Porcine gastric mucosa

Each cooked egg sample (whole eggs, their egg whites and yolks) were digested under simulated gastro-intestinal conditions involving sequential treatments with pepsin (porcine gastric mucosa; Sigma-Aldrich, Oakville, ON, Canada) and pancreatin (porcine pancreas; Sigma-Aldrich, Oakville, ON, Canada) as described in our previous study [9] (link). The pH and temperature of the samples were maintained constantly during the course of hydrolysis using Titrando (Metrohm, Herisan, Switzerland) and a circulating water bath respectively. The hydrolysis was terminated by raising the temperature to 95°C and the hydrolysates were freeze-dried without centrifugation separation.

Suspensions of 12.2% w/w nanocrystalline cellulose (NCC) and 3% w/w nanofibrillated cellulose (NFC) were purchased from Cellulose Lab Company, Fredericton, NB, Canada. Tween 20 and β-carotene powder (≤95% purity) were purchased from Sigma-Aldrich Company (Sigma-Aldrich, Inc., St. Louis, MO, USA). Soybean oil without any purification was purchased from a local supermarket in Nakhon Pathom, Thailand. Pepsin from porcine gastric mucosa, porcine lipase, mucin from porcine stomach, porcine bile extract, and Nile Red were purchased from Sigma-Aldrich Company (Sigma-Aldrich, Inc., St. Louis, MO, USA). Sodium azide and chloroform were purchased from Ajax Finechem (Ajax Finechem Pty., Ltd., New SouthWales, Australia). Sodium chloride, calcium chloride, monobasic phosphate, and all chemicals used in this study were of analytical grade. Double distilled water was used for the preparation of all solutions.

For immunohistochemistry (IHC), 3.5 μm-thick paraffin-embedded sections of the pellets from the chondrogenic redifferentiation were dewaxed, rehydrated and pre-digested for 30 min at 37°C for antigen retrieval with pepsin (1 mg/ml in 0.5 M acetic acid) (porcine gastric mucosa) (Sigma Aldrich), in case of collagen type II staining. Sections were treated with 3% hydrogen peroxide before starting the staining with the Dako labeled streptavidin-biotin (LSAB) 2 system-horseradish peroxidase (HRP) kit (Dako) and antibodies against collagen type II (AF5710; Acris, Hiddenhausen, Germany, and MA5-13026; Invitrogen). For histological staining of proteoglycans, Safranin O (Fisher Scientific) was used. In all samples, a final staining of cell nuclei by Gill’s haematoxylin No. 3 (Sigma-Aldrich) was performed.

The preparation of digestive juices was described in our previous works [13 (link),31 (link),32 (link)]. The enzymes used here were: α-amylase (porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA), pepsin (porcine gastric mucosa, Sigma-Aldrich, St. Louis, MO, USA), lipase (porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA), pancreatin (porcine pancreas, Sigma-Aldrich, St. Louis, MO, USA), trypsin (porcine pancreas, Fermelo Biotec, Santiago, Chile), and chymotrypsin (bovine pancreas, Fermelo Biotec, Santiago, Chile). Bile extract (porcine, Sigma Aldrich, St. Louis, MO, USA), soy lecithin (Blumos, Santiago, Chile), 1 N HCl (Fisher Chemical, Pittsburg, PA, USA) and 1 N NaOH (Heyn, Santiago, Chile) were also used. Purified water was used for the preparation of all solutions. To simulate the gastrointestinal fluids, simulated salivary fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared, which were adjusted to pH values of 7.0, 3.0 and 7.0, respectively. Simulated fluids were made up of the corresponding electrolyte stock solutions, enzymes, CaCl₂ and water, according to the standardized digestion method. The concentration of the stock solutions was based on the method proposed by the COST Infogest network [29 (link),30 ] (Table 1). For all assays, freshly prepared simulated digestion fluids were used.