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QC 20240205

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications., QC 20240703

QC 20230123

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, fi-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of similar to 7 nm and a width of similar to 11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces., QC 20230425

The blood-brain barrier (BBB) limits the uptake of central nervous system (CNS)-targeting drugs into the brain. Engineering molecular shuttles for active transportation across the barrier has thus potential for improving the efficacy of such drugs. In vitro assessment of potential transcytosis capability for engi-neered shuttle proteins facilitates ranking and the selection of promising candidates during develop-ment. Herein, the development of an assay based on brain endothelial cells cultured on permeable recombinant silk nanomembranes for screening of transcytosis capability of biomolecules is described. The silk nanomembranes supported growth of brain endothelial cells to form confluent monolayers with relevant cell morphology, and induced expression of tight-junction proteins. Evaluation of the assay using an established BBB shuttle antibody showed transcytosis over the membranes with an apparent permeability that significantly differed from the isotype control antibody., Not duplicate with DiVA 1740044QC 20230706

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge., QC 20220110

QC 20220322

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., QC 20221116

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10−23 and 2*10−13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys, QC 20220621

Objective. The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. Methods. More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. Results. Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. Conclusion. These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay., QC 20210816

The extent that antibodies to SARS-CoV-2 may protect against future virus-associated disease is unknown. We invited all employees (n=15,300) at work at the Karolinska University Hospital, Stockholm, Sweden to participate in a study examining SARS-Cov-2 antibodies in relation to registered sick leave. For consenting 12,928 healthy hospital employees antibodies to SARS-CoV-2 could be determined and compared to participant sick leave records. Subjects with viral serum antibodies were not at excess risk for future sick leave (adjusted odds ratio (OR) controlling for age and sex: 0.85 [95% confidence interval (CI) (0.85 (0.43-1.68)]. By contrast, subjects with antibodies had an excess risk for sick leave in the weeks prior to testing [adjusted OR in multivariate analysis: 3.34 (2.98-3.74)]. Thus, presence of viral antibodies marks past disease and protection against excess risk of future disease. Knowledge of whether exposed subjects have had disease in the past or are at risk for future disease is essential for planning of control measures.Trial registration: First registered on 02/06/20, ClinicalTrials.gov NCT04411576., QC 20210419

We show for the first time that 470 nm thick spider silk membranes support co-culturing of endothelial (HDMEC) and smooth muscle cells (SMC). These cell-silk-cell constructs mimic the wall of small blood vessels. The silk membranes are formed through self-assembly at the liquid:air interface of a standing solution of spider silk protein. We show that the silk membranes enable communication between the cells better than commercial tissue culture inserts (TC-inserts)., Part of ISBN 9781733419031QC 20230626

SARS-CoV-2 may pose an occupational health risk to healthcare workers. Here, we report the seroprevalence of SARS-CoV-2 antibodies, self-reported symptoms and occupational exposure to SARS-CoV-2 among healthcare workers at a large acute care hospital in Sweden. The seroprevalence of IgG antibodies against SARS-CoV-2 was 19.1% among the 2149 healthcare workers recruited between April 14th and May 8th 2020, which was higher than the reported regional seroprevalence during the same time period. Symptoms associated with seroprevalence were anosmia (odds ratio (OR) 28.4, 95% CI 20.6-39.5) and ageusia (OR 19.2, 95% CI 14.3-26.1). Seroprevalence was also associated with patient contact (OR 2.9, 95% CI 1.9-4.5) and covid-19 patient contact (OR 3.3, 95% CI 2.2-5.3). These findings imply an occupational risk for SARS-CoV-2 infection among healthcare workers. Continued measures are warranted to assure healthcare workers safety and reduce transmission from healthcare workers to patients and to the community., QC 20201123

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers., QC 20200819

The composition of serum proteins is reflecting the current health status and can, with the right tools, be used to detect early signs of disease, such as an emerging cancer. An earlier diagnosis of cancer would greatly increase the chance of an improved outcome for the patients. However, there is still an unmet need for proficient tools to decipher the information in the blood proteome, which calls for further technological development. Here, we present a proof-of-concept study that demonstrates an alternative approach for multiplexed protein profiling of serum samples in solution, using DNA barcoded scFv antibody fragments and next generation sequencing. The outcome shows high accuracy when discriminating samples derived from pancreatic cancer patients and healthy controls and represents a scalable alternative for serum analysis. Brofelth, Ekstrand et al use DNA barcoded scFv antibody fragments and next generation sequencing for multiplex profiling of proteins in serum from pancreatic cancer patients with high accuracy. This approach can potentially be used in high throughput precision diagnosis., QC 20200820

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., QC 20200702

Three-dimensional (3D) neural tissue cultures recapitulate the basic concepts during development and disease better than what can be obtained using conventional two-dimensional cultures. Here, we use a recombinant spider silk protein functionalized with a cell binding motif from fibronectin (FN-silk) in combination with a human recombinant laminin 521 (LN-521) to create a fully defined stem cell niche in 3D. A novel method to assemble silk blended with LN-521 together with human pluripotent stem cells (hPSC) is used to create centimeter-sized foams, which upon cultivation develop into 3D cell constructs supported by a microfibrillar network. After initial cell expansion, neural differentiation was induced to form a homogenous layer of continuous neuroectodermal tissue that allows further differentiation into neuronal subtypes. The silk-supported 3D cell constructs could then be detached from the bottom of the well and cultured as floating entities, where cells appeared in distinctive radial organization resembling early neural tube. This shows that the neural progenitors retain their cellular self-organization ability in the FN-silk/LN-521-supported 3D culture. Calcium imaging demonstrated spontaneous activity, which is important for the formation of neuronal networks. Together, the results show that hPSCs integrated into FN-silk/LN-521 foam develop into neural progenitors and that these stay viable during long-term differentiations. FN-silk/LN-521 also supports morphogenesis mimicking the human brain development and can serve as base for engineering of hPSC-derived neural tissue., QC 20200603

Pancreatic islet transplantation has not yet succeeded as an overall treatment for type 1 diabetes because of limited access to donor islets, as well as low efficacy and poor reproducibility of the current procedure. Herein, a method to create islets-like composite clusters (coclusters) from dispersed endocrine cells and supportive cells is described, attempting to improve compatibility with the recipient and more efficiently make use of the donor-derived material. To mimic the extracellular matrix environment, recombinant,spider silk functionalized with cell binding motifs are used as 3D support for the coclusters. A cell binding motif derived from fibronectin (FN) was found superior in promoting cell adherence, while a plain RGD-motif incorporated in the repetitive part of the silk protein (2R) increased the mobility and cluster formation of endocrine cells. Self-assembly of a mixture of FN/2R silk is utilized to integrate endocrine cells together with endothelial and mesenchymal cells into islet-like coclusters. Both xenogenic and allogenic versions of these coclusters were found to be viable and were able to respond to dynamic glucose stimulation with insulin release. Moreover, the endothelial cells were found to be colocalized with the endocrine cells, showing that the silk combined with supportive cells may promote vascularization. This method to engineer combined islet-like coclusters allows donor-derived endocrine cells to be surrounded by supportive cells from the recipient, which have the potential to further promote engraftment in the host and considerably reduce risk of rejection., QC 20200325

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers., QC 20200819

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers., QC 20200819

In this work we present three different ways to characterize the mechanical properties of spider silk nanomembranes. The nanomembranes are formed by self-assembly at the liquid:air interface of a standing solution from which they can be lifted. The mechanical properties are evaluated by (1) manually dropping lead bullets onto the nanomembrane, (2) motorized lowering of a cylindrical indenter to record force-deformation characteristics, and (3) using a standard bulging experiments. Using these methods we show that the nanomembranes are both strong and flexible opening up for applications as pneumatic actuators in MEMS microvalves, or as cell layer actuators in organ-on-a-chip., QC 20220301

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers., QC 20200819

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers., QC 20200819

QC 20190918

Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-Z(VEGFR2)) was formatted into a tetrameric construct (tetra-Z(VEGFR2)) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-Z(VEGFR2) induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-Z(VEGFR2). In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-Z(VEGFR2) silk and tetra-Z(VEGFR2)-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-Z(VEGFR2) -silk and tetra-Z(VEGFR2) -silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-Z(VEGFR2)-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended., QC 20200108

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 (silk(wt)) 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 silk(wt) 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., QC 20190809

Full-thickness cutaneous wounds, such as deep burns, are complex wounds that often require surgical interventions. Herein, we show the efficacy of acellular grafts that can be made available off-the-shelf at an affordable cost using silk biomaterials. Silkworm silk fibroin (SF), being a cost-effective and natural biopolymer, provides essential features required for the fabrication of three-dimensional constructs for wound-healing applications. We report the treatment of third-degree burn wounds using a freeze-dried microporous scaffold of Antheraea assama SF (AaSF) functionalized with a recombinant spider silk fusion protein FN-4RepCT (FN-4RC) that holds the fibronectin cell binding motif. In order to examine the healing efficiency of functionalized silk scaffolds, an in vivo burn rat model was used, and the scaffolds were implanted by a one-step grafting procedure. The aim of our work is to investigate the efficacy of the developed acellular silk grafts for treating full-thickness wounds as well as to examine the effect of recombinant spider silk coatings on the healing outcomes. Following 14-day treatment, AaSF scaffolds coated with FN-4RC demonstrated accelerated wound healing when compared to the uncoated counterpart, commercially used DuoDERM dressing patch, and untreated wounds. Histological assessments of wounds over time further confirmed that functionalized silk scaffolds promoted wound healing, showing vascularization and re-epithelialization in the initial phase. In addition, higher extent of tissue remodeling was affirmed by the gene expression study of collagen type I and type III, indicating advanced stage of healing by the silk treatments. Thus, the present study validates the potential of scaffolds of combined silkworm silk and FN-4RC for skin regeneration., QC 20191002

Complex cutaneous wounds like diabetic foot ulcers represent a critical clinical challenge and demand a large-scale and low-cost strategy for effective treatment. Herein, we use a rabbit animal model to investigate efficacy of bioactive wound dressings made up of silk biomaterials. Nanofibrous mats of Antheraea assama silkworm silk fibroin (AaSF) are coated with various recombinant spider silk fusion proteins through silk-silk interactions to fabricate multifunctional wound dressings. Two different types of spider silk coatings are used to compare their healing efficiency: FN-4RepCT (contains a cell binding motif derived from fibronectin) and Lac-4RepCT (contains a cationic antimicrobial peptide from lactoferricin). AaSF mats coated with spider silk show accelerated wound healing properties in comparison to the uncoated mats. Among the spider silk coated variants, dual coating of FN-4RepCT and Lac-4RepCT on top of AaSF mat demonstrated better wound healing efficiency, followed by FN-4RepCT and Lac-4RepCT single coated counterparts. The in vivo study also reveals excellent skin regeneration by the functionalized silk dressings in comparison to commercially used Duoderm dressing and untreated wounds. The spider silk coatings demonstrate early granulation tissue development, re-epithelialization, and efficient matrix remodelling of wounds. The results thus validate potential of bioactive silk matrices in faster repair of diabetic wounds., QC 20190802

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., QC 20190624

QCR 20210113

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., QC 20180628

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., QC 20181030

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., QC 20180808

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., QC 20180927

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., QC 20180529

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., QC 20180529

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., QC 20180529

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., QC 20180529

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., QC 20180529

QC 20210113

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., QC 20171017

QCR 20210113

QCR 20210113

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed., QC 20201020

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein., QC 20201020

QC 20231115

QC 20231115

Future success of cell therapy for type 1 diabetes (T1D) relies on robust protocols for the differentiation of stem cells to pancreatic islets. However, currently established protocols generate stem cell-derived pancreatic islets (SC-islets) which contain non-endocrine cell populations and have immature functionality. Herein we report on the development of a new and robust protocol for the generation of potent SC-islets enriched in functional endocrine cells from all 8 tested human pluripotent stem cell (hPSC) lines. We show that differentiation to the endocrine progenitor (EP)stage on 2D is significantly enhanced by laminin-521, and a short duration of the previous pancreatic progenitor (PP) stage. Importantly, we find that self-aggregation of EP cells greatly reduces unwanted proliferative and non-endocrine cells to a minimal level. Subsequent suspension cultures generate mono-hormonal SC-islets with strong in vitro glucose responsiveness, similar to human islets. Upon transplantation into the anterior chamber of the eye (ACE) of a diabetic mouse model, the SC-islets undergo further maturation in vivo and re-establish normal glycemic control. The SC-islets before and after transplantation are to a major extent free of non-endocrine cell populations. In conclusion, our novel differentiation protocol results in pure and efficiently functioning SC-islets that should be well suited for transplantation into patients with T1D., QC 20240827

Type 1 diabetes is a life-threatening disease characterized by lifelong insulin dependency and a reduced quality of life. Pancreatic islet transplantation is a promising treatment but is limited by donor shortages and significant islet loss during the procedures. The usage of pancreatic islet-like aggregates derived from pluripotent stem cells (SC-islets) could solve the problem of shortage of islets. Incorporating these SC-islets into a supporting scaffold could protect them during handling.FN-silk, a recombinantly produced spider silk protein functionalized with a cell adhesion motif from fibronectin, can generate 3D networks that mimic the extracellular matrix (ECM). We herein demonstrate a reproducible method for integrating SC-islets well-distributed within stable 3D networks of FN-silk. These SC-islets showed high viability and maintained functionality, with increased glucagon expression and improved beta cell maturation as compared to free SC-islets. Additionally, the support of FN-silk networks enables cultivation of SC-islets under conditions needed for scale-up. Moreover, the integration of the SC-islets into sheets of FN-silk networks facilitates handling during transplantation into the anterior chamber of the eye (ACE) of rabbits. Such transplanted FN-silk incorporated SC-islets were found vascularized six weeks post-transplantation, with sustained insulin and glucagon expression., QC 20240827

Cryopreservation is crucial for making cell therapies more accessible by enabling long-term storage and facilitating transplantation logistics. However, preserving cells in 3D models implies significant challenges, including inhomogeneous penetration of cryoprotectants and non-uniform heat transfer during freezing and thawing. Vitrification is a novel cryopreservation approach that improves the survival of living cells by providing optimal cooling and rewarming rates to eliminate ice crystal-induced mechanical injury. Nevertheless, sterility issues can arise from direct contact with liquid nitrogen (LN2). The twisted vitrification method addresses this by separating the cells and LN2 into two different compartments, ensuring a sterile vitrification process.In this study, we optimized the cryopreservation of stem cell-derived pancreatic islets (SC-islets) by combining twisted vitrification with the supporting biomaterial FN-silk. We compared the viability of free SC-islets to those supported by a FN-silk network during cryopreservation using either conventional slow freezing or twisted vitrification. Our results showed a higher post-recovery for SC-islets incorporated in FN-silk using twisted vitrification, outperforming both conventional slow freezing and twisted vitrification of free SC-islets. Additionally, insulin and glucagon expression were maintained after vitrification within the FN-silk network., QC 20240827