Characterization and Cytotoxic Activity of Microwave-Assisted Extracted Crude Fucoidans from Different Brown Seaweeds.

Microwave-assisted extraction (MAE) is recognized as a green method for extraction of natural products. The current research aimed to explore the MAE for fucoidans extraction from different brown seaweeds, including Fucus vesiculosus, F. spiralis, and Laminaria saccharina. Following several solvent-extraction pre-treatment steps and MAE optimization, the algal biomasses were extracted in a ratio of 1:25 in 0.1 M HCl containing 2 M CaCl2 for 1.0 min. The results showed that L. saccharina's extract was different from the others, regarding the highest sugar content reached 0.47 mg glucose equivalent/mg extract being confirmed by monosaccharide composition analysis and the lowest fucoidan content and sulfation degree at 0.09 mg/mg extract and 0.13, respectively. Moreover, these findings were confirmed by tentative structural elucidation based on Fourier-transform infrared spectrometry which also showed a different spectrum. However, the MAE enhanced melanoidins formation in products, which was confirmed by the intense band at 1420 cm-1. Interestingly, the results of monomeric composition showed that fucoidan extract by MAE from F. vesiculosus belonged to sulfated galactofucans which are known for their potential bioactivities. Furthermore, the cytotoxic activity of the four fucoidans in concentrations ranging from 4.9 µg/mL to 2500 µg/mL was investigated and correlated with the chemical characterization showing that F. vesiculosus_MAE fucoidan was the most potent and safest. The current research revealed the chemical heterogeneity of fucoidans regarding taxonomical class and used greener extraction method of fucoidans toward the achievement of the UN sustainability goals.

Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels.

Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

Azido-functionalized gelatin via direct conversion of lysine amino groups by diazo transfer as a building block for biofunctional hydrogels.

Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio-based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide-alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side-chain extension. Approximately 84-90% of the amino groups are modified as shown by 1 H-NMR spectroscopy, 2,4,6-trinitrobenzenesulfonic acid assay as well as Fourier-transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido-functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG-DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.

Highly Ordered Gelatin Methacryloyl Hydrogel Foams with Tunable Pore Size.

In this study, we present a fast and convenient liquid foam templating route to generate gelatin methacryloyl (GM) foams. Microfluidic bubbling was used to generate monodisperse liquid foams with bubble sizes ranging from 220 to 390 µm. The continuous phase contained 20 wt % GM and 0.7 wt % lithium phenyl-2,4,6-trimethylbenzoylphosphinate as photoinitiator. Gelation was achieved via UV-initiated radical cross-linking of GM. After cross-linking, the hydrogel foams were either swollen in water or freeze-dried. The pore sizes of the dry foams were 15-20% smaller than the bubble sizes of the liquid templates, whereas the pore sizes of the swollen porous hydrogels were in the range of the bubble sizes of the liquid templates. Compared to commonly used methods for the fabrication of biopolymer scaffolds, our route neither involves cryogenic treatment nor toxic chemicals or organic solvents and potentially allows for the photoencapsulation of cells.

Advanced formulation of methacryl- and acetyl-modified biomolecules to achieve independent control of swelling and stiffness in printable hydrogels.

Biobased hydrogels are considered to mimic native extracellular matrix due to their high water content and are considered as adequate matrices for cell encapsulation. However, the equilibrium degree of swelling (EDS) and stiffness of simple hydrogel formulations are typically confined: Increasing polymer concentration results in increasing stiffness and simultaneously decreasing EDS. The aim of this contribution was to decouple this standard correlation between polymer content, stiffness and EDS as well as the assembly of hydrogels with graded composition of hydrogels by layer-wise printing. We investigated two sets of formulations, which consisted of three different compositions with increasing total biopolymer concentration (10.6%, 11.5%, 13.0%). Within these compositions the amount of gelatin methacryloyl acetyl (GMA) was constant (10%), whereas the proportion of methacrylated hyaluronic acid and chondroitin sulfate increased. In the first set of formulations GMA with one fixed degree of methacryloylation (DM) was used, whereby the storage modulus (G') increased from ~10 to ~25 kPa and the EDS decreased from ~700 to ~600%. In the second set of formulations we gradually lowered the DM of the GMA in parallel to increase of polymer concentration and achieved an increase of both, G' from ~11 to ~18 kPa and EDS from ~690 to ~790%. By dispensing these compositions, we created a glycosaminoglycan-graded hydrogel. We proved the cytocompatibility of the dispensing process, the used photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and layer-wise UVA irradiation. Glycosaminoglycan gradient was proved stable for 28 d,encapsulated chondrocytes were viable and produced new matrix.

Physical Interactions Strengthen Chemical Gelatin Methacryloyl Gels.

Chemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking.