monograph series no. 22 - equine musculoskeletal biomarkers

8/12/2019 Monograph Series No. 22 - Equine Musculoskeletal Biomarkers

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Proceedings of a Workshop on

EQUINE MUSCULOSKELETALBIOMARKERS

30th October 2nd November 2005Colorado, USA

Editors: C. W. McIlwraith and J. F. Wade

H a v e m e

y e

r F ou n d a t i

o n

Havemeyer FoundationMonograph Series No. 22


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Havemeyer Foundation Monograph Series No. 22

CONTENTS

E DITORS FOREWORD ....................................................................................................................Page v

Use of synovial fluid and serum biomarkers in equine bone and joint disease: A reviewC. W. McIlwraith................................................................................................................................Page 1

Proteoglycan metabolites as biomarkers of cartilage degeneration in degenerative joint diseases B. Caterson, C. E. Hughes and C. B. Little ......................................................................................Page 6

Proteoglycan metabolites as markers of cartilage breakdown in equine jointsC. Little and B. Caterson...................................................................................................................Page 9

Prospective clinical study assessing serum biomarkers for musculoskeletal disease in23-year-old racing Thoroughbreds

D. D. Frisbie, E. Duffy, R. Arthur, J. Blea, V. Baker, R. C. Billinghurst and C. W. McIlwraith.....Page 11

Does inflammation trigger tissue destruction or is tissue destruction activating inflammationin joint disease?

D. Heinegrd ...................................................................................................................................Page 13

What biomarkers are telling us and the challenges aheadS. Laverty .........................................................................................................................................Page 14

Involvement of the cartilage/bone junction in equine osteoarthritis (OA) of the middle carpal jointS. Ekman ..........................................................................................................................................Page 16

Bone biomarkers in horses: where are we now and future prospects? J. Price and B. Jackson ...................................................................................................................Page 18

Are bone biomarkers alone a useful tool to follow treatment or exercise regimens in horses?O. M. Lepage ...................................................................................................................................Page 20

Cartilage oligomeric matrix protein (COMP) in the equine middle carpal joint in relation tofractures and load

E. Skildebrand ...............................................................................................................................Page 23

Molecular markers of tendon injury: clinical aid or research tool? R. K. W. Smith, P. Onnerfjord, M. R. Smith, J. Dudhia and D. Heinegard.....................................Page 24

Imaging biomarkers: where are we going?C. E. Kawcak ...................................................................................................................................Page 26

Infrared spectroscopy - a new tool for the study of synovial fluidC. B. Riley........................................................................................................................................Page 32

Where are we with population genetics in the horse: what can be learned from studies of thehuman genome and where can it take us?

M. Vaudin.........................................................................................................................................Page 34


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Genetic markers: use of microsatellite repeats in equine disease association studies L. Hillyer, M. Binns, M. Vaudin and J. Price ..................................................................................Page 36

Genetic markers in equine orthopaedic diseaseP. Clegg............................................................................................................................................Page 38

Molecular markers in the diagnosis of equine disease E. M. Eastman .................................................................................................................................Page 40Equine gene expression array: applications

A. L. Bertone ....................................................................................................................................Page 42

L IST OF PARTICIPANTS ..................................................................................................................Page 45

AUTHOR INDEX ..............................................................................................................................Page 47

Equine Musculoskeletal Biomarkers


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EDITORS FOREWORD

Over the past 10 years there has beenconsiderable progress in the evaluation of synovial fluid and serum biomarkers andtheir usefulness in the management of musculoskeletal disease. Biomarkers have thefollowing potential uses: 1. Improving ourknowledge of the pathogenesis of equine jointdisease, and associated pathological conditions of cartilage, bone and tendon; 2. Diagnosis of earlydisease in these tissues; and 3. The ability tomonitor objectively responses to therapy both inexperimental models and clinical cases. The stateof knowledge in this area was originally assessedin a workshop on molecular markers of cartilageand bone metabolism in the horse inNorthampton, UK in 2000. This workshop wasorganised by Joanna Price, Wayne McIlwraith,Stina Ekman and Leo Jeffcott. Pioneers of

biomarkers in human orthopaedics, Robin Poole,Bruce Caterson, Mike Bayliss, Dick Heinegard,and Patrick Garnero also participated.

That workshop was sponsored by theHorseracing Betting Levy Board UK and BayerAnimal Health US, as well as the ResearchCommittee of the Swedish Horse RacingTotalizator Board (ATG).

Since 2000, considerably more progress has beenmade and this symposium was organised to bringback key workers in this area to assess howprogress had been made and where trulybiomarkers fitted into our clinicalarmamentarium. This workshop was sponsored bythe Dorothy Russell Havemeyer Foundation, withsecondary assistance for travel by IDEXX.Considerably more work had been done by equineresearchers and we were also fortunate to be ablecoax Professors Bruce Caterson and DickHeinegard back to this symposium to correlateparallel advances of biomarkers in humanorthopaedics. The willingness of the HavemeyerFoundation to support workshops of this nature isapplauded by all concerned and the additionalsponsoring of a monograph to ensure theinformation gained is disseminated to the widest

possible audience is also appreciated. I expresssincere thanks to the Foundation, in particular, toMr Gene Pranzo, President of the Foundationwhose continued enthusiasm and encouragementare much appreciated. Mr Pranzo was soimpressed by the quality of the meeting, as well asthe social programme that another symposium isplanned in 4 years time.

v

Wayne McIlwraithWorkshop Organiser



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HAVEMEYER SCIENTIFIC WORKSHOPS

1981 First International Workshop on Lymphocyte Alloantigens of the HorseOctober - New York City, USAOrganiser: Dr D. F. Antczak

1982 Second International Workshop on Lymphocyte Alloantigens of the HorseOctober - Cornell University, Ithaca, New York, USAOrganiser: Dr D. F. Antczak

1983 Third International Workshop on Lymphocyte Alloantigens of the HorseApril - New Bolton Center, University of Pennsylvania, USAOrganiser: Dr D. F. Antczak

1984 First International Symposium on Equine Embryo TransferOctober - Cornell University, Ithaca, New York, USAOrganisers: Drs D. F. Antczak and W. R. Allen

1985 Fourth International Workshop on Lymphocyte Alloantigens of the HorseOctober - University of Kentucky, USAOrganisers: Drs D. F. Antczak and E. Bailey

1986 Workshop on Corynebacterium equi Pneumonia of FoalsJuly - University of Guelph, CanadaOrganiser: Dr J. F. Prescott

1987 Fifth International Workshop on Lymphocyte Alloantigens of the HorseOctober - Louisiana State University, USA

Organisers: Drs D. F. Antczak and J. McClure

1989 Second International Symposium on Equine Embryo TransferFebruary - Banff, Alberta, CanadaOrganisers: Drs D. F. Antczak and W. R. Allen

1990 International Workshop on Equine SarcoidsApril - Interlaken, SwitzerlandOrganisers: Dr D. F. Antczak and Professor S. Lazary

1992 Workshop on Equine Neonatal MedicineJanuary - Naples, FloridaOrganisers: Drs D. F. Antczak and P. D. Rossdale



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Third International Symposium on Equine Embryo TransferFebruary - Buenos Aires, ArgentinaOrganisers: Drs D. F. Antczak, W. R. Allen, J. G. Oriol and R. Pashen

1995 Equine Perinatology

July - Cambridge, EnglandOrganiser: Dr P. D. Rossdale

Second International Equine Leucocyte Antigen WorkshopJuly - Lake Tahoe, California, USAOrganisers: Drs D. F. Antczak, P. Lunn and M. Holmes

First International Workshop on Equine Gene MappingOctober - Lexington, Kentucky, USAOrganisers: Drs D. F. Antczak and E. Bailey

Erection and Ejaculation in the Human Male and Stallion: A ComparativeStudyOctober - Mount Joy, Pennsylvania, USAOrganiser: Dr S. M. McDonnell

Bone Remodelling WorkshopOctober - Corcord, Massachusetts, USAOrganiser: Dr H. Seeherman

1997 Second International Workshop on Equine Gene MappingOctober - San Diego, California, USAOrganisers: Drs D. F. Antczak and E. Bailey

Maternal Recognition of Pregnancy in the MareJanuary - Dominican RepublicOrganisers: Drs W. R. Allen and T. A. E. Stout

Uterine Clearance

March - Gainesville, Florida, USAOrganiser: Dr M. M. LeBlanc

Trophoblast DifferentiationSeptember - Edinburgh, ScotlandOrganisers: Drs D. F. Antczak and F. Stewart

1998 Third International Genome WorkshopJanuary - San Diego, California, USA

Organisers: Drs D. F. Antczak and E. Bailey



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Third International Workshop on Perinatology: Genesis and Post NatalConsequences of Abnormal Intrauterine Developments: ComparativeAspectsFebruary - Sydney, AustraliaOrganiser: Dr P. D. Rossdale

Horse Genomics and the Genetic Factors Affecting Race Horse PerformanceMarch - Banbury Center, Cold Spring Harbor, New York, USAOrganisers: Drs D. F. Antczak, E. Bailey and J. Witkowski

Allergic Diseases of the HorseApril - Lipica, SloveniaOrganisers: Drs D. F. Antczak, S. Lazary and E. Marti

Equine Placentitis WorkshopOctober - Lexington, Kentucky, USAOrganisers: Drs D. F. Antczak, W. R. Allen and W. Zent

Septicemia II WorkshopNovember - Boston, Massachusetts, USAOrganiser: Dr M. R. Paradis

1999 Equine Genome ProjectJanuary - San Diego, California, USAOrganisers: Drs D. F. Antczak and E. Bailey

Third International Equine Genome WorkshopJune - Uppsala, SwedenOrganisers: Drs D. F. Antczak, E. Bailey and K. Sandberg

Fourth International Meeting of OIE and WHO Experts on Control ofEquine InfluenzaAugust - Miami, Florida, USAOrganiser: Dr J. Mumford

European Equine Gamete Workshop

September - Lopuszna, PolandOrganisers: Drs W. R. Allen and M. Tischner

Fetomaternal Control of PregnancyNovember - Barbados, West IndiesOrganisers: Drs T. Stout and W. R. Allen

2000 Equine Genome ProjectJanuary - San Diego, California, USAOrganisers: Drs D. F. Antczak and E. Bailey

Uterine Infections in Mares and Women: A Comparative StudyMarch - Naples, Florida, USAOrganiser: Dr M. M. LeBlanc



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5th International Symposium on Equine Embryo TransferJuly - Saari, FinlandOrganiser: Dr T. Katila

2001 USDA International Plant & Animal Genome ConferenceJanuary - San Diego, California

Equine Immunology in 2001January - Santa Fe, New MexicoOrganiser: Dr D. P. Lunn

Asthma and Allergies IIApril - HungaryOrganisers: S. Lazary and E. Marti

From Elephants to AidsJune - Port Douglas, AustraliaOrganiser: Professor W. R. Allen

International Equine Gene MappingJuly - Brisbane, AustraliaOrganiser: K. Bell

Second Meeting of the European Gamete Group (EEGG)September - Loosdrecht, The NetherlandsOrganiser: Dr T. A. E. Stout

Foal Septicemia IIIOctober - Tufts University European Center, Talloires, FranceOrganiser: M. R. Paradis

Infectious Disease Programme for the Equine Industry and VeterinaryPractitionersOctober - Marilyn duPont Scott Medical Center, Morvan Park, Virginia, USAOrganisers: Drs J. A. Mumford and F. Fregin

From Epididymis to EmbryoOctober - Fairmont Hotel, New Orleans, USAOrganiser: Dr L. H-A. Morris

2002 USDA International Plant & Animal Genome ConferenceJanuary - San Diego, California

Comparative Neonatology/PerinatologyMarch - Palm Springs, CaliforniaOrganiser: P. Sibbons



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Stallion Behaviour IVJune - Reykjavik, IcelandOrganisers: S. McDonell and D. Miller

Rhodococcus Equi II

July - Pullman, WashingtonOrganiser: J. Prescott

Equine Orthopaedic InfectionAugust - Dublin, IrelandOrganiser: E. Santschi

Inflammatory Airway DiseaseSeptember - Boston, USAOrganiser: Dr E. Robinson

2003 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, California

Embryonic and Fetal NutritionMay - Ravello, ItalyOrganiser: S. Wilsher

Genomics and the Equine Immunity SystemJune - Ithaca, New YorkOrganiser: D. F. Antczak

Fifth International Gene Mapping WorkshopAugust - Kreuger Park, South AfricaOrganiser: E. Baily and E. Vandyke

Equine Recurrent Laryngeal NeuropathySeptember - Stratford-upon-Avon, UKOrganisers: P. Dixon and E. Robinson

Transporting Gametes and EmbryosOctober - Brewster, MassachusettsOrganiser: E. Squires

Third Meeting of the European Gamete Group (EEGG)October - Pardubice, Czech RepublicOrganiser: J. and Z. Mller

Nosocomial Infections and Biosecurity in Equine HospitalsOctober - Lexington, USAOrganiser: F. Bain and J. Taub-Dargatz



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2004 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, California, USA

Equine Viral Herpes Virus WorkshopJune/July - Tuscany, ItalyOrganiser: P. Lunn

Equine Embryo Transfer VI WorkshopAugust - Rio de Janiero, BrazilOrganiser: M. Alvarenga

Sporting Injuries in Horses and Man: A Comparative ApproachSeptember - Lexington, USAOrganiser: E. J. L. Soulsby

Maternal Recognition of Pregnancy in the Mare IIINovember - Barbados, West IndiesOrganiser: T. A. E. Stout

2005 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, California, USAOrganiser: J. Mickelson

Comparative PlacentologyApril - Victoria, CanadaOrganiser: P. Sibbons

Sixth International Gene MappingJuly - Dublin, IrelandOrganisers: E. Bailey and J. Flynn

World Equine Airway SymposiumJuly - Ithaca, USAOrganisers: D. Ainsworth, E. Robinson, N. DuCharme, B. McGorum and L. Viel

Genetic Relatedness Between Different Breeds of Horses using MolecularMarkersAugust - PolandOrganisers: M. Binns, G. Lothran and B. Graiak

International Equine Gamete GroupSeptember - Khlungsborn, GermanyOrganisers: H. Alm, H. Torner, K. Hinrichs and E. Squires

Equine Musculoskeletal BiomarkersOctober/November - Colorado, USAOrganiser: W. McIllwraith


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Equine Influenza and Cross Species TransmissionNovember - Florida, USAOrganiser: J. Mumford

Uterine Infection in Mares & Women: A Comparative Study IINovember - South Carolina, USAOrganiser: M. M. LeBlanc


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HAVEMEYER MONOGRAPH SERIES

The following are monographs available to date at a cost of 9.95 each.

Series No 1P ROCEEDINGS OF THE F IRST M EETING OF THE E UROPEAN E QUINE G AMETE G ROUP (EEGG)Editors: W. R. Allen and J. F. Wade5th8th September 1999

Lopuszna, Poland

Series No 2P ROCEEDINGS OF A W ORKSHOP ON F ETOMATERNAL C ONTROL OF P REGNANCYEditors: T. A. E. Stout and J. F. Wade14th16th November 1999

Barbados, West Indies

Series No 3P ROCEEDINGS OF THE 5TH INTERNATIONAL SYMPOSIUM ON E QUINE E MBRYO T RANSFEREditors: T. Katila and J. F. Wade6th9th July 2000Saari, Finland

Series No 4P ROCEEDINGS OF A W ORKSHOP ON E QUINE IMMUNOLOGY IN 2001Editors: D. P. Lunn and J. F. Wade24th28th January 2001Santa Fe, New Mexico

Series No 5P ROCEEDINGS OF THE SECOND M EETING OF THE E UROPEAN G AMETE G ROUP (EEGG)Editors: T. A. E. Stout and J. F. Wade26th29th September 2001

Loosdrecht, The Netherlands

Series No 6 P ROCEEDINGS OF A W ORKSHOP ENTITLED F ROM E PIDIDYMIS TO E MBRYOEditors: L. H-A. Morris, L. Foster and J. F. Wade18th21st October 2001

New Orleans, USA

Series No 7 F OURTH INTERNATIONAL M EETING OF OIE AND WHO E XPERTS ON C ONTROL OF E QUINE

INFLUENZA

Editors: J. A. Mumford and J. F. Wade3rd5th August 1999Crowne Plaza Hotel, Miami, Florida, USA

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Series No 8P ROCEEDINGS OF A W ORKSHOP ON C OMPARATIVE NEONATOLOGY /P ERINATOLOGYEditors: Dr P. Sibbons, L. Foster and J. F. Wade13th15th March 2002Palm Springs, California, USA

Series No 9P ROCEEDINGS OF A W ORKSHOP ON INFLAMMATORY A IRWAY D ISEASE :DEFINING THE SYNDROMEEditors: A. Hoffman, N. E. Robinson and J. F. Wade30th September3rd October 2002

Boston, USA

Series No 10P ROCEEDINGS OF A W ORKSHOP ON E MBRYONIC AND F ETAL NUTRITIONEditors: S. Wilsher and J. F. Wade15th18th May 2003

Ravello, Italy

Series No 11P ROCEEDINGS OF A W ORKSHOP ON E QUINE R ECURRENT L ARYNGEAL NEUROPATHYEditors: P. Dixon, E. Robinson and J. F. Wade7th10th September 2003Stratford-upon-Avon, UK

Series No 12P ROCEEDINGS OF A W ORKSHOP ON T RANSPORTING G AMETES AND E MBRYOSEditors: E. Squires and J. F. Wade2nd5th October 2003

Brewster, Massachusetts, USA

Series No 13P ROCEEDINGS OF THE T HIRD M EETING OF THE E UROPEAN G AMETE G ROUP (EEGG)Editors: J. Mller, Z. Mller and J. F. Wade12th15th October 2003Pardubice, Czech Republic

Series No 14P ROCEEDINGS OF THE VI I NTERNATIONAL SYMPOSIUM ON E QUINE E MBRYO T RANSFEREditors: M. Alvarenga and J. F. Wade4th6th August 2004

Rio de Janerio, Brazil

Series No 15P ROCEEDINGS OF A W ORKSHOP ON SPORTING INJURIES IN H ORSES AND M AN :A C OMPARATIVE APPROACHEditors: Lord Soulsby and J. F. Wade23rd25th September 2004

Lexington, USA

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Series No 16 PROCEEDINGS OF A W ORKSHOP ON M ATERNAL R ECOGNITION OF P REGNANCY IN THE M ARE IIIEditors: T. A. E. Stout and J. F. Wade13th16th November 2004

Barbados, West Indies

Series No 17 PROCEEDINGS OF A W ORKSHOP ON C OMPARATIVE P LACENTOLOGYEditors: P. Sibbons and J. F. Wade21st24th April 2005Victoria, Canada

Series No 18PROCEEDINGS OF THE F IRST INTERNATIONAL E QUINE G AMETES G ROUP W ORKSHOPEditors: A. Hannelore, H. Torner, K. Hinrichs, E. Squires and J. F. Wade18th-21st September 2005

Rostock, Germany

Series No 19PROCEEDINGS OF A W ORKSHOP ON UTERINE INFECTION IN M ARES & W OMEN :A C OMPARATIVE STUDY IIEditors: M. M. LeBlanc and J. F. Wade9th-13th November 2005South Carolina, USA

Series No 20PROCEEDINGS OF A W ORKSHOP ON E XERCISE -INDUCED P ULMONARY H AEMORRHAGE : S TATEOF C URRENT K NOWLEDGEEditors: D. J. Marlin, K. W. Hinchcliff and J. F. Wade9th-12th March 2006 Vancouver, Canada

Series No 21PROCEEDINGS OF A W ORKSHOP ON E MBRYONIC AND FETAL NUTRITIONEditors: S. Wilsher, W. R. Allen and J. F. Wade29th May-1st June 2006

Ravello, Italy

If you wish to order copies, please contact R & W Communications, Suites 3 & 4, 8 Kings Court, WillieSnaith Road, Newmarket, Suffolk CB8 7SG, UK, Tel: +44 1638 667600, Fax: +44 1638 667229,e-mail: [email protected]

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USE OF SYNOVIAL FLUID AND SERUMBIOMARKERS IN EQUINE BONE AND JOINTDISEASE: A REVIEW

C. W. McIlwraith

Colorado State University, Fort Collins, Colorado 80523, USA

SUMMARY

Considerable progress has been made in the use of synovial fluid in serum biomarkers in thediagnosis of equine bone and joint disease. Thismaterial is from a larger review article publishedin 2005 in the Equine Veterinary Journal, andpresented as an initial lecture at a HavemeyerSymposium on Biomarkers in the Horse,sponsored by the Dorothy Russell HavemeyerFoundation, IDEXX and the OrthopaedicResearch Centre at CSU.

INTRODUCTIONSignificant economic losses due to a highprevalence of lameness in racehorses (the majoritybeing associated with osteoarthritis (OA) are wellrecognised). Any improvement in prevention,diagnosis, treatment, and prognostication in thisarea is welcome and biomarkers are potentiallyuseful for this purpose.

The osteoarthritic joint is characterised bydamage to and loss of articular cartilage matrix

components, along with reduced joint function. Itis now well established that the natural disease inthe horse is variable, but synovial membrane,fibrous joint capsule, articular cartilage,subchondral bone, and intra-articular ligamentscan be involved in primary and/or secondary roles.The inflammatory reaction in the synovialmembrane and capsule, any alteration in thedynamic equilibrium between the biosyntheticphase (chondrocytes synthesise and storeextracellular matrix (ECM)) and degradationphase (proteolytic enzymes are activated) havebeen implicated in the pathogenesis of human OA(Sandell and Aigner 2001). Inflammatory

proceses result in an increase in levels of inflammatory mediators, with release of micromolecules and their fragments into synovialfluid and serum following the anabolic andcatabolic processes in the cartilage. Investigationsat the ORC have emphasised a primary role forsubchondral bone microdamage in thepathogenesis of traumatic joint disease and earlydetection of primary change in the subchondralbone is also desirable.

The terms biomarker, biochemical marker,and molecular marker have all been used todescribe either direct or indirect indicators of abnormal skeletal tissue turnover (Billinghurst2001). These biomarkers are often molecules thatare the normal products and byproducts of themolecular processes occurring within the skeletaltissue. In disease, alterations occur and the balancebetween the anabolic and the catabolic processeswithin the skeletal tissues and consequently,concentrations of biomarkers may increase ordecrease. Most of the biomarkers we currently usein the horse are related to either the synthetic ordegradative processes involvingtype II collagenand/or the proteoglycan molecules in the cartilagematrix. In addition, synthetic and degradativemarkers oftype I collagen are used for earlydetection of bone problems. Biomarkers canpotentially be used to: 1) clarify pathologicalprocesses in the joint; 2) dif ferentiatediagnostically between affected and non-affected joints and distinguish the degree of degradation inarticular cartilage; and 3) monitor the response totherapy.

According to the way they are detected,biomarkers can be subdivided into biochemicaland immunological markers. A good example of abiochemical marker is the dimethyl methyleneblue (DMMB) assay used for detecting


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glycosaminoglycans (GAGs) in the synovial fluidand serum. Immunological markers provide asensitive means to identify types and utilisemonoclonal and polyclonal antibodies producedagainst various epitopes on fragments liberatedboth from normal and degenerating joint tissue.The term biomarkers has more recently beenapplied to imaging techniques as well as geneticmarkers and microarrays. The review hereaddresses traditional biochemical andimmunological markers that form the bulk of theliterature.

DIRECT AND INDIRECT BIOMARKERS

Direct biomarkers originate principally fromcartilaginous or bony structures or are enzymesthat are active only in these tissues. They providespecific information about alterations in cartilagematrix anabolism or catabolism. An example of adirect biomarker that detects collagen degradationis depicted in Figure 1.

Indirect biomarkers are not derived principallyfrom the tissues that make up the joint, but havethe potential to influence the metabolism of these

tissues or the integrity. Indirect markers includecytokines such as IL-1, matrix metalloproteinases,eicosanoids (PGE2 is a commonly used examplein the equine synovial fluid), insulin-like growthfactors, hyaluronan (HA), and C-reactive proteins(CRP).

INDIVIDUAL DIRECT BIOMARKERS OFCARTILAGE METABOLISM

Carboxypropeptide of type II collagen (CPII)

Biomarkers of anabolic processes includecarboxypropeptide of type II collagen. Antibodiesagainst CPII are a useful measure oftype II collagen synthesis. Although CPII concentrationshave been found to be not significantly higher insynovial fluid of joints with osteochondralfragmentation, the levels were significantly higherin the serum (Frisbieet al. 1999). It has also beenshown that repeated use of IA methylprednisoloneacetate leads to potentially harmful inhibition of CPII synthesis and also an increased release of degradation products of aggrecan from articularcartilage (Robionet al. 2001).

Exposedepitope

Hiddenepitope

TropocollagenA m i n o -terminus

Nonhelicaltelopeptide region

Collagenase

Conformational epitope(triple helix dependant)

Exposure of hidden epitopes

Collagenase

cleavage site

Unwinding aftercleavage toproduce chains& exposure ofnormally hiddenepitopes

Composed ofthree identicalhelical chains

Carboxy-terminus

Nonhelicaltelopeptide regionCross- link

Collagen fibril

Fig 1: Structure of fibrillar type II collagen to show the composition of a collagen fibril with cross-links between thenon-helical telopeptide regions of individual tropocollagen molecules and the helical regions of adjacent molecules.

Each tropocollagen molecule is composed of a triple helix of three identical chains. The cleavage site of collagenaseis indicated. The cleaved triple helix unwinds to expose hidden epitopes on chains that are not detectable byantibodies in the native triple helix. Non-helical (telopeptide) and conformational (triple helical-dependent) epitopesare also indicated (Poole 1992).


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Chondroitin sulphate (CS)

Chondroitin sulphate is a major GAG of aggrecanand has proven to be a useful biomarker foraggrecan synthesis. An epitope called CS-846 thatis normally found in fetal and OA cartilage and isalmost absent in healthy mature articular cartilage,has been measured in a number of species. In astudy in the horse, CS-846 levels weresignificantly higher in joints with osteochondralfragmentation than in control joints, and serumlevels were also significantly higher (Frisbieet al.1999). Discriminate analysis using a combinationof serum CS-846 and CPII concentrations allowed79% of horses to be correctly classified as havingosteochondral damage.

BIOMARKERS OF CATABOLIC PROCESSES

Type II collagen fragments

Biomarkers of catabolic processes includetype II collagen fragments. Measuring the degradation of type II collagen with biomarkers has proven to beof benefit in monitoring OA as well asosteochondritis dissecans (OCD). Antibodies havebeen developed to identifytype II collagen

fragments that have been cleaved or denatured,exposing previously inaccessible regions(neoepitopes) of the molecule (Hollanderet al.1994; Fig 1). Using these antibodies, significantelevations in levels of degradedtype II collagenhave been demonstrated in synovial fluid andserum samples from horses, dogs, and rabbits withexperimental OA (Billinghurstet al. 1997). Ourequine options now includes the COL2-3/4Cshortimmunoassay for detecting collagenase-cleavedcollagen fragments (types I and II ) as well as more

recently, a specifictype II collagen degradationassay that is specific for the horse (Billinghurst2001), which is designated as 234CEQ.

In a recent study of skeletal markers in OCDin foals (Billinghurstet al. 2004), a combinationof significantly higher serum levels of CPII,higher levels of COL2-3/4Cshort and lower levelsof 234CEQ correlate with high osteochondrosisscores (radiographically). This study suggestedthere is an increased collagen turnover in OCD,but by measuring the serum levels of specificbiomarkers of collagen metabolism, it is possibleto identify foals with OCD and predict theirclinical outcome (Billinghurstet al. 2004).

Glycosaminoglycans (GAGs)

The 1,9 DMMB assay for GAGs has been usefulin a number of studies, and in recent work, is oneof the markers that can distinguish osteoarthritis inan exercising horse from normal increases withexercise alone. Breakdown products of keratansulphate have also been developed, but havelimited usefulness in the horse. There are a seriesof monoclonal antibodies that have beendeveloped to recognise epitopes in the chondroitinsulphate - GAG chains (Catersonet al. 1983 and1999) and these are useful.

Cartilage oligomeric matrix protein (COMP)

Cartilage oligomeric matrix protein (COMP) is anabundance noncollagenous protein constituent of articular cartilage. A number of studies have beendone with COMP in both joint disease and tendondisease. It is still uncertain where this product fits.Monoclonal antibody technologies have also beendeveloped to recognise neoepitopes associatedwith breakdown of the aggrecan molecule byaggrecanase . Preliminary work in the horse showshigh levels of aggrecanase activity in synovialfluid of diseased joints compared to levels of stromelysin and this has allowed characterisationof the mechanisms by which aggrecan is brokendown in equine joint disease.

INDIVIDUAL DIRECT BIOMARKERS OFBONE METABOLISM

Biomarkers of anabolic processes

Carboxy and amino terminal propeptides (PICPand PINP): Biomarkers of anabolic processes

include carboxy and amino terminal propeptides(PICP and PINP). During normaltype I collagensynthesis, as withtype II , cleavage of PICP andPINP off the procollagen molecule occurs andthese cleaved propeptide fragments can beexploited as markers of bone formation. PICPhas been measured and levels decreasesignificantly with age and increase with exercisewhen compared to non-exercised control horses(Price et al. 1995a, b). In a preliminary study,PICP was shown to have potential value asmolecular marker for monitoring changes inmatrix turnover following tendon injury (Jacksonet al. 2003).


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Osteocalcin: Osteocalcin is a small non-collagenous protein associated with boneassembly and turnover. A recent study in thehorse, in which serum markers were used todifferentiate concentrations of osteocalcin andCS846 concentrations of osteocalcin and CS846provided the best correlation to the modifiedMankin score and clinical degree of pain (Frisbieet al. 2003). Serum osteocalcin concentration wasalso af fected by rate of growth in weanling horsesof similar age, whereas there was no change withserum bone alkaline phosphatase (Petersenet al.2001).

Biomarkers of catabolic processes

Type I collagen nonhelical telopeptide (ICTP) :Biomarkers of catabolic processes includetype I collagen nonhelical telopeptide. This fragment isused as a marker of bone resorption in humanarthritis. However, levels of ICTP have beenmeasured in the horse in relation to age, exercise,and breed differences, but have not been shown tobe of value in detecting pathological processes.(Priceet al. 1995a,b; Kawcak 1998).

Type I collagen C-telopeptides (CTX) : Type I collage C-telopeptides have been proven to be auseful marker of specific bone resorption inhumans. In a study in the horse done at the ORC,CTX was less useful than other cartilagebiomarkers (CS846, CPII and GAG) in predictingwhether serum was from a control exercised or anosteoarthritic horse (Frisbieet al. 2002).

Bone sialoprotein (BSP) : Bone sialoprotein iselevated in human patients with clinical OA.Recent work in the horse established that there is

an increase in BSP at the cartilage/bone interfacewhen degenerative changes of the bone andcartilage are compared to the morphologicallyintact cartilage and bone. The challenge still is tomeasure BSP in the synovial fluid and serum of racehorses.

C ONCLUSIONS

In conclusion, we are not yet at the stage of havinga magic marker to diagnose the degree of articular cartilage or bone disease in a single jointwith 100% accuracy. However, much progress hasbeen made. Various marker molecules have

already been used successfully and an increasingnumber are coming onto the market in the form of ready-to-use kits. Although currently still usedmainly in research settings, the time is not faraway that these kits will be used widely in clinicalpractice. For a good assessment of the conditionof the cartilage and other tissues of the joint, acombination of the determination of (repeatedlycollected) selected markers with other diagnostictechniques, such as arthroscopy and/or MRI,seems more promising.

R EFERENCES

Billinghurst, R.C., Dahlberg, L., Ionescu, M., Reiner, A.,Bourne, R., Rorabeck, C., Mitchell, P., Hambor, J.,Diekmann, 0., Tschesche, H., Chen, J., van Ward,H., Poole, A.R. (1997) Enhanced cleavage of type IIcollagen by collagenases in osteoarthritic articularcartilage. J. Clin. Invest. 99, 1534-1545.

Billinghurst, R.C., Brama, P.A., van Weeren, P.R.,Knowlton, M.S. and Mcllwraith, C. (2004)Evaluation of serum concentrations of biomarkersof skeletal metabolism and results of radiography asindicators of severity of osteochondrosis in foals.

Am. J. vet. Res. 65, 143-1500.Billinghurst, R.C. (2001) Biomarkers of Joint Disease.

In:Current Therapy in Equine Medicine 5 , Ed: N.E.Robinson, Saunders, Philadelphia. pp 513-520.

Caterson, B., Hughes, C.E., Johnstone, B. and Mort, J.S.

(1999) Immunological markers of cartilageproteoglycan metabolism in animal and humanosteoarthritis. In: Articular Carti lage and Ostecoarthritis Eds: K.E. Kuettner, R.Schleyerbach, J.G. Peyron & V.C. Hascall. NewYork, Raven Press pp 415-427.

Caterson, B., Christner, J.E. and Baker, J.R. (1983)Identification of a monoclonal antibody thatspecifically recognises corneal and skeletal keratansulfate. J. Biol. Chem. 258 , 8848-8854.

Frisbie, D.D., Ray, C.S., Ionescu, M., Poole, A.R.,Chapman, P.L. and McIlwraith, C.W. (1999)Measurement of the 846 epitope of chondroitin

sulfate and of carboxy propeptides of type IIprocollagen for diagnosis of osteochondralfragmentation in horses. Am. J. vet. Res. 60 , 306-309.

Frisbie, D.D., Al-Sobayil, F., Billinghurst, R.C. andMcIlwraith, C.W. (2003a) Serum markersdifferentiate exercise from pathology and correlateto clinical parameters of pain in an osteoarthriticmodel.Proceedings Orthopaedic Research Society .

Frisbie, D.D., Al-Sobayil, F., Billinghurst, R.C. andMcIlwraith, C.W. (2003b) Synovial fluidbiomarkers distinguish exercise from osteoarticularpathology.Proc. Am. Ass. Equine Practnrs. 49, 116-

117.Hollander A.H., Heathfield, T.F., Webber, C., Iwata, Y.,Bourne, R., Rorabeck, C. and Poole, A.R. (1994)Increased damage to type II collagen in


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osteoarthritic articular cartilage detected by a newimmunoassay. J. Clin. Invest. 93 , 1722-1732.

Jackson, B.F., Smith, R.K.W. and Price, J.S. (2003) Amolecular marker of Type I collagen metabolismreflects changes in connective tissue remodelingassociated with injury to the equine superficialdigital flexor tendon. Equine vet. J. 35 , 211-213.

Kawcak, C.E (1998) Effect of Loading on Subchondral Bone of the Equine Carpal and Meta-carpophalangeal Joints. PhD Dissertation,Colorado State University.

McIlwraith, C.W. (2005) Use of synovial fluid andserum biomarkers in equine bone and joint disease:A review. Equine vet. J. 37, 473-482.

Petersen, E.D., Sicilano, P.D., Turner, A.S., Kawcak,C.E. and McIlwraith, C.W. (2001) Effect of growthrate on serum-bone-specific alkaline phosphataseand osteocalcin in weanling horses. In:Proc. 17thSymposium Equine Nutrition and Physiology ,Lexington, KY. pp 123-124.

Poole, A.R. (1992) Immunology of cartilage. Eds: R.W.Moskowitz, D.S. Howell, V.M. Goldberg & H.J.

Mankin. In: Osteoarthritis Diagnosis and Management, Edn 2 . Orlando, Florida, WBSaunders, pp 155-189.

Price, J.S., Jackson, B., Eastell, R., Goodship, A.E.,Blumsohn, A., Wright, I., Stoneham, S., Lanyon,L.E. and Russell, R.G.G. (1995a) Age-relatedchanges in biochemical markers of bone metabolismin horses. Equine vet. J. 3, 201-207.

Price, J.S., Jackson, B., Eastell, R.,Wilson A.M., Russell,R.G.G., Lanyon, L.E. and Goodship, A.E. (1995b).The response of the skeleton to physical training: Abiochemical study in horses. Bone 17, 221-227.

Robion, F.C., Doiz, B., Bour, L., Marcoux, M.,Ionescu, M., Reiner, A., Poole, A.R. and Laverty, S.(2001) Use of synovial fluid markers of cartilagesynthesis and turnover to study effects of repeatedintra-articular administration of methylprednisoloneacetate on articular cartilagein vivo . J. Orthop. Res.19 , 250-258.

Sandell, L.J. and Aigner, T. (2001) Articular cartilageand changes in arthritis: Cell biology of osteoarthritis. Arthritis Res. 3, 107-113.


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PROTEOGLYCAN METABOLITES AS BIOMARKERSOF CARTILAGE DEGENERATION IN DEGENERATIVE

JOINT DISEASES

B. Caterson, C. E. Hughes and C. B. Little*Connective Tissue Biology Labs, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, Wales, UK; *Raymond Purves Bone & Joint Research Laboratories, University of Sydney Institute of Bone & Joint Research, Royal North Shore Hospital, St Leonards, New South Wales2065, Australia

Over the past 25 years The authors laboratory haspioneered the production and use of monoclonalantibody (mAb) technologies to study cartilageproteoglycan structure, function and metabolismin health and disease. Many of the mAbs producedby the authors laboratory recognise structural(protein or carbohydrate) epitopes and enzyme-generated neoepitopes either present or generated,respectively in these cartilage proteoglycans. Inpast and recent studies it has now been realisedthat several of these mAbs can be used torecognise potential biomarkers (changes incartilage proteoglycan biochemical compositionand/or chondrocyte metabolism/phenotype) thatcan be used to monitor or diagnose metabolicchanges that are occurring in the pathogenesis of degenerative joint diseases. This paper provides asummary of past and new studies identifyingcartilage metabolic biomarkers that can bepotentially used to diagnose or monitor theefficacy of therapeutic or surgical treatments of equine degenerative joint diseases.

STRUCTURAL BIOMARKERS

In the mid 1980s detection of cartilage aggrecanmetabolites (keratan sulphate KS) in the serumof arthritis patients was proposed as a potentialbiomarker of cartilage degeneration indegenerative joint diseases (Thonaret al. 1985).In the 1990s early studies investigated KSmetabolites in equine synovial fluid and serum aspotential biomarkers of degenerative joint disease(Alwanet al. 1990). Analyses of KS metabolites,in conjunction with other cartilage biomarkers, arestill being used to evaluate the efficacy of pharmacological and nutraceutical treatments forhorse joint pathology today (Caronet al. 2002;Orthet al. 2002; Celesteet al. 2005).

ANABOLIC NEOEPITOPE BIOMARKERS

In the mid- to late-1980s researchers from ourlaboratory first described the expression of anabolic neoepitopes, manifested as differentchondroitin sulphate (CS) sulphation motifs oncartilage aggrecan glycosaminoglycans, as novelbiomarkers indicative of anabolic (attemptedrepair) responses of chondrocyte metabolism inthe pathogenesis of arthritic disease (Catersonet al. 1990; Viscoet al. 1993; Slateret al. 1995).These mAb reagents have also been used to studyanabolic/repair changes in equine cartilagemetabolism (Todhunteret al. 1996; Dartet al.2003). CS sulphation motif mAbs have also beenproduced by other laboratories and used inanalyses of synovial fluid and serum for diagnosisof osteochondral fragmentation in horses (Frisbieet al. 1999).

C ATABOLIC NEOEPITOPE BIOMARKERS

In the early 1990s researchers in our laboratoryalso pioneered the production and use of mAbtechnologies to detect catabolic neoepitopesgenerated by matrix protease cleavage of cartilageaggrecan in its interglobular domain (IGD) seeCatersonet al. (2000) for review. These mAbreagents were used by researchers at Dupont todiscover the aggrecanases (ADAMTS-4 andADAMTS-5). One of these mAbs (BC-3) can beused to detect and now quantify aggrecancatabolites generated by aggrecanase catabolismof the IGD of cartilage aggrecan in thepathogenesis of arthritic disease. This mAb andother related monoclonal and polyclonalantibodies that recognise protease-generatedneoepitopes in cartilage matrix macromoleculesare showing increasing potential as a means of


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identifying useful biomarkers for degradativeprocesses involved in arthritic disease.

NEW POTENTIAL BIOMARKERS OFDEGENERATIVE JOINT DISEASE

As the result of a side-project emanating fromrecent studies aimed at generating new mAbs thatrecognise keratanase- or keratanase II- generatedstub neoepitopes, we also have produced newmAbs have also been produced that recognise coreprotein structural epitopes on 2 of the smallleucine-rich proteoglycans (SLRPs) first found incorneal matrix stroma; ie keratocan and lumican.We have been aware for a long time that alteredsynthesis of matrix molecules and changes in thephenotypic expression of the hyaline articularcartilage chondrocytes are early signs of changesin the metabolism (and thus function) of articularcartilage with the onset of degenerative jointdiseases. In addition, a recent study (Younget al.2005) also described changes in gene and proteinexpression of biglycan, decorin, fibromodulin, andinterestingly lumican in an ovine meniscectomyanimal model of degenerative joint disease. Wetherefore evaluated the expression of keratocanand lumican as potential biomarkers of degenerative joint disease in human patientsundergoing total hip or knee replacement surgery.Our studies have shown that analysis of gene(using qualitative RT-PCR) and protein (usingWestern blot analysis) expression of the SLRPkeratocan is absent in normal hyaline articularcartilage (from femoral head fractures) butupregulated in pathological cartilage obtainedfrom patients with osteoarthritis (OA) of the kneeor hip. In contrast, lumican gene expressionappeared to be constitutive in both normal andOA cartilage unlike that seen in the ovine animalmodel (Younget al. 2005). However, lumicanexpression at the protein level was absent innormal cartilage and upregulated in OA cartilagesamples similar to that seen in the ovine model of degenerative joint disease (Younget al. 2005). Atpresent, it is not clear what is the function of altered SLRP (particularly keratocan and lumican)expression in the pathogenesis of degenerative joint disease. One possibility is that thisunexpected expression of SLRPs signals a changein the chondrocyte phenotype whereby the cellsattempt to produce atype I collagen, keratocan andlumican regulated, fibrillar scar, in response to thealtered mechanical environment manifest with the

onset of OA. Alternatively, changes in SLRPexpression have been recently linked with theupregulation and occurrence of inflammationwhich is an important factor driving joint tissuedestruction in arthritic diseases (Schaeferet al.2005; Sjoberget al. 2005). The potential forupregulation of keratocan, lumican and otherSLRPs in equine joint pathology still needs to beinvestigated.

M ONOCLONAL ANTIBODIES THATRECOGNISE CHONDROITIN SULPHATE (CS)SULPHATION MOTIFS AS POTENTIALBIOMARKERS IDENTIFY STEM / PROGENITOR CELLS IN MUSCULO -SKELETAL TISSUES

In the mid 1980s (Catersonet al. 1990)researchers in our laboratory produced andcharacterised several mAbs that recognised linearglycosaminoglycan (GAG) sulphation epitopemotifs in CS GAG present on connective tissueproteoglycans. Several of these have been used todetect anabolic/dedif ferentiation changes in OAcartilage from humans and animal models(Catersonet al. 1990; Viscoet al. 1993; Slateret al. 1995). In recent studies (Hayeset al. 2008) 3 of these mAbs [3-B-3(-); 4-C-3 and 7-D-4] have alsobeen used to identify potential chondroprogenitorcells (and further differentiated cell subtypes) inthe superficial zone/layer of hyaline articularcartilage where they are believed to reside(Dowthwaiteet al. 2004). We have also used thesemAbs to demonstrate their ability to identifypotential stem/progenitor cell subpopulations inregions where they occur in developingintervertebral disc tissues and tendon. Preliminarystudies, using chondrocytes from bovine cartilage,also indicate that we were potentially able to useFACS procedures to separate and isolate thesestem/chondroprogenitor cells for their enrichmentand use in tissue engineering technologies formusculoskeletal tissue repair and regeneration.

R EFERENCES

Alwan, W.H., Carter, S.D., Bennett, D., May, S.A. andEdwards, G.B. (1990) Cartilage breakdown inequine osteoarthritis: measurement of keratansulphate by an ELISA system. Res. vet. Sci. 49, 56-

60.Caron, J.P., Peters, T.I., Hauptman, J.G., Eberhart, S.W.and Orth, M.W. (2002) Serum concentrations of keratan sulfate, osteocalcin, and pyridinoline


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crosslinks after oral administration of glucosamineto standardbred horses during race training. Am. J.vet. Res. 63, 1106-1110.

Caterson, B., Flannery, C.R., Hughes, C.E. and Little,C.B. (2000) Mechanisms involved in cartilageproteoglycan catabolism. Matrix Biol. 19 , 333-344.

Caterson, B., Mahmoodian, F., Sorrell, J.M.,Hardingham, T.E., Bayliss, M.T., Carney, S.L.,Ratcliffe, A. and Muir, H. (1990) Modulation of native chondroitin sulphate structure in tissuedevelopment and in disease. J. Cell. Sci. 97 , 411-417.

Celeste, C., Ionescu, M., Poole, A.R. and Laverty, S.(2005) Repeated intraarticular injections of triamcinolone acetonide alter cartilage matrixmetabolism measured by biomarkers in synovialfluid. J. Orthop. Res. 23, 602-610.

Dart, A.J., Little, C.B., Hughes, C.E., Chu, O., Dowling,B.A., Hodgson, D.R., Rose, R.J. and Johnson, K.A.(2003) Recombinant equine growth hormone

administration: effects on synovial fluid biomarkersand cartilage metabolism in horses. Equine vet. J.35 , 302-307.

Dowthwaite, G., Bishop, J.C., Redman, S.N., Khan,I.M., Rooney, P., Evans, D.J., Haughton, L., Bayram, Z., Boyer, S., Thomson, B., Wolfe, M.S. andArcher, C.W. (2004) The surface of articularcartilage contains a progenitor cell population. J.Cell Sci. 117 , 889-897.

Frisbie, D.D., Ray, C.S., Ionescu, M., Poole, A.R.,Chapman, P.L. and McIlwraith, C.W. (1999)Measurement of synovial fluid and serumconcentrations of the 846 epitope of chondroitin

sulfate and of carboxy propeptides of type IIprocollagen for diagnosis of osteochondralfragmentation in horses. Am. J. vet. Res. 60, 306-309.

Hayes, A.J., Tudor, D., Newell, M.A., Hughes, C.E. andCaterson, B. (2008) Chondroitin sulfate sulfationmotifs as putative biomarkers for isolation of articular cartilage progenitor cells. J. Histochem.Cytochem . 56 , 125-138.

Orth, M.W., Peters, T.I. and Hawkins, J.N. (2002)

Inhibition of articular cartilage degradation byglucosamine-HCl and chondroitin sulphate. Equinevet J. Suppl. 34, 224-229.

Schaefer, L., Babelova, A., Kiss, E., Hausser, H.J.,Baliova, M., Krzyzankova, M., Marsche, G., Young,M.F., Mihalik, D., Gotte, M., Malle, E., Schaefer,R.M. and Grone, H.J. (2005) The matrix component

biglycan is proinflammatory and signals throughToll-like receptors 4 and 2 in macrophages. J. Clin. Invest. 115 , 2223-2233.

Sjoberg, A., Onnerfjord, P., Morgelin, M., Heinegard, D.and Blom, A.M. (2005) The extracellular matrix andinflammation: fibromodulin activates the classicalpathway of complement by directly binding C1q.

J. Biol. Chem. 280 , 32301-32308.Slater, R.R., Bayliss, M.T., Lachiewicz, P.F., Visco,

D.M. and Caterson, B. (1995) Monoclonalantibodies that detect biochemical markers of arthritis in humans. Arthritis and Rheumatism 38 ,655-659.

Thonar, E.J., Lenz, M.E., Klintworth, G.K., Caterson, B.,Pachman, L.M., Glickman, P., Katz, R., Huff, J. andKuettner, K.E. (1985) Quantification of keratansulfate in blood as a marker of cartilage catabolism.

Arthritis and Rheumatism 28 , 1367-1376.Todhunter, P.G., Kincaid, S.A., Todhunter, R.J.,

Kammerman, J.R., Johnstone, B., Baird, A.N.,Hanson, R.R., Wright, J.M., Lin, H.C. and Purohit,R.C. (1996) Immunohistochemical analysis of anequine model of synovitis-induced arthritis. Am. J.vet. Res. 57 , 1080-1093.

Visco, D.M., Johnstone, B., Hill, M.A., Jolly, G.A. andCaterson, B. (1993) Immunohistochemical analysis

of 3-B-(-) and 7-D-4 epitope expression in canineosteoarthritis. Arthritis and Rheumatism 36 , 1718-1725.

Young, A.M., Smith, M.M., Smith, S.M., Cake, M.A.,Ghosh, P., Read, R.A., Melrose, J., Sonnabend,D.H., Roughley, P.J. and Little, C.B. (2005)Regional assessment of articular cartilage geneexpression and small proteoglycan metabolism in ananimal model of osteoarthritis. Arthritis, Researchand Therapy 7, R852-R861.


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PROTEOGLYCAN METABOLITES AS MARKERS OFCARTILAGE BREAKDOWN IN EQUINE JOINTS

C. Little and B. Caterson*

Raymond Purves Bone and Joint Research Laboratories, University of Sydney Institute of Bone and Joint Research, Royal North Shore Hospital, St. Leonards New South Wales, Australia 2065;*Connective Tissue Biology, School of Biosciences, Cardiff University, Museum Avenue Box 911,Cardiff CF10 3US, Wales, UK

Progressive degradation of articular cartilage is acentral feature of arthritis irrespective of theinciting cause, and is characterised by loss of aggrecan due to excessive degradation byaggrecanases followed by proteolysis of thecollagen network. This latter event may signify thepoint of irreversible cartilage damage. Aggrecanloss from cartilage in joint disease is drivenprimarily by proteolysis by the A Disintegrin AndMetalloproteinase with ThromboSpondin motif (ADAMTS) family of enzymes (Littleet al.2005). However, in late-stage disease, in concertwith collagen proteolysis by the collagenolyticmatrix metalloproteinses (MMPs), some MMP-cleaved aggrecan metabolites are observed. Thereis evidence that following aggrecan depletion butpreceding, and possibly as a necessary pre-requisite for collagenolysis, other matrixcomponents such as the small leucine rich repeatproteoglycans (SLRPs, decorin, biglycan andfibromodulin) that coat the collagen fibres arereleased from cartilage at leastin vitro(Sztrolovicset al. 1999). Differentiating earlyfrom progressive and late degeneration of cartilage may therefore be possible by evaluatingthe sequential release of cartilage matrixconstituents. We sought to use bothin vitro modelsof progressive cartilage degeneration and synovialfluids from horses with arthroscopically-characterised joint disease, to determine whetherdif ferent aggrecan and SLRP metabolites mightserve as useful markers of progressive cartilagedegeneration.

Articular cartilage was harvested from highand low weight-bearing regions of equine carpal joints with no morphological evidence of jointdisease. Cartilage was cultured for 3 days inDMEM with 10% FCS and then washed andcultured for 28 days in serum free DMEM

containing 10 ng/ml IL-1beta or 1ng/ml IL-1betaplus 50 ng/ml OSM, with media changes every 7days (Littleet al. 2005). Release of proteoglycanand collagen was quantitated using DMMB andhydroxyproline assays, respectively. Western blotanalysis of ADAMTS- and MMP-generatedaggrecan metabolites (BC-3 and BC-14antibodies, respectively), MMP-generated type IIcollagen catabolites (9A4 antibody), and decorin,biglycan and fibromodulin (polyclonal antibodiesto the C-terminus) in cartilage extracts and culturemedia at Days 7, 14, 21 and 28 was undertaken.Synovial fluids were harvested at the time of arthroscopic surgery by Professor WayneMcIlwraith and the cartilage damage and historyfor each animal recorded. Synovial fluids weresubjected to associative CsCl density gradientultracentrifugation, hyaluronidase digestion andWestern blot analysis of aggrecan and SLRPmetabolites.

It has previously been reported thatstimulation of equine cartilage with both IL-1betaalone and IL-1/OSM induced significantADAMTS- but not MMP-dependent aggrecanloss, with most release occurring over the firstweek of culture. The authors also observedincreased link protein release from cartilageinduced by IL-1 alone and IL-1/OSM, with nodif ference in the amount or fragmentation of thereleased link protein. Unlike other species, IL-1beta alone but not IL-1/OSM inducedcollagenloysis from Day 14 onwards in equinecartilage (Littleet al. 2005). The collagen releasewas due to MMP activity and was associated withlate stage C-terminal truncation, but notinterglobular domain cleavage of aggrecan byMMPs. There was early (Day 7 and 14) andpersistent (Day 21 and 28) release of decorin andfibromodulin from cartilage, but importantly this


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only occurred in the cultures in whichcollagenolysis was subsequently induced (ie 10ng/ml IL-1 alone but not IL-1/OSM). In contrastwith other species evaluated, biglycan release wasnot induced from equine cartilage by eithercatabolic stimulus despite its presence in cartilageextracts. The pattern of aggrecan, collagen andSLRP release from cartilage was similar in highand low weight-bearing cartilage.

Analysis of synovial fluid from joints withvarying pathology (chip fractures cartilagedegeneration and ligament tearing) demonstratedmany similarities and some differences from theinvitro cartilage degradation studies. Firstly, onlyADAMTS- and not MMP-generated aggrecanmetabolites were detected. The ADAMTS-generated fragments in joint fluids (BC-3 positive)ranged in size from 60 to >300kDa, with thelargest metabolites not previously observed in thein vitro cartilage degradation studies. The overall(total) immunoreactivity with BC-3 did notcorrelate with glycosaminoglycan levels in thefluid. The pattern of BC-3 positive fragmentsdiffered markedly between horses, particularlywith respect to the presence or absence of the verylargest metabolite. Intact (45kDa) decorin coreprotein was detected in most synovial fluids, whileminor catabolites were only rarely observed.Neither fibromodulin or biglycan core proteinswere detected in any of the joint fluids examined,while MMP-cleaved collagen fragments werepresent in all joints.

Taken together, these results suggest thatseveral cartilage matrix components may be usefulmarkers of cartilage degradation in equine jointdisease. Progressive degradationin vitro wasassociated with ADAMTS-driven aggrecan lossfollowed by decorin and fibromodulin but notbiglycan release, and then collagenolysis byMMPs. Thein vivo pattern of matrix componentrelease is likely to be more complex as varyingstages of cartilage degeneration may besimultaneously present in the one joint.Furthermore, changes in synthesis of aggrecan andSLRPs may occurin vivo which are not observedin thein vitro model where serum free conditionslimit biosynthesis by the chondrocytes. Insynovial fluids, BC-3 immunoreactivity was not

just a surrogate marker of glycosaminoglycanlevels, suggesting that it may be a usefulbiomarker of particular cartilage degenerativeevents. Furthermore, the pattern of BC-3fragments dif fered markedly between joints inassociation with varying degrees of C-terminaltruncation of the aggrecan molecules.Determining the C-terminal sequence of thesefragments and generating new neoepitopeantibodies may provide a better tool formonitoring their presence. Equine cartilageinvitro released decorin and fibromodulin prior toand in association with collagen breakdown,suggesting that these SLRPs may be useful tomonitorin vivo . The reason that biglycan was notreleased from equine cartilage may be related tothe fact that mature adult equine cartilage wasused in the present studies. Unlike thein vitrosituation, only decorin core protein was detectedin synovial fluids of horses with joint disease andwhether this arises from the cartilage or other jointtissues is presently unknown. While all of thesynovial fluids in the present study were from joints evaluated arthroscopically, no control forparameters that may af fect synovial biomarkerlevels (eg age, sex, exercise level, previoustreatment etc) could be imposed limiting theirutility to correlate biomarkers with the stage of cartilage degeneration. Future studies willevaluate changes in synovial fluid in anexperimental model of equine joint disease wherelongitudinal analysis will allow progression of joint disease to be better correlated with changesin aggrecan (BC-3) and decorin metabolites in the joint fluid.

R EFERENCES

Little, C.B., Flannery, C.R., Hughes, C.E., Goodship, A.and Caterson, B. (2005) Cytokine inducedmetalloproteinase expression and activity does notcorrelate with focal susceptibility of articularcartilage to degeneration.Osteoarthritis Cartilage13 , 162-170.

Sztrolovics, R., White, R., Poole, A., Mort, J. andRoughley, P. (1999) Resistance of small leucine-richrepeat proteoglycans to proteolytic degradationduring interleukin-1-stimulated cartilage catabolism.

Biochem J. 339 , 571-577.


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PROSPECTIVE CLINICAL STUDY ASSESSING SERUMBIOMARKERS FOR MUSCULOSKELETAL DISEASE IN23-YEAR-OLD RACING THOROUGHBREDS

D. D. Frisbie, E. Duffy, R. Arthur*, J. Blea, V. Baker**, R. C. Billinghurst andC. W. McIlwraith

Gail Holmes Equine Orthopaedic Research Center, Colorado State University, 2503 Bay Farm Road,Fort Collins, Colorado 80523; *311 E Grandview Avenue, Sierra Madre, California 91024;3320Fairpoint Street, Pasadena, California 91107; **10542 Walker Street, Cypress, California 90630, USA

INTRODUCTION

The use of peripheral blood to assessmusculoskeletal disease has been a goalresearchers have been working towards for over adecade. There have been many studies to validatethe usefulness of serum biomarkers in the horse(McIlwraith 2004). To date, however, only oneother study has assessed their usefulness inclinical cases (Jacksonet al. 2005). This studyshowed significant differences in horses withdorsal metacarpal disease compared to controlsbut no difference in horses that went on to sustaina fracture in this region. The current study wasdesigned to build on previous work by theinvestigators (Frisbieet al. 1999; 2002) thatdemonstrated promising results of biomarkers forthe identification of intra-articular pathology in acontrolled clinical or experimental setting. Thisstudy represents the real world application of 6serum biomarkers for the detection of musculoskeletal injuries.

M ATERIALS AND METHODS

Two- or 3-year-old Thoroughbred racehorses wereentered into the study when they arrived atThoroughbred race tracks in southern California.Each month a lameness examination wasperformed by a study veterinarian and peripheralblood collected and serum stored. Horses wereremoved from the study when (McIlwraith 2004)they were out of training for more than 30 days forany reason, or (Jacksonet al. 2005) they wereenrolled in the study for 10 months. Only horsesthat sustained a single musculoskeletal injury andhad completed at least 2 months in the study wereanalysed. For the purposes of this study, a

musculoskeletal injury was considered one of thefollowing: intra-articular fragmentation (IAF),injury to a tendon or ligamentous structure (TL),incomplete or complete non-articular fracture(ICF) and periostitis (BS).

Serum samples were analysed forglycosaminoglycan (GAG), type I and II collagen(Col short), type II collagen synthesis (CPII), typeII collagen content (Col CEQ), aggrecan synthesis(846), osteocalcin (OC) as a marker of boneformation and (CTX) as a marker of bonedegradation as previously reported (Frisbieet al.2002).

Statistical analysis was performed using theSAS statistical software package, version 8e.Non-parametric measures of assumption weretested using a Chi-squared analysis. All outcomevariables that were concentrations were logtransformed (natural log) to meet assumptions of normality. When direct comparisons were made aleast squares means procedure was utilised and ap-value of


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horses, 23 (31%) sustained an intra-articularfragmentation (IAF), 18 (24%) a tendon orligamentous injury (TL), 13 (17%) incomplete orcomplete non-articular fracture (ICF) and 21(28%) were diagnosed with periostitis (BS).

When samples collected at the time of injurywere compared to control horses the followingobservations were made: a significant drop inserum GAG levels were noted for the IAF and ICFhorses. An increase in CTX was seen in TLhorses. No significant changes were detected inBS horses, although significant increases in GAG,Col Short and decrease in osteocalcin were seenwhen baseline samples were compared to injuredsamples of the same BS horse.

DISCUSSION

The results of this study indicated that biomarkerscan be used as a diagnostic aid in clinicalmusculoskeletal injuries as well as provide insightto the pathogenesis of disease processes. Each of the injuries studied here had a unique biomarkerpattern following injury, suggesting further studyon predictability of injury is warranted.Furthermore the fact that the biomarkers were

indeed significantly altered in clinical casesdespite the confounding effects present in the realworld is promising.

ACKNOWLEDGMENTS

This project was funded by the Grayson JockeyClub Research Foundation.

R EFERENCES

Frisbie, D.D., Al-Sobayil, F., Billinghurst, R.C. andMcIlwraith, C.W. (2002) Serum biomarkersdistinguish exercise from osteoarthritic pathology.Osteoarthritis & Cartilage 10 , S53.

Frisbie, D.D., Ray, C.S., Ionescu, M., Poole, A.R.,Chapman, P.L. and McIlwraith, C.W. (1999)Measurement of synovial fluid and serumconcentrations of the 846 epitope of chondroitinsulfate and of carboxy propeptides of type IIprocollagen for diagnosis of osteochondralfragmentation in horses. Am. J. vet. Res. 60, 306-309.

Jackson, B.F., Lonnell, C. and Verheyen, K.L. (2005)Biochemical markers of bone metabolism and riskof dorsal metacarpal disease in 2-year-oldThoroughbreds. Equine vet. J. 37 , 87-91.

McIlwraith, C.W. (2004) Current state of biomarkers inequine bone and joint disease, in Focus on joints.

Am. Ass. Equine Pract. 109-127.


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DOES INFLAMMATION TRIGGER TISSUEDESTRUCTION OR IS TISSUE DESTRUCTIONACTIVATING INFLAMMATION IN JOINT DISEASE?

D. Heinegrd

Department of Experimental Medicine, BMC, Plan C12, Lund University, SE-22184 Lund, Sweden

Cartilage extracellular matrix consists of 2 majorfunctional entities, the aggrecan moleculescontributing an extreme charge density andtherefore accomplishing an osmotic environmentthat retains water leading to a resistance todeformation. Collagen fibrillar networks provideresistance to tension and distribute load. There area large number of molecules now known thatinteract both in the formation and in themaintenance of these macromolecular assembliesand also in concomitantly interacting with cellsurface receptor relaying information to the cellson the condition of the matrix. Examples of alteredcatabolism, repair attempts and how matrix mayinteract with the cells were discussed.

There is a continuous turnover of these matrixmolecules, necessary for the adaptation to loadand repairing fatigue damage. In pathology thereis both new synthesis in repair attempts andincreased breakdown, putatively different innature, of matrix constituents. Some of thefragments formed are no longer retained anddiffuse into the synovial fluid. There is a potentialfor some of these bioactive fragments to affect, forexample the inflammatory response. Anyfragments activating inflammation have thepotential to cause chronicity and progressive jointdestruction by inducing production of cataboliccytokines. Examples of candidate molecules werediscussed.


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WHAT BIOMARKERS ARE TELLING US AND THECHALLENGES AHEAD

S. Laverty

Universit de Montral, Robin Poole, Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Canada

The measurement of cartilage and bone specificbiomarkers (BM) of matrix synthesis anddegradation in body fluids is not only potentiallyuseful for the prediction, early diagnosis andstaging of joint diseases, but also to define matrixprocesses, whether catabolic or anabolic.Furthermore, they may be used to define a responseto medication and provide specific read-outs fortargeted matrix therapy. A review of results fromselected recently published and ongoing animalstudies in our laboratories using BMs to assesscartilage and bone metabolism is provided.

Major alterations in cartilage aggrecanmetabolism, including the release of markers of both synthesis (846) and turnover (KS), wererecently detected by analyses of synovial fluid(SF) following repeated intra-articular injectionsof a clinically relevant dose of triamcinoloneacetonide in normal horses (Celesteet al. 2005).These results were consistent with results of aprior study where methylprednisolone acetateinjections were evaluated (Robionet al. 2001).

Non-steroidal anti-inflammatory drugs(NSAIDS), are also commonly used to treat thesymptoms of equine osteoarthritis but have beenreported to have adverse effects on both cartilageand bone metabolism. We chose to study skeletalmetabolic ef fects of continuous oraladministration of clinically used doses of NSAIDSby assessing a battery of markers of cartilage andbone synthesis and degradation sequentially inserum and SF. A significant increase inosteocalcin occurred in the sampled joints fromtreated horses and no other changes occurred inthe BM levels over time. These findings areencouraging and suggest that, in horses at least,NSAID administration does not have adverseeffects on cartilage and bone metabolismin vivo(Fradetteet al. 2005).

The cartilage content of BMs may also beassessed in terminal animal studies to evaluate theef fects of medication on cartilage matrixmetabolism. No effects were detected of glucosamine on cartilage type II collagenmetabolism, assessed by specific synthesis (CPII)and degradation markers (Col23/4c short- a markerof cleavage and Col2 3/4m a marker of degradation)in a rabbit model of OA, but a modest protection of GAG content was observed (Tiralocheet al. 2005).Combined, these various results provide support forthe potential use of BM evaluation to assesstherapeutic ef fects of drugs or toxicity.

Parallel to these studies, researchers havecontinued to explore what can be learned aboutosteochondrosis (OCD) from BM assessment. As afollow up to our earlier studies on BMs and OCD,we attempted to discriminate a population of yearlings with stifle OCD (marked joint effusionand a radiographic diagnosis) from a populationwithout lesions based on serum BM analyses.Previously we reported that a differential alterationin aggrecan (decrease in 846 and KS epitopes) andtype II collagen turnover (increase in CP II) wasobserved in young horses (Lavertyet al. (2000)with tarsocrural OCD, based on SF analyses, and aselective increase in type II collagen cleavage bycollagenase in femoropatellar OCD cartilage(Laverty et al. 2002). Despite results of theseprevious studies, we detected no dif ference inserum levels of the cartilage specific BMs analysedin this group of yearlings. We did however detect asignificant increase in serum levels of bone alkalinephosphatase in the animals with OCD whencompared to healthy animals (Laverty unpublisheddata). As it is difficult to obtain client compliancefor synoviocentesis in these cases, it remainsunanswered whether significant changes in the SFBM levels with stifle OCD occurs.


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Many challenges remain ahead on our path toa better understanding of what molecules we aremeasuring and how they may be influenced.

Areas, amongst others, that need to beinvestigated include:

Factors contributing to BM release (collagenversus GAG). Rates of formation, accumulation and

clearance of BM from both normal anddiseased joints.

Routes of systemic clearance and sites of metabolism of BM.

Influence of various degrees of jointinflammation on clearance.

Genetic effects on individual variation inmarker levels.

The relationship between the amount of marker released and the cartilage content asthe amount released may not only depend oncartilage quantity but also quality.

R EFERENCES

Celeste, C., Ionescu, M., Poole, A.R. and Laverty, S.(2005) Repeated intraarticular injections of

triamcinolone acetonide alter cartilage matrixmetabolism measured by biomarkers in synovialfluid. J. Orthop. Res. 23 , 602-610.

Fradette, M., Cleste, C., Richard, H. and Laverty, S.(2005) Effect of oral administration of NSAIDS onbiomarkers of cartilage and bone metabolism. In:

European College of Veterinary Surgeons Meeting , Lyon . Edited, 2005.Laverty, S., Ionescu, M., Marcoux, M., Boure, L., Doize,B. and Poole, A.R. (2000) Alterations in cartilagetype-II procollagen and aggrecan contents insynovial fluid in equine osteochondrosis. J. Orthop.

Res. 18, 399-405.Laverty, S., Okouneff, S., Ionescu, M., Reiner, A.,

Pidoux, I., Webber, C., Rossier, Y., Billinghurst, R.C. and Poole, A.R. (2002) Excessive degradation of type II collagen in articular cartilage in equineosteochondrosis. J. Orthop. Res. 20, 1282-1289.

Robion, F., Doiz, B., Bour, L., Marcoux, M., Ionescu,M., Reiner, A., Poole, A. and Laverty, S. (2001) Useof synovial fluid markers of cartilage synthesis andturnover to study effects of repeated intraarticularadministration of methylprednisolone acetate onarticular cartilagein vivo . J. Orthop. Res. 19, 250-258.

Tiraloche, G., Girard, C., Chouinard, L., Sampalis, J.,Moquin, L., Ionescu, M., Reiner, A., Poole, A. R.and Laverty, S. (2005) Effect of oral glucosamine oncartilage degradation in a rabbit model of osteoarthritis. Arthritis Rheum 52, 1118-1128.


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INVOLVEMENT OF THE CARTILAGE/BONE JUNCTION IN EQUINE OSTEOARTHRITIS (OA) OFTHE MIDDLE CARPAL JOINT

S. Ekman

Department of Biomedicine & Veterinary Public Health, Division of Pathology, Pharmakology &Toxicology, SLU, Box 7028, SE-75007 Uppsala, Sweden

The third carpal bone of the middle carpal joint isexposed to excessive load during thehyperextension phase of the racing stride in thestandardbred trotter (STB; Johnstonet al. 1995)and lameness due to osteoarthritis (OA) in this joint is common (Pool and Meagher 1990).Articular and bone lesions in the radial facet,such as superficial cartilage fraying, cartilageerosion, subchondral bone sclerosis, bonenecrosis, osteocartilagenous interface collapseand incomplete fractures are often found atnecropsy. The simultaneous changes in articularcartilage and underlying bone are of greatimportance in OA (Kawcaket al. 2001). Here,findings of a pilot and a published study (Ekmanet al. 2005) are discussed, concentrating on thecartilage/bone interface in the middle carpal jointof trotters. The definition of equine OA maydiffer among clinicians, radiologists andpathologists and this is also discussed from apathologists point of view.

In a pilot study it was found that morphologicalchanges at the radial facet from STBs differed withage (1-, 2-, 3- and 4-year-old) and training. Asemilunar bonesclerosis developed together withcartilage matrix changes such as, reducedstainability, chondro and bone necrosis,chondrocyte proliferation (clusters) and collapse of the cartilage/bone interface. The lesions in thecalcified articular cartilage and cartilage/boneinterface could often be found without superficialfraying. Mild to moderate synovitis was oftenpresent and lameness had occasionally been seen inall of the horses with lesions, but was impossible tocorrelate to type of lesion.

Possible biomarkers for cartilage/bonechanges, mirroring bone activity in theextracellular matrix could be found among the non-collagenous proteins forming 10% of the organic

bone matrix. Candidates that have been implicatedare; bone sialoprotein (BSP), osteocalcin,osteonectin, osteoadherin, osteopontin, biglycan,decorin. Many of these are, however, not bone-specific and many can not be used as markers foractivity within the cartilage/bone interface.

BSP, a RGD-containing protein, (Franzen andHeinegrd 1985) has been suggested as a markerfor subchondral bone activity in OA (Wollheim1999). It is a cell binding protein and bindsstrongly to hydroxyapatite (Oldberget al. 1988). Itis concentrated in the cartilage/bone interface earlyin experimental OA of guinea pigs and later in thedeeper bone (deBrieet. al. 1997). Anultrastructural immunolocalisation of BSP in theosteocartilagenous interface of the equine thirdcarpal bone showed a marked increase of BSP inthe interface with degenerative changes comparedto the intact cartilage/bone interface (Ekmanet al.2005). BSP immunolabelling was higher in theunderlying intact bone of the normal areascompared to the fragmented bone of thedegenerated areas. The changes found in these joints probably represent early OA and none of thehorses had clinical evidence of lameness from themiddle carpal joint at the time of euthanasia.

The findings suggest that BSP can be used asan in vivo biomarker for juxta articular boneactivity in early OA.

R EFERENCES

deBrie, E., Lei W., Reinholt F.P., Mengarelli-WidholmS., Heinegrd D. and Svensson O. (1997)Ultrastructural immunolocalisation of bonesialoprotein in guinea-pig osteoarthritis.

Osteoarthritis & Cartilage , 5, 387-393.Ekman, S., Skildebrand, E., Heinegrd, D., andHultenby K.. (2005) Ultrastructuralimmunolocalisation of bone sialoprotein in the


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osteoacartilagenous interface of the equine thirdcarpal bone. Equine vet. J. 23, 26-30.

Franzen, A. and Heinegrd, D. (1985) Isolation andcharacterization of two sialoproteins presented inbone calcified matrix. J. Biol. Chem. 232 , 715-724.

Johnston, C., Roepstorff, L., Drevemo, S. and Roneus,N. (1995) Kinematics of the distal forelimb duringthe stance phase in the fast trotting standardbred.

Equine vet. J. 18, 170-174.Kawcak, C.E. (2001) The role of subchondral bone in

joint disease: a review. Equine vet. J. 33, 120-126.Oldberg, ., Franzen, A. and Heinegrd, D. (1988) The

primary structures of a cellbinding bonesialoprotein. J. Biol. Chem. 263 , 430-432.

Pool, R.R. and Meagher, D.M. (1990) Pathologicfindings and pathogenesis of racetrack injuries.Vet.Clin. N. Am. Equine Pract. 6, 1-30.

Wollheim, F.A. (1999) Bone sialoprotein a new markerfor subchondral bone.Osteoarthritis & Cartilage , 7,331-332.


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BONE BIOMARKERS IN HORSES: WHERE ARE WENOW AND FUTURE PROSPECTS?

J. Price and B. Jackson

Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK

The first papers describing the measurement of biochemical markers of bone cell activity inhorses were published in the early 1990s. Theprospect of being able to non-invasively measureequine bone metabolism was very exciting andbone markers were considered to have greatpotential as both clinical and research tools. In thesubsequent years many research groups havedescribed the measurement of bone biomarkers ina variety of contexts and this is now an appropriatetime to review this body of information and toattempt to assess the prospects for the future.

A number of bone markers have beenmeasured in the horse, the majority using assaysoriginally developed for human use and assessedfor their equine cross-reactivity, although someequine-specific assays have been developed.Bone markers measured in horses include;osteocalcin, deoxypyridinoline (DpD), bonealkaline phosphatase (BALP), the carboxy-terminal propeptide of type I collagen (PICP), thecarboxy-terminal telopeptide of type I collagen(CTX-MMP/ICTP), the carboxy-termialtelopeptide of type I collagen (CTX-1) and thetype I-collagen cross-linked N-terminaltelopeptide of type I collagen (NTx). A number of studies have also demonstrated that bone markerconcentrations are influenced by a number of controllable and uncontrollable variables thatinclude age, breed, season, time of day and gender(although the influence of gender remainssomewhat controversial). We have recentlydemonstrated that stage of oestrus can alsoinfluence bone marker concentrations.

Several studies have measured bone markersin order to monitor how different trainingregimens influence equine bone. These reportshave described the measurement of a number of different bone markers in horses of different ages

and exercised using a variety protocols. Thismakes it very difficult to directly compare andinterpret the results of these studies however, whatis clear is that exercise is an important variablethat can influence bone marker concentrations.The most consistent data have been obtained fromwell designed experimental studies in which bonemarkers were compared in horses exposed to verydifferent exercise regimens (eg no exercisecompared to fairly intense exercise). Based on ourearly work in experimental treadmill studies, wethen set out to establish whether bone markerscould be used as a method for identifying thoseexercise regimens that may be osteogeniccompared to those that may have detrimentaleffects on bone. We have now undertaken 2studies in reasonably large cohorts of horses incommercial race training. Our results have lead usto conclude that in the field bone markers havelimited value for informing how traininginfluences bone adaptation because so manyvariables can potentially influence theirconcentrations.

The other important question that needs to beaddressed is whether bone markers can be used asdiagnostic and/or prognostic markers? There havebeen a small number of reports which describechanges in bone marker concentrations inmusculoskeletal diseases, including osteochondrosisand osteoarthritis. Because studies in humans haveshown that bone markers are useful for identifyingpeople at increased risk of osteoporotic fracture, oneof our aims has been to test the hypothesis that bonemarkers could predict fracture in horses. To explorethis hypothesis, we measured 4 bone markers in 4092-year-olds and 365 3-year-olds at the start of theirflat race training (OC, PICP, ICTP and CTX).Disappointingly, the conclusion of this study is thatbiochemical markers have no value for identifying


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horses that are at risk of fracture in the subsequenttraining/racing season. Neither did we find thatmarker concentrations were different in fracturecases compared to controls when measuredlongitudinally. This study also showed that cautionmust be taken when interpreting data from smallnumbers of cases, since preliminary analysis of ourdata indicated that fracture was associated with asignificant difference in specific bone markerconcentrations. On a more positive note, we haveshown that bone markers are potentially useful forthe early identification of 2-year-old racehorseshorses at increased risk of developing dorsalmetacarpal disease (DMD), a fatigue damage injuryassociated with the introduction of high speedexercise. In man the other important application forbone markers is to monitor the effects of differenttreatments for metabolic bone disease. Bonemarkers could therefore prove to be extremelyuseful for monitoring the effects of different drugs ornutraceuticals on the equine skeleton, although verylittle work has been published in this area to date.

C ONCLUSIONS

In the last 15 years research on equine bonemarkers has led to the development and validationof a variety of assays and the identification of anumber of variables that affect bone markers (age,breed etc). There is no doubt that this research hashelped contribute to our understanding of equineskeletal physiology and in experimental situationsbone markers have also been shown to have valuefor monitoring the effects of exercise on bone cellactivity. However, the jury remains out on whetherbone markers have value as diagnostic orprognostic tools. Well designed clinical researchstudies are now required to address this issue. Theuse of bone markers in combination with geneticmarkers to assess risk of injury may also bepossible in the future. An area that also deservesfurther study is the use of bone markers toobjectively assess the effects on bone cell functionof novel and established treatments for equinemusculoskeletal disease.


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ARE BONE MARKERS ALONE A USEFUL TOOL TOFOLLOW TREATMENT OR EXERCISE REGIMENS INHORSES?

O. M. Lepage

Gremeres Lyon Equine Research Centre, Universit de Lyon, F-69003; Ecole Nationale Vtrinairede Lyon, F-69280 Marcy lEtoile, France

INTRODUCTION

To finely tune bone equilibrium during growing of afoal, training or drug intervention, veterinariansneed appropriate biomarkers and defined specificsites on weight-bearing structures to get informationsuch as bone mineral density (BMD) or speed of sound (SOS) values (Lepageet al. 2001). Thisabstract summarises different studies performed toassess the effect of a non-aminobisphosphonates(Varela et al. 2002), and flat racing-exercise(Carstanjenet al. 2003a,b; Carstanjenet al. 2005) ondifferent bone biomarkers.

M ATERIALS AND METHODS

1) The effect of training was assessed on 2- to 4-year-old Thoroughbred horses subjected to flatracing-exercise according to 3 training intensities:slow gallop, canter and high speed work.

2) The effect of the non-aminobisphosphonatestiludronic acid (Tildren CEVA, France) via ivadministration at 1 mg/kg during 30 min, was

assessed on normal adult Standarbred horses.

Biochemical biomarkers

Biochemical biomarkers were investigated inserum using commercially available radio-immunoassays.

For bone formation: osteocalcin (OC:DSL-6900, DSL Inc, Webster, Tx, USA) and bone-ALP(Tandem-R Ostase, Immunotech, Belgium) weretested.

For bone resorption: carboxy-terminal cross-linked telopeptide of type I collagen generated bymatrix metalloproteinases (CTX-MMP:Orion

Diagnostica, Espoo, Finland); C-telopeptide of type I collagen cross-links (CTX-I: Crosslaps,Elecsys, Roche Diagnostics, Switzerland) andCathepsin K (Oxford Biosystems, England) wereused. For Cathepsin K, levels obained were toolow for good detection and it was concluded thatthis assay was not working in horses.

Bone imaging biomarkers

Speed of Sound (SOS; m/s) values were obtainedwith an ultrasound device using a probe with anaxial transmission mode (Omnisense, SunlightLtd, Israel). Superficial dorsal, lateral and medialcortical bone of the third metacarpal bone (MCIII) was assessed with this method. (Lepageet al.

monograph series no. 22 - equine musculoskeletal biomarkers

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