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ns(ACLT) free moving rats, and 3 exercise groups (slight, moderate and intense) subjected to running training. Rats were killed 14 and 28 days

Elsafter surgery.

Results: On D14 histological assessment demonstrated a benecial inuence of a slight and a moderate exercise vs control ACLT group.Hsp70 increased signicantly in the moderate group vs controls. On D28, histological lesions strongly decreased in the slight and moderateexercise groups vs ACLT group, while an intense effort abolished this benecial trend. Interestingly, the concomitant course of apoptoticevents (caspase 3-positive cells) and the co-expression of Hsp70 in the various groups varied, when signicant, in an inverse manner. In theintense group this overexpression was not noted, as a burn out appeared, thus leading to a loss of this protective effect.

Conclusion: This study shows that a calibrated slight or moderate exercise exerts a benecial inuence on the severity of chondral lesions inACLT rats. Conversely, a strong effort abolishes this chondroprotective effect. This effect could be related to a reduced level of chondrocyteapoptosis through anti-apoptotic capacities of stress-induced Hsp70 overexpression. 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Key words: Cartilage, Chondrocyte, Apoptosis, Exercise, Heat shock protein, Experimental osteoarthritis, Apoptosis.

Introduction

Osteoarthritis (OA) is characterized by a progressive de-generation of articular cartilage associated with remodelingof the subchondral bone, marginal osteophyte formationand progressive symptomatic loss of mechanical function.As the etiology of OA remains incompletely understood,a variety of animal models have been developed with theaim of characterizing the features of the early phases of OA,studying its progression and evaluating new drugs and/ororiginal therapies1. Experimental OA can be induced invarious ways2: abnormal biomechanical forces resultingfrom joint destabilization, displaced loading or structuralalterations resulting either from a degradation of theextracellular matrix by physical, enzymatic means (papain,

collagenase) or from a disturbance of chondrocyte metab-olism (e.g. iodoacetate, vitamin A).Anterior cruciate ligament transection (ACLT) model has

widely been studied in various animal species (rabbits3,4,and dogs5,6) and more recently in the rat7, thus providingnew insights into pathogenic mechanisms and impact ofloading on hyaline cartilage. Biomechanical calculationssuggest that damage of the surface zone leads to increasedloading of the cartilage matrix and higher stresses on theunderlying cartilage, leading to a sequence of events inwhich the degeneration of the supercial zone develops intobrillations of the cartilage and eventually results inerosions and ulcerations. In this model, there is an increasein the number of cells exhibiting signs of degeneration oreven death, often related to chondrocyte apoptosis8,9, aspreviously shown in the dog10 and in the rabbit11. Ourpreliminary data also suggest that chondrocyte caspase-dependent apoptosis also occurs in ACLT-induced OA inthe rat, and that a moderate impact exercise in ACLT rats isassociated with decreased severity of chondral lesions andapoptotic events12.Caspases are a family of proteases that have been

demonstrated to play a prominent role in determining DNAdamages and ancillary caspase-dependent cell-death. Theinitial death signals can activate the apoptotic pathwaywhich involves a cascade of highly regulated hierarchical

1This study was supported by grants from Pole Europeen deSante, CPRC CHU Nancy, and GIP Fonds de recherches HMRAVENTIS (FR99RHU037).* Address correspondence and reprint requests to: Pr. Pierre

Gillet, MD, PhD. UMR 7561 CNRS - Nancy I, Physiopathologieet Pharmacologie Articulaires, Faculte de Medecine, BP 184,Avenue de la Foret de Haye, F54505 Vandoeuvre-Les-Nancy,France. Tel: 33-383-683-950; Fax: 33-383-683-959; E-mail: Pierre.Gillet@medecine.uhp-nancy.frReceived 4 August 2003; revision accepted 12 June 2004.Doseresponse relationship for exerosteoarthritis in rats: a pilot study1

Laurent Galois M.D.yz, Stephanie Etienne M.Sc.y,Ph.D.y, Christel Cournil-Henrionnet Ph.D.y, DamiePh.D.y, Didier Mainard M.D.yz and Pierre Gillet MyUMR 7561 CNRS - Universite Nancy I, FrancezDepartment of Orthopaedic Surgery, Universitary Hos

Summary

Objective: To investigate the inuence of a calibrated exercise oosteoarthritis (OA) in the rat, and to explore the effect of exercise on

Methods: The OA model was induced by anterior cruciate ligament tra

OsteoArthritis and Cartilage (2004) 12, 779e786

2004 OsteoArthritis Research Society International. Published bydoi:10.1016/j.joca.2004.06.008779se on severity of experimental

aurent Grossin Ph.D.y, Astrid Watrin-PinzanoLoeuille M.D., Ph.D.y, Patrick Netter M.D.,., Ph.D.y*

tal of Nancy, France

he progression of structural lesions in an experimental model ofe level of chondrocyte caspase-dependent apoptosis and of Hsp70.

ection (ACLT). Rats were placed in 4 experimental groups: operated

evier Ltd. All rights reserved.

InternationalCartilageRepairSociety

ismolecular events13. These events are mediated by a pro-teolytic cascade in which upstream activator caspasesinitiate and amplify the maturation of effector caspases that,in turn, cleave a discrete subset of cellular polypeptides tomanifest the apoptotic phenotype. Caspase 3 in its activeform is one of the key mediators of apoptosis in itsexecution phase and its expression may herald imminentapoptosis better than TUNEL assay14 and may act asa surrogate specic marker for early chondrocyte apopto-sis15,16. As done in the clinics, we routinely use activecaspase 3 immunostaining in evaluating experimentalOA12, because TUNEL is less specic and apoptosis isoverestimated by TUNEL assay compared with caspase 3detection17.On the other hand, a number of other factors, like heat

shock proteins (Hsps), have been shown to exert a pro-tective effect against chondrocyte apoptosis. Some Hspmembers are constitutively expressed, and the expressionlevels increase in response to stress, while others areinduced after exposure to stress. Hsp70, especially in itsinducible form, is now the focus of scientic attention asa protein that can inuence the apoptotic process throughphysical interaction with key components of the machin-ery18, and could obstruct the activation of caspase 3 duringNO- or staurosporine-induced apoptosis in primary chon-drocytes19. Because exercise training has been consistent-ly shown to increase the expression of Hsp in varioustissues20, and especially skeletal and cardiac muscles21 inboth dose-dependent22 and age-related23 manners, itseems plausible to hypothesize that exercise training isalso able to decrease the level of apoptotic events inweight-bearing articular cartilage.As Hsp70 has been suggested to play an important role

in the early stages of adaptation of chondrocytes tobiomechanical joint constraints24, the primary aim of thisstudy was to extend our previous observations on the effectof a calibrated exercise on the histological and immuno-histochemical changes of experimental OA in rats. In thisdoseeresponse pilot study, we rst studied the inuenceof various levels of exercise on the natural course of thedisease (histological score). In a second step, we haveassessed whether stress-induced Hsp70 overexpressionwas associated with the magnitude of caspase 3 activation.

Materials and methods

ANIMALS

Male Wistar rats (200 g; 8 weeks old) were obtained fromCharles River Laboratories (St Aubin les Elbeuf, France).The maintenance and care of the experimental rats were inaccordance with the guidelines of the NIH for AnimalWelfare Act. Rats were kept in individual plastic cages ina 12:12 lightedark cycle (light-on period, 6:00 AMe6:00PM) in a controlled temperature chamber on sawdustbedding. They were fed a standard diet and had access totap water ad libitum. Body weight was recorded at regularintervals.

SURGICAL PROCEDURE

Rats underwent an ACLT under anaesthesia (i.p. in-jection of a mixture of acepromazine 1.25 mg/kg + ketamine38 mg/kg). According to Williams et al.25, a para-patellarskin incision was performed on the medial side of the rightknee joint, and thereafter on the medial side of the patellar

780 L. Galotendon. Patella was then dislocated laterally to provideaccess to the joint space and ACL was transected in theexed knee. A positive anterior drawer test validatedcomplete transection of the ligament. The joint was thenirrigated with sterile saline to avoid ancillary inammatoryprocess, and a purpose-made suture was processed. Anaive group (sham group) undergoing arthrotomy withoutACLT was included as an internal control for characterizingthis experimental model on various key points.

CALIBRATED EFFORT

Rats that had received ACLT were then assignedrandomly to various groups, a control group, and 3 exercisegroups. In the control group, ACLT rats were allowed tomove freely in standardized cages. According to ourprevious data, in the exercise groups, ACLT rats weresubjected to running training once a day 5 days a week for2 or 4 weeks on a motor driven treadmill (LE 8700, LSILetica(r), Barcelona, Spain) for rodents with a constantspeed of:

30 cm/s for 15 min (slight exercise), leading to a dis-tance of 7.5 km over 28 days;

30 cm/s for 30 min (moderate exercise), leading toa distance of 15 km over 28 days;

30 cm/s for 60 min (intense exercise), thus leading toa total distance of 30 km over 28 days.

HISTOLOGICAL GRADING

Animals were killed by cervical dislocation under anaes-thesia. Whole knee joints were dissected, xed in 4%paraformaldehyde ( pH 7.4), decalcied with Rapid Decal-cifiant Osseux (RDO, Apex, Canada), dehydrated througha descending series of ethanol with the use of anautomated tissue processing apparatus. After embeddingin parafn, serial sections with a thickness of 5 mm wereprepared for histological examination and immunohisto-chemistry. The sections were stained with hematoxylineeosin to observe cellularity, and toluidine blue to assessproteoglycan content.The severity of OA lesions was graded on a scale

adapted from Mankins score by two independent observers(LG and SE). This score ranged from 0 to 15 according tostructure, cellularity, toluidine blue staining, thickness ofhypertrophic chondrocyte layer, bone remodelling andosteolysis. Structure was graded from 0 to 5 (0Z Normal,1Z Pannus and surface irregularities, 2Z Clefts to tran-sitional zone, 3Z Clefts to radial zone, 4Z Clefts tocalcied zone, 5Z Complete disorganization). Cellularitywas graded from 0 to 3 (0Z Normal, 1Z Diffuse hyper-cellularity, 2Z Cloning, 3Z Hypocellularity). Toluidineblue staining was graded from 0 to 3 (0Z Normal,1Z Slight reduction, 2Z Moderate reduction, 3Z SevereSevere reduction). Thickness of hypertrophic chondrocytelayer graded from 0 to 2 (0Z Normal, 1Z Moderatedecrease, 2Z Total decrease). Bone remodelling andbone osteolysis were graded from 0 to 1, respectively, with0Z No and 1Z Yes. This score was determined in fourcompartments: medial and lateral part of the tibia andmedial and lateral part of the femur (the patella was notassessed) thus leading to a maximal score of 60 per knee.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry was performed on the serial

et al.: Doseresponse relationship on severity of rat OAparafn sections as previously described12. Tissue sections

The primary antibodies used in this study were: rabbit (19.2G 1.6 on D7, 34.0G 4.7 on D14, and 39.8G 7.4 on

Linpolyclonal active Caspase 3 antibody (R & D Systems,Abington, U.K.) diluted at a ratio of 1:300 and rabbitpolyclonal Hsp70 antibody (StressGen Biotechnologies,Victoria, Canada) diluted at a ratio of 1:300. The presenceof antigen was estimated by determining the number ofspecic chondrocytes staining positive in the supercialzone (supercial and upper intermediate cartilage layers)and in deep zone (lower intermediate and deep layers).Each zone was divided into 4 different sections. The cellcount scores were determined separately for the medial andlateral sides of condyles and plateaus. The total number ofchondrocytes and the number of chondrocytes stainingpositive for the specic antigen were determined at 40!magnication for the supercial and the deep zones,respectively.

STUDY DESIGN AND STATISTICAL ANALYSIS

A preliminary pilot descriptive study was performed ina small number of rats (3 ACLT and 3 sham on D7, 14 and28) to verify in vivo the occurrence of both apoptotic eventsand overexpression of Hsp70 in this model. Next, for

Fig. 1. Time course of histological lesions (medial tibial plateaus) in AC(bottom images), cellularity, and surface integrity by HematoxylineEos

was observed as a structural alteration (hypercellularity). On D14, s

decreased compared with D7. On D28, subchondral bone modiD28). On D7, a mild transient synovitis inherent to thesurgical procedure of arthrotomy was noted. Structuralalterations appeared in the ACLT group, predominating inthe medial condyle. Slight proteoglycan depletion anddiffuse hypercellularity were present in all compartments(Fig. 1). On D14, condyle lesions were stable. Structuralalteration of cartilage increased in medial and lateral tibialplateaus. This tibial alteration consisted of clefts toradial zone, leading in some cases to bone exposure.A dramatic decrease in cellularity was observed in thewhole cartilage. Proteoglycan depletion was stable and thebrosis persisted. On day 28, degenerative lesions werehomogeneous in all compartments ( plateaus and con-dyles). Mirror-image lesions were observed specially inmedial compartment. Subchondral remodelling was morepronounced at this stage.Apoptotic events, depicted by active Caspase 3 immu-

nostaining (Fig. 2), were increased throughout the exper-iment (12e14% on D7, D14 and D28) when compared withsham-operated knees (4e9%). Apoptotic events predomi-nated on D7 in the supercial zone of cartilage and onD14 in the deep zone. On D28, apoptotic events were

T rats. Proteoglycan content was evaluated by toluidine blue stainingHES staining (upper images). On D7, a slight proteoglycan depletionwere deparafnized and rehydrated. The sections were pre-treated with chondroitinase ABC (0.25 U/ml in PBS, pH 8.0;Sigma, St. Louis, MO, USA) for 90 min at 37(C.Permeability was enhanced by using 0.3% Triton X-100 inPBS (Sigma, St. Louis, MO, USA) for 30 min. Endogenousperoxidase activity was blocked by incubating sections withfreshly prepared 3% hydrogen peroxide (Sigma, St. Louis,MO, USA) for 30 min. Non-specic staining was blocked byincubation of the sections with blocking serum supplied byNovostain superABC kit (Novocastra, Newcastle, UK) for60 min. Sections were incubated overnight at 4(C withprimary antibodies in a humidied chamber. A biotin-labelled goat anti-rabbit IgG (Novostain superABC kit)was used as a secondary antibody for 45 min. A biotineavidin detection system (Novostain superABC kit) was usedaccording to the manufacturers recommendations. Theperoxidase was detected using liquid diaminobenzidinesubstrate kit (Novocastra, Newcastle, UK). After counter-staining with methyl green, slides were dehydrated andmounted with Eukitt (Labonord, France).

studying the doseeresponse relationship for exercise, 10ACLT rats were compared to 5 slight, 5 moderate and 5intense rats only on D14 and D28, since our previousresults indicated no statistical inuence of exercise on D7.Data are expressed as meanG standard error of the mean(S.E.M.). One-way ANOVA followed by a Students t testwas used to determine the statistical signicance of the dif-ferences between ACLT group and ACLT group withcalibrated effort. P values lesser than 0.05 were consideredsignicant. A total of 68 rats were used in this study.

Results

HISTOLOGICAL CHANGES AFTER ACLT

Histological changes

Articular cartilage from the sham-operated knee jointswas histologically normal throughout the study (score of 0).In ACLT rats, histological score progressively increased

781Osteoarthritis and Cartilage Vol. 12, No. 10upercial lesion reached radial zone. The cellularity progressivelycations appeared. Clefts were more pronounced at this stage.

inonf Apo

isINFLUENCE OF A CALIBRATED EFFORT ON THE

COURSE OF ACLT MODEL

On D14 histological assessment demonstrated a bene-cial inuence of a slight and a moderate exercise vs controlACLT group (P!0:05). In contrast, no signicant benecialor detrimental inuence was noted in the intense group.Apoptotic events (10% caspase 3-positive cells in thecontrol group) were not signicantly decreased in slight,moderate and intense groups. Hsp70 increased signif-icantly in the moderate group vs controls (22.4% vs 12.8%,P!0:05). On the other hand, a benecial trend wasobserved in the slight group (16.7%, NS) and no inuencewas present in the intense group (11.2%, data notshown).On D28, histological lesions strongly decreased in the

slight and moderate exercise groups vs ACLT group, whilean intense effort abolished this benecial trend (Fig. 3).Typically, ACLT group was characterized by clefts reachingthe radial zone and strong proteoglycan depletion. In theslight group, clefts were less pronounced, limited to thetransitional zone. Additionally, as shown in Fig. 4, pro-teoglycan depletion was less marked. In the moderategroup, when compared with ACLT group, cartilage pre-sented a dramatic hypercellularity and less brillationslocated only in the supercial zone. Conversely, nobenecial or detrimental effect was observed in theintense vs ACLT group: clefts reached the radial zoneand proteoglycan content was strongly decreased.Interestingly, the concomitant course of caspase-de-

was strongly signicant in the medial compartment, mostlyinvolved in this experimental model. In this particular group,concomitant Hsp70 overexpression was more pronounced,in both supercial and deep zones, suggesting that thisanti-apoptotic factor could, at least in part, contribute tothis benecial effect. The lack of signicant negative

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atsingthte (6grOA is the most common joint disorder in a large numberof people older than 65 years. Knee OA is more commonlyassociated with disability than OA of the other joints. Manytreatment programs have been developed including med-ications with NSAIDs, chondroprotective drugs, physicalmodalities and therapeutic exercises. Therapeutic exercisein OA may prevent accelerated degeneration caused bydisuse without causing further degeneration, as suggestedby recent longitudinal clinical studies26,27. Ambivalenteffects of training on cartilage is well known in the clinics28:it is established that OA is associated with heavy lifting,farming and elite sport activity, and, on the other hand,moderate exercise does not seem to increase the risk ofOA, and under certain circumstances, even could preventknee OA. This ambivalent effect has previously beenobserved experimentally: a high load effort has a detrimentaleffect on the operated knee in meniscectomized rat29, andto a lesser extent, in naive rats30, but in other situations itmay prevent spontaneous knee OA in the hamster31.Additionally, recent experimental studies in equine articularcartilage also showed ambivalent effect of strenuous vsmoderate exercise on the metabolism and aspect ofarticular cartilage32,33.This study has been conducted in rats because the use of

a training mill makes it possible to measure the effortapplied to weight-bearing joints, and, to the best of our

synovial uid for nutrition. Cyclic loading induced byphysiological and overuse activities produces deformations,pressure gradients and uid ows within the tissue.Laboratory investigations, performed in vitro and in vivo,have shown that mechanical stress has a direct effect onchondrocyte metabolism, and could, under certain con-ditions, induce anti-apoptotic factors such as Hsp70, asdemonstrated herein in ACLT rats. Conversely, overuse, orexcessive stress, can induce an excess of apoptosis39. Thisambivalent effect is highlighted by the fact that regulardistance running seems to have experimentally no adverseeffect on normal joints, contrasting with the fact that highimpact joint loading may exert joint degeneration in healthyand experimental OA cartilages29, as if a burn outappeared.As observed in the clinics, chondrocyte death, either

necrotic or apoptotic, is also observed in this particular ratmodel of OA following ACLT40, as shown in meniscectom-ized rats41. Apoptotic events are noted in 12e14% ofchondrocytes vs 4e9% in controls, probably due to thematuration process. Consequently, therapeutical modula-tion of apoptotic caspases could be of great benet duringearly phases of OA42. Previous studies have demonstratedthat the chondrocytic expression of Hsp70 is positivelycorrelated with the clinical severity of OA39,43, and thatHsp70 played a role in cell protection from stress, especiallyin the early events. Mechanical stress24, heat stress orcorrelation between caspase 3- and Hsp70-positive cells inall compartments (Pearson r 0:42, PZ 0.08, not quitesignicant) probably reects the complexity of the interac-tion between pro and anti-apoptotic stimuli, thus accountingfor the lack of linearity of the dose response, the probableexistence of a threshold, as well as a small therapeuticindex between efciency and inefcacy of Hsp inductionagainst apoptosis. Additionally, in the intense group thisoverexpression was not noted, as if a burn out appeared,thus leading to a loss of this protective effect.

Discussion

Fig. 4. Histological analysis of cartilage lesions during ACLT model in rwere evaluated with HematoxylineEosin (HES, upper gures) stainstaining (TB, bottom images). For the slight exercise group (15 min),decreased (note the synovial pannus, bottom image). For the moderasupercial brillation and a hypercellularity. After an intense exercise

to those observed in the ACLT

Osteoarthritis and Cartilage Vol. 12, No. 10knowledge, these results demonstrate for the rst time thebenecial inuence of a calibrated moderate exercise onthe natural course of experimental rat OA, while a strongeffort was without benecial outcome. Furthermore, ACLTmodel mimics some features of human OA, like earlyinammatory synovitis34, in some case leading to synovialpannus-like tissue35 (Fig. 4), changes in proteoglycancontent and collagen structure, cartilage erosions36 pre-dominating in the medial compartment, late osteophytosis,subchondral bone remodelling, and chondrocytic apopto-sis37 with caspase 3 activation38, thus reinforcing theclinical relevance of our experimental approach.Basically, cartilage is an avascular tissue, and chondro-

cyte metabolism depends on diffusion and convection of

undergoing a calibrated effort (D28). Cellularity and surface integrityand proteoglycan content was assessed by using toluidine blue

e lesions reached the transitional zone and the proteoglycan contentexercise group (30 min), the cartilage was characterized by a slight0 min), the erosion reached the radial zone and lesions were similaroup without exercise (sham).

783cytokines are presumed to act as stress on OA cartilage.

isWhen OA progresses, due to matrix disruption, compres-sive load increases, especially in the medial compartment,and subsequently Hsp70 can be induced in cartilage, aspreviously reported in rat quadriceps muscle followingACLT and exercise training44 and spontaneous OA inC57 black mouse45. Additionally, it has been reported thatthe overexpression46 or the induction47 of Hsp70 protectschondrocytes from cell death in vitro44 and in vivo45. Thispresumption is supported by our nding that Hsp70-positivecells were mainly found (Fig. 5) in the areas where apoptoticevents (caspase 3-positive cells) were dramatically less-ened. This negative imbalance between apoptotic eventsand expression of different stress proteins has already beenobserved in chondrocytic cells of the growing plate in the rattibia48, as a key process in the promotion or the regulationof bone calcication. Unfortunately, as observed in the

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Fig. 5. Caspase 3 and Hsp70 expressions assessed by immunos-taining on D28. Caspase 3 expression was less pronounced inmoderate exercise group in both supercial and deep zones(P!0:05) in the medial compartment. No signicant differenceswere observed in slight and intense exercise when compared to theACLT group. A concomitant overexpression of Hsp70, an anti-apoptotic factor was noted in the moderate exercise group(P!0:05) in both compartments in supercial and deep zone.(P values were determined by t-test, n 10 rats for ACLT group,

n 5 rats for other groups, mean scoreG standard error).

784 L. Galopresent study, overexpression of Hsp70 to mechanicalconstraints is not dose dependent (minimal and maximalthresholds) and a too-high load impact could lead to a burnout of this machinery, leading to override these protectiveeffects, with a restored apoptosis as an end result.

LIMITATIONS OF THE STUDY

The use of a quadruped model for the study of exercise ofan unstable joint may introduce a bias in extrapolating thesedata to bipeds. Nevertheless, this model has been validatedin the rat by our group and others: it mimics over a shortperiod (28 days) some features of early OA, like surfaceerosion, synovitis and subchondral bone remodeling, evenin young animals. It was thus interesting for us to study theinuence of a calibrated effort on early stages of OA,sensitive to pharmacological modulation. Additionally,numerous studies on endurance training have beenperformed in young rats for studying the inuence of stresson Hsp expression and its inuence on apoptotic events,thus allowing us to minimize the number of rats sacricedby using calibrated protocols. In addition, rat (8e10 weeksof age) is the archetypal animal for preclinical pharmaco-logical studies: ACLT, even in young rats, seems thusa validated, reproducible, low-cost candidate for combiningboth physiopathological and therapeutical approaches inexperimental OA.

In conclusion, a calibrated slight or moderate exerciseexerts a benecial inuence on the severity of chondrallesions in ACLT rats. Conversely, a strong effort abolishesthis chondroprotective effect. In these conditions stress-induced Hsp70 overexpression may exert chondroprotec-tive properties through its anti-apoptotic capacities. Hsp70exerts cytoprotective and biosynthetic functions in acting asa chaperone within chondrocytes, but concomitant pro-inammatory (e.g., cytokines and eicosanoids) and apopto-tic signals (e.g., NO, Fas-L) as well as co-expression ofother Hsps, may counteract these benecial effects in someinstance during OA. Given the fact that Hsp70 haschondroprotective effects increasing intracellular expres-sion of Hsp70 may be a highly effective approach to preventapoptotic cell death. Therefore, future studies should bedirected toward gene delivery of Hsp70 to chondrocyte inexperimental OA models.

Acknowledgements

The authors thank Venkatesan Narayanan, Bernard Terlainand Jean Yves Jouzeau for their expert advices and MichelThiery for taking good care of animals.

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786 L. Galois et al.: Doseresponse relationship on severity of rat OA

Dose-response relationship for exercise on severity of experimental osteoarthritis in rats: a pilot studyIntroductionMaterials and methodsAnimalsSurgical procedureCalibrated effortHistological gradingImmunohistochemistryStudy design and statistical analysis

ResultsHistological changes after ACLTHistological changes

Influence of a calibrated effort on the course of ACLT model

DiscussionLimitations of the study

AcknowledgementsReferences