Removal of glycosaminoglycans affects the in situ mechanical behavior of extrafibrillar matrix in bone

Previous studies have shown that glycosaminoglycans (GAGs) in bone matrix, coupling with water in bone matrix, may play a significant role in toughening bone tissues. Since GAGs are most likely present only in the extrafibrillar matrix (EFM) of bone, we hypothesized that GAGs in EFM would have a major impact on bone tissue toughness. To confirm this conjecture, we removed GAGs ex vivo from human cadaveric bone samples using a protein deglycosylation mix kit and then examined the in situ mechanical behavior of mineralized collagen fibrils (MCFs) and the surrounding EFM of the samples, using a high-resolution atomic force microscopy (AFM). By testing the bone samples before and after removal of GAGs, we found that under the wet condition removal of GAGs resulted in an increase in the elastic modulus of both EFM and MCFs, whereas a significant decrease in plastic energy dissipation was observed mainly in EFM. In contrast, under the dry condition the removal of GAGs had little effects on the mechanical properties of either MCFs or EFM. These results suggest that both MCFs and EFM contribute to the plastic energy dissipation of bone, whereas in the presence of matrix water removal of GAGs significantly reduces the capacity of EFM in plastic energy dissipation, but not MCFs. In addition, GAGs may affect the elastic modulus of both EFM and MCFs. These findings give rise to new understanding to the underlying mechanism of GAGs in toughening of bone tissues.