4.5.1. In Vitro Enzymatic Resistance

In vitro enzymatic resistance of the F1- and F3-derived SCMs and nc-SCMs was determined by evaluating their stability in native fetal bovine serum (FBS, Euroclone, Milan, Italy) and in the presence of a commercial bacterial collagenase. For the FBS stability test, F1- and F3-derived SCMs (6 mg) were incubated with 1 mL of FBS at 37 ◦C in a humidified atmosphere for 15 day. The collagenase stability evaluation was determined, as already described [44]. Briefly, collagenase from *Clostridium histolyticum* in a ratio of 1:10 (enzyme:substrate) was used in 1 mL of PBS at 37 ◦C for 6 day. The enzymatic solution was refreshed daily to ensure continuous enzymatic activity on the SCMs. As a control, a commercial porcine collagen membrane (25 × 25 mm), called Bio-Gide® (Geistlich Pharma AG, Wolhusen, Switzerland), which is widely used in dental and bone surgery, was submitted to digestion as well. Experiments were performed three times in duplicate.
