Hyaluronic acid sodium salt from streptococcus equi

Hyaluronic acid (HA) sodium salt from Streptococcus equi (8–15 kDa), HAp nanoparticle, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2,4,6-trinitro-benzensulfonic acid (TNBS), dexamethasone, β-glycerophosphate disodium salt hydrate, L-ascorbic acid, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Gelatin from porcine skin (250 Bloom) was obtained from Geltech (Busan, Korea). Human bone marrow-derived mesenchymal stem cells (hBMSCs) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Minimum essential medium alpha (MEM-α), fetal bovine serum (FBS), penicillin–streptomycin, and Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) were purchased from Cytiva (Logan, UT, USA). The Actin Cytoskeleton and Focal Adhesion Staining Kit was purchased from Merck Millipore (Burlington, MA, USA). The Mouse Osteoprotegerin enzyme-linked immunosorbent assay (ELISA) Kit was purchased from Biomatik (Wilmington, DE, USA). Human TNFSF11/RANKL/TRANCE (Sandwich ELISA) ELISA Kit was obtained from LSBio (Seattle, WA, USA). Other reagents and solvents were commercially obtained and used as received.

Gold (III) chloride trihydrate (HAuCl₄·3H₂O), L-ascorbic acid (L-AA), rosmarinic acid (RA), silver nitrate (AgNO₃), hyaluronic acid (HA) sodium salt from Streptococcus equi, oleic acid (OA), and bovine serum albumin (BSA) were acquired from Sigma-Aldrich (St. Louis, MO, USA). All remaining reagents and solvents were of analytical purity grade. Cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The water used in all the experiments was purified to 18.2 MΩ·cm at 25 °C through a Millipore system (Millipore, Burlington, MA, USA).

Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was purchased from Sigma-Aldrich (Poole, UK). Methocel A15LV (methylcellulose, MC) was supplied by Dow Chemical Company (Midland, MI, USA). Hyaluronic acid (HA) sodium salt from Streptococcus equi was purchased from Sigma-Aldrich (Poole, UK). Transwell cell culture inserts (polycarbonate filter, 1.1 cm² diameter, 0.4 μm pores) were obtained from Corning (Corning, NY, USA). Hank’s Balanced Salt Solution (HBSS), phosphate-buffered saline (PBS), trypsin, Dulbecco’s Modified Eagle Medium (DMEM), Triton X-100, and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich (Poole, UK). AlamarBlue^® cell toxicity agent was supplied by Thermo Fisher Scientific (Waltham, MA, USA).

Gold (III) chloride trihydrate (HAuCl₄·3H₂O), hyaluronic acid (HA) sodium salt from Streptococcus equi (MW 7000–250,000 g/mol), oleic acid (OA) (MW 282.46 g/mol), trypsin, fetal bovine serum (FBS), and penicillin/streptomycin were all supplied from Sigma-Aldrich (Steinheim, Germany). Dulbecco’s modified Eagle medium (DMEM) was supplied by Biowest (Nuaillé, France). The human keratinocyte cell line, HaCat, the human melanoma cell line, A375, and the murine melanoma cell line, B16F10, were provided by Cell Line Service GmbH (Eppelheim, Germany). Recombinant human epidermal growth factor (EGF) was purchased from Life Technologies (Waltham, MA, USA). The water used in all the experiments was purified through a Millipore system (Millipore, Burlington, MA, USA). All the remaining chemicals and substrates used were of analytical grade.
Intramuscular administration of a mixture of ketamine (Ketamidor^®, Richter Pharma, Wels, Austria) and chlorpromazine (Largactil^®, Laboratórios Vitória, S.A., Amadora, Portugal) (10:2 v/v) at a dose of 2 mL/kg was used as anesthetic protocol for the in vivo experiments.

Poly(L-Lysine hydrobromide) (PLL, Mw 10,000 Da) was purchased from Alamanda Polymers. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), 4-Imidazoleacetic acid, Sodium Hydroxide (NaOH), Hyaluronic acid sodium salt from Streptococcus equi (1.5–1.8 × 106 Da), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from Sigma Aldrich without further purification. Dialysis tubing benzoylated (cut off 2000) was purchased from Sigma Aldrich. Water was ultra-pure grade (type I).
MISSION^® microRNA Mimic (has-miR-34a-5p) was purchased from sigma Aldrich. The miR-34a sequence was: UGGCAGUGUCUUAGCUGGUUGU.