Recombinant Spider Silk Bioinks for Continuous Protein Release by Encapsulated Producer Cells.

Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of Gaussia princeps as a reporter domain. eADF4(C16) and eADF4(C16)-RGD hydrogels provide structural and mechanical support, promote HEK293 cell growth, and allow fusion protein production by the latter. Bioink-captured HEK293 producer cells continuously release functional TNFR2-Fc-GpL over 14 days. Thus, the combination of biocompatible, printable spider silk bioinks with drug-producing cells is promising for generating implantable 3D constructs for continuous targeted therapy.

Recombinant Spider Silk Gels Derived from Aqueous-Organic Solvents as Depots for Drugs.

Hydrogels are widely used in various biomedical applications, as they cannot only serve as materials for biofabrication but also as depots for the administration of drugs. However, the possibilities of formulation of water-insoluble drugs in hydrogels are rather limited. Herein, we assembled recombinant spider silk gels using a new processing route with aqueous-organic co-solvents, and the properties of these gels could be controlled by the choice of the co-solvent. The presence of the organic co-solvent further enabled the incorporation of hydrophobic drugs as exemplarily shown for 6-mercaptopurine. The developed gels showed shear-thinning behaviour and could be easily injected to serve, for example, as drug depots, and they could even be 3D printed to serve as scaffolds for biofabrication. With this new processing route, the formulation of water-insoluble drugs in spider silk-based depots is possible, circumventing common pharmaceutical solubility issues.

Factors Influencing Properties of Spider Silk Coatings and Their Interactions within a Biological Environment.

Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.

Rheological and Biological Impact of Printable PCL-Fibers as Reinforcing Fillers in Cell-Laden Spider-Silk Bio-Inks.

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.

Impact of Cell Loading of Recombinant Spider Silk Based Bioinks on Gelation and Printability.

Printability of bioinks encompasses considerations concerning rheology and extrudability, characterization of filament formation, shape fidelity, cell viability, and post-printing cellular development. Recombinant spider silk based hydrogels might be a suitable material to be used in bioinks, that is, a formulation of cells and materials to be used for bioprinting. Here, the high shape fidelity of spider silk ink is shown by bioprinting the shape and size of a human aortic valve. Further the influence of the encapsulation of cells has been evaluated on spider silk hydrogel formation, hydrogel mechanics, and shape fidelity upon extrusion based bioprinting. It is shown that the presence of cells impacts the gelation of spider silk proteins differently, depending on the used silk variant. RGD-modified spider silk hydrogels are physically crosslinked by the cells, while there is no active interaction between cells and un-tagged spider silk proteins. Strikingly, even at cell densities up to ten million cells per milliliter, cell viability is high after extrusion-based printing, which is a significant prerequisite for future applications. Shape fidelity of the printed constructs is demonstrated using a filament collapse test in the absence and presence of human cells.

Bioselectivity of silk protein-based materials and their bio-inspired applications.

Adhesion to material surfaces is crucial for almost all organisms regarding subsequent biological responses. Mammalian cell attachment to a surrounding biological matrix is essential for maintaining their survival and function concerning tissue formation. Conversely, the adhesion and presence of microbes interferes with important multicellular processes of tissue development. Therefore, tailoring bioselective, biologically active, and multifunctional materials for biomedical applications is a modern focus of biomaterial research. Engineering biomaterials that stimulate and interact with cell receptors to support binding and subsequent physiological responses of multicellular systems attracted much interest in the last years. Further to this, the increasing threat of multidrug resistance of pathogens against antibiotics to human health urgently requires new material concepts for preventing microbial infestation and biofilm formation. Thus, materials exhibiting microbial repellence or antimicrobial behaviour to reduce inflammation, while selectively enhancing regeneration in host tissues are of utmost interest. In this context, protein-based materials are interesting candidates due to their natural origin, biological activity, and structural properties. Silk materials, in particular those made of spider silk proteins and their recombinant counterparts, are characterized by extraordinary properties including excellent biocompatibility, slow biodegradation, low immunogenicity, and non-toxicity, making them ideally suited for tissue engineering and biomedical applications. Furthermore, recombinant production technologies allow for application-specific modification to develop adjustable, bioactive materials. The present review focusses on biological processes and surface interactions involved in the bioselective adhesion of mammalian cells and repellence of microbes on protein-based material surfaces. In addition, it highlights the importance of materials made of recombinant spider silk proteins, focussing on the progress regarding bioselectivity.

Structure-Property Relationship Based on the Amino Acid Composition of Recombinant Spider Silk Proteins for Potential Biomedical Applications.

Improving biomaterials by engineering application-specific and adjustable properties is of increasing interest. Most of the commonly available materials fulfill the mechanical and physical requirements of relevant biomedical applications, but they lack biological functionality, including biocompatibility and prevention of microbial infestation. Thus, research has focused on customizable, application-specific, and modifiable surface coatings to cope with the limitations of existing biomaterials. In the case of adjustable degradation and configurable interaction with body fluids and cells, these coatings enlarge the applicability of the underlying biomaterials. Silks are interesting coating materials, e.g., for implants, since they exhibit excellent biocompatibility and mechanical properties. Herein, we present putative implant coatings made of five engineered recombinant spider silk proteins derived from the European garden spider Araneus diadematus fibroins (ADF), differing in amino acid sequence and charge. We analyzed the influence of the underlying amino acid composition on wetting behavior, blood compatibility, biodegradability, serum protein adsorption, and cell adhesion. The outcome of the comparison indicates that spider silk coatings can be engineered for explicit biomedical applications.

Flow Simulation and Gradient Printing of Fluorapatite- and Cell-Loaded Recombinant Spider Silk Hydrogels.

Hierarchical structures are abundant in almost all tissues of the human body. Therefore, it is highly important for tissue engineering approaches to mimic such structures if a gain of function of the new tissue is intended. Here, the hierarchical structures of the so-called enthesis, a gradient tissue located between tendon and bone, were in focus. Bridging the mechanical properties from soft to hard secures a perfect force transmission from the muscle to the skeleton upon locomotion. This study aimed at a novel method of bioprinting to generate gradient biomaterial constructs with a focus on the evaluation of the gradient printing process. First, a numerical approach was used to simulate gradient formation by computational flow as a prerequisite for experimental bioprinting of gradients. Then, hydrogels were printed in a single cartridge printing set-up to transfer the findings to biomedically relevant materials. First, composites of recombinant spider silk hydrogels with fluorapatite rods were used to generate mineralized gradients. Then, fibroblasts were encapsulated in the recombinant spider silk-fluorapatite hydrogels and gradually printed using unloaded spider silk hydrogels as the second component. Thereby, adjustable gradient features were achieved, and multimaterial constructs were generated. The process is suitable for the generation of gradient materials, e.g., for tissue engineering applications such as at the tendon/bone interface.

Enhanced vascularization andde novotissue formation in hydrogels made of engineered RGD-tagged spider silk proteins in the arteriovenous loop model.

Due to its low immunogenic potential and the possibility to fine-tune their properties, materials made of recombinant engineered spider silks are promising candidates for tissue engineering applications. However, vascularization of silk-based scaffolds is one critical step for the generation of bioartificial tissues and consequently for clinical application. To circumvent insufficient vascularization, the surgically induced angiogenesis by means of arteriovenous loops (AVL) represents a highly effective methodology. Here, previously established hydrogels consisting of nano-fibrillary recombinant eADF4(C16) were transferred into Teflon isolation chambers and vascularized in the rat AVL model over 4 weeks. To improve vascularization, also RGD-tagged eADF4(C16) hydrogels were implanted in the AVL model over 2 and 4 weeks. Thereafter, the specimen were explanted and analyzed using histology and microcomputed tomography. We were able to confirm biocompatibility and tissue formation over time. Functionalizing eADF4(C16) with RGD-motifs improved hydrogel stability and enhanced vascularization even outperforming other hydrogels, such as fibrin. This study demonstrates that the scaffold ultrastructure as well as biofunctionalization with RGD-motifs are powerful tools to optimize silk-based biomaterials for tissue engineering applications.

Data for microbe resistant engineered recombinant spider silk protein based 2D and 3D materials.

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].