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  • 1. Chouhan, Dimple
    et al.
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Mandal, Biman B.
    Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts2018In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, no 28, p. 23560-23572Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Horak, Josef
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Dev, Apurba
    Uppsala Univ, Ångström Lab, Solid State Elect, Uppsala Box 534, SE-75121 Uppsala, Sweden..
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Behnam, Kiarash
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Linnros, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Recombinant Spider Silk as Mediator for One-Step, Chemical-Free Surface Biofunctionalization2018In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 28, no 21, article id 1800206Article in journal (Refereed)
    Abstract [en]

    A unique strategy for effective, versatile, and facile surface biofunctionalization employing a recombinant spider silk protein genetically functionalized with the antibody-binding Z domain (Z-4RepCT) is reported. It is demonstrated that Z-silk can be applied to a variety of materials and platform designs as a truly one-step and chemical-free surface modification that site specifically captures antibodies while simultaneously reducing nonspecific adsorption. As a model surface, SiO2 is used to optimize and characterize Z-silk performance compared to the Z domain immobilized by a standard silanization method. First, Z-silk adsorption is investigated and verified its biofunctionality in a long-term stability experiment. To assess the binding capacity and protein-protein interaction stability of Z-silk, the coating is used to capture human antibodies in various assay formats. An eightfold higher binding capacity and 40-fold lower detection limit are obtained in the immunofluorescence assay, and the complex stability of captured antibodies is shown to be improved by a factor of 20. Applicability of Z-silk to functionalize microfluidic devices is demonstrated by antibody detection in an electrokinetic microcapillary biosensor. To test Z-silk for biomarker applications, real-time detection and quantification of human immunoglobulin G are performed in a plasma sample and C1q capture from human serum using an anti-C1q antibody.

  • 3.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Expanded knowledge on silk assembly for development of bioactive silk coatings2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Silk is a fascinating natural material made from proteins that self-assemble through structural rearrangements into one of the toughest materials known. As silk is protein-based, durable and elastic, it has many features that makes it suitable as a scaffold material for tissue engineering. Natural silk proteins are complex and thus difficult to produce synthetically. Therefore, partial silk proteins have been designed for production in heterologous host cells such as expression strains of Escherichia coli. This thesis presents investigations of the properties of one such partial spider silk protein, 4RepCT, and its assembly process, and describes the development of bioactive silk coatings and their properties. The focus has been to develop coatings for implant surfaces to prevent infections and improve interactions with cells.

    In Paper I, the intrinsic properties and contribution to the self-assembly process of the two protein parts 4Rep and CT were investigated separately, in a mixture (4Rep+CT) and as a fusion protein (4RepCT). The results showed that assembly occurs both at the liquid-air and liquid-solid interfaces. CT reached the interface fast but did not refold to form β-sheets, characteristic for silk, on its own. 4Rep adsorbed rapidly, and extensive intermolecular interactions were formed, although unorganized. Covalent linkage between 4Rep and CT, as in 4RepCT, and thus close proximity between the two silk parts, was found to be crucial in order to obtain both conversion into β-sheet rich structures and a nanofibrillar topography of the adsorbed proteins.

    The finding that 4RepCT self-assembles into nanofibrillar coatings on solid surfaces could be useful for various applications, for example to improve implant surfaces. The coating process was thus further evaluated in Paper II, showing that the silk coatings were chemically resistant and could also be made from silk protein variants where additional peptide motifs had been fused to 4RepCT at the genetic level. Silk with a cell-binding motif (FN-silk) and an antimicrobial peptide (Mag-silk) could assemble onto titanium, stainless steel and hydroxyapatite, respectively, materials that are commonly used for implants. Fibroblasts and endothelial cells were successfully cultured on FN-silk coatings and proliferated well. Finally, coatings of Mag-silk were evaluated for their ability to prevent adhesion of Staphylococcus aureus.

    In Paper III, silk from silkworms were used to construct materials in three different formats suitable for wound healing applications. Microporous scaffolds, electrospun mats and thin coatings of silkworm silk could all be coated with 4RepCT. They thereby gained the functions added via 4RepCT fusion proteins with a cell-binding motif (FN-silk), an antibody binding domain (Z-silk) or an enzyme (Xyl-silk). This shows upon a versatile method for functionalization of materials in different formats with bioactive motifs and domains.

    In Paper IV, the aim was to develop dual-functional silk coatings to promote osseointegration and prevent bacterial adhesion to orthopedic and dental implants. Coatings of regular silk (4RepCT) and FN-silk were given additional functions by using the transpeptidase Sortase A to mediate conjugation with the biofilm dispersal enzyme Dispersin B, or the endolysins PlySs2 and SAL-1. The obtained coatings showed a reduced adhesion of S. aureus compared to regular silk and FN-silk. Moreover, osteosarcoma cells adhered and proliferated well on coatings of FN-silk also when conjugated with enzymes.

    Altogether, the work presented in this thesis suggests that 4RepCT silk coatings are valuable as a base for construction of bioactive surfaces. The coatings can be applied on many different surfaces, and the bioactive coatings developed herein show potential for wound healing applications and prevention of biomaterial-associated infections.

  • 4.
    Nilebäck, Linnea
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Arola, Suvi
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Kvick, Mathias
    AlbaNova Univ Ctr, Spiber Technol AB, S-10691 Stockholm, Sweden..
    Paananen, Arja
    VTT Tech Res Ctr Finland Ltd, Tietotie 2, FI-02150 Espoo, Finland..
    Linder, Markus B.
    Aalto Univ, Sch Chem Engn, Dept Bioprod & Biosyst, POB 16100, FI-00076 Aalto, Finland..
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism2018In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 39, p. 11795-11805Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Nilebäck, Linnea
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Chouhan, Dimple
    Jansson, Ronnie
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Widhe, Mona
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Mandal, Biman B.
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Silk-Silk Interactions between Silkworm Fibroin and Recombinant Spider Silk Fusion Proteins Enable the Construction of Bioactive Materials2017In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 9, no 37, p. 31634-31644Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Nilebäck, Linnea
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Hedin, Jesper
    Widhe, Mona
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Floderus, Lotta S.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Krona, Annika
    Bysell, Helena
    Hedhammar, My
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study2017In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 18, no 3, p. 846-854Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Thatikonda, Naresh
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Kempe, Adam
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in Vitro Cell Culture: A Step toward Creation of Artificial ECM2018In: ACS Biomaterials Science and Engineering, ISSN 2373-9878, Vol. 4, no 9, p. 3384-3396Article in journal (Refereed)
    Abstract [en]

    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.

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