An in vitro model to test the contribution of advanced glycation end products to bone biomechanical properties

We developed an in vitro model which provides the ability to test the effects of advanced glycation end products (AGEs), specifically pentosidine (PEN) and one of its inhibitors, the aminoguanidine (AMG), on cortical bone. This model allows modification of the extent of collagen cross-linking, while controlling other factors known to influence bone strength. In this in vitro model, young bovine cortical bone specimens were incubated in phosphate-buffered saline (PBS) ± ribose (RIB, an inducer of AGEs formation) ± AMG for 15 days at 37 °C. The mineral and organic matrix as well as biomechanical properties were examined.We found that (i) incubation ± treatments did not induce collagen denaturation compared to specimens that were not incubated; (ii) neither treatment or incubation time effected the concentration of trivalent enzymatic cross-links pyridinoline and deoxypyridinoline. The non-enzymatic cross-link PEN was undetectable in specimens that were not incubated or that were incubated in PBS or AMG alone. However, PEN concentration increased significantly in specimens incubated with RIB, whereas ribose-induced PEN formation was markedly inhibited by AMG. (iii) Incubation ±treatments did not change the mineral maturity, crystallinity or microhardness assessed by X-ray diffraction, X-ray microscopy analyses, FTIRM and micro-indentation tests. (iv) PEN concentration was not associated with biomechanical properties assessed by 3-point bending.In conclusion, this in vitro incubation model of young bovine cortical bone induced physiologic concentrations of PEN in the RIB + AMG group and is the first to show that AMG inhibits ribose-induced formation of PEN cross-links in bone while not affecting the organic and mineral phases. AGE concentration did not influence bending mechanical properties; however, the simple 3-point bending test we used was likely inadequate to demonstrate effects of AGEs on mechanical properties.