Abstract
Networks of crosslinked keratin filaments are abundant in epithelial cells and tissues, providing resilience against mechanical forces and ensuring cellular integrity. Although studies of in vitro models of reconstituted keratin networks have revealed important mechanical aspects, the mechanical properties of crosslinked keratin structures remain poorly understood. Here, we exploited the power of electron beam irradiation (EBI) to crosslink in vitro networks of soft epithelial keratins 8 and 18 (k8–k18) filaments with different irradiation doses (30 kGy, 50 kGy, 80 kGy, 100 kGy, and 150 kGy). We combined bulk shear rheology with confocal microscopy to investigate the impact of crosslinking on the mechanical and structural properties of the resultant keratin gels. We found that irradiated keratin gels display higher linear elastic modulus than the unirradiated, entangled networks at all doses tested. However, at the high doses (80 kGy, 100 kGy, and 150 kGy), we observed a remarkable drop in the elastic modulus compared to 50 kGy. Intriguingly, the irradiation drastically changed the behavior for large, nonlinear deformations. While untreated keratin networks displayed a strong strain stiffening, increasing irradiation doses shifted the system to a strain softening behavior. In agreement with the rheological behavior in the linear regime, the confocal microscopy images revealed fully isotropic networks with high percolation in 30 kGy and 50 kGy-treated keratin samples, while irradiation with 100 kGy induced the formation of thick bundles and clusters. Our results demonstrate the impact of permanent crosslinking on k8–k18 mechanics and provide new insights into the potential contribution of intracellular covalent crosslinking to the loss of mechanical resilience in some human keratin diseases. These insights will also provide inspiration for the synthesis of new keratin-based biomaterials.
Highlights
Filamentous biopolymers constituting the cytoskeleton and extracellular matrix (ECM) are the essential ingredients of life, maintaining cell shape and mechanically supporting cells and tissues
The linear viscoelastic properties of keratin gels can be quantified by the frequency (ω) dependent complex shear modulus G*(ω) = G (ω) + iG”(ω), where G and G” are the elastic and viscous moduli, respectively. k8–k18 filament networks at 1.5 mg/mL were irradiated at five different doses (30 kGy, 50 kGy, 80 kGy, 100 kGy, and 150 kGy)
We have shown the feasibly of electron beam irradiation (EBI) to covalently crosslink k8–k18 filament networks and investigated the rheology and architecture of the resultant crosslinked keratin gels
Summary
Filamentous biopolymers constituting the cytoskeleton and extracellular matrix (ECM) are the essential ingredients of life, maintaining cell shape and mechanically supporting cells and tissues.
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