Tài liệu Osteoporosis in Men: The Effects of Gender on Skeletal Health Second Edition pptx

Sclerostin, an osteocytes specific protein, inhibits osteoblast differentiation and, based on the sig-nificant increase in bone mineral density in the sclerostin knockout mouse [10], is

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10 11 12 13 10 9 8 7 6 5 4 3 2 1

RobeRt A AdleR, Hunter Holmes McGuire VA Medical

Center and Virginia Commonwealth University School of

Medicine, Richmond, VA, USA

MAtthew R Allen, Departments of Anatomy and Cell Biology,

Indiana University School of Medicine, Indianapolis, IN, USA

ShReyASee AMin, Division of Rheumatology, College of

Medicine, Mayo Clinic, Rochester, MN, USA

diAnA M Antoniucci, University of California, San

Francisco; Physicians Foundation of California Pacific Medical

Center, Division of Endocrinology, Diabetes and Osteoporosis,

San Francisco, CA, USA

AndRe b ARAujo, New England Research Institutes, Inc.,

Watertown, MA, USA

lAuRA A.G ARMAS, Creighton University Osteoporosis

Research Center, Omaha, NE, USA

GiAMpieRo i bARoncelli, Department of Obstetrics,

Gynecology and Pediatrics, 2 nd Pediatric Unit, ‘S Chiara’ Hospital,

Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy

SilvAno beRtelloni, Department of Obstetrics, Gynecology

and Pediatrics, 2 nd Pediatric Unit, ‘S Chiara’ Hospital, Azienda

Ospedaliero-Universitaria Pisana, Pisa, Italy

ShAlendeR bhASin, Section of Endocrinology, Diabetes

and Nutrition, Boston University School of Medicine and Boston

Medical Center, Boston, MA, USA

john p bilezikiAn, Department of Medicine, Division of

Endocrinology, Metabolic Bone Diseases Unit, College of Physicians

and Surgeons, Columbia University, New York, NY, USA

neil c binkley, University of Wisconsin, School of Medicine

and Public Health, Madison, WI, USA

Steven boonen, Center for Musculoskeletal Research,

Department of Experimental Medicine, Katholieke Division of

Geriatric Medicine, Leuven University Hospital, Department

of Internal Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

Adele l boSkey, Starr Chair in Mineralized Tissue Research and Director, Musculoskeletal Integrity Program, Hospital for Special Surgery, New York; Professor of Biochemistry, Weill Medical College

of Cornell University; Professor, Field of Physiology, Biophysics and Systems Biology, Graduate School of Medical Sciences of Weill Medical College of Cornell University; Professor, Field of Biomedical Engineering, Sibley School, Cornell Ithaca; Adjunct Professor, School of Engineering, City College of New York, NY, USA RoGeR bouillon, Laboratory of Experimental Medicine and Endocrinology (LEGENDO), Katholieke Univeriteit Leuven (KUL), Leuven, Belgium

dAvid b buRR, Departments of Anatomy and Cell Biology and Orthopaedic Surgery, Indiana University School of Medicine; Department of Biomedical Engineering, IUPUI, Indianapolis, IN, USA

Melonie buRRowS, Department of Orthopaedics, University

of British Columbia; Centre for Hip Health and Mobility, Vancouver, Canada

Filip cAllewAeRt, Center for Musculoskeletal Research, Leuven University Department of Experimental Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

GeeRt cARMeliet, Laboratory of Experimental Medicine and Endocrinology (LEGENDO), Katholieke Univeriteit Leuven (KUL), Leuven, Belgium

luiSellA ciAnFeRotti, Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy

juliet coMpSton, University of Cambridge School of Clinical Medicine, Cambridge, UK

FeliciA coSMAn, Regional Bone Center Helen Hayes Hospital, West Haverstraw, New York; Department of Medicine, Division of Endocrinology, Metabolic Bone Diseases Unit, College of Physi- cians and Surgeons, Columbia University, New York, NY, USA SeRGe cReMeRS, Division of Endocrinology, Department of Medicine, Columbia University, New York, NY, USA

i x

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k ShAwn dAviSon, Laval University, Quebec City, PQ, Canada

dAvid w deMpSteR, Department of Pathology, College of

Physicians and Surgeons, Columbia University, New York, NY, USA

john A eiSMAn, Bone and Mineral Research Program, Garvan

Institute of Medical Research; University of New South Wales; St

Vincent’s Hospital, Sydney, NSW, Australia

GhAdA el-hAjj FuleihAn, Calcium Metabolism and

Osteo-porosis Program, American University of Beirut Medical Center,

Beirut, Lebanon

eRik Fink eRikSen, Department of Endocrinology and Internal

Medicine, Aker University Hospital, Oslo; Spesialistsenteret

Pilestredet Park, Oslo, Norway

MuRRAy j FAvuS, Section of Endocrinology, Diabetes, and

Metabolism, University of Chicago, Chicago, IL, USA

dieteR FelSenbeRG, Zentrum Muskel- & Knochenforschung,

Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin,

Freie Universität & Humboldt-Universität Berlin, Berlin, Germany

SeRGe FeRRARi, Service of Bone Diseases, Department of

Rehabilitation and Geriatrics, WHO Collaborating Center for

Osteoporosis Prevention, Geneva University Hospital, Geneva,

Switzerland

dAvid p FyhRie, David Linn Chair of Orthopaedic Surgery,

Lawrence J Ellison Musculoskeletal Research Center, Department

of Orthopaedic Surgery, The University of California, Davis; The

Orthopaedic Research Laboratories, Sacramento, CA, USA

pAtRick GARneRo, INSERM Research unit 664 and Synarc,

Lyon, France

luiGi GennARi, Deparment of Internal Medicine, Endocrine,

Metabolic Sciences, and Biochemistry, University of Siena, Italy

piet GeuSenS, Department of Internal Medicine, Subdivision

of Rheumatology, Maastricht University Medical Center,

Maastricht, The Netherlands; Biomedical Research Institute,

University Hasselt, Belgium

vicente GilSAnz, Director, Childrens Imaging Research

Program, Childrens Hospital Los Angeles, Professor of Radiology

and Pediatrics, University of Southern California, Los Angeles,

CA, USA

MonicA GiRotRA, Memorial Sloan-Kettering Cancer Center;

Joan and Sanford I Weill Medical College of Cornell University,

New York, NY, USA

AndReA GiuSti, Department of Gerontology &

Musculo-Skeletal Sciences, Galliera Hospital, Genoa, Italy

AndReA GiuStinA, Department of Endocrinology &

Metabolic Diseases, Leiden University Medical Center, Leiden,

The Netherlands

SteFAn GoeMAeRe, Ghent University Hospital, Department

of Endocrinology and Unit for Osteoporosis and Metabolic Bone

Diseases, Gent, Belgium

deboRAh t Gold, Duke University Medical Center, Durham,

dAvid j hAndelSMAn, Department of Andrology, ANZAC Research Institute, Concord Hospital, University of Sydney, Sydney, NSW, Australia

elizAbeth M hAney, Oregon Health and Science University, Portland, OR, USA

dAvid A hAnley, University of Calgary, Calgary, AB, Canada RobeRt p heAney, Creighton University Osteoporosis Research Center, Omaha, NE, USA

RAvi jASujA, Section of Endocrinology, Diabetes and Nutrition, Boston University School of Medicine and Boston Medical Center, Boston, MA, USA

helenA johAnSSon, WHO Collaborating Centre for Metabolic Bone Diseases, University of Sheffield Medical School, Sheffield, UK

john A kAniS, WHO Collaborating Centre for Metabolic Bone Diseases, University of Sheffield Medical School, Sheffield, UK jeAn-MARc kAuFMAn, Ghent University Hospital, Department of Endocrinology and Unit for Osteoporosis and Metabolic Bone Diseases, Gent, Belgium

RobeRt klein, Bone and Mineral Unit, Oregon Health & Science University and Portland VA Medical Center, Portland,

OR, USA StAvRoulA kouSteni, Division of Endocrinology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA

diAne kRueGeR, University of Wisconsin, Madison, WI, USA kiShoRe M lAkShMAn, Section of Endocrinology, Dia- betes, and Nutrition, Division of Endocrinology & Metabolism, Boston University School of Medicine, Boston Medical Center, Boston, MA, USA

thoMAS F lAnG, Professor in Residence, Department of Radiology and Biomedical Imaging, and Joint Bioengineering Graduate Group, University of California, San Francisco, San Francisco, CA, USA

bRuno lApAuw, Ghent University Hospital, Department of Endocrinology and Unit for Osteoporosis and Metabolic Bone Diseases, Gent, Belgium

joAn M lAppe, Creighton University Osteoporosis Research

Center, Omaha, NE, USA

benjAMin z ledeR, Endocrine Unit, Department of Medicine,

Massachusetts General Hospital and Harvard Medical School,

Boston, MA, USA

willeM leMS, Department of Rheumatology, Vrije Universiteit

Amsterdam; VU Medisch Centrum, Amsterdam, The Netherlands

X SheRRy liu, Departments of Medicine and Biomedical

Engineering, College of Physicians and Surgeons, Columbia

University, New York, NY, USA

Shi S lu, Regional Bone Center, Helen Hayes Hospital, West

Haverstraw, New York, NY, USA

heAtheR M MAcdonAld, Schulich School of Engineering,

University of Calgary, Calgary, Canada

chRiStA MAeS, Laboratory of Experimental Medicine and

Endocrinology (LEGENDO), Katholieke Universiteit Leuven

(KUL), Leuven, Belgium

Ann e MAloney, Maine Medical Center Research Institute,

Scarborough, ME, USA

peGGy MAnnen cAwthon, San Francisco Coordinating

Center, California Pacific Medical Center Research Institute, San

Francisco, CA, USA

clAudio MARcocci, Department of Endocrinology and

Metabolism, University of Pisa, Pisa, Italy

lynn MARShAll, Department of Medicine, Bone and Mineral

Unit, Department of Public Health and Preventive Medicine,

Oregon Health & Science University, Portland, OR, USA

GheRARdo MAzziotti, Department of Medical and Surgical

Sciences, University of Brescia, Italy

euGene v MccloSkey, WHO Collaborating Centre for

Metabolic Bone Diseases, University of Sheffield Medical School,

Sheffield, UK

heAtheR A MckAy, Department of Orthopaedics, University of

British Columbia; Centre for Hip Health and Mobility; Department

of Family Practice, University of British Columbia, Vancouver,

Canada

chRiStiAn MeieR, Division of Endocrinology, Diabetes and

Clinical Nutrition, University Hospital Basel, Basel, Switzerland

pAul d MilleR, University of Colorado Health Sciences

Center, Medical Director, Colorado Center for Bone Research,

Lakewood, CO, USA

biSMRutA MiSRA, College of Physicians and Surgeons,

Columbia University, New York, NY, USA

SteFAno MoRA, Departments of Radiology and Pediatrics, Childrens Hospital Los Angeles, Los Angeles, California, USA; Laboratory of Pediatric Endocrinology, BoNetwork, San Raffaele Scientific Institute, Milan, Italy

tuAn v nGuyen, Bone and Mineral Research Program, Garvan Institute of Medical Research; University of New South Wales; St Vincent’s Hospital, Sydney, NSW, Australia

AndeRS oden, WHO Collaborating Centre for Metabolic Bone Diseases, University of Sheffield Medical School, Sheffield, UK clAeS ohlSSon, Center for Bone Research, Department

of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

teRence w o’neill, Epidemiology arc Unit, University of Manchester, Manchester, UK

eRic S oRwoll, Bone and Mineral Unit, Oregon Health & Science University, Portland, OR, USA

SocRAteS e pApApouloS, Department of Endocrinology & Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands

René Rizzoli, Division of Bone Diseases [WHO Collaborating Center for Osteoporosis Prevention] Department of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland

cliFFoRd j RoSen, Maine Medical Center Research Institute, Scarborough, ME, USA

MARtin RunGe, Aerpah Clinic Esslingen, Esslingen, Germany john t SchouSboe, Park Nicollet Health Services, Minneapolis; Division of Health Policy & Management, School of Public Health, University of Minnesota, MN, USA

eGo SeeMAn, Endocrine Centre, Heidelberg Repatriation Hospital/Austin Health, Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia

MARkuS j Seibel, Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, NSW, Australia deboRAh e SellMeyeR, Metabolic Bone Center, The Johns Hopkins Bayview Medical Center, Baltimore, MD, USA

elizAbeth ShAne, Columbia University College of cians & Surgeons, New York, NY, USA

Physi-jAy R ShApiRo, Bone and Osteogenesis Imperfecta Programs, Kennedy Krieger Institute; Department of Physical Medicine and Rehabilitation, Johns Hopkins University, Baltimore, MD, USA Shonni j SilveRbeRG, Division of Endocrinology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY, USA

x i i

StuARt l SilveRMAn, Cedars-Sinai/UCLA and the OMC

Clinical Research Center, Los Angeles, CA, USA

RAjAn SinGh, Section of Endocrinology, Diabetes and

Nutrition, Boston University School of Medicine and Boston

Medical Center, Boston, MA, USA

eMily M Stein, Columbia University College of Physicians &

Surgeons, New York, NY, USA

thoMAS w StoReR, Section of Endocrinology, Diabetes

and Nutrition, Boston University School of Medicine and Boston

Medical Center, Boston, MA, USA

pAwel Szulc, INSERM Research Unit 831, Hôspital Edouard

Heriot, Lyon, France

MAhMoud tAbbAl, Calcium Metabolism and Osteoporosis

Program, American University of Beirut Medical Center, Beirut,

Lebanon

youRi tAeS, Ghent University Hospital, Department of

Endocrinology and Unit for Osteoporosis and Metabolic Bone

Diseases, Gent, Belgium

chARleS h tuRneR, Department of Orthopaedic Surgery,

Indiana University School of Medicine, Indianapolis; Department of

Biomedical Engineering, IUPUI, IN, USA

lieSbeth vAndenput, Center for Bone Research, Department

of Medicine, Sahlgrenska Academy, University of Gothenburg,

Gothenburg, Sweden

diRk vAndeRSchueRen, Center for Musculoskeletal

Research, Leuven University Department of Experimental

Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

kAtRien venken, Center for Musculoskeletal Research, Leuven University Department of Experimental Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

lieve veRlinden, Laboratory of Experimental Medicine and Endocrinology (LEGENDO), Katholieke Universiteit Leuven (KUL), Leuven, Belgium

AnneMieke veRStuyF, Laboratory of Experimental Medicine and Endocrinology (LEGENDO), Katholieke Universiteit Leuven (KUL), Leuven, Belgium

QinGju wAnG, Endocrine Centre, Heidelberg Repatriation Hospital/Austin Health, Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia

connie M weAveR, Department of Foods and Nutrition, Purdue University, West Lafayette, IN, USA

FeliX w wehRli, Department of Radiology, University of Pennsylvania, Philadelphia, PA, USA

Sunil j wiMAlAwAnSA, Professor of Medicine, crinology & Metabolism; Director, Regional Osteoporosis Center, Department of Medicine, Robert Wood Johnson Medical School, New Brunswick, NJ, USA

Endo-kRiStine M wiRen, Bone and Mineral Unit, Oregon Health & Science University; Portland VA Medical Center, Portland,

OR, USA RoGeR zebAze, Department of Endocrinology and Medicine, Austin Health, University of Melbourne, Melbourne, Victoria, Australia

huA zhou, Regional Bone Center, Helen Hayes Hospital, West Haverstraw, New York, NY, USA

x i i i

The field of osteoporosis has grown enormously over the last

4 decades, with a focus upon the issues that relate to skeletal

health in women It was only about 15 years ago that the

sci-entific community began to acknowledge that osteoporosis

in men is also important The first edition of Osteoporosis in

Men, published in 2001, was a seminal event in that it called

attention to the problem in an organized series of articles on

male skeletal health and bone loss Now, with this second

edition of Osteoporosis in Men, further progress in this area

is emphasized with particular emphasis on new knowledge

that has appeared during the last decade

Osteoporosis in men is heterogeneous with many

eti-ologies to consider besides the well known roles of aging

(Sections 1-4) and sex steroids (Sections 6-8) The roots of

the problem in some individuals can be back dated to the

pre-pubertal and pubertal growth periods that determine the

acquisition of peak bone mass

In addition, Osteoporosis in Men, second edition, deals

exhaustively with important clinical issues Nutritional

con-siderations, the clinical and economic burden of fragility

fractures, and diagnostic approaches are particularly strong

aspects of the text (Sections 5, 7, 9) These chapters

tran-scend, in part, the specific focus of the volume, making it a

useful resource and a valuable reference for an audience not

necessarily well-informed in bone and mineral disorders

The last section of Osteoporosis in Men, second edition,

highlights therapeutic approaches Treatment options are less

well defined in men than in women because virtually all of

the clinical trials involving men have been much smaller and

shorter in duration with surrogate, instead of fracture, points With this smaller database, it nevertheless appears that men respond to available pharmacological approaches

end-to osteoporosis in a similar manner end-to women (Section 10) Available clinical data support the efficacy of these therapies

in men with both primary and secondary osteoporosis

Finally, Osteoporosis in Men, second edition provides

a view of the future, underscoring a number of unresolved issues to be included in the agenda for future research in this area These include discussions related to an appropriate BMD-based definition for male osteoporosis, a further under-standing of the factors implicated in age-related bone loss and idiopathic osteoporosis in men, and randomized-controlled studies directly assessing fracture risk reduction, particularly for non vertebral fracture In all these areas, more definitive information is needed

This thorough and comprehensive book integrates new, accessible and informative material in the field It will help investigators, as well as practitioners and students, to improve their understanding of male skeletal health and bone loss The additional knowledge, assembled in such a readable manner, should help us achieve one of our ultimate goals-better care of men with osteoporosis

Gerolamo Bianchi, MDDepartment of Locomotor SystemDivision of RheumatologyAzienda Sanitaria Genovese

Genova, Italy

The first edition of Osteoporosis in Men was published

in 1999, about 15 years after the earliest publications on the

subject Over the past decade, we have witnessed a surge

of further interest in the subject of male osteoporosis This

second edition of Osteoporosis in Men is, thus, timely

In the second edition, we have made major additions to

reflect increased areas of new knowledge, including

genet-ics and inherited disorders Previous topgenet-ics are updated and

extended to make them timely also New topics include:

l Important basic processes including bone biochemistry

and remodeling

l Mechanical properties and structure

l Genetics and inherited disorders

l Growth and puberty

l Nutrition, including calcium, vitamin D, protein and

other factors

l Sex steroids in muscle and bone

l Assessment of bone using DXA, CT, ultrasound,

bio-chemical markers

l Sarcopenia and frailty

l Diagnostic approaches

l Treatment approaches including bisphosphonates,

parathy-roid hormone, androgens and SARMS and newer agents

A key element of the book continues to be sex

differ-ences in bone biology and pathophysiology that can inform

our understanding of osteoporosis in both men and women

The increased scope of the book is the result of tions from prominent experts in the field, including many who contributed chapters to the first edition New authors also have provided novel insights for the second edition Editorial responsibilities were shared by the three of us

contribu-As was the goal before, Osteoporosis in Men, Second Edition, is meant to be useful to a broad audience, including students of the field as well as those already knowledgeable

We have sought to summarize a compendium of tion intersecting general and specific areas of interest This volume will make apparent that information available con-cerning osteoporosis in men still lags behind what we know about osteoporosis in women On the other hand, major advances in our understanding of the male skeleton in health and in disease are being translated into practical approaches

informa-to their clinical management We hope this second edition provides a valuable reference source for you and that it also will serve to stimulate further advances in the field

Eric OrwollPortland, OregonJohn BilezikianNew York, New YorkDirk VanderschuerenLeuven, BelgiumPreface to the Second Edition

x v

As detailed throughout this book, osteoporosis is

charac-terized by increased risk of fracture due to changes in the

‘quality’ of bone [1] To appreciate why bone becomes

weaker or less resilient to fracture with age in both men

and women and in individuals of different races, a

gen-eral knowledge of bone development and age-dependent

changes is necessary In line with the theme of this book,

it is noted that there are both age- and sex-dependent

dif-ferences in bone properties and composition, some related

to the rate at which bones develop in boys and girls, some

related to the impact of genes on the X-chromosome which

produce proteins important for bone development and/or

metabolism and some due to the direct effect of sex

ster-oids on bone cells [2] To appreciate the discrete

differ-ences between bone structure and composition in men and

women this chapter reviews the basics of bone

composi-tion and organizacomposi-tion and the mineralizacomposi-tion process from

the point of view of sexual dimorphism, where such

differ-ences between men and women are recognized Emphasis

is placed on those factors that contribute to bone strength;

geometry, architecture, mineralization, the nature of the

organic matrix and tissue heterogeneity

Bone organIzatIon

Bone Heterogeneity

The structure of bone appears different depending on

the scale at which it is examined At the centimeter level,

whole bone can be viewed as an organ, for example, the

tubular (long and short) bones such as the femur and digits, respectively, and the flat bones, such as the calvaria in the skull Slightly better resolved, at the millimeter level, are the components of the bones, the cortices that surround the mar-row cavity, the cancellous bone within the marrow cavity, the marrow cavity itself, the cartilaginous ends, etc At the micrometer to millimeter level are the individual intercon-necting struts of the trabeculae, the lamellae and the osteons that surround the vascular canals The cells and the com-posite matrices also can be visualized as part of this micro-structure Finally, at the nanometer level, bone consists of an organic matrix made mainly from collagen fibrils and non-collagenous proteins, lipids, nanometer size mineral crystals (discussed below) and water There is also heterogeneity

in both the size of the collagen fibrils and the composition and sizes of the crystals deposited on this matrix [3, 4] This heterogeneity is important for the mechanical competence

of the tissue [5] To understand the process of tion, knowledge of the cells and the extracellular matrices

the Biochemistry of Bone: Composition and Organization

Adele l Boskey

Starr Chair in Mineralized Tissue Research and Director, Musculoskeletal Integrity Program, Hospital for Special Surgery, New York; Professor of Biochemistry, Weill Medical College of Cornell University; Professor, Field of Physiology, Biophysics and Systems Biology, Graduate School of Medical Sciences of Weill Medical College of Cornell University; Professor, Field of Biomedical Engineering, Sibley School, Cornell Ithaca; Adjunct Professor, School of Engineering, City College of New York, USA

Osteoporosis in Men



osteoblasts line the surface of the mineralized bone They

synthesize new matrix and regulate the mineralization and

turnover of that matrix Once these osteoblasts become

engulfed in mineral they become osteocytes and connect

with one another by long processes (canaliculae) (see Figure

1.1) The osteocytes are the cells that sense mechanical

sig-nals and then convey them through the matrix Osteocytes

produce many of the same proteins as osteoblasts, but the

relative concentrations of these proteins are not the same

and the ways in which these cells use regulatory pathways

differ As reviewed in detail elsewhere [8], the osteoblasts

use the WNT/beta-catenin pathway [9] to regulate synthesis

of new bone; the osteocytes use the WNT/beta-catenin

path-way to convey mechanical signals Osteoblasts synthesize

more alkaline phosphatase, more type I collagen and more

bone sialoprotein than osteocytes, while osteocytes

specifi-cally produce sclerostin, a glycoprotein that is a WNT and

BMP antagonist, and produce high levels of dentin matrix

protein 1 [8] Sclerostin, an osteocytes specific protein,

inhibits osteoblast differentiation and, based on the

sig-nificant increase in bone mineral density in the sclerostin

knockout mouse [10], is believed to be important in

deter-mining the high bone mass phenotype [11] This increase in

bone mass was noted to be comparable for both sexes [10]

There is sexual dimorphism in the density of osteocytes, as

females gain osteoclast lacunar density with increasing age,

while males show a decrease in this parameter [12] This

may explain why bone loss in women results in a decrease

in trabecular number, while in males there is a thinning of trabeculae [13] Some of the other functions of osteoblasts and osteocyte proteins will be discussed later

The cells responsible for the turnover of bone, the clasts, are of hematologic and macrophage origin [14] As seen in the electron micrograph in Figure 1.2, these multi-nucleated giant cells attach to the surface of the bone via a

osteo-‘ruffled border’ They receive signals from osteoblasts that control bone remodeling and regulate the turnover of the mineralized matrix They remove bone by producing acid and couple that with the transport of chloride out of the cell The acid dissolves the mineral (see below) and, after the mineral is removed, release proteolytic enzymes that degrade the matrix During the dissolution of the matrix, signaling molecules communicate with the osteoblasts and new bone formation is triggered Androgens and estrogens inhibit osteoclast activity to different extents [15] explain-ing some of the sexual dimorphism in osteoclast activity.There are a number of other cells in bone, marrow stromal cells, pericytes, vascular endothelial cells, fibroblasts, etc that function as stem cells [16] but their properties are beyond the scope of this chapter and will not be discussed here

skel-a bone thskel-at is optimskel-ally designed to beskel-ar the loskel-ads imposed

on it [18] In the long and short tubular bones, endochondral

Osteoblast

FIgure 1.1 Transmission electron micrograph showing

oste-oblasts lining the bone surface in an adult male Sprague-Dawley

rat Inside the bone are the osteocytes, connected to one another

by canaliculae The banded pattern of the collagen is also visible

Magnification is marked on the figure Courtesy of Dr Stephen B

Doty, Hospital for Special Surgery, New York.

Osteoclasts

Bone

50 Microns

FIgure 1.2 Transmission electron micrograph of an osteoclast

on the bone surface of a 70-year-old woman The ruffled borders sealing the cell to the mineralized surface are indicated along with the magnification Courtesy of Dr Stephen B Doty, Hospital for Special Surgery, New York.

ossification, in which a cartilage model becomes calcified

and is replaced by bone, provides the basis for longitudinal

growth, while widening of the bones takes place by

apposi-tion on already formed bone in the periosteum concurrent

with removal of the inner (endosteal) surfaces

Endochondral ossification starts during embryogenesis

and continues throughout childhood and into adolescence,

peaking during the ‘growth spurt’ The rate at which

changes in bone geometry occur depends on genetics, the

environment and hormonal signals [19, 20] With the

excep-tion of individuals with rare genetic mutaexcep-tions, the process

of endochondral ossification terminates during adolescence

with the closing of the growth plate This generally occurs

in girls around age 13 and in boys around age 18 [21] In

contrast, there is a report of a man who had a bone age of

15, based on bone mineral density (BMD), at age 28 and

lacked closed epiphyses and had continued linear growth

into adulthood due to a mutation in his estrogen-receptor

alpha (ERalpha) gene [22] His testosterone levels were

reported as normal Other related cases with abnormalities

in the ability to synthesize estrogen (aromatase deficiency)

had a similar phenotype, but longitudinal growth could be

modulated with estrogen treatment [23]

During aging, at least in mice [24] and, most likely, in

humans [25], there is a decrease of bone formation

(osteo-genesis) and an increase of fat cell formation (adipo(osteo-genesis)

in bone marrow There is also a difference between aging

pat-terns in bones of men and women In general, in both sexes,

bone strength is maintained by the process of remodeling,

removal of bone by osteoclasts and formation of new bone

by osteoblasts These coupled processes [26] are not

equiva-lent in men and women Testosterone decreases this pathway

in men [27], perhaps contributing to the delayed start of

age-dependent bone loss in males relative to females In women,

menopause-related estrogen deficiency leads to increased

remodeling [28] and, with age, bone loss is accelerated and

bone loss exceeds formation, causing cortices to being

thin-ner and more porous and trabeculae to become disconnected

and thinner In men, the changes in remodeling lead to bone

loss occurring later in life [29] Concurrent bone formation on

the periosteal surface during aging occurs to a greater extent

in men than in women, thus diminishing some of the bone

loss [30] In a cross-sectional study of older men and women

[29], men had significantly larger cross-sectional bone sizes

than women which, in turn, was associated with decreased

compressive strength indices at the spine, femoral neck and

trochanter and bending strength indices at the femoral neck

Bone compoSItIon: tHe Bone

compoSIte

Independent of age, state of development, race and sex,

bone is a composite material consisting of mineral crystals

deposited in an oriented fashion on an organic matrix The organic matrix is predominately type I collagen, but there are also non-collagenous proteins and lipids present The non-collagenous proteins account for a small percentage of the bone matrix, yet they are important for regulating cell–matrix interactions, matrix structure, matrix turnover and the biomineralization process Knowledge about the func-tions and critical status of these proteins has come from studies of mutant animals (naturally occurring and those made by genetic manipulation), cell culture studies [31] and analyses of the proteins’ activity in the absence of cells

the mineral

The mineral component of the bone composite is an logue of the naturally occurring mineral hydroxyapatite Bone hydroxyapatite is comprised of nanometer sized crystals [32] These crystals have the approximate chemical composition Ca5(PO4)3OH but are carbonate-substituted and calcium and hydroxide deficient [33] The individual crys-tals have a broad range of sizes, depending on the age of the bone and the health of the subject, but are always oriented parallel to the long fiber axis of the collagenous matrix (Figure 1.3) There is a broad distribution of the amount of mineral in the matrix, again varying with age, environment and disease The average amount of mineral in the matrix can be measured by burning off the organic matrix (ash weight) or by radiographic measurement of density (bone mineral density or bone mineral content) There is some sexual dimorphism in the ash weight in bones of egg-laying

ana-1.5 µm

FIgure 1.3 Transmission electron micrograph of a section of

bone from the tibia of an adult male mouse The electron dense mineral crystals can be seen to lie parallel to the collagen fibril axis Courtesy of Dr Stephen B Doty, Hospital for Special Surgery, New York.

Osteoporosis in Men



chicks, with males having, on average, a greater mineral

content in any given bone than age matched female bones

[34] but, in humans of the same race, the ash content of

adult male and female bones is similar [35], perhaps because

there is a well defined maximum amount of mineral that can

fit into the bone matrix Only in osteomalacia and related

diseases is the mineral content reduced and that occurs in

both sexes Bone mineral density measured by computed

tomography, tends to be higher in males than females at

each stage of life, but differences are removed when

cor-rected for bone length and cortical thickness [29, 36, 37]

The composition of bone hydroxyapatite varies with

age, diet and health due to the substitution of foreign ions

and vacancies into the crystal lattice and to the absorption

of these ions on the surface of the crystals The substituted

ions also have been reported to differ when male and female

mouse bones are compared, although the number of such

studies is limited When attention is paid to the sex of the

animal, compositional studies show differences in mineral

content and composition [38] The effects of sex steroids on

bone development can explain many of these differences

For example, assessing the effects of sex hormones on bone

composition Ornoy et al [39] compared a variety of

com-positional parameters in gonadectomized mice treated with

male and female sex steroids While the investigators found

that tibial mineral content (ash weight) was comparable in

all the groups, Ca and P content increased after ovariectomy

Estradiol treatment increased mineral content and bone Ca

and P in ovariectomized and in intact females and

orchiect-omized mice, while testosterone had smaller effects

the extracellular matrix

Collagen provides the oriented template or scaffold upon

which these mineral crystals are deposited The collagen

is predominately type I, a triple helical collagen, with the

individual chains having the amino acid sequence

(X-Y-Gly)n, where X and Y are any amino acids, often proline

and hydroxyproline, and glycine is the only amino acid

small enough to fit in the center of the triple helix [40] The

importance of type I collagen for the proper mineralization

of the matrix is seen in the different osteogenesis imperfecta

diseases, a set of diseases, reviewed elsewhere [41], caused

by mutations that lead to altered quantity or quality

(com-position) of type I collagen and result in brittle bones There

are also other collagen types in bone, including fibrillar type

III collagen and non-fibrillar type V collagens [42] No sex

dependent differences in the distribution of collagen types

have been reported, however, there are differences in the

non-collagenous proteins that are found associated with the

collagen matrix In the next section, these non-collagenous

proteins will be presented as families, with emphasis on

their roles in mineral formation and turnover and other

ways in which they might affect sexual dimorphism in bone

strength

the non-collagenous proteins: gla proteins

The most abundant non-collagenous protein in vertebrates

is a small protein, osteocalcin, also known as bone gla tein [40] This small (5.7 kDa) protein has three gamma-carboxy-glutamic acid residues, with a high affinity for hydroxyapatite and calcium as demonstrated by its crys-tal and nuclear magnetic resonance (NMR) structures [43, 44] Osteocalcin is frequently used as a biomarker for bone formation [45], although it is also released from bone and hence can reflect remodeling rather than only forma-tion In studies where bone tissue osteocalcin levels and serum osteocalcin levels were compared as a function of age and sex, the levels in men exceeded those in women

pro-at all ages until age 60, when levels in women increased and then decreased, reflecting age-dependent increases in bone remodeling [46, 47] This most likely is an estrogen- determined effect as, in the rat, estrogen treatment is associ-ated with a decrease in osteocalcin [48]

Knockout mice lacking osteocalcin have thickened bones and, thus, it was initially suggested that osteocalcin was important for bone formation [49] Further studies led to the suggestion that osteocalcin was important for osteoclast recruitment [50], a suggestion supported by in vitro and in vivo assays [40] Most recently, Karsenty’s group has sug-gested, from studies in wildtype as well as osteocalcin knockout mice, that the uncarboxylated form of osteocalcin acts as a hormone, regulating glucose levels in cultures of pancreatic cells and in the skeleton [51] The role of osteo-calcin in glucose metabolism is suggested by the observation that osteoblastic bone formation is negatively regulated by the hormone leptin Leptin, secreted by fat cells (adipocytes), has multiple hormonal functions including, but not limited to: appetite suppression, initiation of puberty in girls and acceleration of longitudinal bone growth in mice, although the data on bone formation have suggested a bimodal pat-tern [52] In humans, a recent report showed postmenopausal women with type 2 diabetes had reduced osteocalcin levels [53] In addition to the identification of osteocalcin as a hor-mone with a postulated role in metabolic syndrome, readers are reminded that the osteocalcin knockout has a bone phe-notype, there is some sex specificity to osteocalcin’s action

in bone [48] and polymorphisms in the osteocalcin gene have been associated with osteoporosis [54–56]

The second gamma-carboxyglutamic acid containing tein in bone (predominantly in cartilage) and in soft tissues

pro-is matrix-gla protein (MGP) MGP pro-is a hydrophobic protein [40] containing five gamma-carboxyglutamate residues that is important for inhibition of soft tissue calcification, as can be seen in the knockout mice where, when MGP is ablated, the animals have excessive cartilage calcification, denser bones and young animals succumb to calcification of the blood ves-sels and esophagus [57, 58] Both the full length protein and its component peptides can inhibit hydroxyapatite forma-tion and growth in culture [59] MGP is more abundant in

soft tissues than in bone, hence it is not surprising that

poly-morphisms in MGP are not associated with bone density or

fracture risk [56]

non-collagenous proteins: Siblings

There is a family of proteins found in bone that have been

named the SIBLING proteins (small integrin binding ligand

N-glycosylated) [60] These proteins are all located on the

same chromosome, all have RGD-cell binding domains, all are

anionic and all are subject to multiple post-translational

modi-fications including phosphorylation and dephosphorylation,

cleavage and glycosylation [61] Each is found in multiple

tis-sues in addition to bone and each has signaling functions in

addition to interacting with hydroxyapatite and regulating

min-eralization (Table 1.1) The SIBLING proteins include

osteo-pontin (bone sialoprotein 1), dentin matrix protein 1 (DMP1),

bone sialoprotein (BSP2), matrix extracellular

phosphoglyco-protein (MEPE) and the products of the dspp gene, dentin

sialoprotein (DSP) and dentin phosphoprotein (DPP)

Osteopontin is the most abundant of the SIBLING teins and has the most widespread distribution In solution [73, 74], in a variety of cell culture systems [75, 76], in ani-mals in which gene expression has been ablated [71] and in models of pathologic calcifications [77], bone osteopontin

pro-is an inhibitor of mineralization When thpro-is glycoprotein pro-is highly phosphorylated it can promote hydroxyapatite forma-tion, most likely due to small conformational changes occur-ring on binding to the mineral surface [78] Osteopontin is also involved in the recruitment of osteoclasts and in regu-lating the immune response [79] Bone specific conditional knockout of osteopontin results in impaired osteoclast activ-ity at all ages [72], but sexual dimorphism was not noted.Dentin matrix protein 1 is a synthetic product of growth plate chondrocytes and of osteocytes, although it was first cloned from dentin [40] DMP1 is not usually found in an intact form but rather it is found as three smaller peptides, an N-terminal peptide, a C-terminal peptide and an N-terminal protein that has a glycosaminoglycan chain attached [65] It

is the only one of the SIBLING proteins to date that has been

taBle 1.1 Bone non-collagenous matrix proteins* whose modification (deletion (KO) or

overexpression (tG)) creates a bone phenotype

Increased crystal size in young animals Females less affected

Regulation of mineralization

Signaling

Thinner collagen fibrils

Regulation of collagen fibrillogenesis Dentin matrix protein-1

[64, 65]

Altered osteocyte function

Regulation of mineralization Signaling response to load Phosphate regulation Dentin sialophosphoprotein

gene (dspp) [66]

KO Increased collagen maturity and

crystallinity in young male and female mice

Regulation of initial calcification Matrix gla protein [57] KO Excessive vascular and cartilage

Osteocalcin [49, 50] KO Thicker bones, smaller crystals suggest

impaired turnover Males/females differ

Regulation of bone turnover Glucose regulation

fibrillogenesis Bone specific KO Decreased bone density, increased bone

* Enzymes, growth factors and cytokines that affect bone are excluded from this table.

Osteoporosis in Men



associated with a bone disease (autosomal hypophosphatemic

rickets) [80] The intact protein appears to inhibit

mineraliza-tion, as does the glycosylated N-terminal fragment, but the

phosphorylated cleaved fragments can promote

mineraliza-tion [81, 82] The knockout mouse has defective

mineraliza-tion, supporting a role for DMP1 as a nucleator [64], although

it appears equally important as a signaling molecule [8]

Bone sialoprotein (BSP) is a specific product of bone

forming cells There are low levels in other mineralized

tissues, such as calcified cartilage and dentin In solution,

BSP is a hydroxyapatite nucleator [83, 84], implying a role

in in situ mineralization In culture, BSP facilitates

osteo-blast differentiation and maturation [85] and thereby

stimu-lates mineralization The BSP knockout is viable, but has

a variable phenotype In the youngest animals, the bones

are shorter, narrower and less mineralized, supporting the

in vitro findings As the animals age, the mineralization

normalizes, but the mice have impaired osteoclast activity,

as they are resistant to bone loss by hind-limb suspension

[63] These data support the hypothesis that because

min-eralization is such an important process, it is crucial to have

multiple pathways to support mineralization BSP

activ-ity may be different in males and females as knockdown

of the estrogen receptor alpha gene in a model of cartilage

induced osteoarthritis resulted in decreased expression of

BSP, implying some gender specificity to the expression

of this protein [86] and studies in chick osteoblasts had

previously demonstrated a response of BSP expression to

estrogen-like molecules [87]

Matrix extracellular phosphoglycoprotein (MEPE) is

made in bone, dentin and also exists in serum as smaller

peptides [67] The MEPE peptides are effective inhibitors

of hydroxyapatite formation and growth, while unpublished

studies show the intact protein, in phosphorylated form,

promotes hydroxyapatite formation Following gene

abla-tion, the knockout animals have excessive mineralization

while the transgenic animal, in which MEPE is

overex-pressed is hypomineralized [67] This protein is one of the

substrates for PHEX (phosphate regulating hormone with

analogy to endopeptidase on the X-chromosome) PHEX is

defective in hypophosphatemic rickets, presumably because

where normally PHEX binds to MEPE and degrades its

inhibitory peptides, in the mutant, this ability to degrade

the peptides is absent and the inhibition persists [68] Thus,

MEPE is an important regulator of calcification Because

PHEX is on the X-chromosome, hypophosphatemic rickets

is more prevalent and more severe in males than in females,

although the female HYP mice have a bone phenotype, but

it is less severe than that of the males [88]

Dentin sialophosphoprotein is expressed as a gene, dspp,

but an intact protein has not yet been isolated Its major

components, dentin sialoprotein (DSP) and dentin

phos-phophoryn (DPP) are found mainly in dentin, but the gene

is expressed in bone [61], and the dspp gene knockout has

a detectable bone phenotype [66] Both DSP and DPP can

regulate mineralization in vitro, thus it is not surprising that the knockout has impaired mineralization both in bone and

in dentin

non-collagenous proteins: SlrpS

Small leucine rich proteoglycans (SLRPS) are the major bone glycoproteins [40] While small amounts of large aggregating proteoglycans (such as aggrecan and epiphican) are resident

in bone as part of residual calcified cartilage, the majority of the bone proteoglycans are smaller These SLRPS include decorin (the major SLRP produced by osteoblasts), bigly-can, osteoadherin, lumican, fibromodulin and mimecan [89] Each of these proteins binds to collagen and regulates col-lagen fibrillogenesis, thus they have an important effect on the bone composite and the mechanical strength of bone In addition, biglycan and decorin are important for regulating cellular activity, perhaps due to the binding of growth factors, and decorin, biglycan and mimecan can regulate hydroxy-apatite formation [90] The properties and functions of these proteins in bone as adapted from these reviews are summa-rized in Table 1.2, while Table 1.1 includes the properties of the knockouts that had bone phenotypes

non-collagenous proteins: matricellular proteins

Another protein family whose members are found in bone are the so-called ‘matricellular proteins’, named so because they regulate the interactions between the cells and the extracellular matrix The members of this family found

in mineralized bone (as distinct from cartilage) include: osteonectin (SPARC), the matrillins, the thrombospondins, the tenascins, the galectins, periostin and osteopontin and BSP (SIBLINGs) Each of these proteins is expressed in higher amounts during development than in adult life, but they are all upregulated during wound repair (callus for-mation) in the adult As noted from studies of mice lack-ing these proteins, or combinations thereof, matricellular proteins affect postnatal bone structure and turnover when animals are challenged by aging, ovariectomy, mechanical loading and fracture healing regeneration but do not have a visible phenotype during normal development [96]

non-collagenous proteins: other

In addition to the families of bone matrix proteins noted above, there are other extracellular matrix proteins that are found in glycosylated and phosphorylated form in bone These include BAG-75 (which is found at the initial sites

of mineralization in culture) [97], SPP24 (that regulates the formation of bone via inhibition of BMP-induced osteo-blast differentiation) [98] and others proteins that serve as signaling molecules or have other functions that are still being investigated [40]

other matrix components

Within the extracellular matrix are other proteins

includ-ing enzymes (Table 1.3), growth factors and other signaling

molecules, as well as lipids that are important for

regulat-ing cell–cell communication and mineral deposition The

actions of lipids in bone are reviewed in detail elsewhere

[40, 103, 104] The importance of lipid rafts (caveolin) is

seen in the caveolin knockout mouse that has increased

bone density and matures more rapidly than control mice

[105] There have not yet been reports of sex-dependent

differences in these mice, although lipid metabolism is

different in men and women

How BoneS cHange wItH age

A key event in the transition from the embryo to the adult

is the development of mineralized structures The cells

that deposit the matrix, regulate the flux of ions and

con-trol the interaction between the matrix components

orches-trate these processes As shown by Figure 1.3, the mineral

in bone is deposited in an oriented fashion on the collagen

matrix It is widely recognized, as reviewed elsewhere

[33, 40], that the collagen provides a template for mineral

deposition, but the extracellular matrix proteins regulate

the sites of initial mineral deposition and control the extent

to which the crystals can grow in length and in width The collagenous matrix is mineralized to a certain extent dur-ing development (primary mineralization) and, as the indi-vidual ages, the rest of the matrix becomes mineralized (secondary mineralization) A variety of signals, discussed elsewhere in this book, activate the osteoclast to remove bone and this removal exposes stimuli that activate osteo-blasts to lay down a new bone matrix, with the matrix pro-teins mentioned above regulating these processes With age, the resorption process exceeds the formative one and this occurs earlier in women then in men

Mouse models in which specific matrix proteins are ablated or inserted provide information both on the sex-ual dimorphic responses of these proteins, but also on the age-related changes Mice, in general, achieve their peak bone mass at 16–18 weeks of age, depending on the sex and background Although the functions of many of these proteins are redundant, because they are so essential for the development of the animal, examining knockout and transgenic animals (see Table 1.1) and the phenotypic appearance of their bones provides clues into the activi-ties of these proteins The only knockouts that totally lack bone are the osterix [106] and the Runx2 knockouts [107], although the retinoblastoma tumor suppressor gene knock-out has severely impaired osteogenesis [108] The knockout

taBle 1.2 Small leucine rich proteoglycans (SLrps) found in bone*

Cell differentiation Initiates mineralization Expression depressed in patient’s with Turner’s syndrome Decorin Generally 1 GAG chain/protein core Regulates collagen fibrillogenesis

Binds and releases growth factors Osteoadherin [91] Keratan sulfate proteoglycan Facilitates osteoblast differentiation and maturation

Regulates HA proliferation Fibromodulin 4 Keratan sulfate chains in its leucine

rich domain

Regulation of collagen fibrillogenesis Asporin [92] Possesses a unique stretch of aspartate

residues at its N terminus

Negative regulator of osteoblast maturation and mineralization

Osteoglycin/mimecan Derived from bone tumor

Also called osteogenic factor

Induces osteogenesis Regulation of collagen fibrillogenesis Regulation of mineralization Lumican Keratan sulfate proteoglycan Regulation of collagen fibrillogenesis

Regulation of mineralization Osteomodulin [93] Keratan sulfate proteoglycan Regulates osteoblast maturation

Periostin (osteoblasts-specific

factor 2) [94]

SLRP made in primary osteoblasts Regulates intramembranous bone formation

Regulates collagen fibrillogenesis Tsukushin [95] 353 amino acid protein upregulated by

estrogen – has phosphorylation sites

BMP inhibitor Regulates mineralization

* Adapted from OMIM: On Line Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/sites/entrez/OMIM unless otherwise noted.

Osteoporosis in Men

1 0

and overexpression of other bone proteins and ‘critical’

signaling pathways have altered bone properties but none

seem to be mandatory, most likely due to the redundancy

of the function of these proteins However, from the

analy-ses of the cell culture and altered phenotype in the animals

having too little or too much of these proteins, the

follow-ing can be identified as important for the formation of the

mineralized matrix: type I collagen, bone sialoprotein,

dentin matrix protein1, BAG-75, osteopontin, PHEX and

alkaline phosphatase The sequence in which they act is not

yet clear

acknowledgmentS

Dr Boskey’s data as reported in this review were supported

by NIH Grants DE04141, AR037661, AR041325 and

AR046121 Dr Boskey appreciates the collaboration of

Dr Steven B Doty who provided the images for this chapter

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Bone remodeling is a fundamental process by which the

mammalian skeleton tissue is continuously renewed to

maintain the structural, biochemical and

biomechani-cal integrity of bone and to support its role in mineral

homeostasis The process of bone remodeling is achieved

by the cooperative and sequential work of groups of

func-tionally and morphologically distinct cells, termed basic

multicellular units (BMUs) or bone remodeling units

(BRUs) Changes in the population and/or activities in any

component of the BMUs disrupts the harmony of the

cellu-lar efforts and leads to changes in bone mass and strength

The cellular activities of bone remodeling units vary within

and among the different bones of the skeleton and this

vari-ation changes with age, underlying the mechanism of

age-related bone loss This chapter reviews current concepts of

bone remodeling with respect to its cellular mechanism,

physiological functions and anatomic variation in cellular

behavior

cellular mechanIsm of bone

remodelIng

Bone remodeling takes place on bone surfaces and is

achieved by multicellular units, BMUs [1, 2] or bone

remodeling units, BRUs [3], the latter term being used

here The process of remodeling consists of four sequential

and distinct phases of cellular events: activation,

resorp-tion, reversal and formation [2, 4, 5] (Figure 2.1A–E) The

microanatomic basis of BRUs is osteonal units in

intra-cortical bone (Figure 2.1G) and discrete osteonal units or

packets in endocortical and cancellous bone (Figure 2.1F),

where removal of old bone is coupled in space and in time

by replacement by new bone [6, 7]

of microcracks and have conveyed signals to the surface

to initiate targeted remodeling However, most remodeling sites are likely to be random [13, 14]

resorption

Osteoclasts affix themselves to the bone matrix through integrins such as 3 [15, 16] The adherence to bone induces ruffled membrane formation and creates an annular

Bone remodeling: Cellular activities

in Bone

Hua ZHou1, SHi S Lu1 and david W dempSter2

1 Regional Bone Center, Helen Hayes Hospital, West Haverstraw, New York, NY, USA

2 Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY, USA

(A) (B)

MB

O L

(E)

Cancellous bone remodeling unit

Formation Resorption

(F)

Reversal

fIgure 2.1 Light photomicrographs of the principal phases of the remodeling cycle in cancellous bone of human iliac crest biopsy

specimens (A) Resorption Several multinucleated osteoclasts are seen in excavating a Howship’s lacuna (B) Reversal The Howship’s lacuna contains no osteoclasts but small mononucleated cells in contact with the scalloped surface (C) Formation A sheet of plump osteo- blasts is seen depositing osteoid (O) on top of mineralized bone (MB) Note the reversal line (L) and osteocyte lacunae (arrowheads) in the mineralized matrix (D) A later stage of formation where the osteoblasts have become flattened lining cells Matrix production has ceased, but a thin layer of osteoid still remains to be mineralized (E) Resting No remodeling activity is in progress but a layer of attenuated cells lines the surface Cross-sectional diagrams of BRUs in cancellous bone (F) and cortical bone (G) The arrows indicate the direction

of movement through space Note that the cancellous BRU is essentially one half of the cortical BRU (A–E, from Dempster DW Bone remodeling In Disorders of bone and mineral metabolism 2nd edn, (eds) Coe F, Favus MJ, pp 315–343, 2002 Lippincott Williams & Wilkins, Philadelphia: with permission F,G, from Seibel MJ, Robins SP, Bilezikian JP (eds) Dynamics of bone and cartilage metabolism, 2nd edn, pp 377–389, 2006 Academic Press, New York with permission).

C h a p t e r 2 Bone remodeling: Cellular activities in Bone 1 7

sealing zone, forming a hemivacuole between the osteoclast

itself and the bone matrix and isolated from the

surround-ing extracellular space (Figure 2.3A, B) By means of

membrane-bound proton pumps and chloride channels,

the osteoclast secretes hydrochloric acid, as well as acidic

proteases such as cathepsin K, TRACP, MMP9, MMP13

and gelatinase into the hemivacuole (see Figure 2.3A, B)

[17, 18] The acidified solution in the resorbing

compart-ment mobilizes the mineralized component of the matrix

and the proteolytic enzymes, which are most active at low

pH, degrade the organic constituents of the matrix This

pro-cess creates the crescent-shaped resorption cavities called

Howship’s lacunae on the cancellous bone surface (see

Figure 2.1A and F) and the cutting cones of the evolving

Haversian systems within cortical bone (see Figure 2.1G)

Generally, the resorption is accomplished by multinucleated

osteoclasts, but both in vivo and in vitro evidence suggests

that mononucleated cells are also capable of excavating bone

and forming resorption cavities and cutting cones [19, 20]

The fate of the osteoclast at the conclusion of the resorption

phase is unclear, but at least some undergo apoptosis [21]

reversal

During this phase, the resorption lacuna is occupied by

mononuclear cells, including monocytes, osteocytes that

were liberated from bone by osteoclasts and pre-osteoblasts that are being recruited to couple the resorption phase with the formation phase (see Figure 2.1B, F, G) [22] The mechanism of osteoblast coupling and the exact nature of the coupling signals are currently undefined, but there are

a number of interesting hypotheses One plausible theory

is that osteoclastic bone resorption liberates growth factors from the bone matrix and that these factors serve as chemo-attractants for osteoblast precursors and then enhance osteoblast proliferation and differentiation Bone matrix-derived growth factors, such as transforming growth factor- (TGF-), insulin-like growth factors I and II (IGF-I and II), bone morphogenetic proteins (BMPs), platelet-derived growth factors (PDGF) and fibroblast growth factor (FGF) are all possible contenders for such coupling fac-tors [23–27] Another attractive premise is that the cou-pling of bone formation to resorption is a strain-regulated phenomenon [28] As bone remodeling units penetrate through cortical bone, strain levels are reduced in front of the osteoclasts, but are increased behind them Similarly, in cancellous bone, strain is posited to be higher at the base of the Howship’s lacunae and lower in the surrounding bone

It is argued that this gradient of strain leads to sequential activation of osteoclasts and osteoblasts, with osteoclasts being activated by reduced strain and osteoblasts, in turn,

by increased strain This hypothesis may account for ment of osteons along the dominant loading direction of the

align-Prostaglandins, multiple hormones, cytokines, ILs and vitamin D

Stromal/osteoblastic

RANKL TNFα

IFNγ

TGFβ

E2

OPG, RANKL, M-CSF

c-Fms–

RANK– c-Fms+RANK– c-Fms+RANK+

M-CSF + RANKL T OPG

HSC

M-CSF

TNFα, IL-1, IL-6, IL-7, other ILs

T T T

fIgure 2.2 Role of cytokines, peptide and steroid hormones and prostaglandins in the osteoclast formation and activation

Hematopoietic stem cells (HSCs) express c-Fms (receptor for M-CSF) and RANK (receptor for RANKL) and differentiate to osteoclasts Marrow mesenchymal cells respond to a range of stimuli by secreting a mixture of pro- and anti-osteoclastogenic factors, the latter con- sisting primarily of OPG (From Ross FP Osteoclast biology and bone resorption In Primer on the metabolic bone diseases and disorders

of mineral metabolism, 6th edn, (ed.) Favus MJ, pp 30–35, 2006 American Society for Bone and Mineral Research, Washington, with permission).

bone [29, 30] Furthermore, osteoclast to osteoblast forward and reverse signaling has recently been implicated in the coupling mechanism [31, 32].

formation

Osteoblasts are recruited and differentiate from mal precursors There is a gradient of differentiation as the osteoblastic precursors reach the bone surface to refill the resorption cavity and the osteoblast phenotype becomes fully expressed (Figure 2.4A) [33] Bone matrix formation

mesenchy-is a two-stage process in which osteoblasts initially size the organic matrix, called osteoid, and then regulate its mineralization (Figure 2.4B) Osteoid consists of collagenous proteins, predominantly type I collagen, accounting for 90%

synthe-of the organic matrix, with non-collagenous proteins ing up the remaining 10%, including glycoproteins (i.e alkaline phosphatase and osteonectin), Gla-containing proteins (i.e osteocalcin and matrix Gla protein) and oth-ers (e.g., proteolipids) [34] Osteoid is deposited on the bone surface in curved sheets called osteoid lamellae, following the contours of the underlying mineralized bone (see Figure 2.4B) Once the collagenous organic matrix is synthesized, osteoblasts trigger the mineralization process, which occurs after a delay of about 20 days, called the mineralization lag time This is accomplished by the release of small, membrane-bound matrix vesicles that establish suitable conditions for initial mineral deposition by concentrating calcium and phosphate ions and enzymatically degrading inhibitors of mineralization, such as pyrophosphate and proteoglycans that are present in the extracellular matrix [35] During this period, the osteoid undergoes a variety of biochemical changes that render it mineralizeable The mineral content

mak-of the matrix increases rapidly to 75% mak-of the final mineral content over the first few days, called primary mineraliza-tion, but it may take as long as a year for the matrix to reach its maximum mineral content, called secondary mineraliza-tion [36] The mineral crystals within bone are analogous

to the naturally occurring geologic mineral, hydroxyapatite (Ca10[PO4]6[OH]2), including numerous ions which are not found in pure hydroxyapatite, such as HPO42, CO32,

Mg2, Na, F and citrate, adsorbed to the hydroxyapatite crystals [34]

As bone formation continues, osteoblasts that have reached the end of their synthetic activity embed them-selves in the matrix, becoming osteocytes (see Figure 2.4A) Osteocytes are regularly dispersed throughout the mineralized matrix and maintain intimate contact with each other, as well as to the cells on the bone surface, through gap junctions between their slender, cytoplasmic processes

or dendrites, which pass through the bone in small canals called canaliculi (Figure 2.5) Osteocytes function as an extensive 3-dimensional network of sensor cells, or ‘syn-cytium’, which can detect a change in mechanical strain

in bone and respond by transmitting signals to the lining

fIgure 2.3 (A) Transmission electron microphotograph of a

multinucleated osteoclast in rat bone Note the extensive ruffled

border, sealing zones and the partially degraded matrix between

the sealing zones (B) Diagram illustrating the primary

mecha-nisms of osteoclastic bone resorption (From Ross FP Osteoclast

biology and bone resorption In Primer on the metabolic bone

dis-eases and disorders of mineral metabolism, 6th edn, (ed.) Favus

MJ, pp 30–35, 2006 American Society for Bone and Mineral

Research, Washington, with permission).

Sealing

zone

(A)

Sealing zone

Signaling

ruffled membrane

Bone αvβ3

C h a p t e r 2 Bone remodeling: Cellular activities in Bone 1 9

cells on the bone surface to initiate targeted remodeling or

to regulate resorption and formation in the newly initiated

bone remodeling cycle [37] Osteocytes die by apoptosis,

which occurs with aging, immobilization, microdamage,

lack of estrogen, glucocorticoid excess and in association

with pathological conditions, such as osteoporosis and

osteoarthritis [38] Osteocyte apoptosis has also been

sug-gested to play an important role in targeting bone

remod-eling following the observation that osteocyte apoptosis

occurs in association with areas of microdamage and that

this is followed by osteoclastic resorption to begin the

replacement of the mechanically challenged bone [39]

Osteoblasts suffer one of three fates during and at the

end of the bone formation phase of the remodeling cycle:

many become incorporated into the matrix they formed

and differentiate into osteocytes; some convert into lining

cells on the bone surface at the termination of formation; and the remainder die by apoptosis Bone lining cells were once thought to serve primarily to regulate the flow of ions into and out of the bone extracellular fluid serving as the blood–bone barrier It has recently been appreciated that, under certain circumstances, for example, stimulation by PTH or mechanical force, bone lining cells can revert back

to functional osteoblasts [40, 41] Another recently covered important function of the lining cells is to create specialized compartments in cancellous and cortical bone where bone remodeling takes place [42] (Figure 2.6).The end result of a completed remodeling cycle by a BRU is the production of a new osteon (Figure 2.7A, B) The remodeling process is similar in cancellous and cortical bone with the remodeling unit in cancellous bone being equivalent

dis-to half of a cortical remodeling unit [43] (see Figure 2.1F, G)

(B) (A)

OB pOB

fIgure 2.4 (A) Light photomicrograph of a human bone biopsy stained with Goldner’s trichrome Osteoblastic lineage in a gradient

dif-ferentiation: osteoblastic precursors (pOB) reach the bone surface → mature osteoblasts (OB) filling in a resorption cavity → pre-osteocytes (pOCY) become incorporated into osteoid (OS) matrix → osteocytes (OCY) embedded within the mineralized bone (MB) (B) Fluorescent photomicrograph of dog bone Two steps of bone formation: osteoid matrix forming on bone surface (OS), mineralizing surface (MS) and mineralized bone (MB) (See color plate section).

fIgure 2.5 (A) Transmission and (B) scanning electron micrographs showing osteocyte processes communicating with cells on

the bone surface (From Marotti G et al The structure of bone tissues and the cellular control of their deposition Ital J Anat Embryol

1996;101:25-79, with permission).

The difference between the volume of bone removed by

osteoclasts and replaced by osteoblasts during BRU

remod-eling cycle is termed ‘bone balance’ As will be discussed

later, the bone balance varies with the anatomical location of

the bone surface as well as with gender, age and disease

PhysIologIcal functIons of bone

remodelIng

The primary functions of bone remodeling are presumed to

be maintenance of the mechanical competence of bone by

continuously replacing fatigued bone with new, mechanically

sound bone and to preserve mineral homeostasis by

continu-ously mobilizing the skeletal stores of calcium and phosphorus

to the circulation It has also been suggested that there must

be other, as yet known functions or reasons why the human skeleton undergoes such extensive remodeling [44]

Like all load-bearing structural materials, the skeleton is subjected to fatigue damage as it ages and undergoes repeti-tive mechanical challenges Older bone displays increased mineralization density as secondary mineralization con-tinues and the water content diminishes, which causes the matrix to become more brittle [45] In addition, aging is associated with biochemical changes in the bone matrix constituents, such as accumulation of non-enzymatic glyca-tion end products [46] and increased cross-linking of col-lagen [47] These changes render the bone more susceptible

to mechanical damage and fracture It has also been onstrated that osteocytes that have undergone apoptosis leave empty lacuna that may become occluded by miner-alized debris [48] and that fatigue microcracks increase in number with bone age and are spatially associated with missing osteocytes [49] Moreover, the fact that resorption cavities are frequently located close to bone microcracks [50, 51] provides compelling evidence that targeted remod-eling is activated in response to the appearance of such microcracks

dem-The skeleton is the greatest repository of mineral ions, such as Ca, Mg and P, in the human body and plays an important role in mineral homeostasis by coordinated interplay with the intestine, the site of net ionic absorp-tion, and the kidney, the site of net ionic excretion Long-term mineral homeostasis is achieved by the BRUs, which mobilize skeletal mineral to blood during bone resorption and return the mineral back to the skeleton during bone for-mation However, at least two other mechanisms allow the skeleton to participate in mineral homeostasis: the blood–bone barrier maintained by the bone lining cells and the percolation of bone extracellular fluid through osteocyte lacuno-canalicular network

OC

fIgure 2.6 Light photomicrograph of a human bone biopsy

stained with toluidine blue An osteoclast (OC) is resorbing bone

within a specialized compartment formed by a dome-shaped layer

of lining cells (arrows) (See color plate section).

fIgure 2.7 (A) Completed basic structural units in cancellous bone and (B) cortical bone The arrowheads delineate reversal lines

(From Dempster DW Bone remodeling In Osteoporosis: etiology, diagnosis, and management, 2nd edn, (eds) Riggs BL, Melton LJ,

pp 67–91, 1995 Raven Press, New York, with permission.) (See color plate section).

C h a p t e r 2 Bone remodeling: Cellular activities in Bone 2 1

In the regulation of calcium homeostasis, a fall in serum

Ca2 concentration is detected by the parathyroid cell

plasma membrane Ca-sensing receptor (CaSR), which leads

to an increase in parathyroid hormone release Parathyroid

hormone acts to mobilize calcium by three mechanisms:

PTH regulates the outflow of calcium from bone by

stim-ulating the resorptive activity in existing BRUs, an acute

response, and by stimulating the activation of new BRUs

and increasing bone turnover, a long-term response [52]

PTH also stimulates renal tubular reabsorption of calcium

and regulates the calcium blood–bone equilibrium through

the lining cells on quiescent bone surfaces Finally, PTH

increases intestinal absorption of calcium by enhancement

of 1,25 dihydroxyvitamin D synthesis [53, 54]

The involvement of the skeleton in phosphate

homeo-stasis is achieved in a similar manner A fall in plasma

phosphate concentration stimulates 1,25-dihydroxyvitamin

D production in the kidney which, in addition to

increas-ing phosphate absorption from gut, stimulates bone

remod-eling to mobilize phosphate from the skeleton Clearly,

phosphate cannot be withdrawn from the skeleton without

being accompanied by calcium and vice versa However,

the unnecessary increase in calcium or phosphate,

respec-tively, can be compensated by the enhanced renal excretion

of calcium or phosphate

As discussed earlier in the chapter, minerals are

trans-ferred into bone during the formation phase of the

remod-eling cycle Because bone formation is usually tightly

coupled with bone resorption, bone remodeling does not

generally lead to a net transfer of mineral to or from the

blood in the long run However, an increase in remodeling

rate does transiently mobilize significant amounts of

min-eral into the blood, because it takes a much longer time

for newly formed bone to reach the same mineral content

as that removed during bone resorption [55] By analogy

to financial transactions, when calcium is urgently needed,

it may be withdrawn rapidly from the bone bank and then

paid back gradually later This allows the skeleton to

par-ticipate in calcium homeostasis without permanently

com-promising its structural integrity However, with advancing

age, the intestinal absorption of calcium declines and,

ulti-mately, the mechanical competence of the skeleton is

com-promised to maintain an adequate serum calcium level

VarIatIon In bone remodelIng

actIVIty throughout the skeleton

The bone turnover rate varies substantially within and

among the different bones of the skeleton It has often been

asserted that cancellous bone has higher turnover than

corti-cal bone [56], which is true when one compares central

can-cellous bone with peripheral cortical bone This is generally

attributed to the four to five times higher surface-to-volume

ratio in the typical cancellous bone than in the typical cal bone [57, 58] and to the close correspondence in cancel-lous bone tissue between marrow cellularity, blood flow and remodeling activity [59] But this view fails to consider the geometrical and biological factors that influence bone turn-over [56, 59] The subdivisions within the bone consist of four distinct surfaces or ‘envelopes’: periosteal, Haversian

corti-or intraccorti-ortical, ccorti-ortical-endosteal corti-or endoccorti-ortical and cellous [4, 56] The evaluation of the activity of BRUs on each subdivision provides a histological estimation of bone turnover with the measurement and calculation of the tetracycline-based bone formation rate and activation fre-quency Such data are available for the ilium and the rib in the human skeleton In the iliac bone of healthy postmeno-pausal white women, bone turnover is 8.4% per year in the subperiosteal envelope, 5.9% per year in the intracortical envelope and 33.7% per year in the subendocortical enve-lope The bone turnover in the total cortical bone is 7.7% per year and in the cancellous bone it is 17.7% per year [56,

can-58, 60, 61] In the cortical bone of the sixth rib, mean bone turnover after 50 years is 4% per year [62]

Differences in cellular activity in BRUs among the tomic subdivisions within the bone determines the net dif-ference between the volume of bone removed and replaced

ana-by each BRU In the Haversian or intracortical envelope, the net bone balance is slightly negative, particularly in the inner half of the cortex, which leads to a decrease in the radial rate of closure of osteons [62], an increase in the Haversian canal diameter, a decrease in osteon wall thickness and an increase in the number of resorption cavities that are aban-doned in the reversal phase and remain unfilled [63–66] In the periosteal envelope, each BRU deposits slightly more bone than it removes Conversely, in the endocortical enve-lope, less bone is laid down than resorbed and the deficit here is greater than the slight positive balance in the perio-steal envelope, which reduces the thickness of cortex [60,

67, 68] In the cancellous bone envelope, there is a shortfall

in the amount of bone replaced compared to that removed, which causes thinning of trabeculae making them more vulnerable to perforation by osteoclasts [60, 67, 68] With aging, the effects of these small increments and decrements

of bone mass accumulate

The net bone balance on each envelope provides a based explanation for the long-established facts concerning the changes in three-dimensional geometry of bones as a function of age [4] Both the positive bone balance on the periosteal surface and the negative balance on the endocor-tical surface increase their respective circumferences, with the latter moving outward at a greater rate than the former, which consequently reduces cortical thickness The cor-tical porosity increases by 1–2% in the outer half of the cortex and by 5–10% in the inner half due to the negative bone balance on the Haversian surface The increase in the osteoclast resorption cavity depth, together with the nega-tive bone balance on the endocortical surface and the

BRU-increase in cortical porosity, leads to the creation of large

voids in the inner third to half of the cortex Ultimately, the

inner cortex resembles the cancellous bone in structure, a

process called cortical bone cancellization, which

contrib-utes to the thinning of cortex In cancellous bone, the

nega-tive bone balance in BRUs is manifested in a reduction of

the completed wall thickness of cancellous bone packets,

which is partially the cause of the gradual age-related bone

loss that occurs in both sexes [69, 70]

There have been relatively few assessments of the

regional variation in bone remodeling and turnover

through-out the skeleton Some attention has been given to the

rela-tionship between the standard biopsy site in the iliac crest

and other skeletal sites [71] As evaluated by

histomor-phometry, the bone turnover rate in ilium is about double

that in the vertebral body [3] Based on the measurement

of osteoid and osteoblast-covered surface, Krempien and

colleagues found a marked disparity among four different

skeletal sites, with an implied rank order of remodeling

rate as follows: iliac crest  lumbar vertebra  femoral

head  distal femur [72] The histomorphometric analysis

of tetracycline-labeled bone samples has been the most

reli-able way to assess regional differences in remodeling rate

but, obviously, is not practical for studies in living subjects

However, there is one case report [73] of an elderly

osteo-porotic woman who died suddenly before a scheduled bone

biopsy for which she had been pre-labeled with

tetracy-cline Twenty-four skeletal sites were sampled at autopsy

and bone formation rates were found to vary widely from a

high of 37% per year in the iliac crest to a low of less than

2% per year in the 10th thoracic vertebra Significant

varia-tions were also found between bones within fairly localized

regions of the skeleton, e.g from one vertebra to the next,

or between the right and left iliac crest The

tetracycline-based bone formation rate in cortical bone of rib, a

once-favored biopsy site, is 3–4% year, which is about twice that

in cortical bone elsewhere in the skeleton [3]

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Bone remodeling is the result of two opposite activities,

the production of new bone matrix by osteoblasts and the

destruction of old bone by osteoclasts The rate of bone

production and destruction can be evaluated by either

measuring predominantly osteoblastic or osteoclastic

enzyme activities or by assaying bone matrix components

released into the bloodstream and eventually excreted in the

urine They have been separated into markers of formation

and resorption, but it should be kept in mind that, in

dis-eases where both events are coupled in time and space at

the level of the basic multicellular unit and change in the

same direction, any marker will reflect overall rate of bone

turnover

The clinical utility of biochemical markers has been

extensively evaluated in postmenopausal osteoporosis [1, 2],

but data in men osteoporosis are more limited At present,

the biochemical markers which are the most specific and

established for bone formation include serum osteocalcin,

bone alkaline phosphatase (bone ALP) and the procollagen

type I N-terminal propeptide (PINP) [1, 2] For the

evalua-tion of bone resorpevalua-tion, most assays are based on the

detec-tion in serum or urine of type I collagen fragments, which

account for 90% of the organic bone matrix These include

the cross-links pyridinoline (PYD) and deoxypyridinoline

(DPD), the telopeptides of type I collagen generated by

cathepsin K (CTX, NTX) and by matrix-metalloproteases

(CTX-MMP or ICTP) and fragments of the helical portion

of type I collagen molecule (helical peptide) [2] The

indi-vidual measurement of most of these biochemical markers

can be achieved with high throughput and analytical

preci-sion on automated platforms [3, 4]

new bIochemIcal markers of bone metabolIsm and new assays

Although current biochemical markers have demonstrated clinical utility in the differential diagnosis of metabolic bone diseases and in predicting fracture risk and response

to treatment in postmenopausal osteoporosis, they do have some limitations Current biochemical markers of bone resorption are based primarily on type I collagen, which

is not bone-specific but rather widely distributed in several other body tissues Some of the type I collagen-based bone resorption markers are characterized by significant intra-patient variability, which impairs their use in individual patients The systemic levels of biochemical markers reflect global skeletal turnover and do not provide distinct infor-mation on the remodeling of different bone envelopes, i.e trabecular, cortical and periosteal Further, their relative contribution to skeletal turnover may vary with aging, dis-ease and treatment Finally, current markers mostly reflect quantitative changes of bone turnover and do not provide information on the structural abnormalities of bone matrix properties which are an important determinant of bone fragility, especially toughness Recently, new biochemi-cal markers have been investigated to address some of these limitations (Table 3.1), although their clinical utility

in assessment of bone turnover abnormalities in pausal and male osteoporosis is limited

postmeno-non-collagenous bone Proteins

Although the vast majority of bone matrix is composed of type I collagen molecules, about 10% of the organic phase

is comprised of non-collagenous proteins, some of them

assessment of Bone turnover in Men Using Biochemical Markers

Patrick Garnero1,2 and Pawel Szulc3

1 INSERM Research Unit 664, Lyon, France

2 Synarc, Lyon, France

3 INSERM Research Unit 831, Lyon, France

being almost specific for bone tissue It has been suggested

that the measurement of these proteins or fragments thereof

could represent specific biochemical markers of bone

turnover

Bone sialoprotein (BSP) is an acidic, phosphorylated

glycoprotein of 33 kDa (glycosylated: 70–80 kDa) which

contains an RGD integrin binding site Although BSP is

relatively restricted to bone, it is also expressed by

tropho-blasts and is strongly upregulated in a variety of human

primary cancers, particularly those that metastasize to the

skeleton [5] A small amount of BSP is released in the

cir-culation and, as such, is a potential marker of bone turnover

[6] Serum BSP levels are increased in malignant bone

dis-eases, in postmenopausal osteoporosis and are decreased by

antiresorptive treatments [6] However, because of its tight

association with circulating factor H, accurate measurement

of serum BSP remains challenging

Other non-collagenous proteins include osteopontin,

which belongs to the small integrin-binding ligand N-linked

glycoprotein (SIBLING) family like BSP [7] and periostin, a

secreted glutamic acid (Gla) adhesion molecule with

prefer-ential distribution in the periosteum envelope They have been

identified specifically as potentially useful in cancer-induced

bone diseases [8] Clinical data in other metabolic bone

dis-eases, including male osteoporosis, are currently unavailable

Although most of the newly synthesized osteocalcin is

captured within bone matrix, a small fraction is released

into the blood where it can be detected by immunoassays

Circulating osteocalcin comprises different immunoreactive

forms including the intact molecule and fragments of

dif-ferent sizes [9] The majority of these fragments is

gener-ated from in vivo degradation of the intact molecule and,

thus, also reflects bone formation In vitro studies suggest,

however, that some osteocalcin fragments could also be

released from osteoclastic degradation of bone matrix [10]

and, thus, may reflect, in part, bone resorption Elevated

lev-els of urinary osteocalcin fragment levlev-els were reported in

osteoporotic postmenopausal women and values decreased after one month of treatment with the bisphosphonate alen-dronate [11] This contrasts with the absence of significant change in serum total osteocalcin levels Urinary osteocal-cin fragment levels were found to be associated with BMD loss and fracture risk in older post-menopausal women [12, 13], but no data have been reported in men

osteoclastic enzymes tRAcP 5b

Acid phosphatase is a lysosomal enzyme which is present primarily in bone, prostate, platelets, erythrocytes and spleen Bone acid phosphatase is resistant to L ()-tar-trate (TRACP), whereas the prostatic isoenzyme is inhib-ited by TRACP Acid phosphatase circulates in blood and shows higher activity in serum than in plasma because of the release of platelet phosphatase activity during the clotting process In normal plasma, TRACP corresponds to isoen-zyme 5 Isoenzyme 5 is represented by two subforms, 5a and 5b TRACP 5a derives mainly from macrophages and den-dritic cells, whereas TRACP 5b is more specific for osteo-clasts [14] These two subforms differ by their carbohydrate content including sialic acid and mannose residues [15], optimal pH and specific activity TRACP 5a is a monomeric protein, whereas TRACP 5b is cleaved into two subunits.Total plasma TRACP activity is measured by colori-metric assays However, the lack of specificity of plasma TRACP activity for the osteoclast, its instability in frozen samples and the presence of enzyme inhibitors in serum are drawbacks which have limited the development of clinically useful enzymatic TRACP assays To overcome these limita-tions, different immunoassays for serum tartrate TRACP, which preferentially detect isoenzymes 5a and 5b, have been developed The first assay for TRACP 5b which was developed uses antibodies that recognize both intact and fragmented TRACP 5a and 5b The selectivity of this assay

table 3.1 New candidate biochemical markers of bone metabolism by category

non-collagenous proteins of

bone matrix and fragments osteoclastic enzymes

regulators of osteoclast-osteoblast differentiation/activity collagen posttranslational modifications

Bone sialoprotein

Osteopontin

TRAPC5b OPG/ RANK-L (osteoclast) Non enzymatic glycation – mediated modifications

of collagen (eg pentosidine, vesperlysine, GOLD, MOLD, CML)

Urinary mid-molecule

osteocalcin fragments

Wnt signaling molecules (Dkkl/sFRP)

Sclerostin (osteoblast) Type I collagen C-telopeptide isomerization (/

CTX ratio) TRACP5b: tartrate resistant acid phosphatase isoenzyme 5b OPG: osteoprotegerin; RANK-L: receptor activator of nuclear factor kB ligand;

Wnt Wingless, Dkk-1: Dicckops-1, sERP: soluble Frizzled-related protein, GOLD (glyoxal-derived lysine dimer), MOLD (methylglyoxal-derived lysine dimer), CML (carboxymethyllysine).

C h a p t e r 3 assessment of Bone turnover in Men Using Biochemical Markers 2 7

for TRACP 5b is partly achieved by performing

measure-ments at optimal pH for TRACP 5b activity [16] More

recently, a new immunoassay using two monoclonal

anti-bodies raised against purified bone TRACP 5b with limited

cross-reactivity for TRACP 5a has been described [17] One

antibody captures active intact TRACP 5b while the second

eliminates interference of inactive fragments We found

that this new enzyme-linked immunosorbent assay (ELISA)

for TRACP 5b is highly sensitive to detect increased bone

turnover following menopause and is also very responsive

to alendronate therapy [18]

Serum TRACP 5b is likely to reflect mostly the number

and the activity of the osteoclasts It may thus provide

infor-mation on the bone resorption process which is

comple-mentary to that provided by collagen-related markers [19]

Another advantage of serum TRAPC 5b relates to its limited

diurnal variation and negligible effect of food intake These

features result in lower intra-patient variability for TRACP 5b

than for collagen-based biochemical markers However,

the magnitude of changes observed following

bisphospho-nate treatment in postmenopausal women is also lower for

TRACP 5b than for collagen markers [16]

Data on serum TRACP 5a isoenzyme are more limited

and there is no yet commercially available assay A recent

study showed that serum TRACP 5a was significantly

increased in patients with rheumatoid arthritis (RA),

espe-cially in those presenting with nodules, whereas TRACP 5b

was only marginally increased and was not associated with

nodules [20] It has also been reported that the alendronate

induced a marked decrease in serum TRACP 5b, but had no

effect on serum TRACP 5a [21] These data indeed support

the view that TRACP 5a is likely to reflect inflammatory

macrophage activity, whereas TRACP 5b is an indicator of

osteoclast activity

Cathepsin K

The enzyme cathepsin K is a member of the cysteine

pro-tease family that, unlike other cathepsins, has the unique

ability to cleave both helical and telopeptide regions of type

I collagen [22] The enzyme is produced as a 329 amino

acid precursor procathepsin K, which is cleaved into its

active form with a length of 215 amino acids This cleavage

event takes place in vivo within the low pH bone

resorp-tion lacunae Commercially, two assays measuring

respec-tively the enzymatic activity and the protein concentration

of cathepsin K in serum are available Clinical data on

serum cathepsin K are still limited In both healthy women

and men, serum cathepsin K decreases with age,

contrast-ing with age-associated increased bone resorption [23]

Increased serum cathepsin K levels have been reported in

patients with active RA [24], patients with Paget’s disease

of bone [25] and in postmenopausal women with

fragil-ity fractures [26] Because circulating concentrations of

cathepsin K are very low and current available assays lack

sensitivity, accurate measurements of this enzyme remain challenging

regulators of osteoclastic and osteoblastic activity

RANK-L and OPG

The RANK-L/RANK/OPG system is one of the main lators of osteoclast formation and function [27] In healthy men, serum OPG increases with age and modestly cor-relates with parathyroid hormone (PTH) and total deoxy-pyridinoline, but not with BMD [28] Although the major contribution of this pathway in postmenopausal bone loss has been clearly established in various animal and clini-cal models, the serum measurement of RANK-L and OPG remains difficult Indeed, at present it remains unclear what proportion of circulating OPG is monomeric, dimeric or bound to RANK-L and which of these forms is the most biologically relevant to measure The same issues arise for the measurements of circulating RANK-L which, in its free form, has barely detectable levels in healthy individuals It

regu-is also unlikely that circulating levels of OPG and RANK-L reflect adequately local bone marrow production These limitations probably explain the conflicting data available

on the association of circulating OPG and RANK-L with BMD and biochemical markers of bone turnover in post-menopausal women and elderly men [29]

of which interacts with a single transmembrane low density lipid (LDL) receptor-related protein 5/6 (LRP5/6) Different secreted proteins, including soluble FRP-related proteins (sFRP), Wnt inhibitory factor-1 (WIF1) and Dickkopfs (Dkk) – isoforms 1, 2, 3, and 4 – prevent ligand-receptor interactions and consequently inhibit the Wnt signaling pathway Alterations of the Wnt signaling pathway and its regulatory molecules including Dkk-1 and sFRP have been shown to play an important role in bone turnover abnormal-ities associated with osteoporosis, arthritis, multiple mye-loma and bone metastases from prostate and breast [31].Immunoassays for circulating Dkk-1 have recently been developed Serum Dkk-1 levels have been reported to be increased in clinical situations characterized by depressed bone formation such as multiple myeloma [32] Circulating levels are also higher in diseases characterized by focal osteolysis, such as multiple myeloma [32], bone metas-tases from breast or lung cancer [33, 34] and RA [35] In

RA patients, we found that increased levels were associated with a faster radiological progression [36] Conversely, in

patients with osteoarthritis of the hip, a clinical situation

characterized by focal sclerosis of subchondral bone, lower

serum Dkk-1 levels have been shown to be associated with

a decreased risk of joint destruction [37] At the present

time there are no data on circulating Dkk-1 in

postmeno-pausal or male osteoporosis Similar to the assessment of

OPG and RANK-L, it is possible that circulating Dkk-1

does not reflect adequately local bone dynamics Another

issue with the determination of serum Dkk-1 is the fact that

Dkk-1 is in platelets and, thus, can be released in the serum

during the process of clotting [38], confounding the

inter-pretation of circulating levels

More recently, an immunoassay measuring sclerostin, an

osteocyte secreted factor inhibiting the Wnt signaling

path-way, has been developed on a multiplex platform (Meso

Scale Discovery, Gaithersburg, MA), but no data on

circu-lating levels have yet been reported

Post-translational modifications of bone type I

collagen

Post-translational modifications of type I collagen,

espe-cially those derived from non-enzymatic age-related

proc-esses, have been suggested to reflect age-related changes of

the mechanical properties of bone tissue Non-enzymatic modifications include the advanced glycation end prod-ucts (AGE) such as the cross-link pentosidine and the isomerization of aspartic acid residues This latter modifi-cation results in the conversion of the native alpha form of CTX ( CTX) to its beta isomerized peptide ( CTX) (Figure 3.1) A series of ex-vivo studies [39] performed on animal or human bone specimens has shown that changes

in pentosidine and CTX isomerization were associated with mechanical properties independently of BMD The ratio between urinary  CTX and  CTX provides a non-invasive tool which allows for the detection of alterations in bone matrix maturation Increased urinary / CTX ratio has been reported in conditions characterized by localized increased bone turnover, such as Paget’s disease and meta-static bone diseases [40, 41], consistent with the presence

of ‘younger’ poorly matured type I collagen molecules in the affected bone sites Immunohistochemistry studies also showed altered CTX isomerization in the abnormal woven matrix, which is comprised of younger, poorly matured col-lagen molecules [40, 41] A recent study found increased urinary / CTX ratio in the type I collagen genetic dis-order osteogenesis imperfecta, which may be indicative

of the qualitative defects of bone tissue observed in these

PYD DPD

K

O

O O

H N

N H α

β

CTX sequence: EKAHDGGR

OH α1

α1

N+

N

fIgure 3.1 schematic representation of c-telopeptide isomerization in type I collagen molecules Type I collagen is constituted by the

association in triple helix of two alpha 1 and one alpha 2 chains except of the two ends (N and C-telopeptides) In bone matrix, type I collagen

is subjected to different post-translational modifications including (1) the trivalent crosslinks by pyridinoline (PYD) and deoxypyridinoline (DPD) which make bridges between 2 hydroxylysine residues within the telopeptides of one collagen molecule and one hydroxylysine (PYD)

or lysine (DPD) in the helical region of a second collagen molecule and the non-enzymatic isomerization of aspartic acid (D) occurring in the

8 amino acid sequence (CTX) within the C-telopeptides of alpha 1 chains Isomerization is a spontaneous posttranslational modification which converts the native CTX (CTX) to its  isomerized (CTX) form in which the peptide bond between D and the adjacent glycine (G) is made through the carboxyl group in position  The urinary ratio / CTX provides a biological index of type I collagen maturation.

C h a p t e r 3 assessment of Bone turnover in Men Using Biochemical Markers 2 9

patients [42] In postmenopausal women, it was found that

increased urinary / CTX ratio was significantly

associ-ated with increased fracture risk independently of both hip

BMD and overall bone turnover More recently, a high /

CTX ratio was shown to be predictive of non-spine and hip

fracture, independent of hip BMD, in elderly men

partici-pating in the Mr Os study (Table 3.2) [43]

The effects of antiresorptive therapy and PTH on urinary

/ CTX ratio in postmenopausal women have recently been

investigated in post hoc analyses of interventional studies

The bisphosphonates, alendronate at a dose of 10 or 20 mg/

day and ibandronate, both induce a decrease of / CTX

ratio, suggesting increased bone collagen maturation [44]

Such changes were not observed with treatments that are

less potent suppressors of bone turnover, such as raloxifene

[44] or calcitonin [45] Conversely, treatment with PTH

1-84 for 1 year, followed by 1 year of placebo or alendronate

was associated with an increase of / CTX ratio [46],

sug-gesting the formation of younger, less mature bone matrix

with PTH All together, these data suggest that the urinary

/ CTX ratio may indeed reflect alterations of bone

col-lagen maturation in women and men with osteoporosis and

provide additional information on bone strength that is not

captured by BMD or conventional bone turnover markers

new technologIes for dIscovery

and assay bone markers

The currently available bone markers have been developed

using a conventional candidate approach based on known

physiopathological pathways, enzymes from osteoblast

or osteoclast and proteins purified from bone matrix It is

likely that the improvement of proteomic technologies, such

as surface enhanced laser desorption ionization (SELDI)

and matrix-assisted laser desorption time-of-flight mass

spectrometry (MALDI-TOF), coupled with bioinformatics,

will provide a means to analyze a broad array of proteins

and to identify novel markers Such a strategy has recently

been applied to define a serum biomarker profile which

was able to differentiate postmenopausal women with high

or low bone turnover [47] Ultimately, such strategies may result in identifying a panel of a few independent markers which, when combined, may improve the sensitivity and specificity to detect patients at high fracture risk Multiplex automated technologies allowing the simultaneous measure-ment of these biochemical markers in a low sample volume will then be required for easy use Illustrating this point, an automated platform with high analytical precision has been recently developed for the simultaneous measurements of osteocalcin, CTX, PINP and PTH in only 20 microliters of serum [48]

factors InfluencIng btm levels In men

Bone turnover is subject to the influence of many factors, some of which have been comprehensively examined Strong correlations between bone turnover marker (BTM) levels in male twins indicate that hereditary factors are an important determinant of the bone turnover rate in men [49] By contrast, ethnic differences in the BTM levels are relatively weak [50]

Bone turnover demonstrates circadian variation For most BTM, acrophase (peak time) is similar in both sexes

In contrast, the average concentration (mesor) and nitude of diurnal variation (amplitude) vary between men and women [51] Levels of most BTMs increase during the night and attain highest values between 04:00 and 06:00 hours Circadian variation is greater for bone resorption than for bone formation In men, diurnal variation is simi-lar for different bone resorption markers Variation of bone formation is lower – 5 to 20%

mag-Mechanisms that govern circadian variability of BTM are not known Food consumption accentuates circadian varia-tion of bone resorption, whereas fasting significantly attenu-ates the circadian pattern probably due to the increased secretion of glucagon-like peptide-2 which is stimulated

by food intake [52] It suggests that the circadian variation

of bone resorption is only partly inherent and, to a major extent, determined by the exogenous nutritional regulation

table 3.2 type I collagen and the risk of fracture in older men: the Mr Os study

rr of fracture (95% cl), age and clinic adjusted rr of fracture, age, clinic and hip bmd adjusted

creatinine and the / CTX ratio From Bauer et al Osteoporosis Int 2008; 19, supp 2;S244 (with permission).

Data on the seasonal variation of BTM in men are

lim-ited However, some studies show increased bone

resorp-tion in winter in older but not in younger men [53] The

winter increase in bone turnover is believed to be related to

vitamin D deficiency and secondary hyperparathyroidism

Therefore, it is more prominent in the elderly who are more

likely to become vitamin D deficient Low

25-hydroxyvita-min D and high PTH levels are also associated with higher

BTM levels regardless of the season in older men This

association is observed especially in institutionalized

per-sons who have severe vitamin D deficiency, high PTH

con-centration and markedly increased BTM levels [54]

Sex steroid hormones are major determinants of bone

turnover in older men Low concentration of the bioavailable

fraction of 17-estradiol is strongly associated with higher

levels of the markers of bone formation and bone resorption

and this effect is observed in men from the general

popula-tion [55, 56] In contrast to the bioavailable fraction, total

17-estradiol is not correlated with BTM levels Men with

overt hypogonadism, as defined by a decreased

concentra-tion of total, free or bioavailable testosterone, had slightly

higher levels of bone resorption markers but not of bone

formation markers [57] By contrast, in the general

popu-lation, the association between the testosterone level (free,

bioavailable) and the BTM levels is weak or not significant

Interventional studies also show that 17-estradiol inhibits

bone resorption in men much more strongly than

testoster-one [58] In this short-term study (3 weeks), it could also be

shown that 17-estradiol and, to a lesser extent, testosterone

are direct stimulators of bone formation

Tobacco smoking was associated with slightly higher

levels of bone resorption markers but not of bone

forma-tion markers [59] It suggests that elevated bone resorption

not matched by a parallel increase in bone formation could

underlie tobacco-induced bone loss However, specific

mechanisms responsible for the increase in bone

resorp-tion in smokers are not elucidated This mechanism could

include vitamin D deficiency, hypercortisolemia, increased

catabolism of 17-estradiol, stimulation of bone resorbing

cytokines by components of smoke, as well as other factors

which are more frequently observed in smokers, although

not induced by smoking itself (e.g low body mass index,

high alcohol intake, sedentary lifestyle)

Most studies show that alcohol abuse is associated mainly

with the inhibition of bone formation (confirmed by bone

histomorphometry) and lower concentrations of bone

for-mation markers (especially OC) [60, 61] By contrast, data

on bone resorption markers levels are divergent These data

suggest that an imbalance between bone resorption and

low-ered bone formation may underlie the low BMD observed in

those who abuse alcohol However, data on bone resorption

should be interpreted cautiously Heavy drinkers often have

lower muscle mass associated with lower urinary creatinine

excretion In these persons, adjustment of the urinary

excre-tion of bone resorpexcre-tion markers for urinary creatinine may

falsely increase their levels Ethanol withdrawal results in

a progressive normalization of bone turnover rate [62] The actions of alcohol on bone include the following potential mechanisms: a direct effect of ethanol on bone cells, under-nourishment, hepatic cirrhosis, hypogonadism, vitamin D deficiency, hypercortisolemia [63] Any one or combination

of these discrete mechanisms could be responsible for tive calcium balance in alcoholism

nega-Immobilization is associated with an acceleration of bone turnover, especially of bone resorption The rapid increase in bone resorption is observed very early during experimental bed rest in young healthy men and is not fol-lowed by a parallel increase in bone formation [64] During the first weeks after acute spinal cord injury, bone resorp-tion increases dramatically to several times higher than the upper limit of the normal range, whereas bone formation increases only slightly [65] It suggests a severe imbalance between these two processes which probably underlies the rapid bone loss associated with immobilization In elderly persons with prolonged, very low physical activity (includ-ing the sick and bedridden), bone turnover rate is higher – both bone formation and bone resorption [66, 67] In this group, there are probably several mechanisms responsible for the increase in bone turnover: immobility itself, under-lying diseases, undernourishment or vitamin D deficiency due to a very low sunlight exposure

Leisure daily physical activity has no major effect on bone turnover By contrast, regular intensive sport train-ing in young healthy men (long distance runners, premier league soccer players) is associated with accelerated bone turnover [68, 69]

Prostate cancer is frequent in men During its natural course, bone metastases often develop and are associated with higher BTM levels, mainly ICTP, bone ALP, NTX and

--CTX (native non-isomerized CTX-I reflecting the most recently synthesized type I collagen molecules) and, to a lesser extent, P1NP, -CTX or OC [70] In these patients, bone resorption is increased to a greater extent than bone formation High ICTP levels suggest an important role of matrix metalloproteinases in the formation of bone metas-tases [71] BTM levels increase sharply with the spread of bone metastases Elevated BTM levels (NTX, bone ALP) were associated with a higher risk of the skeletal-related events (e.g pathological fractures, spinal cord compres-sion) regardless of the presence of bone metastases or treat-ment status [70, 72]

Some kinds of androgen deprivation therapy (ADT) used in the therapy of prostate cancer (analogues of lutein-izing hormone-releasing hormone, orchiectomy) promptly increase bone turnover resulting in rapid bone loss In men without bone metastases, BTM increases during the first months, then stabilize at the higher level [73] Bone resorp-tion increases more than bone formation Anti-resorptive treatment (neridronate, pamidronate) initiated simultane-ously with ADT prevented the increase in BTM [74] In men who had previously received ADT, bisphosphonates decreased elevated BTM levels in men who did not have

C h a p t e r 3 assessment of Bone turnover in Men Using Biochemical Markers 3 1

progression of bone disease, but not in men who

experi-enced a progression of disease [75, 76] Other

anti-resorp-tive medications (estrogens, raloxifene, diethylstilbesterol)

also decreased BTM (or prevented their increase) in men on

ADT Also, in men with metastatic prostate cancer treated

with ADT, bisphosphonates induced rapid and protracted

inhibition of bone resorption [77]

Corticosteroids are the principal group of drugs

increas-ing the risk of osteoporosis They promptly inhibit bone

formation, a fall in the OC concentration being

consist-ently most significant and most rapid followed by a delayed

and weaker decrease in the levels of PICP, PINP and bone

ALP [78, 79] Bone resorption can increase, especially after

treatment exceeding 3 months, but data are less consistent

Interestingly, OC concentration increased significantly and

normalized after withdrawal of the long-term corticosteroid

therapy [80] Inhaled corticosteroids induced a small but

statistically significant decrease in the OC concentration

but did not influence other BTM levels [81] In the

anal-ysis of the effect of corticosteroids on BTM, it should be

noted that their effect depends on the age of patients and

the underlying disease In bronchial asthma, changes in the

BTM reflect the undesirable effect of corticosteroids on

bone turnover, while chronic inflammatory diseases, such

as rheumatoid arthritis, may themselves induce changes

in bone turnover In these patients, higher bone

resorp-tion may confound the effect of corticosteroids on BTM

Corticosteroid-treated patients usually have more severe

underlying disease than patients who do not receive

cor-ticosteroids In the longitudinal studies, changes in BTM

reflect mainly both the pharmacologic effect of

corticoster-oids and the severity of the disease at baseline

clInIcal aPPlIcatIons of bone

turnover markers In male

osteoPorosIs

association of bone mineral density and bone

loss with the btm levels

Young men achieve their peak areal bone mineral density

(aBMD) in young adulthood Attainment of peak BMD

(growth arrest, consolidation) is associated with a reduction

in bone turnover and a decrease in BTM levels However, the

age of peak aBMD varies according to the skeletal site (from

20 to 25 years at the hip, up to 40 years at distal radius) This

is probably the reason why, in young men, the correlation of

BTM with aBMD and trabecular microarchitectural

param-eters is weak or, most frequently, not significant [82, 83] In

older men, who are in the phase of bone loss, BTM levels

are weakly but significantly correlated with aBMD [82, 83]

The older the age group, the stronger is the correlation,

prob-ably signifying age-related acceleration of bone loss in men

The difference between average aBMD in men with low and

high BTM levels was greater at the predominantly trabecular

skeletal sites than for the predominantly cortical sites In this group, BTM correlated weakly but negatively with trabecu-lar bone volume and trabecular number

In older men, higher BTM levels are associated with slightly more rapid bone loss in some [84, 85], but not all [86] studies These data suggest that bone loss in men is determined mainly by an acceleration of bone turnover driven

by a slight increase in bone resorption which is not matched

by a parallel increase in bone formation This imbalance results in age-related bone loss However, the link between BTM levels and remodeling events were studied mainly in women and it is not certain that they can be directly extrapo-lated in men In some men, osteoblast insufficiency may be a main determinant of bone loss To our knowledge, no study has reported lower aBMD and slower bone loss in men with low concentrations of bone formation markers

Continuous periosteal apposition influences bone size, aBMD and calculated rates of bone loss However, it is not reflected by BTM levels Therefore, increased BTM levels seem to reflect mainly bone loss at endosteal surfaces [84] However, methodological limitations of the applied approach should be recognized The calculation of endo-steal bone loss has been based on the geometric approxima-tions and has not been confirmed by a more direct method

In men, bone loss is slow, especially before the age of

70 Therefore, its individual values during a short-term low up may be biased by a measurement error, especially in men less than 70 By contrast, a single BTM measurement does not necessarily reflect the bone turnover rate during

fol-a long-term follow up BTM levels reflect the overfol-all rfol-ate

of bone turnover, whereas the rate of bone loss may vary according to the skeletal site At every skeletal site, bone metabolism is influenced by systemic factors (e.g hor-mones) and by local factors (e.g mechanical load) which may differently affect the bone turnover and the rate of bone loss according to the skeletal site Furthermore, data concerning the lumbar spine are inconclusive because its aBMD increases with age due to progression of osteoarthri-tis Thus, the weak overall correlation between bone loss and BTM in men may result from both biological determi-nants and methodological limitations

Prediction of the fragility fractures by btm in elderly men

Few studies have assessed the use of BTM for the tion of osteoporotic fractures in men In a prospective nested case-control study from the Dubbo cohort (cases – 50 men with incident symptomatic low trauma fractures; controls – 101 men free of any bone disease who did not take any medication affecting bone disease and had not had fractures in the past), increased serum ICTP concentration (fourth quartile) was associated with an almost threefold higher risk of fracture [87] ICTP remained predictive of fractures after adjustment for aBMD and other confound-ers It predicted all fractures analyzed jointly as well as

predic-hip fractures, vertebral fractures and other fractures

ana-lysed separately Serum CTX and PINP concentrations

did not predict fractures However, this study has several

limitations Exclusion from the control group of men with

prevalent fractures and concomitant major diseases, mainly

bone diseases, can overestimate the difference between the

groups Ascertainment of the incident vertebral fractures

was suboptimal Time of blood sampling was not

standard-ized, which can affect markedly CTX levels

In the MINOS cohort composed of 790 men aged 50–85

years and followed up from 3 to 90 months, 77 incident

fractures (including 27 radiographically determined

verte-bral fractures) occurred in 74 men [84] None of the large

panel of BTM measured at baseline (OC, bone ALP, PINP,

serum and urinary CTX, free and total DPD) predicted

frac-tures regardless of the statistical model used (continuous

log-transformed BTM levels, various thresholds,

adjust-ment for confounders including aBMD, analysis limited to

major fragility fractures) The principal limitation of this

study was the low number of the incident fractures The

large dropout for longitudinal spine x-rays could

underesti-mate the number of incident vertebral fractures

In two prospective nested case-control analyses from the

Mr OS cohort composed of men aged 65 years and older and

followed up for 5 years on average (cases – 427 men with

incident non-spine fractures; controls – 943 and 1013

ran-domly selected men, respectively), increased serum

concen-trations of PINP, TRACP5b and CTX as well as increased

urinary excretion of  CTX and of  CTX (highest quartile)

were each not independently associated with the risk of hip

or non-spine fractures in multivariate models adjusted for

other confounders including femoral neck aBMD [43, 85]

By contrast, as previously indicated, increased / CTX

ratio was associated with a twofold higher risk of hip and

non-spine fracture also after adjustment for BMD [43]

BTM levels reflect overall bone turnover rate, whereas

the  / CTX ratio is supposed to reflect the degree of

colla-gen maturation The fact that the increased  / CTX ratio,

but not the conventional BTM, predicted fracture suggests

that impaired collagen maturation may be associated with

an increased bone fragility in elderly men independent of

BMD and overall bone turnover rate

Increased levels of conventional BTM do not seem to

be useful to predict fractures in older men in contrast to

women Several possible hypotheses can be put forward

to explain this observation Negative results may be related

to the markers themselves In older men, bone formation

markers remain stable or increase only in very old men and

are much lower than in postmenopausal and older women

[88] Importantly, even in women, bone formation markers

were less predictive of fracture than bone resorption

mark-ers [1, 2] Bone resorption increases with age in men, but

urinary DPD excretion is markedly lower in older men than

in women of similar age [88] It suggests that few men can

have BTM levels sufficiently high to affect substantially

bone strength Thus, the so-called ‘increased bone turnover’ defined by the highest quartile may correspond to lower BTM levels and lower rate of bone turnover in absolute terms (e.g number of bone remodeling units) and, consequently,

to a lower damage of bone tissue in men than women (smaller bone loss from the peak bone mass, less cortical thinning, less deterioration of the trabecular microarchitec-ture, smaller deficit in bone mineralization)

Furthermore, it has not yet been proven that the ship between bone turnover rate and the loss of bone mass and strength is the same in men and in women It is not clear whether the same BTM reflect similarly the degrada-tion of bone matrix in both sexes Urinary DPD excretion increases with age in men This increase is also observed for DPD adjusted for the glomerular filtrate volume, which indicates that it is not an apparent increase due to the age-related decrease in muscle mass and urinary creatinine [83] By contrast, age-related increase in the serum and uri-nary levels of NTX and CTX is weaker or not significant in men (in contrast to women) [83, 89], whereas serum ICTP increases [86] Thus, enzymatic mechanisms and principal products of degradation of bone type I collagen may be different in men and in women

relation-Bone turnover rate reflects mainly metabolic status

of the trabecular bone However, men have larger bones and higher cortical mass which can have a strong protec-tive effect but is not reflected by the BTM levels The peri-osteal apposition can also reduce the loss of bone strength and again, it is not reflected by the BTM levels [84] Furthermore, the above studies have assessed mainly or exclusively non-spine fractures However, vertebral frac-tures may be more dependent on bone fragility, whereas non-spine fractures may depend more on the trauma It should be also recognized that a fracture is a rare event and a long-term follow up is needed to collect a number of fractures high enough to attain sufficient statistical power However, the predictive power of a single BTM measure-ment may decrease with time in long-term studies

Other studies also need to be mentioned In men aged

70 and over, baseline carboxylated OC (COC) to total OC ratio (COC/TOC) was lower in those who subsequently sus-tained a fracture than in men who did not [90] In men and women analyzed jointly, low COC/TOC predicted fractures

in a short-term but not in long-term study, however, these data were not adjusted for aBMD Vitamin K is necessary for the gammacarboxylation of OC Therefore, decreased COC/TOC may reflect vitamin K deficit However, it is not clear if this association reflects the effect of vitamin K

on bone metabolism or is a by-stander of the association between nutritional deficits and increased bone fragility.Homocysteine (Hcy) is not a marker of bone turnover, however, it has appeared as an indicator of fracture risk and this association was stronger in men than women [91, 92] However, high Hcy concentration was predictive of frac-tures mainly in old and frail elderly High Hcy concentration

C h a p t e r 3 assessment of Bone turnover in Men Using Biochemical Markers 3 3

was associated mainly with increased risk of hip fracture

and some of these analyses were not adjusted for aBMD

Thus, Hcy may simply reflect poor health status, unhealthy

lifestyle and nutritional deficits that influence aBMD,

risk of fall and mortality Hcy has been supposed to be a

marker of the ultrastructure of collagen, because it

inhib-its lysyl oxidase, an enzyme necessary for the synthesis of

cross-links [93] Impairment of collagen cross-linking may

interfere with bone mineralization, compromise trabecular

organization and reduce bone strength [94] Hcy also

stimu-lates differentiation and function of osteoclasts [95] Thus,

the mechanism underlying the association between the Hcy

level and fracture risk remains to be elucidated: impairment

of the ultrastructure of bone collagen matrix, increased bone

turnover, lower aBMD, nutritional deficits, or frailty due to

the poor general health status and propensity to fall?

In summary, the currently available BTM levels are not

independently related to the risk of fragility fracture in men

From the pathophysiological point of view, it suggests that

higher bone turnover rate (as assessed in comparison with

the levels observed in older men) is not associated with the

increased fragility in older men From the clinical point of

view, it means that the measurement of BTM levels cannot be

recommended for the assessment of the fracture risk in older

men in clinical practice Biochemical assessment of tive modifications of bone matrix, which may be associated with higher bone fragility in men, needs further studies

qualita-effect of antI-osteoPorotIc treatment on btm In men

Data on the changes induced by anti-osteporotic treatment

in men are limited because there are few studies on the pharmacological treatment of osteoporosis in men

testosterone replacement therapy (trt)

Effect of testosterone replacement therapy (TRT) on bone turnover in hypogonadal men depends on the initial hormo-nal status, normalization of testosterone level during treat-ment and the treatment duration TRT is efficient in men with overt hypogonadism, but not in men with borderline decreased testosterone concentration TRT is not efficient if testosterone level has not been normalized The effect of TRT may also depend on the initial BMD and bone turnover rate.TRT reduces bone resorption moderately but promptly and significantly (Figure 3.2) [96, 97] The decrease was

Day

fIgure 3.2 Effect of transdermal testosterone (T) gel and testosterone patch treatment on urinary N-terminal crosslinking telopeptide

of type I collagen / creatinine ratio and serum osteocalcin concentration in 227 hypogonadal men aged 19 to 68 (mean  SE) The subjects were initially (days 0 to 90) randomized to three groups: T patch (closed triangles), T gel 50 mg/day (closed squares), and T gel 100 mg/day (closed circles) (left panel of each graph) Based on the serum T levels, the dose of T gel was adjusted upwards or downwards to 75 mg/day

at day 90 if the serum T level was below or above the adult male range, respectively: T gel 50 to 75 mg/day (open squares), T gel 100 to

75 mg/day (open circles) (right panel of each graph) (Wang et al Effects of transdermal testosterone gel on bone turn over markers and

bone mineral density in hypogonadal men From Wang et al., Clin Endocrinol 2001; 54:739–750 (with permission).

significant for more specific bone resorption markers such

as DPD or NTX-I, but not for hydroxyproline which is

not specific for bone and poorly sensitive Decrease in

the urinary excretion of bone resorption markers per mg

urinary creatinine is partly related to the increase in

mus-cle mass induced by testosterone Therefore, data expressed

per glomerular filtrate volume and serum markers of bone

resorption can be more reliable, although experimental

data are limited The overall effect of TRT on bone

forma-tion markers, as presented in the metaanalysis of Isidori et

al., was not significant [98] However, these data should

be interpreted cautiously Apart from the aforementioned

general limitations, the dynamics of bone formation during

TRT should be taken into account Bone formation

mark-ers increase during the first months of TRT, then plateau

[96, 97] This increase may reflect the direct

stimula-tory effect of TRT on bone formation Later, TRT-induced

decrease in bone resorption was followed by a decrease in

bone formation which may reflect general slow down of

bone turnover These studies present certain limitations

Groups are small and heterogeneous (etiology, age of

diag-nosis of hypogonadism, age at the beginning of the study,

duration of TRT before the study, doses of TRT and way of

administration, degree of normalization of the testosterone

level, duration of TRT during the study) A placebo group is

not always included TRT-induced increase in aBMD may

reflect mainly the stimulation of bone formation in young

men and the inhibition of bone resorption in the elderly

anti-resorptive treatment

Studies in men concern principally alendronate and

risedro-nate, which increase aBMD and decrease BTM Both

eugo-nadal and hypogoeugo-nadal men were recruited for these studies

Bisphosphonates induce a rapid decrease in BTM levels,

detected after 1 month of treatment [99, 100] After 3–12

months, decrease in BTM levels attains 50–60% for bone

resorption and 15–40% for bone formation (Figure 3.3)

[99, 101] Then, BTM levels remain stable Decrease in

BTM is comparable for 5 and 10 mg alendronate and 5 mg

risedronate In osteoporotic men, treatment with 35 mg

rise-dronate weekly decreased serum bone ALP concentration

by 25–30%, serum CTX concentration by about 50% and

urinary NTX excretion by about 35% [102] This decrease

was observed after 3 months of treatment (earliest time

point tested), then BTM levels remained relatively stable

In patients receiving at least 7.5 mg oral prednisone

daily, alendronate and risedronate decreased BTM in

patients receiving glucocorticoids for less than 3 months

and in patients treated for more than 6 months However,

BTM were analyzed jointly in both sexes In hypogonadal

osteoporotic men receiving an adequate TRT, alendronate

decreased urinary DPD excretion promptly and rapidly

[103] In men with Klinefelter’s syndrome, intravenous

ibandronate decreased the bone turnover rate and increased

aBMD [104] However, after withdrawal of the treatment, BTM levels returned to the pretreatment levels and aBMD decreased In a group of human immundeficiency virus (HIV)-infected men treated with highly active antiretroviral therapy who had BMD T-score  0.5, annual zoledronate administration decreased urinary NTX excretion by about 60% and serum concentrations of OC and CTX by 50–60% compared to the placebo group (OC and CTX-I were not measured at baseline) [105]

In 28 men with idiopathic osteoporosis, nasal calcitonin

200 IU daily administered for 1 year reduced bone turnover [106] Decrease in bone resorption was significant after 3 months and attained 50% after 12 months It was followed

by a milder decrease in bone formation markers which became significant after 6 months

50 40 30 20 10

12 8 4

fIgure 3.3 Effects of alendronate on biochemical markers of

bone resorption in 259 patients (top panel) and bone formation in

264 patients (bottom panel) receiving an average daily dose of at least 7.5 mg of prednisone (or its equivalent) All values are means (SE) The solid horizontal lines indicate the mean reference val- ues for premenopausal women, and the dotted horizontal lines 1

SD above and below the mean The values were significantly decreased at 48 weeks in the patients receiving 5 mg of alen-

dronate and those receiving 10 mg (P  0.001) From Saag et al

N Engl J Med 1998; 339:292–299 (with permission).

C h a p t e r 3 assessment of Bone turnover in Men Using Biochemical Markers 3 5

treatment with bone formation stimulating

agents

Effect of recombinant human parathyroid hormone (1–34)

(rhPTH-[1–34]) on BTM levels was assessed in two

rand-omized placebo-controlled studies [107, 108] Markers of

bone formation increase rapidly with a significant

incre-ment of PINP after 1 month of treatincre-ment followed by an

increase in bone resorption markers (Figure 3.4) This rapid

increase in bone formation indicates that rhPTH-(1–34)

directly stimulates osteoblastic cells After 6–9 months

of treatment, BTM attain the maximum (50–250% above

baseline), then slightly decrease but remain elevated

By contrast, during combined treatment (alendronate and

rhPTH-[1–34]) started 6 months after the beginning of the

anti-resorptive treatment, the increase in the serum

concen-trations of the bone formation markers induced by

rhPTH-(1–34) were lower and the increase in aBMD at the spine

and femoral neck was less than after rhPTH-(1–34) alone [109] (Figure 3.5) In growth hormone (GH) deficient men, recombinant human GH increases bone resorption and bone formation [110, 111] BTM increase after 4 days of treat-ment, attain peak values (50–300% above baseline) after 6–

12 months, then decrease BTM decrease despite sustained elevated histomorphometric parameters of bone formation and resorption During GH therapy, changes in BTM and aBMD did not correlate, probably because BTM increase from the beginning of treatment, whereas aBMD decreases slightly then increases A similar pattern of changes in BTM levels (increase then decrease) was found in adults inde-pendent of the etiology of GH deficiency The studies on GH treatment present several limitations including small groups, both sexes analyzed jointly, no placebo group in most cases, different doses of GH, different regimens and treatment dura-tion Thus, these results should be interpreted cautiously

–20 0 PICP

P = 0.06 vs placebo

Placebo TPTD20 TPTD40

P < 0.001 vs placebo –20

P = 0.021 vs placebo

fDPD/CR

Months

P � 0.001 TPTD20 vs 40 (Bone ALP, PICP, NTX, CTX); P < 0.01 (TDPD) at all timepoints after baseline

fIgure 3.4 Median percent changes from baseline in biochemical markers of bone formation (top) and resorption (bottom) from baseline

to endpoint for observed cases at 1, 3, 6, and 12 months (Bone ALP – bone alkaline phosphatase, PlCP – procollagen I carboxy-terminal, NTX/CR – urinary N-telopeptide/creatinine ratio, fDPD/CR – free deoxypyridinoline/creatinine ratio, TPTD20 – teriparatide 20 g; TPTD40 – teriparatide 40 g) From Orwoll et al J Bone Miner Res 2003; 18:9–17 (with permission).