RESUMEN
Triple-negative breast cancer (TNBC) is the most invasive type of breast cancer with high risk of brain metastasis. To better understand interactions between breast tumors with the brain extracellular matrix (ECM), a 3D cell culture model is implemented using a thiolated hyaluronic acid (HA-SH) based hydrogel. The latter is used as HA represents a major component of brain ECM. Melt-electrowritten (MEW) scaffolds of box- and triangular-shaped polycaprolactone (PCL) micro-fibers for hydrogel reinforcement are utilized. Two different molecular weight HA-SH materials (230 and 420 kDa) are used with elastic moduli of 148 ± 34 Pa (soft) and 1274 ± 440 Pa (stiff). Both hydrogels demonstrate similar porosities. The different molecular weight of HA-SH, however, significantly changes mechanical properties, e.g., stiffness, nonlinearity, and hysteresis. The breast tumor cell line MDA-MB-231 forms mainly multicellular aggregates in both HA-SH hydrogels but sustains high viability (75%). Supplementation of HA-SH hydrogels with ECM components does not affect gene expression but improves cell viability and impacts cellular distribution and morphology. The presence of other brain cell types further support numerous cell-cell interactions with tumor cells. In summary, the present 3D cell culture model represents a novel tool establishing a disease cell culture model in a systematic way.
RESUMEN
In the context of tissue engineering, biofabrication techniques are employed to process cells in hydrogel-based matrices, known as bioinks, into complex 3D structures. The aim is the production of functional tissue models or even entire organs. The regenerative production of biological tissues adheres to a multitude of criteria that ultimately determine the maturation of a functional tissue. These criteria are of biological nature, such as the biomimetic spatial positioning of different cell types within a physiologically and mechanically suitable matrix, which enables tissue maturation. Furthermore, the processing, a combination of technical procedures and biological materials, has proven highly challenging since cells are sensitive to stress, for example from shear and tensile forces, which may affect their vitality. On the other hand, high resolutions are pursued to create optimal conditions for subsequent tissue maturation. From an analytical perspective, it is prudent to first investigate the printing behavior of bioinks before undertaking complex biological tests. According to our findings, conventional shear rheological tests are insufficient to fully characterize the printing behavior of a bioink. For this reason, we have developed optical methods that, complementarily to the already developed tests, allow for quantification of printing quality and further viscoelastic modeling of bioinks.
Asunto(s)
Bioimpresión , Hidrogeles , Impresión Tridimensional , Ingeniería de Tejidos , Bioimpresión/métodos , Ingeniería de Tejidos/métodos , Hidrogeles/química , Reología , Humanos , Andamios del Tejido/química , ViscosidadRESUMEN
The development of bio-inks capable of being 3D-printed into cell-containing bio-fabricates with sufficient shape fidelity is highly demanding. Structural integrity and favorable mechanical properties can be achieved by applying high polymer concentrations in hydrogels. Unfortunately, this often comes at the expense of cell performance since cells may become entrapped in the dense matrix. This drawback can be addressed by incorporating fibers as reinforcing fillers that strengthen the overall bio-ink structure and provide a second hierarchical micro-structure to which cells can adhere and align, resulting in enhanced cell activity. In this work, the potential impact of collagen-coated short polycaprolactone-fibers on cells after being printed in a hydrogel is systematically studied. The matrix is composed of eADF4(C16), a recombinant spider silk protein that is cytocompatible but non-adhesive for cells. Consequently, the impact of fibers could be exclusively examined, excluding secondary effects induced by the matrix. Applying this model system, a significant impact of such fillers on rheology and cell behavior is observed. Strikingly, it could be shown that fibers reduce cell viability upon printing but subsequently promote cell performance in the printed construct, emphasizing the need to distinguish between in-print and post-print impact of fillers in bio-inks.
Asunto(s)
Tinta , Seda , Seda/química , Hidrogeles/farmacología , Hidrogeles/química , Polímeros , ReologíaRESUMEN
Biomimicry applies the fundamental principles of natural materials, processes, and structures to technological applications. This review presents the two strategies of biomimicry-bottom-up and top-down approaches, using biomimetic polymer fibers and suitable spinning techniques as examples. The bottom-up biomimicry approach helps to acquire fundamental knowledge on biological systems, which can then be leveraged for technological advancements. Within this context, we discuss the spinning of silk and collagen fibers due to their unique natural mechanical properties. To achieve successful biomimicry, it is imperative to carefully adjust the spinning solution and processing parameters. On the other hand, top-down biomimicry aims to solve technological problems by seeking solutions from natural role models. This approach will be illustrated using examples such as spider webs, animal hair, and tissue structures. To contextualize biomimicking approaches in practical applications, this review will give an overview of biomimetic filter technologies, textiles, and tissue engineering.
Asunto(s)
Materiales Biomiméticos , Arañas , Animales , Materiales Biomiméticos/química , Biomimética , Seda/químicaRESUMEN
Janus fibers are a class of composite materials comprising mechanical and chemical to biological functionality. Combining different materials and functionalities in one micro- or even nanoscale fiber enables otherwise unreachable synergistic physicochemical effects with unprecedented opportunities for technical or biomedical applications. Here, recent developments of processing technologies and applications of polymeric Janus fibers will be reviewed. Various examples in the fields of textiles, catalysis, sensors as well as medical applications, like drug delivery systems, tissue engineering and antimicrobial materials, are presented to illuminate the outstanding potential of such high-end functional materials for novel applications in the upcoming future.
RESUMEN
Biotechnological production is a powerful tool to design materials with customized properties. The aim of this work was to apply designed spider silk proteins to produce Janus fibers with two different functional sides. First, functionalization was established through a cysteine-modified silk protein, ntagCys eADF4(κ16). After fiber spinning, gold nanoparticles (AuNPs) were coupled via thiol-ene click chemistry. Significantly reduced electrical resistivity indicated sufficient loading density of AuNPs on such fiber surfaces. Then, Janus fibers were electrospun in a side-by-side arrangement, with "non-functional" eADF4(C16) on the one and "functional" ntagCys eADF4(κ16) on the other side. Post-treatment was established to render silk fibers insoluble in water. Subsequent AuNP binding was highly selective on the ntagCys eADF4(κ16) side demonstrating the potential of such silk-based systems to realize complex bifunctional structures with spatial resolutions in the nano scale.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Fibroínas/metabolismo , Seda/metabolismo , Animales , Proteínas de Artrópodos/química , Fibroínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Seda/química , ArañasRESUMEN
The discovery of penicillin started a new era of health care since it allowed the effective treatment of formerly deadly infections. As a drawback, its overuse led to a growing number of multi-drug resistant pathogens. Challenging this arising threat, material research focuses on the development of microbe-killing or microbe repellent agents implementing such functions directly into materials. Due to their biocompatibility, non-immunogenicity and mechanical strength, silk-based materials are attractive candidates for applications in the biomedical field. Furthermore, it has been observed that silks display high persistency in their natural environment giving reason to suspect that they might be attractive candidates to prevent microbial infestation. The current review describes the process of biofilm formation on medical devices and the most common strategies to prevent it, divided into effects of surface topography, material modification and integrated additives. In this context, recent state of the art developments in the field of natural and artificial silk-based materials with microbe-repellant or antimicrobial properties are addressed. These silk properties are controversially discussed and conclusions are drawn as to which parameters will be decisive for the successful design of new bio-functional materials based on the blueprint of silk proteins.
Asunto(s)
Antiinfecciosos , Arañas , Animales , Antibacterianos , Antiinfecciosos/farmacología , Materiales Biocompatibles/farmacología , Biopelículas , SedaRESUMEN
Data presented in this article describe bacterial and fungal repellent properties of 2D-films and 3D-hydrogels made of different recombinantly produced spider silk proteins based on consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4). Here, the attachment, growth, and microbial colonization of Streptococcus mutans (S. mutans) as well as Candida albicans (C. albicans) on plane and micro-patterned films were visualized by scanning electron microscopy (SEM). Also, microbial viability data are provided of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) on hydrogels made of eADF4(C16) and eADF4(C16)-RGD, quantified using the Alamar blue assay. Experimental results, design of a post-operative contamination model of microbes with mammalian cells, and methods in the data article refer to the research paper "Engineered spider silk-based 2D and 3D materials prevent microbial infestation" published recently [1].
RESUMEN
There has been a significant increase in the use of sensitive biological components, e.g., growth factors or enzymes, in implanted scaffolds/devices. To prevent diffusion away from the targeted area and to maximize access of the biological agent to the desired target, it is necessary to provide a supportive substrate to immobilize and protect biological agents from the environment. For this purpose, nanofiber fabrics are highly promising due to their high porosity, capacity for solution flow-through and high surface-to-volume ratio. However, electrospinning often requires harsh processing conditions, such as the use of volatile solutions, which can result in loss of activity of the incorporated biological components. In this study we developed a mild process for electrospinning of eADF4(C16), a recombinant spider silk protein. eADF4(C16) is non-cytotoxic, displays excellent stability against hydrolytic and enzymatic degradation and opens the opportunity for genetic addition of bioactive factors. Therefore, an aqueous spinning dope of eADF4(C16) was loaded with either green fluorescence protein (GFP) or the recombinant fusion protein GFP-eADF4(C16). The fluorescence activity of GFP is dependent on its proper folding, which does not occur in organic solvents, making it an attractive model protein. We were able to demonstrate the usability as well as the significance of the all-aqueous processing conditions for the activity of GFP in electrospun spider silk scaffolds.
Asunto(s)
Fibroínas/química , Agua/química , Animales , Fibroínas/genética , Fibroínas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Seda/metabolismo , Espectrometría de Fluorescencia , ArañasRESUMEN
The surgically induced angiogenesis by means of arteriovenous (AV) loops represents a powerful method to significantly enhance vascularization of biomaterials. Regarding tissue engineering applications, spider silk is a promising biomaterial with a good biocompatibility and slow biodegradation. This study aims at investigating vascularization as well as de novo tissue formation of fibrous matrices made of electro-spun (ES) or wet-spun (WS) engineered ADF4(C16) spider silks in the rat AV loop model. Either ES or WS spider silk fibrous matrices were filled into Teflon chambers. Intrinsic vascularization was induced by means of an AV loop. After 4 weeks of vascularization, de novo tissue formation and biocompatibility were analyzed. Regardless of their significantly differing fiber diameters, both ES and WS eADF4(C16) fiber matrices displayed a good biocompatibility and initiated de novo tissue formation as well as vessel formation. Both matrices demonstrated partial vascularization originating from the AV loop, with more vessels in spider silk matrices with lower fiber diameters. We were able to demonstrate intrinsic vascularization of spider silk fibrous matrices by means of the AV loop. Moreover, our study indicates that the adjustment of the fiber diameter of engineered spider silks enables new possibilities to optimize vascularization. Impact Statement Spider silk is a promising biomaterial demonstrating excellent biocompatibility and biodegradation. Biotechnology allows the high-volume production of recombinant spider silk proteins, such as eADF4(C16), with the required purity for biomedical applications. In this study, eADF4(C16) fibrous matrices were produced by either electro- or wet-spinning, resulting in different fiber diameters. Forming an arteriovenous fistula, surgical vascularization of the scaffolds was induced. After 4 weeks, both silks demonstrated a good biocompatibility and tissue formation. The thinner electro-spun fibers displayed a faster biodegradation and vascularization, indicating that the adjustment of the fiber diameter is a valuable tool to fine-tune vascularization and biodegradation.
Asunto(s)
Arterias/fisiología , Modelos Biológicos , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/farmacología , Seda/farmacología , Arañas/química , Venas/fisiología , Actinas/metabolismo , Animales , Arterias/efectos de los fármacos , Masculino , Implantación de Prótesis , Ratas Endogámicas Lew , Ingeniería de Tejidos , Venas/efectos de los fármacos , Microtomografía por Rayos XRESUMEN
Natural silk fibers represent one of the most advanced blueprints for (bio)polymer scientists, displaying highly optimized mechanical properties due to their hierarchical structures. Biotechnological production of silk proteins and implementation of advanced processing methods enabled harnessing the potential of these biopolymer not just based on the mechanical properties. In addition to fibers, diverse morphologies can be produced, such as nonwoven meshes, films, hydrogels, foams, capsules and particles. Among them, nanoscale fibrils and fibers are particularly interesting concerning medical and technical applications due to their biocompatibility, environmental and mechanical robustness as well as high surface-to-volume ratio. Therefore, we introduce here self-assembly of silk proteins into hierarchically organized structures such as supramolecular nanofibrils and fabricated materials based thereon. As an alternative to self-assembly, we also present electrospinning a technique to produce nanofibers and nanofibrous mats. Accordingly, we introduce a broad range of silk-based dopes, used in self-assembly and electrospinning: natural silk proteins originating from natural spinning glands, natural silk protein solutions reconstituted from fibers, engineered recombinant silk proteins designed from natural blueprints, genetic fusions of recombinant silk proteins with other structural or functional peptides and moieties, as well as hybrids of recombinant silk proteins chemically conjugated with nonproteinaceous biotic or abiotic molecules. We highlight the advantages but also point out drawbacks of each particular production route. The scope includes studies of the natural self-assembly mechanism during natural silk spinning, production of silk fibrils as new nanostructured non-native scaffolds allowing dynamic morphological switches, as well as studying potential applications. This article is categorized under: Biology-Inspired Nanomaterials > Peptide-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.
Asunto(s)
Nanofibras , Nanotecnología , Ingeniería de Proteínas , Seda , Animales , Bombyx , Células Cultivadas , Técnicas Electroquímicas , Humanos , Proteínas RecombinantesRESUMEN
Silk is a protein-based material which is predominantly produced by insects and spiders. Hundreds of millions of years of evolution have enabled these animals to utilize different, highly adapted silk types in a broad variety of applications. Silk occurs in several morphologies, such as sticky glue or in the shape of fibers and can, depending on the application by the respective animal, dissipate a high mechanical energy, resist heat and radiation, maintain functionality when submerged in water and withstand microbial settling. Hence, it's unsurprising that silk piqued human interest a long time ago, which catalyzed the domestication of silkworms for the production of silk to be used in textiles. Recently, scientific progress has enabled the development of analytic tools to gain profound insights into the characteristics of silk proteins. Based on these investigations, the biotechnological production of artificial and engineered silk has been accomplished, which allows the production of a sufficient amount of silk materials for several industrial applications. This chapter provides a review on the biotechnological production of various silk proteins from different species, as well as on the processing techniques to fabricate application-oriented material morphologies.
Asunto(s)
Proteínas Recombinantes/síntesis química , Seda/química , Seda/síntesis química , Animales , Ingeniería de Proteínas/métodos , Ingeniería de Proteínas/tendenciasRESUMEN
Spider dragline silk shows the highest toughness in comparison to all other known natural or man-made fibers. Despite a broad experimental foundation concerning the macroscopic silk thread properties as well as a thorough simulation-based molecular understanding, the impact of the mesoscale building blocks, namely nano-/submicrometer-sized filaments, on the mechanical properties of the threads remains the missing link. Here, we illustrate the function of these mesoscaled building blocks using electrospun fibers made of a recombinant spider silk protein and show the impact of ß-sheet content and fiber hydration on their mechanical performance. Specifically elucidating the interplay between ß-sheet-cross-linking (fiber strength) and structural water (fiber extensibility), the results bridge the gap between the molecular and the macroscopic view on the mechanics of spider silk. It is demonstrated that the extensibility of the here used single (MaSp2-like) protein system is in good accordance with the simulated extensibilities published by other groups. Furthermore, sufficient hydration of the fibers is shown to be a prerequisite to obtain a toughness in the range of that of natural dragline silk. Preliminary studies on electrospun fibers of the MaSp2-based recombinant spider silk proteins used in this work have indicated their basic applicability in the technical field of filter systems as well as in regenerative medicine. The presented work provides a fundamental understanding of the mechanical performance of such fibers under different wetting conditions, a prerequisite to further specify their potential for such applications.
RESUMEN
The performance of biomaterials largely depends on the materials biocompatibility, which is directly related to unwanted side effects like foreign body responses and inflammation, and the potential of interaction of cells with its surface, for example, cell adhesion. In the distinct application of catheters, low or even no cell adhesion is eligible. To influence the properties of existing and commonly used biomaterials and to further increase their biocompatibility, a coating with a recombinantly produced spider silk protein as outer layer was applied on three selected catheter polymers (polyurethane, polytetrafluoroethylene, silicone) and evaluated based on cell adhesion. The tested cell types, HaCaT keratinocytes (epidermal cells), B50 neuronal cells, C2C12 myoblasts (muscle cells) and BALB/3T3 fibroblasts (connective tissue), exhibited low or no adhesion on the silk-coated materials. In combination with the lack of toxicity, the good biocompatibility, and the low body response, it could be shown that silk coatings have a high potential as a biomedical coating material, e.g., for catheters.
RESUMEN
Using a self-assembly of recombinant spidroins, biomimetic spinning dopes are produced and wet-spun into fibers. Upon varying the molecular design of the underlying recombinant spidroins, the influence of the amino- and carboxy-terminal domains, as well as the size of the repetitive core domain on fiber mechanics, is determined. Fiber toughness upon biomimetic processing equals and even slightly exceeds that of natural ones.
Asunto(s)
Productos Biológicos , Materiales Biomiméticos/química , Fibroínas/química , Fenómenos Mecánicos , Arañas , Animales , Proteínas Recombinantes/químicaRESUMEN
Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh. Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable ß-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD). The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.
Asunto(s)
Filtros de Aire , Proteínas de Artrópodos/química , Fibroínas/química , Arañas , Animales , Proteínas Recombinantes/químicaRESUMEN
A polyester polycaprolactone-based polyurethane elastomer (PU) and poly-(l-lactide) (PLLA), two common biomaterials, were electrospun to produce membranes with fibres either randomly orientated or aligned. PU was used to produce membranes consisting of smooth fibres. PLLA was used to prepare fibres with a textured surface. Contact angle measurements of the PU and PLLA cast films reveal that they were both below 90 degrees and fully wetted in less than 60 s. These membranes were investigated for the effect of fibre topography and fibre alignment on cell adhesion, using mouse L929 fibroblasts. It was found that the alignment of electrospun fibres controls the directional spreading of fibroblast independent of fibre being smooth or textured.