Background Staple line leakage and bleeding are the most common problems associated with the use of surgical staplers for gastrointestinal resection and anastomotic procedures. These complications can be reduced by reinforcing the staple lines with buttressing materials. The current study reports the potential use of cholecyst-derived extracellular matrix (CEM) in non-crosslinked (NCEM) and crosslinked (XCEM) forms, and compares their mechanical performance with clinically available buttress materials [small intestinal submucosa (SIS) and bovine pericardium (BP)] in an ex vivo small intestine model.Methods Three crosslinked CEM variants (XCEM0005, XCEM001, and XCEM0033) with different degree of crosslinking were produced. An ex vivo peristaltic inflation model was established. Porcine small intestine segments were stapled on one end, using buttressed or non-buttressed surgical staplers. The opened, non-stapled ends were connected to a peristaltic pump and pressure transducer and sealed. The staple lines were then exposed to increased intraluminal pressure in a peristaltic manner. Both the leak and burst pressures of the test specimens were recorded.Results The leak pressures observed for non-crosslinked NCEM (137.8 +/- 22.3 mmHg), crosslinked XCEM0005 (109.1 +/- 14.1 mmHg), XCEM001 (150.1 +/- 16.0 mmHg), XCEM0033 (98.8 +/- 10.5 mmHg) reinforced staple lines were significantly higher when compared to non-buttressed control (28.3 +/- 10.8 mmHg) and SIS (one and four layers) (62.6 +/- 11.8 and 57.6 +/- 12.3 mmHg, respectively) buttressed staple lines. NCEM and XCEM were comparable to that observed for BP buttressed staple lines (138.8 +/- 3.6 mmHg). Only specimens with reinforced staple lines were able to achieve high intraluminal pressures (ruptured at the intestinal mesentery), indicating that buttress reinforcements were able to withstand pressure higher than that of natural tissue (physiological failure).Conclusions These findings suggest that the use of CEM and XCEM as buttressing materials is associated with reinforced staple lines and increased leak pressures when compared to non-buttressed staple lines. CEM and XCEM were found to perform comparably with clinically available buttress materials in this ex vivo model.