Recombinant spider silk coatings functionalized with enzymes targeting bacteria and biofilms.

Bacteria forming biofilms on surgical implants is a problem that might be alleviated by the use of antibacterial coatings. In this article, recombinant spider silk was functionalized with the peptidoglycan degrading endolysin SAL-1 from the staphylococcal bacteriophage SAP-1 and the biofilm-matrix-degrading enzyme Dispersin B from Aggregatibacter actinomycetemcomitans using direct genetic fusion and/or covalent protein-protein fusion catalyzed by Sortase A. Spider silk assembly and enzyme immobilization was monitored using quartz crystal microbalance analysis. Enzyme activity was investigated both with a biochemical assay using cleavage of fluorescent substrate analogues and bacterial assays for biofilm degradation and turbidity reduction. Spider silk coatings functionalized with SAL-1 and Disperin B were found to exhibit bacteriolytic effect and inhibit biofilm formation, respectively. The strategy to immobilize antibacterial enzymes to spider silk presented herein show potential to be used as surface coatings of surgical implants and other medical equipment to avoid bacterial colonization.

Bioactive Silk Coatings Reduce the Adhesion of Staphylococcus aureus while Supporting Growth of Osteoblast-like Cells.

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.

Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.

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.

Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism.

The mechanism of silk assembly, and thus the cues for the extraordinary properties of silk, can be explored by studying the simplest protein parts needed for the formation of silk-like materials. The recombinant spider silk protein 4RepCT, consisting of four repeats of polyalanine and glycine-rich segments (4Rep) and a globular C-terminal domain (CT), has previously been shown to assemble into silk-like fibers at the liquid-air interface. Herein, we study the interfacial behavior of the two parts of 4RepCT, revealing new details on how each protein part is crucial for the silk assembly. Interfacial rheology and quartz crystal microbalance with dissipation show that 4Rep interacts readily at the interfaces. However, organized nanofibrillar structures are formed only when 4Rep is fused to CT. A strong interplay between the parts to direct the assembly is demonstrated. The presence of either a liquid-air or a liquid-solid interface had a surprisingly similar influence on the assembly.

Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin 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.

Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in Vitro Cell Culture: A Step toward Creation of Artificial ECM.

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.

Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials.

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.

Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study.

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.