Water residing in small ultrastructural spaces plays a critical role in the mechanical behavior of bone.

Water may affect the mechanical behavior of bone by interacting with the mineral and organic phases through two major pathways: i.e. hydrogen bonding and polar interactions. In this study, dehydrated bone was soaked in several solvents (i.e. water, heavy water (D2O), ethylene glycol (EG), dimethylformamide (DMF), and carbon tetrachloride(CCl4)) that are chemically harmless to bone and different in polarity, hydrogen bonding capability and molecular size. The objective was to examine how replacing the original matrix water with the solvents would affect the mechanical behavior of bone. The mechanical properties of bone specimens soaked in these solvents were measured in tension in a progressive loading scheme. In addition, bone specimens without any treatments were tested as the baseline control whereas the dehydrated bone specimens served as the negative control. The experimental results indicated that 22.3±5.17vol% of original matrix water in bone could be replaced by CCl4, 71.8±3.77vol% by DMF, 85.5±5.15vol% by EG, and nearly 100% by D2O and H2O, respectively. CCl4 soaked specimens showed similar mechanical properties with the dehydrated ones. Despite of great differences in replacing water, only slight differences were observed in the mechanical behavior of EG and DMF soaked specimens compared with dehydrated bone samples. In contrast, D2O preserved the mechanical properties of bone comparable to water. The results of this study suggest that a limited portion of water (<15vol% of the original matrix water) plays a pivotal role in the mechanical behavior of bone and it most likely resides in small matrix spaces, into which the solvent molecules larger than 4.0Å cannot infiltrate.