κ carrageenan

Collagen was purchased from Chrono-log (Havertown, PA, USA). ASA, Fura-2/AM, κ-carrageenan, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ATP Assay Kit was obtained from Biomedical Research Service Center (Buffalo, NY, USA). Fibrinogen Alexa Fluor 488 conjugate was obtained from Molecular Probes (Eugene, OR, USA). Antibodies against phospho-p44/42, p44/42, phospho-p38, p38, phospho-SAPK/JNK, SAPK/JNK, phospho-Akt, and Akt were acquired from Cell Signaling Technology (Beverly, MA, USA). HPLC-grade reagents, acetonitrile, and water were obtained from J. T. Baker (Phillipsburg, NJ, USA). All chemicals were of reagent-grade.

Calcium chloride hydrate and κ-carrageenan were purchased from Sigma Aldrich (St. Louis, MO, USA). Cerium oxide nanopowder (CeO₂ NPs) was bought from US Research Nanomaterials. Glutaraldehyde 25% solution in water was supplied from Titrachem. HCl fuming 37% also was purchased from Merck (Rahway, NJ, USA). The chemicals used were all analytical grade.

LDL was isolated from human plasma obtained from DRK-Blutspendedienst NSTOB, Springe, Germany (All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee of the Hannover Medical School. All donors signed the informed consent) and oxidized with CuSO₄ as previously described^{38 (link),39 (link)}. oxLDL was characterized by the protein concentration measured by DC-protein assay (Biorad, Munchen, Germany) and lipid peroxidation measured by the TBARS assay (Cayman chemicals, Michigan, USA). oxLDL concentration of 0.43 µM/mg protein (~10 ug/mL protein), 0.86 µM/mg protein (~20 ug/mL protein) and 1 µM/mg protein (~25 ug/mL protein) was used. Stimulation of the osteoclast with oxLDL and inhibition of the several receptors (CD36, LOX-1, TLR-4 and SRA-1) were all done at the beginning of osteoclast differentiation i.e. when RANKL was added. CD36, LOX-1, TLR-4 and SR-A were inhibited by 10 µM of sulfosuccinyloleate (SSO) (Cayman chemicals, Michigan, USA), 250 µM of κ-Carrageenan (Sigma, Steinheim, Germany), 5 µM of CLI-095 (Invivo-Gen) and 100 µg/mL of dextran sulphate (Sigma, Steinheim, Germany) respectively.

The κ-Carrageenan, calcium carbonate (CaCO₃), Sodium carboxymethyl cellulose (NaCMC), (+)-Catechin hydrate, Zinc nitrate hexahydrate, Magnesium nitrate hexahydrate, and gelatin, which have been used in this study were procured from Sigma-Aldrich (Jefferson City, MS, USA). All these materials were applied as received without any additional purification. Distilled water was applied in the synthesis of the hydrogel.

κ-Carrageenan and lipoxygenase type I-B from soybean were purchased from Sigma (St. Louis, MO, U.S.A.). For the in vivo experiments, Wistar rats (180–220 g, 3–4 months old) were kept in the Centre of the School of Veterinary Medicine (EL54 BIO42), Aristotelian University of Thessaloniki, which is registered by the official state veterinary authorities (presidential degree 56/2013, in harmonization with the European Directive 2010/63/EEC). The experimental protocols were approved by the Animal Ethics Committee of the Prefecture of Central Macedonia (no. 270079/2500).

Lyophilized HEWL and polysaccharides were purchased from Sigma-Aldrich and used without preliminary purification: hen egg-white lysozyme (lot L6876), chitosan (lot 448877), κ-carrageenan (lot 22048) and sodium alginate (lot 180947). The β-(1,4)-galactan, a polysaccharide of the rhamnogalacturonan I side chains, was purchased from Megazyme (P-GALPOT, 120501c).
HEWL fibrils were prepared by incubating 5 mL of 15 mg/mL⁻¹ (1 mM) protein solution containing 25 mM NaCl and 10 mM HCl at pH 1.9 at 65 °C in a thermostat, with stirring. The solution was kept under the described conditions for 5 days. The growth of the fibrils was confirmed using spectrofluorimetry, with a thioflavin T (ThT) fluorescent indicator.
Mature fibrils were dialyzed against pure 400 mL of distilled water, and the solution was changed after 2, 4, 6, 8, and 24 h (total volume used amounted to 2 L). The quality of the dialysis was monitored by the electrical conductivity of the dialysate. The convergence of conductivity changes was considered as the complete removal of salt excess. Then dialyzed samples were centrifuged. The water-soluble fraction was used for further studies. The concentration of the water-soluble fraction of protein fibrils was controlled by UV adsorption intensity at 280 nm (extinction coefficient ε₂₈₀ = 2.65 mg/mL⁻¹/cm⁻¹) [45 (link)].