Effects of antiinflammatory drugs on soft agar growth of rabbit articular chondrocytes

Effects

of Antiinflammatory Drugs on Soft Agar Growth of Rabbit Articular Chondrocytes

By Yukio Kato, Katsuhiko

Sato, Tatsuya

Atsushi Shimazu, INDEX

WORDS:

matory

drugs.

Chondrocyte;

soft

agar:

antiinflam-

ESTRUCTION of articular cartilage is a feature of chronic inflammatory joint diseases such as osteoarthritis and rheumatoid arthritis. Nonsteroidal antiinflammatory drugs (NSAIDs) are used extensively for treatment of chronic inflammatory joint diseases. However, little information about the effect of NSAIDs on chondrocyte replication is currently available.

D

Koike, Kazuhisa Nakashima, and Masahiro

Wei Qun Yan,

lwamoto

Ideally, NSAIDs should not have any adverse effect on the proliferation of articular chondrocytes because chondrocyte replication is required for cartilage repair. Chondrocyte cultures may be useful to test the effect of antiarthritis drugs on chondrocyte replication. Chondrocytes maintained on plastic dishes readily transform into motile cells (Fig 1A) morphologically indistinguishable from fibroblasts,‘.2 and hence may not respond to potentially effective drugs. Chondrocyte dedifferentiation in conventional cultures may be due to the cells’ adaptation to the artificial substrates. Chondrocytes grown in the absence of a tissue culture surface in soft agar3 or in agarose gels (Fig 1B)4 retain the ability to synthesize large amounts of cartilage-characteristic proteoglycans3 and type II collagen4 for more than 3 weeks. In the present study we examine the effects of NSAIDs on the growth of rabbit articular chondrocytes in soft agar and in agarose gels. MATERIALS

AND

METHODS

Materials Basic fibrobiast growth factor (FGF) was purified from bovine pituitaries’ and generously supplied by Dr Denis Gospodarowicz (University of California, San Francisco). Bacto agar was purchased from Difco Laboratories (Detroit). Agarose (low gelling temperature, type VII) was from Sigma Chemical Co (St Louis). Fetal bovine serum was obtained from Gibco Laboratories (Grand Island. NY). Ham’s F- I2

Fig 1. Morphologic appearance of rabbit articular chondrocytes maintained on plastic dishes IA) or in agarose gels (B).

From the Departments of Biochemistry and MaxilloFacial Radiology, Faculty of Dentistry, Osaka University; and the Department of Orthopaedics. School of Medicine. Osaka City University, Osaka, Japan. Yukio Kato, DDS, PhD: Assistant Professor; Katsuhiko Sato: Graduate Student; Tatsuya Koike, MD: Research Associate; Kazuhisha Nakashima: Graduate Student; Wei Qun Yen, MD: Visiting Research Associate;Atsushj Shimazu: Graduate Student; Masahiro Iwamoto. DDS, PhD: Research Associate. Address reprint requests to Yukio Kato, DDS. PhD, Department of Biochemistry, Faculty of Dentistry. Osaka University, I-8, Yamadaoka, Suita, Osaka 565. Japan. :r) 1989 by Grune & Stratton, Inc. 0049-0172/89/1803-1003$5.00/O

SeminarsUIArthritisandRheumatism, Vol 18, No 3, Suppl 1 (February), 1989: pp 7-10

KATO ET AL

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Fig 2. Effects of NSAlDs on colony formation by rabbit articular chondrocytes in soft agar with (CL q) and without (HI FGF. 0, Number of small colonies whose diameter is between 0.1 and 0.2 mm; q. number of large colonies whose diameter is larger than 0.2 mm: n, number of colonies whose diameter is larger than 0.1 mm in cultures without FGF. Values are averages f SD for three to six cultures.

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medium was from Nissui Pharmaceutical Co (Tokyo). Plastic culture dishes were purchased from Terumo Co (Tokyo).

Chondrocytes Chondrocytes were isolated from articular cartilage of 4-week-old New Zealand rabbits with 0.1% collagenase (Sigma, type IA) as described by Shimomura et al.6

Colony Formation in Soft Agar or in Agarose Gels A I-mL base layer of 0.72% Bacto agar or agarose in Ham’s F-12 medium supplemented with 10% fetal bovine serum and antibiotics (medium A) was added to a 35-mm plastic Petri dish. Chondrocytes (2 or 5 x 10s) were suspended in 1 mL of 0.41% Bacto agar or agarose in medium A and used as an overlay.’ FGF (0.4 ng/mL) was added two days after cell seeding and every fourth day thereafter. NSAIDs (tiaprofenic acid, naproxen sodium, indomethacin, and acetylsalicylic acid) were dissolved in dimethyl sulfoxide (1 rL) and added once to the appropriate concentration three days after cell seeding. Control cultures received dimethyl sulfoxide (1 pL) alone. Cultures were maintained at 37OC under 5% CO, in air for seven or 14 days, and then the number of colonies was counted. In the present study, a cell colony is defined as a cluster of cells whose diameter is between 0. I and 0.2 mm (small colony) or larger than 0.2 mm (large colony). Previously, we showed that FGF induces the growth of differentiated chondrocytes in soft agar.’ In the absence of FGF, no colony formation by chick embryo and rabbit chondrocytes was observed. FGF induced high levels of colony formation within 2 weeks.’ We have also shown that poorly differentiated chondrocytes treated with vitamin A or bromodeoxyuridine did not form colonies in soft agar even in the presence of FGF.’ Therefore, colony formation in soft agar is a hallmark of differentiated chondrocytes. In contrast

to FGF, platelet-derived growth factor, insulinlike growth factor, and epidermal growth factor induced very low levels of colony formation.’ In the present study, we therefore used FGF to establish the mitogenic response in soft agar of rabbit articular chondrocytes.

Monolayer Cultures For preparation of low-density cultures on plastic tissue culture dishes, chondrocytes were seeded at IO’ cells per 35mm plastic dish and maintained in 2 mL of Eagle’s minimum essential medium supplemented with 10% fetal bovine serum and antibiotics. NSAIDs (in 1 pL of dimethyl sulfoxide) were added to give a final concentration of lo-’ mol/L three days after cell seeding. Control cultures received I rL of dimethyl sulfoxide alone. DNA content of cultures was measured on day 7.

DNA DNA was determined by a fluorometric method.’ RESULTS

Indomethacin, acetylsalicylic acid, naproxen, or tiaprofenic acid alone had little effect on colony formation by rabbit articular chondrocytes in soft agar in the absence of FGF (Fig 2). However, in the presence of FGF, indomethacin ( 10m6 to 10m4 mol/L), acetylsalicylate ( 10m4 mol/L), and naproxen ( 10d4 mol/L) decreased by 20% to 55% the efficiency in colony formation by chondrocytes in response to FGF (Fig 2). Furthermore, at the high concentrations, these drugs decreased colony size; a 40% to 65% decrease in the number of large colonies was observed (see Fig 2, solid bars). Tiaprofenic acid,

EFFECTS OF NSAIDS ON CHONDROCYTE GROWTH

Fig 3. Effects of NSAlDs on colony formation by rabbit articular chondrocytes in agarose gel. Chondrocytes 12 x 103) were maintained for seven days in agarose gel in the presence of various concentrations of NSAlDs with or without FGF. as described in Marerials and Methods. Values are averages for duplicate cultures.

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even at high concentrations (10m4 mol/L), had little effect on chondrocyte proliferation in soft agar in the presence of FGF (Fig 2). The effect of NSAIDs on chondrocyte proliferation in agarose gel was also examined. In agarose gel, rabbit articular chondrocytes rapidly proliferated even without FGF (in the presence of 10% fetal bovine serum) and formed colonies seven days after cell seeding (Fig 3), probably because pure agarose gel, unlike Bacto agar, does not inactivate serum growth factors.

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M Indomethacin, at concentrations higher than 10e5 mol/L, depressed the efficiency in colony formation of chondrocytes in the presence and absence of FGF by 60% to 80%. However, other NSAIDs including tiaprofenic acid, even at 10m4 mol/L, had little effect on chondrocyte proliferation in agarose gel (Fig 3). Rabbit articular chondrocytes were also seeded at low density on plastic dishes and maintained in 10% serum for seven days with or without 1O-~4mol/L NSAIDs. Figure 4 shows

Fig 4. Effects of NSAfDs on the growth of rabbit articular chondrocytes on plastic culture dishes. Chondrocytes were seeded IlO cells/3bmm dish) and maintained with 10% fetal bovine serum in the presence of lo-’ mol/L NSAIDs. Drugs were added three days after cell seeding. DNA content was determined on day 7. Values are averages for duplicate cultures.

10

KATO ET AL

that all drugs tested had no effect on the proliferation of chondrocytes on plastic dishes. DISCUSSION

The present study showed that indomethacin, naproxen, and acetylsalicylate suppress colony formation by chondrocytes in soft agar, although they have little effect on proliferation of chondrocytes on plastic tissue culture dishes. Chondrocytes grown on the artificial substrate became elongated (Fig 1A),whereas chondrocytes in soft agar or in agarose gels maintained a spherical configuration (Fig 1B). The morphologic alteration or loss of chondrocyte phenotypic expression in conventional cultures on plastic may account for the lack of the effect of the drugs on chondrocyte replication. Alternatively, certain drugs may suppress the synthesis of an extracel-

lular matrix that is required as an anchoring support for growth in the semiliquid medium.3 The present study also demonstrated that tiaprofenic acid, even at high concentrations, had little effect on chondrocyte growth in soft agar, in agarose gels, and in monolayer cultures on plastic dishes. This suggests that tiaprofenic acid might not directly inhibit proliferation of articular chondrocytes in vivo. Thus, important differences among antiinflammatory drugs with respect to their effect on chondrocyte proliferation are evident. Osteoarthritis and rheumatoid arthritis produce complex changes in cartilage metabolism, the pathophysiology of which is not fully understood. Studies of rabbit articular chondrocytes in soft agar or in agarose gels may be useful in analyzing responses of chondrocytes to inflammation and to antiinflammatory drugs.

REFERENCES 1. Chacko S, Abbot J, Holtzer H: Loss of phenotypic traits by differentiated cells. VI. Behaviour of the progeny of a single chondrocyte. J Exp Med 130:417-430, 1969 2. Kato Y, Gospodarowicz D: Effect of exogenous extracellular matrices on proteoglycan synthesis by cultured rabbit costal chondrocytes. J Cell Biol 100:486-495, 1985 3. Kato Y, Iwamoto M, Koike T: Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar. J Cell Physiol 133:491-498, 1987 4. Benya PD, Shaffer JD: Dedifferentiated chondrocytes

reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215-224, 1982 5. Gospodarowicz D, Cheng J, Lui G-M, et al: Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography: Identity with pituitary fibroblast growth factor. Proc Nat1 Acad Sci USA 81:6963-6967, 1984 6. Shimomura Y, Yoneda T, Suzuki F: Osteogenesis by chondrocytes from growth cartilage of rat rib. Calcif Tissue Int 19:179-187, 1975 7. Hinegardner RT: An improved fluorometric assay for DNA. Anal Biochem 39:197-201, 1971