to young beagles - a eular & bmj rheumatology journal

Annals of the Rheumatic Diseases 1993; 52: 369-377

Adaptation of canine femoral head articularcartilage to long distance running exercise inyoung beagles

Mikko J Lammi, Tomi P Hakkinen, Jyrki J Parkkinen, Mika M Hyttinen, Matti Jortikka,Heikki J Helminen, Markku I Tammi

Department ofAnatomy,University ofKuopio,PO Box 6,SF-70211 Kuopio,FinlandM J LammiT P HakkinenJ J ParkkinenMM HyttinenM JortikkaH J HelminenM I TammiCorrespondence to:Dr Mikko Lammi,Department ofAnatomy,University of Kuopio,PO Box 1627,SF-70211 Kuopio,Finland.

Accepted for publication25 January 1993

AbstractObjective-To study the effects of longterm (one year), long distance (up to 40km/day) running on the metabolism ofarticular cartilage the biosynthesis ofpro-teoglycans was examined by in vitrolabelling of anterior (weight bearing) andposterior (less weight bearing) areas ofthefemoral head from young beagles.Methods-Total sulphate incorporationrates were determined and distribution ofthe incorporated sulphate was localisedby quantitative autoradiography. Concen-tration and extractability of the proteo-glycans were determined, and proteoglycanstructures were investigated by gel fil-tration chromatography, agarose gel elec-trophoresis, and chemical determinations.Results-In the less weight bearing area

the amount of extractable proteoglycanswas decreased (p-0.02), simultaneouslywith an increased concentration of re-

sidual glycosaminoglycans in the tissueafter 4 M GuCl extraction (p-0 05). Incontrol animals proteoglycan synthesiswas most active in the deep zone of thecartilage, whereas exercise increased syn-thesis in the intermediate zone. There wasa tendency to a lower keratan: chondroitinsulphate ratio in the running dogs. Nomacroscopical or microscopical signs ofarticular degeneration or injury were

visible in any ofthe animals.Conclusion-The articular cartilage ofthefemoral head showed a great capacity toadapt to the increased mechanical load-ing. The reduced proteoglycan extract-ability in the less weight bearing area

changed it similar to the weight bearingarea, suggesting that the low extractabilityof proteoglycans reflects the long termloading history of articular cartilage. Thecongruency of the femoral head withacetabulum seems to protect the cartilagefrom the untoward alterations that occurin the femoral condyles subjected to asimilar running programme.

(Ann Rheum Dis 1993; 52: 369-377)

The continuous wellbeing of articular cartilageis vital for the joint as it is almost totally unableto recover from trauma. Weight bearing hasbeen shown to be important for the main-

tenance of normal articular cartilage, becauseamputation of the leg below the knee (allowingfree movement of the joint) could not maintainnormal composition of the cartilage.' Physicaltraining has often been considered to havebeneficial effects on the properties of articularcartilage, although catabolic responses to heavyexercise have also been reported.2 3 Anatrophied cartilage was not able to recover ifrunning exercise was started immediately afterimmobilisation.4Loading of an instable joint causes severe

changes in the structure and function ofarticular cartilage.5 Cutting of the anteriorcruciate ligament or meniscectomy"9 leads tothe appearance of osteoarthritic lesions. It hasbeen suggested that the onset of osteoarthritisis related to increased turnover of proteo-glycans.' Besides erosion and fibrillation in thetibial joints, meniscectomy causes enhancedextractability of proteoglycans in the femoralhead, which was thought to be due to thedeficiency of hyaluronan in the tissue.'0

In our earlier experiments with animalmodels we have studied the role of immobil-isation, remobilisation, and running exerciseon the health and maintenance of normalarticular cartilage." These studies showed thatthe effects of immobilisation varied in differentarticular areas. In general, immobilisation leadsto a remarkable local loss of proteoglycansfrom the matrix. Remobilisation caused theconcentrations to revert to normal but notin all of the joint surfaces.'2 13 On the otherhand, moderate running (4 km/day) increasedproteoglycan content and enhanced the bio-mechanical properties of the cartilage.14 15These changes, regarded as positive, werepartially lost in another running experiment(20 km/day), apparently owing to the higherdegree of local strain and weight bearing.'6Whether a long distance running exercise

(40 km/day, 15 weeks) affects the rate ofproteoglycan synthesis was studied here in thecanine femoral head. The incorporation rate ofsulphate into articular cartilage was measuredby in vitro labelling. The distribution ofsynthesis within the tissue was assessed byquantitative autoradiography. The specimenwas divided into a weight bearing (anteriorpart) and a less weight bearing (posterior part)area to study possible differences due to weightbearing. The structure of the proteoglycanmolecules was further characterised by bio-chemical analyses.

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Materials and methodsANIMALS

Female beagle dogs of pure breed wereprovided by the National Laboratory AnimalCentre (Kuopio, Finland) and by ShamrockLtd (Hereford, UK). The dogs lived instandard cages with a floor area of 0 9x 1-2 m(height 77 cm), according to National Instituteof Health recommendations. For each experi-mental animal (n= 10) a control of the same agewas chosen from the same litter.

EXERCISE PROTOCOL

The dogs started the running exercise at theage of 15 weeks on a treadmill adjusted to 15°uphill inclination. The daily running distancewas increased gradually, so that at the ageof 55 weeks it was 40 km/day at a speed of6 km/h (fig 1). The animals were killed at theage of 70 weeks.

SAMPLE PREPARATION

The dogs were killed with an overdose ofbarbiturate and the femoral head was dissectedfree of muscles with special care taken not towound the cartilage surface. A dentist's drillequipped with two cutting discs and a 1 mmspacer was used to produce three cartilageslices. The slices were divided into anterior(weight bearing) and posterior (less weightbearing) sectors by sawing a cutting linehorizontally at the superior part of the joint.The sampling method was chosen based onreported results of the directions of measuredmaximum hip joint forces in dogs withinstrumented endoprosthesis.17 The citedreference showed that the main loads acted atmedioventral directions. The samples wereremoved from the femoral head, placed intoculture dishes filled with ice cold Eagle'smedium (EMEM, Flow Laboratories, Irvine,Scotland), supplemented with Earle's salts andantibiotics (100 U/ml penicillin, 100 ,ug/mlstreptomycin), and transported to the tissueculture laboratory on an ice bath.

Running schedule

At maximum 40 km/day150 uphill inclination

0

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Figure 1 Schematic presentation of the running programme. After an adaptation periodthe 10 dogs ran daily (five days a week) 40 km/day with 15° uphill inclination for15 weeks.

CHEMICAL ANALYSIS

Proteoglycans were quantified as uronic acid"8and elution of proteoglycans or glycosamino-glycans in chromatographic runs was assayedby the Safranin 0 precipitation method'9 andits autoradiographic application to quantifymacromolecular, radiolabelled glycosamino-glycans.20 Concentration of DNA was deter-mined from a proteinase K digest with calfthymus DNA as the standard.2'

INCORPORATION OF 35S-SULPHATEOne batch of 35S-sulphate was diluted and usedthrough the whole experiment (AmershamInternational, Little Chalfont, UK). The slicesof explants were transferred into ice cold, freshEMEM supplemented with Earle's salts andantibiotics. The tissue was preincubated forone hour at 4°C to ensure even distribution ofthe radioactive isotope precursor (1 -8 MBq/ml) in tissue before labelling at 37°C in a 5%CO2 atmosphere for four hours. Aliquots weretaken to measure the radioactivity of thelabelling medium. The synthetic activity wasstopped by replacing the medium with ice coldphosphate buffered saline. One tissue block,containing the underlying bone, was cut fromeach sample for quantitative autoradiographyand cartilage from the rest of the tissue (formeasurement of sulphate incorporation) wasdissected free from the bone and its wet weightwas determined.

QUANTITATIVE AUTORADIOGRAPHY OF TISSUE

SECTIONS

In vitro labelled tissue blocks (about 1 mm3)were prepared for the autoradiography asdescribed earlier22 modified by an additionalstep for decalcification (4 13% EDTA in 0-1 Mphosphate buffer, pH 7*4, for two days at 4°C).The grain density in developed tissue sectionswas assessed by image analysis. A colour CCDvideo camera WV-CD 132 (Panasonic, with574X581 pixel resolution) was connected toa Leitz microscope (Wetzlar, Germany) and40 images (total magnification X 1730 on themonitor) captured by QuickCapture DT 2255frame grabber (Data Translation, Marlboro,MA) were averaged by image analysis software(Image; public domain software by WayneRasband, NIH) on a Macintosh fx micro-computer (Apple, Cupertino, CA) to producethe primary image. The grains concentrated inthe centres of cells had to be thresholded intoa separate binary image as the succeedingfiltering excluded them. The primary imagewas then smoothed by a median filter,convoluted by a 5 X 5 high pass filter, anderoded before thresholding into anotherbinary image. These binary images weresummed by a logical OR operator to givethe final image showing the pixels coveredby grains. Each microscopical view wasdivided into three fields of the same size(107 ,um width X 34 ,um height respectively)and the relative proportion of total grain areain each field was counted. Consecutive views(four to five) starting from the superficial zone

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Effects of long term running on articular cartilage

and ending in the deep zone were captured andquantified. For each animal, six sections wereanalysed.

MEASUREMENT OF SULPHATE INCORPORATIONIncorporation rates were estimated fromproteinase K (type XXVIII, Sigma, St Louis,MO) digests of cartilage. Tissue pieces weredigested for 17 hours at 60°C by 0 05% pro-teinase K in 0 1 M phosphate buffer (pH 7 4),10 mM EDTA included. Aliquots of digestswere eluted through Sephadex G-25 gel fil-tration columns (PD-10, Pharmacia, Uppsala,Sweden) to remove unincorporated sulphateprecursor and radioactivity was measured byliquid scintillation (LKB, Bromma, Sweden) induplicate aliquots from PD-10 eluates andfrom medium samples dissolved in a watersoluble scintillation cocktail (OptiPhase,Pharmacia, Turku, Finland).

SULPHATE INCORPORATION INTO KERATAN

SULPHATE

The sulphate incorporation into keratansulphate was estimated by gel chromatographicseparation of glycosaminoglycan chains anddisaccharides produced after specific cleavageof chondroitin sulphate by chondroitinaseABC. Proteinase K digested samples, separ-ately pooled in each group, were transferredinto deionised water by PD-10 desaltingcolumns and reduced with alkaline boro-hydride (1 M NaBH4 in 0 05 M NaOH for 17hours at 45°C). The samples were neutralised,lyophilised, and digested for six hours bychondroitinase ABC (Seikakagu Co, Tokyo,Japan) in 0G1 M TRIS-acetate, pH 8-0. Theenzyme was heat denatured, the sampleswere chromatographed on a Sephacryl S-300column (0 5X20 cm, eluted with 0G1 Msodium phosphate, pH 7 0, 0 5% CHAPS,0-15 M NaCl, and 10 mM Na2SO4) and thefractions were analysed by liquid scintillationand Safranin 0 to determine the radioactivityin the macromolecular keratan sulphate anddisaccharides produced from chondroitinsulphate.

EXTRACTION OF PROTEOGLYCANSCartilage was sliced into small pieces andproteoglycans were extracted with 4 M GuCl(Fluka, Buchs, Switzerland) in 50 mM sodiumacetate, pH 5-8, including the bacterial growthand protease inhibitors: 10 mM disodiumEDTA (Sigma), 100 mM E-amino-n-caproicacid (Sigma), 5 mM benzamidine HCI (Sigma),and 0-02% sodium azide (w/v; Merck,Darmstadt, Germany). Extraction was con-tinued for 60 hours.The non-extractable fraction was digested

by 0-05% proteinase K as described andtransferred into deionised water by a desaltingcolumn and the residual uronic acid contentwas measured. The samples in each group werepooled and the relative glycosaminoglycanchain lengths were estimated by SephacrylS-300 gel chromatography after keratanase and

chondroitinase ABC digestions as describedlater.

SODIUM DODECYL SULPHATE (SDS) AGAROSEGEL ELECTROPHORESIS OF THE

PROTEOGLYCANS

Extracted proteoglycans were transferred intoassociative conditions by PD-10 gel chromato-graphy (50 mM sodium acetate, pH 5 8,including the same inhibitors as describedpreviously) and precipitated in 75% ethanol.Each sample was run in duplicate by agaroseelectrophoresis as described earlier.'3 Relativemobilities were compared with a chondroitinsulphate standard (from shark cartilage;Sigma) and bovine articular cartilage Al pro-teoglycan, applied into the same gel.The fixed and stained gels were dried on

Gel-Bond and scanned with a grey-scalescanner (256 shades of grey) connected to aMacintosh fx microcomputer (Apple). Thescanned images of the stained gels werequantified by image analysis software (Image).Autoradiography films exposed to gels for twoweeks were developed for five minutes (DL-9,Eastman Kodak, Rochester, NY), washed fortwo minutes in water, then fixed for 11 minutes(AL-4, Eastman Kodak) followed by a washunder tap water. The films were then scannedand mean film density of the lanes wasmeasured by image analysis.

SEPHACRYL s-500 GEL FILTRATION

CHROMATOGRAPHY

Extracted proteoglycans in each group werecombined and triplicate samples (500 1d)were applied to a Sephacryl S-500 column(1 X 30 cm) equilibrated with extraction buffer.The column was eluted with extraction buffer(20 ml/h) and fractions were analysed forproteoglycans.

ASSOCIATIVE SEPHACRYL s-1000 GEL

FILTRATION CHROMATOGRAPHY

Aliquots of the pools of proteoglycans (100 ,ugglycosaminoglycan) were transferred into0 05 M Na2PO3, pH 7-0, including 0-15 MNaCl, 0 5% CHAPS, 10 mM Na2SO4 andprotease inhibitors by a PD-10 column, andincubated with 2% hyaluronan (w/w of totaluronic acid, Healon, Pharmacia) overnight at4°C. Triplicate Sephacryl S-1000 chromato-graphy (1 cmX30 cm column) was performedfor each sample to estimate the proportion ofproteoglycans able to reaggregate with hya-luronan. The flow rate was 20 ml/hour andfractions (500 ptl) were analysed for proteo-glycans and radioactivity.

RELATIVE GLYCOSAMINOGLYCAN CHAINLENGTHSThe relative chain lengths of glycosamino-glycans remaining after keratanase and chon-droitinase ABC treatments were compared bySephacryl S-300 gel chromatography. Residualand GuCl extracted glycosaminoglycans were

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studied separately. The extracts and proteinaseK digested residues were desalted, lyophilised,and reduced by borohydride as describedearlier. Specific enzyme digestions were thenperformed after another desalting andlyophilisation. A Sephacryl S-300 gel column(1X30 cm) was eluted with 01 M Na-phosphate, pH 7 0, 0/5% CHAPS, 0-15 MNaCl, and 10 mM Na2SO4.

CHONDROITIN SULPHATE DISACCHARIDE

ISOMERS

Extracted proteoglycan samples (10 ,ug ofuronic acid) were digested with chondroitinaseAC II (from Arthrobacter aurescens, EC 4.2.2.5;Seikagaku Co). Chondroitin sulphate isomersand hyaluronic acid were separated by thinlayer chromatography23 and molar proportionsmeasured from scans obtained by a ShimadzuCS-930 chromatogram spectrophotometer(Kyoto, Japan) at 232 nm. The proportion oflabelled chondroitin 6-sulphate and chon-droitin 4-sulphate were estimated from Hyper-film p-max film (Amersham Int) after a twoweek exposure. The digitised image of the filmwas analysed by Image software.

GLUCOSAMINE: GALACTOSAMINE RATIO

The rest of the GuCl extracted sample poolswere hydrolysed in 2 M HCl for 17 hoursat 1 03°C to produce an optimal yield ofhexosamines from glycosaminoglycan chains.24The acid was evaporated by air flow andinternal standard (deoxyglucose) and waterwas added, dissolving the hydrolysate into2 ml. The hydrolysates were analysed on aCarbopak PAl column by a Dionex HPLCsystem equipped with a pulsed amperometricdetector and computer interface (Dionex,Sunnyvale, CA). The monosaccharides wereseparated by isocratic elution with 16 mMNaOH (flow 1-0 ml/minute, run time 20minutes).

STATISTICAL ANALYSIS

The statistical significance between the run-ner and control groups was analysed byWilcoxon's matched pairs signed ranks test.

ResultsNo difference in the body weights was foundbetween control (mean 9 9 (SD 1-0) kg) andexperimental groups (9*7 (1*1) kg), althoughfood consumption of the runners was 50%higher than that of controls. The running itself

was considered aerobic as the pulse rate of therunners did not exceed 130 beats/min.25The articular surface of the femoral head was

checked for the quality and possible damagecaused by the running exercise. No macro-scopic injury, osteophytes, or fibrillation couldbe seen in any of the animals. Concentrationof DNA varied a lot in individual specimens(table 1), as noted earlier,26 but the meanvalues were similar in every group.The rate of sulphate incorporation (pmol/mg

wet weight/hour) was a little higher in theweight bearing area (fig 2), but only slightdifferences (not statistically significant) werenoticed between groups. The rates were ingood agreement with those obtained earlier incanine knee joints.8 Uronic acid concentrationswere similar to those reported previously.27Statistically significant changes were found inthe posterior area of the femoral head (lessweight bearing, table 1). A loss of extractableproteoglycans in the runner group (p<0.02)was compensated for by an increase in proteo-glycans resistant to extraction (p<0 05). Astatistically significant decrease in theextraction percentage was noted in the lessweight bearing area also (pz,005). Runningapparently raised non-extractable proteo-glycans in the less weight bearing area close tothat of the weight bearing area.

Quantitative autoradiography was usedto obtain the distribution profile of newly

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Figure 2 Sulphate incorporation ratesfrom individualdogs (nine in each group) after one year of running exercise.Posterior (less weight bearing (LWB)) and anterior (weightbearing (WB)) areas were studied separately and the resultswere correctedfor tissue wet weight. The mean of the ratesin each group is shown by a horizontal line.

Table1

Mean (SD) of the chemical measurements and incorporation rates in the low weight bearing (LWB) and highweight bearing (WB) areas of the canine femoral head (n=8-10)

LWB control WB control LWB runner WB runner

,tg DNA/mg wet weight 0 49 (0-24) 0 50 (0-21) 0-48 (0-28) 0-49 (0 26)Incorporation rate (pmol S04/mg/h) 14-1 (5-7) 17-6 (6-8) 15-8 (6 5) 16-8 (5 2)Uronic acid/mg wet weight (extractable) 10-1 (2 8) 7-3 (3-1) 9-4 (2.7)** 9-0 (1-9)Uronic acid/mg wet weight (residual) 2-0 (0 4) 2-4 (1-3) 2-7 (0-6)* 2-6 (1-4)Extractability (%) 83-1 (3-9) 75-4 (4 2) 78-5 (4 2)* 77.7 (6-5)

*po0-05, **ps0o02, Wilcoxon's matched pairs signed ranks test (control and runner groups from each area were testedseparately).

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Surface Tidemark (representing keratan sulphate) was foundin the runner group (table 2). The results

-0- LWB control thus suggest a reduced synthesis and content*LWBrunner /|>t

of keratan sulphate (relative to chondroitinsulphate) after long term running.The size distribution of proteoglycan mono-

mers of individual specimens was examinedby SDS agarose gel electrophoresis and frompooled samples by Sephacryl S-500 gelchromatography under dissociative conditions.Toluidine blue stained agarose gel showed two

slowly migrating bands consisting of the largeproteoglycans and a higher mobility band ofsmall proteoglycans at Rf 0-8 in all groups(fig 4). Table 3 presents the percentage pro-

WB control portions of the large and small proteoglycans*WB runner h j t determined from toluidine blue stained gels. A

different pattern was produced by autoradio-graphy of the same gels (fig 4). Most of thenewly synthesised proteoglycans were in theslower migrating band of the large proteo-glycans (fig 4), and a much higher proportionof the radioactive precursor was incorporatedinto the small proteoglycans, as would beexpected on their amount present in the tissue,suggesting a relatively higher turnover rate forsmall proteoglycans. Running slightly dim-o 2 4 6 8 10 12 14 inished the percentage of radioactive labelling

Field of the small proteoglycans (NS).... . . .. . . . ~~~~~Sephacryl S-500 gel chromatography ofDistribution of sulphate incorporation in the posterior (less weight bearing Saplave ghls lroflsor otand anterior (weight bearing (WB)) areas analysed by quantitative samples gave roughly similar profiles for both

Pography. Relative grain areas in consecutive fields extendingfrom cartilage surface groups. As a whole, the results were in goodark were measured by image analysis and the percentage distribution profiles were agreement with those acquired from agarose

gel electrophoresis. The differences were smalland no significant increase in the size of proteo-

synthesised proteoglycans in the tissue. Suc- glycans of the runners could be noticed.cessive fields from surface to tidemark were The proportion of proteoglycans able toanalysed and the results were normalised to form aggregates was studied by Sephacrylshow the labelling density of each field in S-1000 gel chromatography with excess

relation to the sum of all fields from surface to hyaluronan added to proteoglycan samples.tidemark. As seen from the profiles (fig 3) the Samples pooled separately for each group gavehighest labelling density was noticed in the similar results in triplicate runs (fig 5). Somedeep zone of the cartilage. After long distance 50-60% of the tissue proteoglycans were ablerunning the synthetic activity in the inter- to aggregate, whereas only about 25% of themediate zone was slightly increased in both less newly synthesised did aggregate. The groupsweight bearing and weight bearing areas, did not differ from each other in their aggre-although the change did not reach statistical gation properties.significance. The relative chain lengths of glycosamino-

Specific enzymatic digestions were used to glycans were compared by Sephacryl S-300 gelestimate the percentage of sulphate incor- chromatography of keratanase and chon-porated into keratan sulphate of the proteo- droitinase ABC digests (fig 6). Safranin 0glycans. Table 2 shows that a decrease in the precipitation was used to quantify therelative amount of newly synthesised keratan distribution of resident proteoglycans and thesulphate to chondroitin or dermatan sulphate radiolabel of both the GuCl extracted andwas detected by Sephacryl S-300 gel chromato- residual glycosaminoglycans. In the weightgraphy as a consequence of running exercise. bearing pool, extracted proteoglycans of theThe ratio of glucosamine to galactosamine in runners had somewhat larger chondroitinthe extracted proteoglycans was similar in theweight bearing and less weight bearing areas ofcontrol animals. A decrease in glucosamine

sulphate chains than controls whereas in lessweight bearing extracts the elution ofchondroitin sulphate of the runner group

Table 2 Percentage proportions of sulphate incorporation into chondroitin and dermatan sulphate and keratan sulphateestimated by Sephacryl S-300 gel chromatography after enzymatic digestions by keratanase and molar ratio ofglucosamineto galactosamine in extracted proteoglycans


Chondroitin+dermatan sulphate (%/6) 74-1 80 6 76-6 82-2Keratan sulphate/chondroitin sulphate ratio 0-35 0-24 0 30 0-22Glucosamine/galactosamine ratio 0-28 0-29 0-26 0-24

LWB=less weight bearing; WB=weight bearing.

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proteoglycans, especially in the less weightbearing samples (table 4), but the changewas not statistically significant. The valuesobtained for the hyaluronan percentage of totalglycosaminoglycans were in good agreementwith an earlier estimate from canine kneejoints.28

Figure 4 Proteoglycans analysed by sodium dodecyl sulphate (SDS) agarose gelelectrophoresis. Gels were stained with toluidine blue and an autoradiographyfilm wasexposed to the radioactivity of the gelfor two weeks. A representative densitometric graph isshown for toluidine blue and autoradiography from the same sample lane. The area coveredby slowly migrating large proteoglycans (PG-Is) is presented in the densitogram, and itsmigration position is shown in the gel. The position of the small proteoglycans (small PGs)is also shown (C=control and R=runner animal, L WB=posterior, less weight bearing areaand WB=anterior, weight bearing area).

Table 3 The results ofSDS agarose gel electrophoresis ofproteoglycans


Toluidine blue staining:Proteoglycan proportions:

Large proteoglycans 98-8 (0-6) 98-8 (0 6) 98-8 (0 2) 98-7 (0 9)Small proteoglycans 1-2 (0 6) 1.2 (0 6) 1-2 (0-2) 1-3 (0 9)

Large proteoglycans% PG-Is 66 2 (6 6) 68-2 (5-9) 64 8 (5 7) 70-3 (5 8)

Autoradiography:Proteoglycan proportions:

Large proteoglycans 91-9 (2 8) 92-1 (1-8) 92-7 (4.2) 93-6 (2 4)Small proteoglycans 8-1 (2 8) 7-9 (1-8) 7-3 (4-2) 6-4 (2 4)

Large proteoglycans:% PG-Is 73-1 (7 3) 74 0 (7 7) 75-1 (8 0) 75-7 (7-0)

In each group, all samples (n=10) were run in agarose gels, stained with toluidine blue andautoradiography films were exposed to the gels. The definition of PG-I is explained in fig 4.

remained much the same as the control.Keratan sulphate chains were heterogeneous incontrol animals, but changed into a morehomogeneous population after the runningexercise. Residual glycosaminoglycans of bothgroups were similar in their elution profiles.The proportions of chondroitin sulphate

isomers and hyaluronan disaccharides wereexamined by thin layer chromatography.23 Nosignificant alterations were noticed in any ofthe disaccharides (table 4). The percentage ofhyaluronan in the residual glycosaminoglycansin control and runner groups was about twiceas high as that found in GuCl extracted proteo-glycans.13 This supports our own findings thata large proportion of the hyaluronan remainsin the tissue after GuCl extraction of 20 pumthick sections (unpublished data). The ratioof chondroitin 6-sulphate to chondroitin4-sulphate was higher in the newly synthesised

DiscussionThe adaptation of the femoral head articularcartilage of young beagles to long distancerunning was remarkably good as only minorbiochemical changes were found in thestructure and metabolism of the proteoglycansof the extracellular matrix after one year oftraining with the final daily running distance of40 km with 150 uphill inclination. Thesefindings are by contrast with an earlier reportby Vasan that showed fibrillation and wear ofthe canine femoral head after six to eight miles(9-6-12 8 km) of daily running for eightmonths (one hour a day) with 200 uphillinclination.3 A different running programme(faster speed and steeper inclination ofthe treadmill) possibly explains the severedegenerative alterations produced in hisexperiment.3 The breed and especially the ageof the dogs (not reported by Vasan) may alsosignificantly affect the response of cartilage toexercise.The chemical composition and tensile

properties vary according to depth29-3' andtopographical site of the articular carti-lage.26 32-34 On the other hand, concentrationof collagen seems to be relatively constant atdifferent depths of the cartilage.35 The tensilestiffness of cartilage correlates well with thehydroxyproline: hexosamine ratio.36 Inden-tation tests have indicated that the highestcompressive stiffness in the femoral head ofhumans resides in a region complementary tothe loaded area of acetabulum,37 whereas inquadruped animals weight bearing is focusedmore to the anterior part of the caput.Y8

Different responses in proteoglycan contentafter the running were apparent in the lessweight bearing and weight bearing areas.Running increased the amount of GuClresistant glycosaminoglycans particularly in theless weight bearing tissue. A similar increaseof GuCl resistant glycosaminoglycans wasreported in rabbits after both physical exerciseand increased weight bearing,39-4' whereasdecreased weight bearing in a splinted legclearly reduced non-extractable glycosamino-glycans.4' This could indicate the presence ofa stiffer collagen network after increasedweight bearing spatially entrapping proteo-glycans in the collagen network. Also, in-creased amounts of type IX collagen, which iscovalently linked to collagen type II,42 can beinvolved in the accumulation of residualglycosaminoglycans, as type IX collagencontains chondroitin or dermatan sulphate.43Conversely, in osteoarthritis the amount ofnon-extractable glycosaminoglycans was dim-inished.7 An intact collagen network is crucialto the function of cartilage. When exposed toleucocyte elastase (attacking the non-helical

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Figure S Aggregation capacity of extracted proteoglycans (PGs) analysed on Sephacryl S-1000 gelfiltration column.Proteoglycans were allowed toform aggregates in the presence ofexogenous hyaluronan (2% wlw of uronic acid) overnightat 4°C. Fractions were analysedfor total and newly synthesised proteoglycans by the safranin 0 precipitation methods.LWB=less weight bearing; WB=weight bearing.

Chondroitin sulphateLWB

Safranin 0

Keratan sulphate

Safranin O

Autoradiography Autoradiography

00 0.2 04 06 08 1.0 00 02 04 0-6 0-8 1 0

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Autoradiography Autoradiography

0-0 0-2 0-4 0-6 0-8 1-0 0.0 0-2 0-4 0-6 0-8 1-0

Figure 6 Glycosaminoglycan (GAG) chain size distribution of chondroitin or dermatansulphate and keratan sulphate of the extracted proteoglycans. Proteoglycan samples were

reduced by alkaline borohydride and digested separately by keratanase and chondroitinaseABC respectively before analysis on Sephacryl S-300. Glycosaminoglycans in the fractionswere determined by the safranin 0 precipitation method. (0) represent control and(0) runner group. L WB=less weight bearing; WBD=weight bearing.

terminal regions of type II and possibly typeIX collagen) reduction of the elastic stiffness ofcartilage was caused."

In this study, the highest synthetic activity incontrol animals was in the deep layers of thecartilage revealed by quantitative autoradio-graphy and an increase in the intermediatezone was noticed after running exercise. Asimilar statistically significant change in thedistribution of proteoglycan synthesis has beenfound in young guinea pigs after 18 weeks oftreadmill running exercise (Hyttinen et al,unpublished data). In our earlier in vitroexperiments, cyclic mechanical compressionpromoted sulphate incorporation by cartilageexplant in the area under loading, an increaselocalised mainly in the intermediate zone.45The ratio of glucosamine to galactosamine is

a good indicator of the proportion of keratanand chondroitin sulphates, even in crudeextracts. A decreased ratio in the runner groupsuggested a relative increase of chondroitinsulphate, a trend similar to the results obtainedin sheep by increased weight bearing.4" Acontradictory finding with increased keratansulphate after enhanced loading was made inrabbits.4' In Sephacryl S-300 chromatographyslightly longer chondroitin sulphate chainswere noticed in the runner group, which maywell explain the trend towards an increase inthe chondroitin:keratan sulphate ratio. Inexperimental canine osteoarthritis, proteo-glycans containing more chondroitin sulphaterelative to keratan sulphate appeared in thetissue.7 Whether the change in keratan:chondroitin sulphate ratio, noticed in thisexperiment, reflects an increased stress andweight bearing due to the inclination of therunning or an early osteoarthritic processremains an open question.

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Lammi, Hdkkinen, Parkkinen, Hyttinen, _Jortikka, Helminen, Tammi

Table 4 Percentage proportions of chondroitin sulphate isomer and hyaluronandisaccharides produced by chondroitinase AC and separated by thin layer chromatography(TLC). The ratio of radioactively labelled sulphated disaccharides was obtained by anexposure ofan autoradiography film to the TLC plate

LWB control WB control LWB rlunner WB runner

UV absorbing disaccharides:CS-6 61-6 (7 3) 62-8 (6 6) 63-0 (5 3) 60-6 (4 9)CS-4 18-6 (2 2) 21 0 (3 8) 20 4 (2-9) 22 2 (2 5)CS-0 8.7 (3 5) 8-5 (3-1) 7 5 (4-2) 9-2 (3 6)HA 11.1 (4 6) 7-7 (3 8) 9.1 (2 9) 8-0 (2 4)CS-6/CS-4 3 4 (0 6) 3 1 (0 7) 3 1 (0 5) 2-8 (0 4)

Radiolabelled disaccharides:CS-6/CS-4 4 4 (1 3) 3 4 (1 2) 4-6 (1-1) 3.8 (1-0)

There was no other evidence of osteo-

arthritic alterations due to the runningprogramme. The lower extractability ofproteoglycans and unchanged incorporationrates rather suggested a tendency opposite to

degeneration. ' '° 47 We suggest that theenhanced load distributed the stress over a

larger articular surface area and initiated an

adaptation reaction particularly in the pre-

viously less weight bearing sites. The findingsby Volpi and Katz38 suggest that cartilage hasthe capacity to respond to various mechanico-chemical histories, as the potential of super-

ficial collagen fibrils to "hyperswell" was

noticed to develop only in the anterior, weightbearing region of bovine femoral heads inresponse to different loading environments.The general shape of the hip joint is

congruent, which minimises the local impacts.With the same running programme, the more

incongruent femoral condyles of the kneeshowed a depletion of proteoglycans in thesuperficial zone after long distance running(Arokoski et al, unpublished data). Thisemphasises the importance of a local response

and the congruency of the joint surface in themaintenance of the normal cartilage. Theresults also suggest that congruency, bycontrolling the strength of local impacts, is an

important factor in determining the response

of cartilage to exercise.

We are indebted to Mrs Eija Antikainen for preparing thephotographs, to Mrs Eija Rahunen for preparing autoradio-graphy sections, and to Mrs Elma Sorsa for the skilful laboratoryassistance. This work was supported by a grant from the FinnishCultural Foundation and its Fund of the North Savo, theResearch and Science Foundation of Farmos, the PauloFoundation, the Academy of Finland, and the Finnish ResearchCouncil for Physical Education and Sports, Ministry ofEducation.

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to young beagles - a eular & bmj rheumatology journal

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