Clinical, diagnostic and immunological characteristics of patients with possible neuroborreliosis without intrathecal Ig-synthesis against Borrelia antigen in the cerebrospinal fluid

The diagnosis of neuroborreliosis is not always straightforward. Intrathecal immunoglobulin (Ig) synthesis against Borrelia antigen may not be detected, at least early in the disease course. Also other neurological and infectious diagnoses have to be considered. We have studied patients with clinical possible neuroborreliosis without intrathecal Ig synthesis against Borrelia antigen in the cerebrospinal fluid (CSF) (n=17). Diagnosis was based on typical clinical history and at least one of the following findings; mononuclear leucocytosis in the CSF (n=4); typical erythema migrans >5 cm in diameter in relation to debut of symptoms (n=8); prompt clinical response to antibiotic teratment (n=14). Also other possible diagnoses had to be excluded. Seventeen patients first investigated because of suspected neuroborreliosis but later confirmed with other diagnoses were used as controls. All patients had a lumbar puncture. Borrelia specific IFN-γ and IL-4 secretion was investigated in peripheral blood (PBL) and CSF with an ELISPOT assay. Polymerase chain reaction (PCR) was used to reveal any Borrelia antigen in the CSF. Six of 17 patients with possible neuroborreliosis showed high IFN-γ secretion in peripheral blood, otherwise we found no statistically significant differences between the groups. PCR did not reveal any Borrelia antigen in CSF. The diagnosis and treatment of possible but not confirmed neuroborreliosis is a clinical challenge. The clinical response to treatment may be the best option in these cases

Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs

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.Peer ReviewedPostprint (published version

Recombinant Spider Silk Functionalized with a Motif from Fibronectin Mediates Cell Adhesion and Growth on Polymeric Substrates by Entrapping Cells During Self-Assembly

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

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 outcome, 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

Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive andosteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano- and microporosity thatresembles natural bone and is paramount for biological processes such as protein adhesion, which can laterenhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allowcell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions throughtheir membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity andcell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell bindingmotif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds withmultiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features ofHA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration anddifferentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA inthe 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

Ultrastrong and Bioactive Nanostructured Bio-Based Composites

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 structuralcomponent 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 stiff ness 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 strengthand functionalities and bring down our dependence on fossil-based resources

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 <i>Antheraea assama</i> and <i>Bombyx mori</i> 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

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