ABSTRACT To identify effective drugs for breast cancer treatment, it is essential to establish physiologically relevant models. Traditional models based on 2D cultures or 3D scaffold‐free spheroids lack the crucial cell–extracellular matrix (ECM) interactions, known to significantly impact drug sensitivity. A newly developed 3D culture format using a network of the recombinant spider silk protein FN‐silk has demonstrated ECM‐like interactions and maintenance of subtype‐specific marker expression in breast cancer cells. In the current study, chemotherapy drug treatment experiments were conducted on the breast cancer cell lines MCF‐7, MDA‐MB‐231, and SK‐BR‐3 cultured in 2D, spheroids, and FN‐silk networks. The results suggest that FN‐silk networks hold promise as a base for an in vitro 3D model assessing the effects of chemotherapeutic agents. Comparing drug responses revealed that cells cultured in FN‐silk networks were generally less sensitive to the drugs tested—as compared to 2D cultures—suggesting that FN‐silk‐supported 3D culture could have higher clinical relevance. Moreover, the results showed improved reproducibility with FN‐silk networks compared to spheroids. This study concludes that FN‐silk networks have the potential to support the establishment of a valuable 3D tumor model for the development of personalized breast cancer treatments and thereby contribute to improved success rates in drug development.

This study advances sustainable pharmaceutical research for endometriosis by developing in vitro 3D cell culture models of endometriotic pathophysiology that allow antifibrotic drug candidates to be tested. Fibrosis is a key aspect of endometriosis, yet current cell models to study it remain limited. This work aims to bridge the translational gap between in vitro fibrosis research and preclinical testing of non-hormonal drug candidates. When grown in a 3D matrix of sustainably produced silk protein functionalized with a fibronectin-derived cell adhesion motif (FN-silk), endometrial stromal and epithelial cells respond to transforming growth factor beta-1 (TGF-β1) in a physiological manner as probed at the messenger RNA (mRNA) level. For stromal cells, this response to TGF-β1 is not observed in spheroids, while epithelial cell spheroids behave similarly to epithelial cell FN-silk networks. Pirfenidone, an antifibrotic drug approved for the treatment of idiopathic pulmonary fibrosis, reverses TGF-β1-induced upregulation of mRNA transcripts involved in fibroblast-to-myofibroblast transdifferentiation of endometrial stromal cells in FN-silk networks, supporting pirfenidone's potential as a repurposed non-hormonal endometriosis therapy. Overall, endometrial stromal cells cultured in FN-silk networks-which are composed of a sustainably produced, fully defined FN-silk protein-recapitulate fibrotic cellular behavior with high fidelity and enable antifibrotic drug testing.

The recombinant functionalized silk protein FN-silk, including a cell adhesion motif from fibronectin, can form networks suitable for 3D culture of adherent cells. Such FN-silk networks have previously been shown to support the growth and differentiation of a wide array of cell types. Herein, we have developed a user-friendly methodology for the creation of free-floating FN-silk networks in 96-well plates with both mature human primary cells and stem cells. We show that human mesenchymal stem cells (hMSC) cultured in FN-silk networks form both cell-cell and cell-matrix contacts, resulting in tissue-mimicking 3D cultures. Viability and expression analysis revealed that hMSC in FN-silk networks have an initial proliferative phase with high cell viability and significantly lower hypoxia and apoptosis, compared to when cultured as scaffold-free spheroids. The FN-silk networks were shown to support differentiation of hMSC into adipocyte-like cells with well-maintained viability during the 3-week-long differentiation period, in contrast to the very poor long-term viability of scaffold-free 3D cultures. Improved adipogenesis was confirmed by lipid droplet staining, quantification of intracellular triglycerides, and secreted adiponectin levels, as well as expression analysis of multiple bona fide adipose markers. Lastly, we show that primary human hepatocytes maintain important functions and phenotypic markers when cultured in FN-silk networks, features that are lost rapidly during conventional 2D culture. We therefore propose FN-silk networks as a valuable scaffold for 3D human cell cultures, providing support for cell proliferation, differentiation, and the maintenance of critical tissue-specific functionality.

Functionalization of biomaterials with extra protein domains will expand their functional roles in biomedical research. The recombinant spider silk protein FN-4RepCT has been shown able to adapt various formats like coatings, nanowires, and macroscopic fibers. Functionalizing these various formats of FN-4RepCT in a site-specific manner will provide the next generation of biomaterials. The current study reports an enzymatic (sortase A) coupling method to site-specifically functionalize various formats of FN-4RepCT with target proteins. The approach is demonstrated with three different functional proteins: the IgG-binding Z-domain, a single-chain variable fragment with specificity for CD38 (scFvCD38), and the antibacterial endolysin Sal-1. The target proteins were produced with an LPETGG sortase recognition tag at the C-terminus to enable coupling. Moreover, a comparative analysis of sortase coupling efficiency of the target proteins was performed using two different silk protein variants, FN-4RepCT with one N-terminal glycine (G-silk) and five N-terminal glycines (G5-silk). The functionalized silks were assessed by using protein gel electrophoresis, fluorescence microscopy, surface plasmon resonance, and a biochemical assay. Results showed that G5-silk is more efficient for sortase coupling of the target proteins in solution as well as to silk coatings, when compared to G-silk. In all cases, the target proteins, the Z-domain, the scFvCD38 fragment, and Sal-1, retained their specific activity after sortase coupling. To conclude, the sortase coupling strategy is a mild and efficient approach to functionalize various silk formats with small (Z-domain) or larger (scFvCD38, Sal-1) functional molecules.

This paper presents the generation and evaluation of a novel potential drug delivery platform for biologics, based on recombinant spider silk. Targeting CD40 for activation of antigen presenting cells, in order to overcome tumor induced T cell tolerance, have shown promising results in cell and animal models. However, further trials have gained limited results due to severe side reactions. To overcome this, we have investigated a strategy for a localized CD40 activation. A CD40 agonist based on a single chain variable fragment (scFvCD40) was enzymatically coupled to silk structures, that were then used to stimulate cells in vitro. A reporter cell line responsive to CD40 agonists was used to evaluate the bioactivity of the developed scFvCD40-silk, and to optimize the method. Once the bioactivity was confirmed, human primary B cells derived from healthy donors were stimulated with the scFvCD40-silk construct. The resulting B cell response was characterized both by upregulated surface expression of the activation marker CD86 (3 fold), suggesting an improved antigen-presenting capacity, and by B cell proliferation (4 fold) generating an expanded B cell population. The detected upregulation of the costimulatory molecule CD86 on the B cells implies a potential of the functionalized silk to steer the tumor-specific T cell response from tolerance to immune activation, including the onset of appropriate effector functions. Finally, we investigated the usability of the novel silk format microspheres for CD40-mediated cell activation in vitro. Here, we were able to demonstrate that scFvCD40-coupled silk microspheres gave a pronounced activation of the CD40-expressing reporter cell line, supporting the suitability of silk microspheres for the delivery of biologics with immune modulatory purposes.

Physiologically relevant human skin models that include key skin cell types can be used for in vitro drug testing, skin pathology studies, or clinical applications such as skin grafts. However, there is still no golden standard for such a model. We investigated the potential of a recombinant functionalized spider silk protein, FN-silk, for the construction of a dermal, an epidermal, and a bilayered skin equivalent (BSE). Specifically, two formats of FN-silk (i.e. 3D network and nanomembrane) were evaluated. The 3D network was used as an elastic ECM-like support for the dermis, and the thin, permeable nanomembrane was used as a basement membrane to support the epidermal epithelium. Immunofluorescence microscopy and spatially resolved transcriptomics analysis demonstrated the secretion of key ECM components and the formation of microvascular-like structures. Furthermore, the epidermal layer exhibited clear stratification and the formation of a cornified layer, resulting in a tight physiologic epithelial barrier. Our findings indicate that the presented FN-silk-based skin models can be proposed as physiologically relevant standalone epidermal or dermal models, as well as a combined BSE.

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.

Orthopedic and dental implants are associated with a substantial risk of failure due to biomaterial-associated infections and poor osseointegration. To prevent such outcomes, a coating can be applied on the implant to ideally both reduce the risk of bacterial adhesion and support establishment of osteoblasts. We present a strategy to construct dual-functional silk coatings with such properties. Silk coatings were made from a recombinant partial spider silk protein either alone (silkwt) or fused with a cell-binding motif derived from fibronectin (FN-silk). The biofilm-dispersal enzyme Dispersin B (DspB) and two peptidoglycan degrading endolysins, PlySs2 and SAL-1, were produced recombinantly. A sortase recognition tag (SrtTag) was included to allow site-specific conjugation of each enzyme onto silkwt and FN-silk coatings using an engineered variant of the transpeptidase Sortase A (SrtA*). To evaluate bacterial adhesion on the samples, Staphylococcus aureus was incubated on the coatings and subsequently subjected to live/dead staining. Fluorescence microscopy revealed a reduced number of bacteria on all silk coatings containing enzymes. Moreover, the bacteria were mobile to a higher degree, indicating a negative influence on the bacterial adhesion. The capability to support mammalian cell interactions was assessed by cultivation of the osteosarcoma cell line U-2 OS on dual-functional surfaces, prepared by conjugating the enzymes onto FN-silk coatings. U-2 OS cells could adhere to silk coatings with enzymes and showed high spreading and viability, demonstrating good cell compatibility.

Presentation of immobilized growth factors with retained bioactivity remains a challenge in the field of tissue engineering. In the present study, we propose a strategy to covalently conjugate a pleiotropic growth factor, basic fibroblast growth factor (bFGF) to a partial spider silk protein at gene level. The resulting silk-bFGF fusion protein has the propensity to self-assemble into silk-like fibers, and also surface coatings, as confirmed by quartz crystal microbalance studies. Functionality of the silk-bFGF coating to bind its cognate receptor was confirmed with surface plasmon resonance studies. As a step toward the creation of an artificial ECM, the silk-bFGF protein was mixed with FN-silk, an engineered spider silk protein with enhanced cell adhesive properties. Bioactivity of the thereby obtained combined silk was confirmed by successful culture of primary human endothelial cells on coatings and integrated within fibers, even in culture medium without supplemented growth factors. Together, these findings show that silk materials bioactivated with growth factors can be used for in vitro cell culture studies, and have potential as a tissue engineering scaffold.

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) fibrillar network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into fibrillar networks under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed fibrils. In the resulting 3D scaffold, the cells proliferate and spread out with tissue-like morphology. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk fibers with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables both differentiation of stem- or precursor cells in 3D and facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a fibrillary ECM-like network, with potential as base for engineering of functional tissue.

In vitro endothelialization of synthetic grafts or engineered vascular constructs is considered a promising alternative to overcome shortcomings in the availability of autologous vessels and in-graft complications with synthetics. A number of cell-seeding techniques have been implemented to render vascular grafts accessible for cells to attach, proliferate, and spread over the surface area. Nonetheless, seeding efficiency and the time needed for cells to adhere varies dramatically. Herein, we investigated a novel cell-seeding approach (denoted co-seeding) that enables cells to bind to a motif from fibronectin included in a recombinant spider silk protein. Entrapment of cells occurs at the same time as the silk assembles into a nanofibrillar coating on various substrates. Cell adhesion analysis showed that the technique can markedly improve cell-seeding efficiency to nonfunctionalized polystyrene surfaces, as well as establish cell attachment and growth of human dermal microvascular endothelial cells on bare polyethylene terephthalate and polytetrafluoroethylene (PTFE) substrates. Scanning electron microscopy images revealed a uniform endothelial cell layer and cell-substratum compliance with the functionalized silk protein to PTFE surfaces. The co-seeding technique holds a great promise as a method to reliably and quickly cellularize engineered vascular constructs as well as to in vitro endothelialize commercially available cardiovascular grafts.

Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.

Functionalization of biomaterials with biologically active peptides can improve their performance after implantation. By genetic fusion to self-assembling proteins, the functional peptides can easily be presented on different physical formats. Herein, a chemical-free coating method based on self-assembly of the recombinant spider silk protein 4RepCT is described and used to prepare functional coatings on various biomaterial surfaces. The silk assembly was studied in real-time, revealing the occurrence of continuous assembly of silk proteins onto surfaces and the formation of nanofibrillar structures. The adsorbed amounts and viscoelastic properties were evaluated, and the coatings were shown to be stable against wash with hydrogen chloride, sodium hydroxide, and ethanol. Titanium, stainless steel, and hydroxyapatite were coated with silk fused to an antimicrobial peptide or a motif from fibronectin. Human primary cells cultured on the functional silk coatings show good cell viability and proliferation, implying the potential to improve implant performance and acceptance by the body.

Nature's design of functional materials relies on smart combinations of simple components to achieve desired properties. Silk and cellulose are two clever examples from nature-spider silk being tough due to high extensibility, whereas cellulose possesses unparalleled strength and stiffness among natural materials. Unfortunately, silk proteins cannot be obtained in large quantities from spiders, and recombinant production processes are so far rather expensive. We have therefore combined small amounts of functionalized recombinant spider silk proteins with the most abundant structural component on Earth (cellulose nanofibrils (CNFs)) to fabricate isotropic as well as anisotropic hierarchical structures. Our approach for the fabrication of bio-based anisotropic fibers results in previously unreached but highly desirable mechanical performance with a stiffness of ∼55 GPa, strength at break of ∼1015 MPa, and toughness of ∼55 MJ m-3. We also show that addition of small amounts of silk fusion proteins to CNF results in materials with advanced biofunctionalities, which cannot be anticipated for the wood-based CNF alone. These findings suggest that bio-based materials provide abundant opportunities to design composites with high strength and functionalities and bring down our dependence on fossil-based resources.

Natural silk is easily accessible from silkworms and can be processed into different formats suitable as biomaterials and cell culture matrixes. Recombinant DNA technology enables chemical-free functionalization of partial silk proteins through fusion with peptide motifs and protein domains, but this constitutes a less cost-effective production process. Herein, we show that natural silk fibroin (SF) can be used as a bulk material that can be top-coated with a thin layer of the recombinant spider silk protein 4RepCT in fusion with various bioactive motifs and domains. The coating process is based on a silk assembly to achieve stable interactions between the silk types under mild buffer conditions. The assembly process was studied in real time by quartz crystal microbalance with dissipation. Coatings, electrospun mats, and microporous scaffolds were constructed from Antheraea assama and Bombyx mori SFs. The morphology of the fibroin materials before and after coating with recombinant silk proteins was analyzed by scanning electron microscopy and atomic force microscopy. SF materials coated with various bioactive 4RepCT fusion proteins resulted in directed antibody capture, enzymatic activity, and improved cell attachment and spreading, respectively, compared to pristine SF materials. The herein-described procedure allows a fast and easy route for the construction of bioactive materials.

Event Abstract Back to Event A fibronectin mimicking motif enhances cell adhesive properties of spider silk Mona Widhe1, Nancy Dekki-Shalaly1 and My Hedhammar1, 2 1 Royal Intitute of Technology, KTH, Division of Protein Technology, School of Biotechnology, Sweden 2 Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry, Sweden Introduction: The cell binding motif RGD is the most widely used peptide to improve cell binding properties of various biomaterials, including recombinant spider silk. We have previously shown a positive effect on cells when RGD was genetically coupled to the recombinant spider silk protein 4RepCT[1]. In the present study we enhanced the cell binding capacity even further by introducing the integrin binding motif from fibronectin, which contains RGD on a constrained loop. Experimental Set-up: 4RepCT was genetically functionalized with the RGD-containing cell binding motif from fibronectin. To enable loop formation, two amino acids flanking the RGD sequence was substituted for cysteines. The positions of cysteines were carefully selected to achieve a 3D mimic of the turn loop seen in the full length fibronectin molecule. The resulting silk protein was denoted FNCC. Linear variants without cysteines were used as controls. Primary cells of human origin (keratinocytes, endothelial cells and mesenchymal stem cells) were used to evaluate cell culture matrices of FNCC-silk. Adhesion, formation of stress fibers and focal adhesion points, as well as integrin α5β1 expression were analyzed. To survey applications, proliferation and migration assays were performed. Results: The FNCC-silk protein allowed efficient assembly into foam and fibers, and could even be transformed into free standing films. Human primary cells cultured on FNCC-silk showed increased attachment, spreading, stress fiber formation and focal adhesions, not only compared to RGD-silk, but also to the linear FN-silk controls. Cell binding capacity of FNCC-silk was equal to bovine fibronectin coatings, was shown to involve the α5β1 integrin, and to support proliferation and migration of keratinocytes. Figure 1. Keratinocytes after 60 min adhesion onto a surface coated with WT-silk, RGD-silk or FNCC-silk. (a) 10x magnification. Scalebar 50µm (b) Area measurement of attached cells. Figure 2. Confocal scans of keratinocytes after 3h adhesion onto FNCC-silk @63x magnification, f-actin red, dapi blue. Focal adhesions green: (left) vinculin (middle) ligand bound integrin α5 (right) activated integrin β1. When culturing keratinocytes on the free standing films of FNCC-silk a monolayer could be formed. We are currently developing novel combinations of FNCC-silk and other biomaterial candidates. Conclusion: A material based on FNCC-silk would make out a suitable candidate for coatings aiming to improve cell attachment, e.g. on stents, or in situations where cells need to be transferred as a cell sheet to e.g. a wound area. The results also suggests that FNCC-silk can efficiently attract inherent cells for migration into e.g. a wound area during healing. The Swedish Research Council; FORMAS; Knut & Alice Wallenberg Foundation; Spiber Technologies AB (for supply of soluble proteins)References:[1] Widhe et al 2013, Biomaterials 34:8223-8234 Keywords: Cell Adhesion, Biomimetic, RGD peptide, biological motif Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Cellular migration and biomaterials Citation: Widhe M, Dekki-Shalaly N and Hedhammar M (2016). A fibronectin mimicking motif enhances cell adhesive properties of spider silk. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00905 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mona Widhe Nancy Dekki-Shalaly My Hedhammar Google Mona Widhe Nancy Dekki-Shalaly My Hedhammar Google Scholar Mona Widhe Nancy Dekki-Shalaly My Hedhammar PubMed Mona Widhe Nancy Dekki-Shalaly My Hedhammar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The cell binding motif RGD is the most widely used peptide to improve cell binding properties of various biomaterials, including recombinant spider silk. In this paper we use genetic engineering to further enhance the cell supportive capacity of spider silk by presenting the RGD motif as a turn loop, similar to the one found in fibronectin (FN), but in the silk stabilized by cysteines, and therefore denoted FNCC. Human primary cells cultured on FNCC-silk showed increased attachment, spreading, stress fiber formation and focal adhesions, not only compared to RGD-silk, but also to silk fused with linear controls of the RGD containing motif from fibronectin. Cell binding to FNCC-silk was shown to involve the α5β1 integrin, and to support proliferation and migration of keratinocytes. The FNCC-silk protein allowed efficient assembly, and could even be transformed into free standing films, on which keratinocytes could readily form a monolayer culture. The results hold promise for future applications within tissue engineering.