Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135 degrees C and 200 degrees C, with another reversible event at similar to 60-80 degrees C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140 degrees C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2O remaining within freeze-dried cartilage that is released progressively upon heating up to 200 degrees C. Spectra obtained after exposure to high temperature and rehydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.