Validation of Fourier transform infrared microspectroscopy for the evaluation of enzymatic cross-linking of bone collagen

Enzymatic cross-linking of the bone collagen is important to resist to crack growth and to increased flexural strength. In the present study, we proposed a new method for assessment of enzymatic cross-link based on FTIR microspectroscopy that takes into account secondary structure of type I collagen. Briefly, femurs were collected from sham or ovariectomized mice and subjected either to LC-MS or embedded in polymethylmethacrylate, cut and analyzed by FTIR microspectroscopy. FTIR acquisition were recorded before and after UV exposure or acid treatment. In addition, femurs from a second animal study were used to compare gene expression of Plod2 and Lox enzymes and enzymatic cross-links determined by FTIR microspectroscopy. We evidenced here that intensities and areas of subbands located at [~]1660 cm-1, [~]1680 cm-1 and [~]1690 cm-1 were positively and significantly associated with the concentration of pyridinoline (PYD), deoxypyridinoline (DPD) or immature dihydroxylysinonorleucine (DHLNL) / hydroxylysinonorleucine (HLNL) cross-links. Seventy-two hours exposure to UV light significantly reduced by [~]86% and [~]89% the intensity and area of the [~]1660 cm-1 subband. Similarly, 24 hours of acid treatment significantly reduced by 78% and 76% the intensity and area of the [~]1690 cm-1 subband. Plod2 and Lox expression were also positively associated to the signal of the [~]1660 cm-1 and [~]1690 cm-1 subbands. In conclusion, our study provided a new method for decomposing the amide I envelope of bone section that positively correlates with PYD and immature collagen cross-links. This method allows for investigation of tissue distribution of enzymatic cross-links in bone section.