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

Lechner, Annika; Trossmann, Vanessa T.; Scheibel, Thomas

doi:10.1002/mabi.202100390

Cited by 14 publications

(18 citation statements)

References 40 publications

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“…The recombinant spider silk variant eADF4(C16) consists of 16 repeats of the so-called C-module, inspired by the repetitive core domain of the natural Araneus diadematus fibroin 4, and was genetically modified with the integrin-binding peptide RGD improving the interaction with cells and with the integrin-binding inactive RGE as a control . As eADF4(C16)-based proteins could be processed into physically cross-linked, nano-fibrillar hydrogels without the need of additional cross-linking agents, encapsulation and cultivation of cells inside these gels is possible . Additionally, due to their viscoelastic properties and shear-thinning behavior, 3D bioprinting of acellular hydrogels and bioinks made of spider silk proteins together with cells could be conducted. , Furthermore, it has been previously shown that different biologicals and drugs could be encapsulated inside and released from recombinant spider silk hydrogels with their activity maintained. , …”

Section: Introductionmentioning

confidence: 99%

“…As eADF4(C16)-based proteins could be processed into physically cross-linked, nano-fibrillar hydrogels without the need of additional cross-linking agents, encapsulation and cultivation of cells inside these gels is possible . Additionally, due to their viscoelastic properties and shear-thinning behavior, 3D bioprinting of acellular hydrogels and bioinks made of spider silk proteins together with cells could be conducted. , Furthermore, it has been previously shown that different biologicals and drugs could be encapsulated inside and released from recombinant spider silk hydrogels with their activity maintained. , …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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“…The majority of materials used for cell patterning are based on the use of synthetic polymers specifically tailored to the selected patterning technique, especially when immobilization of biologically active components is required. , As a result of this customization, these materials are chemically different from the bulk materials used in tissue engineering and implants in general, making it difficult to directly link cell responses between microscopic and bulk platforms. In the case of the recombinant spider silk protein, the fibrous nature of bulk hydrogels, reflecting the ECM matrix, has become the basis for biofabrication approaches in tissue engineering. ,, Here, we applied spider silk self-assembly using the fibrous, hydrogel-like morphology that persists on the surface , to expose specific biotargeting functions represented by DNA interactions as a model. Moreover, any insights gained on the microstructured platforms based on the presented nanohydrogels will facilitate and accelerate the transfer of knowledge toward bulk hydrogels suitable for 3D printing of scaffolds or coatings of implants.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of the recombinant spider silk protein, the fibrous nature of bulk hydrogels, 59 reflecting the ECM matrix, has become the basis for biofabrication approaches in tissue engineering. 39,60,61 Here, we applied spider silk self-assembly using the fibrous, hydrogel-like morphology that persists on the surface 41,42 to expose specific biotargeting functions represented by DNA interactions as a model. Moreover, any insights gained on the microstructured platforms based on the presented nanohydrogels will facilitate and accelerate the transfer of knowledge toward bulk hydrogels suitable for 3D printing of scaffolds or coatings of implants.…”

Section: Discussionmentioning

confidence: 99%

DNA Functionalized Spider Silk Nanohydrogels for Specific Cell Attachment and Patterning

et al. 2022

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Nucleated protein self-assembly of an azido modified spider silk protein was employed in the preparation of nanofibrillar networks with hydrogel-like properties immobilized on coatings of the same protein. Formation of the networks in a mild aqueous environment resulted in thicknesses between 2 and 60 nm, which were controlled only by the protein concentration. Incorporated azido groups in the protein were used to “click” short nucleic acid sequences onto the nanofibrils, which were accessible to specific hybridization-based modifications, as proved by fluorescently labeled DNA complements. A lipid modifier was used for efficient incorporation of DNA into the membrane of nonadherent Jurkat cells. Based on the complementarity of the nucleic acids, highly specific DNA-assisted immobilization of the cells on the nanohydrogels with tunable cell densities was possible. Addressability of the DNA cell-to-surface anchor was demonstrated with a competitive oligonucleotide probe, resulting in a rapid release of 75–95% of cells. In addition, we developed a photolithography-based patterning of arbitrarily shaped microwells, which served to spatially define the formation of the nanohydrogels. After detaching the photoresist and PEG-blocking of the surface, DNA-assisted immobilization of the Jurkat cells on the nanohydrogel microstructures was achieved with high fidelity.

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“…[28][29][30] Using chemically modified surfaces with the protein [31] or ultrathin nanofilms thereof, [32] the spider silk nanofibrils assembled on top into immobilized physically crosslinked networks, which displayed swelling and softening in the water, that is, properties similar to the bulk spider silk hydrogels. [33,34] The nanofibrillar nature of these nanohydrogels enabled a higher modification density and greater substrate accessibility compared to monolayers, whilst the mild aqueous self-assembly conditions in combination with the mechanical and chemical robustness of the nanohydrogels provided excellent scaffolds for embedding sensitive components such as enzymes and aptamers. [32] Spider silk proteins could be modified using both chemical [32,35] and genetic methods.…”

Section: Introductionmentioning

confidence: 99%

Dual Patterning of Self‐Assembling Spider Silk Protein Nanofibrillar Networks

Lamberger

Kocourková

Minařík

et al. 2022

Adv Materials Inter

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well as cell-cell and cell-ligand interactions. [10,11] Due to their reduced size and compact construction, these require fewer resources for their production, reducing their costs, whilst also minimizing the amount of analyte. This enables a much higher throughput of samples and their readouts than with more conventional macroscopic methods. [10,12] Photolithography represents a commonly used approach for microstructuring, whereby substrates are covered by a photosensitive resin, onto which illumination with a specific pattern imparts spatially defined soluble/insoluble areas, thus enabling selective deprotection of the surface and subsequent modifications at microscales. [10,13] Nonetheless, such microstructuring is often limited to the usage of covalently coupled synthetic polymers, due to the relatively harsh conditions involved in the photoresist processing.Milder methods have been developed, such as polymer grafting using photocatalyzed coupling reactions, [14,15] but 3D structuring and additional functionalization of the networks require ever more complex polymers increasing associated synthesis costs. The polymerization of dopamine could also be used to generate a pattern with high fidelity, which can be further modified, [16,17] but the spectrum of reactions that can be employed, is limited to the dopamine moiety, requiring elaborate chemistry if desiring to spatiotemporally implement several different functions.An approach to multilayering different functional substrates is presented by self-assembling protein films, which adhere to the prior layer using non-covalent interactions. [18] Such approaches are more versatile in the functionality and modification alternatives, but the introduction of specific functions on different spots of the substrate is often problematic. The non-covalent bonding, which predominates among the film layers, must also be resilient to breaking, limiting the variety of available interactions dependent on the application conditions, and impeding the development of a generally applicable system.A strong non-covalent bonding interaction is found in silks, where the intrinsically random coil domains self-assemble into β-sheet-rich formations, whereby they are stabilized in a network of the same proteins. [19] The difference in solubility and the inducible nature of the conversion could be used for micropatterning. [20][21][22][23][24] Yet, a method, which provides the duality of being able to introduce high fidelity, high-resolution micropattern with a plethora of possible functionalization Self-assembly of a recombinant spider silk protein into nanofibrillar networks in combination with photolithography is used to produce diversely functionalized micropattern. Amino-modified substrates coated with a positive tone photoresist are processed into 1 µm deep arbitrarily shaped microwells, at the bottom of which spider silk proteins are covalently coupled to the deprotected aminated surface. The protein layer serves to seed the self-assembly of nanofibrils from the same protein in the ...

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

Cited by 14 publications

References 40 publications

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

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

DNA Functionalized Spider Silk Nanohydrogels for Specific Cell Attachment and Patterning

Dual Patterning of Self‐Assembling Spider Silk Protein Nanofibrillar Networks

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