There is increasing evidence that osteoporotic fragility depends not only on decreased bone mass exclusively but also on other factors, such as microarchitecture, remodeling rate, and intrinsic bone material quality [
1‐
4]. This suggests the possibility that osteoporotic fractures might be favored by material properties of the bone tissue altered with age or osteoporosis. Bone material is a stiff and tough nanocomposite composed of staggered mineral crystal platelets with a thickness of a few nanometers, embedded within organic collagen-rich matrix. The two components—the mineral and the organic matrix—have extremely different mechanical properties, and the intrinsic properties of the composite material are, in part, determined by the degree and homogeneity of mineralization [
5] and by the mechanical properties of the organic matrix [
6].
Previous studies on transiliac bone biopsy samples have shown that bone mineralization density distribution (BMDD), as assessed by quantitative backscattered electron imaging (qBEI) [
7], is constant in normal adult trabecular bone and deviation from the normal distribution is associated with various diseases, such as increased bone fragility [
7,
8], osteoporosis [
9,
10], and osteogenesis imperfecta [
11,
12]. Very recently, differences in BMDD between cortical and trabecular bone have been reported and it was shown that in cortical bone there are more skeletal site–specific variations [
13]. However, the consequences of these observations for bone strength are not known, but they may potentially have a great impact since cortical bone plays a crucial role in maintaining mechanical competence at important skeletal sites such as the femoral neck.
In general, the thickness and shape of the cortical shell are determined by endocortical remodeling and periosteal apposition to optimize bone strength [
14]. In the particular case of the femoral neck, greater periosteal apposition and presumably less endocortical resorption in the inferior segment produce a thicker cortex than superiorly [
15‐
18]. Hip fractures account for nearly 30% of osteoporotic fragility fractures [
19] and are likely to arise primarily from structural changes in the cortical bone geometry [
20]. Indeed, it has been shown for women who sustained a hip fracture that the superior cortex, which is mainly subjected to tensile stress, revealed half of the thickness, while the inferior cortex rather increased its thickness compared to normal [
21,
22]. Reduced cortical thickness in hip fracture cases was associated with about 40–50% reduction of the calculated elastic stability, giving evidence for a higher susceptibility for local buckling in hip fracture cases, as reported very recently by Thomas et al. [
23]. At the material level, it was reported that the mean mineralization density is lower in the superior region compared to the inferior and, further, generally reduced in patients with hip fractures [
22].
Discussion
The present study was undertaken to explore whether femoral neck fragility could also be associated with changes at the bone material level. We used a unique combination of qBEI and nanoindentation, giving access to local mineral concentrations and mechanical properties at identical cortical sites at the micrometer level. sSAXS measurements provided additional information on the size of the mineral particles within the organic matrix.
Consistent with the data of Loveridge et al. [
22], we found generally that the superior cortex was significantly less mineralized than the inferior region. This is described by the parameter Ca
Peak, corresponding to an average mineral content in a large specimen area. This finding was also mirrored by the values of Ca
Ind measured locally at the exact positions of the nanoindents. These regional differences of mineral content may arise from local differences in bone turnover in the femoral neck [
4] and might be associated with different mechanical requirements. Indeed, the superior region is mainly loaded in tension, while the inferior region is predominantly loaded in compression [
21,
30,
31]. Other cortices principally loaded in compression were also found to be more highly mineralized [
32].
The measured decrease in bone mineralization density in the present fracture cases is in line with other reports on osteoporotic hip fractures [
22,
33]. To shed some light on the possible origins of this decrease, we note that BMDD is generally determined by two processes, bone turnover and mineralization kinetics [
8]. A change in either of these processes might, therefore, lead to a shift in the mean calcium content. For example, a shift toward lower mineralization has been reported for trabecular bone in postmenopausal osteoporosis [
9,
34] as a result of elevated bone turnover. Total bone turnover of the femoral neck was not evaluated in our set of biopsies, but previous studies gave evidence that there were no marked differences between cases and controls [
4]. In a more recent study, the lower mineral content found in the hip fracture cases could not be explained by altered bone turnover [
22]. Consequently, the authors speculated that inherent defects in the collagen matrix leading to altered mineralization kinetics might be responsible for the decreased mineralization [
22]. In this context, it is particularly striking for the fracture patients in this study that the reduction of mineral content was not accompanied by a corresponding reduction in Young’s modulus and hardness as measured by nanoindentation. Since these material properties depend on a combination of the structure and interaction of the mineral and the organic phase, some structural changes have to compensate for the reduced mineral content. Given that the size of mineral crystals is also unchanged, it becomes very likely that the organic part of the bone nanocomposite material is stiffer in the fracture cases, compensating for the lowered mineral content. A possible cause for this could be an altered cross-link pattern or a different mineral–organic interface, but the present study does not allow us to differentiate between such possibilities [
35]. In any case, it is plausible that a stiffer organic matrix will increase the bone material brittleness and, thus, predispose to the observed hip fractures. It should be noted that, during mineralization, the space of free exchangeable water is gradually replaced by mineral; thus, a lower mineral content could also correspond to a higher water content in the bone matrix. This could in principle lead to an altered PMMA-to-collagen ratio during embedding of a specimen with lower mineral content, contributing to the observed behavior of nanoindentation. However, densely mineralized bone matrix has been shown to be hardly altered by the embedding procedure since PMMA surrounds the bone matrix more than penetrating it [
36].
Of course, the conclusions outlined above depend on the fact that the measurements of elastic moduli are sensitive enough to actually reveal differences in elastic moduli if they exist. Indeed, in principle, a higher mineral content is expected to increase the elastic modulus and vice versa [
5,
37‐
40]. In this respect, it is quite rewarding to consider the observed differences in elastic modulus between the inferior and superior regions in each specimen. These differences are fully consistent with the increased mineral content in the inferior compared to the superior region. This means that differences in elastic modulus due to varying mineral content are measurable and, thus, indirectly supports the conclusions about the altered organic matrix in the fracture cases. The reason the correlation between mineral content and modulus remains intact in a specimen is that an altered collagen matrix in a given patient affects the superior and the inferior regions in the same way.
Nevertheless, it has to be emphasized that in mature lamellar osteonal bone the correlation between calcium content and elastic modulus is affected by a number of factors: (1) the range of Ca-concentration values is rather narrow, making a correlation analysis difficult and uncertain; (2) there is an inherent variation in apparent calcium levels due to the counting statistics accompanying the BE signal; and (3) there can be variations of modulus at given mineral content due to the mechanical anisotropy of the mineralized collagenous matrix. Indeed, it has been shown that the stiffness of mineralized collagenous tissue is higher in the direction of the collagen fibrils than perpendicular to it [
41]. However, within our line scans, hundreds of mechanical data of different lamellae were obtained and we can assume that, on average, the influence of the orientation of the organic matrix had only a minor effect on the outcome of the measurements. Hence, the observed lack in E and H reduction in the patients with fractures, though the mineralization density decreased, seems to be a robust finding. Further, it should be noted that nanoindentation was performed using fixed and embedded bone material, a procedure which in principle modifies the mechanical properties of the bone matrix. However, as with previous studies, all specimens were fixed in exactly the same way and, therefore, comparison between the samples was possible [
11,
27,
42].
In contrast to our findings, lower mineralization densities were reported very recently together with lowered microhardness values in the iliac crest from patients with idiopathic osteoporosis [
43]. However, these reported findings cannot be directly compared due to different cohorts of patients and different skeletal sites which were measured. Moreover, microhardness and hardness from nanoindentation measure mechanical properties at different size scales, a fact which is also reflected by differences in their absolute values.
There is growing evidence that the organic matrix plays an important role in affecting the mechanical parameters of bone and that age-related alterations of collagen properties are leading to increased fracture susceptibility [
40,
44]. Also, genetic influences on collagen structure, like by COL1A1 Sp1 alleles, have been discussed [
33]. Several studies have demonstrated that abnormalities in posttranslational modifications of collagen in patients with osteoporosis may lead to decreased bone strength [
45‐
47]. FTIR analysis has revealed differences in the spatial distribution of pyridinium/reducible collagen cross-links in the ilium of patients with osteoporotic fractures [
48], while decreased enzymatic cross-linking but excessive nonenzymatic cross-links have been reported in fracture cases compared to controls [
49].
In conclusion, our findings support the hypothesis that a modification of the organic matrix may be at the origin of a more brittle bone material facilitating bone failure in hip fracture cases. Indeed, the organic matrix is usually held responsible for reducing the brittleness of bone [
50,
51], and a stiffening of this matrix might compensate for the lower mineral content found in bone from hip fracture cases. A stiffer matrix would increase the brittleness of the bone material and, hence, the fragility of the bone tissue. Thus, it is essential that studies of bone fragility in aging, in osteoporosis, or in other bone diseases take into account the contribution of the organic phase in addition to the bone mineral.
Acknowledgments
We thank Gerda Dinst, Phaedra Messmer, and Sonja Lueger for careful sample preparations and qBEI measurements at the bone material laboratory of the Ludwig-Boltzmann-Institute of Osteology (Vienna, Austria). This study was supported by the AUVA (research funds of the Austrian Workers Compensation Board), by the WGKK (Viennese Sickness Insurance Funds), the FWF Projects P16880-B13 and P20646-B11 (Austria), the National Osteoporosis Society, the British Council, and the MRC.