Skin substitutes based on gellan gum with mechanical and penetration compatibility to native human skin.

The study reports on a simple system to fabricate skin substitutes consisting of a naturally occurring bacterial polysaccharide gellan gum. Gelation was driven by the addition of a culture medium whose cations induced gellan gum crosslinking at physiological temperature, resulting in hydrogels. Human dermal fibroblasts were incorporated in these hydrogels and their mechanical, morphological, and penetration characteristics were studied. The mechanical properties were determined by means of oscillatory shear rheology, and a short linear viscoelastic regime was noted up to less than 1% of strain amplitude. The storage modulus increased with an increasing polymer concentration. The moduli were in the range noted for native human skin. After 2 weeks of fibroblast cultivation, the storage moduli showed signs of deterioration, so that a culture time of 2 weeks was proposed for further studies. Microscopic and fluorescent staining observations were documented. These depicted a crosslinked network structure in the hydrogels with a homogeneous distribution of cells and an assured cell viability of 2 weeks. H&E staining was also performed, which showed some traces of ECM formation in a few sections. Finally, caffeine penetration experiments were carried out with Franz diffusion cells. The hydrogels with a higher concentration of polymer containing cells showed an improved barrier function against caffeine compared to previously studied multicomponent hydrogels as well as commercially available 3D skin models. Therefore, these hydrogels displayed both mechanical and penetration compatibility with the ex vivo native human skin.

Antimicrobial and physicochemical characterization of 2,3-dialdehyde cellulose-based wound dressings systems.

Biobased and biodegradable films were prepared by physically mixing 2,3-dialdehyde cellulose (DAC) with two other biopolymers, zein and gelatin, in three different proportions. The antimicrobial activities of the composite blends against Gram-positive and Gram-negative bacteria increase with the increase of DAC content. Cell viability tests on mammalian cells showed that the materials were not cytotoxic. In addition, DAC and gelatin were able to promote thermal degradation of the blends. However, DAC increased the stiffness and decreased the glass transition temperature of the blends, while gelatin was able to decrease the stiffness of the film. Morphological analysis showed the effect of DAC on the surface smoothness of the blends. The contact angle confirmed that all blends were within the range of hydrophilic materials. Although all the blends showed impressive performance for wound dressing application, the blend with gelatin might be more suitable for this purpose due to its better mechanical performance and antibacterial activity.

Polysaccharide-based skin scaffolds with enhanced mechanical compatibility with native human skin.

We report a custom-made technique to synthesize process-convenient skin scaffolds by tuning the mechanical properties of hydrogels based on a few naturally occurring polysaccharides to match the rheological properties of previously established benchmarks, i.e., the ex vivo native human skins. We studied the mechanical parameters using oscillatory shear rheology. At small strain amplitudes, the intrinsic elastic modulus showed an almost linear dependence in the middle and a changing rate profile at the two ends with concentration of the principal hydrogel component variant, i.e., kappa (κ)-carrageenan. At large strain amplitudes, the hydrogels demonstrated intercycle strain-softening behavior, the onset of which was directly proportional to the κ-carrageenan concentration. We observed a concentration match for the intrinsic elastic modulus of the benchmark within this sigmoidal curve fit. Contextually, we need to explore other potent polymeric hydrogel systems to achieve mechanical affinity in terms of multiple rheological parameters derived from both strain amplitude and angular frequency sweeps. Additionally, we carried out diffusion experiments to study caffeine permeation attributes. The hydrogels show improved barrier features with increasing κ-carrageenan concentration. In terms of the penetration flux and total cumulative amount of permeated caffeine, this enhanced mechanical adherence demonstrates comparable penetration features with the commercial 3D skin model.

Custom-made rheological setup for in situ real-time fast alginate-Ca2+ gelation.

There is a growing interest in the in situ gelation of the alginate-Ca2+ system due to its remarkable applications. In this work, we record and evaluate the fast gelation kinetics of alginate-Ca2+ using a custom-made rheometric setup. This enables us to inject CaCl2 into the alginate while we perform the rheological measurements. We successfully measure the in situ gelation reaction from the early stages. As the alginate concentration is increased up to 3 wt.%, we observe a systematic increase of the elastic modulus, G'. Similarly, higher concentrations and injected volumes of CaCl2 increase the magnitude and initial growth rate of G'. At longer times, the growth rate of G' is lower. It decreases further very slowly, indicating that the chemical reaction requires quite a considerable amount of time to be completed. Finally, from the rheometric data, we estimate the average rates of the elastic modulus during the initial and quasi-steady-state stages.

Nonlinear rheological behavior of gelatin gels: In situ gels and individual gel layers filled with hard particles.

HYPOTHESIS: Previously, we examined the impact of two preparation procedures on the mechanical properties of native gelatin gels. In this work, we extend our research by considering hard spherical particles as fillers. We expect that the presence of these fillers significantly affects the viscoelastic response. EXPERIMENTS: We prepared fresh in situ gels and individual gel layers filled with glass micro-beads up to 1% w/w and aged for 24 and 48 h. The hydrogels were made of 3 and 5% w/w gelatin at 5 °C, and we performed large amplitude oscillatory shear (LAOS) tests. We analyzed the intracycle linear and nonlinear response using normalized Lissajous-Bowditch curves. Utilizing the MITlaos software, we decomposed the total intracycle stress into elastic and viscous contributions and calculated the Chebyshev harmonics coefficients. FINDINGS: The fresh in situ gels exhibit severe progressive stiffening during the strain sweeps and a subsequent sharp decrease of both moduli. The filled layers show smoother yielding than the in situ gels. The fillers increase the dynamic moduli, affect the terminal LAOS regime, and enhance the intracycle nonlinearities at higher concentrations. The Lissajous-Bowditch curves of the aged layers indicate elastoplastic behavior, which is more pronounced for the 48 h filled gel layer than the native counterpart.

Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi.

It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

Nonlinear rheological behavior of gelatin gels: In situ gels and individual layers.

The gelation procedure and the gelation time of gelatin gels may lead to apparently similar materials, however, with different rheological fingerprints under small and large oscillatory shear deformations. Here, in the first paper of this series, gelation of 3 and 5% w/w gelatin solutions at 5 °C was performed in situ on the rheometer plate and in custom-made casting modules to obtain individual gel layers. Large amplitude oscillatory shear (LAOS) tests were performed. The nonlinear deformation regime was qualitatively analyzed using normalized Lissajous-Bowditch curves. The MITlaos software was employed to decompose the total intracycle stress response and to calculate the Chebyshev coefficients ratios. The dynamic moduli of the fresh gels were measured directly after the in situ preparation and within a time frame until 1.5 h. In the strain sweeps, we observed intense stiffening followed by yielding above 200% strain. However, the individual gel layers aged for 24 and 48 h show different LAOS fingerprints. The extensive loops in the viscous Lissajous-Bowditch curves indicate an elastoplastic material response. Based on the overall nonlinear rheological response, we propose a structural formation that describes the behavior of the gels for the conditions studied here. In the second paper of this series, we give the impact of hard micro-fillers (glass beads) and we describe the nonlinear characteristics of the filled gels.

Nonlinear viscoelastic properties of native male human skin and in vitro 3D reconstructed skin models under LAOS stress.

This work discusses the first set of rheometric measurements carried out on commercially accessible juvenile and aged skin models under large amplitude oscillatory shear deformations. The results were compared with those of native male whole human and dermis-only foreskin specimens, catering to a few ages from 0.5 to 68 years, including specimens from a 23-year-old male abdomen. At large strains, strain thinning was more pronounced for the dermis of the young skins and for their whole skin counterparts. An inverse qualitative tendency was observed for the adult skins and the skin models. This can be explained by the high dermal collagen compactness associated with an incomplete epidermal proliferation. The qualitative Lissajous plots as well as the quantitative dimensionless indices analyzed using the MITlaos software indicated predominant nonlinear intracycle elastic strain stiffening and viscous shear thinning for all the native specimens at the maximum deformation. For the full thickness models, we have evidence of structure collapse and yielding under similar conditions. The whole skin specimen from the 68-year-old male showed smaller age-dependent nonlinear elastic contributions than the dermis, which we relate to the epidermal degeneration taking place during aging. Regardless of the age group, the models manifested more pronounced intercycle and intracycle elastic nonlinearities, and their magnitudes were significantly larger. The nonlinear elastic trends will serve as advanced standards for understanding and delineating the mechanical limits of destructive and non-destructive deformations of such unique biomaterials.

Shear Banding in 4:1 Planar Contraction.

We study shear banding in a planar 4:1 contraction flow using our recently developed two-fluid model for semidilute entangled polymer solutions derived from the generalized bracket approach of nonequilibrium thermodynamics. In our model, the differential velocity between the constituents of the solution allows for coupling between the viscoelastic stress and the polymer concentration. Stress-induced migration is assumed to be the triggering mechanism of shear banding. To solve the benchmark problem, we used the OpenFOAM software package with the viscoelastic solver RheoTool v.2.0. The convection terms are discretized using the high-resolution scheme CUBISTA, and the governing equations are solved using the SIMPLEC algorithm. To enter into the shear banding regime, the uniform velocity at the inlet was gradually increased. The velocity increases after the contraction due to the mass conservation; therefore, shear banding is first observed at the downstream. While the velocity profile in the upstream channel is still parabolic, the corresponding profile changes to plug-like after the contraction. In agreement with experimental data, we found that shear banding competes with flow recirculation. Finally, the profile of the polymer concentration shows a peak in the shear banding regime, which is closer to the center of the channel for larger inlet velocities. Nevertheless, the increase in the polymer concentration in the region of flow recirculation was significantly larger for the inlet velocities studied in this work. With our two-fluid finite-volume solver, localized shear bands in industrial applications can be simulated.

Linear viscoelastic and microstructural properties of native male human skin and in vitro 3D reconstructed skin models.

The study reports first ever account of measurements of linear viscoelastic moduli under small amplitude oscillatory shear deformations, for commercially available juvenile and aged in vitro 3D reconstructed skin models. The results were compared with those of native male whole human and dermis-only foreskin samples, catering to a wide age group from 0.5 to 68 years, including samples from a 23-year-old male abdomen. In the strain sweep tests, the dermis of the juvenile/young age group assumed a higher intrinsic elastic modulus than the whole skin. A reverse qualitative trend was noted for the adult/aged age group. Confirmed by the histological examination of the stained cross-sections, this is attributed to the nascent epidermal differentiation and the high fiber density of dermal collagen. The oscillation frequency sweeps exposed a greater dependence of the elasticity on the frequency for the native male dermis foreskin samples as compared to the whole skins, irrespective of age. This is anticipated since the extremely structured epidermis confers higher resistance to the whole skins towards intracycle deformations compared to the dermis, thereby storing smaller elastic energy. The 3D skin models examined in this work exhibited a broader linear viscoelastic region, a larger viscoelasticity, and much higher dynamic moduli, compared to the native skin. The rheological trends are a significant addition to the literature and may be used as a reference for the design of next generation of scaffolds.