Annals of the Rheumatic Diseases 1995; 54: 831-835

Proteoglycan depletion and size reduction inlesions of early grade chondromalacia of thepatella

Urho Vaatainen, Tomi Hakkinen, Ilkka Kiviranta, Heikki Jaroma, Ritva Inkinen,Markku Tammi

AbstractObjective-To determine the content andmolecular size of proteoglycans (PGs)in patellar chondromalacia (CM) andcontrol cartilages as a first step in investi-gating the role ofmatrix alterations in thepathogenesis of this disease.Methods-Chondromalacia tissue from 10patients was removed with a surgicalknife. Using identical techniques, ap-parently healthy cartilage of the same sitewas obtained from 10 age matchedcadavers (mean age 31 years in bothgroups). Additional pathological cartilagewas collected from 67 patients with gradesII-IV CM (classified according to Outer-bridge) using a motorised shaver underarthroscopic control. The shaved cartilagechips were collected with a dense net fromthe irmrgation fluid of the shaver. Thecontent of tissue PGs was determined bySafranin 0 precipitation or uronic acidcontent, and the molecular size bymobility on agarose gel electrophoresis.Results-The mean PG content of the CMtissue sampled with a knife was dramatic-ally reduced, being only 15% of that incontrols. The cartilage chips collectedfrom shaving operations of grades II, III,and IV CM showed a decreasing PGcontent: 9%, 5%, and 1% of controls,respectively. Electrophoretic analysis ofPGs extracted with guanidium chloridefrom the shaved tissue samples suggesteda significantly reduced size ofaggrecans inthe mild (grade II) lesions.Conclusion-These data show that thereis already a dramatic and progressivedepletion of PGs in CM grade II lesions.This explains the softening of cartilage, atypical finding in the arthroscopicexamination of CM. The PG size re-duction observed in grade II implicatesproteolytic attack as a factor in the patho-genesis ofCM.

(Ann Rheum Dis 1995; 54: 831-835)

Softening of articular cartilage in the patella,frequently described as chondropathy orchondromalacia of the patella, varies from asoft blister to total destruction of the cartilage.Although chondromalacia of the patella is acommon phenomenon, its aetiology is unclear;mechanical factors have been consideredimportant, however.'

The matrix of cartilage consists of a colla-genous network and interfibrillar aggregatesformed by proteoglycans (PGs) with hyaluro-nan. PGs are extremely hydrophilic and adsorbwater into the compartment ramified by thecollagenous network. As a consequence ofwater absorption, the matrix remains turgid,creating the resilient nature of the cartilage.IAPGs are therefore essential for the compressivestiffness of cartilage matrix, while the collagenfibres are responsible for the tensile strength ofarticular cartilage.3The chondrocytes synthesise and secrete

PGs into the matrix and, in conditions ofnormal turnover, degraded PGs are releasedfrom articular cartilage into synovial fluid.5-7 Inexperimental osteoarthritis, the total content ofPGs decreases despite their increased bio-synthesis, indicating an abnormally rapid lossof PGs.>'2 Focal loss of PGs has been foundin canine patellar cartilage after acute trans-articular patellofemoral loading.3 Reducedconcentrations ofPG also occur in the articularcartilage after immobilisation of the canineknee.'4 1' With aging and often in osteo-arthritis, the electrophoretic mobility of PGmonomers is increased, indicative of areduction in their molecular size.'6 17However, little is known about the PGs in

chondromalacia of the patella, though it is themost frequently diagnosed cartilage lesion inthe knee joint. The purpose of the presentstudy was to examine the PG content ofchondromalacia lesions of different grades, andto begin to characterise the structuralalterations of PGs in the diseased matrix.

Materials and methodsThe study was approved by the ethics com-mittee of the Kuopio University Hospital andall the subjects gave their informed consent totake part in the study.

CARTILAGE SAMPLESPatients with anterior knee pain were examinedclinically and knee joint roentgenograms weretaken. Those patients giving a strong suspicionof a patellofemoral problem, but without radio-logical signs of osteoarthritis, were recruited tothe study. Athroscopy of the knee was per-formed in 130 patients, and 77 showedchondromalacia ofthe patella (CM) as the onlyintra-articular cartilage lesion. At operation,the fibrillated cartilage of the CM lesion was

Department ofSurgery,Kuopio UniversityHospital,Kuopio, FinlandU VaatainenI KivirantaH JaromaDepartment ofAnatomy,University ofKuopio,Kuopio, FinlandT HakkinenR InkinenM TammiCorrespondence to:Dr Urho Vaatainen,Department of Surgery,Kuopio University Hospital,P.O. Box 1777,FIN-70211 Kuopio,Finland.Accepted for publication23 May 1995

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shaved smooth to reduce pain, and the shavedtissue taken for PG analysis. Sixty seven ofthese cartilage samples were taken arthro-scopically, and 10 were taken during openarthrotomy with a knife. At arthroscopy, CMlesions were classified into four gradesaccording to Outerbridge. 8 We recruited to thestudy only those patients with chondromalaciaof the patella who showed intact cartilagesurfaces in the other areas of the knee joint.

Cartilage tissue from CM lesions of 10patients (six women, four men; mean age 31years, range 21-45) was removed in as intacta condition as possible, using a surgical knifeand cutting perpendicularly to the articularsurface down to the subchondral bone, whichwas left intact. Open surgery in these patientswas indicated for lateral release in fourpatients, patellar realignment in four, and peri-osteal transplantation in two patients. Usingidentical techniques, apparently healthycartilage from the same sites was obtainedwithin 24 hours after death from 10 agematched cadavers (mean age 31 years, range19-42). Cadavers with known autoimmunediseases, haemophilia, and metabolic disorderswere excluded. Additional pathological carti-lage was collected from 67 patients (mean age35 0 (SD 9 9) years) with grades II-IV CMusing a motorised shaver (Stryker, StrykerEndoscopy, Sunnyvale, USA) with an aggress-ive meniscus cutter blade of 5 mm diameterunder arthroscopic control. Forty eight percentof the chondromalacia lesions were located onmedial facet of the patella, 39% were on thecentral area (diameter of 15 mm), and 13%were located on the lateral facet of the patella.As the cartilage lesion was smoothed, theshaved cartilage tissue was collected with adense cotton net from the irrigation solution(0'9% sodium chloride) leaving the joint. Thesamples were put into one millilitre Eppendorftubes and frozen at -420C.

Bovine knees were obtained from the localabattoir and sampled with a shaver and knifeusing exactly the same technique as for thehuman cartilages.

HISTOLOGY

Cartilage samples were taken for histologyfrom 10 CM samples and from 10 cadavercontrol samples. The tissue was fixed inbuffered formaldehyde solution, dehydratedthrough an alcohol series to xylene andembedded in paraffin. Sections 3 ,um thickwere cut and stained with haematoxylin-eosin.

EXTRACTION OF PROTEOGLYCANSThe wet cartilage samples were weighed.Those from cadavers and those taken frompatients with a knife were sliced with a micro-tome to a thickness of 20 iim at -20°C, andlyophilised. Cartilage samples obtained fromshaving operations in 26 patients in the CMgroup (mean age 35 0 (SD 9 0) years) wereextracted without further cutting.PGs were extracted from cartilage samples of

71 9-406 9 mg wet weight with 1.5 ml of

4 mol/l guanidinium chloride (GuCl, Fluka,Buchs, Switzerland) in 50 mmol/l sodiumacetate, pH 5-8 at 4°C. Disodium-EDTA10 mmol/l (Sigma Chemical Co, St Louis,MO, USA), 100 mmolI/l e-amino-n-caproicacid, 5 mmolIl benzamidine hydrochloride(Sigma), and 0.02% (weight/volume) sodiumazide (Merck, Darmstadt, Germany), wereused as protease and bacterial growthinhibitors.

After extraction, all the samples were trans-ferred into deionised water through a desaltingcolumn (PD-10, Pharmacia, Sweden). PGswere quantitated by Safranin 0 precipitation'9using chondroitin sulphate C (Sigma) as astandard, or by uronic acid determination.20

AGAROSE GEL ELECTROPHORESISEach sample was analysed by electrophoresis inhorizontal, submerged 3 mm thick 0 75%(w/v) agarose slab gels as described pre-viously.2' Relative mobilities were comparedwith a chondroitin sulphate standard (sharkcartilage, Sigma), and with bovine articularcartilage Al PG, electrophoresed in the samegel.The gels were stained with toluidine blue,

dried on a Gel-Bond film (FMC BioProducts,Rockland, USA) and scanned with a tabletopgrey scale scanner (256 shades of grey)connected to a microcomputer (Macintosh II,Apple, Cupertino, Ca, USA). The scannedimages of the gels were analysed for staindensity and graphically averaged by imageanalysis software (Image, public domainsoftware byW Rasband, NIH, USA).

STATISTICAL METHODS

Mann-Whitney U test was applied to test thedifferences between control and chondro-malacia groups. To examine correlations,Pearson's squared correlation test was used.

ResultsKnee joint cartilage surfaces were carefullystudied to exclude as far as possible anyvariance associated with joint diseases otherthan chondromalacia of the patella. If othercartilage lesions were observed at arthroscopy,the samples were not accepted for the study.Patients with irritated plicas were excludedalso.

Control cartilages showed a normal histo-logical appearance (fig 1A). Cartilage changesin chondromalacia included fibrillation ofthe joint surface and erosion of the cartilage(fig 1B-D).

PROTEOGLYCAN CONTENT IN

C0HTONDROMALACIA

The concentration of uronic acid in chondro-malacia cartilage was decreased on average to15% of that in controls (table 1). A largernumber of samples from shaving operationswere used to estimate the relationship betweenPG concentration and grade of lesion. The

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A,.a

I&

Figure 1 Haematoxylin and eosin stained histological sections. A: Control cartilage showing normalfeature of cartilagestructure; B: grade II chondromalacia-superficial zone partly lost; C, D: grades III and IV-superficial zone and part ofthe intermediate zone have been eroded. Original magnification x 10.

decrease in the concentration of uronic acidwas greatest in the most advanced lesions(grade IV), but no statistically significantdifferences between the grades were apparent(table 2).The overall concentration of uronic acid was

smaller in the samples harvested from shavings,compared with those taken with the knife,apparently as a result of swelling of the shavedtissue chips and possibly, also, some extractionof the PGs in the irrigation fluid.

AGAROSE GEL ELECTROPHORESIS OF

PROTEOGLYCANSFigure 2 shows the graphically averaged PGprofiles of control and chondromalacia samples(grades II-IV). The broad peak in all groupscorresponds to aggrecan, which is very hetero-geneous in human articular cartilage.22-24 Thesmaller PGs (decorin and biglycan) migrate at

Table 1 Uronic acid concentration in the patellar cartilageofpatients with chondromalacia (CM) of the patella, andin apparently normal, age matched cadaver controls

Group Sex Uronic acid % of(FIM) (Aglmg wet wt) control

Controls (n = 10) 1/9 3-61 (1-78) 100CM (n = 10) 6/4 0-53 (0 62)*** 15

Values are mean (SD).***p < 0-001, Mann-Whitney U test.

Table 2 Uronic acid concentration (mean (SD)) indifferent grades ofchondromalacia (CM) of the patella,measured in samples harvestedfrom the irrigation fluidduring shaving ofcartilage lesions

Grade of Sex Uronic acidCM (F/M) (gg/mg wet wt)

II 13/12 0 33 (1-06)III 14/16 0-12 (0-14)IV 10/2 0-05 (0-04)

2t g

3

RelativIVilt

22 AC/)

Gradeve mob lit

Figre2 Agrs e lcrphrsso atlg

proteoglycans from grades II-IV ofchondromalacia patellae,(---) and controls (-). Densitograms oftoluidine bluestained gels, expressed as percentage of total stain intensityin a sample. Each line represents a graphic average ofindividual samples. Vertical lines definefive segments forpurposes ofcomparing mobilities (see text and table 3).

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Table 3 Relative mobility (RF) of the proteoglycans from different grades (CMII-CMIV)ofchondromalacia of the patella and controls (C) in agarose gel electrophoresis: fiveprofile segment as shown in figure 1

Group Mobility (RF)

SO-40 >0 40 >0 55 >0 65 >0 78A0 55 -0 65 20 78

C (n = 7) 15 (17) 46 (15) 26 (11) 9 (11) 3 (6)CMII (n = 6) 1 (1)* 20 (13)* 40 (6)* 32 (11)** 7 (6)CMIII (n = 16) 5 (8) 35 (17) 33 (9) 22 (13)* 4 (7)CMIV(n=4) 3(2) 48(8) 31(6) 15(2) 3(6)

Values are percentage (SD).*p < 0 05, **p < 0-01, compared with controls (Mann-Whitney U test).

RF 08-09 in the electrophoretic system used.To allow quantitative comparison of PGmobilities between control and chondro-malacia samples, the profiles were split into thefive segments shown in figure 2. The integratedstain intensity in each segment was calculatedto provide the values shown in table 3. Thedata show a significant decrease in the lowmobility segments (RF <055) in grade IIchondromalacia, and a corresponding signifi-cant increase in the high mobility segments (RF0O55-078). In grade III chondromalacia, thehigh mobility segment (RF 0O65-078) was

significantly increased (table 3). Statisticallysignificant differences of PG mobilitiesbetween controls and grade IV chondromalaciawere not observed.

EFFECT OF SHAVING ON PROTEOGLYCAN SIZE

Electrophoretic analysis of paired cartilagesamples obtained from bovine knee articularcartilage with a knife and shaver revealed no

difference in PG size, thus excluding any arti-ficial PG degradation arising from the shavingprocedure.

DiscussionThese data have demonstrated a severe andprogressive depletion of PGs in chondro-malacia of the patella grade II-IV lesions. Thisexplains the typical finding of a softening ofcartilage in arthroscopic examination of CM.The smaller size of PGs extracted from themild lesions may reflect proteolysis as a factorin the initiation of the lesion.The importance of a sufficient content ofPG

and an intact collagen network to the normalstiffness of articular cartilage has been reportedin several studies.29 27 Our results support thesefindings, and indicate that the softeningregularly noted in chondromalacia is related todisappearance of PGs from the tissue.Softening of articular cartilage is a dominantfeature even of grade I chondromalacia with an

intact cartilage surface, indicating that PG lossis an early phenomenon in the degenerativeprocess.The association between cartilage softening

and PG loss can be used to detect early changesin cartilage matrix by measuring its stiffness(elastic modulus) with a novel indentationdevice applicable during arthroscopy.2" 29 Thisshould provide quantitative follow up measure-

ments on the condition of the cartilage matrixeven in grade I cases.

The turnover of cartilage matrix involves theproteolytic cleavage of the core protein ofaggrecan, the predominant cartilage PG (on amass basis).3" Uncontrolled proteolysis maywell account for the PG loss in chondro-malacia. The present results demonstrated asignificant size reduction in the large PGswhich is in agreement with the concept that, atleast in the initial stages of the disease, thetissue and its aggrecans have been subject tosevere attack by proteinase(s). However, untilmetabolic data are available, it is possible thatlong term inhibition of aggrecan synthesis mayexplain the greatly reduced quantities ofapparently degraded aggrecans present in thechondromalacia patellae lesions.The absence of size reduction of PGs in

severe (grade IV), compared with early,chondromalacia may reflect a complete loss ofthe PGs originally present in the tissue andtheir replacement by molecules newly synthe-sised through activated repair, or moleculesfrom the healthy rim of the lesion. Thisconcept is supported by observations in osteo-arthritic cartilage, which show degradation ofPGs in the initial phase, followed later byreplacement of PGs with larger aggregatingmolecules.3' In addition, large aggrecans arepresent particularly in new, osteophyticcartilage.3'Our findings clearly establish the major role

of the PG matrix in chondromalacia of thepatellae, and warrant further studies on factorsleading to the PG depletion.

This study was supported by grants from the North SavoRegional Fund of the Finnish Cultural Foundation and theFoundation of Finnish Orthopaedics and Traumatology.

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