Fibrillar Nanomembranes of Recombinant Spider Silk Protein Support Cell Co-culture in an In Vitro Blood Vessel Wall Model.

Basement membrane is a thin but dense network of self-assembled extracellular matrix (ECM) protein fibrils that anchors and physically separates epithelial/endothelial cells from the underlying connective tissue. Current replicas of the basement membrane utilize either synthetic or biological polymers but have not yet recapitulated its geometric and functional complexity highly enough to yield representative in vitro co-culture tissue models. In an attempt to model the vessel wall, we seeded endothelial and smooth muscle cells on either side of 470 ± 110 nm thin, mechanically robust, and nanofibrillar membranes of recombinant spider silk protein. On the apical side, a confluent endothelium formed within 4 days, with the ability to regulate the permeation of representative molecules (3 and 10 kDa dextran and IgG). On the basolateral side, smooth muscle cells produced a thicker ECM with enhanced barrier properties compared to conventional tissue culture inserts. The membranes withstood 520 ± 80 Pa pressure difference, which is of the same magnitude as capillary blood pressure in vivo. This use of protein nanomembranes with relevant properties for co-culture opens up for developing advanced in vitro tissue models for drug screening and potent substrates in organ-on-a-chip systems.

Surface Functionalization of PTFE Membranes Intended for Guided Bone Regeneration Using Recombinant Spider Silk.

Alveolar bone loss is usually treated with guided bone regeneration, a dental procedure which utilizes a tissue-separation membrane. The barrier membrane prevents pathogens and epithelial cells to invade the bone augmentation site, thereby permitting osteoblasts to deposit minerals and build up bone. This study aims at adding bioactive properties to otherwise inert PTFE membranes in order to enhance cell adherence and promote proliferation. A prewetting by ethanol and stepwise hydration protocol was herein employed to overcome high surface tension of PTFE membranes and allow for a recombinant spider silk protein, functionalized with a cell-binding motif from fibronectin (FN-silk), to self-assemble into a nanofibrillar coating. HaCaT and U-2 OS cells were seeded onto soft and hard tissue sides, respectively, of membranes coated with FN-silk. The cells could firmly adhere as early as 1 h post seeding, as well as markedly grow in numbers when kept in culture for 7 days. Fluorescence and scanning electron microscopy images revealed that adherent cells could form a confluent monolayer and develop essential cell-cell contacts during 1 week of culture. Hence, functionalized PTFE membranes have a potential of better integration at the implantation site, with reduced risk of membrane displacement as well as exposure to oral pathogens.

Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.

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.

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.