Infrared attenuated total reflection spectroscopic surface analysis of bovine-tail intervertebral discs after UV-light-activated riboflavin-induced collagen cross-linking.

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross-links. Loss of collagen cross-linking combined with other age-related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross-linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state-of-the-art method to assess the extent of collagen cross-linking in tissues requires destructive procedures and high-performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR-ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV-light-activated riboflavin (B2)-induced collagen cross-linking in bovine IVD samples. Thirty-five fresh bovine-tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV-light only and (e) UV-light-B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR-ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real-time assessment of exogenous IVD cross-linking.