Stimulation of glycosaminoglycan production and

Annals of the Rheumatic Diseases, 1984; 43, 313-319

Stimulation of glycosaminoglycan production andlysosomal activity of human synovial cells in cultureby low environmental pHB. J. CLARRIS, J. R. E. FRASER, K. D. MUIRDEN, L. P. MALCOLM,M. W. A. HOLMES, AND K. ROGERS

From the University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Victoria 3050,A ustralia

SUMMARY Glycosaminoglycan production, acid hydrolase activity, proliferation, and morphologywere examined in human synovial cells subjected to low environmental pH. The amount and themolecular size of newly synthesised glycosaminoglycan (GAG) were increased without signifi-cant change in the rate of cell proliferation. Lowered pH produced an increase in the size ofcytoplasmic organelles. Some of these possessed ultrastructural features of lysosomes, but otherswere clearly nonlysosomal and were of uncertain identity. Intracellular activity of the lysosomalacid hydrolase N-acetyl-B-glucosaminidase (NAG) was not altered by low pH, but a markedincrease occurred in extracellular NAG activity, indicating enhanced release.

Low pH values have been reported in synovial fluidsfrom inflamed joints of patients with RA.'-3 In themost extreme cases this could enhance the activity offree lysosomal enzymes in synovial fluid, particularlyfor example cathespin B, which is active in the pHrange of 6-7.' A further possibility is that increasedhydrogen ion concentrations in rheumatoid jointsmight induce functional changes in synovial intimalcells, which could account for a number of thepathological features of RA, including synovial pro-liferation, increased activity of lysosomal acid hyd-rolases in synovial tissue56 and fluid,7- 0 and reducedhyaluronic acid viscosity.'" To examine thishypothesis glycosaminoglycan production, lysosomalactivity, and proliferation of human synovial intimalcells were studied in culture conditions designed tosimulate the pH changes which can occur inrheumatoid joints.

Materials and methods

Synovial cells. Primary cultures were prepared bytrypsinisation of synovial tissue within intact joints ofnonarthritic cadaver donors, as described previ-ously."2 Cell lines derived from confluent primarycultures were used in early passages while theirbehaviour was still typical of euploid cells.'3Accepted for publication 10 May 1983.Correspondence to Dr B. J. Clarris.

Culture media and pH control. Eagle's basalmedium (EBM) was used throughout, supplementedwith fetal bovine (FS) or human (HS) serum or both.Sera were heated 56°C for 30 min before use. Mediawere buffered by dilution of 1 M stock solutionsof sterile NaH2PO4, NaHCO3, and the organicbuffers Pipes (piperazine-N,N'-bis(2-ethanesulphonicacid)), Bes (N,N-bis (2-hydroxymethyl)-2-aminoe-thanesulphonic acid), Hepes (N-2-hydroxyethyl-piperazine-N'-ethanesulphonic acid), Tes ((N-trishydroxymethyl)methyl-2-aminoethanesulphonicacid), and Epps (N-2-hydroxyethyl-piperazine prop-anesulphonic acid) in phosphate-free EBM asdescribed by Ceccarini.'4 The pH of complete mediawas adjusted with 1 M NaOH at 22°C, using aRadiometer (Copenhagen) 26 pH meter. pH valuesat 37°C were less than 0 1 pH units higher than at22°C. Final concentrations of the individual organicbuffers ranged from 5 to 20 mM. None of the buffersinhibited the growth of synovial cells at these con-centrations. At the termination of experimentssupernatant media were sealed in plastic tubes andthe final pH was measured at 22°C.EBM and salt solutions (Hanks's balanced salt sol-

utions-HBSS; Dulbecco phosphate bufferedsodium chloride-PBS) were obtained from Com-monwealth Serum Laboratories, and FS and trypsinfrom Flow Laboratories Australasia. Lipid-freebovine serum albumin (BSA) was supplied by the

313

314 Clarris, Fraser, Muirden, Malcolm, Holmes, Rogers

Sigma Chemical Co. and the organic buffers byGrand Island Biological Co.Morphology and ultrastructure. Living cultures

were examined with phase contrast illuminationunder an Olympus PMB6 tissue culture microscope.Cultures were prepared for transmissiom electronmicroscopy (EM) by fixation in situ with 2%glutaraldehyde in 0-2 M cacodylate buffer, pH 7 4,for 10 min at room temperature, after rinsing withPBS. The cells were detached with a soft scraper,centrifuged into pellets, washed in cacodylate bufferwith 7 5% sucrose, and stained with 2% osmiumtetroxide for 30 min at room temperature. The pel-lets were dehydrated stepwise in ethanol, embeddedin Epon, sectioned, and stained with lead citrate.Measurement of cell proliferation. Replicate cul-

tures were prepared in polystyrene culture flasks (25cm2 culture surface area) after dispersal of stock cul-tures with 0 25% trypsin in Ca++/Mg++-free PBS.Each flask received 4 ml EBM + 10% FS containingapproximately 3 x 105 cells. The vessels were sealedand the cells allowed to attach and spread at 37°C for18-24 h. Cultures were allocated to treatments byrandom numbers. Before test media were added onegroup of cultures was selected for counting to obtaininitial cell numbers. The culture vessels were sealedwithout adjustment of CO2 in the atmosphere. Initialand final cell numbers were determined with a Coul-ter counter after complete dispersal with 0 5 % tryp-sin in Ca++/Mg++-free PBS.Assay of NAG. Extracellular activity was

measured in 0 5 ml aliquots of the culture supernat-ants. After the final count 10 ml of the trypsin-cellsuspension was centrifuged and the cells disrupted byfreezing and thawing 3 times. The broken cells weremixed with 001 M tris-HCl, pH 7 0, containing 0 25M sucrose. Intracellular NAG activity was measuredin 0 5 ml of the cellular extracts after clarification bycentrifugation. NAG was assayed colourimetricallyas described previously."5Measurement of glycosaminoglycan (GAG) syn-

thesis. Synovial cultures established in plastic flasks asdescribed above were incubated for 48 h with [3H]-sodium acetate (specific activity 3 8 Ci per mmol;Radiochemical Centre, Amersham) at a final con-centration of 20 ,uCi per ml (0 74 mBq per ml). Thesupernatants were aspirated and 0 25 ml aliquotsincubated with 112 units of pronase for 60 min at37°C. After brief centrifugation 20 ,l samples wereapplied to Whatman 3M chromatography paper andtreated with 0 1 % cetylpyridinium chloride asdescribed by Castor et al. 16 After washing with 0 1 MNaOH [3H]-labelled GAG was measured by liquidscintillation.Molecular weight analysis ofGAG. Culture media

containing [3H]-labelled GAG were centrifuged for

20 min at 37 000 g on a Sorvall RC2-B centrifuge.The supematants were ultracentrifuged at 170 000 gaverage for 20 h at 4°C on caesium chloride gradients(initial density 1 51 g per ml). The bottom one-thirdof the gradient was dialysed exhaustively against0 07 M S0rensen's phosphate buffer, pH 7 2. Themolecular weight distribution of the labelled GAGwas analysed by chromatography in SephacrylS-1000 (Pharmacia Fine Chemicals), with a column70 cm in length and 1 6 cm diameter. The column waseluted with S0rensen's buffer, pH 7-2, at a flow rateof 16 7 ml per h and radioactivity in 2 ml fractionsdetermined by liquid scintillation counting. The elu-tion profile was compared with molecular weightcalibration curve provided by courtesy of Dr K.Granath (Pharmacia AB, Uppsala).

Lactic acid dehydrogenase. This was measured inculture supernatants by the Department ofBiochemistry at Royal Melbourne Hospital.

Results

Stability ofpH. Despite the organic buffers a drift ofup to 0 3 pH units was shown by the difference be-tween initial and final readings (Fig. 1). Single pHvalues shown on subsequent figures therefore repre-sent the final reading only.

Effect ofpH on cell proliferation. Multiplication of

200finm

CO x(h,0

2 100xn =

n(J)61

w

=. I ..

z

±- -F80-

40.

i)

Initial pH 6.807-03728 760Final pH 7 087247-557-74

(a)

6-72 7596-76 768

(b)Fig. 1 Effect ofenvironmental pH on proliferation ofsynovial cells: (a) and (b) are results from separateexperiments. The cells were cultured for 48 h in EBM +10% HS. Vertical bars are mean increases in cell numbersfrom 4 replicate cultures (± SEM). Initial counts were2*73 x 105and 2 94 x IO5cells perculture for (a)and (b)respectively.

oi

Stimulation ofglycosaminoglycan production 315

synovial cell-lines was previously found to beserum-dependent and proportional to serum con-centration."7 In the present study serum concentra-tion was standardised at 10% (v/v). Human serumwas used in preference to FS since the latter contri-butes a high background in the NAG assay. As shownby Fig. 1(a), a variation in pH from 7-74 to 7-08produced no significant alteration in growth rate in48 h. Longer exposures at pH values of approxi-mately 7- 0 usually resulted in stow loss of cells fromthe culture surface and littering of the medium withdebris and floating cells. Further reduction in pHbelow7T0 produced suppression of growth, but asshown by Fig. 1(b) some net increase in cell numbersstill occurred in 2-day experiments at pH values aslow as 6- 8. Without serum, proliferation of synovialcells ceases, but cultures can be maintained for atleast 48 h without appreciable loss of cells by replac-ing serum with BSA at a concentration of 4 mg perml. As shown by Table 1, variation of pH from 7 8 to6 6 in this medium produced no appreciable changein cell numbers over 48 h.GAG synthesis. Decrease in pH produced a highly

significant and reproducible increase in GAG synth-esis (Fig. 2). The GAG produced at low pH was ofexceptionally high molecular weight, with a narrowdistribution of polymer sizes (Fig 3).Morphology and ultrastructure. Macrophage-like

cells present in the primary phase of isolation arestrongly adherent to culture surfaces and do notreadily detach with trypsin. Hence after 2-3 passagessynovial cell lines consist entirely of elongatedfibroblast-like cells. These normally contain manysmall and widely distributed cytoplasmic organelleswhich can be seen by phase contrast microscopy (Fig.4A). Culture at low pH appeared to produce anincrease in the size of the cytoplasmic organelles andclustering of these bodies round the nucleus (Fig.4B), but a marked change was not evident in theirnumber. In living cells there was no evidence ofenlarged vacuoles, unusual nuclear features, or pro-

Table 1 Effect of variation ofpH on the numbers ofsyno-vial cells persisting in serum-free medium* for 48 h

Initial cell numbers Initial Final cell numbers Final(mean + SEM) x 10-3 pH (mean + SEM) pH(n = 4) x lO-3

283-9 3-6 6 50 292-9 4-6 6-616-95 265-9± 11-6 7-197-37 251-5 16-3 7-627 55 28552+- 1-6 7-717-74 288-5 4-4 7-82

* Serum was replaced by BSA at 4 mg/ml.

164

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a

E

aU.(9

U

12L

4-

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Initial pH 690 753Finial pH 671 746

Fig. 2 Effect ofdecreasing pH on GAG production bysynovial cells. The composition of the medium andduration of the experiment were as for Fig. 1.[3H]-sodium acetate was included in the medium at a finalconcentration of20 ,uCi per ml. The assay was asdescribed in 'Materials and methods.' Vertical barsrepresent means ofdeterminations from 3 replicatecultures (+ SEM). The difference is statisticallysignificant (Students t test, p<0-002).

nounced changes of cell shape in response to low pH.Typical electron microscope fields showed highlyelongated cells in control cultures (Fig. SA), whereasin cultures exposed to low pH many less elongated,more macrophage-like cells occurred, often with welldeveloped marginal filipodia (Fig 5B). Lysosomeswere sparse in the controls, and in many fields nonecould be seen, as in Fig. SA. In contrast, the cellsexposed to low pH contained many lysosomal bodiestending to occur in clusters. Hence the large bodiesseen by phase contrast microscopy of living cellsmight represent lysosomal aggregates rather thangrossly swollen individual lysosomes, though themean diameter of single lysosomes was greater than


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0 1b 6 10 10 4

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40 60 80 100 120 140VOLUME (ml)

Fig. 3 Effect ofpH on molecular weight ofGAGproduced by synovial cells. The cells were cultured for48 h in medium containing [3H]-sodium acetate as for Fig. 2at pH 7-46 (0) or 6-71 (0). The supernatants werechromatographed in Sephacryl S-i 000 and analysed asdescribed in 'Materials and methods'.

those occurring in control cells. The phenomenon oflysosomal clustering could also explain the appar-antly unchanged numbers of organelles noted in theliving cells. The synovial cells exposed to low pH alsocontained a number of amorphous, clearly non-lysosomal bodies, with approximately the same sizerange as the lysosomes, which remain unidentified(see example marked U in Fig. 5B).NAG activity. Decrease in pH produced no

significant alteration in intracellular activity ofNAGin synovial cells (Fig. 6).However, NAG levels increased in the supernat-

ant media. NAG activity levels remained unchangedwhen the media were subsequently dialysed to pH7-0 (Fig. 7), indicating that the increase was due toenhanced release from the cells rather than to anyeffect on buffering in the enzyme assay. Similarresponses occurred in nonproliferating cultures pro-duced by substituting BSA (4 mg per ml) for serum(Table 2).LDH. None of the treatments caused appreciable

release of LDH from the cells.

Discussion

Evidence from studies in RA1-3 suggests that tissues

Fig. 4 Morphological effects ofdecreasing pH onsynovial cells. The cultures were photographed after 48 hin medium at (A) pH 7 50, (B) pH 7-08. Note theenlarged cytoplasmic organelles in (B). Negative phasecontrast. (x 444).


F

Fig. 5A Fig. 5B

Fig. 5 Ultrastructure ofsynovial cells culturedfor48 h in medium ofpH (A) 750 (controls) and (B) 7-08 (low pH). Controlcells are elongated, with sparse lysosomes (none evident in (A)). In (B) a macrophage-like cell is shown with enlargedlysosomes (L), mainly in clusters, marginal filipodia (F), and an example of an unidentified amorphous granule (U).(x 14 780).

within the joints in inflammatory arthritis are fre-quently, and perhaps chronically, subjected to highconcentrations of hydrogen ions. In the present studywe found that pH changes similar to those observedin rheumatoid synovial fluids can produce alterationsin the behaviour of synovial intimal cells, includingenhancement of GAG production and activation ofthe lysosomal system. The wide variations in pHalone had no appreciable effects on growth rates, andthe hyperplasia commonly seen in rheumatoidsynovia is therefore likely to be due to other stimuli.

Initially stimulation ofGAG production by low pHwas indicated by a gross and consistent increase in theamount of precipitate formed in low pH media in the'mucin clot' test,'8 which is used as a qualitative indexof GAG production by synovial cells. Quantitativemeasurement by the isotopic procedure demon-strated that the differences were due largely to the

comparitively high molecular weight of GAGreleased from the cells in conditions of low pH.Studies in this and other laboratories indicate that atleast 90% of the GAG secreted by human synovialcells in culture hyaluronic acid (HA).'6 '" A similarresponse to low pH in rat embryonic fibroblasts20supports the view that increase in HA productionmight be a typical response of GAG-producingcells to lower pH. Our findings indicate that thelow viscosity of synovial fluid in rheumatoidpatients" is unlikely to be a consequence of thedecreased pH often noted in the joints of thesepatients. On the other hand the molecular weight ofGAG secreted by synovial cells at pH values near tonormal was considerably lower than expected fromour experience with these cells cultured in standardbicarbonate-buffered medium, though variability iscommon in these conditions. At this stage it is not


80.

-

:z

_ 60-

u 40.0-zC-

c) 20-

O,

Ext race ll Ilar

7-08 724 7.55 7-74

Fig. 6 Influence ofpH on intra- and extrcactivity ofNAG. The cells were cultured for+ 10% HS. NAG was determined as in 'Mmethods'. Vertical bars are means from 4 rcultures (+ SEM).

80-

I-

z

60_l-

c-

u 40-

0-

a 20nr-

0A6-5 7-0

pHFig. 7 NAG activity in culture supernataniand after (i) 48 h dialysis at 4°C against 0tris-HCI, pH 7-0, with 0-15 M NaCI andCaCI2. The dialysis equalised each supemaPoints are means of determinations from 4cultures (± SEM).

Iiit iace lILi ar

708 7-24 7.55 7.742cellular48 h in EBM

Table 2 Effect of variation pH in culture media on intra-and extracellular activities ofNAG from synovial cellscultured in serum-free medium *

Initial Final NAGtpH pH

Intracellular Extracellular Total(mean + SEM) (mean t SEM)(n = 4) (n = 4)

6 50 6-61 107-4 ± 8 5 76-7± 2-7 184-16 95 7-19 135-6 + 5 2 61-2± 2-1 196-87-37 7-62 125-5 ± 07 43-3 07 168-8755 7-71 133-8 4-7 32-4 1-5 165-27-74 7-82 146-2+ 2-3 26-6± 1-2 172-8

* The medium contained BSA at 4 mg/ml in EBM. Duration of theincubation was 48 h.t Activity of N-acetyl-B-glucosaminidase (NAG) was assayed bydeterminingp-nitrophenol released from the substrate as describedin 'Materials and methods'.

faterials and possible to specify whether the smaller moleculeseplicate produced at higher pH reflect impaired polymerisa-

tion during synthesis or extracellular breakdown.Degradation due to hyaluronidase is an unsatisfac-tory explanation, since release of hyaluronidase fromsynovial cells cannot be induced, even by substanceswhich produce gross stimulation of the lysosomalsystem."5 Another possibilty is the absorption ofenergy from beta emission in the particular condi-tions of pH and buffer composition gave rise toenhanced generation of free radicals, which can causedegradation of HA.21 From a practical viewpoint theenhanced secretion at low pH suggests a means foroptimising the producton of high-grade HA for otherexperimental purposes.The swollen organelles seen by phase contrast in

synovial cells grown in low pH media included struc-tures clearly identifiable by electron microscopy aslysosomes, and also bodies of about the same size asthe lysosomes, but lacking recognisable features.These bodies bore a resemblance to the so-calledtype 1 inclusions noted by Lie et al.22 in human skinfibroblasts, which, however, were produced in mediaof unusually high pH. In both cases the accumulationof these obscure bodies in the cytoplasm might signifyincipient degenerative changes, though in the case of

F ***t the synovial cells such changes would not be consis-7-5 8.0 tent with the sustained growth rates. The apparent

swelling of lysosomes in response to low pH occurredwithout increase in intracellular activity of NAG.However, there was a pronounced increase in

ts before (0) extracellular NAG, and the fraction released there->.0 M fore appears to be newly synthesised enzyme sec-0-001 M reted without storage. The mechanism might involveztant to pH 7*0. passive loss in association with increased pinocytosisreplicate or a controlled exocytosis. This is an area for further

investigation.


We have previously found that release of NAG byhuman synovial cells can be promoted by adenosine23and also by E or F type prostaglandins,24 which areputative mediators of inflammation. More recently asimilar reaction was found to occur in response to asoluble factor produced by human peripheral bloodmonocyte-macrophages (Clarris and Hamilton,unpublished results).The total lysosomal response in the inflamed joint

might therefore be due to many factors, perhapsamplified by interactions. Though the lysosomalcomplex contains proteolytic enzymes such ascathepsin B, which is potentially capable of acting inthe breakdown of cartilage matrix,4 25 the activity ofthese enzymes is in general likely to be blocked afterrelease owing to unfavourable pH, except perhaps inthe most extreme cases of pH depression. On theother hand, as suggested by Dingle,26 much lower pHvalues are possible in microenvironments betweenclosely opposed tissues at cartilage-pannus junctions.Lowered pH in these locally protected sites mightinduce the release and enhance the activity oflysosomal enzymes from intimal cells while partiallysuppressing the activity of neutral proteases such ascollagenase and plasminogen activator. An analo-gous model for this hypothetical situation has beendescribed by Vaes,27 in which degradation of matrixby osteoclasts correlates more closely with lysosomalhydrolases released into protective resorption zonesthan with enzymes favoured by neutral pH.

This work was supported by a grant from the National Health andMedical Research Council of Australia. The authors thank Dr K.Granath, Pharmacia AB, Uppsala, for permission to use calibrationdata for determination of the molecular weight of hyaluronic acid.

References

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2 Treuhaft P S, McCarty D J. Synovial fluid pH, lactate, oxygenand carbon dioxide partial pressure in various joint tissues.Arthritis Rheum 1971; 14: 475-84.

3 Farr M, Kendall M J, Bold A M, Hawkins C F. Significance ofpH in synovial fluid. Ann Rheum Dis 1981; 40: 621.

4 Eeckout Y, Vaes G. Further studies on the activation ofprocollagenase, the latent precursor of bone collagenase. Effectsof lysosomal cathepsin B, plasmin and kallikrein. Biochem J1977; 166: 21-31.

5 Muirden K D. Lysosomal enzymes in synovial membrane inrheumatoid arthritis. Ann Rheum Dis 1972; 31: 265-71.

6 Wegelius 0, Klockars M, Vainio U. Acid phosphatase activity inrheumatoid synovium. Acta Med Scand 1968; 183: 549-51.

7 Smith C, Hamerman D. Acid phosphatase in human synovialfluid. Arthritis Rheum 1962; 5: 411-4.

8 Beckman G, Beckman L, Lempery R. Acid phosphatase activity

in the synovial fluid of patients with rheumatoid arthritis andother disorders. Acta Rheumatol Scand 1971; 17: 47-56.

9 Jacox R F, Feldmahn A. Variations of B-glucuronidase concen-tration in abnormal human synovial fluid.J Clin Invest 1955;34:263-67.

10 Caygill J C, Pitkeathly D A. A study of 1-acetylglucosaminidaseand acid phosphatase in pathological joint fluids. Ann RheumDis 1966; 25: 137-44.

11 Castor C W, Prince R K, Hazelton M J. Hyaluronic acid inhuman synovial effusions; a sensitive indicator of altered con-nective tissue cell function during inflammation. ArthritisRheum 1966; 9: 783-94.

12 Fraser J R E, McCall J F. Culture of synovial cells in vitro. AnnRheum Dis 1965; 24: 351-9.

13 Clarris B J, Fraser J R E. Relationship between chromosomalchanges and alterations in the behaviour of a strain of humansynovial cells during its life history in vitro. Ann Rheun Dis1968; 27: 597-603.

14 Ceccarini C. Effect of pH on plating efficiency, serum require-ment and incorporation of radioactive precursors into humancells. In Vitro 1975; 11: 78-86.

15 LeMarshall J, Fraser J R E, Muirden K D. Lysosomal activationby neutral saccharides in cell cultures of synovium. Ann RheumDis 1977; 36: 130-8.

16 Castor C W, Bignall M C, Hossler P A, Roberts D J. Connectivetissue activation XXI. Regulation of glycosaminoglycanmetabolism by lymphocyte (CTAP-I) and platelet (CTAP-III)growth factors. In Vitro 1981; 17: 777-85.

17 Clarris B J, Fraser J R E. The effects of homologous andheterologous whole serum upon multiplication of recently-isolated human synovial cells in culture.AustJ Exp Biol Med Sci1967; 45: 549-60.

18 Grossfeld H, Meyer K, Godman G. Differentiation of fibro-blasts in tissue culture as determined by mucopolysaccharideproduction. Proc Soc Exp Biol Med 1955; 8: 31-5.

19 Baxter E, Fraser J R E, Harris G S. Fractionation and recoveryof secretions of synovial cells synthesized in culture with radioac-tive precursors. Ann Rheum Dis 1973; 32: 35-40.

20 Von Kittlich P-D, Neupert G. Experimentelle Beeinflussung derSynthese Saurer Mucopolysaccharide (Glykosaminoglykane) inFibroblastenkulturen. IV Einfluss des pH-Wertes auf Zell-proliferation und MPS-muster. Ex Pathol (Jena) 1972; 7:117-24.

21 McCord J M. Free radicals and inflammation. Protection ofsynovial fluid by superoxide dismutase. Science 1974; 185:529-31.

22 Lie S 0, Schofield B H, Taylor H A, Dory S B. Structure andfunction of the lysosomes of human fibroblasts in culture.Dependence on medium pH. Pediatr Res 1973; 7: 13-19.

23 Fraser J R E, Clarris B J, Baxter E. Patterns of induced variationin the morphology, hyaluronic acid secretion, and lysosomalenzyme activity of cultured human synovial cells. Ann RheumDis 1979; 38: 287-94.

24 Clarris B J, Malcolm L P. Effect of prostoglandins E,, E2 and F,aon N-acetyl-#-glucosaminidase activities of human synovial cellsin culture.Ann Rheun Dis 1983; 42: 187-91.

25 Burleigh M C, Barrett A J, Lazarus G S. Cathepsin B1. BiochemJ 1974; 137: 387-98.

26 Dingle J T. The secretions of enzymes into the pericellularenvironment. Philos Trans R Soc Lond (Biol) 1975; 271:315-24.

27 Vaes G. Collagenase, lysosomes and osteoclastic bone resorp-tion. In: Woolley D E, Evanson J M, eds. Collagenase in normaland pathological connective tissues. Wiley and Sons, 1980:185-207.

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Stimulation of glycosaminoglycan production and