Alginate-Lysozyme Nanofibers Hydrogels with Improved Rheological Behavior, Printability and Biological Properties for 3D Bioprinting Applications.

In this study, alginate nanocomposite hydrogel bioinks reinforced with lysozyme nanofibers (LNFs) were developed. Alginate-LNF (A-LNF) suspensions with different LNF contents (1, 5 and 10 wt.%) were prepared and pre-crosslinked with 0.5% (w/v) CaCl2 to formulate A-LNF inks. These inks exhibit proper shear-thinning behavior and good recovery properties (~90%), with the pre-crosslinking step playing a crucial role. A-LNF fully crosslinked hydrogels (with 2% (w/v) CaCl2) that mimic 3D printing scaffolds were prepared, and it was observed that the addition of LNFs improved several properties of the hydrogels, such as the morphology, swelling and degradation profiles, and mechanical properties. All formulations are also noncytotoxic towards HaCaT cells. The printing parameters and 3D scaffold model were then optimized, with A-LNF inks showing improved printability. Selected A-LNF inks (A-LNF0 and A-LNF5) were loaded with HaCaT cells (cell density 2 × 106 cells mL-1), and the cell viability within the bioprinted scaffolds was evaluated for 1, 3 and 7 days, with scaffolds printed with the A-LNF5 bioink showing the highest values for 7 days (87.99 ± 1.28%). Hence, A-LNF bioinks exhibited improved rheological performance, printability and biological properties representing a good strategy to overcome the main limitations of alginate-based bioinks.

Multifunctional nanofibrous patches composed of nanocellulose and lysozyme nanofibers for cutaneous wound healing.

Cutaneous wounds frequently require the use of patches to promote healing, nevertheless, most commercial products are fabricated with non-biodegradable synthetic substrates that pose environmental problems upon disposal. Herein, the partnership between two biobased nanofibrous polymers, namely a polysaccharide (nanofibrillated cellulose (NFC)) and a protein (lysozyme nanofibers (LNFs)), is explored to design sustainable fibrous patches with good mechanical performance and biological functionalities for wound healing applications. Two patches with different morphologies were prepared by vacuum filtration of a water-based suspension of both nanofibers and by sequential filtration of the separated suspensions (layered patch). The resultant freestanding patches exhibited high thermal stability (up to 250 °C), mechanical performance (Young's modulus ≥3.7 GPa), and UV-barrier properties. The combination of the bioactive LNFs with the mechanically robust NFC conveyed antioxidant activity (76-79% DPPH scavenging) and antimicrobial activity against Staphylococcus aureus (3.5-log CFU mL-1 reduction), which is a major benefit to prevent microbial wound infections. Moreover, these patches are biocompatible towards L929 fibroblast cells, and the in vitro wound healing assay evidenced a good migration capacity leading to an almost complete wound occlusion. Therefore, the partnership between the two naturally derived nanofibrous polymers represents a potential blueprint to engineer sustainable multifunctional patches for cutaneous wound healing.

Tuning lysozyme nanofibers dimensions using deep eutectic solvents for improved reinforcement ability.

Deep eutectic solvents (DESs), a novel generation of solvents, have recently been described as efficient and timesaving fibrillation agents for proteins. In this context, the present work aims at assessing the effect of the hydrogen bond donor (HBD) of cholinium chloride ([Ch]Cl):carboxylic acid based DESs on the dimensions (length and width) of lysozyme nanofibers (LNFs). Mono-, di- and tri-carboxylic acids (acetic, lactic, levulinic, malic and citric acids) were used to prepare different DES formulations, which were successfully used on the fibrillation of lysozyme. The results showed that the carboxylic acid (i.e. the HBD) plays an important role on the fibrillation efficiency and on the length of the ensuing LNFs with aspect-ratios always higher than those obtained by fibrillation with [Ch]Cl. The longest LNFs were obtained using lactic acid as the HBD with an average length of 1004 ±â€¯334 nm and width of 31.8 ±â€¯6.8 nm, and thus an aspect-ratio of ca. 32. The potential of these protein nanofibers as reinforcing additives was evaluated by preparing pullulan (PL)-based nanocomposite films containing 5% LNFs with different aspect-ratios, resulting in highly homogenous and transparent films with improved mechanical performance.