the wavelength interval 493–540 nm (live cells) and 614–702 nm
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(dead cells).
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3. RESULTS AND DISCUSSION
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Amyloid fibril scaffolds were prepared as schematically described in Figure 1A. First BLG
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monomers were self-assembled into high aspect ratio fibrils12 as imaged by TEM in Figure 1C.
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Thereafter, ice-templating, using two different freezing gradients, followed by freeze-drying was
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used to prepare porous, dry amyloid fibril aerogel scaffolds. Since untreated freeze-dried fibrils
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immediately re-disperse when added to water, we used a solid-state cross-linking process to
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stabilize the scaffolds. For this reaction, BTCA29 was added to the fibril dispersion together with
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SHP which was used as a catalyst30 prior to templating and drying to allow a good distribution of
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cross-linker among the fibrils. This means that, in the heat curing step following the freeze-
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drying, they were in immediate proximity to the fibrils allowing for an efficient and
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homogeneous cross-linking of the fibril scaffold. Re-dispersibility tests as well as SEM were
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used to determine the optimal weight ratio of amyloid fibril to cross-linker (1:0.2), as well as the
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optimal heat curing time (180 s at 150 °C) (Figure S1–S3 in the Supporting Information). By
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varying the freezing temperature, and thereby the freezing gradient, it is possible to control the
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shape and size of the ice crystals formed31 and with that, the final pores created in the material
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(Figure 1B,E). In this study, the concentration of amyloid fibrils in the dispersion has been varied
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and two different freezing gradients (-20 °C and -196 °C) were utilized to create 6 different
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material combinations (Figure S4). In general, the larger freezing gradient produced stratified
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pores with a smaller pore size than the lower freezing gradient sample, which showed a more
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cellular structure. The apparent densities and porosities were found to be similar for both
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freezing gradients and increased proportionally to the total concentration of fibrils used in the
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materials (ρapp ~ 20, ~30 and ~50 mg cm-3 and ϕ ~ 96–98% for 1, 2 and 4 wt% materials
3
respectively). Closer examination of the pore walls by high resolution SEM revealed that the
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morphology of the intertwined amyloid fibril network remained, as seen by the appearance of
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fibril aggregates on the otherwise smooth pore surface, after the scaffold ice-templating and
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cross-linking (Figure 1D).
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Figure 1. Schematics of amyloid fibril scaffold preparation and electron microscopy of amyloid
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fibrils and amyloid fibril scaffolds. A, Schematic of amyloid fibril cross-linking mechanism and
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preparation of scaffolds templated by ice crystals. B, Scanning electron micrograph of a dry 2
4
wt% amyloid fibril aerogel scaffold prepared using a -20 °C freezing gradient. C, Transmission
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electron micrograph of amyloid fibrils. D, Scanning electron micrograph at high magnification
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showing the fibrillar structure of the pore walls of the fibrillar aerogel scaffolds. E, Scanning
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electron micrograph of a dry 2 wt% amyloid fibril aerogel scaffold prepared using a -196 °C
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freezing gradient.
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The solid-state cross-linking reaction used here has the advantage of requiring a minimum
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amount of post treatment (only short heat treatment) of the already formed amyloid aerogel
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scaffolds. Therefore, the ice-templated pore morphology is intact and the cross-linking as such
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does not influence the resulting macro-gel structure. From the re-dispersibility tests (Figure S1–
14
S2), it is already evident that the cross-linking has a clear stabilizing effect on the amyloid
15
aerogel scaffolds. In fact, at sufficient amount of cross-linker relative to fibril (≥ 1:0.2) and heat
16
curing time (≥ 60 s), the scaffolds are stable in water for more than two years. FT-IR in
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attenuated total reflection (ATR) mode was performed to further verify the cross-linking
18
mechanism. In Figure 2A, the spectra of an amyloid fibril scaffold is compared to BTCA cross-
19
linked scaffolds for an increasing amount of heat treatment in the full wavenumber range
20
considered in the experiment. Upon cross-linking, the most clearly seen differences occur in the
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lower wavenumber region between 1200 and 800 cm-1. Assigning the low wave number peaks
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first, the vibration at 810 cm-1 represents the H-P-H wagging mode of the SHP and the vibrations
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at 1040 cm-1 and 1080 cm-1 represent C-O-C stretching vibrations of the cyclic anhydride formed
24
as an intermediate during the BTCA cross-linking reaction.32,33 The other bands are overlapping
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with the bands from the protein, and are therefore difficult to uniquely assign. In order to observe
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the spectroscopic differences more clearly, Figure 2B compares the reference sample with the
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sample used in this study (3 min heat treatment) before and after washing, where data is in the
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range 1800 to 600 cm-1 and normalized by the intensity of the common peak around 1635 cm-1.
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Firstly, it is noted that the washing step effectively removes the peaks related to the SHP and
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intermediate structures of BTCA still remaining in the sample. These results are, however, not
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quantitative and while BTCA has been found non-toxic up to an intake of 500 mg/kg/day based
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on in vivo studies in rats34 and SHP is frequently used as a food additive, further work using
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NMR should be performed to verify the absence of residual chemicals before advancing the use
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of the materials in vivo. An increased intensity shoulder for the vibrations at 1720 cm-1 is also
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observed, representing the ester carbonyl bands formed as a result of the cross-linking reaction.35
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To resolve this peak more clearly, the difference spectra of the cross-linked samples and
12
reference sample are plotted in Figure 2C. From this, a gradual increase in intensity for
13
increasing heat treatment time can be seen around 1700 cm-1, indicating an increasing
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concentration of carboxylic acid groups35 resulting from the attached BCTA crosslinker
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molecules. Still, considering the sufficient integrity in the re-dispersibility tests, 3 min heat
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treatment was used for the samples in this study to also minimize the potential degrading effects
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of the heat treatment on the amyloid fibrils.
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Figure 2. FTIR spectra of amyloid fibrils before and after cross-linking with BTCA. A, FTIR
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spectra in the range 4000–600 cm-1 with broken axis at the spectral region blocked by the ATR
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crystal for untreated amyloid fibrils (black line) and BTCA cross-linked amyloid fibrils with
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increasing heat curing times. B, FTIR data normalized by the 1635 cm-1 peak for amyloid fibrils
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before (black line), after cross-linking (red line) and after cross-linking and washing (dotted red
8
line). C, Difference FTIR spectra in the range 1800–1650 cm-1 for increasing heat curing times.
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The mechanical performance of the amyloid fibril aerogel scaffold was furthermore evaluated
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using compression tensile testing. Figure 3A shows representative compressive stress strain
12
curves for the two different freezing gradients. Interestingly, at the same total fibril concentration
13
these curves display a different behavior. The Amyloid-20 sample has a higher stiffness than the
14
Amyloid-196 sample, and follows a qualitatively different compression pattern. This is attributed
15
to the differences in microstructure between the samples (Figure 1B,E) where the more evenly
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distributed cellular like structure of Amyloid-20 is expected to be more efficient in distributing
2
the initial load on the sample, deforming first by cell wall bending (initial linear region) followed
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by cell wall buckling (plateau like region).36 This behavior is different compared to the more
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sheet like morphology of the Amyloid-196 samples allowing an easy compression of the parallel
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layers. At sufficient compression, however, the initial microstructure no longer matters and the
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two samples follow a similar slope defined by the amount of fibrils present in the materials,
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indicating a similar compressed microstructure in the two samples. Figure 3B shows the elastic
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modulus for the different sample types as a function of aerogel density, ρ. For both samples, the
9
stiffness increases with the increased concentration of fibrils as expected. The increase follows a
10
general scaling behavior, ߩ~ܧఈ , where an exponent α of ∼1 and ∼1.8 was found for Amyloid-20
11
and Amyloid-196 respectively. There are, to our knowledge, no comparable data for amyloid
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fibrils, however the same exponent of 1.8 was previously observed for liquid nitrogen frozen and
13
freeze-dried nanocellulose foams.37 To evaluate the mechanical performance of these samples at
14
conditions more relevant for biological applications, mechanical compression measurements
15
were also performed in PBS buffer solution, see Figure 3C. The elastic modulus was evaluated
16
from the stress-strain curves after a 24 h equilibration in PBS buffer. For all fibril concentrations,
17
a lower stiffness was obtained, as expected, based on the plasticizing effect of the water. The
18
data also indicate a slight difference in the scaling exponent of the modulus as a function of
19
density for the two sample types, where α of ∼1.3 and ∼1.7 was found for Amyloid-20 and
20
Amyloid-196 respectively. Even though these differences should be treated cautiously based on
21
the limited amount of data points available, the behavior is interesting since it suggests that the
22
microstructural differences observed in the dry state also lead to different trends in how the
23
modulus scales with fibril concentration in the wet state. Overall, the different sample
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morphologies and fibril concentrations investigated here clearly show that it is possible to
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control the stiffness of the scaffold, and attain values varying within one order of magnitudes
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from ~4 to ~40 kPa. This range matches well to that of stiffer tissues such as fibrotic wound
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tissue, muscle and cartilage suggesting good mechanical compatibility to these biological
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systems.38
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Figure 3. Mechanical properties of the amyloid fibril aerogel scaffolds. A, Compressive stress
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strain curves representative for the amyloid fibril scaffolds in dry state prepared using a -20 °C
4
(black line) and -196 °C (red line) freezing gradient. B–C, Young's moduli extracted from the
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slope of the initial linear part of the compressive stress strain curves for varying amyloid fibril
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concentrations measured in dry state and equilibrated in PBS solution respectively. The data is
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presented as arithmetic means and standard deviations (n=3).
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To evaluate the performance of the amyloid aerogel scaffolds for cell growth, Caco-2 human
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epithelial colorectal adenocarcinoma and HT29 human colorectal adenocarcinoma monolayer
3
cells were seeded into the aerogel scaffolds and incubated for 4 days. Confocal microscopy using
4
live/dead staining (Calcein AM and Ethidium homodimer-1 fluorophore for live and dead cells
5
respectively) was thereafter used to qualitatively monitor the cell viability (see Figure 4). From
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the surface images in Figure 4A,B it is clear that the majority of cells thrived in these conditions
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as shown by the live stain (green channel), and only in rare occasions do we see single dead cells
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(red channel). To investigate cell viability in the bulk of the samples, confocal images were
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acquired at different sample depths measured from the sample surface plane, see Figure 4C and
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Supporting Information Figure S5. From these images, where the transmitted channel is overlaid
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onto the green and red fluorescent channels, it is clear that cells are also alive at depths down to
12
~250 µm in the sample after which it becomes difficult to filter out the fluorescence signals from
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the cells. This observation is consistent with the average size of the two cell types considered
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here (see further discussion below) being smaller than the average pore sizes of the scaffolds, i.e.
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the cells have room to penetrate into the interior of the scaffolds.
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Figure 4. Laser scanning confocal microscopy of the amyloid fibril scaffolds following cell
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growth. A–B, Surface images of Caco-2 (A) and HT29 (B) cells grown on the Amyloid-20 2wt%
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scaffold. C, Slices of the scaffold at different depths from the surface showing growth of HT29
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cells in the bulk of an Amyloid-20 1 wt% scaffold. The cells were stained using a Live/Dead
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staining kit and pixels presented as green and red corresponds to living and dead cells
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respectively. In C the green and red channel are overlaid on a channel showing the light
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transmitted through the sample.
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To quantitatively evaluate the amount of living cells in the different amyloid fibril aerogel
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scaffolds, cell viability was investigated utilizing a lactate dehydrogenase (LDH) assay and the
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results were expressed as the amount of living cells compared to cellular growth within the well
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Biomacromolecules
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in the absence of the amyloid fibrils on the cell culture plate, see Figure 5. The results show that,
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in general, a high number of cells thrived when incubated within the scaffolds, with on average,
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a better performance for the Caco-2 cells compared to the HT29 cells was observed. This may be
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attributed to the differences in size and ultrastructural features of the two colorectal
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adenocarcinoma cell lines. The average size of HT29 and Caco-2 cells are 16.6 ± 0.17 µm39 and
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12.4 ± 0.4 µm40 respectively. Additionally, both cell lines form epithelial monolayers in vitro,
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and tumors in vivo,41 however, enterocytic differentiation of the cells results in different external
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features, specifically, HT-29 cells excrete mucin whereas Caco-2 cells do not, which could also
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affect the attachment of the cells to the amyloid scaffolds. Nonetheless, for all scaffolds a cell
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survival rate above 80% was achieved, demonstrating a good biocompatibility with these
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epithelial cell lines. We also note that there are small variations within the different scaffolds, in
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particular, in the growth of HT29 cells, where growth in the Amyloid-20 4wt% is significantly
13
greater than the 1wt% Amyloid-20 and 196 scaffolds. The increasing cell survival observed for
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the HT29 cell growth in Amyloid-20 furthermore indicates that appropriate scaffold pore sizes
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were maintained at all fibril concentrations in these samples in contrast to the Amyloid-196
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samples where this trend was absent.
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These cross-linked nanomaterials have potential as scaffolds for tissue engineering. To date,
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proposed materials have been based on natural and synthetic polymers, materials that have been
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designed to induce molecular biorecognition as the cells respond to their microenvironment.42
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These nanostructured materials need to permit cell penetration and proliferation, nutrient and
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waste product permeation and new capillary network formation as directed by the exposed
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surface area and porosity of the materials.43 Every tissue requires a defined structure matrix
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design with specific material properties, thus the development of materials whose mechanical
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and chemical properties can be tuned on the time and length scales of cell development,
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exogenously by the user, or endogenously by the cells are desired for the investigation of cellular
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processes. This research demonstrates the first instance of using cross-linked amyloid fibril
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aerogel materials as stable, cost effective and biocompatible scaffolds for cell culture. As these
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nanomaterials are formed by hydrolyzable elements,44 it is expected that the amyloid fibril
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aerogels will be affected by biological components; cells will produce and secrete enzymes and
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other proteins that could modify the fibril microstructure. Ideally, materials used as 3D cell
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culture scaffolds will allow cells to adhere and communicate with one another, and eventually
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degrade and shape according to their final location. In this short term study, the microstructure of
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the fibrils has allowed for cellular proliferation, however, more long term studies would be
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required to determine the effect of biological interactions upon these aerogels in order to fully
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assess their usability as 3D cell culture scaffolds when integrated in vivo. It was also found that
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changing the fibril concentration and freezing rate during preparation furthermore allows tuning
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the pore morphology, density and consequently mechanical stiffness of the scaffolds. This work
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hence provides a route forward for the continued exploration of amyloid fibril aerogels with
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tailored physical properties as scaffolds for cell growth.
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Figure 5. Cell survival data for cells grown within the cross-linked amyloid fibril scaffolds. A–
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B, Cell survival presented as percentage of living cells compared to a PBS buffer reference for
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Caco-2 and HT29 cells respectively (data are n=3,± SEM). Overall, there is significantly greater
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cellular growth of the Caco-2 cells than the HT29 cells in the scaffolds (p
