The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T-max, and the water volume fraction, epsilon (1/T-max = 0.000731epsilon + 0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, Tmax was constant and the fibre remained unchanged, no matter how much excess water was added. This Tmax value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1degreesC and 0.48 for glutaraldehyde treated fibres, 74.1degreesC and 0.59 for HMDC treated fibres, 69.3degreesC and 0.64 for MDA treated fibres, and 65.1degreesC and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking. (C) 2004 Elsevier Ltd. All rights reserved.