12 n hydrochloric acid

The hydroxyproline (Hyp) assay was performed on demineralised dentine blocks from each demineralisation group in order to quantify the amount of denatured collagen. The demineralised dentine samples were transferred into 1 ml PBS at pH 7 prior to being incubated at 37 °C with gentle shaking for 24 hours in 20 mg trypsin (treated with L-1-tosylamide-2-phenylethyl chloromethyl ketone, (TPCK)) (Sigma Aldrich, Poole, UK) in 1 ml PBS at pH 8. Dentine blocks were then rinsed with 1 ml PBS pH 8. Each sample solution (100μl) was transferred to a pressure-tight vial and incubated at 120 °C for 3 hours with 100 μl of 12 N hydrochloric acid (Sigma Aldrich, Poole, UK). Activated charcoal (5 mg) (Sigma Aldrich, Poole, UK) was stirred into the solutions before undergoing centrifugation at 13000 x g (Sigma Aldrich, Poole, UK) for 2 minutes. Each supernatant (10μl) was added to a 96 well plate (Greiner BIO-One, Stonehouse, UK) and the quantity of Hyp was measured per mass of sample, against Hyp standards, using a spectrophotometer at 560nm (Tecan, Switzerland).