FN-silk scaffolds as ECM & basement membrane mimics for 3D tissue engineering applications

QC 2026-03-03

Abstract

FN-silk is a recombinant functionalized silk protein shown to self-assemble into elastic fibrillar 3D matrices that favor in vivo-like cell-ECM interactions. This thesis aims to expand on the current understanding of FN-silk protein as a biomaterial for constructing such physiologically relevant ECM mimics, both of interstitial fibers and basement membrane, for tissue engineering applications. This is examined both in terms of optimizing existing and developing new methods for constructing FN-silk matrices, as well as utilizing them as scaffolds to develop in vitro models of barrier tissues.

Specifically, Paper I describes an optimized, and Paper II a newly developed, method to prepare FN-silk basement membrane mimics. Moreover, a new way of preparing FN-silk 3D networks is described (Paper III) and proposed as an ECM mimic for in vitro (Paper III) and in vivo applications (Paper IV). Both the FN-silk membrane and 3D network are used to construct in vitro skin tissue models presented in Paper III, and the FN-silk membrane alone is used to support an alveolar-capillary in vitro model (Paper V). Lastly, Paper IV describes the use of the 3D network as a dermal replacement scaffold in an in vivo porcine wound healing model.

Paper I established the parameters of preparing FN-silk membranes supported by custom-designed inserts in order to yield a reproducible membrane-insert culture system that could be seeded with cells bilaterally and further used for applications described in Papers III and V.

Paper II explored a different method of forming the FN-silk membrane, this time around hydrogels. Functionalizing hydrogels with FN-silk increased the native tissue mimicry not only by modeling the basement membrane but also by addressing key hydrogel shortcomings such as poor cell adhesion and inadequate mechanical properties as well as unfavorable cell-mediated hydrogel contraction.

Paper III presented and characterized FN-silk-based skin tissue 3D models. Primary human cells were cultured on FN-silk matrices, yielding standalone dermal and epidermal models as well as a bilayered dermal-epidermal one. The models feature dermal ECM production and formation of microvascular-like structures, as well as in vivo-like epidermal stratification and cornification.

Paper IV extended the favorable FN-silk-skin cell interactions in vivo, in a porcine wound healing model. The FN-silk 3D network was compared with a commercial dermal replacement scaffold, achieving a faster re-epithelialization rate and better mechanical properties of the treated wounds.

Lastly, Paper V compared the FN-silk membrane with a commercial synthetic one in the context of supporting an in vitro alveolar-capillary model. The FN-silk-based models attained high tissue mimicry in both morphological and functional characteristics, contrary to the commercial controls.  Notably, in Paper V an in vitro model of alveologenesis was established for the very first time. This was achieved on FN-silk-supported cultures.

Taken together, the work conducted in this thesis builds on the current knowledge of the potential of FN-silk as a biomaterial for in vitro and in vivo applications. It demonstrates that the chemically defined FN-silk-based scaffolds can be used to construct models that closely recapitulate the in vivo microenvironment, holding great promise for drug screening studies as well as platforms for understanding human physiology and disease.

Link to DiVA