The risk of morbidity and even mortality caused by age-related bone fragility fractures is becoming an increasing concern worldwide. In the past, the age-related decrease in bone mineral density (BMD) was used to predict fragility fractures of elderly human bone. However, BMD alone cannot fully explain bone fragility fractures since the mineral is only one of four constituents (i.e. mineral, collagen, non collagenous proteins, and water) of bone, which may also contribute to the mechanical competence of the tissue. Among the constituents, proteoglycans (PGs) are a group of non-collagenous proteins in bone matrix. Glycosaminoglycans (GAGs), the main constituent of proteoglycans, are highly polar and negatively charged. Thus, GAGs have a strong potential of attracting water molecules into the bone matrix, thus indicating a potential for PGs to affect the in situ mechanical behavior of bone.

In this study, we hypothesized that proteoglycans in bone play a critical role in sustaining the in situ toughness of bone in cooperation with water. In addition, we also hypothesized that age-related changes in proteoglycans may compromise its ability to sustain the in situ toughness of bone. In order to test the first hypothesis, a novel nanoscratch test was used to measure the in situ toughness of bone specimens, which were treated with and without PNGase F, an enzyme that specifically removes the Nlinked oligosaccharides of GAGs from core proteins. For the second hypothesis, we tested bone samples from young, mid-age and old aged human donors in terms of the in situ toughness and the amount of GAGs in bone matrix. Biochemical and histochemical assays were used to assess the amount of GAGs in the bone matrix.

The results of this study showed that removal of GAGs led to a significant decrease of the in situ toughness of young and mid-age bone samples. In contrast, the in situ toughness of young and mid-aged bone specimens under dry conditions was not affected regardless of whether they were treated with PNGase F or not. The in situ toughness of old bone specimens did not change with hydration status or treatments by PNGase F. This study, for the first time, suggests that GAGs play a critical role in toughening bone only when water is present. In other words, water functions as a plasticizer in the bone matrix only when GAGs are present. However, the toughening mechanism diminished in elderly bone, suggesting that age-related changes in proteoglycans may be responsible for the age-induced degradation of human bone toughness.