Elsevier

Bone

Volume 110, May 2018, Pages 187-193
Bone

Full Length Article
Bone tissue aging affects mineralization of cement lines

https://doi.org/10.1016/j.bone.2018.02.004Get rights and content

Highlights

  • Assessment of calcium content in both cement lines and corresponding osteon

  • Cement lines have consistently higher calcium content than the enclosed osteon.

  • Cement line mineralization correlates positively with osteonal calcium content.

  • Cement lines' calcium content may reflect a tissue-age related factor.

Abstract

Cement lines are known as thin peripheral boundaries of the osteons. With a thickness below 5 μm their composition of inorganic and organic compounds has been a matter of debate. Here, we hypothesized that cement lines become hypermineralized and their degree of mineralization is not constant but related to the tissue age of the osteon. Therefore, we analyzed the calcium content of osteons and their corresponding cement lines in a range of different tissue ages reflected by osteonal mineralization levels in femoral cortical bone of both postmenopausal women with osteoporosis and bisphosphonate-treated cases. Quantitative backscattered electron imaging (qBEI) showed that cement lines are hypermineralized entities with consistently higher calcium content than their corresponding osteons (mean calcium content: 29.46 ± 0.80 vs. 26.62 ± 1.11 wt%; p < 0.001). Micro-Raman spectroscopy complemented the qBEI data by showing a significantly higher phosphate/amide I ratio in the cement lines compared to the osteonal bone (8.78 ± 0.66 vs. 6.33 ± 0.58, p < 0.001), which was both due to an increased phosphate peak and a reduced amide I peak in cement lines. A clear positive correlation of cement line mineralization and the mineralization of the osteon was observed (r = 0.839, p = 0.003). However, the magnitude of the difference between cement line and osteonal calcium content decreased with increased osteonal calcium content (r = −0.709, p < 0.001), suggesting diverging mineralization dynamics in these osseous entities. The number of mineralized osteocyte lacunae per osteon bone area correlated positively with both osteonal and cement line calcium content (p < 0.01). The degree of mineralization of cement lines may represent another tissue-age related phenomenon, given that it strongly relates to the osteonal mineralization level. Understanding of the cement lines' mineralization and their changes in aging and disease states is important for predicting crack propagation pathways and fracture resistance mechanisms in human cortical bone.

Introduction

In human cortical bone, bone remodeling gives rise to cylindrical structural elements termed Haversian systems or secondary osteons [1]. They are constituted of a central canal surrounded by concentric osseous lamellae, including interlamellarly located osteocyte lacunae and the outermost covering sheath often designated as “cement line” (CL) or “cement sheath”. The cement line is considered the boundary of the osteon [2,3], separating more freshly deposited osteonal bone packets from predominantly older surrounding bone (interstitial bone). Apart from representing a clear boundary, the cement line also serves as a material bond between the osteon and its' surrounding [4,5]. Although cement lines are often considered as impermeable barriers, we recently observed that in younger individuals osteocyte canaliculi often penetrate the cement line and anchor in the surrounding bone, thus establishing communication between bone segments of different tissue ages [6]. This is essential for a successful adaptation of the whole bone to mechanical demands [6]. In contrast, aged individuals show significantly fewer connections between osteocyte lacunae from neighboring osteons or interstitial bone, reflecting impaired communication between tissue structural units [6].

The composition of the cement line was often subject for debate [7], and it was for years unclear if it is mineral-deficient [8,9] or mineral-rich [10,11]. Lack of agreement even about the basic composition of cement lines likely precluded in-depth analyses focusing on their relationship with mineralization processes. However, in a comprehensive study using backscattered electron microscopy Skedros et al. [12] suggested the mineral-rich (and/or collagen deficient) nature of the cement lines. In an early study, Philipson compared the content of sulfur and the arrangement of mineral fraction between the cement line and the adjacent osteon in orangutans and whales but found no significant differences [13]. Several studies have shown that non-collagenous proteins (i.e. osteopontin, glycosaminoglycans, osteocalcin, and bone sialoprotein) are a component of the cement line's matrix [12,14,15].

In general, new bone matrix during osteonal remodeling is produced within a few months [1,16]. Yet, the mineral is deposited at a much slower rate and the secondary mineralization takes place over longer time periods [17,18]. Hence, it was shown that the overall degree of mineralization in the osteon increases with maturation and tissue age [[19], [20], [21]]. Fuchs et al. have shown a linear relationship between osteonal age and secondary mineralization (measured by Fourier-transform infrared imaging - FTIR) in rabbits for osteons up to an age of 350 days [22]. Unfortunately comparative data cannot be obtained for humans easily, since it would require manifold administration of labeling agents. Akkus et al. measured the mineral content of primary bone packets in men with Raman microspectroscopy and found it to increase continuously over two decades in non-remodeled bone packets after which a plateau was reached [23]. However, it is unknown if the cement line mineralization also changes with age and whether a relationship between osteon and cement line mineralization processes exists.

Indeed, bone material is a mosaic of packets with different degrees of mineralization, and the interface between areas with differing material properties has been considered as an important site for crack deflection [12,[24], [25], [26], [27]]. The characteristics of the cement lines are therefore of great importance for fully understanding the paths of crack propagation and deflection through bone [8,12,28,29], as well as general mechanisms of fracture resistance. Understanding the role of cement lines will help to create a more complete view of the complex fracture resistance properties of bone, which can be further influenced by porosity [30]. This is particularly relevant in osteoporotic individuals that often sustain a catastrophic fracture due to extensive crack growth, as well as in bisphosphonate-treated osteoporotic patients where remodeling and mineralization are likely changed [5,31].

Considering that increasing mineralization, e.g. through crystal growth occurs frequently in many biological tissues including bone [32,33], we hypothesized that cement lines are not always equally mineralized, but their composition is related to the osteon's mineralization level – a surrogate marker of osteon's tissue age. To test this hypothesis, we have evaluated the mineralization of osteons and their corresponding cement lines in osteoporotic bone from untreated and bisphosphonate-treated elderly individuals. The understanding of cement line mineralization in relation to osteonal mineralization is important for interpreting and determining crack propagation and fracture resistance mechanisms in human cortical bone.

Section snippets

Specimen collection and preparation

The specimens used in this study were obtained from ten elderly postmenopausal women with osteoporosis, half of which were treatment-naïve (age: 81 ± 5 years) and the other half received antiresorptive treatment with alendronate (age: 86 ± 8 years, treatment duration: 6 ± 1.6 years, dosage: 10 mg/day or 70 mg/week). Osteoporosis was diagnosed by dual-energy X-ray absorptiometry (DXA) measurements at autopsy, and all patients' characteristics were available from medical records and autopsy

Results

The composition of the osteons and their cement lines in the femoral cortical bone in elderly osteoporotic individuals was analyzed (Fig. 1). Quantitative backscattered electron imaging (Fig. 2) confirmed that cement lines are hypermineralized, showing a mean calcium content of 29.46 ± 0.80 Ca wt% (Fig. 2A,D). Moreover, cement lines were constantly higher mineralized than their corresponding osteons in all examined tissue ages (p < 0.001, Table 1, Fig. 2A).

However, there was a clear positive

Discussion

Assessing the calcium content of cement lines belonging to osteons of variable mineralization, we show that the cement lines are indeed hypermineralized structures and that their degree of mineralization may represent another tissue-age related factor in the bone quality framework. Namely, although cement lines had a consistently higher calcium content than the osteon that they are surrounding, they showed a range of mineralization levels that were strongly dependent on the osteonal

Conclusions

Backscattered electron imaging and micro-Raman spectroscopy revealed that cement lines are hypermineralized structures. Moreover, cement lines' calcium content may represent another tissue-age related factor, which should be considered in the bone quality framework, given that it is strongly correlated to the osteonal calcium content, a surrogate marker of tissue age. Further investigation of the nature of cement line mineralization will help to understand fracture resistance mechanisms in

Acknowledgements

The authors acknowledge the support from the German Research Foundation (DFG, BU 2562/2-1/3-1), GRK 1896, cluster of excellence “Engineering of Advanced Materials” at the Friedrich-Alexander-Universität Erlangen-Nürnberg, the European Union (H2020, npSCOPE, 720964), Alexander von Humboldt Foundation (1162414 - HFST-P/2015), Southeast-European Cooperation of the University Medical Center Hamburg-Eppendorf, and the Serbian Ministry of Science (III45005).

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