The Influence of Lifelong Exposure to Environmental on Quality in Humans

Debbie Chachra

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Materials Science and Engineering University of Toronto

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Debbie Chachra Graduate Depamnent ofhIaterkds Science and Enginee~g University of Toronto

The objective of this study was to determine if lifelong esposure to environmental sources of fluoride (including fluoridated water) had an effect on bone quality in humans. Nine.-rsvo femoral heads were obtained fiom individuals undergoing total hip arthroplasty in regions wïth and without fluoridated water (ïoronto and Montreal, respectively) , so that the donors would have had a wide range of fluoride esposure. As the sarnples were obtained at surgery, the femorai heads were affected by osteoarthritis (75), (9) and other diseases. The fluoride content of cancelious bone was assessed by instrumental neutron activation analysis. h nurnber of contnbutors to bone quality were assessed. The compressive and torsional mechanical properties were measured for canceiious cores escised from the cencre of the femoral head. The architecture \vas assessed by image analysis of an s-ray of a 5 mm thick coronal section of the femoral head, as we!J as of histologicai sections taken from the superior (weightbearing) and the inferior (nonweightbearing) surface of the femoral head. The degree of mineralization was measured using backscattered electron imaging and microhardness, again at the superior and the inferior surface. Femoral heads frorn Toronto donors had a greater mean fluoride content than those

from Montreai donors (1033 + 438 ppm ur. 643 Ç 220 ppm). However, the fluoride content of the Toronto donors ranged approsimately twelvefold (1 92-2264 ppm) and entirely contained the range of Montred donors. Therefore, fluoridated water exposure is not the only determinant of fluoride content. The logarithm of the bone fluoride content increased wïth age. No substantive effect of fluoride, independent of age, was observed for the mechanical properùes, Similady, at the inferior surface, the architecture was affected by age but nor by fluoride incorporation but the degree of rnineralizaüon was nor affected by either. However, the degree of mineralization (measured by both backscattered electron ïmaging and microhardness) at the SU~~~OKsurface increased linearly with the fluoride content As osteoarthrïtis results in a reduced degree of minedzation at the superior surface, *is suggests that the presence of fluoride (whïch inueases the degree of mineralization in osteoarrhritis-affected bone) may aid in preventing this loss. Acknowledgments

Edrrcalion is u~hatmrviLpes wben what bas been leurnt bas beznJoptten.

B.F. Skinner (1 964)

Many, many people are owed thanks for their assistance wïth thïs endeavour. First and foremos~1 would iike to express my gratitude to my doctoral advisor, Dr. Marc Grynpas. Between the uncompromisingly high standards he sets for himself and the high espectatïons he has for me, I have much to iïve up to. As a result of his unfailing encouragement, support, and advice over the duration of this research, 11 have to come to believe that 1can. I would also like to thank my cornmittee members, Drs. Hardy Lïmeback, Robert Pilliar, and Zhirui Wang, for their espert advice. A nurnber of individuals provided assistance in recniiting patients into this study, obtaining specirnens, and prepa.ring specirnens. 1would like to thank oucoliaborators, Drs. Aan Gross and Carol Fiutchison at Mount Sini Hospital in Toronto and Drs. David Zukor and Me1 Sch~vartzat the Jewish General Hospital in Montreal. The staff of the Preadmission Clinic at Mount Sinai Hospitai were endlessly helpfd during the process of recnriting patients. The Departmenr of PathoIogy at Mount Sinai Hospital provided considerable assistance in prepating the specimens, Mr. Alan Wolff in particuiar. 1would aIso Like to thank Dr. Rita bdelfor her assistance and expertise. Technical assistance for tbis project was provided by the staff of the Speual Histology Laboratol (Ms. Maria Mendes is owed special thanks), Dr. Ron Hancock and hls. Susan Aufreiter of the Slowpoke Reactor Facility at the University ofToronto, and bk. Doug Holmyard. 1am grateful to Dr. Shelley Bdfor her expert advice on statistical analysis. Within the Grynpas Laboratory, 1would like to acknowledge the assistance of Mr. Richard Cheung. 1 wouid aiso iike to espress my appreciation to my labmates for making the laboratory a welcoming, fnendiy place to do research. Findy, thïs thesis would never have been smrted, much less completed, without the support and encouragement of my Giends and farnily, to whom X am deeply grateful.

3.3.4 Coronal secrions ...... -...... 1 16 3.3.5 Hi~foiogi'taillsectzonr ...... 118 3.36 Bac,& affered eledron imagz'ng ...... 121 3.3.7 ~Wobardne~stedkg ...... 123 3.3.8 S~~mmay:e~eéctof~~ofresiiettce...... 125 3.4 DIFFEREXESBE-N E-iIGH- -LND LOW-FLUORIDE L\!nlDU.-ILS ...... 126

3.4.0 Introdz&on ...... t...... 126 34.1 ~a~/einfomh'onforfhtftvBqzfartr~e~...... 126 . . 3.4.2 Cbenzicaa/ con.onfzon of ~aqbies...... 128 3.4.3 ~\.lechanica/p~erttës...... 131 3.4.4 Coronal sechans ...... 134 3.4.5 HistoiogicaI re&o ns...... 137 3.4.6 Bac,& aitend eiecfron i~naging...... 739 3.4.7 &Iinoharhe~s...... 7 42 3.4.8 Sttrnmary: top zm boifom parh7e offhode content...... 144 3.5 EFFEC~OF GEXDER ON RESPONSE TO FLUORIDE ...... 245 3.5.0 Introdtrctzon ...... 145 3.57 Paizintprojies ...... 745 3.5.2 Age, densiiy and fizioniie content...... 146 3.5.3 Chernical comporiton of cunceliors bone ...... 749 . . 3.5.4 fMecbanicai&mperfe~of cance//oozt~bone ...... 157 3.5.5 Image ana&ziof comnal setth...... 757 3.56 Image and shtanabis o/hizto(gicaisectzons...... 762 3.5.7 Buckrcatered electmn imagkg ...... 1 64 3.5.8 ~\ficrobarAnes.r tesfng ...... 769 3.5.9 St~mmary:efect ofgnder on reqorzse tofihooride ...... 175 3.6 EFFEC~OF DISFISE ON RESPONSE TO FLUORIDE ...... 176 3.6.0 Introdzcizon ...... 776 3.6.7 Pafièntpmj5ie.r and tbe predidor uanabie~...... 176 3.1.2 Cllemical COI* usifion of can ce//o/she ...... 177 3.6.3 illechanical tesritg of cancelhs botte ...... 187 3.6.4 Coronalsectzotz~...... 187 3. 1 .5 HLrtoIog'Gai sectz'onr ...... 193 3.6.6 Backrcattered e/ecfron imagng ...... 201 3.6.7 iVIicrohardneer tessfrzg of bor~e...... 206 3.6.8 Szfmnzaty: eefct ofdisease date on reqonse tofzionlie ...... 17 3.7 EFFEC~OFLOC_~~ON\LT~~FE~~OR-U.HE~...... 213 3.7.0 Introdrrchan ...... 273 3.7.1 Image mza&irof distoogzkai sam$es ...... 2 15 3.7.2 Backcattered eiemon imagihg ...... 7 3.7.3 Mim/I~~rdnessof bone ...... 219 3.7.4 Vanabi/ify Offiotl'de content...... -2.27 3.7.5 Surnmnry: efect if iocahon...... 222

List of Tables

Table 2.8.1 Qualitative grading scale for assessing the severitg of osteoartbritis .....,...... 5 1 Table 3.1.1 Ciy of ongin, gender. age and disease state of donors ...... 58 Table 3.2.1 Elementai composition of canceiious cores ...... 70 Table 3.2.2 Mechanicd properties of cancellous bone ...... 74 Table 3.2.3 Regression information for compressive mechanical testing ...... 75 Table 3.2.4 Image and swtanalysis of coronai sections ...... 83 Table 3.2.5 Regression information for image andysis of coronal sections ...... 84 Table 3.3.1 Chernicai composition of cancellous bone by INM ...... 113 Table 3.3.2 Mechanicd properties of canceiious bone ...... ,...... 115 Table 3.3-3 Image andysis data for coronai sections ...... 117 Table 3.3.4 Image anaiysis data for histological sections ...... 119 Table 3.3.5 Stnit anaiysis data for histologicai sections ...... 120 Table 3.3.6 Backscattered electron imaging data ...... 122 Table 3.3.7 Mïcrohardness of bone ...... 124 Table 3.4.1 Patient data for the high- and low-fluoride qudes...... 127 Table 3.4.2 Chernical composition of cancellous bone ...... 129 Table 3.4.3 Compressive mechanicd properties of canceilous bone ...... 132 Table 3.4.4 Torsional mechanical properties of canceiious bone ...... 133 Table 3.4.5 Image analysis of coronal sections ...... 135 Table 3.3.6 Smtanalÿsis of coronal sections...... 136 Table 3.4.7 Image and stmt analysis of histoIogicai sections ...... 138 Table 3.4.8 Degree of mineralization by backscattered electron imaging...... 140 Table 3.4.9 ~Microhardnessof bone ...... 143 Table 3.5.1 Chemicai composition of cancellous bone, by gender ...... 150 Table 3-52 lvfechanicai propemes of cancellous bone, by gender ...... 153 Table 3.5.3 Mechanical properties and predictor variables ...... 154 Table 3.5.4 Image analysis of coronai sections, by gender ...... 159 Table 3.5.5 Image and strut analysis of histologicd sections, by gender ...... 163 Table 3.5.6 Backscattered electron imaging, by gender ...... 165 Table 3-57 Mcrohardness of bone, by gender ...... 170 Table 3.6.1 Chemicai composition of osteoporotic and osteoarthritic bone ...... 178 Table 3.6.2 Mechanicd properties of osteoporouc and osteoarrhritic bone ...... 183 Table 3.6.3 image and smt analysis of coronal sections ...... 188 Table 3.6.4 Image and stmt anaiysis of histological sections ...... 195 Table 3.6.5 Differences benveen superior and inferior sections ...... 196 Table 3.6.6 Image and stn~tandysis of histological sections, by gender ...... 200 Table 3.6.7 Backscattered electron irnaging, by disease...... 203 Table 3.6.8 Differences between superior and inferior sections ...... 204 Table 3.6.9 Microhardness of femoral heads ...... 208 Table 3.7.1 Image analysis of histoIogical samples, by site ...... ZIG Table 3.7.2 Backscattered electron imaging data, by site...... 218 Table 3.7.3 hiïcrohardness of bone, by site ...... 220 Table 4.1.1 Fluoride content of canceilous bone ...... 233 Table 4.1 -2 Pearson correlations of BSE and microhardness ...... 253 List of Figures

Figure 1.2.1 Factors which collectively comprise bone quality...... 11 Figure 1-3.1 Plasma concentration curve following a bolus of ingested fluoride ...... 15 Figure 1-7.1 Backscattered electron images of fluorouc bone ...... 25 Figure 2.2.1 Schematic of femoral head: sample locaüons ...... 33 Figure 2.5.1 Backscattered elecuon image of cancellous human bone ...... 43 Figure 2.6.1 Schematic of two-dimensiond star volume ...... 46 Figure 2.6.2 Schematic of trabecular struts ...... 47 Figure 2.7.1 Contact s-rays of coronal sections of femoral heads ...... 50 Figure 32.1 Relationship bem-een densiy and age of donor ...... , ...... 64 Figure 3.2.2 Relationship benveen density and fluoride content...... 65 Figure 3.2.3 Relationship between fluoride content and age of donor...... 66 Figure 3.2.4 Relationship between logarithm of F content and age ...... 67 Figure 3.2.5 Relationship between chlorine and fluoride content ...... 71 Figure 3.2.6 Ultirnate compressive stress is correlated \.thdensity ...... 76 Figure 3.2.7 The main at UCS deciined weakly with age ...... 77 Figure 3.2.8 Ultirnate stress is negatively correlated to the F content ...... 78 Figure 3.2.9 Energy to yield deciined with F content ...... 79 Figure 3.2.10 Relationshïp bemeen shear modulus and age ...... 80 Figure 3.2.1 1 Trabecular bone volume decreases with fluoride content ...... 85 Figure 3.2.12 Trabecular separauon increases wïth fluoride content ...... 86 Figure 3.2.13 Nurnber of endpoints increases with densiq- ...... 87 Figure 3.2.14 Image analysis of inferior section: trabecular separation ...... 90 Figure 3.2.1 5 Image analysis of Liferior section: trabecular number ...... 91 Figure 3.2.1 6 Image analysis of infenor section: trabecular volume ...... 92 Figure 3.2.1 7 Image analysis of inferior section: trabecular volume ...... 93 Figure 3-2-28 Strut analysis of inferior section: nodes ...... 94 Figure 3.2.1 9 Strut analysis of inferior section: nodes ...... 95 Figure 3.2.20 Strut analysis of inferior section: free ends ...... 96 Figure 3.2.21 hheralization of superior subchondral bone ...... 99 Figure 3-2-23 Mineralization of cancelIous bone at the superior surface ...... 100 Figure 32-23 Logit of subchondral bone at the superior surface ...... 101 Figure 3.2.24 Logit of cancellous bone at the superior surface ...... 102 Figure 3.2.25 Logits of subchondd bone at the inferior surface ...... 103 Figure 3.2.26 Mïcrohardness of superoprosimal bone: subchondral...... 106 Figure 3.2.27 hiïcrohardness of superoproxhnal bone: cancellous ...... 107 Figue 3.2.28 Microhardness of superodistal bone: subchondral ...... 108 Figure 3-3.1 Fluoride content of bone from Toronto and Monueal ...... 112 Figure 3.5.1 Fluoride content is not related to age in men ...... 147 Figure 3.5.2 Fluoride content is related to age in women ...... 148 Figure 3.5.3 Yield stress vs log of the fluoride content: males only ...... 155 Figure 3.5.4 Shear modulus decreases with age: mdes only ...... 156 Figure 3.5.5 Trabecular separation increases \.ch F content for males ...... -160 Figure 3.5.6 Trabeculas nurnber decreases with F content for males ...... 161 Figure 3.5.7 fierzlization of superior subchondrd bone: males only...... 165 Figure 3.5.8 LMineralization of superior subchondrd bone: females only ...... 167 Figure 3.5.9 Mineraiization of superior cancellous bone: femdes only ...... 168 Figure 3.5.10 Microhardness of subchondral bone: fernales only ...... 171 Figure 3.5.1 1 Mcrohardness of cancellous bone: females ody...... ,...... 172 Figure 3.5.12 Microhardness of subchondrai bone: females only ...... 173 Figure 3-5-13 hficrohardness of canceiious bone: females only ...... 174 Figure 3.6.1 Chlorine concentration increases with fluoride content ...... 179 Figure 3.6.2 Calcium-to-phosphate ratio decreases wïth F content ...... 180 Figure 3.6.3 Ultimate compressive stress of osteoarrhatic sarnples ...... 184 Figure 3.6.4 Compressive modulus fds with age in OA femaies ...... 185 Figure 3.6.5 Energy to yieid and F content: 0Aonly ...... -186 Figure 3.6.6 Trabecular nurnber decreases with age for OA patients ...... 189 Figure 3.6.7 Trabecular separation increases \.th age for OA patients ...... 190 Figure 3.6.8 Trabecular thickness increases with age for OA patients ...... 191 Figure 3.6.9 Trabecular number for osteoarthritic males ...... 192 Figure 3.6.10 Nurnber of nodes for fernale OA patients ...... 197 Figure 3.6.1 1 Superior trabecdar separation of male OA patients ...... 198 Figure 3 -6.12 Merior trabecular number of femaie OA patients ...... 199 Figure 3-6-13 ~Mineralizationof superior cancelious bone ...... 205 Figure 3.6.14 Microhadness of superodistal subchondral bone ...... 209 Figure 3.6.15 Microhardness of superoprosimal subchondral bone ...... 210 Figure 3.7.1 Schematic of femoral head: sarnple locations ...... 214 Figure 4.1.1 Total mineral content decreases with density of the cancellous core ...... 234 Figure 4.1 -2 Contact s-ray of coronal section ...... 241 Figure 4.1 -3 Ultimate compressive stress vs the square of the density ...... 242 Figure 4.1.4 Yield stress increases Lineady \.thcompressive modulus ...... 243 Figure 4.1 -5 Features of the stress-strain cuve for cancellous bone in compression ...... 244 Figure 4.1 .6 Structue of cancellous bone ...... 245 Figure 4-1-7 Node-node sauts and free-free struts: histologicaI section ...... 249 Figure 4.1.8 Node-node struts and free-free struts: coronal section ...... 250 Figure 3.1.9 Microhardness and mineraikation of superior subchondrai bone ...... 254 Figure Al Measured parameters of compressive mechanical tes~g...... 27G 1: Introduction GeneralJack D. Ripper: ~bfandrake,do yon reake that in addirion to fioniialrng rvater, why, there are ~tttdie~z~ndenvay to Jnodate ml.jIozi4 fi~itjzricei,,-oz@, rrrgar, mdk ... ice cream. Ice mam, Mundrake, chi/dren's IGe meam

Group Cap. Lionel Mmdraùe: Lord Jack.

GenewackD. ICIpper: I'oti know when~oaorbegan?

Group CapLiunel Man&ake: I-110, no. I don '4 Jack.

Stanley Kubrick Dr. Strangeelo~e (1964) 1.0 Introduction

Fluonde is an element that occurs naturdy in the hurnan diet and is found in significant concentrations in foods such as fish and other marine products. May water sources also contain fluoride. The interaction of fluoride and mùlerdized tissues in the body was hrst recognized early in this century as a result of tosic fluoride esposure, wîth the dual observations of endernic in regions where the water supplies were naturdly fluoridated at high (>4 ppm) levds pean 19361 and of kcreased radiodensity of the skeleton in individuals esposed to fluoride [Kleerekoper 19961. The observation that increased fluonde esposure led to decreased risk of dental caries (see section 1.2.1, below) led to the wïdespread fluoridation of municipal water supplies at 1 ppm, beginning in 1945 [Horowitz 19911. Sirnilarly, the observation of increased bone mass with fluoride esposure suggested that fluoride rnay be an appropriate therapy for osteoporosis [Rich and Ensinck 19611. Over the last half-century, the effects of moderate to high doses of fluoride on bone have been investigated in animal models, in dinical studies, and in epidemiological smdies. iiithough some controversy remains about the appropriate dosages and treaunent protocols to optimaily increase bone mass and decrease fracture risk, the mechanisms of action and the results of therapeutic fluoride administraüon are generally weil-understood. However, fluoride has a cornples, dose-dependent suite of eff-ects on bone (see below). Moderate to high doses of fluoride, administered over a span of several months to several years, mai result in a similar accumulation of fluoride in bone as esposure to low doses of environmental fluoride (approsimately 1 mg/day) over the course of decades. As the mechanism of action of fluoride on bone varies strongly with the dose, the effects on bone may be quite different. While the assessment of acute esposure lends itself to research in animal models as \veU as clinical studies, research to understand the effect of esposure to environmentai fluoride on bone cm only be performed in speües where the requisite decades of esposure are possible; in practice, oniy in hurnans. W'hiie the effect of environmental esposure to fluoride on bone fracture risk in humans has been assessed by epidemiological studies (see below), the approach taken in this smdy is quice different. The purpose of this research is to determine if nuoride accurnulated through esposure to environmental sources cm alter bone quali5 and if so, to determine what the relaaonship is between the measured parameters and the fluonde content of bone. For this study, humari bone sarnples were obtained in the form of femoral heads retrieved duEng tord hip arrhroplasty fiom patients residing in municipalihes with and without artificidy Buoridated water (Toronto and Montreal, respectiveiy). The mechanical integrity of the canceilous bone (which cm serve as a prosy for fracture risk), together with its nvo major underlying components-the degree of mineralization and the architecture-were each esamined.

1.1 The structure and physiology of bone

1.1.1 The structure of bone

From an engineering perspective, bone is a composite material with a comples, three- dimensional architecture. The material of bone consists of an organic rnatrk reinforced with minerai in approsimately equal volume fractions (aithough in a 1:3 ratio by weight because of the si,pificandy higher density of the mineral). Over 90% of the organic component is type 1 coiiagen; the remainder is primarily non-collagenous proteins and smail molecules. The mineral component consists of poorly crystalline hydrosyapatite, with a stoichiometric formula of Ca,,,(POJ,(OH),. However, the mineral component is not generdy found in this stoichiometric form, as a number of ions substitute for the phosphate and hydroqi groups, induding sodium, carbonate and fluoride. The minera1 Çorms needle-like crystals, 3-6 nm in diameter and 20-40 nrn long. In the composite structure, these crystals Lie parailel to the coliagen fibres [Bouvier 19891. \Yrhile the esact nature of the relationship remains poorly understood, it is likely that the coilagen fibres epitaxïaily nucleate minerai growdi, resulting in a close apposition benveen the nvo components and a strong interface, thereby forming an effective composite with high strength and unparalieled toughness. Bone assue in the body thar is primariiy loaded in compression, such as the spine and the epiphyses (the ends of die long ), has a sponge-like appearûnce and is termed 'cancelious' or 'mbecular' bone. This bone has a porous structure, characterized by a comples three-dimensionai latticework of plates and struts (trabeculae). The architecture of the smcture, including the amount present, the orientation, and the degree of connecuvity, contributes to the mechanical strength of the tissue as a whole. 1.1.2 Remodelling in bone

Unlike the other major rnineralized tissue in the body, the teeth, bone is not a static materiai. Tnroughout the lifetime of the individual, even afier growth is complete, bone tissue is remodeI1ed; that is, material is removed and replaced. The biological activity in bone is mediated by nvo types of ceils. Bone is resorbed by osteoclasts, mdùnucleated giant ceiis derived fkom monocytes, which act by first dissolving the bone mineral by creating a highiy acidic microenvironment benveen the cedl body and the underlying bone tissue, and then digesting the collagen matrix. New bone is then formed by osteoblasts, derived Erom fibroblast-like mesenchymal precursors, mhich Iay down an organic matrk which is dien mineralized. Resorption and formation of bone are normdy tighdy coupled, Gth new bone beïng laid down immediately after resorption is completed. In canceilous bone, such as is found in che femoral head, this process cakes place on the surface of the trabeculae DCaplan et a/. 19941. As closely coupied as resorption and formation are, they are rarely esactly baianced and therefore the amount of bone that forms the skeleton may be increasing or decreasing. DLU%I~growth, the rate of formation esceeds the rate of resorption considerably. However, after the age of approsimately thirty, the balance shifts siïghdy towards increased resorption. This results in a graduai decline of bone mass over the remaining liktime of the individuai

Kaplan et a/.19941. RemodeIling esists for severai reasons. The Eirst is homeostatic; the mirierd in bone serves as a reservoïr for calcium and phosphate. More than 9g0/o of the cdcium and 85% of the phosphorous in the body is sequestered in the bone. Remodeliing provides a mechanism to remove and replace these physiologically important ions as needed. In addition, remodelling aids in maintaking the mechanicd integrity of bone. Constant turnover of bone (on average, al1 the bone in the skeleton is replaced every ten years) ensures that fatigue damage does not have a chance to accumulate to dangerous levels. Additionaliy, there is increasing evidence that dus mai not simply be an unguided, prophylactic process; bone remodehg has been found to increase in areas of severe fatigue damage wori and Burr 19931. In this case, apoptosis of cells in the damaged areas may be the trigger that begins the resorption/formation qde. The remodekg process also provides a mechanism by whîch the shape or die architecture of the bone can respond to changing loading patterns, whether increased loading or disuse. The hypothesis ùiat the structure of bone directly reflects the applied load is termed Wolff s Law pplan et al. 1994; Wolff l986j. In addiaon, the rate of rernodelling is affected by other factors, notably hormones. Ses hormones, such as testosterone and estrogen, are responsible for the gro~vthspurt at puberty as \veU as influencing bone mass throughout life. At menopause, for esarnple, the cessation of estrogen production leads to more rapid turnover of bone, resulting in accelerated bone loss.

1.2 Assessing changes to bone

1.2.1 Bone quality

The concept of bone qualiry \vas introduced in response to the recognition that bone mass or density is an insufficient predictor of fracture risk [Heaney 1992, Heaney 19931. \We the term is somewhat loosely dehed, bone quality is comprised of the parameters of bone chat illustrate or contribute to its mechanical ïntegrity, and therefore to the fracture risk in the patient. These parameters and their interrelationships are illustrated in Figure 1.2.1. In ktro measurements of the mechanical properties of the bone are considered to correspond directly to the fracture risk. The mechanical propemes themselves are a function of both the bone material and the architecture (including both the amount and comectiviq of die bone). The primaq determiriant of the former is the degree of mineralizaaon of the bone. The architecture, or the structure and comectivity of the bone, can be assessed by image analysis. Remodeling pararneters, as measured by histomorphometry, conuribute to both the architecture and to the material properties of the bone.

1.2.2 Changes to mechanicd properties

Among the many roles that bone plays in the body, its mechanical function is certainly the most conspicuous and one of the most important. This is highlighted in patients with osteoporosis, which is characterized not by bone lossper se but by the loss of the mechanical integriy of bone, as manifested by fracture upon minimal loading. The mechanical function of bone depends not only on the amount of bone present, but also its organization at the macroscopic (such as the shape of long bones), the microstructural (the architecture of trabeculae or of Haversian systems) and the ultrastructural (the inamate association of coiiagen and mineral) level. Alterations to any one of them can have profound effects. The mechanical function of bone cm be assessed in &O by mechanical teskg; a sample of bone is loaded to failure in a universal testing machine and the mechanical properties are determined (the methodology is discussed in detaii in section 2.4). The advantage of this technique is that it integrates aIl the changes that occur in the bone into parameters that are closely related to a dinicaliy significant outcorne; the mechanical

properties are considered to be â proxy for the fracme risk. The disadvantage, of course, is

that it is a destructive, in mtro test

1.2.3 Changes to rnineralization: backscattered electron irnaging

Backscattered electron imaging of bone tissue is a powerful technique that ailows visual discrimination of different Ievels of rnineralization, induding cernent lines, newly-formed bone, normaiiy- and hypemrineralized bone [Boyde and Jones 19831. Duhg bone formation or rernodeling, the unmineralized precursor of bone (osteoid) is iniùdy laid down. Prirnary mineralization occcurs immediately and the tissue attains about nvo-thirds to three-quarters of its total mineral concentration at this point Secondary rnineralization occurs as the minerd content increases to its Einal level over the following months [ Lacrois 19711. The result of this is a disnibution of regions with a range of mineralizations [eg Portigliatti Borbos 19831. This distribution, and the resdting rnineraiization profile, can be affected by age, disease state, or fluoride esposure (set below). Briefly, analysis is performed \.th a scanning electron microscope, but rather than coliecting secondary electrons produced as a result of bornbardment with tbe elecuon beam, backscattered electrons (electrons that are deflected almost direcdy bachvards from che sarnple) are coilecred by an annular detector located hostdirectiy above the sample. The probabilitv of this type of collision occ~gincreases with increasing atomic number (that is, with increasing numbers of protons in the nucleus). The tota! observed intensity dl be related both to rhe atornic number of the constituents of the sample and to the density of the nudei. In the case of bone, the constituents of the sample are largely hed (calcium and phosphate, together with oq-gen, carbon and other lighter nuciei). The intensity of the detected electrons, therefore, is related only to the density of the heavier nudei; in other words, ro the amount of calcium (that is the degree of rnineralization) of the region being imaged. Studies using synthetic analogues of bone material as weil as chick bone from a wïde range of ages conbedthe cone1ation benveen observed greylevel and degree of rnineralizauon ES kedros et al: 1993a, 1993bI Relatively Little research using backscattered electron imaging to look at age-related changes in bone has been perfonned. A study evamining the degree of mineralization in ribs from ùidividuals ranging from neonatai to 59 years of age suggest that it increases with increasing age in adults [Reid and Boyde 1987. However, smdies using microradiography and physicai measurements of density suggest that the degree of mineralization of corticd bone increases to approsimately 45 years of age, decreases at GO45 years, and increases again to 80-85 years [Grynpas and Holrnyard 1988, Sirnmons et al. 19911.

1.2.4 Changes to mineraikation: rnicrohardness testing of bone

The hardness of a matenal is defined as its resistance to penetrauon, which corresponds weii to our intuitive notion of what constitutes hardness. From an empiricai perspective, the hardness of a material is measured by irnpressing an indenter into a material, under a aven load, and measuring the size of the indentation forrned. The relationship benveen the two is a measure of the hardness [hprino 19611. Practicaily, however, thïs raises another point; that the indentation produced must be permanent (or at leasr survive long enough to be measured). Therefore, the hardness is also related to the permanence of the deformation pees 1981J. klïcrohardness is usually deGned as the hardness measured under an applied load of less than 200 g, producing an indentation less than 30 pm in ciiarneter PVeaver 19661. Since the eariiest studies of microhardness of bone, it has been recognized that the microhardness correlates very dosely to the degree of mineralization [Carlstrom 19541. Microhardness aiso correlates closely to the Young's modulus and yield strength of the materid (Evans et a[. 1990, Currey and Brear 19901. Ln tuni, the degree of mineralization correlates closely to the mechanical properties of the bone mateùal (the mechanical properties of the tissue as a whole would, of course, also depend on the architecture of the material) podgskinson et O/. 1989, Evans et a/.l99O]. Measuring the microhardness, therefore, provides a link benveen the mineralizauon of the bone and its material properties. While the mineralization of bone was observed to increase somewhat with increasing age, as measured by backxattered electron imaging or microradiography (as discussed above), rnicrohardness testing of cortical and cancellous bone showed relauvely lide change in rnicrohardness with age aher skeletal maturity Peaver 19661. 1.2.5 Changes to architecture: image analysis

The mechanicd properües of bone are the result of nvo integrated factors: one is the matenai propertïes of the bone, discussed above, and the second is the architecture of the bone. Cancellous bone has a comples three-dimensional architecture of intercomected struts and plates. The amount of bone (and b y extension, the size and spaùng of individual trabeculae), the orientation of the trabeculae, and the amount of comectivity of the bone ail contribute to its mechanicai propemes PIosekilde 19981. The structure and co~ecuvityof canceiious bone cm be assessed by image analysis techniques. The general approach is to image a histologicai section, x-ray, or phorograph of bone to a computer and to use ùireshoiding techniques to create a binq image of the structure, Gom which the structurai paarneters (proportion of trabecdar bone, trabecdar rhiclrness, separation, and number) cm be obrained. The image is then skeletonized, resulting in a schematic of the aabeculae, and the multiple points (points where trabeculae intersect) and the fiee ends are identified. From this structure the comectivity parameters are determined [Sema 1988, Grynpas et a/.19931. In healthy young individuais, canceiious bone is dense and well-comected. As individuais age, this comples meshwork alters. The amount of bone decreases over time, with a concurrent loss of connectivity @Iosekilde 19981. Studies of cancellous bone in the vertebra and the iliac crest illustrate an age-related decline of nearly 50°/o beween the ages of 20 and 80 [Thomsen et al. 2000, Dempster et a(. 1993, Grote et al. 1995, hlosekiide 1989, Clarke et al

1996, Schnitzler ei a(. 1990aI. The star volume and the node:te&us ratio (both measures of the comectivity) increase and decrease, respectively, wirh increasing age pomsen et al. 2000, Vesterby et al. 1989, Vesterby 19901.

1.2.6 Changes to remodeling: histomorphometry

Parameters measured in static histomorphometry, as used in the present study, overlap somewhat with the structurai panmeters measured by image analysis. These include the trabecular bone volume, thickness, number and separation. However, the use of Masson's trichrome staining of the histological samples, rather than von Kossa staliing of minerai, dows newly formed, unmineraiized bone-osteoid-to Le distinguished from older bone. The parameters relating to the osteoid (osteoid volume, surface and thickness), together with the eroded surface, provide an assessrnent of the degree of remodehg (bone formation and resorption, respectively) occuning in the specimen. The rate of remodeling bears on the material properties of bone; increased bone formation is associated with a greater proportion of unrnineralized osteoid as weU as a greater proportion of newer (and therefore less mineralized) bone. These changes shifi the rnineralization profile to\vzds Iess-mineralized, and therefore less stiff, bone, As well, of course, a remodeling process that favours resorption results in bone loss, which manifests in che architectural properties as both reduced trabecuiar bone volume and as a loss of connectiviy. As discussed above (section 1-25),normal age-related changes include a loss of bone, with an associated decline in uabecular thickness and number and an increase in the uabecular spackg. At the iliac crest, osteoid parameters show lide change [Clarke et al: 19961 or an increase in osteoid surface and volume [Schnitzler et al 1990a] with age. fracture risk

mechanical properties

material architecture I 1 1 1 1 1 1 1 I 1 I I 1 1 1 1 1 1 1 1 1 j mineralization remodeling structure, 1 1 para meters connectivity i bone quaFty

FLgure 1.2.1 Factors which coffdvefy comprise bone quaLity

Bone quality is a function of the mechanical, material, and architectural properties of bone. 1.3 Fluoride and the body

13.1 Fluoride exposure: water fluoridation

In the 1940s, communiq studies established thar the incidence of dental caries decreased with increasing fluoride concentration in drinking water [Centers for Disease Control 19921.

Given the generaliy poor state of oral health in the population at the Ume [Schlack ef nl. 19461, public health officiais suggested thac communal water supplies be artificiaily fluoridated at 1 ppm, and in 1945, Grand Rapids, Michigan became the hst Ùty to do so, foilowed by many other municipal centres across Noah America fHorowitz 19921. Since its inception, fluondated \vater has dramatically reduced the incidence of caries, and this positive effect has reached across aii socioeconornic groups, and artificial fluoridation of water is generaliy considered to be a successful public health initiarive. Since 1935, however, Buoride has becorne a cornponent of numerous other dental care products, including toothpaste, mouthwash, and topical fluoride preparations. Simultaneously, concern has arisen about the incidence of dental fluorosis and die possible impact of environmental exposure on the skeleton. A recent meta-analysis PIcDonagh er a/. 20001 suggests that the positive effect of fluoride on caries incidence is overstated; they indicate that the median of mean differences in the proporcion of children \vithout caries is approsimately 15%. They also observed that, at 1 ppm, an estimated 12.5% of people esposed suffer from dental fluorosis that they 6nd 'aestheticaily concerning.' These data suggest that municipal water fluoridauon may be a vi& ofits own success; the widespread adoption of fluoride-containingdental care products is reducing the impact of water fluorïdation. Concurrentiy, there is increasing concern about other effects that fluoride has on rnineralized tissues.

1.3.2 Fluoride ingestion

Fluoride is ingested rhrough fluoridated water, food, and oral care products such as toothpaste and fluondated mouthwash. M&cipdy fluoridated water generdy has a fluoride concentration of 1 ppm (1 mg/L), and the average adult consurnption of water is 1.5 L per day. Many foods contain fluonde, notably fish and other marine products, and tea [Singer et a/.198q. Fluoride ingestion from food in individuals over the age of nvelve has remaîned constant at appro'umateiy 0.4 mg/day Purt 19921. Increasingly, however, the diet of individuals king in non-fluoridated areas is comprised of beverages and foods prepared ~5thfluoridated water [Clovis and Hargreaves 19 881. Individuals, especiaiiy children pimard et al. 19891, also ingest fluonde from oral care products, paflcularly toothpaste. On average, 1 g of toothpaste is used per bnishing, and adults typicdy ingest about 25% of it Da7hitford 1994I. In North America, toothpaste is fluondated at 2000-1 100 ppm, resulung in ingesùon of 0.25-0.75 rng/day of fluoride. Fkaily, fluonde supplements are ofien provided in non- fluoridated areas Ilsrnail 19941. Overd fluoride consumption therefore includes other sources as weLi as fluoridated water, with an estirnated addt total intake of fluonde ranging from 1.2 to 2.2 mg/day BYhirford 19941.

1.3.3 Fluoùde metabolism

Figure 1.3.1 presents an overvlew of fluoride metabolism in the body PWiitford 19891. Ingested fluoride is absorbed rapidly and almost completely in the body. iX"hde some absorption occurs in the mouth, this is likely to be an issue ody with topical fluonde preparations that are not swailowed FVhitford 19891. The majority of fluoride absorption occurs by diffusion, prirnarily in the stomach and the proximal small intestine. Approsimately 80 to 90°/o of ingested fluonde is absorbed. The absorption is very rapid, mith a half-life of approsimately 30 minutes, so peak plasma concentrations are reached within an hou [Phipps 19961. Non-absorbed fluoride is generaiIy escreted in the feces Foivin and Meunier 1990). Once absorbed, fluoride enters the plasma. It does not bind to proteins or any other constituent paves 19681. It then distributes between nvo comparunents: the blood and soft tissues, from which it is cleared within a few hous, and the calcified tissues, in which fluoride cm be sequestered with a half-life of years Foivin and Meunier 19901. The concentration in sofi üssues is proportional to the plasma concentration PVhitford et al. 19791. Calcified tissues clear fluonde from the plasma at an estremely high rate; in adults, approsimately half ofingested fluonde becomes associated wîth &e bones within 24 hours (&e remaïnder is escreted). h higher proportion of fluoride is retained in the calcified tissues of young children due to their high bone modeling rates mtford 19941. Fluoride incorporation and its relationship to bone remodehg is discussed in detaii below (section 1-4.1). Fluoride is generally not considered to be under homeostatic control in the body, and therefore the plasma concentration is a fhction of the ingested fluoride. In healthy adults for whom fluoridated drinking water provides the major source of fluoride, the fastinç plasma concentration of fluoride (i pmol/L) is numericdy equivalent to the concentration of the fluoride in the water (in ppm), clearly indicatlig that the plasma concentration of fluoride is not regulated in the body jJ'9hitford 19941. Fluonde that is not taken up by the skeleton is escreted into the urine by the kidneys. Its rend clearance rate is far more rapid than other halogens; Li healthy adults, the clearance rate is approximately 35 mL/mLi, alhough the range can vqconsiderably [l.tford 19941. The rend clearance of fluoride in young children is much lower than that of adults, rangïng from approsimately 4 ro 9 mL/rnin. In addition, here is evidence that the uptake of nuonde into the skeleton may be dose-dependent Fkstrand rt al. 19921. This suggests that children may be more susceptible CO adverse effects of fluoride esposure. Little data is available for the elderly. A number of factors can affect fluoride metaboiism. Fluoride absorption in the stornach is affecred by the presence of elements that form insoluble compounds with fluoride, notably calcium DVhitford 19941. Unsurprisingly, a high-calcium diet is therefore associated with increased fecal escretion of fluoride [Cerklewski and Ridiingron 1987, Whirford 19941, and dus is also why calcium-containing liquids are given to individuals foUowing esposure to a toxic dose of fluoride. Fluoride absorption is also increased with decreasing gastric pH PYhitford 19891 and this may be a variable that pdally accounts for the differential response of individuals to . Findy, the profile of fluoride concentration in the plasma (and therefore the esposlrre of the calufied tissues to fluoride) is a function of how rapidly it is cleared from the svstem and therefore on the rend clearance. Good responders to fluoride therapy for osteoporosis have decreased rend clearance compared to poor responden [ICraenzlin et al. 19901 Increased urïnary pH and f3ow result in increased renal clearance FVhitford 19891. Conversely, the rate of clearance is decreased in individuais with reduced renal function; these individuals are therefore also at greater risk for skeletd fluorosis [Boivii et ai. 19861. A nurnber of other factors can affect fluoride metabolism, including altitude, aùd-base disturbances, and esercise prford 19891. I1 I 1 I HOURS

Figue 1.3.1 Plasma concentration curve folio wu7g a bolus of ingesteci fluozide

[reprinted from Whitford 19891 1.4 Fluoride and bone

1.4.1 Incorporation of fluoride

In general, fluonde is not incorporated into fuliy mineralized bone and accumulates only in bone forrned during the penod of esposure poivin and Meunier 19931. A mathematicai mode1 of fluoride incorporation in the skeleton Fumer et a/.19931 indicates that the total bone fluonde content is linearly related to the daily ingestion of fluoride in adults up ro age 55, afier which ic appears to plateau. However, other research suggests that fluoùde cm continue to be incorporated into bone through to the eighth decade oFlife DXreatherell 1969, Ishiguro et a(. 1993, Richards et a(. 19941. The incorporation of fluoride can be affected by other factors, notably osteoporosis or impaired rend fiction. In osteoporosis, as resorption of bone esceeds formation, fluonde is mliimdy incorporated into bone. Ln fact, if daily intah is low, patients may actudy /ore fluoride fiom bone /JX%tford 19891. Conversely, increased uptake of fluonde by bone results if rend fünction is impaired since ingesred fluoride is not deared rapidly from the system @kstrand and Spak 19901. Therefore, fluoride levels in bone cm greatiy [Weathereli 1969, Ishiguro et d 1993, Richards et a/.19941. The concentration of fluonde in bone is generdy higher in sites with a higher turnover rate, such as canceiious rather than cortical bone PVeathereil 1969, Ishiguro et al 19933, or vertebrae rather than the iliac crest Fumer et a/.19931.

1.4.2 Effect on bone mineral

Regxdless of concentration or dose, fluoride is incorporated into bone mineral during formation, via a p hysicochemical mechanism. Fluoride substitutes for the hydrosyl group in hydrosyapatite, formuig fluorapatite. This substitution, while no means complete (even in highly fluorouc bone, fluoride replaces onlg about a thïrd of the hydrosyl ions) [Boivin et a(. 19891, neverrheless has profound consequences. The £luonde malies the crystal lanice more compact -and stable [Grynpas 19901, and a mixture of fluorapatite and hydrosyapatite has been shown to be less soluble thm either component indivïduaiiy PIoreno et a(. 199q. The onset ofmineralization is also delayed, resulting in increased osteoid formation [Grynpas et a/.19861. The concentrations of other contaminant ions, such as carbonate and magnesiurn, appear to be affected, although the esact effecr remains controversial [Grynpas 19901. Finally, fluoride shifts the mineralization profie of bone (a histogram of density fractions) towards denser, more mature fractions and fluoride appears to be concentrated in these denser &actions [Grynpas et al. 198G].

1.4.3 Effect on bone cells

Wefluoride is passively incorporated into bone rnineral at all concentracïons, it appears to oniy affect bone ceiis at much higher serum levels than would be experienced through dnnking fluoridated water alone Fumer et al. 19931. It is weli boum that high levels of fluoride increase bone mass in patients subjected to fluonde therapy and in fluorotic individuals (discussed fuaher below). This is a result of both the increased resistance to resorption of fluoridated bone mineral, rogether with a mitogenic effect of these levels of fluonde on osteoblasts. This is supported by work both in vivo Foivin etaL 19891 and in culture [Gruber and Baylink 19911. However, the osteoblasts appear to be flattened and moderately active rather than plump, cuboidal and highly secretor).. This suggests that, while fluonde is mitogenic and a promoter of differentiation of osteoblast precursors, it is somewhat tosic to individu4 ceiis at these concentrations [8onjour et a/. 19931. Nevertheless, the overali effect is of increased bone formation. LWe the effect of fluoride on osteoclasts is less weU-understood, there is some in m5ro evidence that sodium fluoride decreases the number of resorption Iacunae as well as the amount of bone resorbed per osteoclast [Okuda et a[, 19901. The net result of these effects is increased bone formation, which accounts for the interest in fluoride as a therapy for osteoporosis.

1.4.4 Effect on bone architecture

Therapeutic administration of rnoderate doses of fluoride (tens of milligrams per day) to patients \.th osteoporosis resuIts in a marked increase in bone mass. However, osteoporosis-associated bone loss results in loss of connectivity as well as thinnïng of the remaining trabeculae and both are thought to be mechanicaüy significant. At present, no biological pathway is knom to restore the comecuvity, and it is therefore thought that ail types of therapy are similarly ineffective at doing so parfitt 2 9821. Accordingly, fluoride therapy has been shown to increase trabeculat chickness, but leave the co~ecuvityunalcered [Aaron et a/.19921. In addition, tlabeculae thus forxned appear to be resistant to perforation by resorption [Aaron et al. l992]. Rais who ingested fluoridated drinking water in a range of concentrations (0,2,4,6 &i/L) displayed sirnilar changes to canceilous bone, and the

Introduction effects were found to be dose-dependent [Cheng and Bader 19903. Findy, biopsies obtained fiom individu& who received fluonde therapy for an average of 39 monchs displayed no change in the uabecular bone volume compared to normal conaols, although diis might be amibutable to their greater age (68 It 9 years vs 55 k 10 years). However, parameters of bone formation (osteoid volume, surface and width) were elevated, indicating that the ûabecuiar bone volume \vas increasing [Grynpas et al. 19901.

1.4.5 Effect on coiiagen-mineral interface

Studies that have assessed the effect of moderate to high doses of fluonde on the mechanicd properties of bone have observed that, while the arnount of bone present may be unchanged or increased, the mechanical strength declines [Turner et al. 1997, Lafage et al. 19951. In association with this, Buoride administration cm result in increased rnineralization, as assessed by density fractionation and microhardness measurements [Chachra et nl. 19991, dthough this \vas not observed by backscartered electron irnaging in a minipig mode1 pratzl elal. 1996, Roschger et al. 1997. In contrast to the observed results, increased mineralization would suggest that the strength and stiffness of the bone should aiso increase. However, both the negative relationship observed bem-een bone minerai cystal width and the femoral failure stress [Turner et a/. 1994 and the results of smd-angle s-ray scattering studies in both fluoride-ueated humans [T;ratzl et al. 19941 and minipigs [Fratzl et al. 19961 suggest that the increased rnineralization bas a deletenous effect on the coUagen-mineral interface. Fratzl and coileagues [1994, 19961 suggest that this is due to the deposition of large, estrafibrillar mineral crystals, which increase the degree of mineralization of bone without contributing to the mechanical propemes. Walsh and Guzelsu [1993] suggest thac minera1 cqstals formed during fluoride esposure (perhaps due to their increased size) do not bond as tightly to the coilagen molecules, resulting in reduced colIagen-mineral interface strength compared to normal bone. These nvo effects on the coilagen-minerai interface may act synergisticalIy to compromise the mechanical properties of bone material in highly fluoridated tissue.

1.5 Fluoride and the mechanical properties of bone

Fluoride has a comples, dose-dependent suite of erfects on bone, including altering the amount of bone, the structure, and the mineral-collagen interface. These result in changes to the mechanicai propertSes of bone, and by extension, to the fracture risk. Techniques such as dual-energy s-oay absorptiometry or histomorphometry, while usefùl, cannot assess these integrated factors. In order to quanti@ changes to mechanical hction, one of two approaches must be used- The first approach is epiderniological; the fracture rate in a given population (such as a chical Mal of patients receiving therapy for osteoporosis) can be measured. The second appraach, commonly used in animai studies, is to use in niro mechanical testing techniques as ap-ay for fracture risk. As discussed in detaii below, the overd picture of the effect of fluode on mechanical properties that has arïsen, based prirnarily on animal studies, tentatiwely suggests a dose-dependent effect on mechanicai properties: improvernent with increasing fluoride content to an ophum point, foilowed by severely comprornised mechanical properties at high concentrations of fluoride (fluorosis).

The evidence that emerges from in vitro mechanical testing of bone from animai studies cleadv indicates that high doses of fluoride are detrimental to mechanical properties. Controversy remaïns over whether there is an optimum fluoride dose or concentration that Leads to improved bone strength. A studv by Turner et a[. Cl9921 established a weak, biphasic response to fluoride ingestion in young (21 day old) rats . The rats were fed a low-fluoride diet, and their drinking water \vas fluoridated at O, 1, 2, 4, 8, 16, 32, 64, or 128 pprn. The rats were sacrificed after four months. The fluoride content eof the vertebrae was assessed, and femora were tested in three-point bending. It was found that the peak bone strength occurred with a fluonde intake of 16 ppm, corresponding tm a bone fluoride content of 1216 ppm, and decreasing thereafter, h segmented regression mode1 \vas postulated to describe this biphasic relationship. This work was in agreement with an earlier study @ch and Feist 19701. A later study by the sarne group Fumer et ai. 19951 again began with 21 day old rats, ingesting water fluoridated at O, 5, 15, or 50 pprn, brut Çoiiowed them through maturïty and senescence: 3,6, 12 or 18 months. In this study, no positive effect of fluoride treatment was observed, dthough there was a negative effecu at higher doses. Xo effect of fluoride was observed in a similar study Finhorn et al. 19921, im which femora from rats esposed to drinking water fluoridated at 0,25, 50 and 75 ppm were tested in torsion. However, the fluonde content of these bones (2026-1 1 72 6 ppm) \vas greater chan the peak fluoride content observed by Turner et al. [1992], mhich may esplain the discrepancy. Beary [1969] found a similar decrease in bone strength in rat femora with high fluoride intake (>45 ppm). Sogaard et al. [1994] esamined the mechanical propemes of vertebsae (primanly cancelious rather than cortical bone). The rats were three months ofage at the start and ingested water fluoridated at 0, 100 and 150 ppm for three months before sacrifice, resultiqg in fluoride levels of 343, 3295 and 4617 ppm respectively. \T'hile no changes to the failure load or stress were observed, the mechanical parameters corrected for ash content (a measurement of bone quality) were adversely affected. Similady, blosekilde et ul. [1987a] found reduced trabecular bone suength in pigs wïth anaverage bone fluonde concentration of 2836 ppm. Findy, a study esamhïng high doses of fluoride in rabbits showed compromised mechanical propemes, despite increased bone mass, rnineralization and hardness [Chachra et a/.1999, Turner et al. 1997. \Tÿhile animal models are useful in providing information about high doses of fluoride, mhich would be difficult to obtain in humans, Siere are two major caveats to be considered prior to estrapolating to the human case. The hst is that the rate of auoride incorporation in other species may not be the same as in humans. For esample, in rats, the rate of fluoride incorporation is an order of magnitude less than in humans, sugges~gthat administration of water fluoridated at 1 ppm to humans would be the equivalent of 10 ppm in rats [Turner et a/. 19921. The difference is partidy accounted for by differences in intestinal absorption benveen rats and humans [hgrnar-Mansson and Khitford 19821. The second difference is that animal studies spicaily examine the effect of high doses of Buoride for short ümes, rather than the low doses and long esposures of humans. ~~e low doses of fluoride are thought to act by altering bone at the level of crystai structure [Grynpas 19901 and the mineral-collagen interface [Walsh and Guzelsu 19931, high doses also affect bone ceUs and therefore remodelhg processes.

To date, data correlating fluoride content and bone quality by in &O mechanical tesung in humans are scarce. Richards et a/. Cl9941 esarnined vercebral trabecuiar bone cylinders from individuals aged 20 to 91, mho were not esposed to artificidy fluondated water, to elucidate changes in bone mechanics and fluoride content with age. They found that the bone mass and the bone strength nomalized for the bone mass decreased with increasing age. ihile the fluoride concentration increased wïth age, it did not affect the bone qualit). in a way that was independent of age and gender effects. At the other limit of fluoride esposure, an assessrnent of the bone quality of iiïac bone biopsies Erom osteoporotic patients

who had received fluonde cherapy [Sogaard tf al. 19941 indicated that there was a reduction in bone strength and bone s trength normalized for ash content, and no increase in bone mass, afier 5 years of fluonde therapy of 40-60 mg/day.

1.5.3 Fluonde and fracture rate in humans

Fluoride administration has been under investigation as a therapy for osteoporosis fûr four decades [Rich and Ensinck 19611. The signal advantage is that fluonde is one of the few therapies known to be anabolic; fluoride administration causes increased bone formation, rather than sirnply maïntaining the amount of bone present. However, fluoride therapy has been complicated by a narrow therapeuac ~vïndowand clifferences in response depending on the formulation of the admliistered fluoade [Grynpas et al. 20001. Fluonde therapy, in conjunction with caIUurn and ~itarninD, is unambiguously associated with increased asial bone mass fHarrïson et al. 19811. However, its use remains controversiai as some smdies have reported no decrease in the rate of fracture [Riggs et al. 1990, subsequently reanalysed and reported in Riggs et al. 19941 or associated Iower iimb PIameUe et al.

1988, Schnitzler et d 19881. The lower limb pain is like1y to be caused by stress fractures [Schnitzler and Solomon1 9851. These hzve been attributed to resorption of estant bone, the initial step in fluonde-induced bone remodeling, Iowering the bone mass and srrength of severely osteoporotic individuais to below the fracture threshold [Schnitzler ef al! 1990b,

1990~1.However, recent studies with low-dose sustained release pak et al. 19951 or intermittent [Schnitzler et al. 19971 fluoride administration sugges t that it may be possible to reduce fracture incidence in osteoporotic patients without the associated side effects. The esperience of fluoride as a therapÿ for osteoporosis highiights the cornples, dose-dependent effect of fluoride on the mechanical properties of bone.

1.6 Epidemiological studies of water fluoridation and fracture risk

Ingestion of water fluondated to 1.0 ppm has been generdy considered to have no detrimental side effects. However, long-term esposure to fluoride can result in significant fluoride accumulation in the skdeton and there is concem that this rnay have adverse effects. In addition, it may result in changes to the structure or degree of mineralization of the bone (as is observed with short-term ingestion of higher doses of fluoride), and this rnay have mechanical consequences. This would, in mm, affect the incidence of osteoporosis, manifested as fracture rate. Despite the large nurnber of published smdies, considerable controversy remains regarding the issue of fluoridated water and fracture risk, wïth studies indcating an increase, decreased, or unchanged risk of fracture with fluorïdated water consurnption. h complete review of such studies can be found elsewhere [Molio and Lehmann 19991 but a description of a nurnber of studies wdl illusuate the point. For esample, nvo studies conelating the regional variation of hip fracture in the United States uacobsen et al. 19921 and in Engiand

[Cooper et& 19911 to local water fluoridaaon showed a slighdy increased risk of fracture. Conversdy, nvo studies comparing fractures rates in fluoridated and unfiuondated communities [Lehmann etaL 1998; Sirnonen and LaiWen 19851 found a reduced ùsk of fracme in the fluoridated cornmunities. One smdy phipps et al. 1997 found a reduced vertebral and hip fracture risk, but an increased risk of wrist fracture. Findy, a nurnber of studies [Cadey et al. 1995, Jacobsen et a/. 1993, Suarez-Almazor et al. 19931 showed no change in the risk of fracture as a result ofwater fluoridation. \We studies such as these must be interpreted with caution because of the Limitations of retrospective epiderrriological studies, two conclusions can be drawn from this mass of data. First, water fluoridation at 1 pprn most iikely has no effect on bone mechanical properties, although it is impossible to esclude a smd effect. This is confirmed by a rneta-analpsis that esarnined 29 studies to determine if a relationship existed benveen skeletal effects of fluoride and water fluoridation; no evidence for such a relationship was found WcDonagh et al. 20001. Second, that in order to determine if there is a 10-15% increase in fracture raie at an appropriate level of certainty would requke more than 400,000 people enrolled in a cohort study4early prohibitive [lacobsen 19921. This suggests that alternate protocols may be a more appropriate way to determine the effect of water fluoridation on bone quaiity. A recent prospective study of the effect of fluoridated water on fracture rate in older women found that there was a slighdy reduced ùsk of hip and vertebral fracture for women who were continously esposed to optïmally fluoridated water for ~vodecades compared to those who were not esposed phipps et a[. 20001. Nevenheless, three important questions have not yet been Myaddressed: the optimum strengd-i of human bone, what bone F levels are associated with this optimum strength and whether the normal accumulation of F in bone has a positive, negative or minimal effect on bone quality. Skeletal fluorosis is endemic in regions where nanirai water supplies contained levels of 10-20 ppm of fluoride, such as in some pms of India Faccini and Teotia 19741. However, there is some evidence that individuals living in areas where the water fluoade content is as lom as 4 ppm may have asymptomatic skeletai fluorosis and are at an increased risk of bone fracture compared to indkiduals in areas with water fluoridated at 1 ppm [Sowers etaL 19911. This is the upper lirnit perniitted in dnnking water, and there some areas in North America where the water fluoride levels are this high [Gordon and Corbin 19921.

1.7 Skeletal fluorosis

At chronically high levels of fluoride exposure, a markedly deleterious effect on the skeleton is observed. These changes are termed skeletal fluorosis. Esposure to escessive fluoride cm occur through a number of routes, three of mhich are the most important. Hydnc fluorosis cm result fiom either prolonged or sporadic exposure to water with a fluoride concentration in escess of 5 pprn, as in certain areas of Indiz Faccini and Teotia

19741 or, notably, Vichy Saint-Yorre water poivin ek a/. 19861. Industrial fluorosis has been obsenred in workers in a nurnber of industries that involve fluoride processing. Iauogenic fluorosis has been obsemed in individuals who have received fluoride as a therapy for osteoporosis or fluoride-containing hgspoivin and Meunier 19901. Fluorosis is usuaily clinicdy identified by observation of increased radiological density of bone (), particularly of the spine. At higher esposures, ectopic calcification of soft tissues such as ligaments and capsules is also obsemed [Boivin and Meunier 19901. The former symptoms become apparent at fluonde concentrations of about 4000 ppm (in dry bone), and is associated with long-term ingestion of escessiveLy fluoridated drinking water. The latter symptoms, associated with pain and immobilization, usuaiiy result from prolonged intake of 20-80 mg/day of duoride [Schiatter 19781. Observed fluoride contents in fluoroac patients range from about 0.56 to 1.33% cf ash weight [Boivin et all 19881. Skeletal fluorosis is characterized by a nurnber of changes to the architecture, remodeling, mineraiization and mechanical properties of bone. Histomorphometry of transiliac biopsies fiom ff uorotic patients shows profound differences comparied to controls. They indude increased cancellous bone volume (which is consistent with the radiologicdy- observed osteosderosis) as well as increases in cortical bone wïdth and porosicy Foivin et al: 1989, Boivin et al: 1990, Boivin 19781. Remodehg in fluorotic bone is s~onglydted in favour of formation, characterked by increased osteoid parameters including perïmeter, width and volume. In patients doubly labelled with tetracycline the mineral apposiaon rate decreased and the rnineralization lag urne increased, which conmibuted to the increased osteoid Foivin et al: 1989, Boivin et al: 1990, Faccîni and Teotia 19741. In a rat mode1 of high fluonde intake combined with rend insufficiency (thereby esacerba~gthe effect of the fluoride), the osteoid volume was increased 20-fold compared to controls; the wide osteoid seams were characteristic of [Turner et a[. 19961. Increased fluoride concentration in bone is associated with increased minerakation in fluoride-treated humans [Grynpas et al: 19941, animal models [Chachra et al. 19991 and fluorotic bone Fang 19781. However, in skeletal fluorosis, the increased mineralization is associated \-thcharacteristic rnineralization defects, as Uustrated in Figure 1.7.1. These include enlarged lacunae and circurnlacunar mineralization defectsCmottied osteons') poyde 1986, Bang 1978, Boivin and Baud 19781 and ILiear formation defects in cancellous bone Foivin et al. 19931. Findv, animal models of skeletal fluorosis indicate that, despite increased rnïneralization, excessive fluoride esposure is associated with decreased mechanical integrity [ Moseliilde et al. 1987, Tumer et a/. 19971. Similar decreases in mechanical properües have been observed for some patients adrninistered fluoride as a therapy for osteoporosis [S ogaard et ar! 1 9 941. F&we l. 7.1 Backscartered elecmimages of ffuoroticbone

As well as increased density, fluorotic bone displays altered mineralization inchding atypical remodeling, large lacunae, and circumIacunar rnineralization defects Boyde et al. 19861 1.8 Osteoarthritis

As the samples for this study were obtained from patients undergoing total hip replacement surgery (Section 3.1.2), they were not normal bone. The majority of the samples were obtained from individuals diagnosed with osteoarthritis, a degenerative disease of the cadage of the . Whde it is considered to be a focal condition, limited to loadbearing regions of the cartilage, there is evidence of involvement of the underlying bone.

1.8.1 Changes to the subchondral bone

There is some evidence that the progression ofosteoarthtitis is associated with increased stiffness of the underlying bone purr and Schaffler 199T]. The thickness of the subchondral bone (bone immediately adjacent to the cardage) was shown to increase in osteoarrhntic specimens; it \vas elevated in the tveightbearing region compared to the non-weiglitbearing region or to normal (old and young) controls. Adjacent canceHous bone showed evidence of increased remodelling [Grynpas et a(. 19911. The subchondral bone in osteoarthritic bone was observed to be less stiffand dense than normal bone, as wdas somewhat hypomineralised and Aspden 1997a, Grynpas et al. 19911.

1.8.2 Changes to the cancelious bone

The observation that osteoarthritis and osteoporosis were rarely Found in the same individual led to the beiief that cancellous bone mass and strength are generally increased in osteoarthrïtic patients, even at sites distant from the affected cartilage, sugges~ga systemic response perstraeten et al. 1991, Gevers et al. 1989, Dequeker et a/.19931. The deep cancellous bone aiso appears to be less mineralized than control bone, although the difference is not as pronounced as at the subchondrai sites. Again, changes in osteoid parameters indicate increased rernodelling, as at the surface [Grynpas et al. 19911. This corresponds to an increase in apparent density compared to controls, as well as increased stiffness and toughness II i and Aspden IgWb]. 1.9 Hypothesis and objectives of this study

The purpose of this smdv was to investïgate the effect of iifelong esposure to environmental fluoride (fluoridated water, toothpaste, dietary sources etc.) on bone qualit). in humans. The mechanical properties of bone speümens were determined, as was the fluoride content. The parameters ttiat contribute to the mechanical integrït). of bone tissue-the degree of mineralization, the architecture of the bone, and the remocieling state-were then studied to deremnine if any of these underlying propedes were affected by fluoride esposure.

1.9.1 Case-control study

The purpose of dis cornponent of the smdy was to deterhe if esposure to fluoridated water alters the mechanical propemes, degree of rnineralization, remodehg state, or architecture of human bone.

1.9.2 Effect of fluoride content

Objective 2 To determine $a reIafionsh@ eewitsbetween tbe pnrnmeterr tht conhilzlte to bone qztakg und thej7ztode content-

The purpose of &discomponent of the study \vas to deterrnine if fluoride accumulation in the skelecon, resulting from environmental esposure, has a dose-dependent effect on the conmibucors co bone qualiry. 1.9.3 Effect of disease States

Objective 3: To ehtnzate the nqonre of hone M disease stafer toj%ode eqîomre.

This component of the smdy aims to determine if there is a differenual response ro fluonde esposure as a result of disease state (osteoarrhntic or osteoporoac), severity of osteoarrhrias, or prosunity to areas of cardage darnage wïthin the osteoarthrïtic fernord head. 2: Experimental Protocols I.P. Pavlov (1936) 2.1 Specimen collection

Femoral heads were obtained from patients undergoing total hip replacement surgery in Toronto and in Montreal. Consent was obtained from patients at th& pre-admission interview, typicdy one to two weeks prior to surgery. At surgery, the femoral head was removed by the surgeon. A coronal section, approsimately 5 mm in ttuckness, was then escised, s-rayed, prepared for histologicai analysis and archived by the Pathology Department at Mount Sinai Hospitai (see detds below). Sections were removed from specimens obtained from Montreal residents by the same staff upon their arrivai at Mount

Sinai Hospital. The ISVO remaining halves of the kmoral head was then stored in a -70°C freezer und testing. A total of 92 speùmens were obtained from patients undergoing surgery. Details of their origin, diagnoses, and gender are provided in Results (Section 3.1.1).

2.2 Sample preparation

2.2.1 Preparation of coronai sections

After their removd from the patient at surgery, femorai heads were given to the Pathology Department at Mount Sinai Hospital, where a coronal section, approsimatdy 5 mm thick, was escised using a band saw. Contact s-rays were made of each section, and a second copy of these were made avaiiable for this research (these s-rays were later used for image analysis; see Section 2.6.1, below). The sections themselves were then decalcified, embedded in paraffin, sectioned and stained for OA grading (see Section 2.8, below).

2.2.2 Dissection of femoral head

Figure 2.2.1 schematicaily illustrates the dissection of the fernord head into sections for analysis. Cores of cancellous bone, G mm in diameter, were excised from the antesor and the posterior hdves of the femorai head, using a diamond coring bit affised in a drill press. A smdi bandsaw w3s then used to cut blocks fur embedding. To bee, the posterior half of the bone \vas then bisected into a superior and an inferior haif. Blocks of bone, appro'dmately 10 mm square and 5 mm thick, were cut kom the inferior and superior faces of the bone. Thcse sarnples were later used for histomorphomeq, image analysis, backscattered electron irnaging, and microhardness testing (see below). Four smaiier sarnples, appro-simately 5 mm cubes, were cut Erom a distai and a prosimal location on the superior and inferior surfaces of the femoral head and embedded for rnicrohardness testkg.

2.2.3 Embedding of specimens

The four blockç for rnicrohardness testing, as well as the superior and inferior block, were embedded without decdcifymg. Sarnples were &ed in 70% ethanol and then dehydrated over three days in a graded series of increasïng concentrations of acetone. They were then gradua& infiltrated with plastic by immersion in increasing concentrations of Spurr resin in acetone: an initial concentration of 50°/o, nvo changes of 80% Spurr, and hdynvo changes of 100% Spurr. Nest, the specimens were transferred to moIds, embedded in fresh 100% Spurr, and aiiowed CO polymerize overnight in an oven at 70°C. Findy, the plastic blocks containhg ernbedded bone were chen removed from the rnolds and nimmed to expose the bone [Schenk etal. 19841 Proximal (towards acetabu hm) 7 Superior (weig htbearing)

4--(1) coronal section

Inferior (non-weightbearing)

Distal ktowardsshaR)

Ky: 2. Coronal secaon: used for image analysis and grading of osteoarrhntic samples 2. Cancelious core: posterior sarnple used for compression tes~g anterior sample used for torsion testing (when possible) 3. Superior and inferior samples: histological sections (mage analysis, histomorphometry) backscanered electron irnaging 4. Superodistal, inferodistal, superoprosimal and superodistal samples: used for rnicrohardness teshg 2.3 Chernical analyses

2.3.1 Sample Preparation

Chernicd anaIysis was performed using neutron activation anaiysis on cores of cancellous bone previously used for compressive testing (see below). The bone samples were washed with a water jet to remove blood and marron- and then defatted ovemight in a 2:l chioroform:merhanol soluaon. The samples were rhen rinsed in sahe, bloned dry, weighed, and placed into poiyethylene specimen containers.

2.3.2 Chernical composition

The elementai composition of all the canceiious bone smples and a preliminary subset of the microhardness specimens (that is, at the centre of the femoral head as weii as at four locations on the surface) were established by instrumental neutron activation analysis (INM) at the SLO\WOKE reactor fadq-at either the Universis. of Toronto or the Royal hLilitary College, Kingston. Sarnples were irradiated for 1 minute with a neutron flus of 1 to 5 x 10" n/cm2/s. The irradiation was foliowed by a 1 minute delay and then a 5 minute count with a soiid-srate gamma ray spectrometer [Grynpas et d 19931. The nec peak areas were autornaucaily calcuiated for the gamma rays that were characteristic of each element and chen converted into concentration. The concenaaaons of calcium and phosphorous (the major constiments of bone mineral) were measured. The concenw~tionsof sodium, chlorine, and magnesium - trace elements - were also measured. The concentrations of iodine, potassium, bariurn and strontium were found to be below the detection limit for this technique. Two other dues were calculated €rom the elemental concentrations. The mass ratio of calcium ro phosphorous (dirnensionless) was determined by talcing the ratio of the nvo percentages. This nurnber is a measure of the degree of stoichiomeay of the mineral (for stoichiometric hydrowapatite, Ca,,(P@J,(OH), the mass ratio of these elements is 2.15). FLiail~r,the total minerai content (%) Kvas estimared by addïng the calcium content (%) to che approxïmate percentage of phosphate, PO,, calcuiated by multiplying the phosphorous content (in '/O) by the factor 95/31, the ratio of the molecular weight of the phosphate ion to the molecdar wveight of phosphorous alone. 2.3.3 Fiuoride content

The measurement of the fluorïde content was similar to diat used for elementai anaiysis. Fluoride contents were measured at nvo reacror sites: the SLO\WOKE reactor at the University oEToronto, and the SLOWPOKE II reactor at the Royal Military CoUege. In dus case, rhe neutron flux used ranged from 2.5 to 10 s 10'" n/cm2/s. Sarnples were irradiated for 20 sec, 10 seconds were dowed to elapse, and then the characteristic gamma ray for fluonde was counred for 10 seconds. Again, the count was converted to mass of fluoride pfernagh et a/.1977. The fluoride content \vas espressed in parts per don(micrograms of fluorïde per gram of bone), fluoride-co-calcium ratio (micrograms of fluoride per mifigram of calcium, aiso measured by KAA), and fluonde content norrnalized to total minerd (calculated as described in section 2.3.2, also espressed as pg/mg). 2.4 Mechanical testing

2.4.1 Compression

Cylinders of canceilous bone, appro-ximatelg.G mm in diameter and G mm long, were escised from the centre of the femoral head. The esact diarneter and length were measured with a pair of digital calipers. The apparatus used \vas upgraded over the course of this research, but in either case the methodology remained the same: the sarnple \vas placed on the platen of either an Instron 1011 screw-driven testing machine or an Instron 4465 servoh@aulic testing machine (Znstron Corp., Canton, kfi) and compressed at 1 mrn/min und failure occuned. The load and the elapsed tirne (which, together with the deforrnaüon rate, was used to calculate the deformation of the sample) were acq~edusing a custom- designed program installed on a personal cornputer. This program was initially written for the Easyest data acquisition and malysis sohvare (Keithley Instruments; Taunton, MA) and then for a sirnilar package, LabVIEW (Naaonal Instruments Corp.; Austin, TX3. The resultant load-de formation curves were then anaiysed using Microso ft Excel (Ncroso ft Corp., Redmond, \VA). The length and the area of the sarnple were used to normalize the stress and the strain applied to the sarnple, according to che standard formuiae [Callister 19971; the strair, in the sample is given by:

where E: engineering suain of the sampIe AL change in length (mm) : gauge length (mm)

The stress in the sample is found using the equation:

where O: stress in sample @Pa) F: force applied to sarnple CN) A: cross-seccional area of sample (mm? The compressive modulus was ciefined as the dope of the linear region of the curve:

where E: compressive rnodulus AG: change in stress va) A&:change in strain

The ultimate compressive sa-ess (UCS) was defiried as the maximum stress prior to the Grst monotonie decrease in load, and the strain at UCS \vas simply the compressive strain in the specimen at this point. The energy absorbed by the sample to fkacture was determined by numericdy integraùng the stress-straïn cuve to the point of masïmum stress. In addition to the Mure properties, the yield stress of the canceilous cores was determined from the stress-strain cuves. The yield stress is dehed as the stress applied to the specimen at the point mhere the stress-srnain cweno longer is linear (the proportional limit). Several methods esist for identitdng this point [Turner and Burr 19933. The method used for this study was as follows: the compressive modulus of each sample was determined by fitüng a regression line to the linear portion of the curve. The equauon of the fine pardel to the rnodulcls line, but whose s-intercept (intersection with the strain axis) mas shifted by 0.2% was then determined, and this line was superimposed on the stress-strain curve of the sample. The proportional limït was then deterrnïned as the point where the this Line intersected the stress-strain curve, and the stress at thïs point was deterrnined as the yield stress [Turner 19891. The area under the curve to this point (resilience) was also calculated. Further information ofi compressive mechanical testing is provided in the Appendix.

2.4.2 Torsion

Cylinders of cancelious bone, approsirnateIy 6 mm in diameter, were escised from the anterior portion of the Çemoral head (as described above) and used for torsion tes~g. Torsion testing was carried out on a custom-built torsional tester; in this apparatus, the test specimen is held betsveen ~vochucks, one of which rotates and one \vhich does not, but which is free to siide longitudindy dong a track. An angIe ceil was afhed to the rotating side and a 20 Ib-in torque cell (approximately 2.26 N-m) was affksed to the non-rotatïng side. The cancelious cores were mounted bem-een brass grips, placed within the chucks of the torsion tester. Each grip had a deep square cavity machined out of it, and mas equipped with four set screws. The cancellous core was aligned with its axïs coiocident with the asïs of rotation and secured wirh the set screws. h doughlike poly(methy1 methacryiate) mkniee \vas then used to 6.U the cavity and allowed to set The gauge length \vas held approsimately constant at 12 mm, chosen to be nvice the diarnetec This was approximately rhe maximum raao possible for the samples, but it is unlikely rhat the samples were tested in pure shear (this is discussed £ùrther in Section 4.1 -4). As the lengths of the cores were variable, depending on the size of the femoral head, rninimizing the gauge length allowed the maximal number of cores to be tested. Once the sample was mounted, the exact diameter at the ends and ac the midpoint were measured, together with the =act gauge length. One of the chucks was rhen rocated at 0.0093 rads-' und failure of the sample occurred. This speed was chosen to minirnize viscoelastic effects [Bruyére Garnier etal 19991. Data from a torque and an angle celi were acquired to a personal cornputer using a cuscom-\vrimn program for the LabVIEW data acquisition system. The torque-angle cwewas then converted to a shear stress-shear srnain cweusing standard formulae [Beer and Johnston 1985]. The shear stress is related to the applied torque through the foiiowing formula:

where T: shear stress r: mean radius of specimen T: appiied torque J: polar: moment of inertia

The polar moment of inertia for a specirnen of constant circular cross-section is:

mhere J: polar moment of inertia r : the mean radius of the specimen As for pure compression, the shear moddus is defined as the dope of the hear region of the stress-strain cuve:

where G: polar moment of inertia AT: change in shear stress @Pa) Ay: change in shear strain

As before, the masimurn shear stress (LA \vas dehed as the masimum value of shear stress obtained before the first monotonic decrease in load, the masimutn shear strain

(y,.-) =-as the corresponding shear strain, and the energy absorbed by the sarnple was the area under the stress-suain cuve to the mzsirnurn shear strain. 2.5 Assessrnent of mineralization

2.5.1 Microhardness testing

Four cubes of bone fiom each femorai head, approsirnately 5 mm long on each side, were prepared for microhardness testing. The sarnples were taken Erom a prosimal and a distai location from each of the superior and the inferior surfaces of the bone (see Figure 2.1). The samples were embedded as discussed above (Section 2.2.2). A Tukon Mode1 300 Micr~har~essTester wittz a Iinoop diamond indenter was used to measure the microhardness. The indentacions were made under a load of 30 grams, wirh a duration of 10 seconds. The indentations thus formed had lengths ranging Erom approximately 80 to 1120 pm. The microhardness was deterrnined from the length of the indentation and the applied force by the folIowing formulae [Cu- 19901:

where: HE Knoop hardness of the material F: test force (N) g: acceleration due to gravïv, 9.8 m/s2 A: pro jecüon area of indentation (mm.)

The projection area of the indentation is defmed as the foliowihg:

A= cd'

where: c: indenter constant d: length of longer diagonal of indentation (mm)

The indenter constant is given by the following formula:

tan -P 3 a 2 tan - 2

where, a: point-apes-point angle of the indenter p: side-apex-side angle of the indenter Inserting all the constants for this apparatus, the followïng formula relates applied force and length of indentation to the aoophardness:

where k is a constant for the apparatus and a function of the indenter geomeq. The microhardness \vas caiculared automaticaily from the measured Iength by an electronic console interfaced ~6ththe microhardness tes ter. Ten iridentauons were made in the subchondral bone, equally spaced dong the lenad of the specimen. A Merten indentations were made at random locations in the trabecular bone of the specirnen. Care was taken to position ail indentations weil away from the edges of the embedded bone to avoid edge effects.

2.5.2 Backscattered electron imaging

Backscattered electron imaging \vas pehrmed on samples of bone from the superior and the inferior surface of the femoral head. Samples were embedded as described above (Section 22.2) and histological sections were removed for image analysis and histomorphometry (see below). Afier this process, blocks of plastic with esposed embedded bone remained. The surface of each block \vas polished to a 1 pm diamond finish, carbon- coated, and mounted on a stub preparatory to backscatrered electron imaging. The methodology foliowed has been described prevïously [Grynpas et al. 19941. Sarnples were imaged in a Hitachi S-2500 (Nissei Sangyo America Ltd., Roliing Meadows, IL) scanning electron microscope. The accelerating voltage \vas 20 kV and the working distance \vas 15 mm. A hkTetra BSE detector (Osford Instruments) was used to image the specimens, and data were coilected using the Link e2U data collection and image analysis sohvare (Osford Instruments). The detector was calibrated at the start of each session using three standards: carbon (Z=G), alurninum (2~13)and alurninurn oxide (&O3; Z,, = 10). An aluminum standard was afLxed to each specimen. Comparing the measured value to the calibration value dowed compensation for the fiuctuating bearn current before and afier analysis of each specimen Povce et a/.19901. Two regions of each bone sample were imaged, at a magnification of40s, nith each image covering an area of approsimîtely 4 mm'. The entire length of the subchondral bone mas imaged with consecutive adjacent images. Also, approsimately 6 fields of the cancellous bone were imaged. The area imaged was the region of bone fixrzhest from the surface. The contrast of the images was set so that the non-mineralized tissue as weU as the plas tic mam~(both of which have low effective atomic number) appeared black. An esample of an image is shomn in Figure 2.5.1. The mineralized bone exhibited a range of greyscde, which was divided into either seven or eight bins (for the canceilous and subchondral bone, respectively). The percentage area ofeach image that mas in each intensity level \vas calculated and a histogram of area as a function of intensiv \vas produced.

The semi-quantitative rnineralization prohle thus formed cmbe described in two ways. A meighted average of the histograxn cm be defined by analogy to the number- or weight- average molecular weighr, used to describe a dismbuüon of polyrner molecular weights PlcCnim et a/.19881. Here, the weighted average is the sum of the bin number multiplied by the traction of the total area in that bin. Seven bins were used for the mineraiizaùon distribution of cancellous bone, and eight for that of the subchondral bone.

A second method of describing the distribution involves the logit, or cumulative log ratio [Bracci et a/, 19981. The logit is dehed as foilows:

logit = log jtod area above the middle bin) (total area below the nidclle bin)

Both values were used for the purpose of statistical analysis. Figure 2-5.1 Backscattered eectron image of cancellous hwan bonc 2.6 Histological sections

2.6.1 Preparation of histological sections

Image analysis of histologicd sections were perforxned on bone from a superior and an inferior site in the femord head. The superior side of the femoral head is weight-bearing, and this bone is direcdy contiguous to the areas of the cdagemost likely to be affected by osteoarthritis. The inkrior, non-weightbearing side is less likely to be dtered by local changes. The samples consisted of blocks of bone, approsimately 10-15 mm square and 5 mm thick, embedded in Spurr resin (see section 2.2.2, above). The blocks were tnmmed to expose the bone and 5 sections were cut using a Reichert-Jung motorized rotary microtome with a tungsten carbide blade. The secuons were placed on gelatinized siides, and incubated in a 60°C oven overnight Püor to staining, the sections were deplasticized in a 3% sodium ethoxide solution. The sections were then stained wïth Von Kossa, which visualizes the minera1 as black [Schenk 19841.

2.6.2 Image and strut anaiysis

The slides were imaged and analysed using a Quantimet 570 (Leica) image processing and analysis system to quantify the arnount, orientation, and comectivity of trabecular struts. Four image analysis parameters were used to quantify the arnount of canceilous bone. The proportion of uabecular bone (Yo) cm be measured directly frorn the image, and the average uabecular thickness (m)cm be calculated indirecdy. The trabecular number (mm") \vas calculated from the trabecular bone volume and the trabecular thickness using the following formula parfin 1987:

where: Tb.N: trabecular number (mm-') TBV: trabecular bone volume (O%) Tb.Th: trabecular thickness (pm) The trabecular separation (mrn), \vas caiculated trom the nabecular number and the trabecdar thickness using the foliowîng reiationship:

where: TbSp: trabecular separation (p) Tb.N: trabecular number (mm") Tb.Th: trabecular thickness (pn)

The nvo-dimensional star volume, a measure of both the mount and the comectivity

of the bone, was calculated using an automated procedure [Vesterby et UL! 19891. A schematic illusuauon of star volume is given in Figure 2.6.1. The star volume is a measure of the average area bounded by uabeculae, and is measured bp randomly placing a point within the marrow space on an image of a bone, estending hes out at random angles from this point, and measuring the length of these lines Lrom their origin to the point where they intersect a boundaq. The two-dimensional star volume is then the average area deiimited by these lines, according ro the foilo\ving formula pomsen et al. 20001:

where: V*: nvo-dimensional star volume (mm-? 4: Iength of line from origin to boundary

Strut anaiysis mas then used to assess the degree of comectiviq- of the bone. The binaq image of the bone (derived from the von Kossa-stained section) \vas skeletonized; that is, the image of the trabeculae network was 'eroded' und it \vas only a single pixel thick. This is a topological invariant process; the skeletonized network has the same topological propemes - in other words, degree of connectiviq - as the original network. Nodes (points where three or more trabeculae converge) and end points (where trabeculae end) were identified. Figure 2.6.2 schematicdy illustrates the suut analysis parameters, including the following: the number of endpoints and of multiple points (mm-?, the amount of free-free, node-free, and node-node sauts (mm/-? [Grynpas et al. 1992, Melhsh et al. 1991, Paxisien et al. 19921. FLgure 2.61 Schematic of WU-dimensional star volurne

The nvo-dimensional star volume is assessed by cirawing Lnes in multiple directions Erom a randornly chosen point on the image. Each line is estended und it comes in contact with an edge of trabecdar bone. The area encompassed by this array of lines is termed the nt-O- dimensional star volume, and is a measure of the degree of comectivi~(or rather, the lack of connecavity) of a trabecdar bone sample. free end

strut

no/ knacfe-free free end free-free strut \ free end

The parameters used for suuc analysis induded the number of end points, the number of multiple points, the amount of Eree-Lee, node-free, and node-node stnits, and the nvo- dimensional star volume. See test for merdetds. 2.7 Image analysis of coronal sections

Coronal sections, approximately 5 mm thick, were escised from the femorai head by the staff of the Mount Sinai Hospital Department of Pathology. Contact s-rays of these specimens were made for hospital records, and copies were made avadable for this study. The s-rays were imaged and analysed using a Qumtimet 570 (Lxica) image processing and andysis system to quantifl the amourit, orientation, and connectivity of trabecular struts throughout the body of the femoral head, using the same techniques as were used for the thin sections (Section 2.6, above),

kfeasured parameters that reflected the mourir: of trabecular bone were the proportion of trabecular bone (O/o), the average trabecular thickness (m),the trabecular number (number of trabeculae per unit area, mm-? and the tmbecular separauon (w).Suut maiysis parameters, which describe the degree of comectivity of the bone, include the foiiowing: the number of endpoints and of multiple points (mm-'), the amount of free-free, node-free, and node-node smts (rnmirnrn-3, and the two-dimensional star volume (mm?.

[Grynpas et a/. 1992, Mellish et a/.199 1, Parisien et al. 19921 As this was a thick section, the s-ray 'coiiapses' the struts throughout the entire thickness into a single image ('overprojection'). Since five millimetres worth of trabeculae are compressed into a single plane, this technique would be espected to overestimate the amount and the corinectivity of the bone compared to the thin sections. Use of thin secions is a more canonicai approach to image analysis; while the coronai sections provided an ove~ewof the femoral head as a whole, the techniques and parameters used were designed for thin sections. In addition, the Çemoral head is much more heterogeneous than other sites that are commonly analysed using this technique (such as vertebrae). This may be esacerbated by the presence of osteoarthntis in many of the speciemens, which is associated with osteosclerosis (an increase in the amount of bone present) as weU as the presence of cysts (voids in the trabecular structure). Esarnples of moderately and severely osteoarthritic femorai heads, drawn from those analysed, are provided in Figure 2.7.1. Cysts and other abnormal regions were rnasked out of the s-ray images of the femoral heads prior to image analysis, thereby iimï~gthe size of the samples andysed. The issues surrounding image and stmt analysis of coronal sections are discussed in more detd in Section 4.1.5. Fe2 Z 1 Con racr x-rays of coronal secrions offernorai heads

A: Femoral head displayïng moderate osteoarrhntic changes (some flanening, osteosclerosis and small cysts).

B: Fernord head displajkg severe osteoarthritic changes (marked flattening, osteosclerosis, and large cysts).

C: Osteoporotic femoral head. 2.8 Grading of osteoarthritic specimens

Coronal sections taken from the femoral heads were decalcified, embedded, sectioned and staLzed wrth haemato~$in/eosinand toluidene blue. These sections were then used to qualitaüvely assess the degree of osteoarrhetis present The scale used was modified Erom hat of Mankin [1971] and others [Carlson e~ al: 1996, Podwomy et al. 19991. Four major categoxïes of articu1a.r cardage degradation were addressed: the structure of the tissue, the degree of safianin O staining (that is, the presence and concentration of cartilage-associated proteoglycans), cells (the presence or absence of chondrocyte doning), and the status of the tidemark. Table 2.8.1 describes the cornponents of the grading scale in detail. The most severe degree of osteoanhritis is indicated by a score of 14 on this grading scale.

III: Safranin O staining

O Normal O Normal 1 Surface 1 Reduced staining 2 Pannus and surface irregularities 2 Focal patchy loss of staining 3 Clefts to transitional zone 3 Diffuse patchy loss of staining 4 Clefis to calcified zone 4 Absent sraining 5 Clefis to calüfied zone G Complete disorganization

II: Ce//-

O Normal O Intact 1 Diffùse hyperceliulariq 1 Osteochondral barrier break 2 Clustering 3 Fïyperceilularity

Table 2-8.1 Qualirative gradkg scaie for assessing the seven-tyof osteo&o's 2.9 Statistical analysis

Stahsacai analysis was performed with Excd (hficrosofi Inc., Redmond, Wh) and SPSS (SPSS Inc., Chicago IL,)sohvare. The data \vas analysed in the iight of several different objectives. Details of the statistical analyses are proovided with the resdts.

2.9.1 Case-control study: Toronto vs Montreal

The first objective of the statistical analysis \vas to determine if any differences exist benveen the rneasured parameters benieen Toronto and montrea ai (cities with fluoridated and non-fluoridated municipal water, respectively). Heteroscedastic, unpaired, nvo-tailed t- tests were used for these comparisons.

2.9.2 Effect of fluonde content

The second objective of this study was to ascertain if a relauonship benveen the measured parameters and the measured fluoride content esist for human bone. Data from borh cities, as weii as di disease States, were merged for these anaiyses, and die bonde content served as a continuous variable. Analysis techniques pùmdg included correlations and regressions. The second element of this component of the study was a comparison of the bone qualiv of individuals in &e highest and in the lowest qudeof bone fluonde content. A systematic effect of fluoride incorporation may be masked in the regessions because of the large amounc of variability in the data, but be apparent when these nvo groups are compared directiy.

2.9.3 Effect of other factors

As the specimens were obtained from a heterogeneous sarnple of individuais @y age, gender, disease state etc.), the thïrd component of the analyses focuses on deterrnining whether differences esist in the response to fluonde incorporation between the groups. Data were segregated by gender to determine if differences benveen rhem or in their response to Buoride were obsewed. In particular, because the majority of the donors were over fie, neady ail of die fernale donors couid be espected to be postmenopausal, and this may affect their response to fluoride incorporation. Nest, data were segregated by disease state, to deterrnine if differences in bone quality exist behveen fimoral heads from individuals with different diagnoses. The analyses considered above (case-conuol and regressions) were repeated for the osteoarthritis subgroup (the Iargest of the subgroups) to detemiine if different relationships were apparent. Additionally, analyses conside~gthe effect of site were also performed on parameters that were measured at nvo or more sites on the femoral head. As osteoarrhritis is a focal disease, the effects of fluonde may manifest differently in different areas of the bone that are affected to a greater and lesser degree by the disease. In addition, anatomical variation in the parameters mai be present. 3: Results Paul Valéry :ldorn/Itéi 3.1 Introduction

3.1.1 Patient i'uifonnation

A tod of 92 specirnens were obtained.

53 donors from Toronto (fluoridated) 39 donors from Montreai (non-fluoridated)

51 femaie 41 male

2 SD) minimum: 23 years maximum: 90 years

Disease state: 75 osteoarrhntis 9 osteoporotic (fractures) 3 rheumatoid arthrius 2 1 osteonecrosis 1 psoriaac arthntis 1 aniqlosing spondyLitis

Table 3.1.1 is a complete List of donors, showing their ug of origin, gender and disease sta te-

The data obtaîned during this study have been analgsed to focus on a number of differenc relationships. The £irst and largesr component is an analysis to determine if the measured parameters of bone quality are related to the amounr of fluoride incorporated into the bone. hncillq to this, the relationships brnveen these parameters and the age of the donor or the physical density zre also esamined. The second component is a case-control- type study, where simple cornparisons of mean values were used to determine if ciifferences in bone qualit). exïsted behveen bones derived fiom Toronto residents and those from Montreai residents. Sirnilarly, the third component compares the parameters of bone quality of bone from individuals in the highest and lowest quartiie of fluoride content, to determine if incorporated fluoride aiters bone qualiiy in a systematic way. Segregating the data both by civ and by the fluoride content wiii aiso enable the effects of incorporatecl fluoride to be esamined direcdy, rather than using fluoride esposure (city of residence) as a pro?. \We the kst thee sections explore the question of whether or not fluoride esposure is associated with altered bone quaiït); the nest nvo esplore how this relationship is affected by other factors: the gender or the disease state of the donor. Finally, parameters measured at more than one site on the femoral head (such as the rnicrohardness and the image anaiysis) were analysed to determine the nature of site-specific differences, if any. Sample Patient City Gender Disease

Al 000724237 Toronto M AN A2 41 0258644 Toronto F OA A3 4431 75070 Toronto F OA A4 803109768 Toronto M AN BI 449381789 Toronto F OA 82 4000921 89 Toronto M OA B3 427526090 Toronto M OA B4 41 0893812 Toronto M OA €35 802865725 Toronto F OA Cl 46037721 1 Toronto F OA C2 800219669 Toronto F OA C3 803048446 Toronto M OA C4 802664011 Toronto F OA C5 8027471 54 Toronto F OA Dl 803093871 Toronto F OA D2 803094309 Toronto F OA D3 230381 658 Toronto F OA D4 8031 42561 Toronto F OA D5 469998769 Toronto F OA El U-371022 Montreal F OA E2 461 833626 Toronto F OA E3 U-140793 Montreal F OA E4 468592712 Toronto M OA E5 U-686918 Montreal F OP FI 41 8488003 Toronto M OA F2 U-103101 Montreal M OP F3 41 9963913 Toronto M RA F4 U-338701 Montreal F OA F5 803551530 Toronto F OP G1 U-676749 Montreal F OA G2 250625449 Toronto F OP G3 U-634113 Montreal F OA G4 803610575 Toronto F OA G5 U-1730 MontreaI M OA Hl 800700459 Toronto M OA H2 U-28589 Montreal M OP H3 466365566 Toronto F OA H4 803639780 Toronto M OA H5 U-655746 Montreal M OA J 1 U-698866 Montreal F OA J2 802837732 Toronto M OA J3 U-230977 Montreal M OA J4 410116313 Toronto M OA J5 U-555787 Montreal M OA KI 803753979 Toronto M OA K2 U-681462 Montreal M OA K3 464820554 Toronto F OA

Table 3.1.1 City of ongin, gmder, age and disease state of donors Sample Patient City Gender Disease

K4 U-699408 Montreal M OA K5 41 1023682 Toronto M OA LI U-294489 Montreal M OA L2 803151 323 Toronto M OA L3 U-56581O Montreal M OA L4 118114222 Toronto M OA L5 U-9155 Montreal F OP Ml 403688724 Toronto F OA M2 U-692897 Montreal F RA M3 88338101 1 Toronto F OA M4 U-659801 Montreal F OP M5 448926535 Toronto F OA N1 U-29852 Montreal F OA N2 4336445 15 Toronto M OA N3 U-427471 Montreal F RA N4 802963579 Toronto M OA N5 U-557266 Montreal F AS Pl U-13697 Montreal F OA P2 803783216 Toronto F OA P3 U-681462 Montreal M OA P4 4591 90062 Toronto F OA P5 803514777 Toronto M OA QI 420643165 Toronto M OA Q2 U-495877 Montreal F OA Q3 804026961 Toronto M OA Q4 U-50963 Montreal M OA Q5 803845841 Toronto M OA RI U-227178 Montreal F OA R2 427239587 Toronto M OA R3 U-70582 Montreal F OA R4 426209621 Toronto F OA R5 U-586392 Montreal F OA SI 802524280 Toronto F OA S2 U-702919 Montreal F OA S3 Li-23751 11 Montreal M OA S4 606924538 Toronto M OA S5 451 192637 Toronto F OA Tl 803804749 Toronto M ON T2 U-360574 Montreal F PA T3 U-232962 Montreal F OP T4 U-687654 Montreal F OA U1 U-604876 Montreal M OP U2 U-740137 Montreal F OA U3 U-463704 Montreal F OA U4 U-56645 Montreal M OA

Table 3.11 (cont.) C2y of ongin, gender, age and drSease state of donors

Re511It5 59 Key to Ta&le 3.2-1:

0A osteoarthntic OP osteoporoac (fracnue) avascular necrosis ON os teonecrosis rheumatic dtis PA psoriatic &us AS ankylosing spondylitïs 3.2 Effect of fluoride incorporation on the parameters of bone quality

3.2.0 Introduction

The purpose of this sectiom of the malysis was to determine if a contïnuous relationship exïsted benveen each of the measured parameters of bone quality (such as ultimate compressive stress, star volume, rniciohardness etc.) and the fluoride content of the specirnen, and to establish the nature of such a relationship. For this component, data for ail 92 specirnens obtained, regardEess of disease state, gender or ci. of origin, were included in the analysis (escept where noted; not ail samples were analysed using ali techniques). In addition to the fluoride content, nvo other parameters were considered as 'independent' or 'predicror' variabIes: the age ofthe donor and the densis. of the canceiious core. The rationale behind this \vas chat many of the properties could be espected to vqas a function of these parameters as weli, and understanding the response to each of these factors individualiy may provide a cleazer picture of the behaviour of the bone. h prelïtninaq step was therefore to ascertain if a relationship esisted benveen any of these three pararnetcrs. A correlation ma& indicated that the fluoride content and the age were correlated, although the dtensity was not correlated with eidier parameter (see Section 32.1). The relationships benveen the density and the age of the donor or the nuonde content are illustrated in Figures 3.2.1 and 3.2.2. In order that the 1inea.r rnodels not be overpararnetrïzed, multiple Linear regressions therefore incorporated density and eitber Euoride or age. The Grst step in the analysis of each parameter was to graph it against each of the three independent paramecers (fluoride content, density and zge). Paramemc and non-parametric correlation mamces were then aeated to observe if any correlations benveen the parameters esisted at d.If so, linear regressions were then performed benveen the measured parameter and each of the correlated prehicror parameters as weii as correlated pairs of pararneters (density and Buoride content orage). Outliers were identified and removed, if appropriate (for esamples, oudiers that were 'pinning' regressions were removed to ensure that a linear relationship did not depend on chat point alone). The assurnptions underlying linear regression (that the variance wars constant, the residuals were normally dismbuted and varied randomiy ~5ththe predicted value, and that consecuâve values were independent) were esamined. If any of these were fdse, an appropriate transform (such as taking the natural logarithm of the data if the variance increased with the predictor variable) was considered to see if a different relationship more closely approsimated the data. The assurnptions were again esamined. A Pearson correlatiorr matn.~\vas created to determine the degree of correlation benveen the fluoride content, fluoride-to-calcium ratio, and fluoride-to-total minerai ratio. The correlation coefficients benveen these three parameters were Eound to be 0.898 (F and F/Ca), 0.907 (F and F/CaPOJ and 0.997 (F/Ca and F/CaPO,), reflecting the approsimately

constant calcium and minera1 composition of the bone. Since all of these were so dosely correlated, the regressions were only perfonned with a single parameter, rather than all three. The fluaide content depended on the fewest nurnber of esperimental measurements (only on the measured mass of fluoride and the mass of the specimen, rather than these bvo as well as the measured mass of calcium and the mass of phosphorous) and it was therefore chosen to be the independent parameter. It should be noted that, mith relative- few exceptions, the adjusted R' values were low even when the p-values were significant. The large amounts of scatter in the data meant that ody relatively smd amounts of the variability were esplained (rypicaily between five and nventy-five percent). However, perforrning the linear regression and obtaining a significant

p-value conhsthat a relationship does esist benveen the measured parameter and the predictor variable. It does not preclude the possibiliq of a different type of relationship also esplaining the data-

3.2.1 Relationship berneen fluoride content and age

The relationship benveen the fluoride content and the age was investigated. A hear relaâonship was not observed (Figure 3.2.3); however, as the variance increased as the age increased, this suggested that a logarithmic transform to stabilize the variance \vas in order.

A linear relationship u/ar observed bent-een the natural logarithm of the fluoride content (in ppm) and the age of the donor, and is illusrcated in Figure 3.2.4. The equation of this line is:

log F &RI) = 5.807 f 0.01276 x age bears) However, while the dope of the line differed significandy from zero @<0.05), the relationship was nevertheless very weak, with an adjusted R' value of ody 0.09, indica~g thar less than 10% of the vdability in the nuonde content is explained by the age of the pauent. Age (years)

Figue 3.2.2 Redationstup between density and age of donor

No systematic relationship was observed between the densiv of the cancellous core (taken from the centre of the fernorai head) and the age of the donor (correlation coefficient = -0.133, p = 0.207). Fluoride content (ppm)

F~gure3.2.2 RelationsI+ berneen density and fluoride content

No systernatic relationship was observed benveen the density and the fluoride content (correlation coefficient = -0.065, p = 0.539). Age of donor (years)

FLg-ure 3.2.3 ReIationshp between fluoride content and age of donor

ITVhile the fluoride content increases with the age of the donor, a significsnt linear relauonship was not obsen~ed.(R2 = 0.064, p = 0.346 for constant) Age of patient (years)

Figure 3.2.4 Relariooship between 1ogarkh.m ofFcontem and age

A weak iinear relationship was observed benveen the namal Iogarithm of the fluoride content (in ppm) and the age of the donor (R~= 0.090, p<0.005). 3.2.2 Chernical composition

Fluoride therapy and skeletai fluorosis are associated with altered concentrations of certain trace elements in bone, as weii as wïth increased minerd content. The purpose of this component of the study is to determine if these changes also occur in response to low-level (environmental) fluoride esposure and incorporation. Accordingly, in addition to the fluoride content, the concenoration of calcium, phosphorous, chiorine, magnesium, and sodium in the cancellous cores were measured by instrumental neutron activation analysis. The weight ratios of calcium to phosphorous (dirnensionless), totai rnineral content (as a percentage, calculating by adding the calcium content and the phosphate content, which in nirn was esümated by multiplying the phosphorous content by the appropriate factor - see Section 2.3.2), fluoride-to-calcium ratio (in units of pg/mg), and normalized fluoride content (norrnalized to the total amount of bone mineral, dso in units of pg/mg) were also determined for each sample. Descriptive infom~itionabout the elemental composition cm be found in Table 3.2.2. The concentration of chlorine was weakly correlated to the fluoride content (in ppm) of the canceilous cores (see Figure 3.2.5), although none of the other trace elements correlated with the fluoride content. Aiso, there was no systernatic variation in the calcium or phosphate content, cdciurn-to-phosphorous ratio, or esumated totai rnineral content with the fluoride content, suggesting that the mineral content of the cancelious core is not affected by fluonde incorporation. No relationships were observed between any of the measured elemental concentrations and the age of the donor, other than the fluoride content, considered in detail in the previous section (Section 32.1). The measured elernental concentrations were also correlated with the density of the specimen. It shodd be noted that this is rn apparent density, not a tissue densiv; thïs is largely governed by the amount of bone present rather than the degree of mineralization of the bone. The calcium, phosphorous, magnesium and total minerai content deneami with increasing density (R2= 0.038, 0.1 86, 0.032 and 0.208, respectively). The calciurn-to- phosphorous ratio increased with increasing densicy (R2= 0.057), indicating chat the phosphorous content is falling more rapidly wïth increasing density than the calcium content. Since the neutron activation anaiysis was perfomed on whole samples, this may reflect the influence of trapped marrow in the denser samples (thereby increasing the weight of the specimen but leat-ing the mass of minera1 unchanged, resulting in a reduced concentration of bone minerai) and it is unlikely that this is an influence of fluoride content. For rhis reason, detailed discussion of these results ~vlube postponed to Section 4.3.3, when the methodo1ogical issues associated with neutron activation analysis dlbe discussed in detail. SEM

Table 3.2.1 Ekmentd composition of canceflous cores 1 O00 2000

Fluoride content (ppm)

Figue 3.2.5 Relarionship berneen chlorine and fluoride content

A ver). weak relationship was observed bemeen the chlosne content of the specimens (in ppm) and the fluoride content (also in ppm) (R' = 0.061, p

The mechanical properties of canceiious bone result from a combination of factors: the arnount of bone present (the apparent densi.), the material properües of the bone tissue, and the architecture of the bone. The mechanical integrity of bone, therefore, is analogous to the bone quaiity in that it integrates all the variable elements of bone tissue into a set of measures that cm serve as a proxy for the clinically significant parameter of fiacture risk. These measured parameters include the compressive moddus (a measure of the saffness of the matenal), the vield stress and the dtünate stress (measures of the strengrh of the materiai), the strain at the dtimate stress, and the energy to yïeld and to failure (both measures of rhe toughness of the material). Descriptive statisucs of the compressive and torsional mechanicd teshg data are given in Table 3.2.2.

Com~remuetesting: Regression information for compressive mecharicd testing is given in Table 3.2.3. As would be predicted, the measured mechanical properàes, with the exception of the strain at UCS, increased Ilieady with the apparent density of the cancellous core. The adjusted R' value vere very low, reflecting the variabdis in rhis data; they ranged from 0.1 11 for the energy absorbed to Mure to 0.190 for the ulümate compressive stress (illustrated in Figure 3.2.6). The suain at UCS (iustrated in Figure 3.2.7), ultimate compressive stress, yield stress and compressive moddus declined weakly with increasing age, suggesting that the mechanical integrity of bone dirninishes somewhat with age. Ir should be noted that the relationships are ail sery weak (adjusted R' ranging from 0.039 to 0.046; less than five percent of the variation in the data is esplained by the dedine in age). Fluoride had a weak but perceptible effect on the mechanical properties: the ultimate compressive stress (Figure 3-23)>yield stress, energy to Mure, and energy to yield (Figure 3.2.9) al1 decreased with increasing Buonde content. However, two of the individuals, with fluoride contents ofgreater than 2000 ppm, were found to be 'pinning' the regressions; if they are escluded, the iinear relationship benveen the energy to Mure is no longer significandy correlated Mth the fluoride content. While the other three relationships are sdi significant, the p-values and R' values are reduced. These nvo individuals have been escluded in the values and graphs provided here. Since the fluoride content increases with age, it is difficult to segegate qe-related changes from fluorîde-related changes. The uitimate compressive stress and the yield stress correlated with both the fluoride content and to its natural logarithm. As cm be recalled 6.om Section 3.2-1, the logarïdun of the fluoride content correlated hearly with the age. This suggests that perhaps these mechanical properties are, in fact, related to the age and not the fluoride content. Esamination of the the R' values and p-values indicates that the correlation benveen these mechanical propemes and either the fluoride content or the logarithm of the fluoride are comparable. However, the relationships benveen these two mechanical propemes and the age are only slighdy weaker. It should be noted that the energy to yield was not significandy correlated wvith either the logan'thm of the fluoride content or the age, suggestïng that the fluoride itself may be a factor. Finaily, multiple regressions were performed to determine the effect oidensity and age, as weil as densiy and fluoride content (as the Buonde content is related to age, using cwvo or more of these parameters would result in overparametrization). The significance values of each of the two terms were esarnined. The ulümate compressive stress and yield stress had irnproved regressions when both the density and the fluoride content were included.

1T,r-.nbmzffe~hig: A total of 27 specimens were tested in torsion (as discussed earlier, the number was iimited by the minimum size requirement for torsionai testin&. The shear modulus, iïke the compressive moduius, decreased iinearly with age (R2= 0.177, pCO.05; see Figure 3.2.10) but noc with the fluoride content, corroborating the effect of age on the stiffness of the bone material. No relationship was obsemed between the maximum shear strain or stress and the age or nuonde content. \VMe it w-ould be espected that the mechanicd propenïes in torsion would be sirnilar to the relationships observed for the compressive mechanical properties and the independent variables, the smaii number of samples may be obscuring any such relanonships. Compressive mechanicd properties:

Parame ter Mean SD SEM

Compressive modulus @Pa) 250 143

Yield stress @Pa) 6.8 0.4 Ultimate stress @Pa) 7.7 4.1

Energy to yield M/m) 0.13 0.12 Energy to faiiure (MJ/m) 0.27 0.28

Torsional properties:

Patame ter Mean SD SEM

Strain at UCS ?/O) 9.1 3.6

Shear moddus @Pa) 75 39

Ultimare shear stress PlPa) 3.3 1.9 Relationship to densîty: p-vahe

Compressive modulus (&Pa) <0.02 positive Ulhate compressive stress (&Pa) (0.05 positive Yield stress (&Pa) <0.005 posi cive Energy ro failure (MJ/m3 <0.01 positive Energy to yield (MJ/d)

Relationship to age: p-vduc Sibon ofcoe&r'uent

Strain at UCS (%) (0.05 nega tive Compressive modulus (&Pa) <0.05 negauve Ulrimate compressive suess (&Pa)

Relationship to fluoride content: p-vahe

Ultimate compressive stress PiPa) (0.05 negative Yield stress (?vlPa) c0.05 negative Energy to yield (MJ/m3 (0.05 nega tive

Relationship to logarithm of fluoride content: lF p-value

Ultimate compressive stress (AlPa) 0.052 4.05 nega tive Yield stress (&Pa) 0.049 (0.05 negative lMuitiple regressions: R' p-value p-value

Ulcimate compressive stress (&Pa) 0.220 Yield stress @Pa) 0.197

Table 3.2.3 Re,oression information for compressive mechanz'cai tesring

75 Figure 3.2.6 UltUnate compressive stress is comelated m*thdensis.

The compressive mechanical propemes of the canceiious bone (wkh the exception of the suah at UCS) were correlated with the densiq. The ulamate compressive stress is illustrated here (R' = 0.190 and p<0.05). Age of donor (years)

Figue 3.2.7 The suaul at UCS declined we&y Nith age

The saain at UCS in compression of the canceilous cores (as weU as the compressive rnodulus and the ultimate compressive stress) decreased with increasing age of the donor (R' = 0.046, pC0.05). 500 1 O00 1500

Fluoride content (ppm)

Figure 3.2.8 Ultimate stress is negatively correIated to the F content

The ultimate compressive stress, as weii as the yield stress, declined with increasing fluoride content of the canceilous core (R' = 0.048, p<0.05). Fluoride content (ppm)

Figure 3.2-9 Energy to pYIdddeched mathF conrenr

The energy ro yield decreases with Licreasing fluoride content (R' = 0.040, p

Age (years)

F~kure32-10 Relationsh> between shearmodulus and age

The shear modulus (measured in torsion) decreased with increasing age of the donor (R' = 0.090, pcO.05). This rriirrored the behaviour of the compressive modulus. 3.2.4 Coronai sections

Contact s-rays were made of thick (5 mm) coronai sections, escised from the Çemoral heads. The images were then acquired to a cornputer, and image and suut analysis of the trabecular architecture \vas performed. A total of 67 coronal sections were anaiysed. The purpose of these analyses \vas to quanti4 the architecrue of the cancellous bone over the entire femoral head, and to determine if any of these measured parameters could be correlated wïth the fluoride content (as well as the age of the donor and the phpical density ofa bone sample). Tt should be noted that the techniques used in these analyses were designed and validated for use wïth Ihin (5 km) histological sections. In addition, the femoral heads are much more heterogeneous chan other anatomical sites, and this is esacerbated by the presence of osteoarthntis in many of the ÇemoraI heads (onlp 28 of the 67 s-rays w-ere grossly normal in appearance; see Section 2.7). This may affect the trends and results noted in this section, and is discussed in greater detail in Section 4.1.5. Image and suut analysis of histologicai secuons was also performed in this study (Section 3.2.3) and those sarnples were used to accurately quanatate the architecture at nvo sites on the Çemoral head. Descriptive information about the measured parameters are provided in Table 3.2.4. Table 3.2.5 contains ail the regression parameters referred to in this section, and graphs of selected reIaaonships are shown. No relationship was observed benveen the arnount of bone present (as measured by the trabecdar volume, thickness, separation or number) and the age of the donor. Homever, the trabecular volume decreased and trabecular separation increased - that is, the arnount of bone present decreased - with the fluoride content (dustrated in Figures 3.2.1 1 and 3.2.12, respectively). The trabecular volume displayed a slightly doser relationship to the logarittim of' the fluoride content - itself linearly related to the age of the donor - than to the fluoride content, so a subtle effect of age may be present. Strut anaiysis \vas used to assess the co~ectivïty.None of the measured parameters displayed a consistent variation with age. The number of endpoints, as weii as the nurnber of node-free struts, appeared to decrease with increasing fluoride content. The number of endpoints also increased with density, dchough the reiationship is not convincingly linear (Figure 3-2-13}.The behaviour of the endpoints is entir* anomalous; the increasing trabecular volume with fluoride content, as established above, would suggest that the number of endpoints should increase \.thdensiv. Srmilady, as the apparent density of the canceilous core increases, the number of endpoints (which is a measure of the disconnectedness of the specirnen) should decrease, not increase. These results are somewhat problematic: ody a minimal relationship benveen the observed vaiues and the age of the donor were observed, counter to what would be espected, and one parameter (the number of endpoints) behaved in a counterintuitive rnanner. These results may be, ar least pdy, due to the application of image analysis techniques designed for thin sections to thick sections and to the heterogeneity of the coronal sections; this wili be discussed in greater detail in Section 4.2.5. Image analysis:

Parameter SEM Range

Trabecular volume ('/O) Trabecular thickness (p) Trabecular nurnber (mm") Trabecular separation (pm)

Strut analysis:

Parme ter SEM Rmge

Free ends (mm-3 Nodes (mm-?

Node-free sauts (mm/mm'-) 1.3 0.69 Nodr-node strucs (mm/&') 0.093 0.050

Star volume (mm? 0.44 0.1 1

Table 3.2.4 Image and strut anaiysis ofcorodsections Relationship to fluoride content:

Paran eter R2 Constant p-value

Trabecular voiurne (%O) 0.069 50.124 p<0.05 Trabecdar separauon (pm) 0.084 275.4 pc0.05 Nurnber of endpoints (mm-? 0.058 8.736 pC0.05 Node-kee suurs (mm/mrn-) 0.062 1.533 pC0.05

Relationship to logarithm of fluoride:

Param eter R' Consranr p-vahe

Trabecular volume ?/O) 0.08 1 66.566 p

Relationship to density:

Parameter R? Constant p-value

Numberofendpoints(rnrn-3 0.149 3.076 p

Table 3.2.5 Reagession inAomatzbn for image analysr's of coronai sections F content (ppm)

F~gure3.Z.Zl Trabecular bone volume decreases w2h Buoride con rem

A meak, negacive linear relationship was obsen~edbenveen the trabecular bone volume (%) and the fluoride content (in ppm) (RZ = 0.069, pc0.05). 1000 2000

Fluoride content (ppm)

A wveak, positive Lnear relaüonship was obsen-ed benveen the aabecular separation (pm) and the ffuoride content (in ppm) (R2= 00.84, pc0.05). The previous graph and thïs one suggrsts that there may be a loss of' bone widi increasing fluonde content. Density (g/cc)

Figure 32.23 Nurnber of endpoints hcreases m*&densiky-

Coun~erintuitively,a weak, positive linear relationship \vas O bserved betsveen the number of endpoints (mm-') and the densiry (in g/cm3 (R' = 0.149, p<0.01). 3.2.5 Histological sections

Two histo1ogical sections fiom each of 79 femoral heads were andysed to determine the amount and comectivity of canceIIous bone. Each specimen \vas approxïmatdy 1.5 cm square. One was escised from the superior (wveightbearing) surface and one boni the inféùor (nonweightbearing) surface. As osteoartliritis is known to alter the amount and architecture of the bone as well as causing cartilage damage (chiefly on the superïor surface), using a section from both regions enabled the localized effects of osteoarttiatis to be differentiated from the normal structure of the bone. Within the superior (weightbearing, and therefore more affected by osteoarrt-intis) section, no relauonship was observed benveen the parameters describing the amount or connecavity of bone and the age, fluoride content, or physical densi. of the cancellous core. In contrast, image analysis of the inferior section indicated that the two derived values, trabecular separation (Figure 3.2.14) and the trabecular number (Figure 52-13) of the inferioï side, correlated with the age of the donor (IL2 = 0.066 and R' = 0.060, respectively; pC0.05) althouph the measured parameters (trabecular volume - iUusu-ared in Figure 3.2.16 - and trabecuiar thickness) did not. The trabecular volume, trabecular number, and trabecular separation of the inferior sections also correlated significantly with the density of the cancellous core (Figure 3.2.17 iiiustrates the relationship between tcabecular volume and density). None of die image analysis parameten conelated Mth the fluoride contenr. Strut analysis of the inferior section, performed in order to assess the comectivity, indicated that the number of nodes (Figure 3-2-18),node-node smts and node-free suuts increased with increasing densi- (R1= 0.117, 0.207 and 0.045, respectively; p<0.05). This increase in connectivity of the bone is consistent with the increase in the amount of bone that was obsenred using image analysis, and also intuitively consistent with increased physicai density. The number of nodes decreased linearly with increasing age (Figure 3.2.19), indicating a Ioss of connectivity which is also consistem wich the decrease in trabecular nurnber and increase in trabecular separation with increasing age that was obsenred. However, the number of free ends (which are associated with dzkonnectedness) aho decreased (Figure 3.2.20). The ratio of nodes to free ends wvas therefore also esamined as a parameter of co~ecùvitythat incorporates both of these rneasured values, but it kvas unchanged with increasing age of the donor, sugges~gchat the nvo parameters were affected similarip by age. No relationship \vas observed between the strut analysis parameters and the fluonde content of bone. 1Wethe arnount and the comectivity of the cancdous bone at the inferior site changed systematicaiiy with the age and the physical density, these relationships were not observed ai the superior site. It WU be also be shown and discussed (in Section 3.6.5) that there is profoündiy more bone present at the superior surface than the inferior surface, with a cornmensurate increase in the connectivity at the superior surface. This persuasively suggests that structure of the canceilous bone at the superior site is govemed by osteoarthritis-related changes, which are ovenvhelming the normal age- and densi-related changes observed at the inferior site. This will be discussed in detail in Section 3.6, which focuses on the effect of disease state on the response of bone to fluoride incorporation. Age of donor (years)

Figure 3.2.14 hage andysis of infenor section: trabecufarsepararion

A weak but significant relationship was observed benveen the trabecular separation of the inferior hisrological section and the age of the donor (R' = 0.066, pC0.05). Age of donor (years)

Figure 3.Z.Z5 Image analysis ofinferior section: trabecular number

A weak but signifrcant reIationship was obsenred benk-een the uabecular number of the inferior hisrologicai section and the age of the donor (R' = 0.060, pc0.05). 20 30 40 50 60 70 80 90

Age of donor (years)

Figure 3.2.26 hage mdysis of u+fenorseca'on: trabecdiu volume

Neither the trabecular volume (iusuated here; R' = 0.021, p = 0.1 14) nor the trabecular thickness of the inferior sections were found to conelate wirh the age of the donor. Density (gkc)

F~kure32-17 image anaiysis ofSenÔrsectimt: trabecular volume

The trabecular volume of the inferior histologicai section (as weU as the trabecular number and trabecular separaaon) was lineady correlated to the physicai density of the cancelious core (in g/cm3 (R' = 0.130, p<0.005). Density (glcc)

Figure 3.2.28 Strut analysis ofinfenor secnon: nodes

The number of nodes per unit area of the canceilous bone was hearly conelated CO the density of the canceilous core at the inferior site (R2= 0.1 17, p40.05). 60 70

Age of donor (years)

Figure 3.2.19 Strut andysis of infenor sechon: nodes

The number of nodes per unit area in the inferior histological section \vas linearly correlated to the age of the donor (in years). One of the donors was 23 years old; \vMe the relationship is Linear with that datapoint induded (R' = 0.052, p = 0.0351, the relationship is nevertheless doser when that outlier is esduded. (R' = 0.085, p = 0.01) and rhat datapoint is escluded from the above graph. Age of donor (years)

Figure 3.2.20 Strut malysis of Senor sec&: fie ends

The number of free ends per unir area in the iderior hiscological section was also negatively correlated to the age of the donor (R2=0.074, pC0.05). 3.2.6 Backscattered electron imaging

Backscanered electron irnaging produces a greyscale image of the bone sample. The bnghtness of different regions of the image correspond to the degree of mineralizaaon of the bone, wïth the dark grev areas beirg the least mineraiized and the bright white areas the most mineralized @la& corresponds to a complete absence of bone). Two parameters are used to describe the resulthg mineralization distributions: the weighted average and the logit of the distribution (see Section 2-52),both ofwhich increase with increasing rnineralization. A total of 79 femoral heads were esamined. Samples of bone from the superior and the inferior surface wsre analysed, and the subchondral and cancellous bone were analysed separately at each site. At the superior site, the weighted averages of the mineraiization distributions at the superior site were found to increase hedywith the fluoride content for both the subchondrai and the cancdous bone (R' = 0.130, pc0.005 and R' = 0.1 14, p<0.005, respectively; see Figures 3.2.21 and 3-2-22)but no relationship benveen the weighted average and the age and density was observed at these sites. The logits of the mineralizaaon distributions for both the subchondral and the cancellous bone at the superior site were linearly related to the natural loag-thm of the fluoride content, although not the fluoride content itself (Figures 3.2.23 and 3-2-34).No relationship was observed benveen the logits of the rnineraiization profile at this site and the other independent parameters. At the infenor site, no relationship \vas observed behveen the weighted averages of the rnineraiization distributions for either che subchondral or the cancellous bone and any of the independent parame ters, including the fluoride content. The logit of the subchondral bone at the inferior site correlated significantly with the age of the donor (correlaaon coefficient 0.234, p<0.05 by Pearson) but the nature of the relationship was not determined (Figure 3.2.25). No other relationships were observed benVeen the logits at the iderior site and any of the independent parameters. The superior and inferior surfaces, therefore, had veq- diffèrent responses to fluoride incorporation. The degree of rnineralization ar: the superior surface increased in response to increasing fluoride, but this response was absent at the inferïor surface. This paraiIels the differential response to age and density that \vas observed for image analysis of histological sections from these two sites. In this case as wd, the observed differences in the reIationship bemeen the degree of mineralkation and the fluoride content for the two surfaces of the bone ma); be a consequence of the osteoarrhritis present in the majori'y of the bones. This possibility d be esplored in greater detail in Section 3.G.G. 1 O00 2000

Fluoride content (ppm)

FIgure 3.221 Mineralization of supebr subchon&ai bone

A positive linear relationship was observed benveen the weighted average of the mineralization distribution of subchondral bone at the superior surface and the fluoride content (in ppm) (R' = 0.130, p<0.005). Fluoride content (pprn)

Figure 3.2.22 Mkerahzation of canceflous bone ar the superior surface

A positive 1inea.r relationship was also obsemed benveen the weighted average of the mineralitauon distribution of canceiious bone at the superior surface and the Buoride content (in ppm) (R1= 0.1 14, pc0.005). log F content (ppm)

The logit of the mineralization profile at the superior subchondral surface was linearly related to the naturai logarithm of the Buoride content (in ppm; R' = 0.089, pc0.05) log F content (ppm)

Figure 32-24 Logi'r of canceUous bone at the supenbr surface

The logit of the mineralization profile at the supenor canceilous surface was hearly relared to the namal logarithm of the fluonde content (in ppm; Et' = 0.066, p

3

2 t .-O -s 1 .-P CL "'O u.- .c O .-c. rn -1 O 1 -2

-3

-4

Age of donor (years)

The logit of the mineralization prohle at the infenor subchondrai surface was correlated with the age of the donor (Pearson correIation coefficient = 0.234, pc0.05) but a significant linear relationship \vas not observed. 3.2.7 Microhardness testing

The mechanical properties of cancellous bone are, in a sense, structural properties. \We the mechanical properties of bone material contribute to the measured mechanical properties, the amount of bone present in the test specimen (corresponding to the density), and the architectural organization of the trabecular bone are also major contributors. The rnicrohardness, by contrast, is a measure of the materra/ propemes of the bone tissue. It is largely governed by the mïneralization of the bone and therefore would be espected to be related to the degree of mineralization as assessed by backscattered elecuon imaging. For the microhardness testing, small blocks comprised of both cortical and canceilous bone were escised from four locations on the femoral head: superodistai, superoprosimal, inferodistal and inferoproxkal (see Fi-oure 2.2.1). 14s \?th the backscattered electron imaging and the histological andysis, the purpose of using multiple samples was to assess differences betsveen sites. A total of 39 blocks were esamined. The microhardness measurernents for the subchondral and canceilous bone at each of the four sites were independentiy correlated with the age of donor, density, and fluoride content of the speumen. At the superoprosimal site, the rnicrohardness of die subchondral and the canceilous bone increased lineariy wïth both the fluoride content and its namral logarithm. In the case of the subchondral bone, a closer fit was obtained to the fluoride content itself than its logarirhm (R' = 0.383, p<0.001 vs. R' = 0.351, pcO.05); the converse was me for the cancelious bone (R' = 0.145, pCO.05 for the relationship with F vs. R' = 0.156, pc0.01 for the relationship with log F). 'T'he relationship of both chese parameters with the fluoride content are iilustrated in Figures 3.2.26 and 3.2.27. At the superodistal site, the rnicrohardness of the subchondral bone increased linsarly with the fluoride content and its natural logmithm; the latter is illustrated in Figure 3.2.34. The microhardness (of either the subchondral or the canceiious bone) did not correlate with the age, density, or fluoride content of the specimen at either of the inferior sites. The microhardness results mirrored the backscattered electron imaging results quite closely, in that the both the degree of rnineralization and the microhardness increased with incorporated fluonde at the superior site but not the iderior site. The microhardness is known to be related ro the degree of mineralization, and it is therefore unsurplising that the relationship to incorporated fluoride is so similar for both. Differences benveen the Mcrohardness at the inferior and supenor surfaces are Wcely to be related to the presence of osteoarrhrius, and wiil be discussed in greater detail in Section 3.6.6. 1 O00 2000

F content (ppm)

Figue 3.2.26 ikiïcrohardness of superoproh*ai bone: subchon drai

A positive linear relationship was observed between the rnicrohardness of the subchondral bone at the superopcoximai site and the fluonde content (in ppm) (R' = 0.383, p

F content (pprn)

Figure 3.2.27 Mïcrohardness of superoproncirnd bone: cancellous

A positive Linear relationship was observed between the microhardness of the cancellous bone at the superoprosimal site and the fluoride content (in ppm) (RZ = 0-145, p

A positive Lnear relationship \vas observed between the rnicrohardness of the subchondral bone at the superodistal site and the natural logarïthm of the fluoride contenc (in ppm) (R' = 0.246, p

A total of 92 femoral heads were obtained from patients undergoing hip replacement surgery in Toronto and Montreal. The specirnens were extremely heterogeneous. The donors ranged in age from 23 to 90. Seventy-five of the 92 patients were osteoarrtintic, and nine were osteoporotic. Fifiy-one of the donors were fernale (di but one ofwhich \vas over fifq years ofage, and therefore peri- or post-menopausal), and the remaining 41 were male (dl but £ive were over fifq years old). The observed fluonde content in the bone ranged from 192 to 3264 ppm, a range of aimost nvelvefold. There was no correlation observed benveen the densiy and the fluoride content, or the densit). and the age of the donor. However, for this population of individuals, the natural logarithm of the fluonde content \vas Linearly correlated with the age of the donor. LVithin the sample population in its entirety, a number of relationships were observed benveen che parameters of bone qualicy and the fluoride content. The chlorine concentration was associated with the fluonde content, but no other trace elements displayed any relationship. The ultirnate compressive stress, veld stress and energ to vield were negaüvely associaced ~viththe fluoride content. The first avo of these aiso decreased with age. LVithin the coronal secuons, a number of parameters were related to the fluoride content (consistent with reduced bone with increased fluoride content). However, thïs \vas not confirmed by the more canonical image analysis of histological sections, although the normal age-related decline in bone mass war observed in the inferior sections. The degree of mineralization, as measured by both backscattered eIectron imaging and microhardness testing, \vas positively associated with the fluoride content at the superior (weightbearing) surface but no t the inferior (nonweightbea.ring) surface of the bone. 3.3 Case-control study: fluoridated vs. non-fiuoridated municipality

3.3.0 Introduction

The purpose of this component of this smdy is to determine if differences esist in the bone quaiity of individuais living in a fluondated and a non-fluorïdated municipality (Toronro and Montreai, respectively). This analysis does not conuol for other factors; it simply compares the mean values for the populauons from each city in their entirety. Accordingly, unpaired t-tests (homoscedastic or heteroscedastic tests, as approprïzte) were

used to compare each of the rneasured parameters of bone quality for each of the ~TOcities in order to determine if differences benveen the means esist. Significance is indicated by a p- value of less than 0.05.

3.3.1 Patient profiles

A total of 39 specimens were retneved from Montreal residents, of which 24 were femaie and the remainlig 15 w-ere male. Twenty-eight of the individuais were diagnosed wvith osteoarrhntis, 7 \.

3.3.2 Fluoride content and chernicd composition

The observed fluoride content of bone samples from Toronto donors ranged from 192 ppm to 2264 ppm, with a mean value of 1033+438 ppm (mean t SD). For bone samples from Montreal donors, the range was much tighter, with a minimum of270 ppm and a rna~umof 1200 ppm. The mean value =-as 643 f 220 ppm (mean ISD). The means were significandy different @<<0.001), indicaring that, on average, individuais residing in Toronto have more Buoride incorporated into their bones. However, as che range of observed values for the Toronto bones indudes the range of observed values for the Montreal bones, this suggests that other factors rather than simply =posure to fluoridated water affect the rate and degree of fluoride incorporation into bone. These data are iliusaated in Figure 3.3.1. Chernical composition data is given in Table 3.3.1. The percentage ofcaluum and phosphorous was greater for the Monaeal specimens than for their Toronto counterpms. The total minera1 content was also greater, although the ratio of calcium to phosphorous was constant for both groups. hongthe tcace elements, there was significantly more chlorine and less rnagnesium incorporared into the high-fluoride voronto) canceiious bone. No difference \vas observed in the concentration of sodium benveen the nvo groups. As would be espected from the large difference in the fluonde contents, the fluoride-to-calcium ratio and the fluoride-to-total-mineral ratio was also much higher for canceiious bone from Toronto donors than Montreal donors. These results indicate chat more minerai is present in the canceiious cores of bones from Monaeal than Toronto. This is counter to the espectaaon diat higher fluoride concentrations aeassociated wïth increased minerai content. 643 ppm I

Toronto

Figure 3.3.1 Fluon'de content of bone fiom Toronto and Montreal

\Vide significandy more fluoride was incorporated into bone from patients residing in Toronto than in Montreal @<0.0001), the range of fluoride contents observed in bone from Toronto paaents included the range of contents observed from Montreal paaents. This suggests chat factors other than fluoride esposure affect incorporation into bone. Toronto

Ca content (%O) P content (O/O) Ca/P ratio Ca+PO, (total mineral, %)

CI content @pm) Mg content (ppm) Na content (ppm)

F content @pm) F/Ca ratio (%/mg) F/CaPO, (Pg/mg)

Table 3.3.1 ~hem'ca.2compositio~ of cancefious borie by

Data presented as mean t SEM.

*The bones from hfontreal residents had, on average, higher calcium and phosphorous concentrations than those from the Toronto residents. While the ratio of the nvo was the same for both groups, the total minerai content was higher in the Montreal group. Among the trace elements, the concentration of chlorine was significandy greater and thac of magnesium significandy less for cancelious bone from Toronto residenrs compared to Montreai residents @

Compre.sxii@en~ecacaoee: Data for the compressive mechanicd properties are given in Table 3.3.2. The mean density of cancelious cores Gom the Toronto specimens (n=53) \vas significantly greater than those ffom Nontreai (n=39) @<0.05). However, as ïndicated in Section 3.2.0, the density of cancellous cores in this study did not correlate close1 wlth either the fluoride content or the age, and it is unclear why the densis. shouid be higher for bones Tom the fluoridated region. The mean strain at ultimate compressive stress (UCS) of bone from the Toronto donors was greater than their Montreai counterparts, as was the energy absorbed to failure @<0.05). As the energy absorbed to the proportional limit was identical in the two groups, &.is suggests that the difference in energy absorption is a consequence of hepost-yield behaviour; the greater straïn at UCS in the Toronto group resdts in a greater energy absorption to failure. This suggests that these bones mai be more ductile than their Montreal counterparts. As would be especred, given their greater mean densiq, the remaining mechanicd parameters were greater for canceilous bone from donors residing in Toronto than from donors residing in Montreal, even though the differences were not significant. This can be observed in the stress at the proportional limit Weld stress), the ultimate compressive stress, and the compressive modulus.

Torsiona/mechanica~p~er/re~:Data for the torsional mechanical properties are given in Table 3.3.2. The number of specimens avaïiable for torsional testing kvas limited by the size of the specimens. A gauge length that \vas mice the diameter was used (12 mm), and only relatively few femoral heads were large enough that a corc of this lengrh could be escised trom the hemisphere of bone remaining afier the corona! section was removed. A total of 27 bones were tested, 15 from Toronto donors and 12 from Montreal donors. No difference was observed in the means of bone from the tlvo ciaes for any of the measured torsional parameters - shear strain to faiIure, ulamate shear stress, or shear moduius - although there mas also a trend tomards increased strain at Mure for the Toronto specimens compared to the Montreal specimens Q~0.09).This again suggests that the Toronto bones are more ductile than the Monmeal bones. The Iack of difference benveen the nvo groups of the ultimate shear stress and the shear moddus is consiscent with the findings for compression. Compressive mechanical testing:

Toronto

Yield stress (&Pa) Energy to yield (MJ/rn?

Strain at ultknate compressive stress ?/O) UItimate compressive stress @Pa) Compressive rnoddus &Pa) Energy absorbed ro failure @IJ/rnJ

Torsional mechanical testing:

Toronto Montceal

Shear saain at fracture Ultirnate shear stress (&Pa) Shear moddus &Pa)

Table 3.3.2 Mechani'cdpperrrés of cancellous bone

Data presented as mean f: SEM.

The mean densis- of the canceilous cores \vas greater for the Toronto specimens than the Montreal specimens. In compression, the strain at fdure and the energy absorbed to Murewere significantly increased in the Toronto specimens compared to their Montreal counterparts. 3.3.4 Coronal sections

Image and strut analysis of contact x-rays of thick (approsimately 5 mm) coronal sections of rhe hmord head were perfomed to assess the amount and comecuviq of the cancellous bone in the fernord head. Data are given in Table 3.3.3. A total of 67 fernorai heads were anatysed, 30 of thern from Montreal and 37 from Toronto. No differences mere observed benveen die nvo groups for an)-of the measured parameters. Toronro Montreal

proportion of trabecdar bone (96) trabecular chicimess (pm) trabecdar number (mm-') uabecdar separation (q)

free ends (mm-? nodes (mm-? fiee-free strun (mrn/mm') node-free struts (-/mm? node-node sauts (mm/mrnl) nvo-dimensional star volume (mm2)

Table 3.3.3 Image andysis data for coronal sectzons

Data presented as mem t SEM. 3.3.5 Histologicai sections

Image and saut analysis of histological sections from the superior and the inferior surface of the femoral head was performed. Foq-nine of the specimens were fiom Toronto residents, and 30 from Montreai residents, for a total of 79 sarnples. Data for pararneters related to the arnount of canceiious bone present are gïven in Table 3.3-4, and strut analysis pararneters (related to the connecuvi~of the canceiious bone) are given in Table 3.3.5. For the supenor surface, the trabecdar number was significantly greater and the trabecular separation was significandy less for the canceiious bone from Toronto residencs compared to those kom LMontreai residents. Correspondinglg, there was a trend towards an greater proportion of trabecular bone @<0.1), and the trabecular duckness \vas greater, on average, for the Toronto specimens compared to the Montreal specimens (even though this difference \vas nor significanr). The NO-dimensionai star volume is also significandy less Li the Toronto specimens than the Montreai specimens, suggesting that the Toronto specimens have greater comectivi~.\me no other sipificant differences were observed in the strut analysis data, the general trends are consistent with more and better-connected canceiious bone in the Toronto specimens compared to the Montreai specïmens (fewer free ends, more nodes, more node-node smin). r\n increase in the amounr of bone measured by image analysis is also consistent with the higher physicai densiq for the canceiious core observed for the Toronto specimens compared to their Montreai counterparts. \We the overali trends observed in the image and strut anaiysis of the inferior sections mirrored those observed for the superior sections, rhere were no signi6cant differences in any of the measured parameters. As can be observed fiom both Tables 3.3.4 and 3.3.5, there are profound differences observed in the amount and the co~ectiviryof bone at the superior surface and the inferior surface, consistent with more canceiious bone at the superior surface. These differences are

IikeIy to be related CO the presence of osteoarrhntis in mang of the specimens and are discussed in detail in Section 3.6.5. Parameters reIatùig to the arnowt of trabecuiar bone:

Toronto Montreal Supehr: proportion of trabecular bone (%) trabecuiar thickness (prn) trabecular nuniber (mm-') trabecular separarion (pm)

Toronto

Infnot: proportion of trabecular bone (!/O) 18 k 2 trabecdar thickness (pm) 141 t 7 trabecdar nurnber (mm-') 1.30 3: 0.05 trabecular separacion (pm) 687 + 39

Table 3.3.4 Image analysis dam for histolobgCal sectioBs

Data presented as mean t SEM.

*For the specimens from the superior surface of the bone, the trabecuiar number \vas significanùv reduced for canceiious bone from Montreal donors compared to that from Toronto donors (pcO.05). There was a trend towards a similar decrease in the proporcion of trabecdar bone @<0.1). There were no observed differences for any- of the paramerers measured on the inferior specimens. Parameters relating to the comectidyof trabecular bone:

Toronto

Supenoc €tee ends (mm-? nodes (mm-') free-free struts (mm/mm-z) node-fiee struts (mm/mm-3 node-node struts (rnm/mrn-3 nvo-dimensional star volume (mm')

Toronto

Idenoz free ends (mm-3 nodes (mm-? free-Eree su-uts (rnm/mm") node-free smts (mrn/mm-') node-node struts (mm/rnm--) nvo-dimensional scar volume (mm')

Table 3.3.5 Smanalysis data for 1Fu'stoIo~olCa.isections

Data presented as mean I SEM.

Thetwo-dimensional star volume of the superior specirnens was signïficantly greater for the bones from Montreal donors than from Toronto donors. There were no obsenred differences for the parameten measured for the inferior specirnens. 3.3.6 Backscattered electron imaging

The weighted average and the logit of the mineralization dismbutions were determined for the bone at two anatomical locaaons (at the supenor and inferior surface of the femoral head). The subchondral bone and the cancellous bone were analysed individuaily. The means of Siese values for specimens fiom Toronto residents (n = 49) and from Montreal residents (n = 30) were compared. No significant differences were observed in the logits and the weighted averages of the distributions (Table 3.3.6), indicating that the degree of rnineraiization of the bone \vas not different benveen these nvo cicies. Site-to-site differences in the degree of mineraikation of the bone wd be assessed and discussed in Section 3.6.5. Weighted average:

Toronto

Supenbr: subchondral cancellous

Infeno~ subchondral canceiious

Logit: Toron to Montreal

Supenor: subchondral canceiio us

Idenor: subchondral cancellous

Table 3.3.6 Backscattered dectron haging data

Data presented as mean -t SEM. 3.3.7 Microhardness testing

Samples used for microhardness cesting came from four different locations on the fernord head and were used to determine if site-specific differences esisted (sec Section 3.7.3). hiïcrohardness data are given in Table 3.3.7. Ac the inferoprosimal site, the microhardness of the subchondral and of the canceiious bone were greater for speümens Gom Toronto donors than their Montreal comterparts, although no other differences were observed. This is consistent with the backscattered electron imaging data; the means of the logits and the weighted averages of die mineralizaüon dismbutions at the inferior site, for both the subchondral and the cancelious bone, were either greater for the Toronto specimens or equal benveen the nogroups, even though no statistical differences were observed. No differences were observed in the degree of mineralizaaon at any of the other sides, as measured by eicher technique. Differences in the microhardness beaveen the four sites are addressed in Section Toronto

Superoproximal: subchondrai cancellous

Supero dstd subchondral cancellous

Inferopr~~uinîd: subchondral canceiious

Infrodr's tak subchondral canceUous

Table 3-3.7 Mcrohardness of bone

Data presented as mean ISEM

*Ac the LnferoprosLnal site, only, the rnicrohardness of both the subchondral and canceiious bone are greater for the specimens from Toronto donors compared to their Montreal coumerparts @

The mean amount of fluoride incorporated into the speùmens kom Toronto residents \vas signi ficantly higher than those from Montreal. However, the range of values observed in the Toronto specirnens entirely inciuded that of the montrea al specimens. The cduurn, phosphorous, and total minera1 content \vas higher for the Montreal specimens. Of the trace elements, the magnesium concentration \vas lower and the chlorine concentration was higher for the Toronto specimens. The density of the Toronto specimens was greater, as was the main at the ulumate compressive stress and the energy absorbed to fdure (measured in compression). No differences were obserç-ed for image analysis of the coronal sections. Image and stnic andysis of the superior histological sections indicated a tendency towards a greater amount of bone in the Toronto specimens- the trabecular separacion and the hvo-dimensional star volume were less, the trabecular number \vas greater, and there was a trend towards a greater trabecular volume compared CO the Montreal specimens. No differences were observed in the degree of rnineralization as assessed by backscattered electron imaging, although the microhardness at one of the four sites esamined was greater for the Toronto specimens compared to their Montreal counterparts. 3.4 Differences between high- and low-fluoride individuals

3.4.0 Introduction

Section 3.2 focused on detemiining the nature of a relaaonship, if any, becween die measured parameters of bone quality and the fluoide content (whether measured direcdy or as a fracaon of the bone minerai) by use of correlation and regression techniques. Unfortunately-, the large amount of variability in the data may be obscuring such a relationship. The approach of this section is somewhat different Rather than attempüng to elucidate a continuous relationship benveen the measured parameters and the bone Buonde content, these analyses attsrnpt to determine if differences in these parameters exist benveen individuals with high and low arnounts of Buonde incorporaced into their canceilous bone. Accordingly, the 92 femoral heads used in this smdy were divided into qudes based on the fluonde content of the canceilous core. Specimens in the top and bottom quade (23 in each quartiie) were identified. For each of the parameters, values for each of these groups were compared using either a homo- or heceroscedastic unpaired t-test (based on Levene's test for equality of variances) or, if a Kolmogorov-Smimov normality test was failed, by the Mann-Whitney non-parametric test. &O, because some gender differences were observed (see Section 3.5), the tests were also performed separately for specirnens frorn male and female donors.

3.4.1 Sarnple information for the two quartiles

The nvo quades of patients differed slightiy in their composition. The major difference was the top qudewas slighdy older, on average, rhan the bonom qude (see Table 3.4.1); this is consistent with the increase in fluonde observed with age. C'nsurprisingly, the majority of individuais in the top quartile resided in Toronto and, of the bottom qude,Montreal. However, six of the low-fluoride individuals (or more than 2j0/o) resided in Toronto, aithough only cwo of the high-fluoride individuais resided in Montreal- The gender distribution and the diagnoses of donors in each quartile \vas remarkably similar: 12 of the donors in the top quartiie were female, 13 in the bottom quade, and 19 of the 23 individuais in each quartde \vas diagnosed with osteoarrhetis. Fluoride content (ppm):

Age of donors:

Mean t SD Range

Gender:

Fende

City of origin:

Disease state: 19 osteoaahritic 19 osteoarthntic 2 osteoporotic 1 osteoporotic 1 rheurnatoid arthgtis 1 rheurnatoid 1 avascular necrosis 1 ankylosing necrosis 1 osteonecrosis

Table 3.4.1 Patient data for the figh- and lo w-ffuon2iequades

*The individuals in the top quade had significandy more ïncorporated fluoride and were significandy older han their bottom-qude counterparts. 3.4.2 Chernical composition of samples

hli data are shown in Table 3-42The fluonde content \vas significantly higher for the top qudethan the bottom quade, as \vas the fluoride-to-calcium ratio and fluoride-to-totd-minera1 ratio (p~~0.001).The mineral content of the cancellous bone was iargely unchanged benveen the quades, dthough the calcium-to-phosphorous ratio was slightly higher for the top qudein the fernales-only group. In the absence of differences benveen the calcium, phosphorous, and total minerai contents, this may sirnply be a result of a siighdy higher calcium content and slightly lower phosphorous content in this group. The concentration of chlorine was also obsemed to be higher in the top quarule than in the bottom quarde; however, this was not observed for the gender-segregated groups. The other trace elements were unaffected by £luoride concentration. Top Qude

Ca content (Oh) ~V1ale.r Fendex Totak

P content (%) iVla/e~ Fermles= Totak

Ca/P ratio i\/kl/es= Fenralex Total-

Total minerai (%) Males: Fe~nale~ Tord

Cl content (ppm) ~Ualer.. Fermier: TotaL-

1Mg content (ppm) ib1afe.r: Fernaie~: TotaL-

Na content (ppm) ~t1aie1: Fenznies: Tord-

Table 3.4.2 Chenumi composition of cancehs bone

1For the femdes only, the Ca/P ratio, but not the calcium, phosphorous, or total minerd concent, is hïgher for the top quartile than the bottom quade of fluoride content (pC0.05).

'The chlorine content is higher for the top quartiie of fluoride content than the bottom (pC0.05) F content @pm) MaLes: Fenza1e.r.- Total-

Table 3.4.2 Chemicd composiaon of canceffousborre (cont'd)

*Unsurprisingly, the fluoride content, ratio of fluoride to calcium, and ratio of fluoride to total mineral are significmtiy greater for the top quartiIe dian the bottom quartile @<<0.0001) 3.4.3 Mechanical properties

Compresnon: Data for compressive mechanical properties are shonm in Table 3.4.3. LWe the density of the cancdous core \vas unchanged benveen the qudes, the ultirnate compressive stress and the yïeld stress were greater for the boctom quade than the top quade. These differences were not observed when the data was segregated by gender. No other differences were observed benveen the qudes. The diçferences observed benveen qudes contrast \.

Tomh: Data for torsional mechanical propemes are shown in Table 3.4.4. A total of 27 samples were tested in torsion, and of these samples, 4 feu into the top qudeby fluoride content and 7 into the bortom quade. Ko &£ferences were observed in the torsionai mechanical propenies of these nvo groups. The number of samples was too smd to permit segregaaon of the quades by gender. Density (g/cm3)

Suain at UCS (%)

Compressive rnodulus (MPa)

Ulümate compressive stress (MPa) Maiex: 7.2 k 1.3 9.3 t 0.9 Femalec 5.0 + 1.2 7.8 2 0.9 TotaL- 6.0 t O.9* 8.4 -t- 0.6*

Yield stress (MPa)

Energy to failure (MJ/m3) ~Wzies: 0.23k0.04 Fen~aies: 0.14 -t 0.04 Total- 0.18kO.03

Energy to yield (M~/rn~)

Table 3.4.3 Compressive mecharukilproperdés of cancehus bone

Mean 2 SEM.

The ultimate compressive stress and the yield stress were higher for the bottom quarde than the top qude. Top qude

Shear straïn at fracture 0.099 k 0.027

Shear rnodulus (MPa) 77 k 13

Ultirnate shear stress (MPa) 2.9 IT 0.7

Table 3.4.4 TorsiondmechafllCalpropeitlksof canceffuusbone

Mean k SEM. No differences were observed benveen the nc-O quades. Too few samples were available (n=3 for top quade, n=7 for bottom quade) to segregate data by gender as weli. 3.4.4 Coronal sections

Jmage and smt analysis paramerers for the coronal section, separated by qude, are given in Table 3.4.5 and Table 3.4.6, respectively. Within the qudes as a whole, the trabecular bone volume \vas less for the top qudethan for the bottom qude,and a similar trend was observed among the females only. The trabecular separation was greater for the top quamle than the bottom qude(which is consistent with reduced bone); this was also observed \,thinthe males ody, and a similar trend vas observed within the females @<0.1). The connectivi~parameters were lugely unchanged. The nurnber of node-free stnits was greater for the bottom qudethan the top quartiie for specimens from fernale donors, but not for those from male donors or for the qudes as a whole. No other differences beween the connectivicy of the top and bottom qudeby fluoride content mere observed. Parameter Top Quartile

Tbvolume (%)

Tbthickness (p)

Tb separation (pm)

Table 3.4.5 Image ana?vsis of coronal sections

Data presented as mean t SEM.

'The trabecular bone volume is grearer for the bottom quade than for the top quartiie. h similar trend @

'Tne trabecular separation was greater for the top quartile than for the bottom- quartiie. This was also observed for the males only, and a sirnilar nend was observed for the fernales (pCO.1). Parameter Top Qude

End points (mm-2)

Nodes (mm') MaLe5: Fenza/e~: Total-

Free-free struts (mm") rMa/ex: Fernales= TotaL-

Node-fige struts Maies: Fernales= Total-

Node-node struts (mm-3 ~Ma(x: Fernales: Tofak

Star volume Males= Female~: Totnk

Table 3.4.6 Stmt andysis of coronal sections

Data presented as mean t SEM.

'For specirnens from fernale donors, the number of node-Çree smts (in mm/rnm') is greater for the bortom qudethan the rop qude(pc0.05). 3.4.5 Histological sections

Data for whole qudes are given in Table 3.4-7. No differences were observed behveen the top and bottom quartile for any of the parameters describing the amount or the connectivity of bone. Within the mdes only, the star volume of the inferior section was greater for che top quade than che bottom quartiie (3.4 i 0.6 vs. 2.0 + 0.3 mmL, respectively; pc0.05). No differences were obsewed for the specirnens derived kom kmale donors. These results contrast with the results obtained for image anaiysis of s-rays of the coronal section (Section 3.4.4), in which it was observed that the uabecdar bone volume was greater and the uabecular separation \vas less for the bottom qudeby fluoride content compared to the top quade. This may be due to the fact that the s-rays are of the femoral head in its entires. and the histological sections are of selected regions, or it may be a consequence of the use of different types of sarnples (s-rays of thick sections in Lieu of thin sections) for ihe analysis. This dibe discussed further in Section 4.1.5. Top qu&e Bottom quartile

Trabecrrirrr vo/rrnle (Oh) Trabeczdar tbickness (,ni) Trabeczdar nzmiber (.lm-'J

Inferior:

Table 3.4.7 Image and srnt analysis of histoIogicai sections

Mean + SEM.

No differences were observed bem-een the whole quades. 3.4.6 Backscattered electron irnaging

The mean logits and weighted averages of the mineraiîzation profles for the top and bottorn quartiies, as weil as the quartiles segregated by gender, are shown in Table 3.4.8. At the superior surface, the top quade displaved a marked increasc in mineraiization compared to the bottom quartile, reflected in the significantly greater weighted zverages and logits of both the cortical and the canceiious bone at this site. No ciifferences with fluonde content were observed at the ;Ifenor surface. Esacdy the sarne pattern of significant differences was observed for the male donors. Within the fernale donors, the means of the weighted averages and logits were greater for the top qude than the bottom quartile as weU, although the differences were not significant. Sirnilarly, for both gender-segregated groups and the total quartiles, the degree of mineralizauon

Lias greater for the top quartile than the botrorn qude, dthough significant differences were not observed. The greater degree of mirieraiization for the top quartile at the superior surface is consistent with the hdings of a linear relationship between the mineraiïzation and the fluoride content (Section 3-25). Weighted average:

Top qude

Males: Females: Total:

Males: Females: Total:

canceiious

Table 3.4.8 Degree of mineralizatïon by backscattered electron imab&g

*The weighted averages and the logits for the rnineraiization profle (that is, the degree of mineralization) at the nvo superior sites were greater for the top quartde than the bottom qude.This was dso observed for the males but not for the fernales. Logit of mineralization distribution:

Top qude

Males: 0.74 t 0.33* Femaies: 1.38 + 0.31 Total: 2-06 t 0.23*

Maies: 1.01 t 0.21" Fernales: 1.20 t 0.38 Total: 1.11 f 0.21"

&Ides: 1.18 k 0.29 Femaies: 1.02 ,t 0.1 8 Total: 1.10 k 0.17

h1ales: 1.02 i 0.36 Fernales: 0.9 1 t 0.27 Total: 0.96 k 0.21

Table 3.4.8 Debgee of mheralization &y backscattered elecmn Unagulg (con Pa')

Mean t SEM.

The weighted averages and the logîts for the mineralization profXe (that is, the degree of mineralization) at the nvo superior sites were greater for the top quartile than the bottom quade. This was dso observed for the males but not for the femaies. 3.4.7 Microhardness

As bodi backscattered electron imaging and microhardness assess the degree of rnineralizaaon of the bone, it is unsurprising that the microhardness data also displayed profound differences benveen the top and bottom qudeby fluonde content. Data by quartile are shown in Table 3.4.9. The microhardness \vas greater for the top quartile than the bottom quartile at the superoprosimai site (both subchondral and canceiious bone), superodistd site and inferoprosimal site (subchondrai bone only) and the inferodistal site (cancellous bone only). Esacdy the same pattern of signihcant differences were observed for the group of femaies ody; however, no differences between quartiles were observed for the maies. Since this is in direct contrast to the resdts of backscartered electron imaging (in which case the dedonors dispIayed the same response as the data as a whole, but thefernnles did not display any significant differences), this is unlikely to be due to ses-specific differences in fluoride response, and is more likeIy to be result from the smail nurnber of samples remaining in each subgroup afier the middle nvo qudesare escluded and the top and bottom quarùles are each subdivided by gender. Top quade Bottom quade

Table 3.4.9 1MTcrohardness of borie

Mean ISEM.

*At these sites, the microhardness \vas greater for the top quade bp fluoride content than it mas for the bottom quartile. 3.4.8 Surnmary: top vs. bottom quartile of fluoride content

The 92 specimens were divided into quades based on the arnount of fluoride accumulation in their bone. The top and bottom quartile were then compared. The mean fluoride content of the top quartile \vas 1434 + 336 ppm, and that of the bottom quartile was 449 + 117 ppm - almost a threefold increase. The top-quade individuais were also older than their bottom-quartile counterparts (70 t il vs 62 f 14 pears). The top qudehad a higher concentration of chlo~eincorporated Lnto the cancellous bone. CY7hile no difference was observed between the density of canceilous cores from the hvo groups, the ultimate compressive stress and the yield stress w-ere greater for the bonom quartile than the top quartile. Despite the lack of difference in density, the trabecular bone volume \vas greater, and the uabecular separation \vas less, for coronal sections from bottom quanile than from the top quartîle. This was not confirmed by image analvsis of histological sections; no diEferences were observed benveen the nvo qudesin the arnount or connectivity of the cancellous bone at either the superior site or the inferior site. Finally, differences in the degree of mineralkation were profound. For the supeüor site, the subchondrai and the canceUous bone were more mineralized (as measured by both logit and weighted average of the mineralkation distribution) for the top qudethan the bottom quartiie. This was mirrored in the increased microhardness of the high-fluoride bone compared to the low-fluoride bone at five of the eight sites esamined (including three of the four superior sites). 3.5 Effect of gender on response to fiuoride

3.5.0 Introduction

The purpose of this component of this study is to determine if gender differences esist in the response of bone to fluoride. In parcicular, the majori- of the females enrolled in the study were more than 50 years oId and therefore couid be expected to be peri- or posc- menopausal. This may lead to a marked difference in response compared to their male counterparts. In Section 3.2, hear regressions were perfomed to assess the relauonship becween the measured parameters of bone quality and the age of donor, densiq- of canceilous bone, or fluonde content of canceilous bone. In ùiis section, the regressions are repeated for each gender individualiy. The regression can change in a nurnber of ways. A hear regression chat is significant for both genders together may also be significant for either gender individually. This suggest that the response is Likely to be independent of gender. Conversely, a linear regression that is significant for both genders together may not be significant for ei'ber gender individudy. Again, however, diis suggests that the reponse to the predictor variable is gender-independent; combining both genders provides enough data points for significance to emerge. Finally, relationships may be significant for either one of the two genders. In this case, the R' value and the p-value are cornpared to that of both genders together. Higher values for the single gender compared to the combined genders suggest that the relationship is gender-specific (that is, adding data from the other gender makes the regression worse, not better). Lower R' and p-values, however, suggest the opposite; thar both genders rnay display the same relationship co the predictor variable, even if ir is more pronounced in one gender chan the other. This approach is used throughout this section as a means of assessing gender-specific effects.

3.5.1 Patient profiles

Slightly more specimens were obtained from female donors than male donors (51 female, 41 male). The patient profiles differed in nvo respects. Füst, the female group was significantly older, on average (mean + SD: 70 k 11 vs 64 + 12; pcO.05 by t-test). Second, proponionately more women were diagnosed with osteoporosis (6 of SI) than men (3 of 41). These two factors, combined with gender differences, most LikeIy conmbute to the significant difference benveen the density of the canceiious cores fiom each of the groups; the rnean den si^ was 0.94 + 0.05 g/cm3for the males and 0.75 k 0.04 g/cm' for the females (p~0.005by t-test).

3.5.2 Age, density and fluoride content

The 'predictor' variables-age, densis; and fluoride content- were analysed to determine if any relationships esisted berneen them. For the data as a whole, the logarïthm of the fluoride content increased lineady with the age of the donor (see Section 3.2.1 and Figure 3.2.4). This relauonship w-as not observed for males (Figure 3-51}; however, it \vas observed for fernales. The femde-only group indudes an outlier by age; a very young individual (23 years old, appro-uimately 30 years younger than the nest-youngest individual). This individual also had the second-lowest fluoride content of aIi specimens frorn female donors. When the spedmen was included, the relaüonship was much stronger as the young sarnple 'pinned' the regression iine (R' = 0.166, p<0.005). When that individual \vas exduded, the R' value was similx for femdes only as for ail individuals and the relationship was much weaker, aibeit still signïficant (R2i'= 0.08 for fernales, R' = 0.09 for aii donors; p

O ther relationships were observed. Age of donor (years)

3.5.1 Fluon-de conten r is not rela ted ro age in men

The namal logarïthm of the fluonde content was not found to correlate @y Pearson test) with the age of the donor for the subset of specimens derived from male donors. Age of donor (years)

FLom-e 3.5.2 Fluon-de content is relared ro age in Nomen

The natural logaüthm of the fluoride content is linearly related to the age of the donor for the fernales-only group, although this relationship is weak (R' = 0.080, pC0.05). This graph and the graph on the previous page are on the sarne scale. 3.5.3 Chexnical composition of canceiious bone

No differences were observed behveen males and fernales for the mean vaiues of any of the rneasured elemental concentrations or ratios (iïustrated in Table 3.5.1). No relauonships were observed benveen any of the elemental concentrations and the fluoride content for either gender individudy. It was observed earlier that the chiorine concentration of the combined data mas found to increase lineady wïth the fluoride content (Figure 3.2.5); however, as this was not observed for either the males or the fernales idividually, this suggests that this response is both very weak (since ail the samples were required for the signïficance to emerge) and gender-independent. No relationship with age was observed for any of the elementai concentrations with the exception of the fluonde content, aiready considered in detail above (Section 3.5.2). Calcium content (?/O) Phosphorous content (Oh) Ca/P ratio Todmineral, CaPO, (O/o)

Chlorine content @pm) Magnesiurn content @pm) Sodium contem @pm)

Fiuoride content (ppm) F/Ca ratio (&mg) F/CaPO, ratio (pg/mg)

Table 3-51 Chemz'cai cornposi~70~of canceUous bone, bygender

Data presenred as mean t SEM.

No significant differences were observed between genders. 3.5.4 Mechanical properties of cancellous bone

Conlp~snon:As discussed eder (Section 3-51), the density of the cancellous cores from male donors was approsirnately 30°/o higher than those fiom fernale donors. This had a comrnensurate effect on the ultimate compressive stress and the energy to failure ofthe cores in compression, mhich were both significantly higher for the males than the females (the UCS was appro-shatelv 25% greater, but the energy to failure \vas more than 70% greater). No other differences were obsert-ed in the compressive mechanical propemes becween genders. Means by gender are shown in Table 3.5.2. A number of the mechanical properties were found to be iinearly related to the densis; age of donor, or fluoride content For the purposes of comparison during the foiiowing discussion, the R' values for both genders together (from Section 3.2.3) are provided for tliese relaaonships in Table 3.5.3, together with the R' values (for signiucant relationships) for the individual genders.

Five of the six measured mechanical properties (the strain at the ultimate compressive stress being the escepuon) were found to vary linearly with the density for the data as a whole (Section 3.2.3. and Figure 3.2.6). \men the data \vas separated by gender, neither the compressive rnodulus nor the energy to veld displayed a significant relationship with the density. The yield stress, ultimate compressive stress, and energy to fdure displayed a significant (or nez-significant; see Table 3.5.3 for p-values) linear relationship only for the fernale, or larger, group. In ail cases, the R' value was smailer than the corresponding value for the data as a whole, suggesting thac the additional data points irnprove the relationship and chat it is therefore not gender-specific.

Similarly, the weak relaaons hip O bserved benveen the ultirnate compressive stress, the srrain at this point, the yield stress or the compressive modulus and age for the whole dataset (see Section 3.2.3 and Figure 3.2.T) was not observed in the smder, gender-segregated groups. Again, this suggests that this relaaonship is not gender-specific. For the data as a whole, the ulhate compressive stress, yield stress and energy to yield decreased weakly with fluoride content (see Section 3.2.3; Figures 3.2.8 and 3.2.9, and Table 3.2.3). No relationships were observed between these parameters and the fluonde content for the gender-segregated data. This suggests that the observed relationships behveen the mechanical properües and the fluoride content are independent of gender. It shouid be reiterated that the mo high-fluoride points (fluoride content >ZOO0 ppm) were escluded £iom this analysis (see Section 52.3 for details). Findy, the nature of the relationship beaveen the mechanical properties and the fluonde content was esplored by detennining whether a re1ationship esisted betxveen the

mechanical properties and the logarithm of the fluoride content. For the data as a whole, 3. linear relationship with the logarithm of the fluonde content esisted for the ultknate compressive stress and the field stress. The y-ield stress was significandy correlated for the males but not the fernales, although the significance \vas higher for both genders rogether. The relationship benveen the ultïmate compressive stress and the fluoride content was no longer significant when the témales were escluded. These results again suggest that the changes observed in the mechanical properties are independent of gender.

Torsion: No differences between mean values of the measured parameters were observed for the maie-only and female-only groups. Of the nventy-seven samples, only sis were kom female donors, most Likely as a resdt of the smder size of the femoral head; the torsion test specimens were limited to those that couid be at least 12 mm long, so the fernord heâd needed to be more than nvice this in diarneter (as a 5 mm thick coronai slice was removed from the centre). Regressions were therefore only performed for the males. For the whole dataset, the shear modulus decreased with increasing age of the donor. This was also observed for the males only; in fact, the R' value was somewhat higher CR' = 0.234 for males only vs. R' = 0.177 for ail individuals). The relationship benveen shear moduius and age for males is illustrated in Figure 3.5.4. Strain at ultimate compressive stress (O/O) 7.6 t 0.3

Compressive modulus @Pa) 266 + 22

Yield stress @Pa) 7.7 + 0-G L%irnate compressive su-ess (&Pa) 8.G I0.7*

Energy absorbed to yield (MJ/rn) 0.15 -t 0.01 Energy absorbed to fracture @IJ/rn) 0.35 k 0.06*

Shear straui to failure Shear modulus @Pa) Ultimate shear stress (&Pa)

Table 3.5.2 Mecham'cal properties of cancdous bone, bygender

Data presented as mean + SEM.

*The ultimate compressive stress and energy absorbed to Mure were significantly greater for the cancelious bone from male donors than from fernale donors. Relationship with density: values and s&nZcance

Parameter Total Mdes Females

Relationship with age: R' values and si&&ïcance

Parameter Totd Males Femafes

Relationship with fluoride content: R7values and s~~cance

Pararneter Total Males Fernales

Table 3.5-3 MechaniCalproperties andpredictor variables

*The significance of this relationship is dependent on the esclusion of a single casewise outlier (a residual of >3 SD from the Line).

Significance is set at p<0.05 for the iinear regressions. 5.5 6.0 6.5 7.0

Logarithm of fiuoride content (ppm)

Figure 3.5.3 Yield stress vs log of t&e tlumi.de content=maies oniy

The veld mess was correlated with the logarithm of the fluoride content for the specimens derived from male donors (R' = 0.072 and pc0.05). 50 60

Age (years)

Figue 3.5.4 Shear mudulus decreases mFVIthage: males udy

The shear modulus of cancellous cores deciined with advancing age in specimens from male donors (R' = 0.234, pC0.05) 3.5.5 Image analysis of coronal sections

With the exception of che trabecuiar ùJchess and the number of endpoints, the distribution of values for the measured image analysis parmeters of coronal sections did not pass the Kolmogorov-Srnirnov norrnality test. Accordingly, a non-paramecric test (Mm- Whitney) was used to assess differences between genders. Means by gender are @en in Table 3.5.4. The cornparison of the mean values for the image and stmt analysis parameters for the males and females displays significanr differences. The trabecuiar volume was the sme for both genders. The trabecular thichess \vas greater for the females. However, the trabecular number and separation (derived from the trabecular volume and the trabecular thichess) were greater and less, respectively, for the males - consistent with more bone in the males. AU of the strut analysis parameters were significantiy or rended to be @<0.1) greater for the male donors than the females - the parameters indicaàng more connectedness (number of nodes and nurnber of node-node smits) and the parameters indicating more disco~ectedness(number of free ends, number of free-free struts, and star volume) alike. Given che increase in both the nurnber of nodes and the number of free ends, it is unsurprising that the number of node-free smts also was greater for the male specimens. These resulcs are largely contradictory and may be due to the large number of heterogeneous, osteoardinüs-affected hmorai heads. This issue d be discussed in greater detail in Section 4.1 .S. Within the data as a whole, the trabecular bone volume and trabecular separation decreased and increased, respectively, Mth increasing fluoride content - that is, the amount of bone present decreased (Section 3.2.4)).When analysed by gender, neither group demonstrated this relationship for the trabecular volume, suggesting that it may be a gender- independent relationship. The trabecular separation increased with increasing Buonde content for the males alone (Figure 3-55]>).In addition, the trabecuiar number decreased wïth increasing fluoride content for the males only (Figure 3.54. No significant relationships with the fluoride content were observed for the males for any orher parameter, and no relationshïp with the fluoride content was observed for any parameter for the fernale-only

€FOUP* Given the differences observed behveen genders, it is difhcult to know how to iriterpret these resdts or what weight shodd be placed on the relauonships with the fluoride content that were observed. hkhodologicd issues rela~gto this method of image anaiysis wïii be discussed in Section 4.1.5. Trabecular bone volume (Yo) Trabecular thickness (pm) Trabecdax number (mm") Trabecular separauon (pm)

Nurnber of endpoints (mm-3 Number of nodes (mm-? Free-free sauts (mm/=") Node-free struts (mm/mm-) Node-node struts (mrn/mm') Srar volume (mm')

Table 3.5.4 Image analysis of coronai seca'ons, &y gender

Data presented as mean 2 SEM.

*Indicates that the value of this pararneter is greater for temales than for males.

**Inclkates chat this pararneter is greater for males than for fernales.

See test for Merinformation. Fluoride content (ppm)

F&gure 3.5.5 Trabeculiusepara Lion hcreases m-th F content for males

The trabecdar separauon, rneasured for coronal sections, increased with increasing fluoride content for specimens for males (R' = 0.1 86, p<0.01) but not for fernales or for al specimen. Fluoride content (ppm)

Fkom-e 3.5.6 Trabecularnumber decreases m.& F content for males

The uabecular number, measured for coronai sections, declined with increasing fluoride content for specïrnens for maies (R2 = 0.178, p<0.01) but nor for females or for ail specirnen. 3.5.6 Image and strut analysis of histological sections

As not all image and stmt analysis paramerers passed a Kolmogorov-Smimov test of normality, unpaired t-tests or Mann-Whitney (non-parametrïc) tests were used, as appropriate, for each compaxïson. Ar the superior sdace, the trabecular bone volume and thïckness was greater for the males han the females. Comrnensurate wïth this, the trabecular separation was reduced and the number of nodes and node-node stnits (measures of comectivicy) were greater. At the inferïor surface, the trabecular number was greater and the trabecular separation was less for the males than the females. The nurnber of node-node struts was aiso greater for the mdes, as was the nurnber of node-free struts. For the individual genders, none of the measured parameters of image or strut analysis were found ro correlate significandy with the age, fluoride content, or densiq At the inferïor section, the trabecdar separation and die trabecular nurnber correlated linearly with the age of the donor when bo th genders were induded (Figure 3-2-14and Figure 3-2-15).Ln addition, the trabecular volume, trabecular number, and trabecular separation of the inferior sections also correlated significantly with the densitv of the cancellous core. The Iack of pardel relationships in either gender lndividudy suggests that they are weak, gender- independent relaaonships. The consistent behaviour of the parameters for the infenor histologicai sections and their relationship to gender, age and density cm be contrasted with the self-contradictory resdts obsenred for the coronal sections (Section 3.5.4). Superior sections:

Mdes

Trabecular bone volume (Yo) Trabecular thickness (pn) Trabecular number (mm-) Trabecular separation (pm)

Number of endpoints (mm-? Nurnber of nodes (mm-? Free-fiee sauts (mm/-? Node-free saits (rnm/rnm-) Node-node sauts (rnm/mm? Star volume (mm?

Iderior sections:

Mdes

Trabecular bone volume ('/O) Trabecular thickness (pm) Trabecular number (mm-? Trabec~darseparatior, (pm)

Number of endpoints (mm") Number of nodes (mm-? Free-fiee stnits (mm/mm4 Node-free smts (rnrn/rnrn? Node-node smts (mm/mm~ Star volume (mm')

Table 3.5.5 Image and strur andysis of fisroIobgCal sections, bygender

Data presented as mean + SEM.

YThesc nvo parameters are significandy different by unpaired t-test or Mann- Whitney, as appropriate. In al1 cases, the differences are consistent with a grearer amount and comectivity of bone for the males than the fernales. 3.5.7 Backscattered electron imaging

No differences were observed between genders for the weighted average or the logit of the rnineralization distribution at any of the four regions rneasured (subchondral and cancelious bone at a supenor site and at an inferior site). Data are shown in Table 3.5.6. The wveighted averages of the data as a whole increased linearly wich the fluoride conrent at the superior subchondrai and cancelious sites (Figures 3.2.21 and 3.2.22). At the subchondrai site, the wveighted averages for the males and females individually correlated Mth the fluoride content (Figures 3.5.7 and 3.58) however, the weighted averages at die cancellous site only correlated significantly with the fluonde content for fernales (Figure 3.5.9). This suggesrs thar the mineralization of bone is more dosely assodated widi the fluonde content wïth women than for men. Findy, the lo@t of the rnineralizacion distribution at the superior subchondd site correlated linearly with the Iogarithm of the fluoride for the data as a whole (Figure 3.2.23) but not for either gender individually. No relationships were observed benveen the descriptors of the mineralization distributions at the superior surface and the age or the densit). of the samples, and no relationships at d were observed benveen the measured and the predictor parameters at the inferior surface. Weighted average of muieralization distribution:

Males

Logit of mineralization distribution:

Mdes Females

Table 3.5.6 Backsca ttered electron hagzhg, by pder

Data presented as mean + SEM.

No differences mere observed benveer, the males 2nd females. Fluoride content (ppm)

The weighted average of the mineralkation distribution of the subchondral bone at the superior site increased with increasing age for fluoride content in the bone for males (R'=0.13 1, p

FI~LLT~3.5.8 iMUlernLization of supen'or subchondral bone: fernales only

The weighted average of the rnineralization distribution of the subchondrai bone at the supenor site increased with increasing age for fluoride content in the bone for fernales (R2=0.104, pC0.05). This grîph is to the same scaie as the previous gaph. Fluoride content (ppm)

Figue 3.5.9 ~eraltzationof superior canceflous banc fernales on&

The weighted average of the mineralkation distribution of the cancelious bone at the superior site increased ~6thincreasing Buonde content in the bone for fernales, but not for males (R2=0.122, p<0.05). 3.5.8 Microhardness testing

While there were no gender-related differences observed benveen the meam of the rnicrohardness data at any site, profound gender-related differences in the response of the microhardness of the bone to the fluoride content. Wken the data as a whofe is esamined, the rnicrohardness of the subchondcrl and canceiious bone at the superopro-&al site and the subchondral bone on* at the superodistal site increased linearly with the fluoride content (see Section 3.2.6).W heeof ùiese displayed a much more pronounced dependence for the fernale-ody group (Figures 3.5.10-12) and no sipnificant relationship with the males alone. In addition, the females also demonstrated a relationship benveen the microhardness and the nacura1 logarithm of the fluoride content at the superodistal canceiious site (Figure 3.5.1 3), although there was also a trend towards a linear relationship wiùi the fluoride content (R'=0.079, p=O.lO4). Mdes

43.3 4 0.7 42.0 + 0.7

44.4 2 1.3 40.9 + 0.7

41.3 f 2.0 41.9 I1.1

44.7 I1: 1.5 39.9 f 0.8

No differences were observed bem-een genders for the microhardness at any of the sites. F content (ppm)

Figure 3.5.20 Microharchess of subchondraf bonc fernales ody

The microhardness of the subchondrd bone ar the superoprosimal site demonstrated a strong dependence on the fluoride content, but only for specimens from fernale donors (R2=0.543, pc0.0005) 1 O00 2000

Fluoride content (ppm)

The microhardness of the canceilous bone at the superoproximal site demonstrated a suong dependence on the fluonde content, but only for specimens fiom fernale donors (R2=0.184, pC0.05) F content (ppm)

The rnicrohardness of the subchondral bone at the superodistal site dso demonstrated a strong dependence on the fluoride content, again only for specimens from fernale donors @'=0.562, p<0.0001) Logarithm of fluoride content (ppm)

Figure 3.5.33 Mïcrohardness ofcancehus bone: fernales only

The microhardness of the canceiious bone at the superodistal site demonstrated a dependence on the natural logarithm of fluoride content, again only for spechens from Çemaie donors (I1'=0.138, p<0.05) 3.5.9 Summary: effect of gender on response to fluoride

The natural Iogarithm of the fluoride content was found to increase lineady with

age for Çemales, but not for males. The femaies were somewhat older and a higher proportion were osteoporotic; this likely conmbutes to their lower density when compared to the males. No substantive differences were observed behveen the chernical compositions of the nvo genders. The ultirnate compressive stress and energy to Mure of the specimens korn maie donors were greater than those from female donors; this likely reflects the greater density. \We the ultimate compressive stress, yield stress, and energy to yield declined

with increasing fluoride content for both genders together, no relations hip kvas O bsemed in either individually, suggesting that the changes are independent of gender. The trabecular number and separation, measured on the s-rays of coronal sections, were found to correlate linearly with the fluoride content for the males but not the fernales. However, the gender-related differences observed for the image and smt anaiysis parameters of the coronal sections contradicted those observed for histological sections. The latter were consistent, \.ththicker struts and more comected trabecuiae obsenred for the males than the females. This suggests that the canonicd technique of using histological sections for image analysis is more reliable than using s-rays of coronal sections. The use of estremelv heterogeneous, osteoarrhritis-affected femoral heads may have been a contributor to dis. No relationship benveen the image and struc analysis parameters and fluoride content, age or density was observed for either gender individuaily. The weighted average of the rnineralizauon distribution at the superior site, rneasured b y backscatrered electron imaging, varied linearly with the fluoride content in both males and fernales for the subchondrai bone, but only in females for the canceilous bone. The microhardness values at three of the four superior sites were found to vary closely with the incorporated fluoride content for females but not for males, suggesting that response of the degree of mineraikation to fluoride incorporation may be influenced by gender. 3.6 Effect of disease on response to fluoride

3.6.0 Introduction

As the speümens for dùs study were obtained fiom individuals undergohg tod hip replacement surgery, none of the femoral heads cm be described as normal. A large majocity of the donors (75 of 92) were diagnosed with osteoarthntis and a handful(9) vith osteoporosis. The purpose of this cornponent of the analysis is, £irst, to deterrnine if there are differences in any of the measured parameters between the osreoarthritic and the osteoporotic group. Second, dùs analysis assesses whether or not fluoride incorporation into osteoarrhotic femoral heads elicits a response that is differenr from that of the femoral heads generdy (that is, irrespective of disease state). A total of 18 of the osteoarthntic femoral heads were graded to assess the degree of osteoarrheas. Out of a possible score of 14 (the most severe osteoarrhntis; see Section 2.9), the mean score of these individuals was 13.3 + 0.4. Only three of the individuals had a score less than the masimurn (MO femoral heads scored 11 and one scored 9). This suggests that, at the urne of surgery, osteoarduitis is already hiriy advanced. However, because gradlig of not di the femoral heads \vas completed (see Section 4-51), no anempt Las made to segregate femoral heads by degree of severity of osteoarthritis.

3.6.1 Patient profites and the predictor variables

As stated above, 75 of the 92 samples obtained were from osteoarrhntic patients. Nine of the femoral heads were from individuals diagnosed as osteoporotic. The rernaining eight patients had a variety of diagnoses (see Section 3.1.1 for full details) with no group esceeding three pauents. The osteoporotic patients were folrnd to be older than the osteoarthrïtic patients (7G + 3 years vs. 68 2 1 years). Rrhile the mean density was slightly less for the osteoporoüc group chan the osteoarrhnuc goup (0.75 f 0.1 1 g/cm3 vs. 0.83 f 0.04 g/cm3), as would be expected, the densis. of the two groups were not statistically disünguishable. Widlln the osteoarrhntis group, 35 of the donors were male and the rernaining 40 were female. Sis fernale donors and 3 male donors comprised the osteoporotic

PUP- LVithin the osteoarrhntic samples, the natural logarithm of the fluoride content varied linearly with age. This was also observed for the data as a whole (Section 3.2.1 and Figure 3.2.4). The R' value \vas slightly less for the osteouchntis-only samples (RZ=0.075) than for d data (R' = 0.090), sugges~gthat this relationship is independent of the disease state of the donor, Again, when the data \vas segregated by gender, the osteoarthritic females displayed a significant linear relauonship benveen the logarithm of the fluoride content and age (R2=0.081, pCO.05) düie the osteoarrhntic males did not The relationship for the osteoarthntic females was approsirnately the same strength as that observed for al1 females (R2=0.080; see Section 3.5.2 and Figure 3-52).

3.6.2 Chernical composition of cancelious bone

Non-parametric (Mann-Whitney) tests were used to determine if differences esisted between the osteoarthritic and osteoporotic groups as the data failed normality tests. The chlorine content was significandy higher for the osteoarthntic group than the osteoporoac group, but no other differences betxveen these nvo groups were observed. As for the data as a whole, the chlo~econcentration was hearly correlated wïth the fluoride content for the osteoarrhritic group (R2=0.084, p<0.01), although thïs relationship did noc achieve significance for either gender individualiy. The relationship \vas doser for the osteoarthrïtis-only samples than ail samples (R.'=0.061 for ali samples; Figure 3.2.5), suggesung that it may be related to the disease state. Within the osteoarthritic males (not the females or the combined groups) the calcium-to-phosphate ratio deched Il-iearly with increasing fluoride content (Figure 3.62). W'hile this relationship was ais0 observed for males in d disease States, the relationship was closer for the OA males only (R'=0.117 for aii males, R'=o. 16~ for osceoardinac males). Osteoporotic

Caiüum content (O/O) Phosphorous content (Oh) Calüurn/phosphorous rauo Total minerai, CaPO, (O/o)

Chio~econtent @pm) Magnesiun content @pm) Sodium contenr: @pm)

Fluoüde content @pm) F/Ca rauo (j.g/mg) F/total mineral (pg/mg)

Table 3.61 Chemi'cai compositio~of osteoporotic and osteoarthn'tr'c bone

Data given as mean + SE,M

*The chlorine content was higher for the osteoarrhnuc bone than the osteoporotic bone. No other differences were obsenred. O 500 1 O00 1500 2000 2500 3000

Fluoride content (ppm)

Figure 3.6.1 Chlorine concentration uïcreases r~l*thfluoride content

The cbilorine concenuation in the cancellous bone was posiuvely correlated with the fluoride content in the osteoarrhetic samples (R' = 0.084, pC0.05) Fluoride content (ppm)

F~&xre3-62 Calcium-to-phosphatera no decreases wit6 F content

The calcium-to-p hosp hate ratio decreased wi th increasing fluoride con tent in canceIious bone from osteoarrhriac males, but not frmm fernales (R2= 0.166, ~4.05) 3.6.3 Mechanical testing of cancellous bone

Cornp-emon: The means of the mechanical propem-es for the osteoarthrïtic and the osteoporotic samples are provided in Table 3-62The compressive mechanical propemes of the nine osreoporotic samples were statisticdy indistinguishable fiom the osteoarrhntic samples (although aii of the parameters, Save for the yield stress, were lower for the osteoporoüc samples). No correlations were obsemed between the mechanical propemes of the osteoporotic samples and the age, densim or fluoride content of the bones, although this may simply be due to the smaü nurnber of samples. The osteoarthritis-speüfic response of the mechanical propeaies were somewhat dichotomous; the response to the predictor variables of the parameters related to energy absorption (the energy absorbed to yield and to failure) and the paramerers related to the strength and stiffness of the bone Oield stress, ulhmate compressive stress, and compressive modulus) diverged when the non-osteoarrhntic samples were esduded. To begin, the relationships benveen density and the yield stress, ultimate compressive saess (illustrated in Figure 3.6.3) or compressive modulus becarne more pronounced (R' = 0.238, 0.252 and 0.147, respecuvely), indicating that the variability was markedly reduced. Each of these relationships \vas much stronger for the data as a whole than for either gender individudy, suggescing that ïr is independenc ofgender. Both the energy to yield and to fadure, however, while still related to the density @<0.05 for rhe slope), no longer displayed a significant value for the intercep t. The relaüonship between the strain at UCS of the cancellous core and the age of the donor, significant for the data as a whole, is no longer so when the OA-ody samples are esamined. The decline in compressive modulus Mth age is strengthened (R' = 0.073), however, and is even more pronounced for the hmales only (see Figure 3.6.4). The decline in the ultimate compressive stress with age is largely unchanged (R2= 0.041 for OA only samples vs R' = 0.044 for ail samples) and indiscemible in the male and fernale specimens individually. A similar weak but gender-independent relationship is observed for the yield stress (R' = 0.053), although this relationship only emerged when the non-osteoarthritic sarnples are escluded. For the sarnples as a whole, a hear relationship was observed benveen the ultimate compressive stress, the Feld stress, or the energy to yield and the Ouoride content. When the non-osteoarcluitic femoral heads were escluded, the relationships betsveen the former nvo parameters and the fluoride content became weaker. For the ultimate compressive stress, the R' = 0.041, compared to 0.048 for ail sarnples. For the yield stress, the R2-value drops from 0.048 for ail samples to 0.039 for just the osteoarthnc sampIes; this is no longer significant @=0.054). In contrast, however, the relationship benveen the energy to yield and the fluoride content was strengthened for the osteoarrh8tic-only samples, its R2-value increasing to 0.073 from an R' of 0.040 that was observed for aii samples (iusuated in Figure 3.6.5). None of these relations hips were significant for either gender individuah, indicatkg that the response was independent of gender. These rcsults suggest that osteoarrhntis may affect the strength and the toughness of bone in different ways. The ultimate compressive stress and yield stress are a measure of the suength of the bone. The energy to yield is related to both the yield stress and the compressive modulus. As the area under the stress-strain cuve to the yield point, this triangle (approsimately) is delineated by the yield stress at the apes and the compressive modulus as the hypotenuse. This suggests that there may be a subtle relationship benveen the compressive moduius and fluoride content as well, as that would account for the stronger relationship that esists benveen the energy to yield and the fluoride content compared to that which exïsts between the yield stress and the fluoride content.

Torsion: Only 27 specirnens were tested in torsion (due to the Limitation of the size of the femoral heads). Of these, 24 were osteoarrhriti~and 2 were osteoporotic. Accordingly, comparisons benveen the disease groups were not performed. When all of the samples were analysed, the shear moduius was observed to decrease wïth increasing age of donor (see Section 3.2.3 and Figure 3.2.10). Wihthis Limited subser of the osteoarthrîtic samples, however, none of the mechanical properties in shear were observed to correlate with the age, density or fluoride content of samples. Compressive modulus (MPa) 246 t 16 232 k 61

Suain at ulhate compressive stress (?h) 7.3 k 0.2 6.5 t- 0.7

Yield stress (&Pa) C'Ltirnate compressive smess (&Pa)

Energy absorbed to proportional limit (MJ/rn? 0.13 $: 0.02 0.13 L 0.02 Energy absorbed to filure @q/rnJ 0.27 t 0.04 0.19 + 0.03

Table 3.62 Mech;uu'Calprope&s of osreoporotic and osteoadin-a'cbone No differences were observed benveen the compressive mechanical properties of osteoarthritic and osteoporouc bone, although chis may simply be because of the low nurnber (9) of osteoporotic specimens. Density (g/cc)

Figure 3.63 Ulrunate compressive stress of osteoart%uï'a'c sa.n@es

The ultimate compressive stress of canceLious cores was significantly correlated with their physical densiv. This was a gender-independent response, and it \vas more pronounced for the osteoarrhnac group alone (R' = 0.252, pc0.05). Age of donor (years)

Figrne 3.64 Compressive modulus fds mFVIthage in OA females

The compressive modulus of compressive cores decreased with increasing age of the donor. This effect was rnost pronounced in the subgroup of femaie donors diagnosed with osteoarrhritis (R' = 0.252, p<0.05). 500 1000 1500 2000

Fluoride content (DD~]

Figure 3-63 Energy to péid and F content: OA ody

The relaaonship bets-veen the energy to yield and the fluoride content was seonger for the osteoarduitis-only specimens than for aii specimens (~'=0.073, pC0.05). 3.6.4 Coronal sections

No difference was obsert-ed in the amount or comectivity of trabecular bone in the coronal sections between the osteoartliritk and the osteoporotic samples (Table 3.6.3). For the data as a whole (ali disease states), no relationship was observed between the measured parameters and the age of the donor. However, within the osteoarthritis-oniy group, the trabecular nurnber decreased and the trabecular separaüon increased \vi& increasing age (Figures 3.6.6 and 3.6.7). In addition, the trabecdar thickness increases with increaslig age (iustrated in Figure 3.6.8; R' = 0.067). None of these three relationships were observed when the osteoarthritic samples were segregated by gender. Again, as for the data as a whole, the trabecular volume of the osteoarthritic samples decreased (R' = 0.056, p

proportion of trabecular bone (O/o) trabecular thickness (pm) nabecdar nutnber (mm'?) trabecular separation (pm) free ends (mm-? nodes (mm-? free-fkee sauts (rnm/mrn-3 node-free srnits (mm/rnm~ node-node strucs (rnrn/mm*l) two-dimensionai star volume (mm?)

Table 3-63 Image and strut analpsis of coronai sections

Data presented as mean + SEM.

No differences were observed be~veenthe osteoarthritic (n=57) and the osteoporotic group (n=4). Age of donor (years)

F12u.r-e3.6 6 Trabccular number decreases Nith age for OA patients

The trabecular nurnber, measured for the coronal sections, decreased significandy with advanüng age of patient for the subset of osteo~thnticpatients (R' = 0.099, pc0.05). This relationship was not obsemed for either gender individuaüy. Age of donor (years)

F1;,@ure3.6 7 Trabecular separation Uicreases Nirh age for OA pauents

The trabecdar separation, rneasured for the coronai sections, increased significantly wïth advancing age of patient for the subset of osteoarthrïtic patients (R' = 0.054, p<0.05). This relationship was not observed for either gender individuaiiy. Age of donor (years)

Figure 3.68 Trabecular thichess hcreases N3Lh age for OA patiknts

The uabecular thickness, measured for the coronal sections, increased si,onificandy widi advancing age of patient for the subset of osteoarrhritic patients (R' = 0.252, p-4.05). This relauonship appeared to be independent of gender. Fluoride content (ppm)

Figure 3.69 Trabecular number for osteodAicmales

The trabecular nurnber of the coronal sections decreased with increasing fluonde content for the subgroup of hmoral heads Gom osteoarduiuc male donors (R' = 0.123, p~0.05).This is consistent with observed increase in trabecular separauon. 3.6.5 Histologicai sections

Image and strut analyses of histologicd sections from the supenor and from the Liferior surface of the fernord heads were performed to determine the amount and the connectivity of the cancellous bone. As the cardage and the underlying bone at the supenor (or weightbearing) side are generdy more affecred by osreoarthrÏas, the purpose of using nvo samples was to be able to differentiate benveen changes to the cancellous bone structure that were a result of osteoarthritis and those that may have other causes. The amount and comectivi~of cancelious bone was significantiy greater for the osteouduitic sarnples than the os~eoporocicsamples, but only at the superior site; no differences were observed at the inferior site (Table 3.6.4). Cornparison of the parameters for the superior and the inhrior site, nithLi a given femord head @y paired t-test), were then performed cable 3.6.5). lWemuch more canceilous bone \vas present at the supenor surface compared to the infenor surface for the osteoarrhetic specimens, no differences were observed for the non-osteoarrhritic samples. These nvo sets of hdings convincingly indicate that cancellous bone formation at the superior surface is in fact stimulated by os teoarthritis. \Vithin the osteoarrhgtic samples, the trabecular nurnber of the inferior section was negatively conelared with age (R2 = O. 116, p<0.05), and this relationship \vas rather closer than that observed for ah the sarnples (R'=o.oGo;see Section 3.2.4). However, for all sarnples, the inferior trabecuiar separation \vas found to increase hearly wirh the age OF the donor (Figure 3.2.14) but this was not observed for the osteoarrhntis-only samples. Fin+, at the inferior site, the number of nodes and the nurnber of node-fiee struts decreased with increasing age of the donor @=0.098 and 0.164, respectively), indicating that the comectivity of the bone decreases with age. The relationship between the nurnber of nodes and the age was more pronounced for the osteoarrhntic females (R'=0.154; Figure 3.6.10) and not significant for the males. No relationship to the age of the donor was observed at the supenor site. The absence of normal age-related decreases in the arnount of bone at the superior surface again suggests pathologïcal bone formation at this sire as a resuit of osteoarthrius. There were marked gender differences in response to density within the osteoarrhntic samples. For males, the superior trabecular number, trabecular separation (Figure 3.6.1 l), and number of nodes were Lneady correlated wich the densiq (R'=0.098, 0.1 46, and 0.120, respectively) in directions consistent with increased bone with increased density. For females, it Kvas the infenbr trabecular number and separation that correlated wïth the densis. (R'=0.127, and 0.105; the former is Uustrated in Figure 3.6.12). No relationship was observed between any of the measured parameters of image or smrt analysis, for either the superior or inferior site, and the fluoride content of the specimen. This is consistent with the findings for the data as a whole. When the osteoarrhritis-only samples mere anaiysed, severai other gender differences emerged. The mean superior trabecular voIurne was greater for males tha for females (pc0.05 by t-test), and the trabecular separauon was greater for females at both the infenor and the superior site (pc0.05). These differefices were not observed when non-osteoartbritic samples mere escluded (see Section 3.5.5). Means bv gender are shown in Table 3.6.6. In sumrnary, mhile normal declines in the amount of bone and its connecnvity were observed at the inferior section, even for the osteoarthntic bone, the amount and comectivity of the canceiious bone at the superior section is govemed by the response of the bone to osteoarthritis. Supenot propomon of trabecular bone (O%) 38 f 2* 19 +3* trabecular thickness (pm) 225 2 8* 138 -t 13* nabecuiar numbe r (mm-') 1.69 I0.03" 1.37 f 0.14' trabecular separation (pn) 385 t 18* 627 k GS*

free ends (mm-? 1.76 + 0.09 2.23 + 0.39 nodes (mm?) 2-1 & 0-l* 1.0 t 0.3* fkee-free smts (mrn/mrn") 0.1 1 k 0-Ol* 0.26 k O-OG* node-free strucs (mm/&') 0.54 I0.02 0.54 _+ 0.08 node-node smts (mrn/rnm3 1.1 6 f 0.06* 0.51 k O.Il* nvo-dimensional star volume (mm-) 2.4 f O.2* 4.1 2 0.P

Lnfenôt proportion of trabecular bone (Yo) 17kl 24 + 6 trabecuiar thickness (pm) 133 k 5 154 + 23 uabecuiar number (mm3 1.25 + 0.04 1.43 + 0.14 trabecular separarion (pm) 724 t 32 582 _+ 89

free ends (mm-) 2.7 + 0.2 2.0 t 0.1 nodes (mm-?) 0.99 + 0.08 1.28 + 0.5 free-Eree sauts (rnrn/rnm-4 0.29 + 0.01 0.25 t 0.05 node-kee struts (rnm/mrn") 0.50 4 0.02 0.56 + 0.05 node-node struts (mm/mm-') 0.39 I0.04 0.63 f 0.25 two-dimensional star volume (mm') 7.1 I0.8 4.1 t 0.9

Table 3.64 hage and svvr andysis of hisroIobol'cd sechs

Data presented as mean t SEM.

*These parameters are significantly different benveen osteoartht-itic and osteoporotic. The direction of the ciifference is, in al1 cases, consistent: with the presence of more bone in the osteoardintic samples &an die osteoporotic samples. proportion of uabecuiar bone (O/o) trabecdar thickness (pn) trabeculv nurnber (mm'-) trabecular separation (pm)

Eree ends (mm-') nodes (-7 Gee-Lee smts (mm/mrn--) node-free smts (mm/rnrn--) node-node smts (mm/mm-3 n.-O-dimensionaistar volume (mm?

Table 3.63 D~~ZCRC~Sberneen supen'or and idenor sections

This table iists the significant clifferences between image and strut analysis parameters as measured for the superior and inferior surface of the femorai head.

OA-osteodritic femoral heads (1142) Non-OA: femoral heads from patients wïth all diseases other than OA (n=12) OP femorai heads Çrom patients diagnosed wïth osreoporosis or fraccure (n=7)

S,I: This parameter was signi6candy greater for the superior surface than the in ferior surface. S This parameter was significantly greater for the inferior surface than the superior surface. NS: No difkrences were observed in this parameter benveen the nvo sites.

The pactern is cIearly consistent with a greater amount of bone presenr at the superior surface than at the inferior surface for osteoardintic femorai heads, but not for non-osteoarrhntic femoral heads. Age of donor (years)

figure 3.610 Niunber ofnodes for fernale OA parJents

At the inferïor site, the nurnber of nodes (an indicator of connectively) decreased with Licreasing age in the samples obtained hmosteoarthritic fexnaies (R' = 0.1 54, p<0.05). Density of cancellous core (g/cc)

Figure 3.6.22 Supenor trabecd' separah ofmale BApatients

The trabecular separation of the superior section was most closely correlated to the densiq for the subgroup of male, osteoardiritic patients (R' = 0.146, pc0.05). Density of cancellous core (gkc)

Figure 3.612 Infenor trabecular number offemale OA paziénts

The trabecuiar number of the inferior secaon was most ciosely correlated to the densiq- for the subgroup of fernale, osteoarrhritic patients (R' = 0.127, p<0.05). Osteoarthritic samples only

Mde

Supenm proportion of trabecular bone (?/O) trabecuiar thickness (pm) trabecuiar nurnber (mm'? wabecuiar separauon (w)

kee ends (mm-? nodes (mm-? free-fiee sauts (mm/rnm-4 node-l.ee struts (mm/rnrnA?) node-node suuts (mrn/rnrn-3 nvo-dimensional star volme (mm2)

Infenot: proporrion of trabecular bone (O/o) trabecular thickness (pm) trabecular number (mm-? trabecular separauon (pm)

free ends (mm'-) nodes (mm-? free-free smts (mm/rnm-4 node-fiee sauts (rnm/rnm'? node-node smts (mrn/mrnA-) nvo-dimensional srar volume (mm')

Table 3-66 image and strut anaiysis of histo~og3'ca.Zsections, by,aender

Data presented as mean f SEM.

%ese parameters were significantly different benveen osteo&tic females and osteoarthritic males. 3.6.6 Backscattered electron irnaging

The mean value of the weighted average and the Iogït of the mineralization distribution were significantly different betlveen the 63 osteoarthritic sarnples and the 7 osteoporotic ones at ody one site: the cancellous bone at the superior site. The mean value of the weighted averxge and the Iogit at this site were 3.63 t 0.07 and 0.35 k 0.13 for the osteoarthnuc bone versus 4.08 t 0.12 and 1.36 t 0.24 for the osteoporotic bone (mean 4 SEM), ïndicating that the osteo~ticbone at this site is less mïneralized than the osteoporotic bone. For the backscattered electron irnaging samples, as for the image analysis of histological sections (Section 3-65), there is concern that the superior, or weightbeiiting, surface of the fernoral head may be displaying pathological changes as a result of osteoarthritis. Accordingly, paired t-tests were perforrned to compare the weighted averages and logits of the degee of mineralization at the superior and inferior site for each femoral head. The data were divided into three groups: osteoarthritic ody, non-osteoarthrïtic (including osteoporotic), and osteoporotic only. \Vidin the osteoarthritic group, the degree of mineralization was significantlv less at the superior surface than its inferior counterpart for both the subchondral and the canceiious bone. For the non-osteoarrhnuc group, there was a trend (pc0.1) towards inmead mineralization for superior canceiious bone compared to inferior cancellous bone. Finaiiy, for the osteoporoac group, no clifferences were observed bem-een sections. This suggests that rhe degree of mineralization of the weightbearing surface of femoral heads is reduced by osteoarthritis. This is consistent wïth the increase in cancellous bone that was observed in Section 3.6.4. As for the data as a whole (see Section 3.2.5 and Figures 3.2.21 and 3-2-22)>the weighted average of the rnineralization distribution correlated lineady with the fluoride content at the superior site for both the subchondral and cancellous bone. The correlations were slightly doser for die homogeneous, osteoarthritis-only group (RL = 0.137 and 0-145, respectively). -4s \vas observed previously (Section 3-56)>the response of the subchondrai bone appeared to be independent of gender but the response of the cancellous bone is more pronounced in females and marginal in males (R' = 0.202, pc0.01 for OA females; illustrated in Figure 3.6.13). The weighted average of the rnineralization distribution at the inferïor subchondral site was also found to correlate significantly with the density of the cancelious cores for the osteoarthPtic fernales (R2i'= 0.127, p<0.05); this relationship was not observed for the data as a whole or anp other subset The log3 of the superior subchondral bone dso correlated with the fluoride contenr for the OA samples (RZ = 0.154, pc0.05). Weighted average:

Osteopor0tic

Supenm subchondral cancellous

Idenor: subchondrai canceilous

Weighted average:

Osreoporotic

Supenm subchondrai cancelious

XBEnor: subchondral canceilous

Table 3.6 7 Backscartered elec-on hagzhg, by disease

Data presented as mean t SEM.

*For canceiious bone at the superior site, the degree of rnineraiization (described by both the weighted average and the logit) \vas greater for the osteoporotic samples than the osteoarrhritic samples. Table 3.6.8 DS~èrencesbetrveen superior and ulfenor secahns

This table is the result of paired t-tests to compare the degree of rnineraiization, described by weighted average and by Iogit, for the superior and inferior surface of the femoral head. Significance was set at p<0.05.

OA: osteoarthntic femorai heads (n=59) Non-OA: femoral heads from patients with al1 diseases other than OA (n=12) OZ! femorai heads from patients diagnosed with osteoporosis or fracture (n=7)

This parameter was significantly greater for the superior surface than the infer& surface. S This parameter was significantly greater for the inferior surface than the superior surface. NS: No differences were observed in this parameter benveen the nvo sites.

The pattern is clearly consistent with less mineralized bone at the superior surface than at the inferior surface for osteoarthritic femoral heads, but not for non- osteoarthritic femoral heads. 1000 2000

Fluoride content (pprn)

Figure 3-623 I).&eraLLzation of supezior cariceflous bone

Ar the superior site, the weighted average of the minerakation distribution of the cancelious bone was was more closely correlared to the fluoride content for the osteoarthriac fernales chan anp other subgroup 02' = 0.202, p<0.01). 3.6.7 Microhardness testing of bone

No significant differences were observed bem-een the rneans of the microhardness values at any of the sites (canceiious and cortical bone at each of four locations: superoprosimal, superodistal, inferoprosimal, or inferodistal) of the osteoarthriac and osteoporotic sarnples (see Table 3.6.9). However, cancellous bone at the superodistal site showed a trend (pz0.06) towards being harder for the osteoporotic bone than its osteoarrhetic counterpart. It is wordi recahg that the cancellous bone at the supenor site had a higher degree of rnineralization (assessed by backscattered electron irnaging) for the osteoporotic bone than the osteoarthritic bone (Section 3.6.5). As for the backscattered electron imaging, the microhardness of paired inferior and superior sarnples were compared to determine if site-speci8c differences existed. The hee groups of samples esamined separately were an osteoarthntis-ody group, a non- osteoarrhntis (ïncluding osteoporosis) group, and an osteoporosis-only group. No differences were obsen-ed benveen sites (data not shown). This may be a resulc of the smaller nurnbers of samples available (n=39 in rotal; 30 osteo'irrhntic and 5 osteoporotic). \Vithin the osteoarthritic samples as a whole, the microhardness of the subchondral and cancellous bone at the superopro?cimai site and the subchondral bone at the superodistal site were found to correlate wvith the fluoride content (R2=0.414, 0.138, and 0.568, respecuvely). These three relationships were also observed for the dam as a whole (Section 3.2.6 and Figures 3.2.26-28). For the nvo subchondral sites, the relaaonships are much doser for the osteoarthrïtis-only samples chan the samples for ali disease States. The relationship benveen the microhardness at the superodistai subchondral site is iilustrated in Figure 3.6.14. For both the subchondral and the canceiious bone at the superoproximal site, the conelauon between rnicrohardness and fluoride content is pronounced for females (Rz=0.605, p<0.0005 for the subchondrai bone - see Figure 3.6.15 - and EZ2=0.208, pc0.05 for the canceiious bone) and absent for males. In contrast to the data for sarnples in aii disease States (Section 3-57}, the relationship between the microhardness of the subchondral bone and the fluoride conrent was observed for both genders at the superodistal site, not just for the females. These resuits suggest that, while osteoarrhntis-related changes to the mean microhardness of bone are not readily discernible, the relationship benveen the microhardness of bone and the fluoride contenr is more pronounced for osteoarthritic specimens than for aU specimens. Furthemore, this reiaaonship is more pronounced still for bone from osteoarrhriüc fernale donors than their male counterpms. Superoproxbat.: subchondral cancelious

Superodrstal= subchondrd cancellous

Inferoprroxid subchondral cancellous

I~feroocli'std: subchondral cancellous

Table 3.6 9 Mcrohardness offemoral heads

Data presented as mean t SEM.

*For canceilous bone at die superodistal site, the microhardness tended to be greater @=0.06) for the osteoporotic bone compared to the osteoarthriuc bone. 60

s-Î 50 (II (II al C F a .c O 5 40

Fluoride content (ppm)

Figure 3.614 Mcrohardness of superodr'srai subchondral bone

At the superodistal site, the microhardness of the subchoodrd bone was was more ctosely correlated to the fluoride content for the osteoarthritic samples than for the data induding ail disease States (R' = 0.430, p<0.0005). Fluoride content (ppm)

F~kure3.625 MTcrohardness of superoprownal subchonaki bone

At the superoprosimal site, the rnicrohardness of the subchondrd bone was was more closely correlated to the fluoride content for the fernale osteoarrhntic samples than for any other group or subgroup (R2 = 0.605, p<0.0005). 3.6.8 Surnmary: effect of disease state on response to fluoride

The measured parameters of bone quality were compared for osteoarthrïtic samples and osteoporotic samples, and the relationships benveen these parameters and the age of the donor, the density of the cancelious core, and the fluoride content of the bone were assessed for the subset of femoral heads derit-ed from osteoaahgtic donors. The relationship benveen the nuonde content and the age of the donor was not improved by escluding non-osteoarrh8tic sarnples, suggesMg that it is independent of the disease state of the donor. Slightiy irnproved relationships benveen the chlorine content or the calcium-to-phosphate raao (for males) and the fluoride content were found for the osteoarthrïtic sarnples. No difference \vas observed in the mechanical properties of the osteoarthetic and the osteoporotic sarnples, although thïs may simply be a resuit of the smd number of osteoporotic specimens. The relationship benveen the compressive modulus and age \vas stronger for the specimens from osteoarthrïtic fernale donors than the data as a whoie. While the ultimate compressive stxess and the jield stress were related to the fluoride content when ail sarnples were included, dus relationship was weakened when the non-osteoarrhgtic samples were escluded. Conversely, the linear relationship becween the energy to yield and the fluonde content was strengthened by exclusion of non-osteoarthrïac specimens. This suggescs that the effect of osteoarrhgtis on the relationship benveen the compressive mechanical properties of bone and the fluoride content may differ for the properties which describe strength and those which describe toughness. These responses were not observed in the gender-segregated groups. No differences were obsenred between the image and strut analysis pararneters of the coronal sections benveen the osteoporotic and osteoarrhetic specimens. The response to fluoride incorporation on the amount and comectiviy of bone as measured in the coronal section that was observed for ail the data was essentiaiiy unchangrd when the non-osteoarthritic samples were escluded. Image and strut analysis of histological sections \vas performed at two sites, a superior and an inferior site. There was markedly more bone present at the superior site than the inferior site for the osteoardi0tic specimens but not the non-osteoarthntic specimens, convincingly codkmkg that osteoaahds increases the arnount of cancellous bone at the weightbearing site. No differences were observed between the osteoarthntic 2nd osteoporotic specimens when oniy the infenor site was considered. No relaaonships were observed benveen the image and strut analysis parameters of the histological sections and the fluoride content, consistent with the findings for the data as a whole. The mineralizatïon of the superior bone \vas rnarkediy less than that at the inferior site for the osteoarthritic bone, but not for the non-osteoarthrïtic bone. Again, chis is convincing evidence that the superior swface is significantly affected by osteoarthrïtis. This ma); also explain why the cancellous bone at the superior site appeared more mineraiïzed for the osteoporotic bone compared to the osteoadritic bone. The relationship behveen the degree of mineralization and the fluoade appeared slightly closer when the non-osteoarrhetic bone was esduded. No differences in the microhardness were observed between the osceoarthritic and osteoporotic specirnens. In contrast to what the findings of backscattered electron imaging would suggest, the microhardness of the osteoardiritic femoral heads did not Vary benveen the superior and the infenor sites. The relationship benveen the microhardness and the fluonde content was at its most pronounced for the osteodritic females and entirely absenc for males, again suggesting that bone in osteoarthntic females is the most responsive to fluoride iricorporation. 3.7 Effect of location within fernoral head

3.7.0 Introduction

A number of parameters were measured at different sites in the fernoral head in order to assess the effect of location oa the measured dues. Ln particular, the presence of osteoarthritis in rnanv of the femord heads suggested that differences in bone qualil malr esist benveen the superior (weightbea~g)surface and the infenor (non-weightbearing) surface, halyses performed at different sites induded image and strut analysis of histoIogical sarnples, backscartered elecnon imaging, microhardness testkg, and instrumental neutron activation analysis. Figure 3.7.1 is a schematic of the femoral head, indicating the matornical location of each speurnen, to be used as reference throughout this section. Proximal (towards acetabulum) 7 Superior

+--(1) coronal section Inferior (non-weightbearing)

F12ure 3.7.1 Schematic offemoralhead sample locations

Kg: 1. Coronal section: used for image analpis and grading O t osteoardiritic sarnples 2. Cancelious core: posterior sample used for compression testing anterior sarnple used for torsion tes~g (when possible) 3. Superior and inferior samples: histological sections (image analysis, his tornorphome~) backscattered electron imaging 4. Superodistal, inferodistal, superoprosimal and superodistd samples: used for rnicrohardness testing 3.7.1 Image analysis of histological samples

Table 3.7.1 presents the means of the image and smtanalysis parameters for the superior and the inferior surface of the femoral head, divided rrito an osteoarrhriac group and a non-osteoarthrïtic group. For the former, there was significantly more cancelious bone present on the superior side of the femoral head than on its inferior counterpart, with correspondingly higher comectivity. This was reflected in the greater proportion of trabecdar bone, trabecdar thickness, trabecular number, number of nodes, and node-node stfuts, as weii as reduced trabecuiar separauon, number of free ends, fiee-free struts, and star volume @<0.0001 by paired t-tests). Only the number of node-free stnits \vas approsimately constant at bot.sites. However, for the non-osteoarthntic samples, no differences in any of the image analysis parameters were observed benveen the superior and the infenor surhces. This suggests that the differences benveen the weightbearing surface and the non- weightbearing surface are a result of osceoarthritis, not of anatomical differences, Osteoarthntic femoral heads (n=62): Supenor

proportion of trabecular bone (3 trabecuiar thickness (pm) trabecuiar nurnber (mm-') trabecular separaaon (p)

free ends (mm-? nodes (mm-') free-free s~~s(mm/ mm-? node-free srmts (mm/mm") node-node sauts (rnrn/mm-3 tsvo-dimensional star volume (mm?

Non-osteoarthritic femoral heads (~147): Supenor In fenor

proportion of trabecuiar bone (O/o) 20 f 2 24 k 4 trabecular thickness (pn) 145 k IO 157 + 16 trabecular number (mm-?) 1.36 f 0.09 1.46 _t 0.09 trabecular separation (prn) 619 i 45 559 2 59

free ends (mm-? 2.27 + 0.26 2.29 4 0.31 nodes (mm-') 1-00 + 0.19 1.39 4 0.32 free-free sauts (mm/mm-3 0.25 k 0.36 0.24 k 0.04 node-free sauts (rnrn/rnm-3 0.52 f 0.05 0.56 + 0.03 node-node sauts (mm/rnm3 0.50 k 0.08 0.67 k 0.18 nvo-dimensional star volume (mm? 3.9 & 0.4 3.7 f 0.7

Table 3.72 Image analysr's ofhistolo~~'caf samples, by site

Data presented as mean f SEM.

*For the osreoarthriuc samples, there was significantly more canceiious bone on the superior side than on the infeuor side, wïth a correspondingly greater connectivity, and this is reflected by ali the parameters escept the nurnber of node-free snuts (p~0.0001by paired t-test). 3.7.2 Backscattered electron irnaging

The degree of mineralitauon was assessed for both the subchondral bone and the cancellous bone at a supenor site and an inferior site. As for the histologicai sections, the specimens were divided into those from osteoarrhntic femorai heads and those form non- osteoarthntic femoral heads, in order to differentiate benveen the effects of osteaahntis and nomai anatornical variation. Data are given in Table 3.7.2. For the osteoaahritic femoral heads, the degree of minerakation \vas significantly less for the supenor surface than the iderior surface; both the weighted average and the Logit vere greater for the inferior subchondral and the inferior cancellous bone compared to their superior counterparts. Also, che weighted average of the degree of mineralkation was greater for the subchondral bone at the infenor site than its undedying cancelIous bone. For the non-osteoarhitic bone, there was a trend towards a greater degree of mineralkation at the superior cancelious site compared to the infenor cancelious site (pc0.1 for both the logit and the degree of mineralizauon). No other differences were observed between the superior and inferior surface, or benveen the subchondral bone and its undedying cancellous bone, for the non-osteoarrhntic specimens. As for the differences observed by image analysis, it appears that the site-to-site differences in mineralitauon for the femorai heads are also a result of the presence of osteoarthrïtis. Osteoarthritic femoral heads (n=62)

Weighred average: ~srrbchondrd cance//om

Non-osteoartfiritic femoral heads (n=7)

Table 3.7.2 Backscartered electron hagktg data, by site

Data presented as mean + SEM.

1For the osteoarthritic bone, the degree of rnineralization is significantly less at the superior surface than the inferior surface (p<0.05).

'For the non-osteoarthritic bone, there was a trend towards a greater degree of mineralization at the superior site compared to the inferior site for the canceiious bone @<0.1 for both the logit and the weighted average).

3 For the osteoarrhritic bone, the weighted average of the degree of rnineralization \vas greater for the subchondral bone at the inferior site than its underlying canceilous bone. 3.7.3 Mïcrohardness of bone

Sarnples were taken kom four sites on the fernord head: a superopro.uima1 site, a superodistal site, an inferoprosimal site and an inferodistal site. The superior and inferior surfaces are the weightbeming and nonweightbearing surfaces, respectïvely, and the prosimal and distal sites are towards the acetabulum and towards the femoral neck (see Figure 3.7.1). Mean dues for each site, grouped by osteoarrhritk and non-ostearrhritic femoral heads, are given in Table 3.7.3. Sarnples were paired and compared to determine if differences esis ted benveen a) the superior and inferior surface, for a given location (distal or prosimal) and type of bone (subchondral or cancel.ious), b) the type of bone for a given site on the femoral head, or c) the location on the femoral head (distal or prosimai), for a given side on the femoral head and type of bone. For the osteoarthriuc group, the microhardness of the canceilous bone was significantly less than that of the overlying subchondral bone at the nvo distal sites (infero- and superodistal). hlso, the cancellous bone at the two proximal sites mas harder than the canceiious bone ac the nvo distd sites (pc0.05 for the inferior site and pz0.06 for the superior site). Taken together, these compaxisons suggest that, for osteoarthritic femoral heads, the canceiious bone at the nvo distal sites is less hard (and possibly less mineralized) than aii other sites. These patterns were not observed in the non-osteoxthritic femoral heads. The only significant difference observed was that the microhardness at the subchondral bone \vas greater at the superodistal site Sian at the superoprosimal site. In surnmq, while sorne site-specific effects of micro hardness were observed, it appears to be largely independent of anatomical location for non-osteoarthntic bone. For osteoarthritic bone, the hardness of the canceiious bone at the distal side of the fernorai head appears to be reduced. Osteoarthritic only (n=31):

subchondrd 43.3 t 0.8 43.1 t 0.8' 43.2 t 1.2 44.6 t 2.2' cancellous 42.1 20.7 40.1 t 0-5' 43.3 t 1.0' 40.4 k 0.6"

Non-osteoarthritic femoral heads (n=8):

subchondral 42.6 t 1.03 45.9 + 1.4~ 40.6 t 1.4 43.2 t 1.0 cancellous 43.6 t 0.7 42.8 t 0.7 42.7 k 1.7 40.6 t 0.9

Table 3.1.3 iM1crohardBe.s~of bone, by sire

Data presented as mean t SEM.

SP: superoprosimai SD: superodistal IP: inferoprosimal ID: inferodistal

or the osteoarth5ic femoral heads, the subchondral bone at both distal locations \vas significantiy harder than the underijing canceiious bone.

or the osteoarthrïtic fernord heads, the canceilous bone at the inferoproxbal site is significandy harder than chat at the inferodistal site.

3 For the non-osteoarrhntic hmoral heads, the subchondral bone at the superodistal site is harder than its superoproldmai coumerpart. 3.7.4 VariabiLity of fluoride content

A preliminaq smdy to deterrnine the effect of intra-femoral head variability of the fluotide content of bone mas performed. For a subset of femoral heads, the Buonde content \vas deterrnined not only for the cancellous core (n=39) but also for each of the four samples that were taken from the surface of the femord head to be used for microhardness testkg: superoprosimal (n= 18), superodistd (n=37), inferoprosimd (n=l8), and inferodistal (nz35). The measured value for the ouonde content of the four surface sites were compared to that of the canceilous core, which \vas removed from the centre of the femord head (see Figure 3.7.1). Since all five samples were taken from a single fernocal head, a repeated-measures ANOVA (the equivalent of a paired t-test for more than cwo variables) was performed to deterrnine if an effect of sire existed on the Buonde content- meit was found to be non- significant @<0.07), the low p-value suggests that there may be some subde effects. As the overd p-\due for signifrcance \vas not bdow 0.05,poxt hoc tests were not performed. 3.7.5 Summq: effect of location

A number of the measured parameters were assessed at nvo or more locations on the femoral head. Wde there may be a normal anatornic variation in some propemes of the bone, osteoarthntis is also known to have localized effects on bone tissue. For this reason, the data was generally segregared in an osteoardintic and a non-ostearrhetic

PUP- \Vidin the os teoarrhntic group, significantly more cancellous bone \vas present at the superior surface compared to the infenor surface, as assessed by image and smit anaiysis of histologicai sections. Also, the subchondral bone at the inferïor site appeared to be more mlieralized than both the underlying cancellous bone and its coumerpart at the superior site. ~vLicïohardnessmeasurernents suggest that the cincellous bone at the distal part of the femoral head (towards the femoral shafi) is less hard than at other sites; both the subchondral bone at the same site and the canceiious bone at proximal sites on the same side of the femoral head were harder. Very few differences were observed betu-een sites for the non-osteoarthritic femoral heads. There was some evidence that the cancelious bone at the inferior site is less rnineralized @y backscattered elecuon irnaging) than its superïor countecpart, and that the subchondral bone at the superodistai site was harder than at the superoprosimal site. No differences were observed in image and saut analysis parameters. Finaiiy, the inaa-femoral head variability of the Ruoride content was assessed, but no significant variacion by location was observed. 3.8.1 Incorporation of fluoride

Fluoride concentrations in cancellous bone from the femoral head ranged from 192 to 2264 ppm, a range of almost twelvefold. Whde residents of a city wîth Buoridated mater had a higher mean fluoride content than those residing in a cicy \vithout fluoride content, the range of fluoride contents overlapped entirely, indicaüng that esposure to fluonde is not the sole determinant of fluoride incorporation. The naturai logarithm of the fluoride content increased wïth age for women, but not for men.

3.8.2 Mechanical properties and fluoride incorporation

The ulamate compressive stress, yield stress, and energy to yield correlated negatively wïth the fluoride content when ail speümens were inchded. The energy to vieid, however, correlated more closely wïth the fluoride content for the osteoarrhritic specimens ody. No substantial differences benveen genders was observed. These results indicate that there may be an effect of fluoride incorporation on mechanical properties, and that it may be mediated by the disease state of the donor.

3.8.3 Architecture of bone and fluoride incorporation

The trabccular bone volume decreased and the trabecular separation increased wïth increasing fluoride content in the femoral heads, as measured on s-rays of coronal sections. However, no effect of fluoride incorporation was observed by image and strut analysis of histological sections. The measured parameters for the image analysis of coronal sections were inremally inconsistent and did not Vary \.

For osteoarthritic femoral heads, the mineralization of the bone at the superior surface displayed a profound relaùonship with the fluoride content. The weighted average of the degree of minerakation of both subchondrai bone correlated dosely with the fluoride content for both males and Çemales; hou-ever, that of the cancellous bone was more pronounced in fernales. Similady, the rnicrohardness of the bone at superior sites were most closely correlated with the fluoride content for female osteoarthritic donors. No such responses were observed at the iderior surface (\vhïch is less affected osteoarthritis). These resuits suggest that the relationship benveen mineralization and fluoride content is associated wïth osteoarthritis-induced bone formation, and is particularly pronounced in these post-menopausai fernales.

3.8.5 Cornparison between high- and low-fluoride groups

The parameters of bone quality for donors from two cities, one with and one without fluoridated municipal water (Toronto and Montreal, respectively) were compared. The mean fluoride content \vas significandy greater for the Toronto cancellous bone. The canceiious cores from the Toronto specimens were denser, more ductile, and tougher compared to their Montreal counterparts. Cornmensurate wïth the increase in density, there was a slight increase in the arnount of bone as measured by image analysis. No substantive differences were observed in the degree of ~eralizauon. Specirnens in the highest and lowest qudeof fluoride content were identified, and the parameters of bone quality from those specimens were compared. The mean fluoride content of the top qudewas approximately three times that of the bottom quartile. Cancellous bone from the bottom quartile had a greater mean yield stress and ultimate compressive stress than the top qude. As measured by image analysis of coronal seüons, the trabecular volume was greater for the bottom quartde than the top quartile; however, this was not confirmed by either image analysis of histological sections or by the physical densiq of the canceilous core. Finally, the degree of mineralization was greater for the top quartile than the bottom quade; this was apparent both by bachcanered electron imaging and by microhardness testing. Comparing the differences obsemd benveen uties with and without fluoridated water and those observed behveen the top and bottom qudeof fluonde incorporation (regardless of uty of ongin), it becomes abundandy- dear that that fluonde expoxz~re cannot be considered to be synonymous with fluonde incorporation or effect. 4: Discussion In nyyordh I regarded the rtniuerse as an open book, ppried in the lavage cfplp'caI eptahmu, rvhereax now if appearJ to me as a te* wriîten in inuzn'UIe ink, ojwhich in oztr rare nmenfr cf grace we are able to den)her a smallfiagment.

Arthur Koestler Bticks to Babel 4.0 Introduction

'This section is divided into four parts. The &et discusses the methodological issues associated with the esperimentd techniques u&sed in this smdy. The second component focuses on the resdts of thïs work; Li particdar, on understanding the interaction benveen the pathological conditions of the donors, the incorporated fluoride, and the measured paramecers of bone quality. The third component deals with the Iarger implications of diis work. The fourth and final section describes some further work that cari be performed to darie or extend the results of thïs study- 4.1 Methodological issues

4.1.1 Introductory remarks

The purpose of this section is to explore some of the methodological issues that may affect or iimit the applicability of this research. Five major areas are discussed below: issues regarding the acquisition and preparation of the femorai heads, an assessment of the use of neutron activation analysis to determine the demental chernical composition, discussion of the approach used for compressive and torsional testing, a cornparison of the assessment of bone by image analysis of thin sections, thick sections, and histomorphometry, and, finally, an exploration of the relationship benveen the microhardness of bone and its rnineralization profile, as determined b y backscattered elecuon imaging.

4.1.2 Acquisition of specirnens

The single most significant issue regarding the acquisition of specimens relates to theîr origin. As the femoral heads were removed from individuais undergoing total hip mhroplascy, thev were, by mydefinition, not normal. The majority of the specimens obtained were from osteoarrhritic patients (75 of 92), with specimens Gom osteoporotic donors (9) a distant second. Whde this is undoubtedly reflects the diagnoses resulting in hip replacement surgery, at least in part, there is also a bias resultïng from the enrolment of patients into surgery. In Toronto, patients were asked to participate in the smdy and provided informed consent at a pre-admission visit; these individuals were al1 therefore undergoing elective surgery. In Montreal, however, patients u-ere enroiled into the study by a member of the surgical staff. This resulted in the enrolment of a greater proportion of individuals who had suffered hip fracture (7 of 39, or 1S0/o, versus 2 of 53, or 4% of those from Toronto). This iikely contrïbuted to the higher mean density of the Toronto canceilous cores cornpared to thek Montreal counterparts; an effect that is probably independent of fluoride content. As elsewhere in this study, care must be taken to understand the effecrs of this type of confounding factor. The consequences of the presence of the pathologies in the femoral heads, of course, comprises much of this document. Afier specimens were acquired, they were stored in a -70°C freezer for variable periods, of up to about sis months. As the femoral heads were obtaïned at the rare of a few per month, storing samples in the freezer util enough were available that they could be analysed as a batch (typically of nventy specimens) was certainly convenient, although not, stricdy speakng, a necessity. Freezing is unliliely to have much effect on architecture or mineralization as long as the bones were in airtight packaging (to minimize dehydration and shnnkage). At least for shorter intervais, freezkg does not adversel affect the mechanical

propemes of bone peker et al. 19841.

4.1.3 Instrumental neutron activation anaiysis of cancellous cores

Instrumental neutron activation analysis (INLIA)was used to measure the chernicd composition and, in particular, the fluoride content of the cancelious cores. Two issues regarding Siis technique bear discussion. The fluoride content üted throughout this study is given as parts per miilion of the wet weight of the cancellous cores that were analysed. The gamma-ray counts that were measured were norrnalized to deterrnine the mass of fluoride in each sample, in micrograms, mhich was then divided by the mass of the sample, in grarns, to produce a value in parts per don.Two other measures of fluoride content could have been used. The fist is the ratio of fluoride content to the calcium content, normaliy espressed in micrograms per milJigram. The second is the ratio of the fluoride content to the total mineral, also in micrograms per dgram. The mass of total minerai is calcdated based on the assumption that it consists of stoichiometric cdcium phosphate (CaPOJ; the mass of calcium is added to the mass of phosphate, estimated by multiplying the phosphorous content by the appropriate factor (the concentrations of magnesiurn, sodium, fluoride etc. - elements normalljr found incorporated into the mineral - are not included in this simple estimate). This measurement (fluoride-to- total-mineral ratio) is comparable to the ash weight of the specimens, an ofien-quoted due. As the fluoride itself is incorporated into the rnineral, the advantage of normalizing it is that changes to the organic component or water content are excluded. If the rnineral content is constant, however, these three measures should be equivaient. In this study, the fluoride- calcium and fluoride-total minerai ratio were highly correlated with the fluoride content (Pearson correlation coefficients ofO.898 and 0.907, respectively). However, both of them were more variable. This is likely due to the fact that they are based on either three or four measured values (the mass of the sample measured on a balance, the mass of the fluoride as measured by FNM, and the mass of the calcium or both the calcium and phosphorous, also measured bg INM) rather than simply the hrst two, as is the case for the fluoride content. The fluoride contents that were measured for canceilous bone in this study were compared to other dues quoted in the literature. As they are variously quoted as a function of wer wveight [Parkins et a/. 19741, dry weight pappalainen et al 19831, or ash weight [Richards et al. 1994, Stein and Granik 2980, Charen et a/.19791, all values were converted to parts per million of wet weight (the measure used in this study), using the composition dam provided in Cowin [1989]. \me neutron activation analysis was used here, fluoride electrodes were used in each of the other studies. The range of fluoride content or the mean determined by these studies are provided in Table 4.2.1, with the fluoride contents Erom the fluoridated and non-fluorïdated groups in this study aiongside for comparison. The obsen~ed values for the non-fluoridated (Montreal) gmup appear comparable to those measured for other geographical regions or anatornical sites by other researchers. However, the mean value quoted for the fluondated region appears somewhat higher than char measured bp Charen et a/. [1979] for a population in a Buoridated region. \XWe this may be a result of anatomic variation or of the pathology of the fernord heads, this may also be due to the fact that individuais in fluoridated regions who participated in this smdy have had an additional nvo decades of esposure to fluoridated water compared to those in the previous study [Charen et a/.19791, Findy, and most significantly, the use of whole canceilous cores appeared to have affected the rneasured values of bone minerai. One of the reasons thar neutron acuvation anaiysis \vas used to assess the chernical composi~onof the bones is that is a non-destructive technique. Accordingly, while the cancellous cores were deaned to remove marrow, they were lefi intact. In the case of Iow-densiv cancelious cores, rernoval of the marrow was clearly complere; visuai inspection of the core revealed an open strutwork of trabeculae. However, the high-densiq- cores appeared solid. Closed ceiis could therefore contain marrow or fat that was not removed by the deaning process, slightly increasing the mass of the speken without increasing the mineral content. The calcium conrent, phosphorous content, and total mineral content all decreased with increasing densiry of the core, while the caiùum/phosphorous raao increased (although it is not clear why the phosphorous content wodd be more affected than the calcium content). Figures 4.1.1 illustrates the decline in total minera1 (calcium phosphate) content with densiq. A second possibiiïry is that the decline in total bone minerai with density (that is, the presence of more bone that is less-rnineralized) results from the presence of osteoarthritis in the femoral heads. Osteoarrhritis is associated wïth increased bone formation and remodeling [Amir et aL 1992, Grynpas et aL 19911. As newly formed bone is less dense than older bone, the presence of osteosclerosis in die femoral heads may lead to a negative relationship benveen the minerai content of the bone materiai and the density of the cancellous core (\vhich is related to both the amount of bone and the density of the bone matecial). Both of these (an artïfactuai dedine in the total minerai content with densiv due to measurement errors, and truc deciine due to the presence of osteoarthrïüs) may be contributbg to the observed relationship beween bone mineral and density. Site Population Value

Richards et al. 1990 vertebrae non-auoridated 256-1356 ppm Lappalainen et a/.1 9 83 iliac crest mked 73-1606 ppm Stein and Granïk 1980 vertebrae mixed 88-1261 ppm Parhs et a/.1974 iliac crest mostly fluoridated 1295-5745 ppm* Charen et a/.1 979 iliac crest fluoridated 706 + 92 ppm

this study fernord head fluoridated 192-2264 ppm (Toronto) (1033 k 438 ppm)

this smdy femorai head non-fluoridated 270-1200 ppm (Mo ntreai) (643 2 220 ppm)

Table 4.2.1 Fluoride content of cancdous bone

Fluoride content as assessed by other studies. The fluonde content is quoted as ppm/wet weight of the bone.

Thevalues measured for this study were reanalysed by Charen et al. 1979 and found to be erroneously high. Density (g/cc)

Fe4 Totdnzineral content decreases mathdensity of the canceUous core

The total minerai content, calculated from the percent calcium and percent phosphorous contents, decreased with increasing density of the canceiious core (R2=0.208, pc0.05). See tesr for detaiis. 4.1.4 Mechanical testïng

Trabecdar oninfation o/lcoqûresnve ~aqle~:Canceiious bone is an anisotropic material [Colvin 19891; the mechanical properties are a function of the direction of loading. Additionally, femord heads display a marked ocientation of the trabeculae, as cm be observed on an s-rajr of a coronai section (Figure 4.1.2). For the femoral heads used in this study, the cend region of the femoral head was removed (the coronal sections were escised to be s-rayed and used for histology by the pathology depmment), and therefore specimens from the central region of compressive loading were unavailable for compressive tes ting. The test specirnens were taken in the antero-posterior direction - that is, pependiczthr to the principal direction of loading and trabecular orientation in the femoral head. The purpose of this section is to understand the ramifications of this. In other stuclies, contact s-rays have been utilised to ensure that the zxis of the test specimen was aligned with the trabecular orientation weaveny et a/.1994, CiareUi el al. 20001. The specimen is then cut dkectly from the s-rayed sample, radier than an adjacent region (as could have been done in chis case). This procedure was used to excise compressive test specimens from human femoral heads [CiareUi etal. 20001. The avis of loading was dong the primary direction of trabecular orientation. A total of 32 specimens from cadaveric bone, approsimately the sarne age of the donors in this study (mean: 75 ÿears) mere tested in compression. The means of the compressive modulus and ultimate compressive stress were substantialiy higher than mere observed in this study (a rnean of approsimately 1200 LMP~ and 15 ;Wa, respectively, compared to 252 hIPa and 8 blpa in this study) although the standard deviations were sirnilar (approsimately 50% in both cases, although the nurnber of samples \vas much higher in the present study). As the apparent densi. of these specirnens was not reported, it is possible that they were denser than the samples used in this smdy, ïïhich would accounr for their improved mechanical propemes. However since the samples used for the present study were escised from a site adjacent to the site of the samples escised bv Ciareiii and CO-workers,it is possible that the densities were sidar and the differences in mechanicd properties resdt Gom a difference in architecme relative to the nvo perpendicular ases of loading. Other papers in the literature that have reported on the mechanical properües of the femoral heads have reported ultimate compressive stress and modulus vdues similar to those found here. For esample, Schoedddi et al. [1974] =amined the compressive mechanical properties of canceilous bone from multiple sites in the human femoral head. In this case,

the test specimens were aii a&ed wittfi the principal direction of trabecular orientation, but were taken from various locations in the femoral head. The range of the ultimate compressive stress and compressive rnodulus were much less than that observed by Ciarelli et a/. [2000], but similar to the vdues &me; the ultimate compressive stress ranged £rom 0.14 to 13.5 hiTa and the mean compressi~emodulus was 344 t 28 (mean k SD). Other dues for the ultimate compressive stress (UCS) and compressive modulus quoted in the literature include 7.61 hDPa and 11.4 hfPa for &e UCS, and 57 bPa and 581 mafor the rnoddus [reviewed in Martens et al: 19831. Findy, one study [-Iartens et al. 19831 esamined the strength of canceIIous cores with th& a-xes aligned in the sarne direction as here; w-hile the sarnples were only obtained frorn nvo femoral heads, the mean age of the donors \vas comparable to the mean age of the donors in this study (67.5 years). The compressive rnodulus was somewhat higher (403.5 1Wa) and the ultimate compressive stress somewhat less (4.9 ?dl?a) than the means of the data presented here (252 MPa and 8 MPa, respectively). As is apparent from this section, the range of ultimate compressive stress and compressive moduli reported in the Lterature is veq- large. There are two factors that contribute to this. One is that the varËabiliy in the mechanical properries tvithin the fémoral head is quite wide, and does not appezu to Vary in a systematic way with location [Schoenfeld ei a/.19741. Secondly, the esact methed of tesûng the compressive properaes ma. have large effects on the measured properties; for esample, the size of the specimen, aspect ratio, and use of lubricated or unlubricated platens are known to affect the modulus and compressive stress [Linde and Hvid 1989, Linde et aj. 1991, Linde et al. l992L However, the values reported here for the mechanicd properties appear to be in keeping ~vithother values reported in the literature.

ReIa~onshtprwithln mecacaoes:Theoretical considerations suggest that the relaüonship between the compressive çtrengtl~and modulus with the apparent densiq should be either hem- or quadratic, depending on whether the structure of cancelious bone is colurnnar or asymmemc, respectivdy [Gibson 19851. Data has been provided in the literarure supportkg both linear [Linde et al: 1991, Schoenfeld et al: 19741 or quadratic relationships [Cowïn 19891. In this study, no evidence \vas observed that would support a quadraüc rather dian a Linear relaüonship. Figure 4.1 -3 illus traces the relationship benveen the dümate compressive stress and the square of the apparent density (that is, the quadratic realtionship), which is indisputably parabolic, supporting the çidings that the mechanical properties vqIineady with the density (eg Figure 3.2.6). Based on the theoreticai arguments, this suggests that cancellous bone from the femoral head that is perpendicular to the principal direction of trabecular orientation (as here) or simply displaced Erom it [Schoenfeld et a/.19741 has a different ceii structure than oriented, dense trabecular bone. A difference in architecture is also consistent with site- and species-specific nature of the relationship between mechanical propemes and density [Keaveny and Hayes 19931. The hear uibate compressive stress-density relationship presented here (Figure 3-2.9) is much Iess steep than that observed for sarnples that were also from femoral heads (albeit in a

different loading direction) by Schoenfeld et al. [1974]; while similar values of ultimate compressive mess are predicted for an apparent density of 0.9 g/cm3 (about 7.5 hPa), the regression line determined for this data predicts an ultimate compressive stress of about 7 &Pafor an apparent density of 0.8 g/cm', whereas the Schoenfeld data would suggest that the dtimate compressive stress should Çaii to appr~~simately4.5 &Pa. However, it should be noted that the Schoenfeld data was both more homogeneous (only four femoral heads were used) and more resmcted (the densit). range presented was only 0.75 to 0.90 g/cm3, compared to the range of 0.3 to 1.7 g/cm3 observed in this study). Nevertheless, the linear relationships observed in both studies suggest thac there may be an underlying similarity in the architecture of the trabecular bone in both directions. Convincing arguments and empirical da= have been forwarded indicating that the yield stress or ultimate compressive stress vary linearly with the Young's modulus. This is thought to result from an evolutionary requirement to maintain a constant Mure suain (to be precise, to maintain a cons tant safety factor betsveen the applied and failure strains) Fumer 19921. It was observed previously that, in the human fernord head, the constant of proportionality benveen the yield mess and the compressive modulus does not vary with loading direction prown and Ferguson 19801. A linear relationship between the yield stress (as well as the dumate compressive stress) and the compressive moddus was in fact observed for this data, and is iiiustrated in Figure 4.1.4). This also suggests that the differences in the mean strain at fracture between the samples from the Toronto group and those fiom the Montreal group may either be an artifact of the testing technique Peaveny et aL 1994,1997] or may reflect differences in the underlying pathologies of the bones in the tsvo groups; there were propoaïondy fewer ostearthriuc specimens in the Monrreal group compared to the Toronto group.

The shpe of the stre~.r-~-trainczirve and cancel/otcs bone rtnrcture: The stress-strain curve denved from one of die compressive test specimens is illusuated in Figure 4.1.5, with characteristic features noted. At the outset, the stress-main cuve is non-linear. The stress initiaiiy increases slowly with increasing stlain, and then more rapidly. This toe region is thought to result frorn serrling of the canceiious core sarnples within the platens of the testïng machine, as the plane ends of the sample corne intu dose apposition with the flat platens. The nest (centrai) region of the stress-strain curve is nearly linear. As canceilous bone is a structure, not sïmply a material, this likely represents elastic (reversible) deformation of bone tissue oriented approsimately in the direction ofloading as weil as bending of individuai trabeculae that are not onented in the direction of loading. As the structure is estremely ïrregular (Figure 4.1.6), these two rnechanisms contribute tu the stress- strain behaviour sirnultaneously. This nearly-linear region is delimited by the proportional Limit PL), which is the point at which the cuve deviates from linearïty. The nest, non-linear region of che mess-strain cweWrely results Erom nvo effects; the fïrst is irreversible plastic deformation (yielding) of bone material, and the second is continued reversible bending of individual struts in the cellular smcnrre of bone. As the relative connibutions of these nvo mechanisms, one reversible and one irreversible, are unknown, the elastic limit (EL), which is the point ar which reversible deformation ends and irreversible deformation begins, is indicated by a dashed he. The bending-and-yield region ends at the point of ulàmate compressive stress (UCS). After this point, the trabeculae composing the cancellous bone struccure begin to fracture, resulcing in decreased load. This 'crus hing' region continues und ail the trabeculae fracture and the bone becomes a compact pellet, at which point the load increases agaLi (analogous to squeezing a sponge). However, the sarnples in this study were not loaded to that point. Torsion textriig: The number of sarnples that were available for torsion testing were evtremely lirnited. The cmcellous cores used for torsional testing, like the compressive sections, were taken fiom the posterior half of the femoral head, in the antero-posterior direction. The cores were 6 mm in diameter, and the gauge length of the specimens was Lsed at ouice thïs, or 12 mm. Since several millimetres were required at each end of the specimen to fk it into the gips, the minimum length of the specimen was approsimatdy 15 mm, setüng a lower Iimit on the diameter of the femoral head to approsimately 35 mm (the ~vohalves of the femoral head plus the 5 mm-thick coronal section). Only 27 of the femoral heads were large enough for a specimen of this size to be removed. This had the effect of biasing the samples towards larger femoral heads, and in partïcular, to mde donors - onllr about a fi& (6 out of 27) of the samples tested in torsion were fkom female donors, even though females made up over half of the total donors (51 out of 92). As the compressive mechanical properties appeared to vary more closely with age and fluoride content for the cancellous bone from female donors than male donors, this may explain why no relationship \vas observed benveen the shear properties and either of these variables. \We the direction of compressive loading in the femoral head is quite weU understood, corresponding wvith the trabecular orientation, the magnitude and direction of loading of cancellous bone in the femoral head in shear is Iess weil-understood. It is therefore much more difficult to assess the effect of orienthg the long asis of the torsional test specimens perpendicular to the principal loading direction of the bone. Data on torsional testing of canceilous bone in general, in act, are scarce, and reports on hurnan canceilous bone, appear to be Limited to a single study: Bruyére Garnier et al. [1999] escised cylinders of cancellous bone from human femorai heads, also removed during total hip arrhroplasty, and tested them in torsion. The zds of the specimens was aligned with the principal trabecular orientation of the specimens. Qualitatively, the shape of the observed stress-suain curves were sirnilar. The measured ultimate shear stress ranged from 1.6 to 10.0 &Pa(mean + SD: 6.1 t 2.7 ma) and the shear modulus from 72 to 499 1Wa (mean k SD: 289 t 140 Pvn?a). The values measured in this study were somewhat lower; the mean ultimate shear stress was 3.3 k 1.9 ma(range: 0.9-8.6 &Pa) and the mean shear stress was 75 f 39 1Wa (range: 19-179 hTPa). This is likely due, in part, because the tested specimens in this study came from a different, less-dense site. Both studies displayed large, relatively similar variabdisr in the measured propemes. The lack of data in the literature on the shear behaviour of canceilous bone suggests that this may be a fertile area of investigation. F~gure4-12 Contact x-ray of coronai section

The principal direction of trabecular orientation in the femoral head can be observed in this x-ray. The canceilous cores that were used for compressive testing were oriented with their ask nomal to the plane of the coronal section. Square of density (gUcc2)

Figue 4.1.3 Uhhate compressive srress vs the square of the tiens*

\We the relationship between the yield stress, ultinate stress, and compressive modulus is reported as being quadratic [Cowin 19891, it is dear chat a straight line is not appropriate for this graph. In contrast, Figure 3.2.6 clearly illustrates thar the relarionship benveen the ulhate compressive stress and the density is Linea.r. Compressive modulus (MPa)

Fe4.1 Yield stress iocr~asesheazly with cumpressive modulus

The yield stress of the compressive test specimens increased linearly \.ththe compressive modulus (R2= 0.653, pc0.05) bending i

0.04 0.06 0.08 0.10 Strain (mm/mrn)

Figue 4.1.5 Fearures of the stress-strain curve for canceflous bone h compression

PL: proportional limit EL: elastic limit UCS: ulhate compressive stress

See test for further details. Figure 4.1.6 Structure of canceLbus bone

Canceilous bone has an irregular cellular structure.

(Scanning elecuon rnicrograph courtesy of D. Holmyard.) 4.1.5 Image analysis of thin and thick sections

Two different methods were used to assess the amount and comectivity of bone. S- rays of coronal sections, approsimately 5 mm thick, were analysed to assess these properiies over the entire hmoral head. Histological sections, only 5 Pm thick, were taken at a superior and an inferior site, and anaijed using the same techniques and determining the same parameters. In addition, the physical density of the canceUous bone was measured at a single site, near the cenue of the femoral head. \Frhile ail three of these techniques are nominally measurhg the same thing (the amount of bone), the acdtechniques and the use of different sites may tead to different results and trends in the data. The amount and connectivity of bone was measured at nvo sites, an inferior site and a superior site. The amount and the connectivity of the bone at the superior site was much greater than that at the inferior site for the osteoarthritic bone, but not the non-osteoarthrkic bone (see Section 3.6.5 and Table 3.6.5). As the superior surface is clearly affected by osteoarthntis, the focus of this section is the inferior surface. First, the data for the iderior sections were esarnined for intemal consistency. Parameuic and non-parametrïc correlation matrices were used to see if relationships esisred benveen measured paramecers. As would be espected, the star volume decreased as the trabecuiar volume increased. The nurnber of node-node stmts and of free-Eree struts were inversely correlated (Figure 4.1.7). The node-free ratio decreased and increased, respcctively, w-ith the nurnber of free-free struts and the nurnber of node-node struts. However, the number of node-node stnits and free-free smts were not correlated with the trabecular volume or the star volume. In addition, for the inferior histological sections, the trabecular volume, trabecular number, uabecdar separation, nurnber of nodes, node-node smts and node-free struts ail correlated in the espected way with the physical densi ty of the canceiious core (see Section 3.2.5 and Figures 3.2.17 and 3.2.18). Finaily, the measured parameters for the inferior histological section were correIated with densiqr in a way consistent with other studies Fazzalari et a/.1983, Crane et al. 19301; the uabecular separation and nurnber were correlated with age of the donor, as was the nurnber of nodes. No relationship was observed benoeen the properües at the superior surface and the age or density (see Section 3-25)>again con&ming that the superior secuon is abnormal. Given that these image analysis techniques were designed for thin sections, and given the regular, predictable behaviours of the measured parameters for the inferior histological secaons when related to each other, the physicai density of the bone, and the age of the donors (see Section 3-25),it seems appropriate to use these results as a baseline, to which the parameters measured for the Y-rays cari be compared. However, the s-ray parameters did not display sïmilar relationships. Within the coronal sections, the star volume decreased wïth increasing trabecular bone volume (as it did for the inferior histoiogical sections). However, in these sections, the number of node-node smts increased with the number of Gee-fiee struts (Figure 4.1 A). In addition, both the fiee-free smts and the node-node stmts increased ~vithincreasing star volume. Similady, both the free-free stmts the node-node struts decreased with increasing trabecuiar bone volume. In cornparison, s-rays of sections of archaeologicai human vertebrai bone were andysed using the same techniques, and the node-node struts and free-free struts were ïnversely correlated (as they were for the histoIogical sections) [S.C. Aganval, personal communication]. Of the measured parameters for the s-ray of the coronai section, oniy the number of endpoints \vas conelated with the density of the canceilous core, increasing iïnearly (see Section 3.2.4 and Figure 32-13). No relationship was observed benveen any of the measured parameters and the age of the donor. Findy, none of the measured parameters for the coronal section were correlateci with their matched parameters for the inferior histological section. The Iack of correlation of the image and smt parameters with the density of the cancellous core and with the inferior section may simply be ascribed to the use of different locations. \%?de normal femoral heads display a decline in the amount of trabecular bone and associated comectivity parameters with age [Fazzalari et a[. 1983, Crane et a/.19901, osteoarthritic bone does not [Crane et al. 19901. It seems likely, therefore, that the coronal sections, which include the osteoaahritis-affected superïor section, do not display a relationship with age because such a relationship is ovenvhelmed by osteoarrhritis effects. However, the Iack of intemalconsistency (in pamcular, the increase in both node-node and free-free struts with increasing trabecular volume) is an issue. One possibility lies in the heterogeneity of the trabecular bone structure. Dolecoronal sections are visibly more heterogeneous structures (see Figure 4.2.2) than vertebrae, wkch have also been subjected to this type of anaiysis Forstjens et a[. 19981, aithough the heterogeneity of vertebrae has also been reported panse et a/. 20011. Ln addition, both osteosclerosis (iicreased bone) and cysts (voids in the bone structure) are characteristic of osteoartbtias [ket a[. 19921 and were observed for many of the femoral heads in thïs study (see Figure 2.7.1). Finally, the variability of the histomorphomemic parameters has been shown to be higher than age- and ses-matched controls [T=azzalariand Parhson 19981. The positive relationship benveen the arnount of node-node smts and the amount of free-free struts may therefore be related to this irihomogeneitj-. One of the consequences of these apparent inconsistencies in die image analpis of x-rays of coronal sections is that it necessarily casts doubt on other fïndings using the same specimen (such as the relationship between the trabecular separauon or the trabecular volume and the fluoride content, Section 3.2.4). In addition, because the whole femoral head \vas used for this analysis, it is difficult to determine whether these relationships to the fluoride content were for the whole femoral head or if they were (for esample), for the supenor surface, which may be dominating the measured parameters. This will be discussed further below (Section 4.2.3). Free-free struts (mm/mm2)

Figure 4.2.7 Node-node struts and fi-ee-dreesrntts: hr'stologicafsection

\Vidin the inferîor histological section, the nurnber of node-node and free-fiee struts were inversely correlated (R' = 0.392, pc0.0001). .s 1:O 1.5

Free-free struts (mm/mm2)

Figue 41.8 Node-node struts and fiee-fiee struts: coronal sec~on

\Vidin the coronal section, the nurnber of node-node and free-free smts were podzi~ebcorreIated (Pearson correlation: 0.426, p<0.005 - the p-value for the intercept was not significant) 4.1.6 Assessing rnineralization: relationship between BSE and microhardness

In this stud';, the degree of mlieralizaùon of the subchondral and canceilous bone of the femoral head w-as measured directly at an inferior and a superior site (at the sarne site as the histological sections - the speümens used for backscattered imaging were the blocks remaining afier sections for image andysis were removed). Backscattered electron imaging \vas used to conspua mlieralization pronles at these locations, which are descnbed by two parameters, the weighted average and the logit. Ln addition, microhardness tesüng was used to deterxnine the hardness at four sites; an inferoproximal, ïnferodistal, superoprosimal and superodistal site. These four sarnples were adjacent to the sarnples used for backscatcered electron imaging (see Figure 3.7.1). As the microhardness is known to be a function of the calcium content of the bone ~odgskinsonet a/. 1989, Currey and Brear 1990, Evans et al. 19901, it seems plausible that a relationship would exist berneen the microhardness and the Iogit or the weighted average of the rnineralization distribution at an adjacent site. To this end, the measured microhardness of the sarnpIes were compared to the weighted average and logit of the adjacent bone of the sarne type: that is, the weighted average and logit of the rnineralization distribution of the subchondral and canceilous bone at the superior site were compared to the microhardness of the subchondral and canceiious bone, respectiveIy, at the superodistal and superoprosimal site, and the analogous correlations were calculated at the inferior site. Pararnemc (Pearson) and non-parametac correlations were used, and the results are proxlded in TabIe 4.1.2. Intripïngly, the pattern of correlations benveen the rnicrohardness and the mineraiization distribution was similar to that of each of hem and the fluoride content, suggesurig that there may be a 1Lik between ail three measurements. At the superior subchondral surface, the correlations were the mos t prono unced; the weighted average and logit correlated significantly with the microhardness of both the cancellous and the subchondral bone for both the distal and the prosimal site. Figure 4.1.9 illustrates the relationship benveen the microhardness of the subchondral bone at the superodistal site and the weighted average of the subchondral bone at the supenor site. However, at the inferior surface, no significant correlations were observed for benveen the microhardness and the mineralization. These results can be compared to the relationship berneen mineraikation and fluoride content (Section 3.2.5) and that between the microhardness and fluoride content (Section 3.2.6). At the superior site, the weighted average and logit were correlated with the fluoride content for both the subchondral and cancellous bone, but no such correlations were observed at the uiferïor site. Similady, the mïcrohardness \vas significantly correlated to the fl uoride content at the superoprosimai site, for botfi the subchondrai and the canceiious bone, and at the superodistai site, but only for the subchondrai bone- Again, no relation was observed bemeen the rnicrohârdness and the fluotide content at any of the inferior sites. The range of observed values for the microhardness, weighted average, and logit at all four sites were sirniiar. As the subchondral and canceilous bone at the superior surface was hypomineralized compared to that at the inferior surface, the iinear relationship rnay simply be emerging fiom the greater range of values. In this case, the conelauon benveen the microhardness or the mir?eralization parameters and the fluoride content may be secondaq- to the changes in remodeling that are a result ofosteoarthrius. The disease state may there fore be the common link relating the microhardness, rnineralization parameters, and fluoride content. The infenor surface, less affected by osteoarthntis, consequently displayed no relationship benveen the rnicrohardness or the rnineralization of the subchondrd or cancellous bone and the fluoride content. This \vill be discussed in greater detail below (Section 4.2.4). It should be mentioned that the relationship behveen calcium content (andogous to the degree of rnineralization) and the rnicrohardness of bone was observed to be non-linear [Currey and Brear 19901, rather than convincingly linear as shown here. However, this may simply result from the restricted range of minera1 content (from a single species, and a single anatomical site) used in this study, compared to the array of speües and sites that were used by Currey and Brear [1990]. The samples used for backscanered electron imaging were prepared very similady to those for microhardness testing; in both cases, the samples were embedded and then polished. Accordingly, as discussed bdow (Section 4.5.1), further work on this study should include microhardness testing of the actuai sampies used for backscanered eiectron irnagïng. Supeno~ subchondral WA logit

cancellous \VA logit

1nfnor.- subchondral \VA logi t

canceiious WA logit

Table 4.2.2 Pearson conelatbns of BSE and m'cmhardrress

\VA: weighted average of the minerakation distribution

NS: correlation is not siçnificant @>0.05)

1 Significant by non-paramemc test (Kendall's tau) but not by pararnemc test (Pearson). Weighted average

The microhardness of the subchondral bone at the superodistal site was linearly reIated to the weighted average of the mineralization distribution of the subchondral bone ac the superior site (R' = 0.371, pc0.0005) 4.2 Understanding the effects of fluoride on bone quality

4.2.1 Fiuoride iincorporation into bone

Fluoride is incorporated into bone formed during die period of esposure [Boivin and Meunier 19931. At environmentai levels of fluoride, this is largely a phy-sicochemical process; fluoride ions substitute for some of the hydrosyl ions in the hydroxyapatite crystal lattice, resulting in the formation of hydroxyfluorapatite [Grynpas 19901. Similady, as bone is resorbed by osteoclasts and the mineral is dissolved, the fluoride that \vas incorporated into the bone is released. The net rate of incorporation of the fluonde into the bone is therefore dependent on a number of factors: the rate of intalie and escretion of fluonde (which is related to the concentration in plasma, which in tum affects the esuaceilular fluid), the concentration of fluoride in the bone, the rate of resorption, the rate of formation, and the fraction of fluoride present in resorbed bone that is reincorporated into new bone. Potentidy, aii of these factors could vqbetween participants in this study. In this study, the fluoride content of bone increased ~4thage of donor (see Figure 3-23), aithough a logarithmic (that is, log F = age) relaaonship was found to be suonger than a hear relationship (Figure 3.2.4). Other studies have aiso observed an increase in bone fluoride content with age, ciung linear relationships benveen the fluoride content and age for individuds living in fluocïdated @?arkins et al. 1974, Charen et al. 1979, hlhava et al. 19801 or

non-fluoridated [hlhava et (1/. 1980, Richards et al. 19941 regions. However, for this snidy, the age of the donors was heavily biased towards older individuais as the sarnples were obtained during total hip arrhroplasty rather than from cadavers. Esamination of the fluoride-age graphs provided in these four studies [Darkins et al. 1974, Charen et al. 1979, Alhava et al. 1980, Richards et al. 19941 shows that the fluoride content appears, qualitatively at least, to increase more rapidly than the fitted Line predicts for individuais greater than approshately 60 years of age. This suggests that a nvo-part regression may be more zppropriate - a linear regression between the fluoride content and age und the age of appro-simately sixry, and a logarihnic reiauonship, as observed here, afier die age of sisty. The non-hear nse in fluoride content at this age may be associated with the increased rate of remodeliag in bone of postmenopausaI women pui et a(. 19821. Xlhava et a(. [l98O] aiso shomed that the increase in fluoride content with age was steeper for women than for men. Similarly, in this smdy, the logarithmic relationship benveen che £luonde content and rhe age was srronger for women aione (R~0.080,Figure 3.5.2) than for both genders (R'=0.064, Figure 3.2.4) or for men alone, which \vas not significant (Figure 3-5-10). Turner et al [1993] proposed a mathematical model for the uptake of fluonde into the shleton. This model predicts that bone fluonde contenc would plateau afier appro.simate1y 55 years of esposure to fluoridated water; as the model staned at age 25, the fluoride content would continue to increase with age und the individual was 80. However, as water has been artificialiy fluoridated for less than four decades in total, the results of ttus study (that fluoride content increases throughout life) are consistent with this mathematical model. Findy, it should be reiterated that the maximum R' value of the relationship benveen the logarithm of fluoride and age \vas only 0.080, indicatïng that the age of the donor accounted for less than 10% of the variance in rhe fluoride content. This ernphasizes the role of factors other than simple esposure in determining the rate and amount of fluoride incorporation into bone. In particdar, a significant question raised b~rthis smdy is what the determinants of fluoride incorporation are. The range of fluoride contents observed for individuais esposed to fluondated water \vas more than twelvefold, and only a smali portion of variation was accounted for by the age of the donor. While fluoride incorporation is hown to affect the degree of incorporation of other aace elements [Zplün 19731, the exact effects remain conaoversial [Gynpas 19901. Of the elements assayed here, oniy the magnesium has been related to the fluoride content [Zipkin 19731. No relauonship between the rnagnesium content and the Buonde content =-as observed in this study. However, the chlorine content mas positively correlated with the fluoride content, albeit weakly (Figure 3.2.5). This is unexpected as the chlorine should be expected to compete with the £luoride for the OH' lattice position. While the lamce structure is stabilized by subsatuuon of F ions, Cl' ions weaken the smcture. In an aqueous environment, therefore, chlorine atoms are discriminaced againsr during formation ~lurnenthal199O]. It is therefore undear why thïs relationship was obsenred. 4.2.2 Mechanical properties of bone

The yield stress, ultknate corripressive stress, and the energ). to yield of a core of cancellous bone were negatively correlated with the fluoride content of the speümen (Section 3.2.3). The relationship benveen the ultimate compressive stress or the yield stress (both related to the strength of the spechen) and the fluoride content was not improved by segregating the data by either gender or by disease, implying chat it is independent of either. However, the Buoride content was associated wïth the age of the donor (Section 3.2.1) and the strength of canceilous bone is known to decrease with age Feaveny and Hayes 1993, Mosekilde et al. l987], which suggests that the ultirnate compressive stress and yield stress of the cancellous bone map in facr be decreasing with increasing age rather than with increased Buoride content. The suength of the relationship benveen these nvo properties and the fluoride content was similar to that of their relationships to either the age or to the logarithm of the Buoride content, itself linearly related to age Fable 3.2.3). While multiple regressions with both the age and the fluonde content were not performed (as they are hown to be related, the mode1 would be overparamerrbed), this suggests that the effect ofage cannot be esduded. Similady, when the top and bottom qudeby fluoride content were compared, the ultimate compressive stress and the Jield stress were pater for the bottom quade (Section 3.4.3). However, the mean age of the donors in the bottom quade was significantly less than those in the top qude(bottom quade: 62 2 3 years, top qude: 70 t 2 years; see Section 3.4.1). Again, the effecr of age-related bone loss therefore cannot be esduded. These findings are consistent with those observed by other researchers. We relationships benveen bone strength and age were consistentiy observed, no independent relarionship benveen the strength and fluoride content was observed for vertebrae from males from a non-fluoridated region [Stein and Granik 19801, iliac crest from both genders segregated by residence in fluoridated and non-fluoridated regions [Aihava et a/.19801, iliac crest from both genders from a rnked-residency population Fappalainen et a/.19831, or vertebrae denved from both genders in a non-fluoridated region [Richards et d 19941. This is aiso consistent with the lack of effect of consurnption of fluondated water at 1 ppm on the fracture rate, as assessed by epidemiological studies [a meta-anaiysis is provided in McDonagh et al. 20001. However, it should be made dear that the results of this study cannot deunitive$ esclude an effecr of fluoride on bone that is independent of age. In an animai rnodel of fluoride esposure, Turner et a(. [1992] found a biphasic reponse of bone strength to bone fluoride content, with a peak strength associated with a bone fluoride content of appro.simately 1200 ppm. This is very dose to the mean fluoride content observed for the bone specimens from the fluoridated region, whkh was 1033 k 438 ppm. However, severai caveats bear considerauon. The hrst is that their study was perforrned in a rat model, and the response of rats to a given dose of fluonde is an order of magnitude less than in humans Fumer et a/.1992, hgrnar-hhsson and Whi tford 19821. The second is that this study \vas carried out in young (21 day old) rats, which were incorporating fluonde Lito their skeleton during formation and growch, and they were only followed out to four months (adulthood). The applicability of dis approach in modeling the effecrs of low levels of fluoride ingestion in humans, oves the span of decades of bone remodeling but not bone formation (in this study, nearly ail participants were adolescents or adults at the Licepaon of municipal water fluoridation), is therefore unclear. As well as the ultimate compressive stress and the yield stress, the energy to yield \vas aiso correlated with the fluoride content. Again, the linear relationship was weak but significant @' = 0.040, pc0.05). However, in contrast to the ultimate compressive stress and the peld sness, the energy to yield was not correlated with the age or to the logarithm of the Buonde content. This suggests that the energy to +id rnay be assouated with fluoride incorporation in a way that is independent of the age of the donor (although, of course, it is possible that these relationships are simply obscured by the variabili- in the data). The relationship between the energy to yield and the fluonde content was not observed to be gender-dependent However, the strength of the association of the energy to yield and the fluonde content increased slightly when the non-osteoarthritic specimens were escluded (see Figures 3.2.9 and 3.6.5), suggesting char this relationship may be associated with the disease state of the specirnens. The energy to yield is the area under the stress-strain cuve to the yield point (for this study, taken as the 0.2O/0 offset; that is, the point of interseccion of a iine parallel to the iinear region of the stress-suain cuve but shified on the strain axis by 0.2O/0 and the stress-straui cuve itself) Fumer 19891. The area can be approsimated as a nght aimgle wich its base dehed by the strain at yield, its height by the yield stress, and the slope of the hypotenuse by the compressive modulus. Therefore, the negative correlation of the energy to yield and the fluoride content may be due to either a decrease in veld stress with fluoride content, a decrease in modulus wïth constant field strain, or an increase in modulus with consrant yield stress. As the observed relationships berneen the energy at yield and the independent parameters did not pardel those observed for the yield stress, this suggests that another parameter (either the 5dd strain or the modulus) is also chmging with the fluoride content, but the reIationship is too subtie to be observed itself. The logical candidate would be the compressive rnodulus, which was observed to correlate significantly with age but not with fluoride content. Two possibilities therefore exist: the hrst is that the yield stress is constant, but the compressive moddus increases slightly with increasing fluonde content, resulting in reduced yield strain and therefore reduced energy to yield. The second possibiliv is that the yield scrain is constant, and that the compressive modulus decreases siightly with increasing fluonde content, resulting in reduced Jrield stress with fluoride content (as is observed) and, again, reduced energy to yield. Given the large scatter in the data, neither hypothesis cm be de&tively conhed or escluded. However, the latter hypothesis has the advantage of being at least partiy conhed (the yield stress does decrease ethincreasing fluoride content). In addition, the slightly stronger association with the osteoarrbntic samples may result from the increased remodeling associated with osteoarthritis [Grynpas et al: 19911. As newly-formed osteoid is Iess stiff than My-mineralized bone @3odgskinson et al: 19891, this may subtly change the compressive modulus. This also may account for the differences in mechanicd properties observed benveen the specirnens from Toronto and Montreal; the strain at fracture and the energy absorbed to fracture (although not the energy to yield) were both greater for the Toronto specirnens than the Montreal specimens (Section 3.3.3). These differences did not coincide with those observed benveen the top and bottom quades by fluoride content (Table 3.4.3), indicating that they cannot be attributed to the difference in mean fluoride content benveen specimens from the two cities. There was a higher proportion of osteoarthritic patients in the Toronto group (89'0) than in the Montreal group (72O/o), and the mineral content of the Toronto specirnens was also less than their Montreal counterparts Fable 3.3.1). However, it should be noted that no differences were observed in the energy to yield (rather &an the post-yield behaviour) benveen the hvo groups. Three of &e measured compressive mechanical properties were shown to decrease with increasing fluonde content: the ultimate compressive stress, the yield stress and the energy to field. The first nvo also were correlated with age, suggesting chat their relationship to the fluoride content was secondary. In contrast, the energy to yield did aot display a significant relationship with age, suggesting that it may have an independent association with the fluoride content. However, the presence ofosteoarthritis in the femoral head may be affecting this relationship. It should also be noted that the observed variability is high and the relationship weak (R' = 0.040 for ali sarnples, indicating that ody four percent of die variability is accounted for by the fluoride content, assurning that there is no effect of age, which is unlikely). Therefore, fkom this smdy, there is lide convincing evidence that the mechanical properties of cancellous bone in the femord head are affected by the fluoride content of the bone.

4.2.3 Architecture of bone

Image analysis was perfomied for both histologicd sections and for an s-ray of a thick coronai section. As discussed above (Section 4.1 S), the observed results were quite different between the superior and inferior histological samples, and benveen the histologicd samples and the coronal slice. At the superior surface of the femoral head, no relationship was observed benveen any of the measured parameters and the age, fluonde content, or physical density of the cancellous core. There \vas considerable evldence that the superior surface \vas dominated by osteoarrhntis-induced osteosclerosis: the arnount and connectivity of bone %-as significandy greater for the superior surface than the inferior surface for the osteoarrh~ucbone but not for the non-osteoarthntic bone, and for the osteoardintic superior sections compared to non-osteoaahntic superior sections (Tables 3.6.4 and 3.7.1). Sirniiar osteoarthritis-induced changes have been previously reported [Crane et a[ 19901. Other studies hâve dso indicated that os teoarthntis-induced changes obscure age effects prane et al. 1990, Fazzalari et aL 19981, dthough negative relationships between the trabecular bone volume at the superior surface and the age of donor for osteoarrhntic bone [Crane et al. 1990, Fazzda.ri et a/. 19831, w-hich were not observed here. At the inferior surface, the trabecular separation and trabecular number correlated with the age of the donor, consistent with the normal age-related deche of bone at the femur and other sites Fundeen et al. 2000, Kawashima and Uhthoff 1991, Mosekilde 19891. In addition, a number of the rneasured parameters (including the trabecular volume) correlated âppropriately with the physical density of the cancellous core as has been previously observed [Fazzalari et a/,1983, Crane ef aL 19901. No relation was observed benveen any of the measured strut or image analysis parameters and the Buonde content (Section 3.2.5)- Findy, no relationship was observed benveen the image and strut andysis parameters, measured for the coronal section, and the age of the donor or the density of the canceiIous core. However, the trabecular volume and the uabecular separation were observed to have a weak correlation with the fluoride content, consistent with increasing bone (Section 3.2.4). As discussed above (Section 4-15), the measured parameters were not internaily consistent. In addition, the results observed for the coronal sections are not consistent wïth the histoIogical sections- Therefore, the possibility that the fluoride dependence observed for the corond sections is artifactual cannot be exduded. However, it is possible that a weak, negative relationship benveen the fluoride content, independent of age, esists for the trabecular bone volume and the trabecular separation of canceiious bone in the femoral head.

4.2.4 Mineralization of bone

The degree of mineralization as measured by backscactered electron imaging (described by the weighted average and the Iogit) and the rnicrohardness (which is related to the degree of rninerdization) were both found to be linearly and posiüvely correlated with the fluoride content at the superior surface but not the inferior surface (Sections 3.2.6 and 3.2.7). This relationship was more pronounced for the osteoarrhetis-only group (particularly for osteoarthritic females) than for the non-osteoardiritic samples (Sections 3.6.6 and 3.6.7). In addition, the degree of mineralization (as measured by both techniques) was greater for the osreoporotic sarnples than for the osteoarrhritic sarnples at the supenor site, although the differences were only found to be significant for nvo of the comparisons (which is likely to be due to the smd number of samples in the osteoporosis group; Tables 3.6.7 and 3.6.9). Finally, the degree of mherîlization and the microhardness at the superior surface were found to be less than their inferior counterparts for the osteoarthritic samples, but not for the non-osteoarthntic sarnpIes (Tables 3.6.8 and 3.7.2). W of these resuits indicate that the mineralization of the supenor surface is affected by osteoarrhntis: the superior surface is hypornineralized compared to the inferior surface or to non-osteoarthritic sarnples, and that the degree of mineralization is positiveiy correlated with the fluoride content. This latter point is fUrrher substanaated by the greater degree of mineralization @y both backscattered electron imaging and microhardness; TabIes 3.4.8 and 3.49, respectively) for the top quade by fluonde content compared to the bottom qude.

The sïrnplest explanation of these observations is that the 105s of mineral at the superior surface is mediated by the fluoride content; that is, the degree of mineralization is reduced in osteoarthriàs but that the arnount of loss is inversely related to the fluoride content, and that this is the mechcuilsm accounting for the positive linear relationship benveen the degree of rnirieralization and the fluoride content. As the fluoride content \vas measured for a cancellous core at the centre of the femoral head (and prelirninaq work indicated that no signifîcant differences were present in the fluoride content betw-een sites), this suggests that the degree of mineraiization at the superior subchondral surface is a function of the fluoride content of the femoral head as a whole, not the reverse. However, if the rnineraiizaüon was associated with the fluoride content in a way that \vas independent of the presence of osteoarthritis, then a positive relationship would be espected at the inferior surface as well as the supenor surface, which \vas not observed. This suggests that the linear relationship between the degree of mineraikation and the fluonde content at the superior surface is therefore related to the presence of osteoarthritis. The resdting spread in the mineralization may dso account for the linear relationship benveen weighred average of the mineralization distribution and the microhardness that was observed at the superior surface but not the inferior surface (Section 4.1 A). A moddating effect of the fluoride content on mineral loss is consistent \.th the stabitizing effect of fluoride on bone rnineral, making it Iess suscepnble to resorption [Grynpas 1990, Moreno et aL 19971. The hypothesized esplanauon for the observed relationship between the degree of xnineralization and the fluoride content is that increased fluoride content decreases resorption and remodeiing of

the bone, thereby limi~gosteoarthrias-associated changes. Fluoride has been shown to have a similar effect of increasing the resistance to loss of rnineral in rats fed a low-calcium diet Pricsson and Ekberg 19751. A second possibility is that the increased remodeling of bone that is assoùated with osteoarthrïus [Grynpas et a/l 19911 resulced in increased fluoride content at both the superior surface and the canceilous core. The fluoride content is likely to be increasing wvith esposure to fluoridated water Fumer et al. 19921, and increased remodeiing may elevate the nuoride levels (depending on the relative amounts of fluoride in the esisting bone and in the newly-

Discussion formed bone). This wouId suggest that a higher fluoride content would be associated with

more remodeling. However, this wouid therefore suggest a negahve relationship benveen the degree ofrninerdizaaon and the fiuonde content. In addition, the top quartiie by fluoride content, if the fluoride content \vas a result of increased remodeling, shoulci have a lover degree of mineralization than the bottom qude. In fact, of course, the opposice is me as the fluoride content was forrrid to be positively correIated \.ththe degree of mineralization. In the study, the mineralization of the superior (weightbearing, and therefore the

mos t affected by osteoarrhritis) surface of the femoral head \vas significantiy reduced compared to the inferior surface or to non-osteoarthntic femoral heads. In addition, the degree of rnineraiization (measured bÿ either backscattered elecuon imaging or by microhardness) was positiveIy and lineariy related to the Buoride content The most probable esplmation of this is that the presence of fluoride limits osteoarduïtis-related minera1 loss. Finally, it shodd be reiterated that no changes in the mineralization or the microhardness with the fluonde content were observed at the inferior (nonweightbearing, and therefore less affected by osteoarthritk) surface of the femoral head. This suggests that environmentai esposure to fluoride does not have a systemic effect on the degree of rnineraiization of bone. 4.3 Implications of this research

4.3.1 Fluoride and osteoarthritis

The therapsuac use of fluoride administration has focused on osteoporosis. However, as fluoride appears to have a pro tecuve effect on the mineralization of weight- bearing bone, it is appropriate to consider if it has a therapeutic role in controihg

~~e osteoartkntis is generally considered to be a progressive, degenerative disease of the cdage, there is increasing evidence rhat the subchondral and deeper bone rnay play a role [Burr and SchaMer 19971. Early signs of osteoarthritis are associated with elevated , suggescing that bone is involved ac the outset, rather than secondq to cdage degradation prune et a/.1 9991. Sidarly, it has been proposed that cartilage fibrillation may be initiated bv a cornpliance mismatch of the underlying subchondral bone; the cartilage rnay be stressed unduly at the boundary benveen regions of less-mineralized, low-stiffness subchondral bone and norrnai subchondral bone Faclin and Rose 19861. Subchondral osteoarthritic bone has been shown to be less mineralized and less stiff than normal or osteoporouc controls [Li and Aspden 1997. This suggests that controhg the rnineralization of the subchondral bone may modulate the progression of osteoarthritis. Preliminary research has been perforrned evaluating the effect of an anùrcsorptive therapy on the severity of inflarnmatory arthritis in an animal mode1 in which localized inflamrnatory arrhntis is induced by carrageenan injections. This protocol is known to result in decreased rnineralization at the site [Lucas et a(. 19921. In a previous study, the rnicrohardness was reduced in the aEfected knee compared to the controls, and this loss \vas partially prevented by administration of the antiresorptive agent. In addiaon, the therapy also resulted in reduced cdagedegradation, SU~'~SM~that the condition of the subchondral bone plays a role in determiring the severity of the arthatis [Podworny et a/. 19991. It is possible that the presence of fluoride in the osteoarthritic femoral heads not oniy partidy prevents against a reduced degree of rnineralization at the subchondral and canceilous bone deep to the affected cartiiage but therefore aiso has a protective effect on the cardage itself. While this is impossible to assess from the present study, further research is certainiy

4.3.2 Direct and indirect cornparisons of fluoride content

A number ofepiderniologicai studies have been performed to attempt to assess the effect of fluoridated water on bone quality. A cornmon approach is to compare the fracture rate benveen nvo cities, one with and one without fluoridated water Fehmann et al. 1998,

Sirnonen and Laitenen 1985, Phipps et al. 1997, Cauley et al. 1995, Jacobsen et ai! 1993, Suarez-Almazor et ai! 19931- One of the weaknesses of these studies is that the nature of the population cannot be controlled, and the possibility that differences in the population esist in addition to the parameter under study (such as fluonde esposure) cmotbe esduded, In this study, the data \vas segregated both by city of residence poronto us. Montreal) and by fluoride content (top vr. bottom quade). The results were illumina~g. The fïrst point of note is the wide variance in the amount of fluoride incorporated into the bone. The original irnpetus to recniit participants in both uties was to obtain a wide range of incorporated fluoride content. Toronto donors were in fact observed to have more fluoride incorporated into their bones, on average. However, the range of observed values for the Toronto residents (192-2264 ppm) cornpletely included that observed for Montreal residents (270-1200 ppm). This conclusively indicates that simple esposure to fluoridated water is only one of a number of factors that influence the amount of incorporated fluoride. This also raises an important question that was not addressed in thic study: in addition to esposure to fluoridated mater and other sources of environmental fluonde, what detemiines the fluoride content of bone? Cornparison of the differences observed between the top and bottom quade by fluoride content (Section 3.4) and those observed benveen Toronto and Montreal residents (Section 3.3) again confirms that city of residence is not a good prosy for fluonde content. For esample, within the mechanical propenies, the strain at failure and the energy absorbed to failure were greater for the Toronto specimens than the montrea al specimens. This would suggest that water fluoridation has a positive effect on mechanical properties (since the energy required to fracture, ~hichis perhaps the most clinicaliy significant mechanical propeq, \vas greater). However, when the sarnples were segregated by fluoride content directly, into top and bottom quartiles, this was not observed. Instead, it was observed that the yield stress and the uitimate compressive stress were lower for the high-fluoride group than the low-fluoride group, Sugges~gthat fluoride incorporation has a deleterious effect on mechanical properties. However, in both sets of cornparisons there were confounding factors; the quades w-ere roughly disease- and gender-matched, but the top-quartile donors were older, on average. The montrea al and Toronto donors were roughly age- and gender- matched, but the distribution of disease States were somewhat different. These results ïndicate that case-control type studies are not appropriate to study the effect of lifelong Buoride exposure on bone. Instead, studies that relate the measured parameters of bone quality to the fluoride content of the bone, as a contïnuous variable, while controlling for age and gender (and preferably with cadaveric samples) shodd be used to provide derutive informarion.

4.3.3 Fluoride and bone quality

Three broad conmbutors to bone quality were assessed in this study: the mechanical properties, the architecture of bone, and the material of bone (in particdar, its degree of rnineralization). However, ody minimal fluoride-related changes to the mechanical properties, independent of age, were observed in this study. Similarly, image and smt analysis of histological sections showed no relationship benveen the amount and co~ecuviryof bone and the fluoride content. \'C"nile some relauonships were obsemed for image analysis of whole coronai sections, the rneasured parameters were interndy inconsistent; the possibiiity that these results are artifactuai therefore cannot be escluded. Finally, the parameters desctibing the rnineralization distribution, as weii as the microhardness, were only correlated with the fluoride content at the superior surface. If osteoarthritis-associatedeffects are escluded, there is no evidence of a relationship between the degree of rnineralization of the bone and the fluonde content. The absence of an observed relationship between the measured parameters of bone quality and the fluoride content cannot defitively esdude the possibility that such a relationship esists. meit is clear that ïndividuals exposed to fluoridated water have more fluonde, on average, incorporated into their bone, there is no evidence suggesting that this has any effect on bone quality. These results are consistent with the Eindings of epidemiologicai studies WcDonagh ef a/.2000]. 4.4 Future work

4.4.1 Analysis of existing specimens

A nurnber of specimens were prepared for anaiysis but not analysed due to tirne constrauits. Three major sets of analyses shodd be completed. The first is the grading of osteoarthritic samples. The severity of osteoarthritis in ody 18 of the 75 osteoarthritic sampIes \vas determined. The results from diese indicated that in the majorïv of cases the osteoarrhritis is very advanced (the mean score was 13.3 out of a possible 14, and only three individuds scored less than 14). However, it is possible that correla~gthe seveaty of osteoarrhritis to the measured parameters of bone quality would clan+ some of the effects, confirrning whether the variations in the parameters (such as the relationship benveen rnineralization and fluoride content at the superior surface) are due to osteoarthnus or to

O ther causes. The second major area where analysis may provide significant insight is in the histomorphomeq of specimens. Whde the amount and dismbution of bone is measured by image analysis of histological sections as weli as histomorphometry, histomorphometry is the only method which measures parameters reflective of the amount of rernodeling, such as percentage of bone covered with osteoid or with eroded surfaces. This may con& the findings observed in other studies that osteoarthrïtis is associated wïth increased bone formation [Grynpas et a/. 19911, which was not frrmly established in this study. Again, evidence of increased remodeling may furrher suggest that the response of bone to fluoride incorporation is dependent on disease state. Also, as the image analysis sarnpIes, his tomorp hometry samples, and backscattered imaging samples were ail derived from a single embedded specimen of bone, this dl enable to us to determine the relationship benveen remodeling, bone formation, and mineralization at the superior and inferior site. Finaily, the embedded bone speurnens that were used for backscatcered elecnon imaging were diarnond-~oiished(to eliminate topographical effects) before being carbon- coated. The polishing procedure is the same as is used for rnicrohardness testing. This suggests that an opportunity esists to direcdy compare these nvo methods of assessing the degree of mineralization @y mechanical means and one by chemicd composition) by measuing the microhardness of the same samples and regions that were previously used for backscattered elecuon irnaging-

4.4.2 Patient information

One underutilized advanrage of this study is that, as it is not an epiderniological study; detailed information about individual donors cm be obtained. At the cime that informed consent was obtained kom participants, hep were asked if they would be willing to be contacted for a follow-up interview and a contact number was obtained. A questionnaire has been drafted that assesses the degree of esposure to fluoride (ïïduding length of residence in a region widi or wïthout fluoridated water, use of fluoride-containing oral hygiene products, dietary preferences for foods and beverages containing relatively high amounts of fluoride, such as tea and seafood, and sdarquestions). This would dow us to explore the role of diet and other factors in the wïde variance in fluonde contents that was

O bserved.

4.4.3 Cadavenc samples

The mos t significant failing of this s tudy is that the femoral heads were obtained from donors at total hip arrhroplasty. Lhile this simplifies issues of sarnple acquisition and consent, nvo major drawbach emerge. The tirsr is thar the population of donors is heavily skewed towards older individuals. The mean age of the donors in this smdy \vas 67- hardly representative of the population as a whole. In addition, because fluoride is incorporated into bone d&g formation, it is Likely that individuais who were exposed to fluoridated water during their childhood and young adulthood (that is, during periods of bone modeiing) would respond differendy to bone than individuals who were fisr exposed to fluoridated water as adults (that is, durl-ig bone remodeI'tz&, as is the case with most of the participants in this study Fumer et a/.1993, Whïtford 19901. The second major drawback is that the femorai heads obrained are not normal. Ln the presenr study, donors were undergoing surgery prirnarily as a resdt of osteoarthritis, with a significant fraction of donors who were diqposed wïth osteoporosis. These diseases ma)- alter the structural, mechanical, and remodeling behaviour of bone (as discussed in Section 4.2.3) in a way that alters its response to fluor.de esposure. The soIutÏon to this is to peïform a studp widi cadaveric bone, as previously reported in the literature pchards efaL 1993, Stein and Granik 19801. In both of rhese studies, normal hurnan vertebrai bodies were renieved from cadavers across a wïde age range. The reason that this was not done in the current study was more pragrnatic than suentific; current regdations in Ontario governing tissue donation for suentific purposes require the consent of nest of kin zit the àme of death. Needless to Say, this is difficult for both the farnily and the researcher. However, one of the advantages of Living donors, lvhich has not been Myesploited in this study, is that detailed information cm be obtained regarding their lifetime fluoride esposure (see above). 5: Summary and Conclusions An e+qerfi~ man ~vhohax made a// the mik'akes, whicb can be made, in a uev nan-oivFe//d. 5.1 Sumrnary of Results

The incorporation of fluoride ùito cancelious bone \vas quantified in this stu.The relationship between three broad conmibutors to bone qualis. and the fluoride content were assessed in this study: the mechanicd propemes, the architecture of bone, and the material properties of bone (in particular, its degree of mineralization). Fluoride incorporauon into cancellous bone at the femoral head ranged from 200 to 2300 ppm. The mean amourit of fluoride incorporated into femoral heads of individuals residing in a fluoridated region (Toronto) \vas greater than in those from a non-fluoridated region (Montreal). However, the range of fluoride contents was ver). large, indicating that factors other than simple esposure are important in determining the amount of incorporated fluoride. The amount of fluoride incorporauon increased with age of donor. Ody minimal fluonde-related changes to the mechanical propemes, independent of age, were observed in this study. The ulàmate compressive stress, yield stress, and energy to yield all declined with the fluoride content. However, the evidence suggests char, for the former nvo paramerers, this dedine is secondary to a decline with age. The energy to yield may potentidy have a very weak relationship to fluoride that is independent of age. Image and suut analysis of histoIogicaI sections showed no relationship benveen the amounr and connecavig of bone and the fluoride content. V(lhile some relationships were observed for image anaiysis of whole coronal sections, the measured parameters were internaily inconsistent; the possibility thac these results are artifactual therefore cannot be escluded. Finaiiy, the parameters describing the mineraikation distribution, as weli as the microhardness, were correlated with the fluoride content ac die superior surface. No relationship was observed behveen the degree of mineralization at the inferior surface and the fluoride content. This suggests chat fluoride does not have a systemic effecc on bone minerdization at rhese levels. However, it does indicate that fluoride may influence osteoarthritis-induced remodeling at the supenor surface.

Summary and Conchmons 5.2 Conclusions

It is necessary to reiterate that the femorai heads for this study came from a subset of individuals that differed significantiy from the population at large: the mean age of the individuals was approsimately 70, and pathologies were present at the femorai head. Care must therefore be taken in estrapolating from this smdy to a population of young individuals, or to normal bone.

Beaxkg those caveats in mind, then, the foilowing conclusions cmbe drawn fiom this smdy:

9 Residing in a city with municipal water fluoridated at 1 ppm results in, on average, increased fluoride incorporation into bone. However, the wide range of fluoride contents observed for individuals esposed to fluoridated water indicates that simple esposure is not the ody determinant of fluoride content in bones. Simple residence in a region with fluoridared water does not substantially alter bone qualiy. ii) Esposure ro fluoridated water (and fluoride incorporation in the range of 200-2300 ppm) does not alter the architecture of bone in a systemaùc way. iü) A weak dependence on the energy to yield and the fluoride content mal- be present, although the influence of osteoarthntis cannot be escluded by this study. No other mechanicai propemes were affected by fluoride incorporation. iv) Fluoride incorporation had no systemic effect on the degee of rnineralization.

V> Incorporated fluonde may have a protecuve effect on subchondral bone in osteoarthntis, limiting the degree of osteoarthritis-induced remodehg and the resultant lowering of the degree of rnineralization of the bone. Appendix: compressive testing This appendis provides information about the compressive mechanicd testing of canceiious bone, further to that provided in Section 2.4.1. The mechanical parameters measured Li tbis study are illusuated in Figure Al. The ultimate compressive stress \vas defined as the peak Ioad achieved pnor to the tirst monotonic decrease in load. As mentioned above, this is associated wïth the onset of trabecuiar fracture. The strain at ultirrîate compressive stress is simply the suain in the sample at this point. The energy to faiiure is the total energ-y absorbed to the point of ulturiate stress, calculated by numerically integrating the stress-strain curve. This is the energ required to deform both the material and the structure itself up to the point where failure occurs. The compressive modulus of the sample was the slope of a regression line fitred through the linear region of the curve. This is associated wïth the stiffness of the trabecular bone structure. The yield point (or a point at which the stress-suain curve deviates from Iinearity by a standardized amount) was dehed using the arbitrary but consistent and standard method of detennining the intersection of the 0.2% offset line and the stress-strain cuve [Turner 19891. The slope and inrercept of the linear regression used to determine the modulus were used to determine the equation of a line paraiiel to rnodulus line (that is, wïth the same slope), but shified towards the right by a strain of 0.2%. This line was then plotted on the smegraph as the acquired stress-strain curve and the stress at the point of intersection kvas reponed as the 'yield stress.' In a solid material, this wodd nomindy represent the onset of plastic deformation. However, as reversible bending of the uabecular structure may also be occurring, this point simply indicates the point of deviarion from linearicy (see Section 4.2.4). The stress-strain cuve was numencally integrated to this point to determine the energy to yield, or the resilience. Tneoreticdy, at Siis point, the energy absorbed by the sample cm be released upon unloading. However, as discussed in Section 4.1.4, reversible bending of the sarnple may aiso occur &eer this point. 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Strain (mmfmm)

F&pre A.2 Measuredparamerers of compressive mechamcal resdng

YS: yield stress UCS: dtimate compressive stress

See test for Merdetaiis.

Appert dix References

Aaron JE, de Vernejoui MC, Kanis JA (1992) Bone hypemophy and crabecular gneration in Paget's disease and in fluoride-treated osteoporosis. Bone Miner 17:399-413 mava EM, Olkkonen H, Kauranen P, E(ari T (1980) The effect of drhhgwater fluoridation on the Buonde content, scrength and mineral density of human bone. Acta On'hop Jcand 5l:413-42O

Allolio B, Lehmann R (1999) Drinking water fluoridauon and bone. E-9 ChEndom'nof Diabetes loi:1 2-20

Amir G, Pirie CJ, Rashad S, Revel1 PA (1992) Remodehg oEsubchondrai bone in osteoarthntis: a histomorphometry study. J Ckn Pufh145:990-992.

Amprino R (1 961) Mïcrohardness testing as a means of analysis of bone tissue biophysical prop erties. In: Biomechatricc11Stzdies of the ~\.Iz~ztldo-Ske/etaLSy~fem. FG Evans, ed. Charles C Thomas, Publisher, Springfield IL.. pp. 20-48 lingmar-Mansson B, Whitford GM (1982) Plasma fluoride levels and enarnel fluorosis in the rat. Canés Res 15:334-339

Bang S (1978) Biophysical study of compact bone tissue in endemic fluorosis. In: Fhodeand Bone. B Courvoisier, h Donath and CA Baud, eds. H. Huber, Berne. pp.77-81

Banse X, Devogelaer JP, hfunüng E, Delloye C, Cornu O, Grynpas M (2001). Inhomogeneity of human vertebral cancellous bone. Bone (in press)

Beary DF (1969) The effects of Buonde and low calcium on the physical propercies of the rat femur. Anat Rec l64:305-3 1G

Beer FI?, Johnston Jr ER (1 985) i\.lechanics o/llltenafs: SI Metni- Enifion. ZvkGraw Hiu Ryerson Ltd., Toronto.

Birkenhager-Frenkel DH, Courpron P, Hupscher EA, Clermonts E, Couanho MF, Schrnitz P, Meunier PJ (1988) Age-related changes in cancellous bone skcture. A dimensional study in the transiliac and iliac cres t biopsy sites. Bone Aldinet-4:l97-2lG

BIurnenthal NC (1 990) The in vitro uprake of trace elements by hydrohyapatite. In: Trace LW.and Fhon'de in Bones and Teeth. ND Priest and FL Van De Vyver, eds. CRC Press, Boca Raton. pp. 307-313

Boivin G (1975) iMorphomemc analysis of fluorotic bone tissue. In: Fhoride andBone. B Courvoisier, A Donath and CA Baud, eds. H. Huber, Berne. pp. 42-46 Boivin G, Baud CA (1978) Osteocyte lacunae of fluoro tic bone tissue: rnicroradiographic, ultxasuuctural and morphomemc studies. In: Ffioride and Bone. B Counroisier, A Donath and CABaud, eds. H. Huber, Berne. pp. 47-55

Boivin G, Chavassie= P, Chapuy MC, Baud CA, Me=?ier PJ (1986) Profil histomorphométrique de la fluorose osseuse induite par l'lngesuon prolongée d'eau de Vichy Saint-Yorre. Comparaison avec Ie taux de fluor osseux. PdBio134:33-39

Boivi? G, Chapuy M-C, Baud CA, Meunier PJ (1988) Fluoride content in human iliac bone: results in controls, patients with fluorosis, md osteoporoùc patients ueated with fluoride. J Bone Miner Res 3:497-502

Boivin G, Chavassieus P, Chapuy MC, Baud CA, Meunier PJ (1989) Skelemi fluorosis: histomorphometrïc analysis of bone changes and bone fluonde content in 29 patients. Bone IO: 89-99

Boivin G, Chavassieux P, Chapuy MC, Baud CA, Meunier PJ (1990) Skeletal fluorosis: histomorphome~chdings. J Bone Miner &.JS(Suppl1): S 185-189

Boivin G, Meunier hiJ (1990) Fluoride and bone: tosicological and therapeutic aspects. In: The ~Metabolicand Mo/edar Ban> ofAcqrrired Diseare, RD Cohen, B. Lewis, I

Boivin G, Meunier PJ (1993) Effects of fluoride on bone minerai. Res ChFonrtr.~ 1513-19

Boivin G, Duriez J, Chapuy M-C, Flautre B, Hardouin P, Meunier PJ (1993) Relationship benveen bone fluoride content and histological evidence of calcification defects in osteoporotic women treated long tem svith sodium fluoride. Osteoporo~izInt3:204-208

Bonjour JP, Caverzasio J, Rizzoli R (1993) Effects of fluoride on bone cells. Res Clin F0nm.r 15:13-19

Bouvier ?VI(1989) The biology and composition of bone. In: Bone Mechanicz. SC Cowin, ed. CRC Press, Boca Raton. pp.1-13

Boyce TM, Bloebaum RD, Bachus KN, Skedros JG (1990) Reproducible method for calibrating the backscattered electron signal for quantitative assessment of mineral content in bone. Jcan ~Microx4:59 1-603

Boyde A, Jones SJ (1983) Back-scattered electron imaging of skeletd tissues. Metab Bone Dix MRer 5:l45-l5O

Boyde A, hfacomachie E, Reid SA, Deliing G, ~MundyGR (1986) Scanning electron rnicroscopy in bone pathologr;: review of methods, potennal and applications. Jcan Efectron iVfiirox TV:lS37-1554

Bracci PM, Bull SB, Grynpas MD (1998) Analysis of compositional bone density data using log ratio transformations. Biome~k54:337-349 Brown TD and Ferguson Al3 (1980) Mechanical properiy dismbutions in the canceilous bone of the human prosimal femur. Acta Odqû Scand 51:429437

Bruno RJ, Sauer PA, Rosenberg AG, Block J, Sumner DR (2999) The pattern of bone minerd densis. in the prosimal femur and radiographie signs of early joint degeneraüon. ] RSez/mafol26:636-640

Bruyére Garnier K, Dumas R, Rumehart C, Mot ME (1999) Mechanical characterization in shear of human femoral canceilous bone: torsion and shear tests. khi! Etig Phy 21:641- 649

Burr DB, Schaffler LW(1997) The involvement of subchondral rnineralized tissues in osteoarthrosis: quantitative microscopic evidence. ~'LlimoosRes Tech 37343-357

Burt BA (1 992) The changing patterns of systernic fluoride intake. J Dent R~J-7l:lZ8-lZ7

Callis ter Jr \KI3 (1 997) LLIa~enahSnènce and Engilleen'ng: AnIntmdt~cr~On. John Wdey and Sons, New York.

Carlson CS, Loeser RF, Purser CB, Gardin JF, Jerome CP (1996) Osteoaahritis in cynomolgus macaques. III. Effects of age, gender, and subchondd bone thickness on the severity of disease. J Bone Min Rer 11:1209-17

Cdstrom D (1954) Mcrohardness rneasurements on single haversian systerns in bone. Expenénfla 10:1 7 1

Carter DR, Hayes YUC (1 976) Bone compressive strength: the iduence of density and strain rate. Snénce 194:1174-1176.

Cauley JA, Murphy PA, Riley TJ, Buhari -*I (1995) EÇfects of fluoridated drinking water on bone rnass and fractures: the study of osteoporotic fractures. J Bone iVher Re.s 10:1076- 1086

Centers for Disease ControI (1991) Public health service report on fluoride benefits and risks. j Am k1edAs.j-oc 266:lOGi-1062, 1066-1067.

Chachra D, Turner CH, Dunipace AJ, Grynpas MD (2 999) The effect of auoride treatrnent on bone mineral in rab bits. Ca/nzThfeInt 64:345-35 1

Charen J, Taves DR, Stamrn JW, Parkins FM (1979) Bone fluoride concentrations associated with fluoridaced drinking water. CalnYTizme Int 27:95-99

Cheng PT,Bader SM (1 990) Effects of fluoride on rat canceiious bone. Bone Miner 11: 153- 161 CiareUi TE, Fyrhie Dl?, Schaffler ?dB, Goldstein SA (2000) Variation in three-dimensional cancellous bone architecture of the pro-ximal témur in female hip fractures and in contxols. J Bone r&er Rer 15:32-40

Clarke BL, Ebeling PR, Jones JD, Wahner HW, 07FallonWM, Riggç BL, Fitzpamck LA (1996) Changes in quantitative bone histomorphometry in aging healthy men. J Ciin Endoniploi ~Lletab81:2264-70

Clovis J, Hargreaves JA (1988) Fluoride intake Erom beverage consumpüon. Comnzz~nDent O ra/ Epidenzioi16:1 1 - 15

Cooper C, Wickham CA, Barker DJ, Jacobsen SJ (1991) Water fluoridaaon and hip Eracture. ]Am Med Assac 266:5 13-514

Cow-in SC (1 9 89) Bone ~Mecbanicj.CRC Press, Boca Raton.

Crane GJ, Fazzalari NL, Parkinson LH, Vernon-Roberts (1990) Age-related changes in femoral trabecuhr bone in arthrosis. Atl'a Orthop Srand 61:421-6

Currey JD, Brear K (1990) Hardness, Young's modulus and yield stress in mamrnalian rnineralized tissues. J Mater JL~Mater A4ed 2: 14-20

Dean HT (1 9%) C hronic endemïc dental fluorosis (mo ttled enarnel). J Am ibIed Asxoc 107:1269-1273

Dequeker J, hfohan S, Finkelman RD, Aerssens J, Baylink Dj (1993) Generalized osteoardintis associated with increased insulin-like growxh factor types 1 and II and transforrning growth factor in cortical bone Gom the iliac crest: possible mechanism of increased bone density and protection against osteoporosis. An%Rhez~m 36:1702-1708

Dempster DW, Ferguson-Peli MW, Meilish RVFE, Cockran GVB, Xie F, Fey C, Horbert W, Horbert W, Parisien M, Lindsay R (1993) Relationships between bone structure in the iliac crest and bone structure and snength in the lurnbar sphe. Orteopor Int 3:90-96

Einhorn TA, Wakley GJX, Linhart S, Rush EB, Maloneÿ S, Faierman E, Baylink DJ (1942) Incorporation of sodium fluoride inro cortical bone does not impair the mechanicd properties of the appendicular skeieton in rats. CakfTirrne Int 5i:127-131

Ekstrand J, Spak CJ (1990) Fluoade pharrnacokineucs: its implications in the fluoride treatment of osteoporosis.] Bone miner Res 5:S53-SG1

Ekstrand J, Ziegler E, Fomon S (1992) Pharmacokinetics of fluoride in infants (abstract).] Dent Res 71 (Spec Iss):52S

E~CSSO~Y, Ekberg O (1 975) Dietetically provoked general and alveolar in rat and its prevenüon or cure bp calcium and £luonde.] Periodon Res 10:256-269 Evans GP, Behiri JC, Currey JD, Bonfield W (1990) hiïcrohardness and Young's modulus in comcal bone exhibitkg a wide range of mineral volume fractions, and in a bone analogue. ]~Wer Jk Mater Med l:3 8-43

Faccini JM, Teoua SPS (1974) Histopathological assessement of endemic skeletal fluorosis. Calcf Ti~xtie&~es16:45-57

Fazzalasï NL, Darracott J, Vernon-Roberts B (1 983) A quantitative description of selected stress regions ofcanceiIous bone in the head of the femur using automatic image anaiysis. Metab Bone Dir Mat Res 5:1 19-25

Fazzaiari NL, Darracoct j, Vernon-Roberts B (1985) Histomorphometrïc changes in the trabecular structure of a selected stress region in the femur in patients with osteoaahritis and fracture of the femorai neck. Bone 6:125-133

Fazzalari NL,Fornvood MEZ, Smith K, Manthey BA, Herreen P (1998) Assessment of cancellous bone quality in severe osteoarthrosis: bone minera1 density, mechanics, and microdamage. Bone 22:381-388

Fazzalari NL, Parkinson IH (1998) Femoral trabecuiar bone of osteoarthritic and normal subjects in an age and sex matched group. Orteoa&nt Carhï6:377-82

Fratzl P, Roschger P, Eschberger J, Abendroth B, Naushofer K (1994) Abnorrnai bone mïneralization after fluoride treament in osteoporosis: a smd-angle x-ray scatte~g study. ]Bone miner Res 9:1541-1549

Fratzl P, Schreiber S, Roschger P, Lafage M-H, Rodan G, Maushofer K (1996) Effects of sodium fluoride and alendronate on the bone mineral in rninipigs: a smaii angle s-ray scattering and backscattered electron imaging study. J Bone Miner Res 11:248-253

Gevers G, Dequeker J, Geusens P, Nyssen-Behets C, Dhem A (1 989) Physical and hisromorphologicai characteristics of iliac crest bone differ according to the grade of osteoadxitis at the hand. Bone 10:173-177

Gibson LJ (1 985) Mechanical behaviour of canceiious bone. J Biomech l8:3l7-328

Gordon SL, Corbin SB (1992) Summaq of workshop on d~kingmater fluoride influence on hip hacture on bone hedth. O~teopomsz5Int 2:109-117

Grote Hj, Amling M, Vogel M, Hahn M, Posl N, Delling G (1995) Intervertebral variation in trabecuiar microarchitecture throughout the normal spirie in relation to age. Bone l6:3Ol-308

Gruber HE, Baylink DJ (1991) The effects of fluoride on bone. Ch Orthop 267:264-277

Grynpas MD, Simrnons ED, Pritzker KP, Hancock RV, HarrisonJE (1 986) 1s fluoridatcd bone different Erom non-fluoridated bone? In: Cell~MediateACaInicafton andiMatnk VesirIes, S. Yousef Ali, ed. Elsevier Science Publishers. pp. 409-414 Grynpas LW,Holmprd D (1988) Changes in quaiiq of bone mineral on aging and in disease. Jcarz Mims 2:1045-1054

Grynpas MD (1990) Fluoride effects on bone crystais. J Bone Miner Res 55169-SI75

Grynpas MD, Alpert B, Katz 1, Liebennan 1, Pritzker KPH (1991) Subchondral bone in osteoarthritis. CaLn~TiweInt 4920-26

Grynpas MD, Acito A, DU~T~U~U31, Mertz B, Very J (1992) Changes in bone rnineralization and mechanicd properties due to long-tenn (1 year) administration of pamidronate (hPD) to adult dogs. Osteopor Int 2:74-8 1

Grynpas MD, Hancock RGV, Greenwood C, Turnquist J, Kessler MJ (1993) The effects of diet, age, and ses on the minera1 content of primate bones. CaIaf Tisme Int 52:399-405

Grynpas MD, Holrnyard DP, Pntzker KPH (1994) Bone mineralizaüon and histornorphometry in biopsies of osteoporotic patients treated wirh fluoride. Celx Mater 4:287-297

Grynpas MD, Chachra DyLimeback H (2000) The action of fluonde on bone. In: The OsteoporonJ Prinlr. JE Henderson and D Goltzman, eds. Cambridge University Press. pp. 3 18-330

Harrison JE, LMcNeilI KG, Sturuidge \VC, Bayley TA, LMurray TM,Williams C, Tarn C, Fornasier V (1 981) Tnree-year changes in bone minerd mass of postmenopausal osteoporoüc patients based on neuuon activation andysis of the central tbird of the skeieton, J ChEndonino1 hietab S2:75 1 -758

Heaney RP (1992) The natural history of vertebral osteoporosis. 1s low bone mass an epiphenomenon? Bone 13 (Suppl2):S23-S26

Heaney EW (1 993) 1s there a role for bone quality in fiagilit). fractures? Ca/nJfTrjmeInr 53 (Suppl 1):S3-S5.

Hodgskinson R, Currey JD, Evans GP (1989) Hardness, an indicator of the mechanical competence of cancelious bone. J Orlhop Res 7:754-758

Horowitz HS (1991) Appropriate uses of fluoride: considerations for the '90s. Inwoducrory paper. ]Pd -Hea/th Dent 1:20-22

Hui SL, Wiske PS, Norton JA, Johnston Jr CC (1982) A prospective study of change in bone mass with age in posmenopausai women. ] Chron Dis 357 2 5

Ishiguro, 11, Nakagala, H., Tsuboi, S., Narita, N., IGto, IC, Li, J., Kamei, H., Yoshioka, I., hiiyauchi, EC, and Hosoe, H. (1993). Distribution of fluoride in cortical bone in human rib. Caln~TissueInt 52:278-282 Ismail AI (1994) Fluoride supplements: curent effectiveness, side effects, and recomendations. Commw Dent OralEpidemiol22:164472

Jacobsen SJ, Goldberg J, Cooper C, Lockvood SA (1992) The association betureen wacer fluoridation and hip fracture among white women and men aged 53 years and older. A national ecologic study. Ann Epidemiol2:6 2 7-626

Jacobsen SJ, O'Fdon V7A4, Melton LJ III (1 993) Hip fracture incidence before and afier the fluoridation of the public water supply, Roches ter, R/Linnesota. Am/ PzMc Healll83:743- 745

Kaminsliy LS, Mahoney MC, Leach J, Melius J, Mïüer MJ (1990) Fluoride: benefits and risks of exposure. Cnt Rev Oral Biol~W1:261-281

Kaplan FS, Hayes WC, Keaveny TM, Bos key A, Einhom TA, Iannotti JP (1 994) Form and function of bone. Ln: On%cpaedicBasri- SnPnce. SR Simon, ed. ilmerican Academy of Orthopaedic Surgeons. pp. 127-184

Kawashima T, Uhthoff HK. Pattern of bone loss of the proximal femur: a radiologie, densitometric, and histornorphomemc study.] Orlhop Res 9:634-640

Keaveny TM, Hayes WC (1993) A 20-year perspective on the mechanical propemes of trabecular bone. Tram AJLVE115534-542

Keaveny TM,Guo XE, Wachtel EF, MuIIahon TA, Hayes WC (1994) Trabecdar bone eshibits Mylinear elastic behavior and yields at low strains. ]Biomecb 27:1 127-1 136

Keaveny TM, Pinilla TP, Crawford RP, Kopperdaid DL, Lou A (1997) Systematic and random enors in compression testing of trabecular bone. J Odop EèS 15:102-11O

ICleerekoper M (1996) Fluoride and the skeleton. Cnt Rev Ckn Lab Sn' 33:139-16 1

Kors tjens CM, Spruijt RJ, Mosekilde L, Geraets WGM, van der Stelt PF (1998) Agreement beween radiograp hic and pho tograp hic trabecuiar patterns. Acta Radio1 39:625-63 1

Kraenzlin ME, Kraenzlin C, Farlev SMG, Fitzsirnmons RJ, BayLLilr DJ (1990) Fluoride pharmacokinetics in good and poor responders to fluoride therapy. ]Bone Miner Res 5 (Suppl 1):S49-S52

Lacrois P (1 971) The internai remodeling of bones. In: The Biocbemistry and Plynolo~of Bone (pded) Vohm~eIII. GH Bourne, ed. Academic Press, New York. pp. 119-144

Lafagc M-H, Bdena Ti Bade MA, Shea M, Seedor JG, Nein H,Hayes WC, Rodan GA (1995) Comparison of aiendronate and sodium fluorcide effects on cancellous and comcal bone in minipigs: a one-year study. ] Clin Inves~95:2127-2133 Lappalainen R, Ehuttila MyLarnmi S, Aihava EM (1983) Fluoride content related to the elemend composition, mineral density and strength of bone in healthy and chronicaüy diseased persons. J Cbron Dis 36:707-713

Lees S (1981) A model for bone hardness. J Biomech 14561-567

Lehmann R, Wapniarz M, Hofmann B, Piper B, Haubie 1, Wolio B (1998) DniikLig warer fluoridation: bone minerai density and hip fracture incidence. Bone 22:273-278

Li B, Aspden RM (1997a) ~Mechanicaiand material propemes of the subchondd bone plate hrom the femoral head of patients widi osteoarrhritis or osteoporosis. Ann Rhezm Dis 56:247-254

Li B, Aspden (199%) Cornposiaon and mechanical propedes of canceiious bone fiom the femoral head of patients with osteoporosis or osteoarrhntis. ]Bone Miner Rer l2:64l- 65 1

Linde F, Hvid 1 (1989) The effect of consuaint on the mechanical behaviour of ~abecular bone specimens. J Biomecb 22485490.

Linde F, Nargaard P, Hvid 1, Odgaard A, Saballe K (1991) Mechanical properties of trabecular bone. Dependency on strain rate. j Biomech 24:803-809.

Linde F, Hvid 1, Madsen F (1992) The effect of specimen geometry on die mechanical behaviour of trabecular bone speürnens. J Biomecb 25:359-368.

Lucas S, Bogoch E, Nespeca R, Grynpas h1D (1992) Bone changes induced in a rabbit model of esperimentai arthritis. Eur] ~VItcsnlfo.rke/Res 1:121-129

Lundeen GA, Vajda EG, Bloebaum RD (2000) Age-related cancellous bone loss in the proximal femur of Caucasian femaies. Orteoporor Int 11:505-511

Lundon K, Dumitriu MyGrynpas M (1994) The long-term effect of ovariectomy on the quality and quanti- of cancelious bone in young macaques. Bone Miner 24:l35-149

Mamelle N, Meunier PJ, Dusan Ft, Guliaume At, Marcin JL, Gaucher A, Prosr A, Zeigler G, Netter P (1 988) Risk-benefit ratio of sodium fluoride treatemnt in primary venebral osteoporosis. Lancet 2:36l-365

Mankin HJ, Dorfman H, Lippieilo L, Zarins A (1971) Biochemicd and metabolic abnormalities in artïcular cartilage from osteoarrhntic hurnan hips.1 Bonejoint Snrg LIS 53523-527

~Martenshf, Vari Audekercke R, Delport P, De Meester P, Mulier JC (1983) The mechanical characterisucs of cancdous bone at the upper femoral region.] Biomecb 16:971-983.

McCrum NG, Bucidey CP, Bucball CB (1 988) Pnnc)fe.s o/Po&zer Engneen'ng. Oxford University Press, N'Y. McDonagh MS, Wting PF, Wilson PM, Sutton AJ, Chestnutt 1, Cooper J, hfisso K, Bradley M, Treasure E, IUeijnen J (2000) Systematic review of water Buoridauon. Bn't Med] 321:855-859

&feUishR, Garrahan N, Compston J (1989) hge-reiated changes in trabecular width and spacing in hurnan Sac crest biopsies. Bone Miner 6:331-338

Memagh JR, Harrison JE, Hancock R, h3cNei.U KG (1977) Measurement of fluonde in bone. hrJAppi RdIsotopes 28581-583

Moreno EC, Kresak hl, Zahradnik RT (1977) Physicochemical aspects of fluoride-apatite systems relevant to the study of dental caries. Caries Res 11:142-171

Morï S, Burr DB (1 993) Increased intracortical remodelling foIlowïng fatigue damage. Bone 14: 103-1O9

Mosekilde L, Kragstrup J, Richards A (1987a) Compressive strength, ash weight, and volume ofvertebral trabecular bone in rsperïmental fluorosis in pigs. Cakf Tiwie Int 40:318-322

Mosekilde L, Mosekilde L, Danielsen CC (1987b) Biomechanical cornpetence of vertebral trabecular bone in relation to ash density and age in normal individuals. Bone 8:79-85

Mosekilde L (1989) Ses differences in age-reIated bone loss of vertebral trabecular bone mass and structure - biomechanical consequences. Bone 10:325-432

Mosekilde L (1998) Osteoporosis - mechanisms and models. In: Ariabokc Tredtmentrfor Odeoporosli. JF i'irhitfield and P Morley, eds. CRC Press, Boca Raton. pp. 31-58

Okuda A, Kanehisa J, Heersche JN (1990) The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. ]Bone Miner Res 5:SZ 15-S 120

Pak CY, Sakhaee EC, Adams-Huet B, Piziak V, Peterson RD, Pointdester JR (1 995) Treatment of posanenopausal osteoporosis \.

Parfitt Xkf (1 982) The coupling of bone formation to : a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metab Bone DIJ Rehr Res 4: 1-6

Pvfitt A, Mathews C, Vilianueva A, Kleerekoper M (1983) Relaaonships benveen surface, volume, and thickness of iliac trabecdz bone in aging and in osteoporosis.J Ckn Inve~t 72:1396-1409

Parfin A, Drezner MyGloseus F, Kanis J, A4alluche H, Meunier P, Ott S, Recker R (1987) Bone histomorphornetry: standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595-6l O Parisien M, ~MeiiishR, Silverberg S, Shane E, Lindsay R, Bitzekïan J, Dempster D (1992) IvLaintenance of cancellous bone comectivity in primary hyperthyroidism: trabecular stmt analysis. J Bone Miner Rer 7:913-919

Parkùls ml, Tinanoff N, Moutinho M, Anstey LW,LT(73zin MH (1974) Relationships of human plasma fluoride and bone fluoride to age. Ca/nJfTik Res 16:335-338

Pelker RR, Friedlander GE, Markharn TC, Panjabi MM, Moen CJ (1984) Effects of fieezing and fieeze-dryhg on the biomechanical propertïes of rat bone. J Ohop Res 1:405-411

Phipps KR (1996) Fluoride. In: Prerent GowMge in ~Vlirhitioon,7<6 ed. EE Ziegler and LJ Filer Jr., eds. ILS1 Press, Washington DC. pp. 329-333

Phipps KR, Onvoll ES, Mason JD, Cauley JA (1997) Community water fluoridation, fractures and bone mineral density. J Botre ~'LIinerRes 12127

Phipps KR, Onvoli ES, Mason JD, Cauley JA (2000) Cornmunity water fluoridation, bone mineral densiv, and Eracrures: prospective study of effects in older women. Brit Men] 321:860-864

Podwomy NV, Kandel RA, Redund RC, Grynpas MD (1999) Partial chondroprotective effect of zoledronate in a rabbit mode1 ofinflarnrnatory aahritis. J Rhezrmatol 26:1972-1982.

Portigliatti Barbos hl, Bianco P, Ascenzi A (1983) Distribution of osteonic and interstitial components in the human femoral shaft with reference to saucnire, calcification, and mechanical properties, Acta Armt 115:178-1 86

Radin EL, Rose Rbf (1986) Role of subchondral bone in the initiation and progression of cardage damage. Clin Odhp 213:34-40

Reid SA, Boyde A (1987) Changes in the mineral density distribution in human bone \.th age: image analysis using backscattered electrons in the SEM. J Bone iMiner Rzs 2:13-22

Rich C, Ensinck J (1961) Effect of sodium fluonde on calcium merabolism of human beings. ~Vatzire191: 184- 185

Rich C, Feist E (1970) The action of fluoride in bone. In: Finode in Medikne, T. L. Vischer, ed. (Bern: Ham Huber), 70-87

Richards A, Mosekilde L, Sogaard CH (1994) Normai age-reiated changes in fluonde content of vertebrai trabecular bone--relation to bone quality. Bone 1521-26

Rias BL, Hodgson SF, O'Faiion WM, Chao EY, Wahner HW, Muhs JM, Cedel SL, Melton LJ (1990) Effect of fluonde treatrnent on the fracture rate in postmenopausal women with osteoporosis. N Engv LW3225302-809 Riggs BL, O'Fdon WM, Lane A, Hodgson SF, Wahner HW, ~MuhsJ, Chao E, Melton LJ (1994) Clinicai trial of fluoride therapy in postmenopausal osteoporotïc women: estended observations and additional analysis. J Bone ~\firnerRer 9:265-75

Roschger P, Fratzl P, Klaushofer K., Rodan (1997) fireralizaaon of cancellous bone after dendronate and sodium fluoride treaunent: a quantitative backscattered electron imaging study on minipig ribs. Bone 20:393-397

Schenk RK, Olah AJ, Herrmann \V (1984) Preparation of calcified tissues for light rnicroscopy. In: Methoh of CaIn9ed Tirme Pqûararion. GR Dickson, ed. Elsevier, N'Y. pp.

Schiack CA, Restarske JS, Dochterman EF (1946) Dental status of 71,O1 5 naval personnel at first examination in 1942. J Am Dentil~~oc33:1141-1146

Schlatter C (1978) Metabolism and tosicoIogy of . In: Fhconiie and Bone. B Courvoisier, A Donath and CABaud, eds. H. Huber, Berne. pp. 1-21

Schnitzler CM, Solomon L (1 985) Trabecular stress fractures during fluoride therapy for osteoporosis. Skeletui Radio114:27G-279

Schnitzler CM, WkgJR, Gear KA, Robson HJ (1 988) Bone fragility of the peripheral skeleton during fluonde therapy for osteoporosis. Cktz Orthop Rel Res 261:268-275

Schnitzler CM, Pettifor JM, MesquitaJM, Bird MD, Schnaid E, Smyth hE (1990a) Histomorphometry of iiiac crest bone in 356 normal biack and white Souch Afncan adults. Bone ~tIiner10:183-199

Schnitzler CM, Mesquita JM, Gear IA, Robson HJ, Moodley GP, Smyth AE (1990b) JXac bone biopsies at the &ne ofperiarticdar stress fractures during fluoride therapy: comparison ~5thpretreatment biopsies. J Bone Miner Re1 5:l4l-l52

Schnitzler CM, Wing JR, Mesquita JM, Gear AK, Robson HJ, Smyth AE (1990~)Risk factors for the development of stress fractures during fluoride therapy for osteoporosis. J Bone Miner Res 5:Sl95-2OO

Schnitzler CM, \VingJR, Rad FJ, van der Menve MT, Mesquita JM, Gear KA, Robson HJ, Shires R (1997) Fewer bone histomorphomemc abnormalities with intermittent than with con~uousslow-release sodium fluoride therapy. Osteopor Int 7:37G-389

Schoenfeld CM, Lautenschlager EP, iMayer PR (1974) Mechanical propemes of hurnan canceilous bone in the femoral head. LWBiol Eng May:313-3 17

Serra J (19 88) Image Ana&i~ and Matbematz'calMo'phoiogy, Vol 7. Academic Press, London.

Simard PL, Lachapelie D, Trahan L, Naccache H, Demers M, Brodeur JiM (1989) The ingestion of fluonde dentifrice by young children. ASDC] Dent Chihi 56:177-I8l Sirnmons ED, Priaker N?H, Grynpas MD (1991) Age-related changes in hurnan corticai bone. J Orfbop Res 9:l5S-l67

Simonen O, Laitinen O (1985) Does fluoridauon of drinking-water prevent bone fragiliq and osteoporosis? Lancet 2:432-434

Singer L, Ophaug RH, Harland BF (1985) Dietary fluoride intake of 15-19-year-old male adults residing in the United States. J Dent Ru 64: 1302-1305

Skedros JG, Bloebaum RD, Bachus KN, Boyce TM (1993a) The meaning of graylevels in backscattered electron images of bone. J Biomeri ~VhtRes 27:47-56

Skedros JG, Bloebaum RD, Bachus KN, Boyce TM, Constantz B (1993) Influence of mineral content and composition on graylevels in backscattered eIectron images of bone. j Biomed Maf Res 27:57-64

Sogaard CH, Mosekilde L, Richards A, Mosekilde L (1994) Marked decrease in trabecuiar bone quaiity after five years of sodium fluonde therapy- assessed by biomechanical testing of sac crest bone biopsies in osteoporotic patients. Bone 15393-399

Sowers Clark MI< Jannausch ML, Wdace FU3 (1991) A prospective smdy of bone mineral content and fracme in comrnunities with differenual fluoride esposure. AmJ Epinemiol133:649-660

Stein ID and Gr& G (1980) Human vertebral bone: relation of strength, porosity and mineralizaaon of fluoride content. Calcc;f Tfsme hr32: 189-1 94

Suarez-hazor ME, Flowerdew G, Saunders LD, Soskolne CL, Russeil, AS (1993) The fluoridaaon of -ng water and hip fracture hospitaiization rates in cwo Canadian communiaes. Amj Public Healfh 83589-693

Taves DR (1968) Electrophoretic mobility of fluoride. ivatctre 220582-583

Thomsen JS, Ebbesen EN, Mosekilde L (2000) A new method of comprehensive static histomorphomctry appiied on human lurnbar venebral cancdous bone. Bone 27:129-138

Turner CH (1 989) Yield behavior of cancellous bone. J Biomech Eng 111: 1-5.

Turner CH, Akhter MI?,Heaney RI? (1992) The effects of Buoridated water on bone strength. J O&op Res 10:581-587

Turner CH (1 992) On \Volff s Iaw of trabecular architecture. J Biomech 25:l-9

Turner CH, Burr DB (1993) Basic biomechanical measurements of bone: a tutorid. Bone 14595-608.

Turner CH, Boivin G, Meunier PJ (1993) A mathematical mode1 for fluonde uptake by the skeleton. CaIc~fTi~sz~eInt 52:130-138 Turner CH, Hasegawa K, Zhang YU, \Vison LM, Li Y, Dunipace AJ (1995) Fluoride reduces bone smengch in older rats-] Dent hs74:f 475-1481

Turner CH, Owan 1, Brizendine EF, Zhang YV, \;Vilson ME, Dunipce AJ (1 996) High fluonde intakes cause osteomalacia and diminished bone strength in rats with rend deficiency. Eone 19: 595-601

Turner CH, Garetto LP, Dunipace Aj, Zhang W, \Vilson ME, Grynpas MD, Chachra D, McClintock R, Peacock MyS tookey GK (1 997) Fluoride treatment increased serum IGF-1, bone turnover, and bone mass, but not bone strength, in rabbits. CaiczzTrjme Inf 61:77-83

Versmaeten A, Van Ermen H, Haghebaen G, Nijs J, Geusens P, Dequeker J (1991) Osteoarrhritis retards the development of osteoporosis. ChOdop 264: 169-1 77

Vesterby A, Gunderson HJG, Melsen F (1989) Star volume of marrom space and urabecdae of the fkst lumbar vertebra: sampling effiuency and biological variation. Bone 10:7-13

Vesterby A (1990) Star volume of marrow space and trabeculae in iliac crest: sarnpling procedure and correlation to star volume of Eirst lurnbar vertebra. Bone 11:249-155

Waish \WR, Guzelsu N (1993) The role of ions and mineral-organic interfacial bonding on the compressive properties of cortical bone. Biomed1LIater Eng 3:75-84

Weathereii JA (1 969) &finerd metabolism in pediauïcs. D Barnveil and \cFZ Burand, eds. Blackveil, Oxford.

YVeaver JI< (1966) The microscopic hardness of bone. J BoneJt hrg48k273-285

Whitford GM, Pashley DH, Reynolds ICE (1979) Fluoride tissue disrribution: short-term kinetics. An/] PhynOl236:F141 -FI 48.

\Xhï tford GM (1 9 89) The Metaborijm and TO~'YIL-Z~of Fh~on'Ae. S. I

\Vhîtford GM (1 990) The p hysiological and toxicological characteristics of fluoride. J Dent Res 69:539-549

\PhMord GLM (1994) Intake and rnetabolism of fluoride. Ah Dent Res 8:5-14

Wolff J (1986) The Luw ofBone Remodei/ing. P Maquet and R Furlong, mans. Sp~ger-Verlag, Berlin.

Zipkin 1 (1973) Fluoridc in the calcified structures. In: Bio/o~~al&her~~li~aho~z.1Zipkin, ed. John Wiley and Sons, New York. pp. 487-505