Micrometer-Sized Magnesium Whitlockite Crystals in Micropetrosis of

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Micrometre-sized magnesium whitlockite crystals in micropetrosis of bisphosphonate-exposed human alveolar bone Furqan Ali A Shah, Bryan E J Lee, James Tedesco, Cecilia L Wexell, Cecilia Persson, Peter Thomsen, Kathryn Grandfield, and Anders Palmquist Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b02888 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Micrometre-sized magnesium whitlockite crystals in micropetrosis of bisphosphonate-exposed human alveolar bone Furqan A. Shah1,2,4, Bryan E. J. Lee3, James Tedesco4, Cecilia L. Wexell1,2,5, Cecilia Persson6, Peter Thomsen1,2, Kathryn Grandfield3,4, Anders Palmquist1,2*. 1

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden

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BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden 3

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School of Biomedical Engineering, McMaster University, Hamilton, Canada

Department of Materials Science and Engineering, McMaster University, Hamilton, Canada

Department of Oral and Maxillofacial Surgery, Public Dental Service, Region Västra Götaland, SÄS, Borås, Sweden 6

Division of Applied Materials Science, Department of Engineering Sciences, The Ångström Laboratory, Uppsala University, Uppsala, Sweden

KEYWORDS osteocyte lacuna; mineralisation; micropetrosis; magnesium; whitlockite; bisphosphonates

ACS Paragon Plus Environment

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ABSTRACT: Osteocytes are contained within spaces called lacunae and play a central role in bone remodelling. Administered frequently to prevent osteoporotic fractures, anti-resorptive agents such as bisphosphonates suppress osteocyte apoptosis and may be localised within osteocyte lacunae. Bisphosphonates also reduce osteoclast viability, and thereby hinder the repair of damaged tissue. Osteocyte lacunae contribute to toughening mechanisms. Following osteocyte apoptosis, the lacunar space undergoes mineralisation, termed ‘micropetrosis’. Hypermineralised lacunae are believed to increase bone fragility. Using nano-analytical electron microscopy with complementary spectroscopic and crystallographic experiments, post-apoptotic mineralisation of osteocyte lacunae in bisphosphonate-exposed human bone was investigated. We report an unprecedented presence of ~80 nm to ~3 µm wide, distinctly facetted, magnesium whitlockite [Ca18Mg2(HPO4)2(PO4)12] crystals and consequently altered local nanomechanical properties. These findings have broad implications on the role of therapeutic agents in driving biomineralisation and shed new insights into a possible relationship between bisphosphonate exposure, availability of intracellular magnesium, and pathological calcification inside lacunae.

Osteoporosis contributes to bone fragility in the ageing population, particularly women, increasing susceptibility to hip and vertebral fractures1. Osteocytes reside within confined spaces called lacunae as the master orchestrators of bone remodelling2. Termed ‘micropetrosis’ (Figure S1), apoptotic osteocyte fragments within the lacunar space are replaced by mineral nodules3, 4, which in osteoporotic human bone are poorly crystalline, magnesium-incorporated hydroxyapatite5. Mineral nodules may coalesce6, giving rise to hypermineralised lacunae which increase the fragility of ageing bone7. Bisphosphonates preserve bone mass and exert an antiapoptotic effect on osteocytes8, may be localised within lacunae9, and in high doses induce a risk


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for developing osteonecrosis of the jaw10. Bisphosphonates also reduce osteoclast viability, and hinder the repair of damaged tissue11. Since osteocyte lacunae contribute towards toughening mechanisms12, it is essential to understand the eventual fate of the lacunar space.

Hypermineralised lacunae in alveolar bone obtained from women who had received bisphosphonates (BP) were identified using backscattered electron scanning electron microscopy (BSE-SEM) (Table S1). These contained equiaxed, ~80 nm to ~3 µm-wide, spherical to markedly rhomboidal mineral nodules (Figure 1). Randomly oriented, acicular nanocrystallites occupied the internodular space and a distinct hypermineralised ring4 frequently enclosed the lacuna. Mineral nodules were ~120% harder and ~50% stiffer than the surrounding bone matrix (Figure 1, Figure S2), substantiating the previously postulated contribution of hypermineralised lacunae to increased brittleness of ageing bone7. Increased prevalence of hypermineralised lacunae would affect mechanical and biological function, however technical difficulties pose significant challenges in accurate determination of their relative proportion. Raman spectroscopy revealed a whitlockite-like structure of these nodules13 with the ν2 PO43- bending vibration at ~407 cm-1 and the ν1 PO43- symmetric stretching mode at ~970–972 cm-1 (Figure 1, Figure S3). For bone apatite, ν2 PO43- and ν1 PO43- bands are typically observed at 432 cm-1 and 958–960 cm1

, respectively. Differences in the crystallographic structures, e.g., intratetrahedral O–P bond

lengths and O–P–O angles, give rise to differences in the Raman spectra of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HAp) where shifts of the PO43- modes are due to deformations in PO43- tetrahedra induced by the surrounding ions14.


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High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) also showed spherical and rhomboidal nodules, and occasionally spherical nodules with a fuzzy exterior, a core-shell structure, and ~5–10 nm porosities throughout the bulk (Figure 2). The rhomboidal nodules were strikingly facetted. Distributed heterogeneously, internodular acicular nanocrystallites were 5–7 nm thick, in agreement with the edge-on dimensions of extrafibrillar apatite platelets15, but no collagen fibrils were detected inside the lacunar space. The hypermineralised ring was 200–500 nm wide, also devoid of collagen fibrils, and comprised of smaller, closely packed, nanocrystallites.

To confirm the crystalline phases, we performed selected area electron diffraction (SAED). Rhomboidal nodules were almost exclusively single-crystals of magnesium whitlockite16,

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(Figure 2, Figure S4, Table S2). Spherical nodules were also highly crystalline, with the exception of those with a fuzzy exterior that were polycrystalline. Internodular acicular nanocrystallites were apatite. In bone matrix, mineralised collagen fibrils aligned parallel to the image plane showed a preferred orientation of the (002) and (004) crystallographic planes, i.e., the c-axis, of bone apatite.

Energy dispersive X-ray spectroscopy (EDX) revealed higher Mg, P, and O content of the mineral nodules with higher Mg/Ca and lower Ca/P ratios than bone matrix (Figure 3). Elemental mapping showed Mg, P, and O enrichment of mineral nodules compared to bone matrix, internodular nanocrystallites, and the hypermineralised ring (Figure 3, Figure S5). Electron energy loss spectroscopy (EELS) confirmed the lower Ca and higher O content of mineral nodules than bone matrix, supporting the EDX analysis (Figure 4, Figure S6). Core-


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losses were observed at characteristic onset energy levels attributable to the presence of C, Ca, and O. The Ca L2,3-edge and O K-edge spectra of mineral nodules and bone matrix showed peaks typical of calcium phosphate phases found commonly in bone. Although it was not possible to directly distinguish between mineral nodules and bone matrix, energy-loss near-edge structure (ELNES) of bone matrix showed higher amorphous carbon from the organic inclusions in nanoporosities and higher mineral carbonate (Table S3).

Additional experiments were performed using bone obtained from healthy, non-osteoporotic women (Ctrl). Hypermineralised lacunae where individual mineral nodules could not be discerned readily by BSE-SEM, primarily contained acicular apatite nanocrystallites (Figure S7). These were oriented randomly with negligible Mg-enrichment compared to bone matrix, similar to those occupying the internodular space in BP bone, and occasionally stacked together as dense aggregations (Figure S8).

We demonstrate the formation of discrete, highly crystalline magnesium whitlockite nodules in bisphosphonate-exposed human bone. Homogeneous distribution of magnesium throughout the nodule bulk, higher magnesium content than internodular acicular nanocrystallites, the distinctive rhomboidal morphology, and their single-crystal-like nature all indicate that these structures are not formed by ion-substitution of bone apatite. Although Mg2+ incorporation into the hydrated hydroxyapatite (HAp) surface is energetically favourable, Mg2+ incorporation into bulk HAp is highly unstable, with respect to Mg2+/Ca2+ exchange with the aqueous environment and with respect to separation into Mg-rich phosphate [Mg3(PO4)2] and hydroxide [Mg(OH)2] phases, and hence dependent on crystal growth kinetics18. The concentrically arranged, core-shell


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structure noted for a few of the nodules suggests that their formation may be associated with a calcification centre, followed by intermittent growth and inhibition, resulting in a heterogeneous, layered structure19.

In addition to highly crystalline mineral nodules, the hypermineralised lacuna contains randomly oriented, acicular apatite nanocrystallites. Isolated whitlockite formations have not previously been identified in bone. During bone development, mineralisation progresses from an amorphous calcium phosphate (ACP) precursor phase towards a highly crystalline mineral20. In vitro and in vivo, the initially precipitated ACP exists as aggregates of primary nuclei which quickly hydrolyse to more stable phases21. However, Mg2+ ions kinetically hinder nucleation and growth of HAp by competing with Ca2+ ions for lattice sites 22. Though less stable than HAp and generally not found in bone, several naturally occurring and synthetic phases having a TCP formula unit, Ca3(PO4)2, are known to exist. Of these, whitlockite is the most common and typically forms under physiological conditions23. The β-TCP structure contains Ca2+ vacancies that are not large enough to accommodate Ca2+ but allow for Mg2+ inclusion thereby stabilising the structure24. In pathological calcifications, whitlockite has been reported in uremic calcifications19, sialoliths25, articular cartilage26, and tuberculous pulmonary tissue27. In contrast, highly crystalline, magnesium-containing HAp particles have been reported in the aortic valve28.

Results from Ctrl bone suggest that in the absence of factors known to suppress apoptosis, i.e., bisphosphonates, it is possible for mineralisation within lacunae to proceed randomly from many nucleation centres, resulting in small acicular crystallites without a preferred orientation. On the other hand, where bisphosphonates were used, nodule-free hypermineralised lacunae are rare.


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With age, a considerable fraction of osteocyte lacunae become partially or completely filled with mineral nodules. Apoptotic osteocytes release vesicles that are able to accumulate Ca2+. Lacunar mineralisation thus results in the appearance of mineral nodules varying in size and morphology. Nodules with high Mg2+ content continue to grow, leading to mineral agglomeration, fusion of nodules, and eventual occlusion of the osteocyte lacuna. Exemplifying pathological calcification, magnesium-containing mineral nodules may exist as isolated, easily identifiable, highly crystalline entities rather than magnesium-enriched calcium phosphate dispersed within bone matrix. Confined to the osteocyte lacuna, these nodules give rise to localised areas of increased stiffness. The absence of collagen in addition to higher Young’s modulus of β-TCP than HAp29 would thus contribute towards decreased strain-to-failure compared to the bone matrix. An explanation for magnesium whitlockite formation may be that lower intracellular pH of apoptotic cells30 persists for longer due to the apoptosis suppression effect of bisphosphonates. Intracellular acidification coupled with high intracellular Mg2+ content thereby provides an environment which favours magnesium whitlockite formation31. Although a direct association between bisphosphonate exposure and magnesium whitlockite crystal formation is not implied, our data provides novel insights into the lacunar mineralisation. The likelihood of whitlockite crystal formation is not excluded for other therapeutic or disease states where sufficient magnesium and an acidic pH environment are available. As whitlockite is less soluble than hydroxyapatite in acidic pH conditions32, whitlockite formation may further contribute towards an already impaired ability of osteoclasts, under the influence of bisphosphonates, to remove the mineral component of bone – a critical driving step in bone remodelling and maintenance of healthy, adaptive, mechanically competent tissue. These findings are fundamental in understanding the processes that occur within the osteocyte lacuna


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long after apoptosis, and point towards structural consequences of anti-resorptive agents in addition to their well-documented biological effects on bone cells.


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Figure 1. Hypermineralised lacunae in BP bone. (a) Equiaxed, spherical (white arrow) and rhomboidal (black arrow) nodules occupy the lacuna (BSE-SEM). (b) Spherical nodules (top) appear porous. Rhomboidal nodules (bottom) are comparatively dense. (c) Internodular acicular nanocrystallites. (d) The hypermineralised ring surrounds a lacuna (top). Complete mineral occlusion of the lacunar space (bottom). (e) Size distribution of mineral nodules measured using


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BSE-SEM (median ± interquartile range). (f) Nanoindentation load-displacement curves of mineral nodules (green) and bone matrix (blue). (g) Hardness (H) and reduced elastic modulus (Er) of mineral nodules are greater than bone matrix (p < 0.0001; Mann-Whitney U test). (h) Mineral nodules exhibit only minor laser-induced damage (arrow) after Raman spectral acquisition. Damage to the sample surface (#) from extended, ~30 min, laser exposure (BSESEM). (i–j) Raman maps of the ν2 PO43- mode characterising whitlockite and hydroxyapatite, respectively. (k–l) Raman spectra of a mineral nodule and bone matrix (as indicated in i–j). Mineral nodules (green) show pronounced shifts of the ν2 PO43- peak towards lower wavenumbers and the ν1 PO43- peak towards higher wavenumbers, compared to bone matrix (blue), resembling reference spectra (black) of whitlockite (in k; RRUFF ID: R080052) and hydroxyapatite (in l; RRUFF ID: R060180), respectively. All scale bars = 2 µm.


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Figure 2. Highly crystalline spherical and rhomboidal nodules. (a–b) Equiaxed, spherical (S) and rhomboidal (R) nodules, and occasionally, spherical nodules with a fuzzy exterior (Sf) are observed (HAADF-STEM). (c) The hypermineralised ring appears collagen-free and is comprised of randomly oriented apatite nanocrystallites. (d) Rhomboidal nodules appear dense.


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Spherical nodules exhibit nanoporosities. (e) Spherical nodules are highly crystalline. Progressively larger nodules (corresponding SAED patterns are labelled 1–3; 600 nm selected area aperture) range from being single-crystal-like to being a polycrystal having a preferred orientation of crystallographic directions. (f) Rhomboidal nodules (labelled 4–6) also exhibit a single-crystal-like nature. In contrast, a spherical nodule having a fuzzy exterior (labelled 7) is polycrystalline. (g–h) Indexed SAED patterns show good agreement with magnesium whitlockite16,

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. (i) Acicular apatite nanocrystallites occupying the internodular space are

inhomogenously oriented (corresponding SAED pattern is labelled 8). (j) In bone matrix, mineralised collagen fibrils (corresponding SAED pattern is labelled 9) aligned parallel to the image plane show a preferred orientation of the (002) and (004) planes, i.e., the c-axis, of bone apatite. Scale bars in a–c = 500 nm; d, e, i, and j = 200 nm; and f = 2 µm. All SAED scale bars = 5 1/nm.


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Figure 3. Mineral nodules are magnesium, phosphorus, and oxygen rich. (a) Mineral nodules (arrows) contain more Mg, P, and O (averaged EDX spectra; n=12), with consequently higher Mg/Ca and lower Ca/P ratios than bone matrix (p < 0.0001; Mann-Whitney U test). (b) EDX elemental maps. Mg, P, and O are distributed homogenously throughout the nodule bulk. All scale bars = 2 µm.


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Figure 4. Mineral nodules contain less carbonate and calcium than bone matrix. (a) In rhomboidal nodules, Ca and O are distributed homogeneously throughout the bulk. The fuzzy exterior of the spherical nodule has higher Ca and lower O than the bulk, resulting in a core-shell structure. C, Ca, and O intensity maps and RGB colour-merge of C (red), Ca (green), and O (blue). (b) EELS elemental line profiles (indicated in a). (c) C K-edge, Ca L2,3-edge, and O K-


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edge spectra representing bone matrix (~1200 pixels), and spherical (~1200 pixels) and rhomboidal (~2400 pixels) nodules. Mineral nodules contain less amorphous carbon, carbonate, and calcium, but more oxygen than bone matrix. A pre-peak feature (~528 eV), noted here only, is likely an electron beam induced artefact. Scale bar in a = 500 nm.


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ASSOCIATED CONTENT Supporting Information Histological appearance of viable osteocytes and hypermineralised lacunae; all nanoindentation load-displacement curves; additional Raman spectroscopy, SAED, STEM-EDX, STEM-EELS data sets; all data from additional experiments performed using Ctrl bone; patient clinical data; measured and reference d spacings for whitlockite; and ELNES peak assignments for the C Kedge. The following files are available free of charge: SupportingInformation.PDF

Corresponding Author * E-mail: [email protected]

Author Contributions F.A.S, K.G, and A.P designed the experiments. F.A.S, J.T, and K.G performed the experiments. F.A.S, B.E.J.L, J.T analysed the data. C.L.W provided the samples and patient data. C.P helped with nanoindentation experiments. F.A.S, K.G, A.P, and P.T wrote the manuscript. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.


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Funding Sources This study was supported by the Swedish Research Council (grant K2015-52X-09495-28-4), the BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Region Västra Götaland, an ALF/LUA grant, the IngaBritt and Arne Lundberg Foundation, the Dr. Felix Neubergh Foundation, Promobilia, the Hjalmar Svensson Foundation, and the Materials Science Area of Advance at Chalmers and the Department of Biomaterials, University of Gothenburg. K.G. acknowledges financial support from the Natural Sciences and Engineering Council of Canada (NSERC) Discovery Grant program.

ACKNOWLEDGMENTS Andreas Korinek, Carmen Andrei, and Travis Casagrande from the Canadian Centre for Electron Microscopy, a facility supported by NSERC and other government agencies, and Dan Wu and Charlotte Skjöldebrand from the Ångström Laboratory, Uppsala University are gratefully acknowledged.

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