Ammonium persulfate

Glycinamide hydrochloride and serine methyl ester hydrochloride were purchased from Watanabe Chemical Industry Co, Ltd. (Hiroshima, Japan). Acryloyl chloride, hydrochloric acid (12 M), potassium carbonate, sodium hydroxide, diethyl ether, methanol, ethanol, tetramethylsilane (TMS), ammonium persulfate (APS), sodium trimethylsilyl propane sulfonate (DSS), N,N-diisopropylethylamine (DIPEA), dichloromethane (DCM), and anhydrous sodium sulfate (Na₂SO₄) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Deuterium oxide (D₂O), 25% glutaraldehyde solution, and Wako Gel^® FC-40 were purchased from FUJIFILM Wako Pure Chemical, Co. (Osaka, Japan). Deuterated dimethyl sulfoxide (DMSO-d₆) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Polyethylene glycol diacrylate (PEGDA) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan) and dialysis membrane (MWCO: 25,000 Da) from Funakoshi Co. (Tokyo, Japan). The glass plates (76 mm × 52 mm) were obtained from Matsunami Glass Industry Co., Ltd. (Osaka, Japan). glass plates were hydrophobically treated with Siliconize L-25 (Fuji Systems Co., Tokyo, Japan) before use. Silicone rubber plates (1 mm) were obtained from As One Co. (Osaka, Japan). All the reagents were used without further purification.

For the preparation of the 5% and 20% EMP hydrogels, the monomer solvent—acryl-EMP (2.83 µmol, 5% or 11.32 µmol, 20%, respectively) with acrylamide (1.07 µmol, 7.6%, Nacalai Tesque, Kyoto, Japan) and N,N′-methylene bisacrylamide (25.9 µmol, 0.4%, Nacalai Tesque, Kyoto, Japan)—in 1 mL of 375 mM Tris-HCl pH 8.8 buffer were polymerized by adding ammonium persulfate (0.1%, Nacalai Tesque, Kyoto, Japan) and TEMED (0.02%, Kanto Chemicals, Tokyo, Japan). After adding ammonium persulfate and TEMED, the monomer solvent was poured in thin cassettes (1 mm thickness and 90 mm width) and polymerized at 20 °C. Non-crosslinked EMP hydrogels (NCE hydrogels) (5% and 20%) were prepared by using EMP instead of acryl-EMP. For EMP-NCE bilayer hydrogel preparation, the same volume of acryl-EMP monomer solvent was polymerized at 20 °C on top of the NCE hydrogel in the same thin cassettes.

FeCl₂·4H₂O was purchased from Avantor Performance Materials, Inc. (Allentown, PA, USA). FeCl₃·6H₂O, toluene, ammonia solution hydrochloric acid, ammonium persulfate, potassium thiocyanate and oleic acid were purchased from Nacalai Tesque (Kyoto, Japan). Hyaluronic acid (molecular weight 3000 kDa) was purchased from Cheng-Yi Chemical Industry Co. Ltd. (Taipei, Taiwan). All other analytical grade reagents and solvents used were purchased from J.T. Baker (Phillipsburg, NJ, USA). αMEM, L-glutamine, fetal bovine serum, and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). Sodium nitrate was purchased from Merck (KGaA, Darmstadt, Germany). Finally, dimethyl sulfoxide, Triton X-100 and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA).