Amyloid Fibrils: Versatile Biomaterials for Cell Adhesion and Tissue

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Review

Amyloid fibrils: Versatile biomaterials for cell adhesion and tissue engineering applications Subhadeep Das, Reeba S Jacob, Komal Patel, Namrata Singh, and Samir K. Maji Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00279 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 28, 2018

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Amyloid fibrils: Versatile biomaterials for cell adhesion and tissue engineering applications Subhadeep Das #1, Reeba S Jacob#1, Komal Patel1, Namrata Singh1, Samir K Maji *1 1. Department of Biosciences & Bioengineering, IIT Bombay, Powai. Mumbai400076. Maharashtra. India. # Equally contributed * Correspondence may be addressed to [email protected]

Abstract Extracellular matrices (ECM) play an enormous role in any living system, controlling various factors and eventually fates of cells. ECM regulates cell fate by providing constant exogenous signals altering intracellular signal transduction for diverse pathways including proliferation, migration, differentiation and apoptosis. Biomaterial scaffolds are designed to mimic the natural extracellular matrix such that the cells could recapitulate natural events alike their natural niche. Therefore, the success of tissue engineering is largely dependent on how one can engineer the natural matrix properties at nano-scale precision. In this aspect, several recent studies have suggested that as long as amyloid fibrils are not toxic, it can be utilized for cell adhesion and tissue engineering applications due to its ECM mimetic surface topography and ability to mediate active cell adhesion via focal adhesions. Although historically associated with human diseases, amyloids have presently emerged as one of the excellent biomaterials evolved in nature. In this review, we focus on the recent advances of amyloid-based biomaterials for cell adhesion and tissue engineering applications. Introduction Amyloids are highly ordered, cross-β-sheet rich structure of protein/peptide aggregates associated with many human diseases including Alzheimer’s, Parkinsons and prion disease.1, 2 More than two dozen human diseases are associated with this aberrant protein folding, which eventually leads to amyloid formation.1 Both folded and natively unstructured protein can misfold and aggregate to a thermodynamically stable and aggregated state through a series of oligomeric state.3 Based on the 1 Environment ACS Paragon Plus

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observation that many proteins/peptides, which are not associated with diseases, can also form amyloids, led to the suggestion that amyloid structure could be the generic4 and lowest energy state fold of proteins/peptides.5 Amyloid fibrils are composed of cross-β-sheet rich structure, where the β-strands are aligned perpendicularly to the fibril axis and individual β-sheets are along with the fibril axis.6-8 This highly repetitive structural arrangement of amyloids made them highly stable similar to silk and resulted in a tensile strength similar to steel.9, 10 Amyloids are also resistant to wide varieties of harsh physical conditions.11 The superior qualities of amyloid state made scientists contemplate whether such a robust protein state has evolved in nature just to create human diseases. Indeed, many studies have shown that amyloid state of protein can perform various native biological functions in the host organism (functional amyloids).11-14 Moreover, the unique stability of amyloid structure has tempted scientists to harness amyloids in different biomaterial and nano-technological applications.15-17 Recently, it was demonstrated that amyloid fibrils from wide varieties of proteins/peptides could be used as a matrix for cell adhesion.16,

18, 19

Amyloid fibrils support cell adhesion and spreading with or without functionalization with cell adhesive motifs like RGD, indicating that cell adhesion could be a generic property of amyloids fibrils.18 Further, understanding the amyloid structure at atomic resolution made it possible to design amyloid fibrils with minimal protein/peptide sequence,20 which could be harnessed for fabricating scaffolds suitable for both soft21, 22

as well as hard tissue engineering.23 In this review, we will discuss the recent developments and advances of amyloid-based biomaterials for cell adhesion and tissue engineering.

Amyloid formation and their unique structural fold. Amyloids are higher order fibrillar aggregates that can be formed by self-assembly of diverse proteins with varied sequence, function and structure.24 Both folded and natively unstructured protein first mis-fold to a partially folded intermediate,25 which then slowly selfassociate to form an aggregation prone nuclei26 (Figure 1A). This phase is often considered as the lag phase of aggregation and is thermodynamically unfavorable. In the next stage called elongation phase, the soluble protein gets added onto the aggregation competent nuclei, which then grows to full-length fibrils. In the final stationary state, fibril formation is complete and it remains in equilibrium with monomeric proteins (Figure 1C). The amyloid state is often characterized by using secondary structural studies such as circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In a CD spectrum, amyloids show single minima at ~218 nm corresponding to β-sheet structure,27 whereas in FTIR spectra these fibrils show a major peak at ~1630 cm-1 in the amide-I region indicating a β-sheet rich

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structure.28 Often FTIR spectra of amyloid fibrils also show an additional small peak at ~1690 cm-1 indicating cross-β-sheet characteristics of protein.28, 29 Amyloids can also bind to dyes such as Thioflavin T (ThT), which generally do not give any fluorescence when proteins are in monomeric or soluble state.30,

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But when the protein reaches the amyloid state, this dye binds to it and therefore produces a strong fluorescence at 480 nm. Hence, this dye is generally used to measure kinetics of amyloid formation.32 Another important assay to measure amyloid formation is using Congo red (CR) dye binding. When CR binds to amyloids, its absorptivity increases along with a red shift in the UV spectrum.33 Moreover, the binding of CR to amyloids also gives a strong green-gold birefringence34 and is considered as one of the goldstandard methods to detect amyloids in vivo. The conformation of an amyloid structure is done mostly using X-ray diffraction (XRD) (Figure 1B). The X-ray diffraction pattern of amyloids generally gives an equatorial reflection at 6-11 Å corresponding to the inter-sheet distance and a meridional reflection at 4.7 Å for interstrand spacing.6-8 The morphology of amyloids under electron microscopy appears as fibrils with a fibril diameter of around 6-12 nm.7 In addition to being mechanically stable, amyloid fibrils also possess stability against a wide range of temperature and pH35, 36. The core of amyloid fibrils is also resistant against various proteases.37 Although slow, reversibility of amyloid fibrils makes them degradable unless highly cross-linked with other biopolymers such as glycosaminoglycans (GAGs)38 and other proteins in the disease state.14, 39-41 Moreover, the stability of resultant amyloid fibrils could also dependent on their protein sequence, where the side chain’s hydrophobicity and ionic nature can dictate the inter-digitation of β-sheet for higher order stability of fibrils.42, 43 Recent studies have suggested that as small as two residue segment and even single amino acid can form amyloid fibrils,44 opening possibilities for the rational design of amyloid fibrils. Association of amyloids with various human diseases. Historically, amyloids were explicitly associated with various diseased conditions such as Alzheimer’s disease (AD), Type II diabetes, prion diseases and Parkinson’s disease (PD).1, 2 In most of these diseases, thread-like fibrillar aggregates from soluble proteins that are extremely resistant to protease degradation were reported.1, 2, 45, 46 To date, approximately 50 different proteinopathies has been identified.47 In each of these disease conditions, specific proteins mis-fold and aggregate to form amyloid fibrils, like Amyloid-β peptide (Aβ) for Alzheimer’s (AD)48, α-synuclein for Parkinsons (PD)49 and Prion protein (PrP) for Creutzfeldt-Jakob disease.50 Clinically amyloidosis is classified as primary, secondary, familial, and finally into isolated types. In a recent review, Dobson and co-workers listed 37 peptides/ proteins found to form amyloid deposits



and involved in different human pathologies, where most of them found in the extracellular space and only 4 of them are in intracellular inclusions.2 In all these diseases, amyloid fibrils were implicated for the diseases causation. However, recent studies suggest that intermediate oligomeric protein assemblies are more toxic48, 51, 52 compared to the mature fibrils, suggesting amyloid fibrils could be less toxic end product of protein aggregation. Functional amyloids. In contrast to disease-associated amyloids, where aberrant protein misfolding leads to protein aggregation, sometimes proteins are also folded to amyloid state to perform normal functions of the host organism.1, 11 The formation of such functional amyloid assemblies is very rapid and highly regulated to avoid the accumulation of toxic oligomers.1, 11-53 The first mammalian functional amyloid, Pmel amyloid, discovered by JW Kelly and co-workers, which rapidly aggregates inside melanosomes and acts as a template for melanin polymerization.53 Recently, it was also shown that protein/peptide hormones aggregate and form amyloids inside the secretory granules for their effective storage.14 Upon signaling, these granules release the active hormone by dis-assembling the amyloid state, thus utilizing the reversible nature of amyloids.14 Microorganisms and other lower life forms such as insects, mollusks and sea animals also utilize the mechanical stability and adhesive nature of the amyloids for their colonization and/or survivals. 54-56 Examples of these include the spider silk of spider web57,

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and amyloids of chorion protein found in the

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eggshell of the silk worm , where the tough and rigid nature of amyloids protect the growing embryo of silk worm from environmental hazards. These studies, therefore, suggest that amyloids are widespread in nature and depending on the function, the natural properties such as stability or reversibility varies.

Amyloids as an adhesive substrate for many lower organisms. Amyloids are capable of binding to various molecules/biomolecules such as proteins, DNA/RNA, biological membranes and small molecules.60-63 This unique surface property of amyloids could be due to the highly repetitive arrangement of charged and uncharged residues on amyloid surface.60-63 The adhesive nature of amyloid is harnessed by many lower organisms for their adhesion to the host surfaces and colony formation.54 55, 56 One of the early discoveries of functional amyloids was done by Chapman et al on the bacterial curli fibrils, a major proteinaceous component of the bacterial biofilm.54 Biofilms are extracellular proteinaceous matrices that aid bacteria in adhesion, resistance to antibacterial agents, invasion and pathogenesis.53, 64-66 The in vivo and in vitro formation of curli amyloid fibrils is very well studied. Similar to mammalian amyloids, curli fibrils are also resistant to protease digestion, denaturing agents such 4 Environment ACS Paragon Plus

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as urea, heat, and detergents.54 Several other bacteria-associated proteins including fimbriae of Salmonella spp 66, pili from M. tuberculosis 67 and harpins of X. campestris and P.syringae 68 are also reported to polymerize into amyloid fibrils. Pili are hair-like bacterial adherence factors present on the bacterial cell surfaces, which help them for adhesion, colonization and infection to the host. Recently it was reported that pili of Mycobacterium tuberculosis could be of amyloidogenic nature due to its fibrillar morphology (as observed in Transmission Electron Microscopy (TEM)) similar to other amyloid fibrils like curli and could bind to amyloid specific dye CR.67 In yeast Candida albicans, the cell adhesion protein termed as Als located on the surface of yeasts are responsible for fungal aggregation.69 The threonine rich domain of Als forms amyloid fibrils and has an important role in the cell-cell aggregation and also the fungi-host adherence.69 Another example is the protozoan parasite Plasmodium falciparum, which in its early merozoite stage uses amyloidogenic merozoite surface protein 2 (MSP2) for adhering to red blood cell (RBCs) for its internalization and subsequent initiation of the disease.70, 71 Thus, amyloids in microbes has functional roles by acting as extracellular matrix and adherence factors that help in colony formation and/or attachment to host to cause diseases, but provides a survival advantage to their respective host organisms.

Amyloids possess cell adhesive properties. In higher organisms, ECM offers physical support and enables interactions between cells to form a functional tissue.72 Cells interact with ECM and cytoskeletal proteins through cell adhesion molecules (CAM), which include selectins, cadherin and integrins.73 These interactions help the cells in performing its function, migration and tissue organization.74 In ECM, proteins such as collagen, laminin or fibronectin are polymerized themselves to interconnecting fibrils, which forms the meshwork that provides mechanical stability and physical scaffolding for tissue formation.75 In addition, these ECM proteins have multiple domains with sequences that can act as integrin recognition motifs like GxOGER (in collagen)76, RGD (in fibronectin)77 or IKVAV (in laminin)78 for enabling the cell adhesion. The successful cell adhesion depends on how cells are able to engage integrins for coordinating the activity of cytoskeleton inside the cells with extracellular matrix.79, 80 Therefore, the strong adhesion of cell on the surface also depends on integrin expression, integrin clustering as well as focal adhesion complex formation.79, 81 Amyloid fibrils can mimic the nano-fibrillar morphology of ECM and these fibrils could be functionalized with ligands to support cell adhesion.16 The first cell adhesion study on amyloid fibrils was performed by Gras and co-workers using RGD modified amyloid fibrils. In their study, Gras and coworkers functionalized the peptide sequence derived from transthyretin with RGD peptides, a well-studied cell



adhesion motif (TTR1-RGD).16 The TTR1-RGD peptide was aggregated such that amyloid fibrils of TTR1-RGD will mimic the ECM and RGD will acts as integrin recognition motif for cell adhesion. The study showed increased cell adhesion for TTR1-RGD amyloid fibrils when compared to unmodified TTR1 fibrils. It is interesting to note that, even though the TTR1-RGD showed higher cell adhesion than TTR1 fibrils, the level of cell adhesion on these unmodified amyloid fibril was not poor.16 Interestingly, the cell attachment to another peptide fibrils TTR1-RAD was very less compared to the TTR1 and TTR1-RGD fibrils.16 Since there was considerable cell adhesion with unmodified TTR1 fibrils, it could be anticipated that amyloid fibrils itself can aid the cell adhesion, but the level of cell adhesion might depend additional factors such as the sequence of the amyloid protein as well as surface topography of the fibrils. To decouple the role of surface topography and surface chemistry of amyloid fibrils, recently Reynolds et al. created surfaces by layering plasma polymer on top of amyloid fibrils.82 The polymer coating masked the chemical groups on the fibrils surface, but without altering the nano-topography of amyloid fibrils.82 The study found that nano-topography influences the cell adhesion to a high degree. One reason for this could be that the amyloid nano-topography helps in deposition of serum proteins on the surface, which eventually helps in the cell adhesion.19 Subsequent study by the same group showed that increasing concentration of lysozyme fibril network induced more cell adhesion. This could be due to the increased surface coverage with the fibrils for the increment in the fibril network. However, cell spreading remained constant up to 40% fibril surface coverage and the area of spread cells substantially increased only when more than 80% of the area was covered by fibrils.19 Moreover, this study also noted that fibril coverage more than the thickness of the individual fibrils influenced the cell adhesion.19 Together, these studies demonstrate that nano-fibrillar topography of amyloids enables cell adhesion and similar to ECM proteins, amyloid fibrils also show density-dependent cell adhesion. Cell adhesion is a generic property of amyloid fibrils. The studies mentioned in the previous section indicate that the unique surface of amyloid fibrils might aid in the mammalian cell adhesion even with the absence of any integrin recognition motif.16, 19 We recently hypothesized that cell adhesion could be a generic property of amyloid fibrils.18 To probe this, we randomly chose more than a dozen of non-toxic proteins/peptide amyloid fibrils possessing different secondary structure and sequence and analyzed the adhesion of mammalian cells on amyloids. We observed that mammalian cells were capable of adhering and spreading on each of amyloid fibrils to different extents, but most of them had higher cell-spread area than the collagen−a native ECM protein.18 In addition, the cell adhesion on amyloid fibrils was also not 6 Environment ACS Paragon Plus

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specific to any cell type; rather different cell types were able to adhere on the amyloid surfaces. Further, we showed that bovine serum albumin (BSA), a commonly used protein to block nonspecific cell adhesion on glass surfaces, when converted to amyloid form, aids in the cell adhesion. To understand the mechanism of cell adhesion on amyloid fibrils, our group studied the actin organization and focal adhesion machinery of cells adhered on the amyloid fibrils.18 The arrangement of actin in cells grown on amyloid surfaces was similar to those on collagen. Moreover, the cells adhered on the amyloid surface were also able to use focal adhesion machinery and facilitate integrin mediated cell adhesions. Interestingly, the cells adhered on amyloid surface were found to have more and larger focal adhesion complexes, more integrin mRNA expression and more integrin protein clustering (Figure 2A and 2B) than the cells that were grown on collagen. This suggest that cells adhered on amyloid fibrils engage the integrin machinery for cell adhesion similar to extracellular matrix proteins.18 When integrin engagement with substrate was blocked with integrin blocking antibody or peptide ligand, significant reduction in cell spreading was observed, corroborating the significance of integrins in cell adhesion on amyloid surfaces. Additionally, NIH 3T3 fibroblasts were found to be highly motile on amyloid surfaces and these cells on varying coating density of fibril showed a biphasic spreading response similar to the effect of ECM density on cell spreading.18 These data suggest that cells on amyloid nanofibrils and ECM could behave in a similar manner. Similarly human mesenchymal stem cells were also able to adhere on amyloid fibrils with integrin engagement and focal adhesion complex formation.18 A key question here is why are amyloid fibril surfaces so suitable for cell adhesion? Also, is cell adhesion on amyloids dependent on the charge of the protein/peptide monomer that constitutes the amyloid fibril? To delineate the roles of charge versus amyloid topography in cell adhesion, charged amino acid polymers like poly-L-Lysine (PLL) and poly L-glutamic acid were utilized. PLL is a charged polymer routinely used to facilitate cell adhesion on the glass surfaces. Cell adhesion was studied on surfaces coated with the soluble form as well as the amyloid fibrillar form of the protein.18 Poly-alanine was used as neutral amino acid polymer control. The results demonstrated that adhesion of cells on charged amyloid polymers were higher than that of their soluble counter parts. We also observed that the amyloid surfaces of these charged amino acid polymers able to recruit higher level of focal adhesion complex formation and higher integrin mRNA expression compared to their soluble polymer counterpart. This study further support that irrespective of protein/peptides sequences and their charges, the amyloid form aid in the adhesion of mammalian cells though integrin recruitment and focal adhesion complex formation similar to ECM proteins.18 Since amyloids are well known to bind to the membrane,83



we hypothesized that lipid-amyloid interaction may be the first step for cell adhesion. It is well known that amyloid interact not only to the planar bi-layered membrane but also to various lipid vesicles. To prove the fact that lipid-amyloid interaction can aid in cell adhesion, we studied the adhesion of RBCs, on amyloid fibrils. RBCs lack integrin machinery and the data from our experiments showed that indeed amyloid fibrils were able to facilitate a concentration dependent adhesion of RBCs. Therefore, the mechanism of cell adhesion on amyloids might be that cells utilize the facile membrane-amyloid interaction as the first step of adhesion on amyloid fibrils, followed by recognition of amyloids as suitable adhesive substrate, resulting in turning on their “outside-in” signaling for integrin expression and clustering (Figure 2C). These integrin expression and clustering then eventually might result in the formation of higher number and larger focal adhesion complex, which further aid in the strong adhesion of cells on amyloids.18 The above-mentioned results posed an another interesting question that how do cells behave on disease associated amyloids, which are implicated in various neurodegenerative diseases such as fibrils of αsynuclein (α-Syn) and Aβ42 associated with Parkinson’s and Alzheimer’s disease, respectively.48, 84 The cell adhesion study on these disease-associated amyloids showed that cells are also able to adhere to these amyloids similar to non-toxic amyloids. However, cell adhesion were less on toxic Aβ(25-35) fibrils.85 Interestingly, a robust cell adhesion was observed Aβ42 fibrils compared to Aβ(2535) fibrils. We hypothesize that cell adhesion and cell death could be independent of each other and the cell death might be due to the exposure of toxic epitope on amyloid fibrils to adhered cells. For example, upon immobilizing on glass substrate the toxic epitope of neurotoxic Aβ(25-35) peptide fibrils might be more exposed compared to Aβ42 fibrils. Thus cell adhesion and toxicity could be two different aspects of amyloid fibrils.18 Moreover, the roughness of the coated fibril surface can also cause death/apoptosis of adherent cells on amyloid surfaces. Overall these studies suggest that though amyloid surface favors cell adhesion, the surface chemistry of the fibrils determines the viability of the adhered cells.86 Self-assembling peptides in tissue engineering. Tissue engineering is an interdisciplinary field focused on development of biomimetic platforms to engineer the damaged tissue of interest.87 It has immense potential in regenerative medicine wherein scientists are targeting to regenerate lost tissue in a living system. A major driving factor in this field is advancement in the understanding of basic stem cell biology and scaffold designing to manipulate cellular response. Self-assembling peptides that form nano-fibrils can be suitably used for such applications such as cell adhesion, differentiation and/or migration.88-92 Recently, with the advent of protein engineering, relatively simple peptide based hydrogels have gained increasing 8 Environment ACS Paragon Plus

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attention in biomaterial development. Peptide based scaffolds in the form of hydrogels have been utilized in multiple ways for various tissue engineering purposes. Hydrogels are three-dimensional networks of polymers cross-linked physically or chemically that can entrap solvents. In chemical hydrogels, chemical cross-linkers covalently link the individual polymers to form a network.93 Thus chemical hydrogels mostly possess irreversible properties. In contrast, the physical hydrogels, like peptide hydrogels, are mostly held by non-covalent interactions such as ionic interactions, hydrogen bonding and hydrophobic interactions.94, 95 In peptide hydrogels, the component peptide monomers non-covalently self-assemble to form an ordered nanostructure. Then multiple of these nanostructures further assembles to form a supramolecular network.94 The gelation behavior of this kind of physical hydrogels can be reversed by external stimuli such as heat, pH, ionic strength and shear force. This is advantageous as gelation can be induced by ambient to physiological conditions, giving way to the development of unique biomaterials. Different properties of hydrogels including swelling capacity, surface properties, mechanical properties and permeability make them promising candidates for a wide variety of applications.96-98 The major advantage of employing proteins/peptides in hydrogels is that their properties could be precisely controlled to achieve the desired functionality. For example, Zhang et al used amphipathic β-sheet peptides that self-assemble into fibrils and form a hydrogel by entrapping solvents.99 The group however initially showed that Ac-(AEAEKAK)2-NH2 peptide (EAK16-II) formed insoluble fibrils of ~ 10-20 nm in diameter, which are β-sheet rich structure.100 Replacement of Glu with Arg and Lys with Asp residues in EAK16-II give rise to Ac-(RARA-DADA)2-NH2 peptide (RADA16-II) that self-assembled into hydrogels in physiological conditions.101, 102 The fibrils of the hydrogel/matrix exhibited Congo red birefringence consistent with amyloid like cross-β structure. The study also found that for amphipathic peptides, self-assembly and hydrogelation properties are dependent on sequence length, charge and hydrophobicity.103, 104 Similar concept of peptide design (H2N-VKVKVKVK-VDPPT-KVKVKVKV-NH2 (MAX1)) was also used to build β hairpin fold to template the intermolecular self-assembly in to a bilayer β-sheet fibrils at pH 9 or in solution of higher ionic strength.105 These engineered hydrogels are often employed as vehicles for cell encapsulation along with suitable therapeutic drugs and growth factors to injury sites for promoting regeneration. Local biochemical signals play significant role in determining the fate of scaffoldencapsulated cells.106 This makes presentation of specific ligands to cells necessary during their development.107 Hydrogels present a unique microstructure to the encapsulated cells along with presenting biochemical cues locally, influencing cell signaling and hence cellular fate. Although complications are faced in dealing with



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inflammatory responses elicited against self-assembled peptide scaffolds, foundational research has shown promising results highlighting the exciting potential to further engineer peptide-based scaffolds for tissue engineering applications. Various types of self-assembled peptides are studied for different types of tissue engineering. For example, in neural tissue engineering, self-assembling peptides containing the laminin epitope IKVAV were tested.108 The nano-fibrous system not only promoted the generation of axons in a mouse model with spinal cord injury but also inhibited the formation of glial scar.108 The RADA 16-I hydrogels filled lesions formed by sciatic axotomy and facilitated cell migration.109 Regeneration was also observed when RADA16-I peptide hydrogel was implanted in the damaged cortex. Peptide based scaffolds also showed considerable promise in the treatment of Duchenne’s muscular dystrophy (DMD). In this case, 3D scaffold based on Fmoc π-β system has been enzymatically triggered to form a hydrogel and deployed in vivo to present the missing protein laminin at the proper site.98 Peptide scaffolds were also utilized in the repair of cartilage, where the bone marrow stem cells (BMSC) was implanted along with transforming growth factor-β1 (TGF-1), dexamethasone and insulin like growth factor 1 (IGF-1) in tripeptide KLD solution.110 The liquid peptide solution was then injected and the assembly reaction was triggered such that the in situ gelation would fill up the defect and initiate healing in a full-size cartilage defect model of a skeletally matured rabbit. Other applications of self-assembled peptides in tissue engineering include gel implantation in eyes for improved and effective recovery post-glaucoma surgery,111 regeneration of myocardium with neonatal cardiomyocyctes,111 homeostatic agents in post-surgery iatrogenic injury,112 imparting rapid vascularization to the damaged tissues.113

and

Amyloid fibrils for tissue engineering application. Amyloid nanofibrils, being excellent cell adhesive material as well as vehicle for drug delivery, was envisioned to be an emerging nano-material for scaffolding in tissue engineering.114 The superior properties of amyloid fibrils, led to intense research in development of scaffolding materials, harnessing the unique properties of amyloid fibrils.15,

115, 116

It was suggested that non-toxic forms of amyloid fibrils could be utilized in designing scaffold for tissue engineering applications.16 The scaffolding could be done by the amyloid fibrils alone or by mixing it with different other components like polyphenols, graphene, carbon nano-tubes etc.19, 23, 116 Given the scope of tuning amyloid based materials, different groups started using either engineered amyloid fibrils derived from short peptides that were non-toxic or full-length proteins that were known to form non-toxic amyloids. For example, hydrogels derived from lysozyme amyloid fibrils were shown to promote cell adhesion, spreading and proliferation.19,


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117, 118

In this study, the gel was developed from full-length lysozyme protein. Interestingly, it was shown that the same protein lysozyme could form non-fibrillar opaque gels at higher pH and temperature.118 Thus, condition of gelation is an important parameter to develop suitable amyloid hydrogel. We have recently reported an amyloid based hydrogel designed from the C-terminus of amyloid-β (Aβ) protein associated with Alzheimer’s disease. These designed short di- and tri-peptides were capped with an Fmoc group at the N-terminus.21 It is proposed that rapid intermolecular association by these peptide monomers leads to formation of amyloid fibrils that eventually entrap the solvent to give a hydrogel (Figure 3). Using NMR studies, it was shown that π-π stacking of the Fmoc capping of peptides are the key factor for initiating the gelation by these peptides. The peptides with same sequence without the Fmoc group at the N-terminus failed to form hydrogel. Another key feature of this class of amyloid hydrogels are their thixotropic properties. Earlier reports by Bolisetty et al also reported the thixotropicity or self-healing nature of amyloid hydrogels derived from β-lactoglobulin fibrils.119 We demonstrated that amyloid hydrogel’s self-healing property helps immensely for easy encapsulation of cells for a 3D cell culture.21 The self-healing property of amyloid hydrogels had been attributed to the hydrophobic patches on the individual amyloid fibrils that form the reversible gel network. This was directly proved by delaying the recovery of hydrogel from the solution using Nile red (NR) dye, which is known to bind to exposed hydrophobic surfaces of protein/peptide aggregates. However, mere amyloidogenicity of hydrogel might not confer thixotropicity of amyloid hydrogels, as Fmoc-FF peptide derived amyloid hydrogel did not show thixotropic properties.21 Fmoc-FF has extensive π-stacking between the Fmoc groups and the phenylalanine rings in the side chain, resulting in formation of a very stiff hydrogel. Thus the driving factor for thixotropicity could be the delicate balance between the π-stacking forces and the hydrophobicity of the amyloid fibrils participating in the formation of the hydrogel network. Further, toxicity assay by 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT) reduction proved that these Aβ derived hydrogels are non-toxic and are suitable for culture of cells and human mesenchymal stem cells (hMSCs). When hMSCs were cultured on these hydrogel, it was shown that hMSCs were preferentially differentiated to neuron without the presence of any growth factors.21 Interestingly, we also found that on stiffer amyloid hydrogels like Fmoc-FF, the stem cells were spread and had morphology like the ones cultured on the glass. This preferential differentiation to the neuron-like lineages could be attributed to the soft nature (

Amyloid Fibrils: Versatile Biomaterials for Cell Adhesion and Tissue

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