Chromic acid

In order to determine the amount of degradation in the alloy, specimens were immersed in a physiological cell culture medium DMEM + Glutamax (Dulbecco’s Modified Eagle’s Medium, (+) 4.5 g/L d-Glucose, (+) Pyruvate, Life Technologies, Darmstadt, Germany) supplemented with 10% FBS (Fetal Bovine Serum, PAA Laboratories, Linz, Austria) in sterilized well-plates. In addition, a set of empty wells with cell culture medium served as control units. For degradation analysis, four specimens per incubation time were considered. Mg-2Ag samples were immersed for 3, 12 and 30 days according to the ASTM NACE/ASTM G31-12a standard. For Mg-0.3Ca, incubation times include the intervals of 1 h till 8 h followed by 1 day, 2 days, and later followed in intervals of 1 week to 6 weeks. In total, 16 mean degradation depth values were calculated for progressing incubation times for the alloy Mg-0.3Ca. Fresh cell culture medium was introduced into the well-plates for every 3–4 days to maintain constancy in pH value. Simultaneous drying process soon after incubation was carried out under argon environment at 37 °C for 24 h. The whole procedure is schematically illustrated in Figure 3.
The developed degradation product layer on the surfaces of specimens was removed with fresh chromic acid (180 g/L in distilled water, VWR International, Darmstadt, Germany). Samples were dipped in the chromic acid medium for 20 min with a gentle stir. In addition, a set of Mg-0.3Ca alloy samples in their initial state were immersed directly in chromic acid for 20 min to check the reactivity of chromic acid towards the alloy. The difference in weights observed before and after this treatment was in the range of limit of detection of the weighing machine. This confirmed the non-reactive nature of chromic acid towards the base Mg-0.3Ca alloy.
The weights of specimens recorded by using a high precision weighing scale (Sartorius, Göttingen, Germany) soon after sterilization and after chromic acid treatments were used to calculate their respective mean degradation depths using Equation (2).

Male C57BL/6J mice (age 4 weeks) were purchased from Harlan (Horst, The Netherlands) and were housed in the light and temperature-controlled animal facility (12/12 (light/dark), 20°C) of Wageningen University. They had free access to water and received standard laboratory chow (RMH-B, Arie Blok BV, Woerden, The Netherlands) for two weeks, followed by the low-fat diet for three weeks to adapt to the purified diets. All experiments were approved by the Ethical Committee on animal testing of Wageningen University. To investigate the effect of dietary fat on development of obesity and insulin resistance and on small intestinal gene expression in C57BL/6J mice, we used low-fat (reference) and high-fat diets that are based on 'Research Diets' formulas D12450B/D12451, with adaptations regarding type of fat (palm oil in stead of lard) and carbohydrates to mimic the fatty acid and carbohydrate composition of the average human diet in Western societies (Research diet services, Wijk bij Duurstede, The Netherlands). Thus, the high-fat diet mimics the ratio of saturated to monounsaturated to polyunsaturated fatty acids (40:40:20) in a human diet. The complete composition of the diets is given in Additional file 1. It should be noted that in these diets the energy density of all nutrients, except fat and starch, is equal.
At the start of the diet intervention, the mice were divided into two groups and were fed a powdered high- or a low-fat purified diet. After 2, 4, and 8 weeks of diet intervention, 6 mice per diet group, per time point were sacrificed after they were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). All mice were sacrificed in the postprandial state and diurnal variability was avoided by harvesting small intestines at the same time of the day for both diet groups. The small intestines were divided into three equal parts along the longitudinal axis (proximal, middle and distal part of the small intestine). Small intestinal epithelial cells were scraped, snap-frozen in liquid nitrogen, and stored at -80°C until RNA isolation. Body weight was recorded weekly. The mice that were sacrificed after 8 weeks of diet intervention were all subjected to an oral glucose tolerance test (OGTT) at week 7. Therefore, after 6-hours fasting, all mice received 0.5 ml of a 20% glucose solution via an oral gavage and blood glucose was measured after 15, 30, 45, 60, 90 and 150 minutes using Accu-Chek blood glucose meters (Roche Diagnostics, Almere, The Netherlands). To determine food intake, non-absorbable chromic oxide was supplemented to the diets for one week (week 5 of diet intervention). At the end of this week feces was quantitatively collected during 48 hours and fecal chromic oxide levels were determined as previously described [15 (link)]. These fecal chromic oxide levels were then used to calculate the energy intake per mouse per day on a high-fat and low-fat diet. An outline of this study design is presented in Additional file 2.
For immunohistochemical analysis, an identical low-fat and high-fat diet intervention study was performed for 2 weeks (n = 12 per diet). Small intestines were again excised, divided into three equal parts, cut open longitudinally, and washed with PBS. Thereafter, the small intestinal parts were fixed in 10% buffered formalin and embedded in paraffin.

Tissues GST enzyme activity: it measures the conjugation of 1-chloro-2,4-dinitro benzene (CDNB) with reduced glutathione that produces a dinitrophenyl thioether which can be detected by spectrophotometer at 340 nm. One unit of GST activity is defined as the amount of enzyme producing 1 mmol of CDNB-GSH conjugate/min under the conditions of the assay according to the method described by Habig et al. [26 (link)].
Tissues GPx enzyme activity: it was measured as IU/gm wet tissue by the reaction between glutathione remaining after the action of GPx and 5, 5-dithiobis-(2-nitrobenzoic acid) to form a complex that absorbs maximally at 412 nm. Glutathione peroxidase activity of 1 U/mg protein was defined as 1 μg of GSH consumed/min/mg protein [27 ].
Determination of tissues CAT enzyme activity: it assayed by the method of Sinha which based on formation of chromic acetate from dichromate and glacial acetic acid in presence hydrogen peroxide, chromic acetate that produced was measures colorimetrically at 570 nm, one enzyme unit was defined as the amount of enzyme which catalyzed the oxidation of 1 μmole H₂O₂ per minute under assay conditions [28 (link)].
Tissues PON1 enzyme activity: PON1 activity towards paraoxon (O,O-diethyl-O-p-nitrophenyl phosphate) was determined by measuring the initial rate of substrate hydrolysis to p- nitrophenol, whose absorbance was monitored at 405 nm in the assay mixture, A PON1 activity of 1 U/mg protein was defined as 1 μmol p-nitrophenol formed per minute per mg protein [29 (link)].