The effect of staining on the monotonic tensile mechanical properties of human cortical bone

Microdamage in the form of microcracks has been observed in cortical bone following in vivo and in vitro fatigue loading. It has been suggested that bone has an inherent ability to repair microdamage at physiological activity levels. If the biological remodelling and repair process cannot keep up with the rate of damage accumulation, as in ageing bone and in individuals such as athletes and military recruits, microdamage may accumulate even at physiological activity levels. Such microdamage accumulation is thought to contribute to stress and fragility fractures. It is therefore important to obtain quantitative data on the rate of damage accumulation so as to understand the etiology of skeletal fractures. Sequential labelling of microdamage using fluorochrome stains at different stages of mechanical loading is becoming standard for assessing damage evolution. Although verification of this staining technique is provided in the literature, it has not yet been reported if the stains change the mechanical properties of cortical bone. In this study, monotonic tensile tests were performed to investigate the effect of the staining on the monotonic tensile mechanical properties of cortical bone. Forty-eight specimens were machined from human femora obtained from three male subjects, aged 52-55 years, and all 48 specimens were systematically divided into one control and three treatment groups. Specimens in the first (n = 12) and second treatment groups (n = 12) were stained with alizarin complexone and calcein (0.0005 M), respectively, for 16 h under 50 mmHg vacuum. Specimens in the third treatment group (n = 12) were kept in calcium-supplemented saline solution under the same conditions of the first and second treatment groups. Specimens in the control group (n = 12) were removed from the freezer prior to testing and allowed to thaw at room temperature in saline solution. Differences among the mean values of the mechanical properties for four testing groups were determined by the Mann-Whitney test at a significance level of P < 0.05. The statistical results indicated that the chelating stains and the staining conditions have no significant effect on the mechanical properties of the cortical bone under monotonic tensile loading. This study demonstrated that microcrack labelling with the chelating stains under aforementioned conditions (stain concentration, staining time, etc.) is a reliable method in that staining cortical bone with alizarin complexone and calcein prior to testing does not affect tensile properties.