Sites of non-enzymatic crosslinks pentosidine and glucosepane in collagen I of human cortical bone

Structural alterations of bone extracellular matrix (ECM) may affect bone quality, which, along with bone mass, affects the ability of bone to resist fracture. In particular, an increase in advanced glycation end-product (AGE) crosslinking of type I collagen has been implicated in contributing to increased bone fragility in diabetes and ageing. However, the location of these crosslinks and their relative abundance is unknown, thus hindering our understanding of how AGE crosslinks impact bone quality. We analyzed physiological crosslinks, glucosepane (GN) and pentosidine (PE), in collagen I from cortical bone of 12 male and 12 female cadaveric femurs using liquid chromatography-tandem mass spectrometry. The analysis identified 11 distinct crosslinks at 8 different sites within the collagen I triple helix. Crosslinks at two sites formed only within the same collagen chain, while the remaining crosslinks formed within the same triple helical molecule and/or between different molecules of the neighboring microfibrils. Most of the GN and PE crosslinks were located to the D-periodic overlap zone of the microfibril. The relative crosslink levels varied significantly at different sites: 0.005% to 2.1% for PE and 0.06% to 24.9% for GN. Female donors had fewer crosslink sites compared to male donors. While total PE and total fAGE levels did not correlate with bone mechanical properties in the present sample size, the level of GN at one site positively correlated with ultimate stress, while the level of PE at another site negatively correlated with post-yield toughness. However, these correlations were weak, and several other correlations between site-specific GN and PE levels and these mechanical properties were not consistent between the sexes. ln conclusion, this study identified the native locations of GN and PE within collagen I of bone, an innovation essential for addressing the question of how the specific structural features of bone ECM affect bone quality.