The recombinant functionalized silk protein FN-silk, including a cell adhesion motif from fibronectin, can form networks suitable for 3D culture of adherent cells. Such FN-silk networks have previously been shown to support the growth and differentiation of a wide array of cell types. Herein, we have developed a user-friendly methodology for the creation of free-floating FN-silk networks in 96-well plates with both mature human primary cells and stem cells. We show that human mesenchymal stem cells (hMSC) cultured in FN-silk networks form both cell-cell and cell-matrix contacts, resulting in tissue-mimicking 3D cultures. Viability and expression analysis revealed that hMSC in FN-silk networks have an initial proliferative phase with high cell viability and significantly lower hypoxia and apoptosis, compared to when cultured as scaffold-free spheroids. The FN-silk networks were shown to support differentiation of hMSC into adipocyte-like cells with well-maintained viability during the 3-week-long differentiation period, in contrast to the very poor long-term viability of scaffold-free 3D cultures. Improved adipogenesis was confirmed by lipid droplet staining, quantification of intracellular triglycerides, and secreted adiponectin levels, as well as expression analysis of multiple bona fide adipose markers. Lastly, we show that primary human hepatocytes maintain important functions and phenotypic markers when cultured in FN-silk networks, features that are lost rapidly during conventional 2D culture. We therefore propose FN-silk networks as a valuable scaffold for 3D human cell cultures, providing support for cell proliferation, differentiation, and the maintenance of critical tissue-specific functionality.

This study advances sustainable pharmaceutical research for endometriosis by developing in vitro 3D cell culture models of endometriotic pathophysiology that allow antifibrotic drug candidates to be tested. Fibrosis is a key aspect of endometriosis, yet current cell models to study it remain limited. This work aims to bridge the translational gap between in vitro fibrosis research and preclinical testing of non-hormonal drug candidates. When grown in a 3D matrix of sustainably produced silk protein functionalized with a fibronectin-derived cell adhesion motif (FN-silk), endometrial stromal and epithelial cells respond to transforming growth factor beta-1 (TGF-beta 1) in a physiological manner as probed at the messenger RNA (mRNA) level. For stromal cells, this response to TGF-beta 1 is not observed in spheroids, while epithelial cell spheroids behave similarly to epithelial cell FN-silk networks. Pirfenidone, an antifibrotic drug approved for the treatment of idiopathic pulmonary fibrosis, reverses TGF-beta 1-induced upregulation of mRNA transcripts involved in fibroblast-to-myofibroblast transdifferentiation of endometrial stromal cells in FN-silk networks, supporting pirfenidone's potential as a repurposed non-hormonal endometriosis therapy. Overall, endometrial stromal cells cultured in FN-silk networks-which are composed of a sustainably produced, fully defined FN-silk protein-recapitulate fibrotic cellular behavior with high fidelity and enable antifibrotic drug testing.

This paper presents the generation and evaluation of a novel potential drug delivery platform for biologics, based on recombinant spider silk. Targeting CD40 for activation of antigen presenting cells, in order to overcome tumor induced T cell tolerance, have shown promising results in cell and animal models. However, further trials have gained limited results due to severe side reactions. To overcome this, we have investigated a strategy for a localized CD40 activation. A CD40 agonist based on a single chain variable fragment (scFv<inf>CD40</inf>) was enzymatically coupled to silk structures, that were then used to stimulate cells in vitro. A reporter cell line responsive to CD40 agonists was used to evaluate the bioactivity of the developed scFv<inf>CD40</inf>-silk, and to optimize the method. Once the bioactivity was confirmed, human primary B cells derived from healthy donors were stimulated with the scFv<inf>CD40</inf>-silk construct. The resulting B cell response was characterized both by upregulated surface expression of the activation marker CD86 (3 fold), suggesting an improved antigen-presenting capacity, and by B cell proliferation (4 fold) generating an expanded B cell population. The detected upregulation of the costimulatory molecule CD86 on the B cells implies a potential of the functionalized silk to steer the tumor-specific T cell response from tolerance to immune activation, including the onset of appropriate effector functions. Finally, we investigated the usability of the novel silk format microspheres for CD40-mediated cell activation in vitro. Here, we were able to demonstrate that scFv<inf>CD40</inf>-coupled silk microspheres gave a pronounced activation of the CD40-expressing reporter cell line, supporting the suitability of silk microspheres for the delivery of biologics with immune modulatory purposes.

Physiologically relevant human skin models that include key skin cell types can be used forin vitrodrug testing, skin pathology studies, or clinical applications such as skin grafts. However, there is still no golden standard for such a model. We investigated the potential of a recombinant functionalized spider silk protein, FN-silk, for the construction of a dermal, an epidermal, and a bilayered skin equivalent (BSE). Specifically, two formats of FN-silk (i.e. 3D network and nanomembrane) were evaluated. The 3D network was used as an elastic ECM-like support for the dermis, and the thin, permeable nanomembrane was used as a basement membrane to support the epidermal epithelium. Immunofluorescence microscopy and spatially resolved transcriptomics analysis demonstrated the secretion of key ECM components and the formation of microvascular-like structures. Furthermore, the epidermal layer exhibited clear stratification and the formation of a cornified layer, resulting in a tight physiologic epithelial barrier. Our findings indicate that the presented FN-silk-based skin models can be proposed as physiologically relevant standalone epidermal or dermal models, as well as a combined BSE.

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.

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.

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.

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.