Effect of dehydrothermal treatment on collagen crosslinking and denaturation

Dehydrothermal (DHT) crosslinking is routinely performed to increase the stiffness and the enzymatic resistance of collagen-based devices. Amide and ester bonds are formed among the collagen macromolecules, as a result of the high temperatures and high vacuum involved in the process. The extent of crosslinking is known to increase with the DHT temperature and duration, but simultaneous collagen denaturation might be induced. The aim of this work was to investigate the extent of crosslinking and denaturation of DHT-treated collagen-based films, by means of thermal and physicochemical analyses. With the ultimate goal of optimizing the DHT process, five different temperatures (110, 120, 140, 160 and 180°C) were used, while the DHT duration was kept constant (24 hours). Differential scanning calorimetry (DSC) was carried out to measure the denaturation temperature (Td) and enthalpy (ΔHd) of the collagen films. The reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBS) with primary amines (-NH2) allowed determining the number of free -NH2 in the collagen films, whereas Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical modifications occurring upon DHT treatment. Higher degrees of crosslinking were attained for increasing DHT temperatures, as demonstrated by reduced number of free -NH2, lower absorbance of amide II band (1545 cm-1) and higher Td values. However, the sharp reduction of ΔHd detected for samples treated at 140, 160 and 180°C indicated a significant denaturation associated to crosslinking. The analysis of the absorbance band at 1236 cm-1 confirmed that collagen denaturation was particularly pronounced for DHT temperatures higher than 120°C, suggesting that, at those temperatures, denaturation might predominate over crosslinking. Further stress relaxation tensile tests and dynamic mechanical analysis (DMA) are currently being performed to measure the stiffness of DHT-treated samples and to estimate the elastically effective crosslink density, according to the rubber elasticity theory.