Coupling Effect of Water and Proteoglycans on the In Situ Toughness of Bone

Abstract

Proteoglycans (PGs) are one type of non-collagenous proteins in the extracellular matrix of bone, which primarily contain a core protein and glycosaminoglycans (GAGs). GAGs are highly polar and negatively charged, thus having a strong tendency in attracting water molecules into the matrix. We hypothesized in this study that PGs in bone play a pivotal role in sustaining the toughness of the tissue only when water is present. To test the hypothesis, we used a novel nanoscratch test to measure the in situ toughness of human cadaveric bone treated with and without PNGase F, an enzyme that specifically removes the N-linked oligosaccharides of GAGs from core proteins. Cortical bone specimens were prepared from the posterior aspect of mid-diaphyseal femurs of six (N=6) male human donors between 51.5±5.17 years old. Biochemical and histochemical assays were used to verify whether N-linked oligosaccharides were removed from bone matrix by PNGase F. By testing wet and dehydrated bone specimens, the coupling effect between water and PGs on the in situ toughness of bone was investigated. The two-way ANOVA analyses showed that removal of GAGs had significant effects on the in situ toughness of wet bone samples. In contrast, the removal of GAGs did not show significant effects on the toughness of dry bone. The results of this study, for the first time, suggest that GAGs play a pivotal role in the in situ toughness of bone only when water is present, and vice versa water functions as a plasticizer in bone only when GAGs are present.

Tissue-Level Mechanical Properties of Bone Contributing to Fracture Risk

“…The work to scratch the bone was found to be less for older donors than for middle aged donors [88]. Also, removing proteoglycans from the surface of human cortical bone caused a reduction in this so-called in situ toughness [89]. Moving forward, advancing existing and emerging techniques is necessary to improve our understanding of how changes in tissue-level mechanical properties contribute to fracture risk.…”

Age‐Related Deterioration of Bone Toughness Is Related to Diminishing Amount of Matrix Glycosaminoglycans (GAGs)

“…However, bound water retained in the extrafibrillar matrix by NCPs (10% of the organic phase), which are highly hydrophilic too, may also significantly affect the mechanical behavior of bone. In fact, recent evidence shows that the effect of hydration on bulk mechanical properties of bone is also manifested in the mechanical response of the mineral phase (32,66) , suggesting that the hydration condition of extrafibrillar matrix is strongly associated with the bulk behavior of bone (35) . It is not surprising because the extrafibrillar matrix, which is primarily comprised of mineral crystals, takes up at least 23% of bone volume (assuming mineralized collagen fibrils are closely packed).…”

“…Briefly, the polished bone cubes were glued onto the holder in a custom-designed chamber filled with PBS solution to ensure a wet condition throughout the test. The nanoscratch tests were performed circumferentially within individual lamellae of osteons on a Nano Indenter XP system (Agilent Technologies, Santa Clara, California, USA) with a cube corner tip, following previously reported procedures (35,46) . Measurements were performed within lamellar layer on six randomly chosen newly formed osteons in the cross section for each test specimen to alleviate effects of possible variations caused by different biological tissue age and local heterogeneity in bone.…”

“…For instance, PGs have been found to regulate the hydrostatic and osmotic pressure, thus affecting the poroelastic behavior of dentine and bone tissues (33,34) . A recent study of our group demonstrates that loss of GAGs may significantly reduce the tissue-level toughness of bone and such effects are most likely induced by the associated loss of bound water in bone matrix (35) . Intriguingly, recent studies show that among the age-related ultrastructural changes (36–39) , the loss of bound water in bone matrix with increasing age may directly lead to significant reduction of the toughness of bone (40–44) .…”

“…(33,34) A recent study of our group demonstrates that loss of GAGs may significantly reduce the tissue-level toughness of bone and such effects are most likely induced by the associated loss of bound water in bone matrix. (35) Intriguingly, recent studies show that among the age-related ultrastructural changes, (36)(37)(38)(39) the loss of bound water in bone matrix with increasing age may directly lead to significant reduction of the toughness of bone. (40)(41)(42)(43)(44) Based on the aforementioned evidence, it could be conjectured that changes in matrix GAGs may contribute to the age-related deterioration of bone quality.…”