The results support our hypothesis that tissue level properties, as measured by nanomechanical properties and tissue mineral density, are different for iliac bone biopsies from long-term bisphosphonate treated SSBT patients with atypical femoral fractures compared to those from age-matched and young normals, as well as from age-matched osteoporotic patients with vertebral fractures. Interestingly, both cortical and trabecular bone plastic deformation resistance were higher in the SSBT group than in the comparison groups. The magnitudes of the differences in plastic deformation resistance ranged from 15 to 22% in cortical bone and 29 to 31% in trabecular bone. SSBT trabecular bone also had higher modulus and exhibited a trend toward higher mineral content than the comparison groups, though the differences in mineral content among the groups were modest (1.7 to 2.4%). The average BFR of the SSBT group was close to zero; in contrast, among the age-matched groups, BFR was 54 fold greater in the osteoporotic group and 48 fold greater in the normal group, compared to the SSBT group. We suggest that differences in SSBT trabecular bone quality are consistent with increased tissue age due to the low BFR of these patients. However the higher resistance to plastic deformation, particularly in cortical bone, may indicate a possible connection between hard tissue plastic deformation properties and toughness properties. Cortical bone mean elastic properties were not different among the groups.
The average mechanical properties and mineral densities of trabecular and cortical bone were not different among the young normal, age-matched normal, and osteoporotic groups. This indicates that the average iliac bone material properties were not detectably altered by age or osteoporosis in our study. In contrast, SSBT cortical bone had higher plastic deformation resistance, and SSBT trabecular bone had higher contact hardness modulus, plastic deformation resistance, and a trend toward higher mineral density, compared to that of osteoporotics. We suggest that SSBT bone quality is different from bisphosphonate-naïve osteoporotics, but the differences cannot be directly attributed to bisphosphonate treatment, SSBT, or the combination of these effects, due to the study design.
Plastic deformation resistance was greater in trabecular bone regions compared to cortical bone regions, except in younger normal subjects. Aging may affect the ability for cortical bone to resist plastic deformation at the material level, relative to trabecular bone. However SSBT bone had greater plastic deformation resistance in both trabecular and cortical regions compared to the other groups. A combination of aging effects, disease and drug interactions may contribute to the ability for bone material to absorb energy through permanent deformation.
Heterogeneity in mechanical properties and mineral densities at the micrometer scale may be beneficial to the mechanical integrity of bone tissue by preventing propagation of cracks [32
]. The relatively low variation in SSBT cortical bone elastic modulus compared to the normal groups is consistent with this hypothesis, as SSBT fractures occurred in regions composed primarily of cortical bone. This finding is also consistent with studies that have shown that bisphosphonate treatment is associated with decreased heterogeneity in the spectroscopic markers mineral/matrix ratio, crystallinity, carbonate/amide I ratio [37
], and mineral density distribution [39
]. Additionally, the trabecular bone of the osteoporotic group had less variable contact hardness properties, and a trend of less variable mineral densities (not shown), than that of the young normal group. A lack of heterogeneity in the cortical hard tissue of SSBT patients and trabecular hard tissue of osteoporotic patients may be associated with atypical and vertebral fractures, respectively, due to decreased resistance to crack propagation. However, the heterogeneity hypothesis was not supported by the plastic deformation resistance variability data. The standard deviation of plastic deformation resistance was high in the SSBT group for both cortical and trabecular bone. One possible explanation is that the positive effect of variability in properties has less impact when the properties are high on average. The mean plastic deformation resistance was higher in SSBT; therefore any beneficial effect of the heterogeneity in properties could be obviated. We did not measure fracture toughness directly nor measure cracking, thus we do not have data to relate cracking or toughness with plastic deformation resistance.
Studies with bisphosphonate-treated dogs have demonstrated changes in trabecular bone properties associated with drug treatment; however the data for cortical bone property changes with bisphosphonate treatment is less clear. In a recent study [41
], dogs were treated for as many as three years with alendronate at one of two dosage levels: 0.2 mg/kg/day, which approximates treatment for postmenopausal osteoporosis on a milligram per kilogram basis, or five times the clinical dose at 1.0 mg/kg/day. Cortical bone mechanical properties were not different between alendronate-treated and vehicle-treated animals as measured by four-point bending of femur specimens. However beagle dogs treated with high dose alendronate were found to have lower toughness in rib cortical bone [42
], indicating that the drug effect on cortical bone properties may be site specific, or evident only at high doses. High dose alendronate treatment was associated with lower toughness and increased strength in the trabecular bone of the vertebrae [12
]. Changes in trabecular bone were also found at the postmenopausal osteoporosis treatment dose, such as increased microdamage [43
], decreased toughness [44
] and increased indentation contact hardness [45
The associations of mechanical properties with bone formation rate in this study are consistent with our previous work [46
] and consistent with the change in mechanical properties of bone tissue with increasing tissue age [47
]. Trabecular contact hardness and plastic deformation resistance both increased with decreasing BFR, and trabecular modulus exhibited a trend of higher modulus in low BFR subjects. Trabecular mineral density increased with decreasing BFR, indicating that changes in mechanical properties may correspond with changes in mineral composition. These results are consistent with previous work in which mechanical properties of iliac biopsies from high and low bone turnover subjects were measured [46
]. Low bone turnover was associated with high contact hardness and modulus in trabecular tissue within both the normal subject group and the vertebral fracture group, while no differences in mechanical properties were observed based on fracture status (fracture/non-fracture). We note that in the present study the BFR effect explained only 12%, 4%, 11%, and 6% of the variability in contact hardness, modulus, plastic deformation resistance, and mineral density, respectively, indicating that there was substantial variability due to effects other than BFR.
Elastic modulus and plastic deformation resistance were only weakly correlated. A study of nanoindentation properties in trabecular bone reported no significant relationship between modulus and plastic deformation resistance [48
]. The weak relationship between plastic deformation resistance and elastic modulus is interesting because it indicates that the two properties may characterize different aspects of the bone hard tissue and provide independent information about the tissue. Trabecular and cortical bone modulus versus plastic deformation resistance relationships were significantly different from each other, with similar slopes but different intercepts. This indicated that for a given plastic deformation resistance value, the predicted modulus was higher for cortical bone than for trabecular bone but that the change in modulus per change in plastic deformation resistance was not different by bone region.
The experimental design was complicated by the fact that our sample set consisted of biopsies from two laboratories, in which biopsies were prepared and embedded by laboratory-specific protocols. The laboratory effect factored significantly in highlighting group differences in trabecular modulus (p=0.002 and p=0.053 with and without laboratory effect, respectively). In the analyses for trabecular and cortical plastic deformation resistance and trabecular contact hardness, the laboratory effect was not significant. In those models, the magnitudes of the group differences were large and likely masked any effect due to the laboratory. It is possible that the laboratory effect was associated with the difference in embedding PMMA properties between laboratories, in which the embedding PMMA for SMC biopsies was less than half as stiff as that used for HFH biopsies. However, the laboratory effect may be a confounding variable for other unmeasured parameters that account for some variability in the elastic modulus data. We suggest that embedding material modulus may be important to monitor when studying a biopsy collection from multiple laboratories or a set of biopsies prepared by multiple embedding protocols, though this effect may be confounded by parameters that were not accounted for in the study design.
We note that interpretation of our results is limited by a cohort effect, since the age-matched normal biopsies, young normal biopsies, and osteoporotic fracture biopsies were collected before 1992, while SSBT biopsies were collected within the last decade. Though two of the three comparison groups were age-matched to the SSBT group, the results may have been influenced by the different birth cohorts. Therefore, generalizations of the current observations should be made with caution.
Another limitation is the variability in the age of the biopsies, from the time of biopsy collection to the time when measurements were made for this study. Storage of biopsies can influence the nanoindenter-measured mechanical properties of embedded bone tissue [49
]. Mittra and colleagues compared the mechanical property measurements of canine cancellous bone embedded in four different epoxies. Elastic modulus of bone was found to increase significantly when measured six months after the tissue was embedded compared to the modulus measured immediately after embedding, while contact hardness did not change over time. While extrapolation of these results to the present study is limited due to the relatively short storage time window monitored, and the fact that PMMA was not one of the materials examined in this study, we note mechanical property differences found in our present study exhibit an opposite trend to the storage time effect. The biopsies from normal subjects and osteoporotic patients in our study had been stored for as long as thirty years, however the SSBT biopsies were stored for less than ten years and had stiffer and harder properties than the biopsies from other groups. Therefore we expect that the mechanical property differences detected in our study to be robust even with the variability in time from embedding to nanoindentation testing.
Regardless of the limitations of the study, differences between SSBT patients and control subjects indicate that fractures at cortical bone sites were associated with differences in tissue properties at a distant site. Plastic deformation resistance was higher in SSBT cortical and trabecular bone, indicating that changes in the plastic behavior of bone tissue may be associated with SSBT and/or long-term bisphosphonate treatment. The differences in mechanical and mineralization properties of trabecular tissue were consistent with trends associated with tissue aging, though these properties were only partially and weakly explained by BFR. Interestingly, cortical bone plastic deformation resistance properties were different in SSBT and not the elastic properties. The collagen component of bone tissue is associated with the post-yield properties of bone [50
], thus future studies may elucidate whether bone quality measures of the organic component, such as crosslinks, can help to explain the SSBT fractures.
Obviously, these associations are statistical rather than causal and must be interpreted cautiously. It is well known that the iliac bone biopsy is a useful but not a perfect model for bone remodeling and mechanical property differences at other sites of the skeleton. Consequently, direct examination of bone mechanical properties at the sites of fractures associated with SSBT will be necessary to make strong conclusions. In addition, comparison of the nanomechanical properties of bisphosphonate-treated patients with and without atypical fractures and/or SSBT must be performed before attributing any causal role for the nanomechanical differences we observed in the current study.