Glutaraldehyde

The cell lines derived from human renal cancer, 769-P, and human breast cancer SKBR3 were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were grown in RPMI 1640, supplemented with 10% fetal bovine serum and a 1% mixture of penicillin–streptomycin antibiotics (all from Gibco, Waltham, MA, USA) at 5% CO₂ and 37 °C in a humidified atmosphere containing 5% CO₂. Cell lines were authenticated with STR DNA Profiling Analysis (GORDIZ, Moscow, Russia). Cell lines were checked with the MycoAlertTM Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and were free of contamination. Bovine serum albumin, ethanol, pepsin, and commercial protease were purchased from Sigma-Aldrich (Saint Louis, MO, USA), while glutaraldehyde was purchased from PanReac AppliChem (Barselona, Spain). Cellular-grade trypsin was purchased from Gibco, while papain was purified in our lab. All the reagents necessary to perform Western blot were purchased from Biorad (Hercules, CA, USA), while the antibodies were purchased from Abcam (Cambridge, UK). Lysosensor green was purchased from Invitrogen (Waltham, MA, USA).

Acrylamide and bisAcrylamide solutions were purchased from Bio-Rad. N,N,N′,N′-Tetramethylethylenediamine (TEMED), N-[3-(Trimethoxysilyl)propyl]ethylene- diamine silane and dichlorodimethylsilane solution were purchased from Sigma. Sodium hydroxide (NaOH) and ammonium persulphate (APS) were purchased from Merck. Glutaraldehyde, ethanol and other solvents were purchased from Panreac AppliChem. Rectangular cover glasses were provided by Thermo Scientific. MEM (E15-888), L-Glutamine, fetal bovine serum and penicillin/streptomycin were purchased from PAA Laboratories (Pasching, Austria).

Iron (II) chloride tetrahydrate (98%) (FeCl₂*4H₂O), glutaraldehyde (25%), and sodium hydroxide (NaOH) (98%) were obtained from PanReac AppliChem (Spain). Iron (III) chloride hexahydrate (97%) (FeCl₃*6H₂O), tetramethylammonium hydroxide (TMAH) (25%), (3-aminopropyl) triethoxysilane (APTES) (98%), and L-cysteine (Cys) (97%) were purchased from Sigma-Aldrich (USA). In addition, 3-(triethoxysilyl) propylsuccinic anhydride (CAS) (95%) was purchased from Shanghai Kayi Chemical Co. (Shanghai, China).

Iron(II) chloride tetrahydrate, iron(III) chloride, 0.25% trypsin-EDTA, 4′,6-diamidino-2-phenylindole (DAPI), phalloidin–tetramethylrhodamine B isothiocyanate (phalloidin-TRITC), 3-isobutyl-1-methylxanthine, dexamethasone, indomethacin, Oil Red O solution 0.5% in isopropanol, 2-phospho-L-ascorbic acid trisodium salt, β-glycerophosphate disodium salt hydrate (99%) and silver nitrate (99%) were obtained from Sigma-Aldrich (Darmstadt, Germany). Citric acid monohydrate was purchased from Acros Organics (Geel, Belgium). Plasmid pWPT-GFP was obtained from Addgene (cat #12255; Cambridge, MA, USA). Dulbecco’s modified eagle medium (DMEM), L-glutamine, and 1% penicillin−streptomycin, dimethyl sulfoxide (DMSO), insulin (human recombinant), Dulbecco’s phosphate-buffered saline (PBS) and ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA; 98.5%) were obtained from PanEco Ltd. (Moscow, Russia). Sodium hydroxide (99.3%) and safranin O were purchased from Dia-M (Moscow, Russia). Heat-inactivated fetal bovine serum (FBS) and StemPro™ Chondrogenesis Differentiation Kit were obtained from Invitrogen (Carlsbad, CA, USA). Glutaraldehyde and paraformaldehyde (PFA) were obtained from Panreac (Barcelona, Spain).

Iron (III) chloride hexahydrate (97%) (FeCl₃∙6H₂O), tetramethylammonium hydroxide (TMAH) (25%), (3-Aminopropyl) triethoxysilane (APTES) (98%) and L-Cysteine (Cys) (97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron (II) chloride tetrahydrate (98%) (FeCl₂∙4H₂O), glutaraldehyde (25%) (GLU), Cd standard solution 1000 mg/L and sodium hydroxide (NaOH) (98%) were obtained from PanReac AppliChem (Barcelona, Spain). Additionally, 3-(triethoxysilyl) propylsuccinic anhydride (CAS) (95%) was purchased from Shanghai Kayi Chemical Co. (Shanghai, China). Commercial aluminum oxide spheres (Alumina) between 600–800 µm of diameter were acquired from Torrecid (L’Alcora, Castellón, Spain).

On day 6, the culture medium was removed from the wells, and cells were fixed with 2.5% glutaraldehyde (Panreac, Barcelona, Spain) and with 0.1% OsO₄ (Sigma-Aldrich, St. Louis, MO, USA). After washing, cell samples were passed through a battery of alcohols of increasing concentration. Samples were sprayed with gold in magnetron EmitechK575XD (Quorum Technologies, Lewis, UK), and analyzed using a TM 3000 microscope (HITACHI, Tokyo, Japan).

Hydrochloric acid (HCl, 37%, CAS 7647-01-0), glutaraldehyde (GTA, 25%, CAS 111-30-8), acetone (99.5%, CAS 67-64-1), glacial acetic acid (99.7%, CAS 64-19-7), and sodium hydroxide (NaOH, 98%, CAS 1310-73-2) were purchased from PanReac AppliChem (Chicago, IL, USA). Low molecular weight (LMW) chitosan (50–190kDa, deacetylation degree of 75–85%, CAS 9012-76-4), phosphate buffer saline (PBS-1X), thiazolyl blue tetrazolium bromide (MTT, CAS 57360-69-7), dimethyl sulfoxide (DMSO, 99%, CAS 67-68-5), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Type B gelatin from the local store Químicos Campota (Bogotá, Colombia). Fetal bovine serum (FBS) was obtained from Biowest (Riverside, MO, USA). Trypsin EDTA was obtained from Lonza (Riverside, MO, USA).