Mona Widhe

Spider silk proteins: recent advances in recombinant production, structure–function relationships and biomedical applications

Spider dragline silk is an outstanding material made up of unique proteins—spidroins. Analysis of the amino acid sequences of full-length spidroins reveals a tripartite composition: an N-terminal non-repetitive domain, a highly repetitive central part composed of approximately 100 polyalanine/glycine rich co-segments and a C-terminal non-repetitive domain. Recent molecular data on the terminal domains suggest that these have different functions. The composite nature of spidroins allows for recombinant production of individual and combined regions. Miniaturized spidroins designed by linking the terminal domains with a limited number of repetitive segments recapitulate the properties of native spidroins to a surprisingly large extent, provided that they are produced and isolated in a manner that retains water solubility until fibre formation is triggered. Biocompatibility studies in cell culture or in vivo of native and recombinant spider silk indicate that they are surprisingly well tolerated, suggesting that recombinant spider silk has potential for biomedical applications.

Recombinant spider silk as matrices for cell culture

The recombinant miniature spider silk protein, 4RepCT, was used to fabricate film, foam, fiber and mesh matrices of different dimensionality, microstructure and nanotopography. These matrices were evaluated regarding their suitability for cell culturing. Human primary fibroblasts attached to and grew well on all matrix types, also in the absence of serum proteins or other animal-derived additives. The highest cell counts were obtained on matrices combining film and fiber/mesh. The cells showed an elongated shape that followed the structure of the matrices and exhibited prominent actin filaments. Moreover, the fibroblasts produced, secreted and deposited collagen type I onto the matrices. These results, together with findings of the matrices being mechanically robust, hold promise not only for in vitro cell culturing, but also for tissue engineering applications.

Current progress and limitations of spider silk for biomedical applications

Spider silk is a fascinating material combining remarkable mechanical properties with low density and biodegradability. Because of these properties and historical descriptions of medical applications, spider silk has been proposed to be the ideal biomaterial. However, overcoming the obstacles to produce spider silk in sufficient quantities and in a manner that meets regulatory demands has proven to be a difficult task. Also, there are relatively few studies of spider silk in biomedical applications available, and the methods and materials used vary a lot. Herein we summarize cell culture- and in vivo implantation studies of natural and synthetic spider silk, and also review the current status and future challenges in the quest for a large scale production of spider silk for medical applications.

Recombinant spider silk with cell binding motifs for specific adherence of cells

Silk matrices have previously been shown to possess general properties governing cell viability. However, many cell types also require specific adhesion sites for successful in vitro culture. Herein, we have shown that cell binding motifs can be genetically fused to a partial spider silk protein, 4RepCT, without affecting its ability to self-assemble into stable matrices directly in a physiological-like buffer. The incorporated motifs were exposed in the formed matrices, and available for binding of integrins. Four different human primary cell types; fibroblasts, keratinocytes, endothelial cells and Schwann cells, were applied to the matrices and investigated under serum-free culture conditions. Silk matrices with cell binding motifs, especially RGD, were shown to promote early adherence of cells, which formed stress fibers and distinct focal adhesion points. Schwann cells acquired most spread-out morphology on silk matrices with IKVAV, where significantly more viable cells were found, also when compared to wells coated with laminin. This strategy is thus suitable for development of matrices that allow screening of various cell binding motifs and their effect on different cell types.

Sterilized Recombinant Spider Silk Fibers of Low Pyrogenicity

We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.

A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices.

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.

Ultrastrong and Bioactive Nanostructured Bio-Based Composites

Natures 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.

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.

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.

Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation

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.