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.

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.

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 cellbinding 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.