CHAPTER 28 MINERALOGY OF BONE
H. CATHERINE \V SKINNER Yale Ullivenit)'
functions of these organs. These discussions illustrate CONTENTS several attributes of the emerging field of medical geology. The scientific information outlined herein is I. Introduction drawn from a diversity of disciplines and expertise, from II. The Skeleton and Mineralized Tissues biophysical and biochemical sciences, from physicians III. Crystal Chemistry of the Mineral in Mineralized Tissues and dentists, and from geologists, mineralogists, and IV. The Crystal Structure of Calcium Apatites engineers. This knowledge enables us to address the V Analysis of Apatitic Biominerals many individual and collective roles that minerals play VI. Osteoporosis in the body. A disorder that affects people on every con VII. Mineral and Mineralized Tissue Research and tinent, osteoporosis, is presented as an example of how Medical Geology information on minerals can be applied. It is only one of the possible targets of opportunity where mineralog ical/geological expertise has, and continues to have, the potential to amel.iorate suffering and promote better global and personal health. Selected classic and recent I. INTRODUCTION references are included to whet the appetite of those who will make contributions to our knowledge in the Medical geology encompasses many scientific endeav future. ors with global activities and impact, but it also includes aspects that are very personal and individual. Local environment is sampled through wh
Copyri g- ht © 201l ) , Elsevier Inc. E,:(CJl liflls 'i" !V/"diml Gcolog)" 667 All ri ghts rcservt.:d . 668 NlTNERALO GY OF BOX"E
ized tissues. Created by the actions of distinct cell tissue age, with nutrition, or with disease (Albright & systems, the tissues are true composites, an intil1hlte Skinner, 1987). All three textures may occur in cortical association of extracellular macro- and microbioorganic bone, the heavily mineralized portions of bone, or in molecules, and inorg,lllie mineral materials. The tissues trabecular bone, the porous and spongy segmentS of the are in dynamic equilibrium with a highly controlled org,l11, and are expressions of their dynamic nature. But fluid whose chemical composition resembles that of the all bioapatite deposits in humans are not normal and ocem: highly oxidized, with a pH around 7 with expected. sodium, Na", and chlorine, Cl-, as the dominant ionic species. The mineral of bones and teeth is a calcium phos phate that closely resembles the naturally occurring B. Pathological Apatitic Deposition mineral, hydroxylapatite (Gaines et aI., 1997), and because of its intimate ,lssociation with biological activ In addition to the normal bones and teeth, bioapatit ~ ity and molecules it is known as bioapatite. The minen11 may deposit pathologically, that is, in the tissues of formed and maintained in the soft tissue or matrix of other organs that would normally not mineralize. bones has peculiar attributes. Each mineralized tissue is Nodular apatitic spherules may be detected throughou a remarkable and unique chemical repository whose the body. One site where bioapatites may occur is in mau1tenance is only beginning to be comprehended as tumors, both benign and cancerous, where rapid cel part of the dynamic skeJet ENAMEL TRABECULAR BONE DENTINE GUM LINE ~ / I I II 1~ CORTICAL BONE / Ilr / MARROW I I 1""\ I I ROOT CANAL tCEMENTUM ROOT 8~~ TRABECULAR FIBROUS BONE SOCKET 1lr:':3 SECONDARY OSSIFICAT'ION CARTILAGE II :··..· ...... ~ CENTER A FIG U R E 1 (A) Sketch of a tooth and a long bone indicating the typical tissues found in these organs. (From Skinner, 2000a, Figure I, p. 356.) (B) Sketch of a longitudinal section and a cross section through a long bone illustrating the tissue types and textures that occur and the changes that take place during the development ofa bone.Arrows indicate the directions ofgrowth and remodeling. (From Albright and Skinner, 1987, Figure 5-16, partA. p. 175.) Numbers at specific sites illustrate the following changes: I. length increase. typical growth direction of a long bone; 2, thickness or diameter increase that also takes place as the organ changes shape and size. Some initial trabec ular tissues become cortical bone tissues. The textures depicted in 2 and 5 are typical of heavily mineralized lamellar bone that results from the remodeling of trabecular tissues into cortical tissues. 3. all bones require local remodeling to achieve final organ size and shape in order to function as part of the skeleton; 4. remodeling is also needed to maintain the internal (marrow) cavity size and shape; 5, extensive remod eling in the mid-shaft of long bones where previous trabeculae have been remodeled into heavily mineralized cortical tissue; 6, the circu lar patterns of haversian bone express the sites of resorption and re-deposition of mineralized tissues which continue throughout life ; 7, an outer protuberance on the bone, probably the site of muscle attachment, which will change as the bone responds to growth and devel opment; and 8, a cross section showing distinct layers of haversian and lamellar bone tissues surrounding the marrow cavity. 670 MINERALOGY OF BO N ' TABLE J. Formulae of Hydroxylapatite. Bioapatite. and Other Calcium Phosphate Phases and the Ca/P Ratios for Their Ideal Compositions Name Formula CalP (wt .7%) Hydroxylapatite Cas(PO.h(OH) 2.15 a Bioapatite (Ca.Na,[ ]) IO(PO., HPO., COJ)6(OH,F,CI,H20,COJO,[ lh 1.33-2.25 Monetite CaHPO. 1.25 Brushite CaHPO.,2 H20 1.25 Octacalcium phosphate Ca8H2(PO.).5 HlO 1.33 Tricalcium phosphate CaJ(PO.h 1.875 Whitlockite Ca9 Mg(POJOH) (PO')6 1.11-1.456 Calcium pyrophosphate dihydrate CalPl0 7·2 HlO 1.25 Tetracalcium phosphate Ca.Pl0 9 2.50 Dahllite (Ca,X) IO(PO.,COJ)6(0.OHh6b COl> 1% F < 1% 2.08 b Francolite (Ca.X)IO(PO.,COl h(0,Fh6 F > 1% COl> 1% 2.08 'An approximate formula containing vacancies [ J. bFormula and composition from McConnell (1973), where X indicates a range of cations. To cover all calcium phosphate mineral species that may be found in the human body is beyond the scope I I I. C RY STAL CHEMISTRY OF THE of this presentation. An introduction to the usual and M INERAL IN M INERALIZED TISSUES predominant mineralizing system (Section ill), the sub micron characteristics of the mineral (Section IV) , and The normal and much of the abnormal or pathological the methodology and techniques used to determine mineral deposits in humans is a calcium phosphate. a the composition, concentration, and distribution of the member of the apatite group of minerals (Gaines et ,11.. mineral in mineralized tissues are presented (Section 1997). The bio-deposits conform crystal chemicall y V). The final section (VI) on the disease osteoporosis most closely to the mineral species hydroxylapatite ifl illustrates how knowledge of the mineral, distinguish the ideal formula Ca,(P0 MOH), (hereafter abbre\i ing normal from the abnormal, and mineral dynamics 4 ated as HA), which is describeu in most introductory have become foci for medical research. mineralogical texts (Klein & Hurlbut, 1985). Howe\'er. Although the techniques for studying bones and the precise composition and crystal structure of th mineral are similar to those employed for all solid mate bioapatite mineral has proved difficult to pinpoint -L rials, mineralized tissues present special problems. tbe term "apatitic" is often used. These difficulti e", Foremost are the difficulties of obtaining sufficient relate to the chemical variability reported from 11l 300°C 2Kbar H20 (wt %) 1. Ca(H 2P04b-H20 + fluid 2. Ca(HZP04)2-H20 + CaHP04 + fluid 3. CaHP04 + fluid 4. CaHP04 + CalO(P04)6(OHjz + fluid 5. CalO(P04)6(OHjz + fluid 7. CalO(P04)6(OHlz + Ca(OHb + fluid P2 05 I \ CaO FIGURE 2 The phase diagram of the system CaO-P20s-H20 at 300°C and 2 Kbars (H20 pressure) to illustrate the several calcium phosphate phases that may OCCUI- with hydroxylapatite under equilibrium conditions. (From Skinner, 1973, Figure I A.) The brackets indicate vacancies in some lattice sites of bone of the structure and bonds predominantly to the solid to achieve a charge-balanced solid phase. calcium, but other cation and anion species, vacancies, This complicated chemical solid can be described and complex molecular groups are usually detected on as follows: bioapatite is predominantly a calcium analysis. phosphate mineral most closely resembling the species hydroxylapatite but usually contains many elements and molecular species other thall calcium and phosphate A. The Mineralizing System: CaO-PzOs-HzO that probably contribute to its physical attributes and reactivity, and should be part of any identification. The The chemical system that defines bioapatite can be sim crystal structure of all apatite minerals is dominated by plified to CaO-P10s-H20. Figure 2 is an experi tetrahedral anions. In the case of hydroxylapatite and mentally determined phase diagram. It is a portion of ioapatite it is phosphate, the phosphorous-oxygen the chemical system that descrihes the mineralization of tetrahedral anionic groups (P04)3-, that forms the back bones and teeth, which is a summary of the solid and 672 ?l11"EILII,OGY OF BO"I: liquid phases that exist ,It equilibrium at a specific tem TABLE II. The Composition of Normal Mineralized perature and pressure in the simplified system (Skinner, Tissues 1973a). The following solids-monetite, CaHP0 , 4 A. Bulk Composition of Bone, Dentine, and Enamel brushite, Cal-IP04 . 2 H 20, and Ca2P/);, all with Ca/P weight ratios that equal 1.25 and mole ratios that equal 1.0-may occur in stable association with H.A when the Bone' Dentine' Enamel' fluid phase is pure water. This phase diagram is the result of experiments at elevated temperatures and pres wt% vol% wt% vol% wt% vol' ; sures to accelerate reaction times and provide crystalline products for accurate an::dyses. It shows the stability Inorganic 70 49 70 50 96 90 fields of the solid phases and fluid compositions for a Water 6 13 10 20 3 8 range of hulk compositions. Brushite ,1ml octacalcium Organic 24 38 20 30 2 phosphate identified in some biomineral analyses are Denisty (avg.) 2.35 2.52 2.92 not found in this diagram as they occur under different (g/cml) temperature and pressure conditions. It should be noted that H.A is the stable phosphate mineral phase through out most of this diagram, but it coexists with other min B. Composition of Major Elements and the erals from pH 4 to 12. Interestingly, only at pH 7 is HA Ca/P Ratio of the Bioapatites in Three the only solid phase associated with a variable compo Tissues (Wt% on a dry, fat-free basis)b sition fluid. At lower pH, the solid phases that occur with HA have higher phosphate content. At a pH greater than 7, the second mineral phase in this simpli ASH 57.1 70.0 95.7 fied experimental system is a mineral known as port Ca 22.5 25.9 35.9 landite, Ca(OH)2' Although the mineral <1l1alyzed after P 10.3 12.6 17.0 Imv temper;H1Jre extraction from tissues (discussed in Ca/P 2.18 2.06 2.11 Section VI) may show a Ca/P higher than that of Mg 0.26 0.62 0.42 hydroA)rlapatite, portLmdite has not been identified Na 0.52 0.25 0.55 0.089 0.09 (Skinner, 1973b). K 0.17 CO2 3.5 3.19 2.35 CI 0.11 0.0 0.27 F 0.054 0.02 0.01 B. Composition of the l\1ineral in 'From Driessens andVerbeeck. 1990. Table 8.2, p. 107; Mineralized Tissues Table 9.4. p. 165; and Table 10.5, p. 183. bFrom Zipkin, 1970, Table 17, p. 72. The bulk chemical composition of the tissues and of the included bioapatites found in them, the dominant human mineralized tissues, are listed in 'E1ble II. 'Elhle 1IA lists the bulk composition of the major components Ca/P ratios than EA. Nlost ofthese minerals may nu ~ _. in these tissues, and Table lIB presents the range of ate and are st;lble in the body fluid-tissue environIl H~ ' major elements and the different Ca/P ratios of the The presence, and a variable amount, of a sec ,.. bioapatites from three different sources. The measured mineral with lower Ca/P that might occur with H .-\ Ca/P wt% range, from 1.3 to 2.2, differs hom the ideal bio-deposits has been cited as a possible reason for [ or stoichiometric value for hydroxylapatite of 2.15. compositional variability measured in tissues. 1 Because the values are both above and below the stoi higher Ca/P ratio may reflect the substi tution chiometric value, several hypotheses for such variations another anion, e.g., CO,"-, for a portion of the PI have been proposed. (discussed in Section rVO). 1, Variations Due to Alllltiple Minerals 2. Variations Due to Nucleation and Alaturatioll One group of investigators suggested that the biomin Another possible reason why the analyses of hioapa eral matter was not a single mineral phase. Table I may not show uniform and stoichjometric compo ~ j shows that there are a number of calcium phosphate is that the mineral precipitate changes over time. J mineral species with different, and in many cases lower, well known that phosphate ions (H2Po'/-, HP(H \ :VII, LilA U)(; Y OF BO.\:E 673 usually oetected in the fluid phase at initial mineral in hones and teetl1. Detailed investigations of the apatite nucleation, This led to the suggestion that nucleation crystal structure, presented in Section T\~ enable us to was a discrete process from growth ano maturation of discuss the ability of this species to incorporate a variety the mineral phase and the composition of the fluid at of chemical species. Each of these incorporations, least locally varied over time as mineralization pro known as solid solution, could alter the Ca/P ratio of ceeoeo (Roherts et aI., 1992). Glimcher (1976) sug the solid. For example, in geological environments, gested that nucleation could be induced hy charges from sodium (Na), lead (Pb), or strontium (Sr) may substi phosphate groups associated with matrix collagen tute for some of the calcium which mea11S an accnrate molecules. Alternatively, because the initial solid was analysis for Ca/P that would be slightly lower than sto so poorly crystalline, Posner (1977) suggested a sepa ichiometric liA. Alternatively, if an anionic group, such rate phase called "amorphous" calcium phosphate as sulfate (S04), became incorporated in place of some mineral, octacalciull1 phosphate was suggested by of the phosphate (P04), the CalP would be above the Brown et al. (1987), and a third suggestion of brushite stoichiometric J-JA value. Calculation of a Ca/P ratio, was made by Neuman and Neuman (1958). All three or a cationlanion ratio, from a chemical analysis will mineral materials were considered intermediates in the depend not onlf on which chemical elements and mineralization process before ,1 mature biomineral species were availahle when the mineral formed but also phase that more closely resembled hydroxylapatite was on the cOll1pleteness of the data used in the calculation. produced, Even during the investigations of the basic If only calcium and phosphate are measlIreo, for mil1eralizing system, the synthetic calcium-phosph,lte example, no l11<1tter how accurately, calculation of a water investigations that delineated the fonm1tion of Ca/P for I,ioapatite may be misleading. The following HA showed this stable and ubiquitous phase which was section on crystal structural details of mineral apatites most often associated with other solid phases and had will allow investigators to more fully appreciate the pos incongruent solubility (Van vVazer, 1958; Skinner, sible uptake and incorporation of specific elements, or 1973<1). molecular species, into bioapatites. A variety of chemical techn.iques for determining the Ca/P ratio in the synthetic chemical system and early mineral deposition confirmed that initial precipitates produced by adding calcium to phosphate-rich solu IV. THE C RYSTAL STRUCTURE tions, or vice versa, was a calcium phosphate m.i.neral OF CALCIU M APATITES \\'ith higher phosphorous (P) content than that of stoi chiometric hydnl:l.,),lapatite (and therefore a lower Ca/P). Although the presence of amorphous and The structure of apatites, a coml11on group of naturally brushite as initial phases h,1s not been confirmed, this occurring Ininerals in lIJaJ1Y rock types throughout the question remains under study (Brown, et aI., 1987; world, is distinguished by hexagonal symmetry (G,1ines Roherts et aI., 1992; K.im et aI., 1995; Aoba et aI., 1998). et ai. , 1997). Figure 3A depicts one projection of the three-dimensional arrangement of the atoms that make up the apatite structure. This view is down the unique 3. Vm-iatiolls Due to Substitutions c- ,1xis and depicts a plane perpendicular to c. The rhom Witbin tbe Nli1'1cral bohedral outlines are the disposition of two of the three Putting aside the nucleation and maturation processes ,1 axes (directjons at 120 degrees apart) and the atoms and identification of the initial precipitates, biomineraJ are preciscly placed conforming to the crystallographic has been mostly thought of as a single calcium phos characteristics of the repeating unit, or lU1it cell, of the phate phase whose variations in Ca/P may be accom apatite stnlcture. modated via substitutions and/or vacancies within the 'It] further illustrate the distribution and importance 1 solid. The opportllnity for elements and molecular of the tetrahedral orthophosphate groups (P04 - ) that species other than c:!lciul11 (Ca), phosphorus (P), oxygen form the b,1ckbone of the ,1patite structure, Figure 3 B (0), and hydroxyl (OH) to occur in I1ClI'Imtl m.ineralized presents another view of the structure. It is a projection tissues is a most important consideration from the per 90° to the c-ax.is along one of the a axes. The yellow spec6ve of medical geology. phosphorus atom is in tetrahedral coordination sur A Illultiplicity of chem.icals exist in the natural envi roul1ded by two white oxygen atoms in the same plane ronment, and it is probable that at least some of them as the phosphorus and two light purple oxygen atoms, Il'ill become part of the dynamic mineral systems found one above the plane, the other below it. Phosphate 674 MINERALOGY Of BO N E u o OH,F •,'" ) J Om o OH,F V Om • OG • Ca2 °G • Ca, • Ca2 p • Ca1 p A C t o OH,F a-diagonal projection I I ) Om c ~ , OG • Ca2 • Ca1 +\ p I I I a-axis projection o B F I G U R E 3 The crystal structure of hydroxylapatite, ideal formula Cas(PO.h(OH). (A) Projection down the c-axis, showing CE distribution of all the atoms in the unit cell. (B) Projection down the Q axis of the unit cell, note the tetrahedral orthophosphate grou s (C) Projection down the Q diagonal, a different view of the three-dimensional arrangement of atoms in the unit cell. (D) Crystals c: hydroxylapatite, note the hexagonal prismatic morphology. :'v'lJ:\ER,\LOGY OF Bo:\F. 675 tions below present examples to show what elements OH,F o find their way into bioapatites and where they may be • Ca2 located. Bioapatite composition mirrors element,ll • Ca1 bioavaiIability. A. Substitutions in the Phosphate Backbone Though the most common minerals are prohably the phosphate apatites, there are arsenate or v,m adate min erals that form with virnJa lly identical apatite-like crys talline structures. The AsO/ -, and VO,'· anions h:lVe a charge and size very similar to the Po.! group. l'\atu rally occurring arsenate or vanadate ,lp,nites usually FLUORAPATITE, ea.(PO.).F contain lea d (Pb) rather than ca lcium. Lead phosphate apatite, the miner,1 1 pyromorphite with ideal formula FIGURE 4 Distr,ibution of the cations in two sites, Ca, and Pb;(PO.);CI, has all cation sites occupied by lead, but in fluorapatite, view down unique c-axis, similar to Figure Caz, 1 3A but with F in place of OH. chlorine, CI -, takes the place of the 011 in the chan nelways (see Section IVC). ()ther tetn1hedral anionic groups, sulfate SO} - ,md anionic groups coordinate w1th clJciuIl1 cations to silicate SiO/-, although of different charge, may ,1J SO produce a charge neutral solid when a singly charged form minerals with apatite structure type, and small 1 species, OH - or Fl-, the blue atom site, is added. FiS'llre amounts of these groups as well as arsenrIte and vana 3C is the view down the diagonal between two tl axes. d,lte may be found in the phosphate ap,nite miner,!Is. )Jote the distribution of the calciulll atoms and the Britholite-Y, a naturally occurring mineral, is one of a 1 1 chm1l1e1ways where the OH - or F - occur par~lllel to the series of phosphate apatites that contain silicon su hsti (-axis direction. The priS1I1 may be cumulative, is not monitored. Lead exposure TAB LE III. Ions That can Substitute in goes unrecognized unless some distinctive disease or Calcium Phosphate Apatites, Their Charge signal, as described in a study on schoolchildren and Ionic Radii Depending on Coordination (iVlielke, 2003), is recognized, evaluated, and related t Number, i.e. VII, IX . ... lead levels. Strontium apatite, ideal formula (Sr,Ca),(P04MOH . Ion Ionic Radius has been designated a separate mineral species and :l member of one of the subsets within the apatite group. l Ca + 1.000,b(VII) because the amount of Sr is greater than 50% of th t I.IS'('IX) total cations present. Studies on naturally o ccurriI1 ~ CdlT 1,14'(VII) rare-earth-elel11ent-substituted apatites show coordi Mg2+ 0,79'(VII) luted substitution: when rare earth elements CREE 0,S9b(VIII) with a charge of 3+ are incorporated in the structur . 4 Sr2• 1.2I '(VII) there lllay he a substitution of Si + for part of the pho,- 1.31 b(IX) phorus. 'iVith both suhstitutions, a charge balance com 2 5 Ba lT 11,3Sb(Vll) parable to the original association of Ca + ;]nd p + in thL 1.4 7b(IX) calciu111 apatite structure can be maintained. Anoth r J Mn l t 0,90WII) coordinated substitution is when an REE + is incorpo l 0,96b(VIII) rated along with Na +. Charge halance 111;]), be achie\'e Na '+ 1,12b(VII) because the cations are distrihuted at both sites ir 1.24b(IX) calcium apatites (Hughes et al., 1991b). K ' ~ 1.46b(VII) Some elements prefer one cation site over the other I.SSb(IX) A study of REE-containing apatite minerals by Hugh Pb2+ 1,23b(VII) et al. (l991b) demonstrated that some ofthe REE pre 1,3S b(IX) ferred the Cal site while others preferred the Ca' sile p5t 0, ~ 7'b(IV) provided charge balance was maintained by suhstitll 5 As - 0,33S'(IV) tions at the anionic sites or with additional and differ Sj4+ 0.26' b(IV) ently charged cations. Site designation for parricu!a elements can be determined using high-resolutior V5+ 0.S4'(VI) X-ray diffraction analyses, paramagnetic resonanCe St.+ 0,12b(IV) thermoluminescence, or inti'ared spectroscopy (Suite 0,29b(VI) et al., 1985). Sb5+ 0.61 b(V'I) Apatite sa mples can have different compositions i All . 0.39' (IV) spite of coming from similar sources or sites. Althoug. l U - 0.9S'(VII) it is possible with modern techniques to show elemem Cel .. 1,07'(VII) at specific sites in the crystal structure of geologi b 1,146 (IX) samples, the tiny crystallites of bioapatite are too sma to ' From Shannon and Prewitt. 1969, for such detailed investigations. It is worthwhile rei bFrom Shannon. 1976, erate that bioapatite composition in one bone may m ' be identical to another bioapatite forming elsewher the same moment or at different times. The Auid-ceU matrix-mineral system composition is unlikely to ~ inside a residence or be from lead-based paint. Some constant from one moment to another, much less fro. from either source could be inges ted and become year to year as the human ages, resides in different gel sequestered in bones or teeth. These ex posures make graphic localities, and ingests water or fo od from dl' lead a "silent" hnard, a potential danger especially for ferent sources. young children who constantly put their fingers in their There is another set of concerns th'lt relate direr ' mouths. The term silent is used hecause Pb is not to cation substitution in bioapatites: bone seeking visible, and detection and amount determined in the emitting radionucLides and ionizing radiation exposUf<. environment or in the body is impossihle without spe of humans. During atomic bomb tests a half century 3= cialized analytical methods and tools. Therefore bio in the southwest United States there was a sca re rehl r logical uptake and sequestration of lead in bones, which to fallout of radioactive nuclides, especially <)O Sr. T l>. MI NE RAl.OGY OF BOl'E 677 ,1l1xiety \vas based on the similarities in behavior of higher amounts of fluorine become available the calcium ami strontiullJ, the half-life of the nuclide (28 element can become incorporated substituting for part, years), the prevailing wind direction toward the more at least, of the OB. For example, 1 mg L- l fluorine heavily populated east, ,md the bct that American added to drinking water, an amount that has become dietary calcium came mostly from milk products with standard for many of the reservoirs that supply water to the largest consumers being children who were actively populations across the United States, appears to reduce putting down uew bone. The worldwide average opoSr caries and may lead to a redncrion in the incidence of \\',lS shown to be about 0.12 microcuries per gram of osteoporosis (,Vatts, 1999). On rhe othe.r hand, the calcium in man or 1/10,000 of the acceptable perm.issi regular ingestion of greater tl1an lOOmgL- I of fluorine ble level ar that time. This snggested that the atomic over a long period of time by humans leads to disease . homb circulating <) OSr was not a global hazard. A Such high amounts of fluorine in local warers and If carbonate, is present in the crystal lattice CO/ -, TABLE IV. The Essential Elements, and other ions, such as triply positively charged cations, it Their Recommended Daily Intake (RDI) might take the place of calcium so that local charge disruption could he balanced by coupled substitution or by vacancies in the lattice. The inclusion of CO) Element RDI could compromise the ideal architecture of the apatite backbone and the channelways. Such destabilization Boron"* (1.7-7.0mg) may account for the very fine-grained nature of Bromine 0.3-7.0mg bioapatites. Calcium** 0.8-1.3g The associ,ltion of CO, with bioapatite is not partic Cesium** 0.1-17.5mcg Chromium**' 130mcg ularly surprising. The molecular species CO2 or bicar bonate, HCO)I-, are produced along with many others Cobalt IS-32 mcg Copper>." ~ 1-2mg during cell metabolism and could adsorb on the high Fluorine';o;, 1.0mgt surface area of the tiny crystallites. Early deposition of Iodine 70-ISOmcg mineral takes place under acidic conditions where Iron** 10-18mg orthophosphate species may aid the nucleation of bio Lithium 730mcg apatite whereas other ions in the fluid, such as bicar Magnesium** 200-450mg bonate, Illay be inhibitory (Glimcher, 1998). Manganese"'* 3.5mg The carbon,lte ion and its distribution is not of major Molybdenum 160mcg concern for this medical geo\(lb'Y purview except that its Nickel** (35-700) presence makes us aware of the necessity to consider Phosphorus*" 0.8-1.3 G both the physical and chemical aspects of the mineral Potassium;"" 3500mg izing system. Carbonate probably aids incorporatiol1 of Selenium 70mcg Silicon** (21-46mg) other elements into the lattice. Bioapatite precipitates Tin** 0.13-12.69 mcg are aggregates of crystallites, which means that the Vanadium (12.4-30.0 mcg) mineral mosaic of many cryst~lilites Gln each present a Zinc** 8-15mg slightly different composition and size. The lower crys tallinity, and variable composition, of carbonate Note: Additional elements detected include the fol containing-bioapatites may be an irritation preventing lowing non-essential elements: Ag. Af, Cd. Pb. and Sp. precise designation of the mineral phase, but it is a **indicates those detected in bone tissues and ( ) indi cates non-essential elements. positive advantage for the biological system. The From the Food and Nutrition Boar·d. National high surface area of the crystallites facilitates their Research COllncil, Recommended Dietary Allowances dissolution as required for the dynamic bone mineral 10th edition, National Academy Press. Washington DC. formation-resorption system. Kature has utilized ,) 1989. the Federal Register #2206, and for elements in solid phase that fulfills several functional roles bone Bronner. 1996 and Skinner et al.. 1972. required for bone (Skinner, 2000b). Bioapatites record exposures of living creatures to the environment and particularly the bioavailability of elemental species in remarkable. M,lny elements and chemicals entering t ' _ our diets. human body may become associated witll, or becorr_ This brief summary does not do justice to investiga part of, tile apatitic mineral matter. tions with a variety of techniques which include elec tron and X-ray diffraction analysis, infrared, polarizecl infrared and Raman spectroscopy, solid state carbon-13 V. ANALYSIS OF APATITIC BIOMINERALS nuclear magnetic re·sonance spectroscopy, and most recently atomic force microscopy. These have been and are used to detect and quantitate the amount of car To ascertain the ranges of inclucled elements and spec bonate within the crystal lattice of a single-phase in bioapatites, the mineral matter must be extracted mineral or bioapatite or as an adsorbed species. concentrated. The techniques devised for separa From the above selected examples and rlable III, the mineral from the associated organic and cellular III very wide range of elements and molecular species that rials can, in many cases, further complicate the as can be accommodated within the apatite crystal struc The other option is to analyze the tissues keeping ture is summarized. Table IV lists tl1e levels of elements the mineral and organic fractions associ FIGU RE 5 Comparison of X-ray powder diffraction pa,· terns of mineral and tissue bioapatites: Ca(OHh, portlanditE B. X-ray and Electron Diffraction WHIT. whitlockite; Tooth, bioapatite; HA hydroxylapatite, sy-· thetic hydrothermal sample (from Skinner, 1973); COrt. ashe : Unambiguous identification of most mineral materials and Cort. ED, cortical bone ashed and ethylene diami r '" utilizes diffraction techniques that can easily determine extracted cortical bone; Valve. human aortic valve pathologiC: the species based on the unique crystal structural char bioapatite deposit. acteristics of the compound (Klug & Alexander, 1954). The powder diffraction method may be used for the the sample gives ,I pattern consistent with all apao[, identification of any crystalline materials. Either X-ray (Skinner, 1968) and focuses attention on the detecti" Or electron diffraction may be employed, and the choice of any additional solids. Enamel is perhaps the only bi n is usually dependent on instrument availability and the material that provides sufficient diffr,lction detail to ca specifics of the sample. Vast databases h,]ve accumulated culate lattice parameters of a calcium apatite. 'TIlbl e over the past hundred years since the techniques were presents the results of calculations from powder ill:.. elucidated. The International Union Committee of fraction data of several clifferent apatites. Electron di ~ Diffraction has a compendium of X-ray diffraction data fraction does not necessarily afford lUore precise resul on ctystalline compounds, both organic and inorganic, over X-ray diffraction as the level of crystalJinity • which c011tains a subset for natural and synthetic the important criterion for producing diffraction. ' mineral materials including the apatites. advantage of electron diffraction is that the beam III The tiny ctystallite size and variable composition of be more finely focused, ancl a small mineral Ct:;'s bioapatites are fulJy expressed in the X-ray di ffraction aggregate within a thin section might be separare analysis of the extracted materials. \Nell-crystallized examined rather than extracting the minenl1 to obtain (without vacancies and >0.5 ~lt11 in average size) hydrox pure mineral sample for povvder diffraction. ylapatite geological samples give many discrete diffrac tion maxima from which one easily calculates unit cell parameters. Comparing a mineral apatite X-ray powder diffraction pattern (Figure 5) with bioapatite, e.g., cor C. Sample Preparation: Mineralized Tissues tical bone mineral, the latter has few and broad maxima. A poorly crystalline compound makes it difficult to 10 examine the milleral phase in its biological ~ determine any compositional details other than to say roundings requires different sample preparation.. > MJ:'.I EIL\LOGY OF B [)~ E 681 TA BL.E V. Calculated Unit Cell (Lattice) Parameters for Synthetic Apatites. and Bioapatites Q-Axis (-Axis Ref. Hydroxylapatite (Synthetic) 315°C, 2 Kbars H20 pressure CaO/P20 S 1.61 9.421 6.882 Skinner (1968) CaO/P20 S 1.12 9.416 6.883 Skinner (1968) 600°C, 2 Kbars H20 pressure CaO/P20 S 1.61 9.4145 6.880 Skinner (1968) CaO/P20 S 1.12 9.4224 6.8819 Skinner (1968) 100°C Precipitation 9.422 6.883 Bell & Mika (1979) (Reitveld calc.) 9.4174 6.885S Young & Holcomb (1982) Mineral 9.418 6.875 Gaines et al. (1997), p. 856 Carbonate Apatites (Synthetic), Low temperature « IOO °C precipitates, air-dried) Direct (adding calcium acetate into a solution of ammonium carbonate+phosphate) 9.373 6.897 Labarthe et al. (1973) 13.8% CO] Inverse (adding ammonium solution into calcium acetate) 10.4% CO] 9.354 6.897 Labarthe et al. (1973) Direct using sodium carbonate 9.440 6.880 LeGeros et al. (1968) Direct using sodium carbonate and fluoride 22.1% CO] 9.268 6.924 LeGeros et al. (1986) High temperatures 1.455 CO]. the maximum determined 9.367 6.934 Rey et al. (1991) Bone Ashed cortical 9.419 6.886 Skinner et al. (1972) Enamel Human 9.421 6.881 Carlstrom (1955) 'The wide variations in the values may be partially attributed to differ'ences in composition of the starting materials and to the methods of prepara tion, e.g .. direct versus inverse, under low temperatures for carbonate apatite synthesis. See Elliott. 1994. p. 234-248 for details on precipitation mechanisms and discussion of IR, X-ray diffraction analyses, and possible locations of the carbonate ion in the apatite lattice. techniques must 11111111111Ze any alteration of the tiny for soft tissue sections exanuned in pathology laborato clystallites while maintaining the organic and cellular ries (NLllluche et aI., 1982). Plastic is required because framework ill which the mineral l11lltter is distributed. the juxtaposition of tiny crystals of hard mineral with Thjn sections are prepared for examination ,vith optical the soft organic matter and cells minimizes differential and electron microscopic techniques so that morpho bardness of the medium put to sectioning. logical relationships typical of the different tissue types After obtaining a fresh tissue sa mple the first act is to and chemical observations on the mineral phase can be soak the pieces in alcohol solution abollt 10 times the assayed (Section VD). sample volume for a few hours or overnight refreshing The techniques to prepare biological tissues as thin the solution several times. Occasiondlly formaldehyde is sections parallel the methods employed in petrology llsed but formalin has to he buffered or the solution will (the study of rocks) (Blatt & Tracy, 1996). The mjner be acidic and at least some mineral cJYstallites may be alized tissue sllmpJe is embedded to obtain a plano lost or dissolve during immersion. The alcohol soak parallel thin section, from 50 to less than 10 11m thick. effectively lowers the water 311d fat content in the tissue, The thickness will depend on the information desired stops further biological degradation, and stabilizes the and the method of analysis. Embedding preferentially organic components. The second stage is to embed the employs plastics, rather than paraffin, the typical media whole sample with not too viscous epoxy materials such 682 J\.11:-lERALOGY OF BO N E A B FIGU R E 6 Transmission X-ray photos of excised samples of bone. (A) Longitudinal section through the upper femur of norma 29-year-old male (from Albright and Skinner, 1987, Figure 5-1). Note the arcuate pattern of the trabeculae. (B) Mid sagittal sections of first lumbar vertebra of a 20-year-old female (above) and 50 year old (below) (from Arnold et al.. 1966, Figure 1.16). The trabecu lae in the older bone show thickening and "reinforcement" accentuating their vertical orientation in the skeleton. as methyl methacrylate, or other commercially available the whole tissue piece IS ready for sectioning an d IM T II plastics such as Spun· or Epon . . The viscosity must polishing. be appropriate to facilitate penetration throughout the Sectioning requires a sharp knife, usually diamond or sample, and probably will require a vacuum to maximize carborundum blades, to cut the plastic embedded tissu ' efficiency. Most mineralized tissue laboratories will without tearing the crystallites from their organ ic have an automated embedding system that takes the matrix. Once cut the thin section is mounted on a gla tissue through the extraction and embedding proce or plastic slide for microscopic study, usually witho dures and applies vacuum and heat and a "hardener" to the addition of a cover slip. Grinding to assure plano ensure a fully homogeneous block of embedded sample. parallel surface for high-resolution electron microprobt Any holes, effectively pockets of air that might incor elemental analysis may be essenti~ll, but it almost aIW,I\ porate dust or foreign materials, must be avoided espe results in smearing and disrupting the Clystallites, <; ( cially if the analysis will use SEM coupled with energy this level of the preparation procedure again requjre- dispersive analysis (SEM/EDXA) to measure the ele care. It is wise to constantly view the section uncler . mental composition of the mineral. Once embedded microscope with at least 40x magnification. Any surbcr ,'V1I K ER!\LOGY OF BONE 683 roughness will cause interference in element,ll analyses, Figure 7 A is an x-ray transmission microradiograph of although background corrections can be applied to the cortical bone tissuc from a dog tibia. It illustrates varia raw data (Reed, 1993). tions inlllineral density in the several osteons (the circu Every laboratory that perf()rms mineralized tissue lar patterns), typical of haversian bone. Variation in thc section analyses, usually the pathology departments of levels of gray in this scction reflect different amounts of hospitals, has their own sterile procedures. l() prepare nuneral per unit arca and detected at higher magnifica uniformly parallel, non-artiLlct-containing hard tissue tion because of the mineral impeding the transmission of thin sections is an art, not a science. Once made, sec x-ray energy. Figure 7B is another view of the same area tions may be stained to iden tify osteoid, cell walls, but illuminated with UV light. Three of the osteons nuclei, etc., or the calcified portions for specialized show two circular dark lines around the central vascular investigations, but such treatments would compromise opening, the pattern resembling an archery target. The analysis of the mineral phase. Careful, sensitive atten lines are due to the incorporation of the antibiotic tetra tion to the preparation of any section is necessary for cycline as the mineral deposits ~lIld the molecule glows accurate documentation of the tissue components, their under lJV light. The two lines mark two separate doses structures, and elemental components. of tetracycline allowing the rate of mineral deposition, and the growth ofosteons in the cortex, to be determined (Skinner and Nalbandian, 1975). D. Histomorphometry Figure 8A is an im B FIGURE 7 Ground section of the cortical portion of a rib from a dog given two doses of the antibiotic tetracycline. The tetracycline is incorporated at the time of deposition of bone tissue (Skinner and Nalbandian, 1975, Figure 3). Magnification 'FIGURE 8 Microradiographs taken with the scanning elec 175x tron microprobe, backscattered images of breast tissue samples. A: In transmitted light, the darker the color the more mineral (A) Calcium phosphate spherules deposited in breast tissue. Note present, an expression of decreased transmission of light. Note the size of the spherules at this high resolution (magnification variations in bone mineral density between the several i1 aversian x 1200) relative to the heavily mineralized calcium phosphate systems in the section. Some contain more mineral than others. deposit (white areas) on the right. Section thickness is 61-lm. 8: Same area in ultraviolet light (tetracycline glows in UV light) (From Poggi, et al., 1998, Figure 3.) (B)The dentin-enamel junction which shows two fluorescent rings (concentriC dark circles) in a tooth showing the typical small crystallites in dentine (left) anc in the haversian systems marking the times when two doses the larger crystallites in enamel (right). (From Goose and Apple of tetracycline were added to the diet as bone tissues were ton, 1982, Figure 2.6.) Enamel crystallites are about 100 I-lm in developing. length. NII:-" E RALOGY OF BONE 685 APPAREN DENSI 0'. W v~ r2xl1Cilhi .'>.PP ARfNT DENS TV II 9 Ie e of :;pcnglcso A F IGURE 9 Osteoporosis examined by transmission X-ray analysis. The first lumbar vertebra from five autopsy subjects illustrates the possibility of quantifying the level of mineral in a bone (from Barzel, 1970, Figure 2B). (A) Top row vertical sec tions through the lumbar vertebrae showing changes in the dis tribution of mineral (white) from homogeneous fully mineralized tissues on left part of the figure to more porous on the right where the trabeculae thicken and appear more vertically ori ented in the samples from older individuals. Middle row trans verse section through the vertebrae shows increased porosity and disorder of the vertical sections. Bottom row transverse sec tions have been extracted and are now fat-free and dry. The porosity increase and the density of the mineral per unit area can be measured, Formula used to calculate the apparent density of columnar sections is weight/volume, glce. (B) Transmission X ray of the entire vertebrae illustrating the apparent density dif ferences that might be visible on radiologic examination (from Barzel, 1970, Figure 4, left half). The several vertebrae depicted in this figure coincide with the mineral density calculated for the sections ,in Figure 9A. volume of bone (Figure 9). The disease has been a topic of investigation for over a hundred years (Arnold, 1966; Barzel, 1970; Riggs & Melton, \995). The tissue reduc tion leads to fragility of the bone organs and Illay cause an osteoporotic individual to sustain a fracture, perhaps without obvious trauma, pain, or other warnings. The main concern is that the usually elderly patient will be B at high risk for additional fractures. In osteoporosis there is a normal mineral/collagen ratio, just less min- 686 NIJ:-.IERALOGY Of BONE eralized tissue per unit arel which distinguishes the dis are so obvious a sign of aging as the bend of the spine. order from osteomalacia where mineral is reduced in also known as dowagers hump, because it is often typical amount relative to the bioorganic matrix. There is, as of older women. Figure 6B ~J\ld Figures 9A and B depict might be expected, no known cure for osteoporosis. It the remarkable di fferences at the tissue level in the dis is part of the normal aging processes and pervasive in tribution of trabeculae ill affected vertebrae. A diagnosis postmenopausal women and older men. The disease is of osteoporosis is inferred from a clinical examination considered a health issue in the United States and in and confirmed by radiologic examination using X-ra~ other developed coulltries. It has become an important radiographic transmission analyses. A radiograph of the area for basic and clinical research with the inciclence of forearm, or leg bones, which is available as a result of fracture considered a signal of disease (Figure 9). Onset an accidental fracture, may present poorly mineralized. of different types of osteoporosis, the relationship to inhomogeneous, or "porous" bone and tissues that are nutrition, exercise, or other potential contributing reminiscent of tllese spine photos. factors, and the results of a variety of treatments are the The density level, or mineral content, of bones cm factors considered in the many epidemiological studies be measured using these radiographs, and such exami in several countries. nations are noninvasive CJ ohnston et al., 1996). Digital Bone mass is genetically programmed for an imiivid radiographic techniques can quanti~ T the mass of any ual but may be modulated qualitatively and quantita bone or portion thereof. The results are compared with tively by environmental factors. Reduction of bone results from persons of like race, staUlre, and age to esti tissue density in the vertebrae, a hallmark of osteo mate the degree of osteoporosis and tlle potential risk porosis, and subsequent vertebral fracture is estimated for future fractures for a speci fic patient. v\That is actu to be as high as 1 out of 4 women by age 65-70 aUy measured is the mineral cOl1centnnion per mass of 0Vasnich, 1996; Eastell, 1(99). This expression of com bone (Fignre 10). promised bone strength results in debilitation and pain High-resolution radiographic analysis methodology. with normal body movements and often premature computerized axial tomography (CAT) scans, has also death. Medical attention now must go beyond bone been used to show local differences in mineral density quantity (mass) into bone quality: architecture and rates and distribution in the cortical bone, or the number. of turnover (Chestnut et al., 2001) that involve molec size, and organization of the trabeculae in a bone. These ular biochemistry and the relationships of the inorganic measurements are a reminder that each bone has its o"n with organic constituents. biomineralization system, which is independent during Osteoporosis, a focus of public attention when bone formation and must be maintained at ,I certain level to loss was recorded for the astronauts subjected to be Age (years) 160 Bone mass (g/cm) 280 < 0.6 140 120 0.60-0.69 (f) ~ OJ > 75-79 C 100 0 0.70-0.79 \'! OJ a. 0 0.80-0.89 0 80 70-74 0 ~ 0.90-0.99 .;:(f) 60 65-69 l!? ~ ti 60-64 u:CIl 40 55-59 20 o ~.-----.-----,-----,-----.-----.------> 1.0 0.90 0.80 0.70 0.60 < 0.60 45 45 50 55 60 65 70 75 2 80 -0.99 -0.89 -0.79 6.9 -49 -54 -59 -64 -69 -74 -79 Bone mass (g/cm) Age (years) FIGURE 10 Graphs comparing the average (over 1000 person years) of fracture risk versus bone mass and fracture risk versus age. (From Johnston et aI., 1996, Figure I.) Decreased secretion or effect present, or alternatively, bone tissue resorption acceler of 1 ,25-(OH)2D or deficiency ated with a hjgh concentration of osteoclasts (Frost, of vitamin D 1973)? These two options are the yin and yang of bone remodeling, the dynamic system that underlies the viability of all skeletal organs (Urist, 1965; Mundy, 19<)9). The onset of osteoporosis is asymptornatic and pre vention is at least partially associated with an adequate diet and the intake, transport, and uptake of calcium, Decreased physical activity protein, and calories as well as vitamin D. The evalua tion of personal cboices, such as consumption of calcium-containing foods or specific trace elements via supplements may not be easily assessed for a particular patient. A variety of elements, hormones, enzymes, and proteins are essential in the compLcated integration of other organs and cycles of the body system for main taining a functional set of dynamic skeletal tissues (Figure 11). From tl1e production of Vitamin D, or parathyroid hormone, to the absorption of calc.ium at Bone loss the intestines, and recycling of phosphorus by the FIG U R E 1 1 Sketch illustrating the proposed contributions kidney, research has shown that all are important and to age-related bone loss in women. The interactions of vitamin must be properly integrated in order to arrive at the best D, hormones, and activity level affect the uptake of calcium, bone treatment for a particular patient with osteoporosis turnover, and cell function, which if not in balance, may cause (Avioli, 2000). bone loss. (From Eastell, 1999, Figure I.) 688 NIINERALOGY OF BONE B. Treatments for Osteoporosis other clinical studies showed no effect and the corticli portion of the long bones decreased in density relati\(> Over the past 50 years investigations from the cell level to an increase in trabecular bone density. The distur to animal model systems have been utilized to assess bance of cortical tissues could be modulated by incrca - treatment regimens. Although some therapies may ing Ca and Vitamin D intake or by arranging F-free appear promising in animals, they may not translate to periods during tre,ltmem (\Vatts, 1999). These inter effective treatments for humans (Jee, 1995). Much of esting insights into the use of fluoride supplements as ;1 the research has involved tests using pharmacologic possible means to increase Auorapatite in bone tissu e agents that act on several body systems and effect the also showed that only some of the fluorine found its \\ - a ~. balance of circulating calcium and phosphorus. This into bioapatite. vVhen new mineral was formed little if seems a sensible approach applicable to all the bones in any fluorine was partitioned into already existing bont the body rather than attempting to construct treatment mineral. The predominant bioapatite phase on bulk for specific bones. analyses remained hydroxylapatite. The gO;] I of osteoporosis tre,ltments is to ,lssist the Fluoride may be rapidly absorbed frol11 the stomach body toward nor1l1al bone grmvth and repair and specif but 50% will be e1iminatecl via the kidneys within a fe" ically to provide for normal mineral formation and hours. Serum fluoride levels between 0.1-0.25 mg L retention. Because the mineral is our foclls, but only one and doses greater than 70mg daily (35mg fluorine cOll1ponent in a complicated and lI1ultif,lceted system, produce grossly abnormal bone (Riggs & Melton. the following three sections on pharnlacologic treat 1995). In areas of China with naturally high Auorine in ments focus on tbe composition and rehltive amounts the environment, telltale signs of brown spots (fluoro of mineral to bring out some of the contributions pro sis) on the teeth and bem legs and bodies attest to its vided by this basic science presentation. interference in the strength and architectural character of normal mineralized tissues (Finkelman et aI., 19(9). There is a narrow therapeutic window for Auorine. 1. Fluoride Domestic water levels at 1!lg-L- l achieve dental bene The first thrust for making fluorine a thempeutic agent fits and may also provide some osteoporotic reLief, but was put forw,Jrcl by dental scientists. Their studies led what is the level and length of treatment, or which them to suggest thilt if the mineral in dental tissues was cooperative treatments might be p,]ired witll fluorine fluorapatite it would be less slIsceptible to car ies as flu ingestion to ensure no abnormal demineralization, and orapatite was the more stable apatite (\fIscher, 1970). To strong bone? incre,lse the formation of Auorapatite would require In shark teeth whose enameloid tissue shows the incrementing the amount of fluorine ingested that highest fluorine mineral content for any vertebrate, the would then become P,lrt of the bone mineraI ,1S the inor uptake unfortunately appears to be related to genetics ganic portion of the tissues should respond to the nu and phylogeny and not to the fluoride concentration in trients supphed. However, it was known that some the aqueous environment (Aoba et al., 1(97). The appro geographic areas with high fluorine in the waters led to priate dose and best schedule for human fluorine inges a disease known as fluorosis. The simple hypothesis of tion have not been fully determined. The possible impact more fluorine belies other aspects of the complicated of fluoride on bone and on other body systems wil! bone tissue system. Fluoride may not only form fluora require much longer and more comprehensive investiga patite, it may affect bone cells and the formation of the tions before fluoride can be part of routine treatment of organic matrix. To obtain the optimum amount of postmenopausal or other types of osteoporosis. fluorine to benefit human bones requires not only bioapatite production and composition, but also consideration of tissue recycling. 2. Bisphosphonates In the presence of smail amounts offluorine there was an increase in mineral matter and therefore the density Bisphosphonates are a group of compounds, analog'ues of bone tissues. One study showed that fluorine was of the pyrophosphates, where the phosphorus atom of mitogenic for osteoblasts and ,1 clinical investigation the phosphate group connects directly to a carbon atom. produced dram,1tic sustained (years) increases in bone The chemical arrangement of several pharmacological milleral density when administereel in doses of 20 bisphosphon ,VII ~ ER .\ LO"Y OF B()~ E 689 OR R[ OR 3. Hormones O=P-C-P=O The most familiar pharmacologic treatment for post OH Rl OH menopansal osteoporosis is to augment the lowered production of estrogens in older women. Although the where R1 ami R2 represent other chemical substituents maximum level of skeletal bone density is affected by (Fleisch, 2000). T hese phosphate-containing chemicals nutrition and usually achieved hy the age of maturity 3dsorb onto mineral surf Albright, ]. A., and Skinner, H. C. \iV. (1987). Bone: Struc tnral Org:ll1ization anel Remodeling Dynamics. In Tbe S(i VII. 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