Silk is mostly known as a luxurious textile, which originates from silkworms first cultivated in China. A deeper look into the variety of silk reveals that it can be used for much more, in nature and ...by humanity. For medical purposes, natural silks were recognized early as a potential biomaterial for surgical threads or wound dressings; however, as biomedical engineering advances, the demand for high‐performance, naturally derived biomaterials becomes more pressing and stringent. A common problem of natural materials is their large batch‐to‐batch variation, the quantity available, their potentially high immunogenicity, and their fast biodegradation. Some of these common problems also apply to silk; therefore, recombinant approaches for producing silk proteins have been developed. There are several research groups which study and utilize various recombinantly produced silk proteins, and many of these have also investigated their products for biomedical applications. This review gives a critical overview over of the results for applications of recombinant silk proteins in biomedical engineering.
Recombinant silk proteins are an interesting class of biopolymers that are well‐suited for biomedical applications. Studies on the use of recombinant silk proteins are summarized, and important conclusions are given. The best features of recombinant silk proteins as a biomaterial are also highlighted, along with exciting future directions.
Immediately following spinal cord injury, further injury can occur through several secondary injury cascades. As a consequence of cell lysis, an increase in extracellular Ca2+ results in additional ...neuronal loss by inducing apoptosis. Thus, hydrogels that reduce extracellular Ca2+ concentration may reduce secondary injury severity. The goal of this study was to develop composite hydrogels consisting of alginate, chitosan, and genipin that interact with extracellular Ca2+ to enable in situ gelation while maintaining an elastic modulus similar to native spinal cord (∼1000 Pa). It was hypothesized that incorporation of genipin and chitosan would regulate hydrogel electrostatic characteristics and influence hydrogel porosity, degradation, and astrocyte behavior. Hydrogel composition was varied to create hydrogels with statistically similar mechanical properties (∼1000 Pa) that demonstrated tunable charge characteristics (6-fold range in free amine concentration) and degradation rate (complete degradation between 7 and 28 days; some blends persist after 28 days). Hydrogels demonstrate high sensitivity to Ca2+ concentration, as a 1 mM change during fabrication induced a significant change in elastic modulus. Additionally, hydrogels incubated in a Ca2+-containing solution exhibited an increased linear viscoelastic limit (LVE) and an increased elastic modulus above the LVE limit in a time dependent manner. An extension of the LVE limit implies a change in hydrogel cross-linking structure. Attachment assays demonstrated that addition of chitosan/genipin to alginate hydrogels induced up to a 4-fold increase in the number of attached astrocytes and facilitated astrocyte clustering on the hydrogel surface in a composition dependent manner. Furthermore, Western blots demonstrated tunable glial fibrillary acid protein (GFAP) expression in astrocytes cultured on hydrogel blends, with some hydrogel compositions demonstrating no significant increase in GFAP expression compared to astrocytes cultured on glass. Thus, alginate/chitosan/genipin hydrogel composites show promise as scaffolds that regulate astrocyte behavior and for the prevention of Ca2+-related secondary neuron damage during acute SCI.
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.
There are currently more than 600 diseases characterized as affecting the central nervous system (CNS) which inflict neural damage. Unfortunately, few of these conditions have effective treatments ...available. Although significant efforts have been put into developing new therapeutics, drugs which were promising in the developmental phase have high attrition rates in late stage clinical trials. These failures could be circumvented if current 2D in vitro and in vivo models were improved. 3D, tissue-engineered in vitro systems can address this need and enhance clinical translation through two approaches: (1) bottom-up, and (2) top-down (developmental/regenerative) strategies to reproduce the structure and function of human tissues. Critical challenges remain including biomaterials capable of matching the mechanical properties and extracellular matrix (ECM) composition of neural tissues, compartmentalized scaffolds that support heterogeneous tissue architectures reflective of brain organization and structure, and robust functional assays for in vitro tissue validation. The unique design parameters defined by the complex physiology of the CNS for construction and validation of 3D in vitro neural systems are reviewed here.
Hydrogels made of polyanionic recombinant spider silk proteins (spidroins) were prepared either in the presence or the absence of Dulbecco's Modified Eagle Medium (DMEM). Mono- and divalent cations ...present in DMEM severely affected the self-assembly process of the spidroins. Although the addition of DMEM had no apparent effect on secondary structure formation, there was a significant effect on the kinetics as well as on the hydrogel network; in the presence of DMEM, gelation occurred more rapidly. Additionally, the hydrogels were stiffer; however, the hydrogels were still shear-thinning. In summary, it can be concluded that there is a significant impact of ionic cross-linking on recombinant spidroin-based hydrogels.
•Characterized spider silk bioinks in the presence of Dulbecco's Modified Eagle Medium.•Examined effects of cations on the behavior of spider silk-based bioinks.•Determined possibility of ionic crosslinking in charged, spider silk-based bioinks.
Bioinks, 3D cell culture systems which can be printed, are still in the early development stages. Currently, extensive research is going into designing printers to be more accommodating to bioinks, ...designing scaffolds with stiff materials as support structures for the often soft bioinks, and modifying the bioinks themselves. Recombinant spider silk proteins, a potential biomaterial component for bioinks, have high biocompatibility, can be processed into several morphologies and can be modified with cell adhesion motifs to enhance their bioactivity. In this work, thermally gelled hydrogels made from recombinant spider silk protein encapsulating mouse fibroblast cell line BALB/3T3 were prepared and characterized. The bioinks were evaluated for performance in vitro both before and after printing, and it was observed that unprinted bioinks provided a good platform for cell spreading and proliferation, while proliferation in printed scaffolds was prohibited. To improve the properties of the printed hydrogels, gelatin was given as an additive and thereby served indirectly as a plasticizer, improving the resolution of printed strands. Taken together, recombinant spider silk proteins and hydrogels made thereof show good potential as a bioink, warranting further development.
Recombinantly produced spider silk proteins have high potential for bioengineering and various biomedical applications because of their biocompatibility, biodegradability, and low immunogenicity. ...Here, the recently described small spider silk protein eMaSp1s is assembled into hydrogels, which can be 3D printed into scaffolds. Further, blending with a recombinantly produced MaSp2 derivative eADF4(C16) alters the mechanical properties of the resulting hydrogels. Different spider silk hydrogels also show a distinct recovery after a high shear stress deformation, exhibiting the tunability of their features for selected applications.
Hydrogels of a novel spider silk protein MaSp1s are successfully produced and characterized. Blending with another spider silk protein changes the mechanical properties of the resulting hydrogels and leads to variances in printability and recovery after shear stress, opening the door for biomedical applications.
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Biofilm formation, especially of antimicrobiotic-resistant microbial strains, are a major problem in health care. Therefore, there is great interest in developing advanced materials ...that are selectively inhibiting microbial adhesion to surfaces, but at the same time promoting mammalian cell growth. In nature, some spider silks have evolved to repel microbes, a feature that could be used in biomaterials. To unravel how microbe repellence can be achieved in engineered spider silk, different recombinant spider silk proteins based on the consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4) were processed into 2D-patterned films and 3D-hydrogels. Strikingly, protein structure characteristics on the nanoscale are the basis for the detected microbe-repellence. Designed spider silk materials promoted mammalian cell attachment and proliferation while inhibiting microbial infestation, demonstrating the great potential of these engineered spider silk-based materials as bio-selective microbial-resistant coatings in biomedical as well as technical applications.
Despite significant investment in tissue engineering over the past 20 years, few tissue engineered products have made it to market. One of the reasons is the poor control over the 3D arrangement of ...the scaffold’s components. Biofabrication is a new field of research that exploits 3D printing technologies with high spatial resolution for the simultaneous processing of cells and biomaterials into 3D constructs suitable for tissue engineering. Cell-encapsulating biomaterials used in 3D bioprinting are referred to as bioinks. This review consists of: (1) an introduction of biofabrication, (2) an introduction of 3D bioprinting, (3) the requirements of bioinks, (4) existing bioinks, and (5) a specific example of a recombinant spider silk bioink. The recombinant spider silk bioink will be used as an example because its unmodified hydrogel format fits the basic requirements of bioinks: to be printable and at the same time cytocompatible. The bioink exhibited both cytocompatible (self-assembly, high cell viability) and printable (injectable, shear-thinning, high shape fidelity) qualities. Although improvements can be made, it is clear from this system that, with the appropriate bioink, many of the existing faults in tissue-like structures produced by 3D bioprinting can be minimized.
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.
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•Electrospinning of spider silk proteins has been accomplished out of aqueous conditions.•Post-treatment of electrospun spider silk fibers has been achieved by water-annealing at 37 °C.•The all-aqueous electrospinning process of spider silk allows for incorporation of biologically active ingredients.•Recombinant technologies allow for permanent incorporation of biologicals within spider silk scaffolds.
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