In vitro degradation of scaffold

Degradation of the scaffold was measured as change in mass of the scaffolds over time in both cell culture media and digestive media. Digestive media consisted of bile salts and pancreatic enzymes (2.4 mg/mL porcine bile extract, B8631; Sigma; 0.4 mg/mL pancreatin, P7545; Sigma; 100 mg/mL NaHCO₃, and dH₂O, pH 6.5). Scaffolds were incubated at 37°C in cell culture media or digestive media. At weekly intervals, scaffolds were removed from solution and weighed. Scaffold degradation was determined according to mass loss.

In a separate experiment, scaffolds underwent seeding with GFP-enteroids. The structural and mechanical properties of the seeded scaffolds, in addition to scaffolds maintained in cell culture media and digestive media, were determined over time (2, 4, and 6 weeks). At each time point, scaffolds underwent rheometer testing (described below, n = 3–4 per group per time point), evaluation with SEM (n = 4), and confocal microscopy (n = 4). Scaffolds for rheometer testing were punched into 25 mm diameter circles. Scaffolds for imaging were punched into 8 mm diameter circles and at the end of each time point were cut in half and processed for either SEM or confocal microscopy.

The microstructural degradation was determined from the SEM images. Scaffold parameters were measured at baseline, 2, 4, and 6 weeks. Images were taken of the scaffolds from the top and side to measure the following parameters: scaffold base thickness, villus height, the width of the villi at the base, the width of the villi at the top, and the spacing between the villi. Measurements were taken using Fiji.³⁴ The number of scaffolds analyzed for each parameter at the baseline time point was 11–12 with an average of 49 images analyzed and an average of 165 independent measurements performed. Similarly, for each time point, an average of four scaffolds was analyzed with an average of 17 images and 58 independent measurements per parameter. Note that only intact villi were analyzed.