Recombinant Spider Silk Forms Tough and Elastic Nanomembranes that are Protein‐Permeable and Support Cell Attachment and Growth

Abstract

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.

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

“…The structural rearrangement of silk proteins to form a nanofibrillar membrane corresponds to a continuous reduction of α-helices in favor of increased β-sheet conformations. 34 The content of β-sheet formation has previously been reported to account for the extensibility of silk proteins, 35 as well as the unfolding and elasticity of, for example, fibrin. 36 The membranes can be lifted from the interface by lowering the custom-made 3D-printed holder ( Figure S1 ) and allowing the membrane to detach from the walls of the well and instead adhere to the holder.…”

“…The thickness of the membrane can be altered by varying the silk concentration or the assembly time. 34 Noteworthily, the thickness of the silk membranes increases over time, from 470 ± 110 nm at day 0 to 690 ± 150 nm at day 7, by keeping them submerged in cell culture media ( Figure S2 ). The serum and growth factor components in the media adsorb onto each side of the silk membranes, thereby adding particular bioactive properties.…”

“… 20 Noteworthily, the two sides of the membrane have different appearances; while the side facing air during formation (from here on the air-side) is smooth, the side facing the solution during formation (from here on the liquid-side) is textured from silk aggregates ( Figure 2 b–c). Despite their difference in appearance, both sides support cell attachment and growth, 34 thereby making them suitable for co-culture applications.…”

“… 33 Yet, recombinant spider silk proteins have recently been shown to form thinner (250 nm) and bioactive membranes that are permeable to human plasma proteins, are mechanically robust, and support the formation of confluent monolayers of epidermal skin cells (keratinocytes). 34 Herein, we report on recombinant spider silk nanomembranes able to support a cell co-culture into an in vitro blood vessel wall model ( Figure 1 ).…”

Progress in the application of spider silk protein in medicine

“…102 What's more, a novel and simple method to produce silk came true by forming a film in air-liquid interface. 105,106 At the same time, the postprocessing of spider silk can also improve the performance of spider silk to a certain extent. 107 Studies on the forms of spider silk protein showed that controlled and characteristic carriers can be obtained by making spider silk into spheres, 108 which can be made into microspheres, 109,110 capsules, 30 particles, 27 foam, 111 films and hydrogels.…”