Aluminum and iron can be deposited in the calcified matrix of bone exostoses.

Aluminum and iron can be deposited in the calci fi ed matrix of bone exostoses

Daniel Chappard

⁎ , Guillaume Mabilleau

, Didier Moukoko

, Nicolas Henric

, Vincent Steiger

, Patrick Le Nay

, Jean-Marie Frin

, Charlotte De Bodman

aGEROM Groupe Etudes Remodelage Osseux et bioMatériaux—LHEA, IRIS-IBS Institut de Biologie en Santé, CHU d'Angers, LUNAM Université,49933, ANGERS Cedex, France

bSCIAM, Service Commun d'Imagerie et Analyses Microscopiques, IRIS-IBS Institut de Biologie en Santé, CHU d'Angers, LUNAM Université, 49933 Angers, Cedex, France

cChirurgie Pédiatrique Orthopédique, CHU d'Angers, 49933, Angers, Cedex, France

dDépartement de Chirurgie Osseuse, CHU d'Angers, 49933, Angers, Cedex, France.

a b s t r a c t a r t i c l e i n f o

Article history:

Received 14 April 2015

Received in revised form 2 September 2015 Accepted 14 September 2015

Available online xxxx

Keywords:

Aluminum Bone matrix Exostosis Hydroxyapatite Mineralization Iron

Exostosis (or osteochondroma) is the most common benign bone tumor encountered in children and adults. Ex- ostoses may occur as solitary or multiple tumors (in the autosomal syndromes of hereditary multiple exostoses).

Exostoses are composed of cortical and medullary bone covered by an overlying hyaline cartilage cap. We have searched iron (Fe) and aluminum (Al) in the matrix of cortical and trabecular bone of 30 patients with exostosis.

Al³⁺and Fe³⁺are two cations which can substitute calcium in the hydroxyapatite crystals of the bone matrix.

The bone samples were removed surgically and were studied undecalcified. Perls' Prussian blue staining (for Fe) and solochrome azurine B (for Al) were used on the histological sections of the tumors. Al³⁺was detected histochemically in 21/30 patients as linear bands deposited by the osteoblasts. Fe³⁺was detected in 10 out of these 21 patients as linear bands in the same locations. Fe³⁺and Al³⁺were not identified in the bone matrix of a control group of 20 osteoporotic patients. Energy X-ray Dispersive Spectrometry failed to identify Fe and Al in bone of these tumors due to the low sensitivity of the method. Wavelength Dispersive Spectrometry iden- tified them but the concentrations were very low. Histochemistry appears a very sensitive method for Fe³⁺and Al³⁺in bone.The presence of these two metals in the exostoses advocates for a disturbed metabolism of osteo- blasts which can deposit these metals into the bone matrix, similar to which is observed in case of hemochroma- tosis with Fe³⁺.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Exostosis (or osteochondroma) is the most frequent benign bone tumor encountered in children or adults. Exostosis may occur as solitary or multiple tumors in the case of an autosomal genetic disorder (the Multiple Hereditary Exostoses—MHE). MHE affects 1/50,000 people and is caused by a mutation in the Golgi-associated heparin-sulfate polymerases EXT1 or EXT2[1]. However, the pathophysiology of isolat- ed exostosis is unknown but it is likely that a dysregulation of osteoprogenitor cells (chondrocytes and osteoblasts) leads to these bony proliferations[2,3]. Exostoses are cartilage-capped tumors which can be sessile or pedunculated. The cartilage covers a shell of cortical bone on which a network of trabecular bone is internally appended.

The center of the exostosis is in continuity with the medullary canal of the bone and contains bone marrow with more or less hematopoietic cells[3].

The last decade has witnessed a considerable interest in the quality of the bone matrix in metabolic bone diseases. Bone quality is“an

umbrella term that describes a set of characteristics that influences bone strength and explain the interrelationships of these characteris- tics” [4]. The different determinants of bone strength have been reviewed elsewhere together with the various methods available to an- alyze them[5]. Histochemistry is a powerful tool to characterize the mineralization of the bone matrix and to identify the presence of certain metal ions abnormally present in it where they can alter bone quality.

The aim of the present study was to analyze the calcified bone ma- trix in a series of human exostoses in search of two metal ions (alumi- num and iron) known to interfere with calcification[6,7]and to alter osteoblast functions[8,9]. Histochemistry and X-ray-based spectroscop- ic methods coupled with Scanning Electron Microscopy (SEM) were used.

2. Patients and methods

2.1. Participants and histological analysis

Between 2010 and 2015, 30 patients were operated on the orthope- dic or pediatric surgery department for one or more exostoses. Tumors were sent to the bone histopathology unit where they were processed undecalcified after embedding in poly (methylmethacrylate). Sections Journal of Inorganic Biochemistry xxx (2015) xxx–xxx

⁎ Corresponding author at: GEROM—LHEA, IRIS-IBS Institut de Biologie en Santé, LUNAM Université Nantes Angers Le Mans, CHU d'Angers, 49933 ANGERS Cedex, France.

E-mail address:daniel.chappard@univ-angers.fr(D. Chappard).

http://dx.doi.org/10.1016/j.jinorgbio.2015.09.008 0162-0134/© 2015 Elsevier Inc. All rights reserved.

Contents lists available atScienceDirect

Journal of Inorganic Biochemistry

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j i n o r g b i o

were cut dry on a heavy-duty microtome equipped with tungsten car- bide knives. They were stained by Goldner's trichrome (for identifica- tion of osteoid and calcified matrix)[10]. Histochemical identification of osteoclasts (bone resorbing cells) was done by the tartrate resistant acid phosphatase method. The Perls' and solochrome azurine B stainings were used for the identification of Fe³⁺and Al³⁺, respectively[11,12].

These histochemical reactions were done in cleaned glass vials and the technicians never used metallic forceps during the staining to avoid con- tamination. Some samples were analyzed by microcomputed tomogra- phy to have a 3D evaluation of these tumors. Morphometric analysis was done but only the osteoid seam thickness will be considered here as a parameter characterizing a mineralization defect (O.Th inμm, normal b15μm).

2.2. Control cases

In our bone laboratory, the histochemical staining of iron and alumi- num is done on all bone samples since a decade. We chosed the 20 most recent bone biopsies refered for osteoporosis as a control group.

2.3. Metal atom characterization by spectroscopy

Poly (methylmethacrylate) blocks containing the bone samples were polished with 0.5μm diamond particles, carbon-coated and ob- served by SEM (EVO LS10, Carl Zeiss Ltd., Nanterre, France) equipped with an Energy Dispersive X-ray spectrometer (EDS–INCA, Oxford, UK). The EDS analysis was performed with the Inca systemfitted with a X-max 20 mm² silicon drift detector (Oxford Instruments, High Wycombe, UK). Prior to quantitative analysis, samples were polished with diamond particles (1-μm thickness) to reach a surface roughness less than 10 nm. The system was calibrated with pure cobalt (Micro- Analysis consultants Ltd., St. Ives, UK) and quantitative analysis was per- formed with an accelerating voltage of 20 keV, a measured probe cur- rent of 500 pA and a working distance of 8.5 mm. During EDX analysis, the specimen is bombarded with a focalized electron beam in- side the SEM. The electrons collide with the atoms' own electrons, ejecting some of them out of their orbit. A position vacated by an ejected electron in an inner shell is then replaced by a higher-energy electron from an outer orbit. To do so, this transferring outer electron liberates some of its energy by emitting an X-ray. The atom of every element re- leases X-rays with unique amounts of energy during the transferring process. In our system, a minimum of 200,000 coups were recorded and the local atomic concentration was calculated with the semiQuant algorithm using the XPP matrix correction. The minimum detectable limit of this setup is of 0.05%–0.1%; (atomic %), that means that an ele- ment with a concentration below 1/2000 atoms will not be detected [13].

The blocks containing the highest amount of aluminum and iron were also examined on a Wavelength Dispersive X-ray spectrometer (WDS, Inca Ware 500, Oxford Instrument, UK) installed on a Merlin SEM (Carl Zeiss Ltd) equipped with afield emission gun. Analyses were also performed at 20 kV. During WDS analysis, the specimen is also bombarded with a focalized electron beam inside the SEM to iden- tify the elemental constituents comprising the sample. This results in X- ray emission in the same way from the bombarded atoms. The wave- length of the X-rays diffracted into the detector are selected by varying the position of an analyzing crystal. Unlike EDS, WDS reads or counts only the X-rays of a single wavelength at time and does not produce a spectrum of wavelengths simultaneously.

3. Results 3.1. Histopathology

The mean age of the patients with exostosis was 23.5 ± 18.4 yr.

(extremes 3 and 80 yr.). There were three patients with MHE; one

patient with six analyzed exostoses and two patients with two ex- ostoses removed during the same surgical intervention (Table 1).

The total number of exostoses was 38 in this series of 30 patients.

The tumors were either sessile or pedunculated and the X-ray as- pect of these exostoses is illustrated inFig. 1. The microCT aspect is depicted inFig. 2and in the video appearing as supplementary material.

The histopathological aspect was consistent with classical de- scriptions of a cartilage covering cap, more or less developed which surmounts the bony part of the tumor (Fig. 3). Cartilaginous columns mimicking a growth plate were observed but the number of trabec- ula was usually reduced, giving broad trabeculae with a central core made of calcified cartilage. Foci of calcified cartilage were some- times observed in the discontinuous cortical shell made of Haversian systems and lamellar bone. The trabeculae were most often com- posed of lamellar bone excepted in the youngest children and in two cases of sub-ungueal exostoses. Trabecular and cortical bones were composed of calcified bone matrix and the closest areas to the cartilage had a high bone remodeling level. Numerous foci of osteo- clasts were observed in these areas. Numerous osteoid seams were observed, but O.Th was never increased (excepted when non- lamellar woven bone was present).

The mean age of the control patients with osteoporosis was (mean age 54.5 ± 16.8 y; range 32–73 y.). There were 12 females and 8 males. Thefinal diagnosis was: glucocorticoid induced osteoporosis (N = 5), mastocytosis (N = 3); post-menopausal osteoporosis (N = 4) and idiopathic male osteoporosis (N = 8).

3.2. Histochemical analysis

The most striking fact was the presence of aluminum and iron in the calcified bone matrix or in the calcified cartilaginous matrix.

Table 1

Clinical description of cases. MHE: patients with a Multiple Hereditary Exostoses disease.

For the semi-quantitative score:−is for an absence of staining, +for the presence of a lim- ited number of stained bands; ++ for numerous stained bands; +++ very high amount of metal bands in the bone matrix.

Case Gender Age Localization Aluminum Iron

1 f 22 Right distal tibia +++ +++

2 m 54 Right scapula − −

3 f 51 Right femur ++ +

4 f 31 Left tibia − −

5 m 45 Right lesser trochanter − −

6 f 27 Right 2nd metatarsal (MHE) − −

7 m 8 Left humerus + −

8 m 22 Right distal tibia + −

9 f 14 Right distal tibia − −

10 m 31 Right peritrochanter ++ +

11 m 14 Left humerus +++ +++

12 m 3 Right scapula − −

13 m 6 Sub-ungueal 3rd metatarsal bone − −

14 m 12 Left tibia +++ +++

15 m 11 Tibia (2 exostoses) +++ +++

16 m 13 Right humerus +++ −

17 f 15 Left scapula +++ +++

18 m 16 Right + left scapulae +++ +++

19 m 80 Right tibia ++ −

20 m 17 6 exostoses (MHE) + −

21 f 19 Left femur +++ +

22 f 16 Right femur+ left tibia (MHE) +++ +

23 m 16 Right toe + −

24 m 8 Left humerus ++ −

25 m 14 Right humerus + −

26 m 40 Left ulna ++ −

27 f 15 Left humerus − −

28 f 16 Right femur right tibia + −

29 m 7 Left iliac bone + −

30 f 63 1st right metatarsal bone − −

Al^{3 +}was detected in the form of deep blue bands and Perls' staining identified Fe^{3 +}as light blue bands (Fig. 4). Iron was never observed in the cartilaginous matrix. Al^{3 +}was detected in 21 patients and iron was found in exactly the same areas in 10 of these patients.

Al^{3 +}and Fe^{3 +}were present in a various number of bands but it was likely that the number of Al^{3 +}bands was always superior to Fe^{3 +}bands. Bands were usually observed in the trabeculae and sometimes in the cortical shell. The bands corresponded to the ce- ment and arrest lines, which indicate a pause or a recovery in oste- oblast activity when they elaborate bone structure units (BSU). In MHE patients, Al^{3 +}and Fe^{3 +}were similar in the different observed exostoses. In the control group of osteoporotic patients, histochem- ical analysis failed to identify Al^{3 +}and Fe^{3 +}in the trabecular and cortical bone.

3.3. Spectroscopic analysis

SEM–EDS failed to identify Al and Fe in all patients analyzed. SEM– WDS could identify the presence of Al and Fe and the concentration ranged between 0.032–0.054% (atomic %) for Fe and 0.030–0.034% for aluminum (Fig. 5).

Fig. 1.A) X-ray of a pedunculated exostosis of the left proximal humerus; B) X-ray of symetric bilateral exostoses in a child with Multiple Hereditary Exostoses—MHE.

Fig. 2.MicroCT images of a sessile exostosis. A) The external view is composed of a cortical bone shell; the upper part corresponds to the calcified cartilaginous part of the exostosis.

B) The internal view shows the presence of a trabecular network.

Fig. 3.Histological analysis. A) Transverse section of a pedunculated exostosis showing the external cartilage cap (c) covered by the perichondrium (pc); the fenestrated cortical shell (cs). In the center, the trabecular network with the bone marrow can be evidenced. The arrowheads indicate the layer of calcified cartilage reproducing a growth plate.

B) Histochemical identification of osteoclasts at the junction calcified cartilage/trabecular bone. Osteoclasts are brown cells, calcified bone is in blue, cartilage is unstained.

4. Discussion

The pathophysiology of MHE is related to mutations in the EXT1 or EXT2 heparan sulfate polymerases which control the development and normal functions of the perichondrium[14,15]. However, in solitary exostoses, the mechanisms are less understood but abnormalities in the osteochondral tissues of bones have been recognized very early[16]. Ex- ternal irradiation can disrupt the architecture of the perichondral ring of Ranvier (an area encircling the growth plate) and provokes an exostosis [17,18]. Osteomyelitis and trauma have also been implicated[19,20]. In the present study, the development of exostosis fulfills the classical criteria in the children. In two adult cases, an exostosis was developed after femoral amputation or knee surgery.

The dysfunction of the osteochondral cells leads to an accumulation of aluminum and iron in about 2/3 of cases. The amount of metal pres- ent in the bone matrix can only be semi-quantitatively observed and the score used here is the reflect of the amount of bands presents in the ex- ostosis. Al³⁺and Fe³⁺bands were observed in cortical bone and mainly in the trabecular envelope of the exostosis. Al³⁺can be present alone in 11 cases and Fe³⁺bands are observed in the same locations. Metal ions are mainly observed in the trabeculae close to the calcified cartilage and

bone at the implantation base of the tumor never contained bands of metal in their constituting BSU. In one case Al³⁺deposits were observed in the calcified cartilage. From a crystallographic point of view, Al or Fe can substitute a Ca atom in the large channel of the hydroxyapatite crys- tal in position (6 h)[21,22]. In this channel centered by a hydroxide group, the six calcium atoms are surrounded by the phosphate groups and when Al is adsorbed, it is linked to the PO₄³⁻groups[23].

Perls' staining is a worldwide admitted histochemical stain for iron.

The method works on soft and hard tissues. In the presence of ferrocy- anide ions, Fe³⁺is precipitated as a highly water-insoluble blue com- plex made of potassium ferric ferrocyanide, also coined Prussian blue.

Fe²⁺is not detected by the method. In bone, it is preferable to avoid the use a counterstain dye if one wants to clearly identify the metal bands in the bone matrix[24–27]. Al and Fe are deposited in the cement or arrest lines when a BSU is formed, either in cortical and/or trabecular bone. These lines are known to contain specific proteins (osteopontin and osteocalcin) that can bind metal ions. It has been shown by a very sensitive method (micro X-rayfluorescence analysis with a synchro- tron) that Zn and Pb ions present in the interstitialfluid can accumulate in the cement lines by uptake in the hydroxyapatite crystal and attach- ment to these proteins[28]. Our histochemical staining methods for Fig. 4.Histochemical identification of aluminum (upper raw A to C) detected by the solochrome azurine B method and iron (lower raw D to F) identified by Perls' Prussian blue. A and D correspond to a patient with a low Al³⁺content and no Fe³⁺; B and E are from a patient with a mid Al³⁺and Fe³⁺; C and F are from a patient with a high Al³⁺and Fe³⁺content. The bars indicate 100μm.

Fig. 5.WDS analysis of the trabecular bone matrix of an exostosis rich en Fe and Al. The method is quantitative (after normalization of the data) but do not show the location of these metals. A) an area imaged and analyzed; B) identification of Fe and Al peaks during measurement.

these two metals also evidenced that Al³⁺and Fe³⁺form bands corre- sponding to these specific area.

Histochemical identification of aluminum in bone was extensively studied in the 1970′when it was found that patients with renal failure developed encephalopathy and osteomalacia[29,30]. The role of alumi- num in the pathogenesis of both diseases was evidenced and histo- chemical identification of Al^{3 +}in the bone matrix was proposed on undecalcified bone sections[31]. Aluminum was most often stained by the aluminon technique using aurine tricarboxylic stain[24,32]. The method was found more sensitive than atomic absorption spectrometry [33]. Atomic absorption spectrometry was not available in the present study; however, a previous report from our group has shown that Al (and other metals) had were present at very low concentrations in the bone matrix and did not increased significantly during aging[34]. Sev- eral authors have described that solochrome azurine B (also termed Mordant blue B or chrome azurol B - CI 43830)[35]gives better results and identifies a larger number of bands in the bone matrix[11,36–38].

The method was compared with atomic absorption spectrometry and was found well correlated. However, this physical method is destructive for the samples although it provides a global estimation of the Al³⁺con- centration inμg/g of bone. In the present study, we tried to identify Fe³⁺

and Al^{3 +}by spectroscopic methods[39,40]. Previous studies have found SEM–EDS suitable to identify Fe and Al only inin vitromodels of calcification in presence of these metal ions[6,7]. The method was also successfully used with other metals (Cr, Co, Ni, Sr) studied in the same conditions[41,42]. It should be noted that, inin vitroexperiments, the concentration in the hydroxyapatite crystals is higher than 0.05%

(atomic %) that is in agreement with the detection limit of the method litterature on semi-quantitative analysis by EDS[13,43,44]. In the present series of exostoses, the method failed to identify Al and Fe in our samples. On the other hand, SEM–WDS confirmed the presence of these two atoms and showed that the local concentrations were very low in the patients. WDS is known to be a very sensitive method to identify traces of metals[40]. Histochemistry is confirmed as a very sensitive method for Al³⁺and Fe³⁺in the bone matrix allowing their precise localization in these tumors. The histochemical reaction was negative in all patients of the control group.

The challenge to face is to understand why these two metals can be deposited in the bone matrix by osteoblasts. Fe³⁺is histochemically identified in bone in cases of iron-related diseases such as hemochro- matosis[45,46],β-thalassemia or drepanocytosis[47–49]. The mecha- nisms are not clearly understood and the role of proteins such as ferroportin and other transporters have been recently reviewed[50].

Clearly, it is possible that these proteins can also serve as transporters for other metallic cations. Co-localization of Al^{3 +}and Fe^{3 +}in bone has been reported in a very limited number of studies[37,51].

The most intriguing problem is to ascertain the origin of aluminum.

In the last decades, aluminum has become omnipresent in modern life, alimentation and environment[52]. Aluminum is present in foods as the additive E 173[53]and has been detected in baby milks[54]and tea[55]. In addition, aluminum, which is one of the most abundant me- tallic elements of the Earth's crust, is also used to make household prod- ucts (dishes, trays, pans, cooking foils, brewage cans…). The major route of exposure to aluminum is through food although a trans- or per cutaneous route has also been identified (antisweating products, vaccines). It is estimated that the aluminum intake can reach up to 13 mg/day[56,57]although the tolerable weekly intake is only 1 mg/kg of body weight[58]. 95% injested aluminum is eliminated by the feces whilst the 5% remaining circulate in the blood, bound to transferrin, albumin and other low molecular weight proteins. The circulating aluminum can mainly localize in two preferential organs:

brain (linked to the phosphoproteins, lipids or phosphate groups of DNA)[59]and bone (bound to the phosphate groups of hydroxyap- atite at the mineralization front)[6,31]. Aluminum localization in the brain is neurotoxic and is implicated in neurodegenerative dis- eases such as Alzheimer, Parkinson and multiple sclerosis[60,61]. A

co-localization of Fe^{3 +}and Al^{3 +}has also been reported in the brain of Alzheimer's disease[62,63].

In bone, low doses of aluminum have been identified as a cause of bone loss in laboratory animals[8]and humans[64]. High doses causes osteomalacia. Aluminum reduces osteoblastic activity and alters bone quality by interfering with calcification and altering the bone levels of important other trace elements such as Zn, Cu, Mn Se, B, Sr…[9].

5. Conclusion

Al and Fe can bind to the phosphate groups of hydroxyapatite crys- tals when an atom of Ca²⁺is replaced by a Fe³⁺or an Al³⁺atom. Histo- chemistry using Perls' and solochrome azurine B methods (for resp.

Fe^{3 +}and Al^{3 +}) are very sensitive techniques and they evidenced a modification of bone quality in this series of exostoses mostly due to these epigenetic factors. The localization of these two cations occurs in the same locations (cement and arrest lines of the bone matrix) in about 2/3 of cases for Al³⁺and 1/3 for Fe³⁺. The mechanism of the de- position of these two ions is not fully understood but a dysregulation of osteogenic cells (chondrocytes and osteoblasts) in these benign tumors seems to be the cause.

Acknowledgments

Authors thank Mr. Yann Borjon-Piron and Mr. Frédéric Christien, Polytech-nantes, Institut des Matériaux de Nantes site Chantrerie for kindly providing WDS analyses on our samples. This work was made possible by grants from the French Ministry of Research, in the Bioregate program. They also thank Mrs. Nadine Gaborit for her skillful assistance with microCT and histotechnology and Mrs. Laurence Lechat for secre- tarial assistance.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.

doi.org/10.1016/j.jinorgbio.2015.09.008.

References

[1] F.M. Vanhoenacker, W. Van Hul, W. Wuyts, P.J. Willems, A.M. De Schepper, Heredi- tary multiple exostoses: from genetics to clinical syndrome and complications, Eur.

J. Radiol. 40 (2001) 208–217.

[2] N.M. Billah, M. Idrissi, R.I. Kaitouni, H. Faraj, M. El Yaacoubi, S. Bouklata, Imagerie des exostoses solitaires, Feuill. Radiol. 53 (2013) 11–20.

[3] J.W. Milgram, The origins of osteochondromas and enchondromas ahistopathologic study, Clin. Orthop. Relat. Res. 174 (1983) 264–284.

[4] D. Felsenberg, S. Boonen, The bone quality framework: determinants of bone strength and their interrelationships, and implications for osteoporosis manage- ment, Clin. Ther. 27 (2005) 1–11.

[5] D. Chappard, M.F. Baslé, E. Legrand, M. Audran, New laboratory tools in the assess- ment of bone quality, Osteoporos. Int. 22 (2011) 2225–2240.

[6] C.N. Degeratu, G. Mabilleau, C. Cincu, D. Chappard, Aluminum inhibits the growth of hydroxyapatite crystals developed on a biomimetic methacrylic polymer, J. Trace Elem. Med. Biol. 27 (2013) 346–351.

[7] P. Guggenbuhl, R. Filmon, G. Mabilleau, M.F. Baslé, D. Chappard, Iron inhibits hy- droxyapatite crystal growth in vitro, Metab. Clin. Exp. 57 (2008) 903–910.

[8] X. Li, Y. Han, Y. Guan, L. Zhang, C. Bai, Y. Li, Aluminum induces osteoblast apoptosis through the oxidative stress-mediated JNK signaling pathway, Biol. Trace Elem. Res.

150 (2012) 502–508.

[9] X. Li, C. Hu, Y. Zhu, H. Sun, Y. Li, Z. Zhang, Effects of aluminum exposure on bone mineral density, mineral, and trace elements in rats, Biol. Trace Elem. Res. 143 (2011).

[10] D. Chappard, in: D. Heymann (Ed.), Bone Cancer: Progression And Therapeutic Ap- proaches, Acad. Press; Elsevier Inc., London 2014, pp. 111–120.

[11] J. Denton, A.J. Freemont, J. Ball, Detection and distribution of aluminium in bone, J.

Clin. Pathol. 37 (1984) 136–142.

[12] M. Kaye, A.B. Hodsman, L. Malynowsky, Staining of bone for aluminum: use of acid solochrome azurine, Kidney Int. 37 (1990) 1142–1147.

[13] INCA, Energy Operator Manual, Oxford Instruments Analytical, High Wycombe, UK, January 2006.

[14] J. Huegel, C. Mundy, F. Sgariglia, P. Nygren, P.C. Billings, Y. Yamaguchi, E. Koyama, M.

Pacifici, Perichondrium phenotype and border function are regulated by Ext1 and heparan sulfate in developing long bones: a mechanism likely deranged in heredi- tary multiple exostoses, Dev. Biol. 377 (2013) 100–112.

[15] B.M. Zak, M. Schuksz, E. Koyama, C. Mundy, D.E. Wells, Y. Yamaguchi, M. Pacifici, J.D.

Esko, Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and long bones, Bone 48 (2011) 979–987.

[16]R. D'Ambrosia, A.B. Ferguson Jr., The formation of osteochondroma by epiphyseal cartilage transplantation, Clin. Orthop. Relat. Res. 61 (1968) 103–115.

[17] R.T. Ballock, R.J. O'Keefe, Physiology and pathophysiology of the growth plate, Birth Defects Res. C Embryo Today 69 (2003) 123–143.

[18] F.D. Murphy, W.P. Blount, Cartilaginous exostoses following irradiation, J. Bone Joint Surg. Am. 44 (1962) 662–668.

[19] A. Vallcanera, A. Moreno-Flores, J. Gomez, H. Cortina, Osteochondroma post osteo- myelitis, Pediatr. Radiol. 26 (1996) 680–681.

[20] P. Bhusari, K.I. Kumathalli, S. Raja, A.K. Mishra, Bony exostosis following a traumatic blow: a case report, J. Indian Dent. Assoc. 5 (2011) 285.

[21] S.V. Dorozhkin, A review on the dissolution models of calcium apatites, Prog. Crystal Growth Charact. Mater. 44 (2002) 45–61.

[22] M. Corno, A. Rimola, V. Bolis, P. Ugliengo, Hydroxyapatite as a key biomaterial:

quantum-mechanical simulation of its surfaces in interaction with biomolecules, Phys. Chem. Chem. Phys. 12 (2010) 6309–6329.

[23]T. Kiss, P. Zatta, B. Corain, Interaction of aluminium(III) with phosphate-binding sites: biological aspects and implications, Coord. Chem. Rev. 149 (1996) 329–346.

[24]A.G.E. Pearse Histochemistry, Theoretical and Applied, Churchill Livingstone, Edin- burgh, 1968.

[25] M.C. de Vernejoul, A. Pointillart, C.C. Golenzer, C. Morieux, J. Bielakoff, D. Modrowski, L. Miravet, Effects of iron overload on bone remodeling in pigs, Am. J. Pathol. 116 (1984) 377–384.

[26] P. Guggenbuhl, P. Fergelot, M. Doyard, H. Libouban, M.P. Roth, Y. Gallois, G. Chales, O.

Loreal, D. Chappard, Bone status in a mouse model of genetic hemochromatosis, Osteoporos. Int. 22 (2010) 2313–2319.

[27] R.W. Horobin, Selection, Evaluation And Design Of Biological Stains, E. Horwood, NY.

Chichester., 1988

[28] B. Pemmer, A. Roschger, A. Wastl, J. Hofstaetter, P. Wobrauschek, R. Simon, H. Thaler, P. Roschger, K. Klaushofer, C. Streli, Spatial distribution of the trace elements zinc, strontium and lead in human bone tissue, Bone 57 (2013) 184–193.

[29] M. Ward, H. Ellis, T. Feest, I. Parkinson, D. Kerr, J. Herrington, G. Goode, Osteomalacic dialysis osteodystrophy: evidence for a water-borne aetiological agent, probably al- uminium, Lancet 311 (1978) 841–845.

[30]M. Wills, J. Savory, Aluminium poisoning: dialysis encephalopathy, osteomalacia, and anaemia, Lancet 322 (1983) 29–34.

[31] H.H. Malluche, Aluminium and bone disease in chronic renal failure, Nephrol. Dial.

Transplant. 17 (2002) 21–24.

[32]R. Lillie, R. Mowry, Histochemical studies on absorption of iron by tissue sections, Bull. Int. Am. Mus. 30 (1949) 91.

[33]M.C. Faugère, H.H. Malluche, Stainable aluminum and not aluminum content re- flects bone histology in dialyzed patients, Kidney Int. 30 (1986) 717–722.

[34] M. Baslé, A. Rebel, Y. Mauras, P. Allain, M. Audran, P. Clochon, Concentration of bone elements in osteoporosis, J. Bone Miner. Res. 5 (1990) 41–47.

[35] R.W. Horobin, J.A. Kiernan, Conn's biological stains: a handbook of dyes, stains and fluorochromes for use in biology and medicine, Biol. Stain Comm. (2002) 208–209.

[36]H. Ellis, M. Pang, W. Mawhinney, A. Skillen, Demonstration of aluminium in iliac bone: correlation between aluminon and solochrome azurine staining techniques with data onflameless absorption spectrophotometry, J. Clin. Pathol. 41 (1988) 1171–1175.

[37] J. Fernandez-Martin, P. Menendez, G. Acuna, A. Canteros, C. Gómez, J. Cannata, Stain- ing of bone aluminium: comparison between aluminon and solocbrome azurine and their correlation with bone aluminium content, Nephrol. Dial. Transplant. 11 (1996) 80–85.

[38]S.A. Romanski, J.T. McCarthy, K. Kluge, L.A. Fitzpatrick, Proc. Mayo Clin. Proc., 68, Elsevier 1993, pp. 419–426.

[39]J. Goldstein, Scanning Electron Microscopy and X-ray Microanalysis, Third edition Springer, US, 2003.

[40]K. Sakurai, H. Eba, K. Inoue, N. Yagi, Wavelength-dispersive total-reflection X-ray fluorescence with an efficient Johansson spectrometer and an undulator X-ray source: detection of 10–16 g-level trace metals, Anal. Chem. 74 (2002) 4532–4535.

[41] J. Beuvelot, R. Filmon, Y. Mauras, M.F. Baslé, D. Chappard, Adsorption and release of strontium from hydroxyapatite crystals developed in simulated bodyfluid (SBF) on poly (2-hydroxyethyl) methacrylate substrates, Dig. J. Nanomater. Biostruct. 8 (2013) 207–217.

[42]G. Mabilleau, R. Filmon, P.K. Petrov, M.F. Baslé, A. Sabokbar, D. Chappard, Cobalt, chromium and nickel affect hydroxyapatite crystal growth in vitro, Acta Biomater.

6 (2010) 1555–1560.

[43] K.F. Heinrich, Electron Beam X-ray Microanalysis, Van Nostrand Reinhold Co., 1981 [44] J. Goldstein, D. Newbury, P. Echlin, D. Joy, C. Fiori, E. Lifshin, Scanning electron mi- croscopy and X-ray microanalysis. A text for biologists, materials scientists, and ge- ologists. Scanning electron microscopy and X-ray microanalysis, A Text For Biologists, Materials Scientists, And Geologists1981.

[45] K. Eyres, E. McCloskey, E. Fern, S. Rogers, M. Beneton, J. Aaron, J. Kanis, Osteoporotic fractures: an unusual presentation of haemochromatosis, Bone 13 (1992) 431–433.

[46]M. Duquenne, V. Rohmer, E. Legrand, D. Chappard, N. Wion Barbot, M.F. Baslé, M.

Audran, J.C. Bigorgne, Spontaneous multiple vertebral fractures revealed primary haemochromatosis, Osteoporos. Int. 6 (1996) 338–340.

[47]P. Mahachoklertwattana, V. Sirikulchayanonta, A. Chuansumrit, P. Karnsombat, L.

Choubtum, A. Sriphrapradang, S. Domrongkitchaiporn, R. Sirisriro, R. Rajatanavin, Bone histomorphometry in children and adolescents with beta-thalassemia disease:

iron-associated focal osteomalacia, J. Clin. Endocrinol. Metab. 88 (2003) 3966–3972.

[48]R.S. Weinstein, C.L. Lutcher, Chronic erythroid hyperplasia and accelerated bone turnover, Metab. Bone Dis. Relat. Res. 5 (1983) 7–12.

[49] L. Rioja, R. Girot, M. Garabédian, G. Cournot-Witmer, Bone disease in children with homozygousβ-thalassemia, Bone Miner. 8 (1990) 69–86.

[50]O. Loréal, T. Cavey, E. Bardou-Jacquet, P. Guggenbuhl, M. Ropert, P. Brissot, Iron, hepcidin, and the metal connection, Front. Pharmacol. 5 (2014) 128.

[51] I. Miyoshi, T. Saito, K. Bandobashi, Y. Ohtsuki, H. Taguchi, Concomitant deposition of aluminum and iron in bone marrow trabeculae, Intern. Med. 45 (2006) 117–118.

[52] C. Exley, Why industry propaganda and political interference cannot disguise the in- evitable role played by human exposure to aluminum in neurodegenerative dis- eases, including Alzheimer's disease, Front. Neurol. 5 (2014).

[53] R.A. Yokel, C.L. Hicks, R.L. Florence, Aluminum bioavailability from basic sodium alu- minum phosphate, an approved food additive emulsifying agent, incorporated in cheese, Food Chem. Toxicol. 46 (2008) 2261–2266.

[54] N. Chuchu, B. Patel, B. Sebastian, C. Exley, The aluminium content of infant formulas remains too high, BMC Pediatr. 13 (2013) 162.

[55]G. Schwalfenberg, S.J. Genuis, I. Rodushkin, The benefits and risks of consuming brewed tea: beware of toxic element contamination, J. Toxicol. 2013 (2013) 370460.

[56] J.A. Pennington, S.A. Schoen, Estimates of dietary exposure to aluminium, Food Addit. Contam. 12 (1995) 119–128.

[57] Y.C. Rajan, B.S. Inbaraj, B.H. Chen, In vitro adsorption of aluminum by an edible bio- polymer poly(gamma-glutamic acid), J. Agric. Food Chem. 62 (2014) 4803–4811.

[58] F. Aguilar, H. Autrup, S. Barlow, L. Castle, R. Crebelli, W. Dekant, K. Engel, N. Gontard, D. Gott, S. Grilli, Safety of aluminium from dietary intake scientific opinion of the panel on food additives,flavourings, processing aids and food contact materials (AFC), EFSA J. 754 (2008) 1–34.

[59]M. Kawahara, M. Kato-Negishi, Link between aluminum and the pathogenesis of Alzheimer’s disease: The Integration of the Aluminum and Amyloid Cascade Hy- potheses, Int. J. Alzheimers Dis. 2011 (2011) 276393.

[60] S.C. Bondy, The neurotoxicity of environmental aluminum is still an issue, Neurotoxicology 31 (2010) 575–581.

[61] C. Exley, T. Vickers, Elevated brain aluminium and early onset Alzheimer's disease in an individual occupationally exposed to aluminium: a case report, J. Med. Case Rep.

8 (2014) 41.

[62] J. Connor, S. Menzies, S.S. Martin, E. Mufson, A histochemical study of iron, transfer- rin, and ferritin in Alzheimer's diseased brains, J. Neurosci. Res. 31 (1992) 75–83.

[63] P.F. Good, D.P. Perl, L.M. Bierer, J. Schmeidler, Selective accumulation of aluminum and iron in the neurofibrillary tangles of Alzheimer's disease: a laser microprobe (LAMMA) study, Ann. Neurol. 31 (1992) 286–292.

[64] D. Chappard, P. Insalaco, M. Audran, Aluminum osteodystrophy and celiac disease, Calcif. Tissue Int. 74 (2004) 122–123.