From Strain Stiffening to Softening—Rheological Characterization of Keratins 8 and 18 Networks Crosslinked via Electron Irradiation

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.

Multiprotein collagen/keratin hydrogel promoted myogenesis and angiogenesis of injured skeletal muscles in a mouse model

“…This properties can be related to the fact that disulfide bonds of keratin was broken following oxidative extraction of keratin and its stiffness is dependent on the side chain chemical groups. It was previously revealed that, for keratin, the strands joining between thick bundles is fragile [60], this weak coupling results more strand slipping under forces and thus does not follow strain stiffening.…”

Systematic altering of semiflexible DNA-based polymer networks via tunable crosslinking

“…This is possibly due to the fact that the GWLC model is designed to explain mutual interactions between filaments in a homogeneous, entangled network of semiflexible polymers. The limitations of the GWLC have been demonstrated in previous studies on networks of the cytoskeletal intermediate fila-ment keratin 15,24,34 and were explained with network architectures which were incompatible with the prerequisites of the theory. We suspect that the inability to account for the jump for the 6 base-pair crosslinkers in this case has a similar cause.…”

“…22 In relation to the engineering of biomaterials, chemical crosslinking, for example via electron beam radiation, can also be used to permanently alter and crosslink collagen networks. [23][24][25] Reconstituted networks of filamentous actin (F-actin) are experimentally well-characterized and were used as an ideal for the development of theoretical models. 9,12,16,21,26,27 The concentration-dependent scaling of the elastic plateau modulus, G 0 ∝ c 7/5 , for entangled F-actin solutions is theoretically described within the frame of the tube model 28 and has been experimentally verified.…”

Revisiting the strain-induced softening behaviour in hydrogels

“…Fig. 4 shows the experimental data obtained for (a) keratin gel, 52 (b) silk fibroin gel, 24 (c) neurofilament-based hydrogel, 22 Table 1 Here, n e , q c and DE are parameters obtained from a fit procedure of eqn (2) to the experimental data extracted from ref. 21, where the stress-strain of samples of PEG hydrogels with different hydrophobic ratios r are presented.…”